Discovery of new imidazole[1,2-a] pyridine derivatives as CDK9 inhibitors: design, synthesis and biological evaluation

Abstract

Colorectal cancer (CRC) is a highly aggressive and extensive malignancy. Presently, targeting the transcriptional regulation of cyclin-dependent kinase 9 (CDK9) is a promising therapeutic approach. Herein, twenty-five compounds (LA-1–LA-13 and LB-1–LB-12) were designed and synthesized with AZD5438 as the lead compound using an imidazole[1,2-a] pyridine skeleton. Compound LB-1 exhibited potent CDK9 inhibition and induced apoptosis in the HCT116 cell line. Moreover, compared with AZD5438, LB-1 demonstrated highly selective CDK9 inhibitory activity, with an IC₅₀ value of 9.22 nM. Accordingly, compound LB-1 could be further developed as a selective, target-oriented CDK9 inhibitor for colorectal cancer.

A promising highly selective CDK9 inhibitor LB-1, with an imidazole[1,2-a] pyridine framework, was discovered for treatment of colorectal cancer.

Introduction

The incidence of colorectal cancer (CRC) worldwide exceeds 2.2 million new cases, which is predicted to rise to 3.2 million new cases by 2024.¹ CRC is typically managed with multimodal therapy, including chemotherapy, radiation and surgery.^2–4 As precision medicine advances, it has been discovered that targeting transcriptional regulation has a significant impact on cancer treatment.⁵ Dysregulated transcription enhances the expression of oncogenes, thus promoting malignant cell development.⁶ Recent progress in understanding transcription regulators and their role in cancer suggests that dysregulated transcription programs are critical in cancer, making cancer cells more sensitive to the inhibition of these regulators than normal cells.⁷ Consequently, targeting transcriptional regulation is a promising therapeutic strategy for solid malignancies, including CRC.⁸

Cyclin-dependent protein kinases (CDKs) are serine/threonine kinases that are mainly divided into two types by function: one type regulates the cell cycle and inhibits proliferation, including CDK1, CDK2, CDK4, and CDK6, and the other type is involved in transcriptional regulation of CDK7 and CDK9.^9,10 CDK9, in complex with cyclin T1, is involved in transcription elongation and collaborates with cyclin T1 to form the positive transcription elongation factor b (P-TEFb).¹¹ Current evidence indicates that CDK9 regulates the expression and activity of several critical tumorigenesis-associated proteins, including myeloid cell leukemia-1 (Mcl-1), myelocytomatosis oncogene (MYC), and X-linked inhibitor of apoptosis protein (XIAP).^12–15 Thus, Mcl-1, a member of the B-cell lymphoma protein 2 (Bcl-2) family, plays a crucial role in many cancers by isolating the apoptosis-inducing proteins BCL2 antagonist/killer (Bak) and Bcl-2-associated X protein (Bax), thereby hindering the activation of apoptosis-inducing proteins.¹⁶ Substantial evidence supports the notion that preventing CDK9 activation might play a crucial role in solid tumour therapy.^17–19 In recent decades, interest in targeting CDK9 as a promising therapeutic strategy for hematopoietic and solid malignancies has increased. Many CDK9 inhibitors, including AZD4573 developed by AstraZeneca,²⁰ VIP152 developed by Bayer,²¹ and GFH009 developed by SELLAS Life Sciences Group,²² have progressed into clinical trials for leukaemia and lymphoma (phase II) (Fig. 1) and exhibit specific cytotoxic effects.

Fig. 1. Structure of representative selective CDK9 inhibitors in clinical trials.

The correlation between CDK9 and CRC has been verified in previous studies.¹⁷ Among three colorectal cancer cell lines, the HCT116 cells exhibited significantly greater expression of CDK9 than normal colorectal cancer cells. Moreover, Western blotting results show that CDK9 is overexpressed in CRC cell lines. Furthermore, targeting CDK9 for the treatment of CRC serves as an effective strategy.

Design of novel CDK9 selective inhibitors

AZD5438 has advanced into phase l trials for solid and hematologic malignancies and demonstrates strong potency against CDK1, CDK2 and CDK9. Based on the structure of AZD5438, we analysed its binding modes with CDK9, which could expand the chemical toolbox for exploring CDK9 as a therapeutic target in CRC. Additionally, these inhibitors could serve as candidates to complement existing paradigms in drug discovery. As shown in Fig. 2, AZD5438 binds with the ATP-binding domain of the CDK9/cyclin T1 cocrystal structure (PDB ID: 4BCF). The imidazole structure could form π–π stacking interactions with Phe103, which is the key amino acid residue of the gatekeeper region. The core scaffold (the aminopyrimidine group) of AZD5438 displayed two strong hydrogen bonds in the hinge binding area with Cys 106. Methyl sulfoxide cannot adapt to the solvent-exposed region in CDK9 because of limited spatial hindrance, which can be used as a structural modification site.

Fig. 2. Binding modes of AZD5438 with CDK9/cyclin T1.

This work focused on the solvent region to design target compounds using a molecular extension strategy, scaffold hopping and mass effects (Fig. 3). First, we retained the basic pharmacophore to maintain biological activity. To improve the binding affinity of CDK9, we induced a substituted benzene moiety to explore how the property of the hydrophobic group affects its inhibitory activity against CDK9. According to a previous study,²³ the CDK family is highly homologous. Although CDK1 and CDK9 are structurally similar, the hinge region, G-loop area, and α-helix structures in the ATP-binding domain of CDK9 are more flexible than those of other CDK subtypes.²⁴ Therefore, various N-containing hydrophilic fragments have been used to adapt the flexible solvent-exposed region, improving the selectivity for CDK9.

Fig. 3. Design strategies for LA and LB series target compounds.

Results and discussion

Chemistry

Scheme 1 outlines the synthetic methodology for synthesizing target compounds LA-1–LA-13. Compounds LA-1–LA-13 were synthesized using a convergent synthesis method. First, in route (l), 2-amino-4-bromopyridine (1a) was used as the raw material, and 1a underwent a condensation reaction to obtain intermediate 3a. Simultaneously, 1a was cyclized to generate key intermediate 2a. In route (II), bromobenzene (4a–4m) with different substituents underwent a Miyaura borylation reaction to give borate compounds 5a–5m with different substituents. Through the Suzuki coupling reaction, intermediate 5a–5m and intermediate 3a to form intermediate 6a–6m. Subsequently, TFA was used to deprotect intermediate 6a–6m to obtain intermediate 7a–7m. The Buchwald reaction between intermediate 2a and intermediate 7a–7m resulted in 8a–8m. Intermediate 9a–9m was prepared through the hydrolytic reaction of Intermediate 8a–8m. Finally, carboxylic acid compounds 9a–9m and morpholine were subjected to amide condensation to obtain target compounds LA-1–LA-13. The synthetic routes of compounds LB-1–LB-12 are shown in Scheme 2. Intermediate 9m was obtained under the same conditions as intermediate 9a–9m general procedure. Carboxylic acid compounds 9a–9m react with different cyclic amines, yielding target compounds LB-1–LB-12.

Scheme 1. Synthesis of compounds LA-1–LA-13. Reagents and conditions: (a) bis(tert-butoxycarbonyl) oxide, t-BuOH, 40 °C, 12 h, 81.4%; (b) ethyl bromopyruvate, EtOH, reflux, 12 h, 72.4%; (c) bis(pinacolato) diboron, Pd(dppf)Cl₂, KOAc, dioxane, N₂, 100 °C, 8 h; (d) Pd(PPh₃)₄, dioxane/H₂O = 5 : 1 (V/V), N₂, 100 °C, 8 h; (e) TFA, DCM, 8 h, r.t.; (f) Pd₂(dba)₃, Xantphos, Cs₂CO₃, dry DMF, N₂, 8 h; (g) NaOH, EtOH, H₂O, reflux, 6 h; (h) morpholine, EDCI, HOBt, dry DMF, 61.2–71.2%.

Scheme 2. Synthesis of compounds LB-1–LB-12. Reagents and conditions: (h) various corresponding amines, EDCI, HOBt, dry DMF, 6 h, 79.5–85.5%.

Structural optimization and structure–activity relationship (SAR) analysis

With insights into the binding modes between CDK9 and ligands, we chose LA-1 as the starting point to develop a new series of CDK9 inhibitors, focusing on the structural diversification of the R₁ and R₂ groups. The inhibitory potency against CDK9 and CDK1/2/9 selectivity was employed as the main indicator for structural optimization and SAR analysis. The antiproliferative effects on HCT116, RKO and HT-29 CRC cells were determined to aid in screening promising candidates.

Although various substituted benzene moieties were replaced with R₁ moieties in the hydrophobic area, LA-13 exhibited remarkable potent activity (Ir = 65.99%, in 20 nM of compounds) compared with the positive control group AZD5438 (Ir = 55.23%). Moreover, LA-13 showed prominent anti-proliferative activity in the HCT116 cell line, with an IC₅₀ value of 1.80 μM, and the results of the cell activity test were consistent with those of the enzyme activity test. As shown in Table 1, compared with that of LA-1, the introduction of fluorine in o- or p-benzene could enhance the inhibitory activity of CDK9. Furthermore, the activity of introducing fluorine at the p-position (LA-4) was more potent than that of introducing fluorine at the o-position (LA-2). Surprisingly, increasing the number of fluorine molecules on the benzene ring had little effect on the improvement in potency, but the introduction of a methoxy group at the 2-position of the benzene ring (LA-5) substantially increased the CDK9 inhibition potency (Ir = 48.26%) and improved the antiproliferation activity against HCT116 cells (IC₅₀ = 2.20 μM). Based on the in vitro activity results, we studied the binding mode of LA-1, LA-4, LA-5 and LA-13 with the CDK9/cyclin T1 complex (PDB: 4BCF) via computer-aided drug design (CADD). As shown in Fig. 4A, LA-1 retains only two hydrogen bonds in the hinge binding area with Cys106 but fails to form π–π stacking interactions with Phe103. When 4-fluorine is on the benzene ring, two key hydrogen bonds remain, except that the 4-fluorine atom on the benzene ring forms an additional halogen bond interaction with Asp 167. The docking mode of LA-5 is consistent with that of the positive control drug AZD5438, in which π–π stacking and hydrogen bonding interactions are retained. When R₁ is disubstituted by 2-OCH₃-4-F, LA-13 retains the key hydrogen bonding in the hinge region and forms π–π stacking interactions with Phe103. Simultaneously, the 4-fluorine on the benzene ring increases the formation of an additional halogen bond with Asp 167, which leads to increased inhibition of CDK9. Furthermore, researchers overlapped the docking modes of LA-1 and LA-13, as shown in Fig. 4B. As expected, both LA-1 and LA-13 could bind with the ATP domain of CDK9 (PDB: 4BCF). However, various substituted benzene groups of LA-1 and LA-13 produce different binding affinities in the hydrophobic cavity inside the ATP-binding domain of CDK9. As a result of the binding mode, LA-13 produces a dihedral angle compared with that of LA-1, which reduces the distance between the benzene ring and ASP167 so that it forms an additional hydrogen bond.

Table 1. Inhibition rates of LA-1–LA-13 against CDK9 and IC₅₀ values of LA-1–LA-13 and AZD5438 against HCT116, RKO and HT-29 cell lines.

Compound	R 1	Ira (%)	IC50 (μM)
			HCT116	RKO	HT-29
LA-1	H	32.38	>5	3.09 ± 0.21	>5
LA-2	2-F	37.84	2.70 ± 0.15	3.29 ± 0.61	>5
LA-3	3-F	25.10	3.60 ± 0.53	2.60 ± 0.47	>5
LA-4	4-F	43.12	2.17 ± 0.85	1.75 ± 0.15	3.76 ± 0.26
LA-5	3-OCH3	48.26	2.20 ± 0.42	2.19 ± 0.34	4.23 ± 0.55
LA-6	4-OCH3	−2.71	>5	2.50 ± 0.25	>5
LA-7	3,4-di-F	24.97	3.61 ± 0.05	2.99 ± 0.18	>5
LA-8	2,4-di-F	42.29	2.45 ± 0.03	3.82 ± 0.23	4.02 ± 0.72
LA-9	3,4,5-tri-F	22.83	3.44 ± 0.12	2.37 ± 0.04	>5
LA-10	4-CH3	−6.71	>5	4.05 ± 0.20	>5
LA-11	3-CH3	3.06	>5	2.21 ± 0.05	>5
LA-12	2-Cl	46.97	>5	2.88 ± 0.24	>5
LA-13	2-OCH3-4-F	65.99	1.80 ± 0.47	2.27 ± 0.13	2.17 ± 0.34
AZD5438b	—	55.23	1.17 ± 0.18	0.85 ± 0.08	3.23 ± 0.33

^aValues are expressed as inhibition rates in 20 nM of compounds.

^b AZD5438 as the positive control.

Fig. 4. (A) Docking modes of LA-1, LA-4, LA-5, LA-13 and CDK9 (PDB: 4BCF). (B) Docking modes of LA-13 and LA-1 in the activity pocket of CDK9 (PDB: 4BCF). The grey represents LA-13 and the purple represents LA-1.

In the LB series, we designed and synthesized a series of compounds by utilizing a subset of R₁ groups employed in the LA series to confirm the summarized SAR at R₁ substituted benzene and position. In particular, the inhibitory activities of LB-1, LB-8 and LB-10 on CDK9 kinase were significantly higher than the positive control. (IC₅₀ = 9.22 nM, 5.25 nM, and 3.56 nM, respectively). As shown in Table 2, LB-1, LB-5 and LB-10 exhibited potent cytotoxicity activity on HCT116 (with high expression of CDK9-related mRNA) compared with AZD5438. (IC₅₀ = 0.92 μM, 1.50 μM, and 1.09 μM, respectively). Furthermore, LB-1 exhibited the best potent cytotoxicity against HCT119 with 0.92 μM.

Table 2. IC₅₀ values (nM) of LB-1–LB-12 against CDK9 and IC₅₀ values (μM) of LB-1–LB-12 and AZD5438 against HCT116, RKO and HT-29 cell lines.

Compound	R 2	IC50 (nM)	IC50 (μM)
			HCT116	RKO	HT-29
LB-1		9.22	0.92 ± 0.41	0.67 ± 0.16	2.94 ± 0.02
LB-2		24.05	1.65 ± 0.32	1.69 ± 0.15	3.65 ± 0.52
LB-3		17.52	1.54 ± 0.46	1.58 ± 0.33	>5
LB-4		242.50	2.60 ± 0.24	1.53 ± 0.28	3.89 ± 0.57
LB-5		20.57	1.50 ± 0.17	1.43 ± 0.51	4.10 ± 0.44
LB-6		385.3	2.36 ± 0.64	1.60 ± 0.14	>5
LB-7		124.8	2.63 ± 0.18	1.29 ± 0.35	>5
LB-8		5.25	3.91 ± 0.20	1.40 ± 0.05	>5
LB-9		15.85	1.78 ± 0.19	1.53 ± 0.07	2.76 ± 0.47
LB-10		3.56	1.09 ± 0.11	3.21 ± 0.43	3.12 ± 0.42
LB-11		27.42	1.73 ± 0.31	1.17 ± 0.05	>5
LB-12		51.61	1.78 ± 0.29	1.57 ± 0.36	>5
LA-13		18.76	2.12 ± 0.09	1.37 ± 0.57	3.24 ± 0.38
AZD5438a	—	19.21	1.38 ± 0.10	0.79 ± 0.54	4.10 ± 0.26

^a AZD5438 as the positive control.

In conclusion, the results indicated that LB-1 exhibited significant inhibitor activity in CDK9 and the strongest antiproliferative activity in HCT116 cells. To determine the CDK selectivity of LB-1, we assayed the inhibition of the activities of protein kinases representing members of different kinase classes (Table 3). Furthermore, the selectivity to CDK9 over CDK1 (>100 fold for LB-1), in which CDK1 and CDK9 have high homology,²⁵ was substantially improved in comparison to AZD5438. Based on these results, we studied the binding mode of LB-1 using a computer-aided drug design (CADD). LB-1 and positive control overlapped (Fig. 5), and this showed that LB-1 and positive control had similar binding mode in the ATP domain of CDK9. Analysis of the binding mode between LB-1 and CDK9 showed that LB-1 introduced a 4-piperidyl morpholine ring used to adapt the flexibility in the solvent-exposed area. Under environmental pH in tumour cells, the N atom of the morpholine ring protonated, which additionally increased the formation of hydrogen bond interaction with Ala 153.²⁶ Moreover, an N atom from the morpholine ring formed hydrogen bond interaction with Asp 109, which was deeply buried within the target, as indicated by the cocrystal. Accordingly, LB-1 is worthy of further investigation into the mechanism of tumour cell death.

Table 3. IC₅₀ values (nM) of LB-1 and AZD5438 against CDK 1, CDK 2 and CDK 9.

Compound	CDK1	CDK 2	CDK9
LB-1	>1000	63.08 ± 0.82	9.22 ± 0.74
AZD5438	13.34 ± 0.45	23.09 ± 1.05	18.25 ± 0.56

Fig. 5. Docking modes of LB-1 and AZD5438 in the activity pocket of CDK9 (PDB: 4BCF). The purple represents LB-1 and the grey represents AZD5438.

Cell colony formation assay

Colony formation was performed to evaluate the effects of LB-1 on the HCT116 cell line. As shown in Fig. 6, LB-1 decreased the number of colonies in a dose-dependent manner, and the results were similar to those of the positive control. At the same concentration, the anti-proliferative effect of LB-1 was slightly better than that of the positive control. This result confirmed the strong anti-proliferative effect of LB-1in vitro. Hence, it could be preliminarily determined that LB-1 had an expected effect on HCT116 cell function.

Fig. 6. Effect of LB-1 and AZD5438 on HCT116 cell colony number. AZD5438 was used as a control. All data are representative of three independent experiments and are shown as mean ± SD. The significance of the differences between the vehicle group and administration groups was determined by the one-way ANOVA test. Columns, means, bars, standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001.

LB-1 induces apoptosis in HCT116 cell

Annexin V-FITC/PI double-staining flow cytometry was used to investigate the mechanism of the effects of LB-1 on the HCT116 cell line. As shown in Fig. 7A, the percentages of total apoptotic cells (Q1-UR + Q1-LR) were 1.44%, 9.19% and 19.80% when the drug concentrations were 0.5, 1.0 and 2.0 μM, respectively. The results showed that LB-1 can induce the apoptosis of HCT116 cells in a concentration-dependent manner. Furthermore, caspase 3 is a key enzyme in the process of apoptosis.²⁷ The results of the GreenNuc living cell caspase 3 assay kit for live cells revealed that the activity of caspase-3 tended to increase as the concentration (0.5 μM, 1.0 μM, and 2.0 μM) increased (Fig. 7B), indicating a caspase 3-dependent mechanism of apoptosis induction.

Fig. 7. (A) Effects of compound LB-1 and AZD5438 on the induction of apoptosis in HCT116 cells at concentrations of 0, 0.5, 1.0 and 2.0 μM for 48 h, followed by loading with Annexin V-FITC /PI fluorescent probe before analysis. (B) Effects of LB-1 on caspase-3 activity in HCT116 cells at concentrations of 0, 0.5, 1.0 and 2.0 μM for 48 h, followed by incubation with GreenNuc™ caspase 3 substrate before analysis. All data are representative of three independent experiments and are shown as mean ± SD. The significance of the differences between the vehicle group and administration groups was determined by the one-way ANOVA test. Columns, means, bars, and standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Intracellular reactive oxygen species (ROS) detection

Mitochondrial dysfunction is often accompanied by increased ROS production. Targeting mitochondria to increase ROS levels is an effective antitumour approach. The fluorescence probe DFCH-DA was used to measure the amount of intracellular ROS in HCT116 cells. This dye is a stable compound that easily penetrates the cell, breaks down by intracellular esterase and converts to DCFH.²⁸ As previously described, after the culture and differentiation of HCT116 cells, LB-1 at different concentrations (0.5–2 μM) was incubated with the cells for 48 hours. According to the flow cytometry results, the level of reactive oxygen species (ROS) increased significantly in a concentration-dependent manner (Fig. 8). These findings preliminarily proved that LB-1 could improve ROS levels in HCT116 cells in a concentration-dependent manner.

Fig. 8. Effects of LB-1 on the ROS levels of HCT116 cells. HCT116 cells were treated with LB-1 at concentrations of 0, 0.5, 1.0 and 2.0 μM for 48 h and loaded with a DCFH-DA fluorescent probe before analysis. All data are representative of three independent experiments and are shown as mean ± SD. The significance of the differences between the vehicle group and administration groups was determined by the one-way ANOVA test. Columns, means, bars, standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Human liver microsomal stability, plasma stability and CYP inhibition

LB-1 was then subjected to in vitro studies to evaluate its metabolic properties. As shown in Table 4, LB-1 exhibits moderate metabolic stability. (t_1/2 = 75.3 min). As illustrated in Table 5, LB-1 has a plasma protein bound rate of 98.4%. Additionally, the inhibitory effects of LB-1 on three key cytochrome P450 (CYP) enzymes—CYP1A2, CYP2D6, and CYP3A4—were evaluated. The findings, as detailed in Table 6, show that LB-1 inhibits CYP2D6 with an IC₅₀ value of 1.57 μM. However, LB-1 did not exhibit any inhibitory activity against CYP1A2 and CYP3A4. Consequently, these results suggest that LB-1 has the potential for drug–drug interactions with medications metabolized by CYP2D6.²⁹

Table 4. Metabolic stability in human liver microsomes^a.

Compound	t 1/2 (min)	Clint (mL min−1 mg−1)	R 2
LB-1	75.3	0.0460	0.9954

^a t _1/2, half-life; Cl_int, intrinsic clearance.

Table 5. Plasma protein binding rate of LB-1.

Compound	F u (%)	F b (%)
Warfarin	0.543	99.5
LB-1	1.640	98.4

Table 6. In vitro CYP inhibition of LB-1.

Compound	IC50 (μM)
LB-1	1A2	2D6	3A4
	>10	1.57	>10

Preliminary evaluation of drug-like properties and in vivo PK study

In vivo pharmacokinetic (PK) properties of LB-1 were further assessed through both intravenous (i.v.) and oral (p.o.) administration routes. The data from these studies, as detailed in Table 7, indicated that LB-1 exhibits higher AUC_0-int (921 h ng mL⁻¹ for i.v. and 185 h ng mL⁻¹ for p.o.) when administered intravenously compared to orally owing to its poor oral bioavailability (F = 4.02%). Consequently, the focus of subsequent research will be on employing chemical modification strategies to enhance the bioavailability of LB-1, while preserving its enzyme inhibitory and antiproliferative activities.

Table 7. Pharmacokinetic parameters in SD rats^a.

Compound	t 1/2 (h)	t max (h)	C max (ng mL−1)	AUC0−t (h ng mL−1)	AUC0-inf (h ng mL−1)	Cl (mL min−1 kg−1)	MRTinf (h)	V ss (L kg−1)	F (%)
LB-1(i.v.)	1.29	—	—	914	921	914	1.19	—	—
LB-1(p.o.)	7.77	1	56.3	172	185	939	5.57	2.58	4.02

^a t _1/2, half-life; t_max, time of maximum concentration; C_max, maximum concentration; AUC_0−t, area under the plasma concentration−time curve from the start of dosing to the last point; AUC_0-inf, area under the plasma concentration−time curve from the start of dosing extrapolated to infinite time; Cl, clearance; MRT_inf, mean residence time from the start of dosing extrapolated to infinite time; and V_ss, steady-state volume of distribution. Dose: p.o. at 10.0 mg kg⁻¹, i.v. at 2.0 mg kg⁻¹ (n = 3 per group).

Conclusion

In conclusion, we successfully synthesized a series of twenty-five compounds, designated as LA-1–LA-13 and LB-1–LB-12, utilizing an imidazole[1,2-a] pyridine framework. These compounds have been identified as potential, selective, and potent CDK9 inhibitors that effectively curb the proliferation of colorectal cancer (CRC) cells. Through molecular docking studies, we discovered that CDK9 possesses a flexible solvent region within its active site, which accommodates the ligand's side chain flexibility, distinguishing it from other CDK subtypes. Our efforts culminated in the identification of LB-1, a compound that demonstrated low-nanomolar inhibitory potency against CDK9 activity and significant cytotoxic effects on HCT116, RKO, and HT-29 CRC cell lines at concentrations of 0.92 μM, 0.67 μM, and 2.94 μM, respectively. Notably, LB-1 showed a high degree of selectivity for CDK9, with an IC₅₀ of 9.22 nM, surpassing the pan CDK1/2/9 inhibitor AZD5438. Furthermore, LB-1 triggers apoptosis through the activation of caspase 3 in HCT116 cells, which is different from traditional CDK inhibitors. In vivo studies, the results exhibited the intravenously injection available property of LB-1. Accordingly, LB-1 might be a promising selective CDK9 inhibitor.

Materials and methods

Chemistry

The chemical reagents and solvents used in the experiment are commercially available chemically pure or analytically pure and have not been further purified except for special requirements. Thin layer chromatography (TLC, GF254, 0.5 mm) was used for reaction monitoring, and 200–300 mesh silica gel was used for column chromatography. The melting point of the compound was determined by applying the BÜCHI B-540 melting point instrument. ¹H-NMR and ¹³C-NMR were measured by applying a Bruker ARX-400/600 nuclear magnetic resonance instrument, and the deuterated reagent was TMS internal standard. High resolution mass spectrometry (ESI-HRMS) was tested by applying an Agilent 6530 accurate mass four-bar time-of-flight mass spectrometer (CV = 30 V).

Synthesis of intermediates of compounds LA-1–LA-13

Synthesis of intermediate ethyl 7-bromoimidazo[1,2-a]pyridine-3-carboxylate (2a)

2-Amino-4-bromopyridine (1a, 1.0 g, 5.78 mmol) was dissolved in 55.0 mL of absolute ethanol. Then, ethyl chloroacetate (1.74 g, 11.56 mmol) was slowly added to the reaction solution in batches while stirring at room temperature. The solution was condensed, refluxed, and stirred for 12 h, and the reaction was monitored by TLC. After monitoring the reaction, the reaction solution was naturally cooled to room temperature, petroleum ether (50 mL) was added to the reaction solution, the solids were precipitated, suction filtration was performed, the filtrate was discarded, and the filter cake was washed with petroleum ether (30 mL × 3). The filter cake was dried to obtain a light yellow solid (1.13 g, 72.4% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 9.10 (d, J = 7.3 Hz, 1H), 8.27 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 7.3, 2.0 Hz, 1H), 4.36 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H); ¹³C NMR (151 MHz, DMSO-d₆) δ 159.81, 148.12, 141.65, 128.30, 121.59, 119.88, 118.39, 115.81, 60.56, 14.40; ESI-MS (m/z): 269.0 ((M + H)⁺), 271.0 ((M + 3H)⁺).

Synthesis of intermediate tert-butyl(4-bromopyridine-2-yl) carbamate (3a)

2-Amino-4-bromopyridine (1a, 10.0 g, 57.80 mmol) was dissolved in 80.0 mL of tert-butanol. Then, di-tert-butyl decarbonate (13.16 mL, 63.58 mmol) was slowly dropped into the reaction solution and stirred at 40 °C for 12 h. After the reaction was monitored by TLC, the filtrate was filtered, the filter cake was washed with petroleum ether (30 mL × 3), and the filter cake was dried to obtain a white solid (12.80 g, 81.4% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 10.09 (s, 1H), 8.14 (d, J = 5.3 Hz, 1H), 8.04 (d, J = 1.8 Hz, 1H), 7.26 (dd, J = 5.3, 1.8 Hz, 1H), 1.46 (s, 9H); ¹³C NMR (151 MHz, DMSO-d₆) δ 153.51, 152.65, 149.19, 132.96, 121.17, 114.67, 80.13, 27.94; ESI-MS (m/z): 273.1 ((M + H)⁺), 275.0 ((M + 3H)⁺).

General chemical experimental synthesis of intermediate 5a–5m

Bromobenzene 4a–4m (1.0 eq.) was dissolved with different substituents in 70.0 mL of dry 1,4-dioxane solution. Then, pinacol biborate (1.5 eq.), potassium acetate (2.0 eq.) and Pd(dppf)Cl₂ (0.05 eq.) were added in turn. After the reaction was monitored by TLC, the reaction solution was naturally cooled to room temperature, and the insoluble impurities were removed by suction filtration. The filtrate was extracted with ethyl acetate (30 mL × 3), and the organic phases were combined. After washing with saturated salt solution (15 mL × 3), it was concentrated to obtain brownish yellow oil. After simple purification by column chromatography (PE : EA = 10 : 1), the palladium residue was removed to obtain a yellow oil 5a–5m.

General chemical experimental synthesis of intermediate 6a–6m

The intermediate 5a–5m (1.5 eq.) was dissolved in the mixed solvent of 1,4-dioxane/water (5 : 1), and the intermediate tert-butyl (4-bromopyridine-2-yl) carbamate (3a, 1.0 eq.), potassium phosphate (2.0 eq.), Pd (PPh₃)₄ (0.05 eq.) were sequentially added. After the reaction was completed, it was monitored using TLC. The reaction solution was extracted with ethyl acetate (30 mL × 3). The organic layers were combined, and Na₂SO₄ was dried overnight. Na₂SO₄ solid was removed by filtering, and the filtrate was concentrated, separated and purified by column chromatography (PE : EA = 15 : 1) to obtain white solid 6a–6m.

Intermediate tert-butyl (4-phenylpyridine-2-yl) carbamate (6a)

White solid, 72.1%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.88 (s, 1H), 8.30 (d, J = 5.2 Hz, 1H), 8.10 (d, J = 1.7 Hz, 1H), 7.72–7.70 (m, 2H), 7.55–7.51 (m, 2H), 7.49–7.46 (m, 1H), 7.33 (dd, J = 5.3, 1.6 Hz, 1H), 1.49 (s, 9H); ESI-MS (m/z): 271.1 ((M + H)⁺).

Intermediate tert-butyl (4-(2-fluorophenyl) pyridine-2-yl) carbamate (6b)

White solid, 66.3%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.92 (s, 1H), 8.34 (dd, J = 5.2, 0.8 Hz, 1H), 8.01 (q, J = 1.2 Hz, 1H), 7.59 (td, J = 7.8, 1.7 Hz, 1H), 7.52 (dd, J = 8.3, 7.1, 5.2, 1.8 Hz, 1H), 7.39–7.35 (m, 2H), 7.21 (dt, J = 5.1, 1.6 Hz, 1H), 1.48 (s, 9H); ESI-MS (m/z): 289.0 ((M + H)⁺).

Intermediate tert-butyl (4-(3-fluorophenyl) pyridine-2-yl) carbamate (6c)

White solid, 67.4%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.93 (s, 1H), 8.32 (d, J = 5.2 Hz, 1H), 8.09 (d, J = 1.7 Hz, 1H), 7.58–7.54 (m, 3H), 7.36 (dd, J = 5.2, 1.7 Hz, 1H), 7.32 (ddt, J = 9.0, 5.9, 2.4 Hz, 1H), 1.49 (s, 9H); ESI-MS (m/z): 289.0 ((M + H)⁺).

Intermediate tert-butyl (4-(4-fluorophenyl) pyridine-2-yl) carbamate (6d)

White solid, 65.9%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.92 (s, 1H), 8.30 (d, J = 5.2 Hz, 1H), 8.08 (d, J = 1.6 Hz, 1H), 7.78–7.75 (m, 2H), 7.37–7.33 (m, 2H), 7.31 (dd, J = 5.2, 1.7 Hz, 1H), 1.48 (s, 9H); ESI-MS (m/z): 289.1 ((M + H)⁺).

Intermediate tert-butyl (4-(2-methoxyphenyl) pyridine-2-yl) carbamate (6e)

White solid, 61.2%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.89 (s, 1H), 8.30 (d, J = 5.2 Hz, 1H), 8.07 (d, J = 1.6 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.33 (dd, J = 5.1, 1.7 Hz, 1H), 7.28–7.25 (m, 1H), 7.22 (t, J = 2.1 Hz, 1H), 7.05 (dd, J = 8.2, 2.5 Hz, 1H), 3.83 (s, 3H), 1.49 (s, 9H); ESI-MS (m/z): 301.1 ((M + H)⁺).

Intermediate tert-butyl (4-(4-methoxyphenyl) pyridine-2-yl) carbamate (6f)

White solid, 63.1%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.82 (s, 1H), 8.25 (d, J = 5.3 Hz, 1H), 8.06 (d, J = 1.6 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.29 (dd, J = 5.2, 1.7 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 3.81 (s, 3H), 1.49 (s, 9H); ESI-MS (m/z): 301.1 ((M + H)⁺).

Intermediate tert-butyl (4-(3,4-difluorophenyl) pyridine-2-yl) carbamate (6g)

White solid, 65.7%. ¹H NMR (600 MHz, DMSO-d₆) δ 9.93 (s, 1H), 8.34–8.30 (m, 1H), 8.07 (d, J = 2.3 Hz, 1H), 7.83 (ddd, J = 11.9, 7.4, 2.0 Hz, 1H), 7.62–7.57 (m, 2H), 7.35 (dd, J = 5.3, 1.7 Hz, 1H), 1.50 (s, 9H); ESI-MS (m/z): 307.0 ((M + H)⁺).

Intermediate tert-butyl (4-(2,4-difluorophenyl) pyridine-2-yl) carbamate (6h)

White solid, 64.1%.¹H NMR (600 MHz, DMSO-d₆) δ 9.93 (s, 1H), 8.33 (d, J = 5.2 Hz, 1H), 7.98 (d, J = 1.5 Hz, 1H), 7.65 (td, J = 8.8, 6.5 Hz, 1H), 7.43 (ddd, J = 11.5, 9.3, 2.6 Hz, 1H), 7.25 (td, J = 8.6, 2.9 Hz, 1H), 7.18 (dt, J = 5.2, 1.6 Hz, 1H), 1.47 (s, 9H); ESI-MS (m/z): 307.1 ((M + H)⁺).

Intermediate tert-butyl (4-(3,4,5-trifluorophenyl) pyridine-2-yl) carbamate (6i)

White solid, 61.9%.¹H NMR (600 MHz, DMSO-d₆) δ 9.95 (s, 1H), 8.34 (d, J = 5.2 Hz, 1H), 8.04 (d, J = 1.7 Hz, 1H), 7.72 (dd, J = 9.0, 6.6 Hz, 2H), 7.37 (dd, J = 5.3, 1.7 Hz, 1H), 1.50 (s, 9H); ESI-MS (m/z): 325.1 ((M + H)⁺).

Intermediate tert-butyl (4-(4-methylphenyl) pyridine-2-yl) carbamate (6j)

White solid, 71.2%.¹H NMR (600 MHz, DMSO-d₆) δ 9.84 (s, 1H), 8.27 (dd, J = 5.2, 0.7 Hz, 1H), 8.10–8.06 (m, 1H), 7.65–7.60 (m, 2H), 7.36–7.30 (m, 3H), 2.36 (s, 3H), 1.49 (s, 9H); ESI-MS (m/z): 285.2 ((M + H) ⁺).

Intermediate tert-butyl (4-(2-methylphenyl) pyridine-2-yl) carbamate (6k)

White solid, 70.3%.¹H NMR (600 MHz, DMSO-d₆) δ 9.89 (s, 1H), 8.29 (d, J = 5.2 Hz, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.53–7.47 (m, 2H), 7.40 (t, J = 7.6 Hz, 1H), 7.31–7.26 (m, 2H), 2.39 (s, 3H), 1.49 (s, 9H); ESI-MS (m/z): 285.1 ((M + H)⁺).

Intermediate tert-butyl (4-(2-chlorophenyl) pyridine-2-yl) carbamate (6l)

White solid, 66.8%.¹H NMR (600 MHz, DMSO-d₆) δ 9.95 (s, 1H), 8.32 (d, J = 5.1 Hz, 1H), 7.86 (s, 1H), 7.62–7.57 (m, 1H), 7.48–7.42 (m, 3H), 7.09 (dd, J = 5.0, 1.6 Hz, 1H), 1.46 (s, 9H); ESI-MS (m/z): 305.1 ((M + H)⁺).

Intermediate tert-butyl (4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) carbamate (6m)

White solid, 64.7%.¹H NMR (600 MHz, DMSO-d₆) δ 9.80 (s, 1H), 8.23 (d, J = 5.1 Hz, 1H), 7.87 (d, J = 1.5 Hz, 1H), 7.36 (dd, J = 8.5, 6.9 Hz, 1H), 7.11 (dd, J = 5.1, 1.6 Hz, 1H), 7.06 (dd, J = 11.4, 2.5 Hz, 1H), 6.89 (td, J = 8.3, 2.5 Hz, 1H), 3.80 (s, 3H), 1.46 (s, 9H); ESI-MS (m/z): 319.1 ((M + H)⁺⁾.

General chemical experimental synthesis of intermediate 7a–7m

Intermediate 6a–6m was dissolved in a dry mixed solvent of DCM/TFA acid (V/V = 1 : 1), stirred at room temperature for 6 h, and monitored by TLC. After the reaction was monitored, the reaction solution was concentrated by rotary evaporation and dissolved by adding water, and the pH was adjusted to 8–9 with 1 N NaOH solution. Filtering, discarding the filtrate, and drying the filter cake to obtain the intermediate 7a–7m were performed, which is a white solid and can be directly used as a reactant for the next reaction without any purification.

General chemical experimental synthesis of intermediate 8a–8m

Intermediate 7a–7m (2.0 eq.), intermediate 7-bromoimidazo [1,2-a] pyridine-3-carboxylate (2a, 1.2 eq.), Pd₂(dba)₃ (0.05 eq.), and 4,5-bis (diphenyl phosphine) were added in turn. After monitoring the reaction, it was cooled to room temperature, extracted with dichloromethane (30 mL × 3), combined with organic layers, washed with saturated saline (15 mL × 3) and concentrated. The metal catalyst was removed by rapid column chromatography (DCM : MeOH = 10 : 1), and the ester intermediates 8a–8m were obtained as yellow solids, which were directly used in the next reaction.

General chemical experimental synthesis of intermediate 9a–9m

Intermediate 8a–8m (1.0 eq.) was dissolved in EtOH, and NaOH (10.0 eq.) aqueous was dropwise added to the reaction solution. After refluxing and stirring for 6 h, the reaction was monitored using TLC. After monitoring the completion of the reaction, the reaction solution was concentrated, water was added, the pH was adjusted to 5–6 with 2 N HCl solution, many solids were precipitated, and suction filtration was performed to obtain carboxylic acid intermediates 9a–9m, which are light yellow solids and can be directly used in the next step without purification.

Synthesis of compound LA-1–LA-13

Synthesis of compound morpholinyl (7-((4-phenylpyridine-2-yl) amino) imidazole [1,2-a] pyridine-3-yl) ketone (LA-1)

Carboxylic acid intermediate 9a (0.5 g, 1.51 mmol) was dissolved in dry DMF. Then, HOBt (0.25 g, 1.82 mmol), EDCI (0.35 g, 1.82 mmol) and morpholine (0.16 g, 1.82 mmol) were added into the reaction solution in turn, and the reaction was monitored by TLC. After monitoring the reaction, the reaction solution was extracted with DCM (30 mL × 3), and the organic layers were combined, washed with saturated saline (15 mL × 3) and concentrated. Separation and purification by column chromatography (DCM : MeOH = 25 : 1) white solid (0.78 g, 88.4%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.73 (s, 1H), 8.81 (d, J = 7.5 Hz, 1H), 8.51 (d, J = 2.3 Hz, 1H), 8.38 (d, J = 5.3 Hz, 1H), 7.89 (s, 1H), 7.73 (dd, J = 7.1, 1.8 Hz, 2H), 7.54 (t, J = 7.6 Hz, 2H), 7.48 (dd, J = 8.5, 6.2 Hz, 1H), 7.21 (d, J = 5.7 Hz, 2H), 7.10 (dd, J = 7.6, 2.3 Hz, 1H), 3.77–3.62 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.75, 156.31, 149.11, 148.88, 148.51, 140.90, 138.10, 138.08, 129.72, 129.67, 128.12, 127.10, 116.34, 114.25, 109.56, 108.70, 99.48, 66.68, 45.28; HRMS-ESI (m/z): calcd. for C₂₃H₂₁N₅O₂, (M + H) ⁺: 399.1695 found, 400.1792.

Synthesis of compound (7-((4-(2-fluorophenyl) pyridine-2-yl) amino) imidazole [1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-2)

LA-2 was under the same condition as compound LA-1. White solid (0.66 g, 86.9%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.75 (s, 1H), 8.81 (dd, J = 7.4, 0.7 Hz, 1H), 8.50 (dd, J = 2.1, 0.8 Hz, 1H), 8.39 (d, J = 5.3 Hz, 1H), 7.89 (s, 1H), 7.62–7.52 (m, 3H), 7.36–7.29 (m, 1H), 7.24 (dd, J = 5.4, 1.6 Hz, 1H), 7.20 (d, J = 1.6 Hz, 1H), 7.10 (dd, J = 7.6, 2.3 Hz, 1H), 3.77–3.62 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.76, 156.33, 148.86, 148.66, 147.74, 140.82, 140.64, 140.58, 138.10, 131.78, 131.73, 128.15, 123.25, 116.50, 116.37, 114.23, 114.07, 113.92, 109.78, 108.70, 99.59, 66.69, 45.33; HRMS-ESI (m/z): calcd. for C₂₃H₂OFN₅O₂, (M+H) ⁺: 417.1601 found, 418.1701.

Synthesis of compound (7-((4-(3-fluorophenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-3)

LA-3 was under the same condition as compound LA-1. White solid. (0.65 g, 85.3%).¹H NMR (600 MHz, DMSO-d₆) δ 9.78 (s, 1H), 8.82 (d, J = 7.5 Hz, 1H), 8.52 (d, J = 2.1 Hz, 1H), 8.41 (d, J = 5.3 Hz, 1H), 7.90 (s, 1H), 7.64 (td, J = 7.8, 1.8 Hz, 1H), 7.53 (dddd, J = 8.7, 7.1, 5.1, 1.8 Hz, 1H), 7.46–7.30 (m, 2H), 7.16 (d, J = 1.6 Hz, 1H), 7.10 (ddt, J = 5.3, 2.9, 1.8 Hz, 2H), 3.77–3.64 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.74, 160.50, 158.85, 155.97, 148.84, 148.23, 144.33, 140.79, 138.08, 131.58, 131.52, 130.84, 130.82, 128.12, 126.31, 126.23, 125.76, 125.73, 116.98, 116.83, 116.36, 116.01, 111.97, 111.94, 108.70, 99.57, 66.68, 45.31; HRMS-ESI (m/z): calcd. for C₂₃H₂OFN₅O₂, (M + H) ⁺: 417.1601 found, 418.1699.

Synthesis of compound (7-((4-(4-fluorophenyl) pyridine-2-yl) amino) imidazole [1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-4)

LA-4 was under the same condition as compound LA-1. White solid. (0.66 g, 87.4%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.77 (s, 1H), 8.82 (d, J = 7.5 Hz, 1H), 8.52 (d, J = 2.3 Hz, 1H), 8.38 (d, J = 5.3 Hz, 1H), 7.91 (s, 1H), 7.80 (dd, J = 8.6, 5.4 Hz, 2H), 7.39 (t, J = 8.7 Hz, 2H), 7.23–7.18 (m, 2H), 7.11 (dd, J = 7.5, 2.3 Hz, 1H), 3.76–3.65 (m, 8H); 13C NMR (151 MHz, DMSO-d₆) δ 164.08, 162.45, 160.74, 156.29, 148.87, 148.55, 148.03, 140.86, 138.08, 134.58, 129.32, 129.27, 128.12, 116.68, 116.54, 116.34, 114.16, 109.49, 108.69, 99.52, 66.68, 45.31; HRMS-ESI (m/z): calcd. for C₂₃H₂OFN₅O₂, (M + H) ⁺: 417.1601 found, 418.1696.

Synthesis of compound (7-((4-(2-methoxyphenyl) pyridine-2-yl) amino) imidazole [1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-5)

LA-5 was under the same condition as compound LA-1. White solid. (0.65 g, 82.8%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.75 (s, 1H), 8.85–8.79 (m, 1H), 8.52 (d, J = 2.3 Hz, 1H), 8.38 (d, J = 5.3 Hz, 1H), 7.90 (s, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.30 (dt, J = 7.8, 1.1 Hz, 1H), 7.27 (t, J = 2.1 Hz, 1H), 7.24–7.20 (m, 2H), 7.11 (dd, J = 7.6, 2.3 Hz, 1H), 7.06 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H), 3.86 (s, 3H), 3.76–3.66 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.72, 160.34, 156.26, 149.01, 148.82, 148.46, 140.95, 139.62, 137.96, 130.82, 128.13, 119.34, 116.35, 115.31, 114.40, 112.49, 109.71, 108.71, 99.41, 66.68, 55.72, 45.30; HRMS-ESI (m/z): calcd. for C₂₄H₂₃N₅O₃, (M + H) ⁺: 429.1801 found, 430.1901.

Synthesis of compound (7-((4-(4-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-6)

LA-6 was under the same condition as compound LA-1. White solid. (0.70 g, 89.6%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.68 (s, 1H), 8.80 (d, J = 7.5 Hz, 1H), 8.50 (d, J = 2.2 Hz, 1H), 8.32 (d, J = 5.4 Hz, 1H), 7.88 (s, 1H), 7.70–7.67 (m, 2H), 7.18–7.15 (m, 2H), 7.10–7.07 (m, 3H), 3.82 (s, 3H), 3.74–3.64 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.76, 160.68, 156.30, 148.92, 148.38, 140.98, 138.09, 130.12, 128.32, 128.09, 116.30, 115.11, 113.81, 108.76, 108.69, 99.39, 66.68, 55.76, 45.31; HRMS-ESI (m/z): calcd. for C₂₄H₂₃N₅O₃, (M + H) ⁺: 429.1801 found, 430.1900.

Synthesis of compound (7-((4-(3,4-difluorophenyl) pyridine-2-yl) amino) imidazole [1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-7)

LA-7 was under the same condition as compound LA-1. White solid. (0.67 g, 84.6%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.75 (s, 1H), 8.81 (d, J = 7.6 Hz, 1H), 8.49 (d, J = 2.1 Hz, 1H), 8.38 (d, J = 5.3 Hz, 1H), 7.89 (s, 1H), 7.87–7.80 (m, 1H), 7.60 (dd, J = 8.6, 5.9 Hz, 2H), 7.22 (dd, J = 5.4, 1.6 Hz, 1H), 7.17 (d, J = 1.6 Hz, 1H), 7.10 (dd, J = 7.6, 2.4 Hz, 1H), 3.77–3.62 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.74, 156.29, 148.83, 148.66, 146.94, 140.81, 138.06, 135.77, 128.16, 124.18, 124.11, 118.87, 118.75, 116.51, 116.40, 116.38, 114.16, 109.70, 108.70, 99.60, 66.68, 45.28; HRMS-ESI (m/z): calcd. for C₂₃H₁₉F₂N₅O₂, (M + H) ⁺: 435.1507 found, 436.1608.

Synthesis of compound (7-((4-(2,4-difluorophenyl) pyridine-2-yl) amino) imidazole [1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-8)

LA-8 was under the same condition as compound LA-1. White solid. (0.71 g, 89.0%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.81 (d, J = 7.5 Hz, 1H), 8.50 (d, J = 2.2 Hz, 1H), 8.39 (d, J = 5.3 Hz, 1H), 7.89 (s, 1H), 7.70 (td, J = 8.8, 6.4 Hz, 1H), 7.45 (ddd, J = 11.5, 9.1, 2.6 Hz, 1H), 7.26 (td, J = 8.4, 2.6 Hz, 1H), 7.12–7.06 (m, 3H), 3.75–3.64 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.77, 160.74, 159.03, 155.99, 148.83, 148.30, 143.51, 140.78, 138.07, 132.21, 132.17, 132.15, 128.15, 123.00, 116.38, 115.93, 113.07, 113.05, 112.93, 112.90, 111.89, 111.87, 108.71, 105.58, 105.40, 105.22, 99.59, 66.68, 55.40; HRMS-ESI (m/z): calcd. for C₂₃H₁₉F₂N₅O₂, (M + H) ⁺: 435.1507 found, 436.1609.

Synthesis of compound (7-((4-(3,4,5-trifluorophenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-9)

LA-9 was under the same condition as compound LA-1. White solid. (0.71 g, 86.6%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.82 (d, J = 7.6 Hz, 1H), 8.58–8.29 (m, 2H), 7.90 (s, 1H), 7.82–7.50 (m, 2H), 7.24 (dd, J = 5.3, 1.7 Hz, 1H), 7.17 (d, J = 1.7 Hz, 1H), 7.11 (dd, J = 7.6, 2.3 Hz, 1H), 3.91–3.51 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.74, 156.27, 152.03, 151.99, 148.81, 148.74, 146.01, 140.72, 138.09, 135.03, 128.17, 116.40, 114.09, 112.19, 112.05, 109.87, 99.69, 66.68, 45.32; HRMS-ESI (m/z): calcd. for C₂₃H₁₈F₃N₅O₂, (M + H) ⁺: 453.1413 found, 454.1517.

Synthesis of compound (7-((4-(4-methylphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-10)

LA-10 was under the same condition as compound LA-1. White solid. (0.63 g, 84.3%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.71 (s, 1H), 8.81 (d, J = 7.5 Hz, 1H), 8.51 (d, J = 2.1 Hz, 1H), 8.37–8.34 (m, 1H), 7.89 (s, 1H), 7.64–7.61 (m, 2H), 7.36–7.32 (m, 2H), 7.19 (dd, J = 3.8, 1.8 Hz, 2H), 7.10 (dd, J = 7.6, 2.3 Hz, 1H), 3.77–3.63 (m, 8H), 2.37 (s, 3H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.76, 156.33, 148.98, 148.91, 148.46, 140.96, 139.33, 138.09, 135.16, 130.30, 128.12, 126.92, 116.33, 114.05, 109.22, 108.70, 99.43, 66.69, 45.31, 21.25; HRMS-ESI (m/z): calcd. for C₂₄H₂₃N₅O₂, (M + H) ⁺: 413.1852 found, 414.1951.

Synthesis of compound (7-((4-(2-methylphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-11)

LA-11 was under the same condition as compound LA-1. White solid. (0.61 g, 81.6%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.72 (s, 1H), 8.81 (d, J = 7.5 Hz, 1H), 8.51 (d, J = 2.4 Hz, 1H), 8.37–8.35 (m, 1H), 7.89 (s, 1H), 7.55–7.51 (m, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.31–7.28 (m, 1H), 7.21–7.19 (m, 2H), 7.09 (dd, J = 7.6, 2.3 Hz, 1H), 3.75–3.65 (m, 8H), 2.40 (s, 3H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.76, 156.31, 149.20, 148.91, 148.47, 140.93, 138.46, 138.09, 138.06, 130.30, 129.62, 128.13, 127.69, 124.20, 116.34, 114.25, 109.58, 108.70, 99.44, 66.68, 55.40, 21.53; HRMS-ESI (m/z): calcd. for C₂₄H₂₃N₅O₂, (M + H) ⁺: 413.1852 found, 414.1952.

Synthesis of compound (7-((4-(2-chlorophenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-12)

LA-12 was under the same condition as compound LA-1. White solid. (0.70 g, 88.4%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.73 (s, 1H), 8.81 (d, J = 7.5 Hz, 1H), 8.50 (d, J = 2.2 Hz, 1H), 8.38 (d, J = 5.3 Hz, 1H), 7.89 (s, 1H), 7.78 (t, J = 1.9 Hz, 1H), 7.69 (dt, J = 7.2, 1.7 Hz, 1H), 7.58–7.53 (m, 2H), 7.23 (dd, J = 5.3, 1.6 Hz, 1H), 7.20 (d, J = 1.4 Hz, 1H), 7.09 (dd, J = 7.6, 2.3 Hz, 1H), 3.74–3.65 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 160.73, 156.30, 148.83, 148.66, 147.54, 140.79, 140.29, 138.06, 134.47, 131.57, 129.46, 128.15, 126.95, 125.85, 116.36, 114.16, 109.82, 108.69, 99.44, 66.68, 45.31; HRMS-ESI (m/z): calcd. for C₂₃H₂OClN₅O₂, (M + H) ⁺: 433.1306 found, 434.1394, (M + 3H) ⁺: 435.1306 found, 436.1398.

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (morpholinyl) ketone (LA-13)

LA-13 was under the same condition as compound LA-1. White solid. (0.68 g, 83.5%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.80 (d, J = 7.6 Hz, 1H), 8.48 (d, J = 2.3 Hz, 1H), 8.31 (d, J = 5.3 Hz, 1H), 7.88 (s, 1H), 7.42 (dd, J = 8.5, 6.8 Hz, 1H), 7.10–7.07 (m, 3H), 6.99 (dd, J = 5.3, 1.6 Hz, 1H), 6.91 (td, J = 8.3, 2.5 Hz, 1H), 3.85 (s, 3H), 3.74–3.64 (m, 8H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.09, 162.47, 162.31, 160.28, 157.79, 157.72, 155.15, 148.44, 147.10, 146.13, 140.53, 137.59, 131.27, 131.20, 127.61, 123.55, 116.45, 115.84, 112.09, 108.21, 107.39, 107.25, 100.41, 100.24, 98.77, 66.21, 56.19, 44.84; HRMS-ESI (m/z): calcd. for C₂₄H₂₂FN₅O₃, (M + H) ⁺: 447.1707 found, 448.1811.

Synthesis of compound LB-1–LB-12

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (4-morpholinopiperidin-1-yl) ketone (LB-1)

The carboxylic acid intermediate 9m (0.5 g, 1.32 mmol) was dissolved in the dry DMF, and HOBt (0.21 g, 1.59 mmol), EDCI (0.30 g, 1.59 mmol) and 4-(piperidin-4-yl) morpholine (0.27 g, 1.59 mmol) were added into the reaction solution in turn. After monitoring the reaction, the reaction solution was extracted with DCM (30 mL × 3), and the organic layers were combined, washed with saturated saline (15 mL × 3) and concentrated. Separation and purification by column chromatography (DCM : MeOH = 25 : 1) gave a white solid (0.71 g, 83.7%). ¹H NMR (600 MHz, DMSO-d₆) δ 9.65 (s, 1H), 8.76 (d, J = 7.5 Hz, 1H), 8.47 (d, J = 2.3 Hz, 1H), 8.31 (d, J = 5.3 Hz, 1H), 7.82 (s, 1H), 7.42 (dd, J = 8.5, 6.7 Hz, 1H), 7.10–7.06 (m, 3H), 6.98 (dd, J = 5.3, 1.6 Hz, 1H), 6.91 (td, J = 8.4, 2.6 Hz, 1H), 4.42–4.38 (m, 2H), 3.85 (s, 3H), 3.57 (t, J = 4.4 Hz, 4H), 3.03 (t, J = 12.2 Hz, 2H), 2.48 (d, J = 4.7 Hz, 4H), 2.46–2.42 (m, 1H), 1.86 (dd, J = 13.2, 4.0 Hz, 2H), 1.44–1.38 (m, 2H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.10, 162.47, 159.89, 157.80, 155.17, 148.30, 147.11, 146.13, 140.38, 137.07, 131.28, 131.21, 127.60, 123.57, 123.55, 116.43, 116.22, 112.07, 108.15, 107.40, 107.26, 100.41, 100.24, 98.78, 66.55, 61.14, 56.20, 49.39, 28.15; HRMS-ESI (m/z): calcd. for C₂₉H₃₁FN₆O₃, (M + H) ⁺: 530.2442 found, 531.2543.

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (4-methoxypiperidine-1-yl) ketone (LB-2)

LB-2 was under the same condition as compound LB-1. White solid. (0.61 g, 81.3%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.67 (s, 1H), 8.76 (d, J = 7.5 Hz, 1H), 8.50 (d, J = 2.3 Hz, 1H), 8.30 (d, J = 5.3 Hz, 1H), 7.83 (s, 1H), 7.43 (dd, J = 8.5, 6.9 Hz, 1H), 7.09 (td, J = 6.2, 2.5 Hz, 3H), 6.99 (dd, J = 5.3, 1.6 Hz, 1H), 6.91 (td, J = 8.3, 2.5 Hz, 1H), 3.98 (dt, J = 13.4, 4.4 Hz, 2H), 3.85 (s, 3H), 3.48 (dt, J = 7.8, 3.7 Hz, 1H), 3.43 (ddd, J = 12.7, 9.4, 3.5 Hz, 2H), 3.28 (s, 3H), 1.90 (ddt, J = 13.6, 7.1, 3.6 Hz, 2H), 1.50 (dtd, J = 12.8, 8.5, 3.8 Hz, 2H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.56, 162.94, 160.38, 158.26, 158.19, 155.59, 148.66, 147.55, 146.60, 141.04, 131.73, 131.67, 128.12, 124.02, 124.00, 116.93, 116.70, 112.56, 108.70, 107.86, 107.71, 100.88, 100.71, 99.07, 75.56, 56.65, 55.53, 31.11; HRMS-ESI (m/z): calcd. for C₂₆H₂₆FN₅O₃, (M + H) ⁺: 475.2020 found, 476.2108.

Synthesis of compound 7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino)-N-(oxetane-3-yl) imidazole[1,2-a] pyridine-3-carboxamide (LB-3)

LB-3 was under the same condition as compound LB-1. White solid. (0.60 g, 82.6%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.71 (s, 1H), 9.22 (d, J = 7.5 Hz, 1H), 8.94 (s, 1H), 8.49 (d, J = 2.1 Hz, 1H), 8.34–8.24 (m, 2H), 7.42 (ddd, J = 8.6, 6.9, 1.8 Hz, 1H), 7.15–7.06 (m, 3H), 6.99 (dd, J = 5.4, 1.6 Hz, 1H), 6.93–6.88 (m, 1H), 5.06 (h, J = 6.9 Hz, 1H), 4.80 (t, J = 6.9 Hz, 2H), 4.61 (t, J = 6.4 Hz, 2H), 3.85 (s, 3H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.09, 162.47, 159.73, 157.79, 157.72, 155.10, 148.81, 147.07, 146.14, 140.69, 131.27, 131.20, 127.25, 123.54, 123.52, 116.62, 116.50, 108.55, 107.39, 107.25, 100.41, 100.24, 98.96, 77.19, 56.19, 43.82; HRMS-ESI (m/z): calcd. for C₂₃H₂₀FN₅O₃, (M + H) ⁺: 433.1550 found, 434.1606.

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (thiomorpholinyl) ketone (LB-4)

LB-4 was under the same condition as compound LB-1. White solid. (0.60 g, 85.01%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.75 (d, J = 7.5 Hz, 1H), 8.48 (d, J = 2.2 Hz, 1H), 8.32 (d, J = 5.3 Hz, 1H), 7.83 (s, 1H), 7.42 (dd, J = 8.5, 6.8 Hz, 1H), 7.10–7.06 (m, 3H), 6.99 (dd, J = 5.3, 1.5 Hz, 1H), 6.91 (td, J = 8.4, 2.5 Hz, 1H), 3.99–3.93 (m, 4H), 3.85 (s, 3H), 2.74–2.70 (m, 4H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.09, 162.46, 160.36, 157.79, 157.72, 155.14, 148.46, 147.09, 146.12, 140.51, 137.23, 131.26, 131.20, 127.61, 123.55, 123.54, 116.45, 116.00, 112.08, 108.20, 107.39, 107.25, 100.41, 100.24, 98.75, 56.19, 27.01; HRMS-ESI (m/z): calcd. for C₂₄H₂₂FN₅O₂S, (M + H) ⁺: 463.1478 found, 464.1587.

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (4-(oxetane-3-yl) piperazine-1-yl) ketone (LB-5)

LB-5 was under the same condition as compound LB-1. White solid. (0.64 g, 79.9%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.74 (s, 1H), 8.77 (d, J = 7.5 Hz, 1H), 8.49 (d, J = 2.2 Hz, 1H), 8.31 (d, J = 5.4 Hz, 1H), 7.85 (s, 1H), 7.42 (dd, J = 8.5, 6.8 Hz, 1H), 7.12–7.07 (m, 3H), 6.99 (dd, J = 5.3, 1.5 Hz, 1H), 6.91 (td, J = 8.3, 2.4 Hz, 1H), 4.55 (t, J = 6.5 Hz, 2H), 4.47 (t, J = 6.1 Hz, 2H), 3.85 (s, 3H), 3.76 (t, J = 5.0 Hz, 4H), 3.45 (q, J = 6.3 Hz, 1H), 2.34 (t, J = 5.0 Hz, 4H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.36, 162.74, 160.34, 158.07, 158.00, 155.45, 148.62, 147.35, 146.39, 140.84, 137.60, 131.53, 131.47, 127.85, 123.85, 123.83, 116.72, 116.26, 108.51, 107.66, 107.52, 100.69, 100.51, 98.97, 74.56, 58.55, 56.46, 49.43, 41.55; HRMS-ESI (m/z): calcd. for C₂₇H₂₇FN₆O₃, (M + H) ⁺: 502.2129 found, 503.2238.

Synthesis of compound (4,4-difluoropiperidine-1-yl) (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) ketone (LB-6)

LB-6 was under the same condition as compound LB-1. White solid. (0.61 g, 80.3%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.69 (s, 1H), 8.78 (d, J = 7.5 Hz, 1H), 8.51 (d, J = 2.3 Hz, 1H), 8.32 (d, J = 5.3 Hz, 1H), 7.93 (s, 1H), 7.43 (dd, J = 8.5, 6.9 Hz, 1H), 7.10 (ddd, J = 7.7, 4.9, 2.5 Hz, 3H), 7.00 (dd, J = 5.3, 1.5 Hz, 1H), 6.92 (td, J = 8.4, 2.5 Hz, 1H), 3.86 (s, 3H), 3.83 (t, J = 5.8 Hz, 4H), 2.10 (tt, J = 13.6, 5.8 Hz, 4H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.10, 162.47, 160.34, 157.79, 157.72, 155.12, 148.48, 147.10, 146.15, 140.67, 137.49, 131.27, 131.20, 127.71, 123.55, 123.53, 116.49, 115.82, 112.10, 108.25, 107.40, 107.25, 100.41, 100.24, 98.68, 56.19, 33.59; HRMS-ESI (m/z): calcd. for C₂₅H₂₂F₃N₅O₂, (M + H) ⁺: 481.1726 found, 482.1780.

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (pyrrolidine-1-yl) ketone (LB-7)

LB-7 was under the same condition as compound LB-1. White solid. (0.58 g, 84.3%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.68 (s, 1H), 9.24 (dd, J = 7.6, 2.0 Hz, 1H), 8.50 (d, J = 2.3 Hz, 1H), 8.32 (dd, J = 5.3, 1.9 Hz, 1H), 8.03 (d, J = 2.0 Hz, 1H), 7.53–7.38 (m, 1H), 7.08 (dt, J = 11.7, 4.4 Hz, 3H), 6.99 (d, J = 5.1 Hz, 1H), 6.92 (tt, J = 8.5, 2.4 Hz, 1H), 3.85 (d, J = 2.1 Hz, 3H), 3.74 (s, 2H), 3.54 (s, 2H), 1.89 (s, 4H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.50, 162.06, 159.43, 157.81, 157.71, 155.14, 148.29, 147.10, 146.12, 140.59, 138.01, 131.28, 131.18, 127.91, 123.57, 123.54, 117.30, 116.45, 112.08, 108.10, 107.42, 107.21, 100.45, 100.19, 98.77, 56.18, 46.61, 25.55; HRMS-ESI (m/z): calcd. for C₂₄H₂₂FN₅O₂, (M+H) ⁺: 431.1758 found, 432.1811.

Synthesis of compound (1,1-dioxidothiomorpholino) (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) ketone (LB-8)

LB-8 was under the same condition as compound LB-1. White solid. (0.67 g, 85.5%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.70 (s, 1H), 8.76 (d, J = 7.5 Hz, 1H), 8.51 (d, J = 2.2 Hz, 1H), 8.32 (d, J = 5.3 Hz, 1H), 7.94 (s, 1H), 7.43 (dd, J = 8.5, 6.8 Hz, 1H), 7.13–7.07 (m, 3H), 7.00 (dd, J = 5.3, 1.5 Hz, 1H), 6.91 (td, J = 8.4, 2.5 Hz, 1H), 4.11 (t, J = 5.2 Hz, 4H), 3.85 (s, 3H), 3.31 (t, J = 5.3 Hz, 4H); ¹³C NMR (151 MHz, DMSO-d₆) δ 163.96, 162.33, 160.48, 157.65, 157.58, 154.95, 148.56, 146.96, 146.02, 140.69, 137.75, 131.13, 131.07, 127.71, 123.40, 123.38, 116.39, 115.26, 111.99, 108.11, 107.26, 107.12, 100.28, 100.11, 98.53, 56.05, 51.01, 43.06; HRMS-ESI (m/z): calcd. for C₂₄H₂₂FN₅O₄S, (M + H) ⁺: 495.1377 found, 496.1426.

Synthesis of compound 7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino)-N-(tetrahydro-2H-pyran-4-yl) imidazole [1,2-a] pyridine-3-carboxamide (LB-9)

LB-9 was under the same condition as compound LB-1. White solid. (0.61 g, 83.4%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.71 (s, 1H), 9.29 (d, J = 7.5 Hz, 1H), 8.49 (d, J = 2.3 Hz, 1H), 8.39–7.99 (m, 3H), 7.43 (dd, J = 8.5, 6.8 Hz, 1H), 7.16–7.05 (m, 3H), 6.99 (dd, J = 5.3, 1.5 Hz, 1H), 6.92 (td, J = 8.4, 2.5 Hz, 1H), 4.04 (tdt, J = 11.6, 8.1, 4.2 Hz, 1H), 3.90 (ddd, J = 11.7, 4.5, 2.1 Hz, 2H), 3.85 (s, 3H), 3.40 (td, J = 11.8, 2.1 Hz, 2H), 1.80 (ddd, J = 12.6, 4.5, 2.1 Hz, 2H), 1.63–1.55 (m, 2H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.09, 162.47, 159.42, 157.78, 157.71, 155.02, 148.37, 147.02, 146.15, 131.27, 131.20, 127.39, 123.52, 123.49, 117.11, 116.53, 112.11, 108.60, 107.39, 107.25, 100.41, 100.24, 66.21, 56.18, 45.00, 32.71; HRMS-ESI (m/z): calcd. for C₂₅H₂₄FN₅O₃, (M + H) ⁺: 461.1863 found, 462.1909.

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (4-methylpiperazine-1-yl) ketone (LB-10)

LB-10 was under the same condition as compound LB-1. White solid. (0.56 g, 75.9%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.81 (s, 1H), 8.77 (d, J = 7.6 Hz, 1H), 8.50 (d, J = 2.1 Hz, 1H), 8.31 (d, J = 5.4 Hz, 1H), 7.84 (s, 1H), 7.42 (dd, J = 8.5, 6.9 Hz, 1H), 7.13–7.08 (m, 3H), 6.98 (dd, J = 5.4, 1.7 Hz, 1H), 6.91 (td, J = 8.4, 2.5 Hz, 1H), 3.85 (s, 3H), 3.72 (t, J = 4.9 Hz, 4H), 2.38 (t, J = 5.1 Hz, 4H), 2.22 (s, 3H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.97, 162.53, 160.55, 158.30, 158.19, 155.70, 148.86, 147.52, 146.57, 140.99, 137.86, 131.74, 131.64, 127.99, 124.09, 124.06, 116.88, 116.50, 112.60, 108.68, 107.89, 107.67, 100.92, 100.67, 99.23, 56.66, 55.12, 46.11, 41.72; HRMS-ESI (m/z): calcd. for C₂₅H₂₅FN₆O₂, (M + H) ⁺: 460.2023 found, 461.2084.

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (4-isopropylpiperazine-1-yl) ketone (LB-11)

LB-11 was under the same condition as compound LB-1. White solid. (0.60 g, 77.2%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.66 (s, 1H), 8.78 (d, J = 7.5 Hz, 1H), 8.48 (d, J = 2.2 Hz, 1H), 8.31 (d, J = 5.3 Hz, 1H), 7.84 (s, 1H), 7.43 (dd, J = 8.5, 6.8 Hz, 1H), 7.11–7.06 (m, 3H), 6.99 (dd, J = 5.3, 1.5 Hz, 1H), 6.91 (td, J = 8.3, 2.5 Hz, 1H), 3.85 (s, 3H), 3.71 (t, J = 5.0 Hz, 4H), 2.69 (t, J = 6.5 Hz, 1H), 2.50–2.45 (m, 4H), 0.98 (d, J = 6.5 Hz, 6H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.56, 162.93, 160.40, 158.26, 158.19, 155.63, 148.81, 147.56, 146.58, 140.90, 137.81, 131.73, 131.66, 128.05, 124.03, 124.02, 116.89, 116.54, 112.55, 108.64, 107.86, 107.71, 100.88, 100.71, 99.24, 56.66, 54.22, 48.74, 18.57; HRMS-ESI (m/z): calcd. for C₂₇H₂₉FN₆O₂, (M + H) ⁺: 488.2336 found, 489.2411.

Synthesis of compound (7-((4-(4-fluoro-2-methoxyphenyl) pyridine-2-yl) amino) imidazole[1,2-a] pyridine-3-yl) (2-oxa-6-aza-spiro (3.3) heptane-6-yl) ketone (2-oxa-3-yl) (LB-12)

LB-12 was under the same condition as compound LB-1. White solid. (0.61 g, 83.1%) ¹H NMR (600 MHz, DMSO-d₆) δ 9.74 (s, 1H), 9.26 (d, J = 7.5 Hz, 1H), 8.54 (d, J = 2.3 Hz, 1H), 8.33 (d, J = 5.3 Hz, 1H), 7.91 (s, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.14–7.08 (m, 3H), 7.01 (d, J = 5.4 Hz, 1H), 6.92 (td, J = 8.4, 2.6 Hz, 1H), 4.72 (s, 4H), 4.62–4.09 (m, 4H), 3.86 (s, 3H); ¹³C NMR (151 MHz, DMSO-d₆) δ 164.50, 162.06, 157.81, 157.71, 155.07, 148.36, 147.08, 146.15, 140.92, 138.07, 131.27, 131.17, 127.52, 123.53, 123.50, 116.54, 115.23, 112.13, 108.54, 107.42, 107.21, 100.45, 100.19, 98.74, 79.70, 56.18, 54.89, 38.17; HRMS-ESI (m/z): calcd. for C₂₅H₂₂FN₅O₃, (M + H) ⁺: 459.1707 found, 460.1787.

Pharmacological assay

The Pharmacological assay methods are shown in the ESI.†

Molecular docking studies

Molecular docking studies methods are shown in the ESI.†

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Author contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Zihan Sun: writing – original draft, methodology, conceptualization. Shijun Sun: methodology development; Xiayu Li: synthesis; Xiang Li: pharmacological assay. Chuang Li: data curation, resource. Li Tang: formal analysis. Maosheng Cheng, Yang Liu: writing – review & editing, supervision, funding acquisition.

Conflicts of interest

There are no conflicts to declare.