Discovery of imidazo[1,2-a]pyridine-thiophene derivatives as FLT3 and FLT3 mutants inhibitors for acute myeloid leukemia through structure-based optimization of an NEK2 inhibitor

Abstract

FMS-like tyrosine kinase 3 (FLT3) with an internal tandem duplication (ITD) mutation has been validated as a driver lesion and a therapeutic target for acute myeloid leukemia (AML). Currently, several potent small-molecule FLT3 kinase inhibitors are being evaluated or have completed evaluation in clinical trials. However, many of these inhibitors are challenged by the secondary mutations on kinase domain, especially the point mutations at the activation loop (D835) and gatekeeper residue (F691). To overcome the resistance challenge, we identified a novel series of imidazo[1,2-a]pyridine-thiophene derivatives from a NIMA-related kinase 2 (NEK2) kinase inhibitor CMP3a, which retained inhibitory activities on FTL3-ITD^D835V and FLT3-ITD^F691L. Through this study, we identified the imidazo[1,2-a]pyridine-thiophene derivatives as type-I inhibitors of FLT3. Moreover, we observed compound 5o as an inhibitor displaying equal anti-proliferative activities against FLT3-ITD, FTL3-ITD^D835Y and FLT3-ITD^F691L driven acute myeloid leukemia (AML) cell lines. Meanwhile, the apoptotic effects of compound supported its mechanism of anti-proliferative action. These results indicate that the imidazo[1,2-a]pyridine-thiophene scaffold is promising for targeting acquired resistance caused by FLT3 secondary mutations and compound 5o is an interesting lead in this direction.

1. Introduction

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase belonging to the PDGF receptor family (class III) [1]. It is characterized by an extracellular immunoglobulin-like domain to which the FLT3 ligand binds, a juxtamembrane (JM) region and cytoplasmic kinase domain with a kinase insert domain [2]. Binding of the ligand induces dimerization of FLT3 followed by autophosphorylation of the kinase domain, which is followed by activation of downstream pathways including RAF-MEK pathway, JAK-STAT pathway and PI3K-AKT pathway [3]. Along with other class III receptor tyrosine kinases (RTKs), FLT3 plays an important role in hematopoiesis and functioning of the immune system [1]. Activating mutations in FLT3, including internal tandem duplication (ITD) and point-mutations in the tyrosine kinase domain, are frequently observed in acute myeloid leukemia (AML) cases [1]. The ITD mutation in which an exon is duplicated in the JM domain interferes with the autoinhibitory activity of the domain, thus rendering it constitutively active [3]. It is observed in approximately 25% of AML patients [1,4]. Additionally, the ITD mutation has been identified as a driver lesion and a valid therapeutic target for AML [5]. Due to its prevalence and therapeutic significance, the FLT3-ITD oncogene has been widely studied in targeted therapy, and multiple small molecule inhibitors of FLT3-ITD have been developed [4,6]. However, acquired resistance due to secondary mutations in the activation loop (D835) and gatekeeper (F691) regions creates a profound challenge for sustained FLT3 inhibition [7–13]. Mutations occurring at activation loop can stabilize the active “DFG-in” conformation and prevent type-II kinase inhibitors from binding [5,8,13]. Meanwhile, mutations at the gatekeeper residue, which is adjacent to the ATP-binding site, can increase bulkiness in the ATP pocket leading to reduction in binding affinity of the inhibitors [14]. Several potent FLT3 inhibitors made good clinical process, but the secondary mutations pose a major challenge. AC220 (quizartinib), a potent type-II FLT3-ITD inhibitor, has been approved in Japan in 2019, but is poorly effective against activation loop or gatekeeper mutations [5,7,8,10,13,15]. Furthermore, gilteritinib, the type-I FLT3 inhibitor approved in 2019, exhibits dramatically reduced activity against F691 mutation [12,13,16]. Additionally, the type-I FLT3 inhibitor, crenolanib, which is currently in phase III clinical trial, only showed mildly reduced sensitivity with the presences of F691L mutation in cellular study [17]. Nevertheless, its clinical activities may be hampered by its short half-life and its dose-limiting toxicity [17,18]. Therefore, the development of novel inhibitors with improved activity against the mutants is of very high clinical significance.

In one of our previous studies, a NIMA-related kinase 2 (NEK2) inhibitor CMP3a showed inhibitory effect on FLT3-ITD^D835V and FLT3-ITD^F691L against a kinase panel [19]. Accordingly, CMP3a provided a validated, hit candidate that can be further optimized for inhibitory profile on FLT3 mutants. However, the cytotoxicity profile of CMP3a is high [19]. Since NEK2 is important for the cell cycle, eliminating the NEK2 inhibition activity is likely to withdraw the cytotoxicity profile of CMP3a.

In this work, by making suitable modification on CMP3a, we eliminated NEK2 activity and discovered an imidazo[1,2-a]pyridine-thiophene based scaffold which inhibits FLT3 and multiple clinically relevant FLT3 mutants. All compounds were tested for NEK2 selectivity. Inhibition kinetics demonstrated that the imidazo[1,2-a]pyridine thiophene compounds are type-I inhibitors of FLT3. Through these studies, compound 5o was identified with equal potency on FTL3-ITD^D835Yand FLT3-ITD^F691L during the continuous effort to discover novel kinase inhibitors [20–24], herein, we discuss the identification and optimization of imidazo[1,2-a]pyridine-thiophene based FLT3 inhibitors through a scaffold remodeling approach.

2. Chemistry

To construct the imidazopyridine core, compounds 1a, 1b, 1c or 1d were cyclized with 2-chloroacetaldehyde in n-butanol under reflux conditions to generate 2a, 2b, 7a and 7b. Then compound 2a or 2b was reacted with various boronic acids under Suzuki coupling conditions to generate the 7-position substituted imidazo[1,2-a] pyridine-thiophene 3a-3b and the 6-position substituted imidazo[1,2-a]pyridine-thiophene 3c-3d. After, compounds 3a-d and 7a-b were subjected to a C—H activating reaction to yield compounds 4a-d and 8a-b. Compounds 4a-d and 8a-b were further derivatized using Suzuki coupling conditions with various boronic acids to generate compounds 5a-q, 6a-g, and 9a-d. The synthetic route for target compounds is depicted in Scheme 1 and Scheme 2.

Scheme 1.

Reagents and conditions: (a). 2-chlorocetaldehyde, n-butanol, 130 °C; (b)boronic acid, Na₂CO₃, Pd(PPh₃)₄, 1,4-dioxane/water, 120 °C; (c) 2,5-dichlorothiophene, K₂CO₃, Pd(PPh₃)₄, Pd(OAc)₂, 1,4-dioxane/water, 120 °C; (d) boronic acid, Na₂CO₃, Pd(PPh₃)₄, 1,4-dioxane/methanol, microwave irradiation, 130 °C.

Scheme 2.

Reagents and conditions: (a). 2-chlorocetaldehyde, n-butanol, 130 °C; (b) 2,5-dichlorothiophene, K₂CO₃, Pd(PPh₃)₄, Pd(OAc)₂, 1,4-dioxane/water, 120 °C; (c) boronic acid, Na₂CO₃, Pd(PPh₃)₄, 1,4-dioxane/methanol, microwave irradiation, 130 °C.

3. Results and discussion

3.1. Design of inhibitors

As mentioned earlier, we chose compound CMP3a as our starting point, the synthesis of which was previously described [19]. To maintain the FLT3 mutant inhibitory activity of CMP3a while simultaneously eliminating the NEK2 inhibitory profile, a series of modifications to the CMP3a structure were performed (Fig. 1). Substitution at the 3-position on thiophene creates key interactions with the glycine-rich loop of NEK2 [19,25]. Consequently, compound CMP4 was synthesized without substitution at the 3-position of thiophene to limit NEK2 activity. Inhibition data demonstrated that removal of the thiophene substitution reduced NEK2 activity while increasing FLT3 potency (Table 1). To solve issues of low in-vivo exposure, CMP4 was further modified to CMP5 by removing the easily oxidized tertiary amine (Fig. 1) [19]. CMP5 exhibited a further reduction in NEK2 potency without a significant change in FLT3 inhibition (Table 1). The amide portion of CMP3a engages in a hydrogen bond network in the back pocket of NEK2 and this moiety was subsequently removed in the final scaffold to further limit NEK2 activity (Fig. 1) [19,25].

Fig. 1.

Structure modification of CMP3a to imidazo[1,2-a]pyridine thiophene.

Table 1.

FLT3 and NEK2 inhibition of CMP3a, CMP4, CMP5.

3.2. Biochemical evaluation

Due to the mutant types of FLT3 are not commercially available, we started our studies with validating the inhibition profile of the scaffold on FLT3-wt in enzymatic level. All synthesized compounds were screened for inhibition of the FLT3 kinase. IC₅₀ values were determined for all active compounds through a biochemical assay, as described in the experimental section. All FLT3 inhibition values were provided in Tables 2 and 3.

Table 2.

FLT3 and NEK2 inhibitions of compounds 5a-5q and 6a-6g.

Table 3.

FLT3 and NEK2 inhibitions of compounds 9a-9d.

Compounds 5a-5j were initially synthesized introducing several substituted phenyl rings and other heterocycles at the R3 position. All the compounds showed improved activity compared to the initial hit. More importantly, all the compounds were selective against NEK2 kinase. The enzymatic data also revealed that halogen substitution may be preferred on the phenyl ring over others. Compound 5e with 3-chlorophenyl substitution on the R3 position showed the best FLT3 inhibition. Based on our previous experience with kinases [22], we replaced pyrazole on imidazopyridine with methylsulfonylphenyl moiety. This replacement showed improved pharmacokinetic properties in an RET kinase targeted project [26]. Upon this substitution, we did not find any significant difference at the enzymatic level. There was only a slight decrease in the activity.

Molecular docking of compound 5e in the structure of FLT3 revealed the binding pose and key interactions. As expected, the imidazopyridine warhead formed the crucial hydrogen bond with Cys694 in the hinge region. This hydrogen bond is ubiquitous in all kinase inhibitors which occupy the ATP binding site. We also observed that the phenyl group at R3 position is buried deep in the pocket and forms a - stacking interaction with Phe691. Additionally, there are several hydrophobic contacts formed by the terminal phenyl group with Phe691 and other residues in this region. These interactions are extremely crucial for better binding. This region of the pocket is deeply buried and hydrophobic. The entropic gain of replacing any water molecules in such pockets with a hydrophobic moiety is very large, resulting in stronger binding [27]. The modeling also reveals that the pyrazole group is more solvent exposed. As depicted in the image, the CH group next to the nitrogen forms good contacts with the backbone carbonyl, which is often referred as an ‘aromatic- ’ bond.

To experimentally validate the importance of the hydrogen bond formed with Cys694, four compounds were synthesized as illustrated in Table 3. Introduction of a methyl group at position 8 on the bicyclic ring increases the distance between N-1 and Cys694 resulting in the loss of the hydrogen bond which results in complete loss of activity from 9a to 9c and 9b to 9d. This result demonstrates that the hydrogen bond plays a crucial role for maintaining the binding interaction of this scaffold.

Further, we investigated the effect of moving the solvent exposed group from 7 to 6 position on the imidazopyridine. Compounds 6a to 6g in Table 2 were synthesized. Kinase inhibition studies showed that while NEK2 inhibitory activity was blocked, but these compounds also showed a significant decrease in FLT3 inhibition. To understand this effect, compounds 6b and 5b were docked in the structure of FLT3. Molecular docking studies revealed that both the compounds adopt a similar binding pose. In fact, the imidazopyridine, thiophene and terminal phenyl group are well overlapping in both poses. Careful examination of interaction of the inhibitors with each of the binding pocket residues indicated that there could be solvation effects playing an important role. Fig. 2A shows that the pyrazole group is heavily solvent exposed. When pyrazole is attached to the C-6 of the imidazopyridine, it is closer to Asp698 and Asn701 which are downstream of the hinge region. In an unbound state, these two residues are expected to be well solvated because of their sidechains. In compound 6b, the pyrazole group is approximately 3 Å away from both these residues, which indicates that to occupy this region, there is a huge desolvation penalty which the ligand should counter for better binding. However, there is no significant enthalpic gain arising from any interactions of the pyrazole. Rather, the aromatic- bond which was otherwise seen when the pyrazole was in 7 position is also lost. These factors could have resulted in the significant decrease in activity.

Fig 2.

(A) Binding pose of 5e in FLT3 model. (B) and (C): Binding pose of 6b in FLT3. Panel (B) Surface representation of the protein shows that pyrazole group is significantly solvent exposed. Panel (C) depicts the close proximity of N-methyl group to Asp698 and Asn701. Hydrogen bonds are depicted in dark blue and aromatic- bond in cyan.

4. Kinase selectivity

As discussed previously, the potent inhibition on NEK2 could induce cytotoxicity. In this work, all synthesized compounds were screened against NEK2. The screening was performed at a single point concentration of 20 μM and the data were presented in Tables 2 and 3 The highest inhibitory percentage at 20 μM was 51% while the remainder were all less than 50%. Removal of NEK2 activity through structure modification of CMP3a suggests a possible improvement in cytotoxic profile on normal cells.

Several known FLT3 inhibitors are multi-kinase inhibitors which explains their poor side-effect profile. To this end, we studied the selectivity of this series of imidazo[1,2-a]pyridine-thiophene derivatives against some of our in-house kinases. The most potent compound 5e and the moderate compound 5g were as representatives to be further screened and the results are presented in Table 4. Compared to the activity on FLT3, compounds 5e and 5g displayed much weaker inhibition on all the other screened kinases (Table 4).

Table 4.

Selected kinases inhibition of compounds 5e and 5g.

Kinase	5e IC50 (μM)	5g IC50 (μM)
FLT3	0.053 ± 0.0091	0.31 ± 0.012
NEK2	>20	>20
RET	6.83 ± 1.78	5.43 ± 0.02
EGFR	>20	>20
CSF-1R	8.56 ± 1.70	2.84 ± 0.026
Aurora A	1.30 ± 0.30	0.97 ± 0.098
NIK	>10	>10

5. Inhibition kinetics studies

As mentioned previously, the D835 mutation in the activation loop prevents the type-II FLT3 inhibitor binding and can induce drug resistance. Therefore, investigation of the mode of inhibition of imidazo[1,2-a]pyridine-thiophene compounds is necessary. Herein, inhibition kinetics of compounds 5e and 5g were studied against FLT3. Based on the biochemical assay data, we generated the Lineweaver-Burk plot which indicated that both 5e and 5g compete with ATP in the FLT3 pocket binding (Fig. 3) and confirmed that imidazo[1,2-a]pyridine-thiophene series of inhibitors were type-I inhibitors of FLT3. As type-I inhibitors, the imidazo[1,2-a]pyridine-thiophene compounds bind FLT3 in “DFG-in” active confirmation, which may afford the possible resistance mechanisms caused by D835 point mutations.

Fig. 3.

Inhibition kinetic study of compounds 5e and 5g.

6. Anti-proliferative activity in leukemia cells

After validating the inhibitory and selectivity profile on FLT3 via different enzymatic assays, the compounds were further tested for inhibition on FLT3 mutants with cellular assays. The FLT3-ITD driven AML cell line, MOLM14, was introduced here. All the compounds were screened on this cell line for anti-proliferative activities. As shown in Table 5 and Table S1, the imidazo[1,2-a]pyridine-thiophene compounds inhibited the cellular proliferation with GI₅₀ ranging from 0.16 to 9.28 μM. As mentioned, the secondary mutations on DFG-loop and gatekeeper residue are the major reasons for resistance, we further studied the anti-proliferative activities of the potent compounds on FLT3-ITD^D835Y and FLT3-ITD^F691L driven MOLM14 cell lines as shown in Table 5. Comparing the anti-proliferative activities on FLT3-ITD, FLT3-ITD^D835Y and FLT3-ITD^F691L mutated MOLM14 cell lines, we identified compound 5o showed balanced inhibitory profile on three cell lines with a respective GI₅₀ values of 0.52 μM, 0.53 μM and 0.57 μM. While, as a control, compared to FLT3-ITD mutation, quizartinib showed decreased potency on FLT3-ITD^D835Y and FLT3-ITD^F691L. Also, as discussed previously, compound CMP3a is cytotoxic on normal cells. To further confirm the improvement of the imidazo[1,2-a]pyridine-thiophene compounds, we tested compound 5o on Ba/F3 cell line, which is a FLT3-negative normal cell line. As shown in Table S2, the selectivity window compared to Ba/F3 cell line was improved to around 200-fold, indicating the improvement of the cytotoxicity profile on normal cells.

Table 5.

Anti-proliferative activities of selected compounds on MOLM14 cells.

	MOLM14-FLT3ITD GI50(μM)	MOLM14-FLT3ITDD835Y GI50(μM)	MOLM14-FLT3ITDF691L GI50(μM)
5a	0.61 ± 0.088	1.68 ± 0.17	3.32 ± 021
5b	0.67 ± 0.079	1.57 ± 0.26	2.75 ± 0.49
5e	0.16 ± 0.029	1.50 ± 0.16	1.29 ± 0.22
5f	0.61 ± 0.060	10.24 ± 1.62	8.89 ± 0.87
5h	0.77 ± 0.068	3.24 ± 0.30	3.03 ± 0.52
5i	0.65 ± 0.11	1.56 ± 0.18	2.91 ± 0.41
5k	0.65 ± 0.060	0.55 ± 0.046	1.35 ± 0.21
5l	0.71 ± 0.084	3.11 ± 0.40	1.58 ± 0.11
5n	0.60 ± 0.083	1.73 ± 0.32	1.40 ± 0.11
5o	0.52 ± 0.062	0.53 ± 0.022	0.57 ± 0.058
5p	0.67 ± 0.054	1.42 ± 0.20	1.23 ± 0.24
5q	0.65 ± 0.056	1.15 ± 0.18	0.95 ± 0.18
Quizartinib	0.00015 ± 0.000015	0.012 ± 0.0026	0.027 ± 0.0035

7. Apoptotic effects of compound 5o on AML cell lines

To further investigate the mechanism of action beyond the anti-proliferative effect of compound 5o against MOLM14 cell lines, we performed AnnexinV/PI staining to examine the potency of apoptosis induction of the compound 5o on FLT3-ITD, FLT3-ITD^D835Y and FLT3-ITD^F691L mutations bearing MOLM14 cell lines using flow cytometry. The cells were exposed to DMSO as control or 1 μM 5o for 24 h and 48 h. After treatment, compound 5o showed apoptotic effects on all three FLT3 mutated cell line and the later time point showed increased potency. As shown in Fig. 4, the FLT3-ITD, FLT3-ITD^D835Y and FLT3-ITD^F691L driven MOLM14 cell lines showed 15.2%, 8.8% and 5.2% more apoptotic cells respectively after 24 h, and showed 34.0%, 16.6% and 19.8% more respectively after 48 h, which indicates that the apoptotic effects of compound 5o contributed to its anti-proliferation activities on FLT3-mutants harboring MOLM14 cell lines.

Fig. 4.

Apoptosis analysis of compound 5o on MOLM14 cell lines.

8. Conclusion

In this study, we identified a series of imidazo[1,2-a]pyridine thiophenes derivatives as FLT3 inhibitors through scaffold modification of CMP3a, a NEK2 inhibitor displaying promising inhibition on multiple FLT3 mutants. Through SAR and molecular docking study, we observed that the nitrogen at the 1-position of the imidazopyridine core engaged in a key hydrogen bond with hinge region which is sterically compromised by substitution at the 8-position. While both 6- and 7-substitutions on imidazopyridine are tolerated, substitution on 7-position resulted in 10 times increased potency than on 6-position. Based on enzymatic screening, the most potent compound of this scaffold was identified with IC₅₀ of 0.058 ± 0.0069 μM on FLT3, which indicated that imidazo[1,2-a]pyridine thiophenes could be good candidates targeting FLT3. With the kinase panel screening of the representative compounds of this scaffold, we identified their good selectivity on FLT3 against NEK2, RET, EGFR, CSF-1R, Aurora A and NIK. Further, the ATP-competitive inhibitory profile was discovered through inhibition kinetic study, which demonstrates that these imidazo[1,2-a]pyridine-thiophenes derivatives as type-I FLT3 inhibitors. Anti-proliferative studies in AML cell lines against clinically relevant FLT3 mutants were performed. Compound 5o was identified as a FLT3 inhibitor maintaining equal potency on FLT3-ITD, FTL3-ITD^D835Y and FLT3-ITD^F691L and having an improved cytotoxic profile on normal cells. Although we claimed the key - stacking interaction with Phe691, which may displace with the presence of the F691L mutation, in the computational model, the significant hydrophobic interactions over that region contribute to retain the activity on FLT3-ITD^F691L. Lastly, via AnnexinV/PI staining, apoptotic effects of compound 5o were identified as the reason for its anti-proliferative activities in AML cell lines. In summary, the imidazo[1,2-a]pyridine-thiophene could serve as a novel scaffold for the discovery and development of potent and selective type-I FLT3 inhibitors, and 5o is an important lead in search of targeted therapy that could overcome acquired resistance for FLT3-driven AML.

9. Experimental

9.1. Chemistry

All the starting materials were obtained from commercial suppliers and used without further purification. Thermo Finnigan LCQ Deca with Thermo Surveyor LCMS System at variable wavelengths of 254 nm and 214 nm was used to monitor the reaction and test the purity of the compounds. The purity of all the final compounds is >95%. The water-methanol gradient buffered with 0.1% formic acid was used as the mobile phase for the HPLC system. NMR spectra was completed on a Varian 400 MHz instrument. The ¹H NMR spectra and ¹³C spectra were recorded at 400 MHz and 101 MHz, respectively. All final compounds were purified using Silica gel (0.035–0.070 mm, 60 Å) flash chromatography, unless otherwise noted. Microwave assisted reactions were completed in sealed vessels using a Biotage Initiator microwave synthesizer (Biotage, Uppsala, Sweden).

9.1.1. General procedure A: synthesis of intermediate 2a-b and 7a-b

To the solution of 1 equivalent of 2-aminopyridine derivatives (1a,1b, 1c or 1d) in n-butanol, 1.2 equivalent of 2-chloroacetaldehyde was added and stirred at 130 °C for overnight. Once the reaction completed, the ethyl acetate was added. Then, dissolved the precipitate after filtering in water. Further, added sodium bicarbonate to the solution till some solid precipitate out. Lastly, extracted the compounds from the solution using DCM and condensed under reduced pressure.

9.1.2. General procedure B: synthesis of intermediate 3a-d

A solution of 1 equivalent of 2a or 2b and 1.2 equivalent of corresponding boronic acid in dioxane/H₂O (4:1) was purged with nitrogen gas. Then 0.03 equivalent of tetrakis (triphenylphosphine) palladium (0) and 2.5 equivalent of sodium carbonate were added to the solution. The reaction mixture was stirred at 120 °C for overnight. After the completion of the reaction, filtered the mixture through a pad of Celite. Collected the solution and condensed it under reduced pressure. Purified the crude through flash chromatography on silica gel using a DCM/MeOH gradient.

9.1.3. General procedure C: synthesis of intermediate 4a-d and 8a-b

1 equivalent of corresponding imidazopyridine (3a-d, 7a-b) and 2 equivalents of 2,5-dichlorothiophene were dissolved in dioxane/H2O (10:1). Then 5 equivalent of potassium carbonate, 0.1 equivalent of tetrakis(triphenylphosphine)palladium (0) and 0.05 equivalent of palladium (II) acetate were added. The mixture was reacted at 120 °C for overnight under nitrogen gas. After completion, the reaction mixture was filter through a pad of Celite and the solution was collected. The crude obtained from the condensed solution was then purified by flash chromatography on silica gel using a DCM/MeOH gradient.

9.1.4. General procedure D: synthesis of final compounds

Dissolved 1equivalent of intermediate 4a-d or 8a-b and 1.5 equivalent of corresponding boronic acid to 1,4-dioxane/methanol (10:1). Following, 0.03 equivalent of tetrakis (triphenylphosphine) palladium (0) and 2.5 equivalent of sodium carbonate were added. Purged the reaction mixture with nitrogen gas and sealed in a microwave vessel. Then heated the reaction to 130 °C by microwave irradiation for 1.5 h. The reaction mixture was filtered after completion, and the filtrate was collected and condensed under reduced pressure. The crude was further purified through flash chromatography on silica gel using a DCM/MeOH gradient.

7-(1-methyl-1H-pyrazol-4-yl)-3-(5-(1-methyl-1H-pyrazol-4-yl) thiophen-2-yl)imidazo[1,2-a]pyridine (5a) The title compound was synthesized from 4a and 1-Methyl-1H-pyrazole-4-boronic acid pinacol ester following general procedure D. Brown solid (23 mg, 88%). ¹H NMR (400 MHz, CDCl₃) δ 8.39 (d, J = 7.3 Hz, 1H), 7.84 (s, 1H), 7.72 (d, J = 5.3 Hz, 4H), 7.59 (s, 1H), 7.17 (d, J = 3.7 Hz, 1H), 7.11 (d, J = 3.6 Hz, 1H), 7.01 (dd, J = 7.1, 1.5 Hz, 1H), 3.98 (s, 3H), 3.95 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 146.92, 137.06, 137.01, 135.71, 133.87, 133.82, 129.68, 127.60, 127.21, 126.43, 124.08, 123.11, 121.22, 119.19, 116.69, 112.26, 112.11, 39.45, 39.31.HRMS (ESI) m/z: [M+H]+ calcd for C₁₉H₁₆N₆S, 361.1157; found 361.1233.

3-(5-(4-fluorophenyl)thiophen-2-yl)-7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (5b) The title compound was synthesized from 4a and (4-fluorophenyl)boronic acid following general procedure D. Light brown solid (21 mg, 80%). ¹H NMR (400 MHz, CDCl₃) δ 8.47 (d, J = 7.5 Hz, 1H), 8.40 (s, 1H), 8.02 (s, 1H), 7.91 (s, 1H), 7.82 (s, 1H), 7.64–7.58 (m, 2H), 7.44 (dd, J = 7.2, 1.7 Hz, 1H), 7.37 (d, J = 3.8 Hz, 1H), 7.35 (d, J = 3.8 Hz, 1H), 7.18–7.11 (m, 2H), 4.00 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.37, 162.71, 161.89, 147.28, 141.94, 137.66, 137.61, 130.72, 129.60, 129.34, 129.30, 128.07, 127.99, 124.98, 124.26, 123.64, 122.68, 120.11, 118.96, 116.57, 116.35, 115.62, 107.80, 39.67. HRMS (ESI) m/z: [M+H]+ calcd for C₂₁H₁₅FN₄S, 375.1002; found 375.1097.

3-(5-(3-fluorophenyl)thiophen-2-yl)-7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (5c) The title compound was synthesized from 4a and (3-fluorophenyl)boronic acid following general procedure D. Yellow solid (24 mg, 73%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (d, J = 7.2 Hz, 1H), 8.38 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 7.87 (d, J = 2.7 Hz, 1H), 7.82 (s, 1H), 7.79–7.76 (m, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.58–7.55 (m, 1H), 7.47 (td, J = 7.9, 2.6 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 7.1 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 146.62, 140.21, 136.67, 135.27, 133.97, 133.90, 131.03, 130.04, 129.72, 128.84, 127.43, 126.03, 125.96, 124.83, 124.66, 123.93, 119.90, 118.44, 118.18, 111.97, 110.73, 38.77. HRMS (ESI) m/z: [M+H]+ calcd for C₂₁H₁₅FN₄S, 375.1002; found 375.1072.

3-(5-(4-chlorophenyl)thiophen-2-yl)-7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (5d) The title compound was synthesized from 4a and (4-chlorophenyl)boronic acid following general procedure D. Light brown solid (19 mg, 59%).¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (d, J = 7.2 Hz, 1H), 8.38 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 7.86 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.70 (d, J = 3.8 Hz, 1H), 7.55 (d, J = 3.8 Hz, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.31 (dd, J = 7.2, 1.5 Hz, 1H), 3.89 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 146.67, 140.79, 136.72, 133.86, 132.18, 129.74, 129.62, 129.22, 128.91, 126.98, 126.20, 125.48, 124.87, 119.94, 112.02, 110.74, 109.66, 38.85.HRMS (ESI) m/z: [M+H]+ calcd for C₂₁H₁₅ClN₄S, 391.0706; found 391.0776.

3-(5-(3-chlorophenyl)thiophen-2-yl)-7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (5e) The title compound was synthesized from 4a and (3-chlorophenyl)boronic acid following general procedure D. Yellow solid (21 mg, 65%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (d, J = 7.2 Hz, 1H), 8.38 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 7.87 (s, 1H), 7.76 (d, J = 3.8 Hz, 1H), 7.62 (d, J = 10.4 Hz, 1H), 7.57 (d, J = 3.8 Hz, 1H), 7.54 (s, 1H), 7.49 (dd, J = 14.2, 7.9 Hz, 1H), 7.31 (d, J = 7.2 Hz, 1H), 7.17 (ddd, J = 8.9, 2.5, 1.2 Hz, 1H), 3.89 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 146.60, 140.55, 136.66, 133.87, 131.26, 131.17, 129.89, 129.71, 128.84, 126.02, 125.88, 124.81, 121.40, 119.90, 118.43, 114.30, 111.96, 111.92, 111.68, 110.72, 38.77. HRMS (ESI) m/z: [M+H]+ calcd for C₂₁H₁₅ClN₄S, 391.0706; found 391.0776.

3-(5-(2-methoxy-4-(trifluoromethyl)phenyl)thiophen-2-yl)-7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (5f) The title compound was synthesized from 4a and (2-methoxy-3-(trifluoromethyl)phenyl)boronic acid following general procedure D. Yellow solid(15 mg, 53%). ¹H NMR (400 MHz, CDCl₃) δ 8.45 (d, J = 7.2 Hz, 1H), 7.92 (s, 1H), 7.84 (s, 1H), 7.79 (s, 1H), 7.72 (s, 2H), 7.61 (d, J = 3.1 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 3.1 Hz, 1H), 7.07 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 4.03 (s, 3H), 3.98 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 157.76, 137.92, 137.05, 134.17, 131.02, 129.66, 129.54, 128.47, 127.57, 126.81, 125.80, 125.21, 124.17, 123.67, 123.39, 123.02, 121.25, 112.34, 112.14, 111.58, 56.07, 39.45. HRMS (ESI) m/z: [M+H]+ calcd for C₂₃H₁₇F₃N₄OS, 455.1075; found 455.1146.

3-(5-(2,6-dimethoxyphenyl)thiophen-2-yl)-7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (5g) The title compound was synthesized from 4a and (2,6-dimethoxyphenyl)boronic acid following general procedure D. Light yellow solid (14 mg, 56%).¹H NMR(400 MHz, CDCl₃) δ 8.47 (d, J 7.0 Hz, 1H), 7.83 (s, 1H), 7.75 (s, 1H), 7.69 (s, 2H), 7.58 (d, J = 3.7 Hz, 1H), 7.26 (t, J = 7.6 Hz, 2H), 6.97 (d, J = 7.1 Hz, 1H), 6.67 (d, J = 9.4 Hz, 2H), 3.96 (s, 3H), 3.88 (s, 6H).¹³C NMR (101 MHz, CDCl₃) δ 157.67, 146.82, 136.99, 134.58, 133.68, 129.68, 129.58, 129.39, 129.06, 127.52, 124.55, 124.29, 121.29, 119.82, 112.14, 111.84, 104.47, 56.10, 39.37.HRMS (ESI) m/z: [M+H]+ calcd for C₂₃H₂₀N₄O₂S 417.1307; found 417.1383.

7-(1-methyl-1H-pyrazol-4-yl)-3-(5-(pyridin-4-yl)thiophen-2-yl)imidazo[1,2-a]pyridine (5h) The title compound was synthesized from 4a and pyridin-4-ylboronic acid following general procedure D. Yellow solid (32 mg, 91%).¹H NMR (400 MHz, CDCl₃) δ 8.45 (d, J = 7.2 Hz, 1H), 7.87 (s, 1H), 7.81 (d, J = 9.6 Hz, 2H), 7.75–7.71 (m, 2H), 7.61 (d, J = 7.0 Hz, 2H), 7.52 (t, J = 6.9 Hz, 1H), 7.27 (s, 1H), 7.22 (s, 1H), 7.04 (d, J = 7.2 Hz, 1H), 3.99 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 147.12, 139.83, 137.02, 134.28, 133.21, 132.97, 131.67, 129.69, 128.70, 128.46, 127.56, 125.51, 124.09, 121.20, 118.91, 112.30, 112.12, 39.41.HRMS (ESI) m/z: [M+H]+ calcd for C₂₀H₁₅N₅S, 358.1048; found 358.1126.

7-(1-methyl-1H-pyrazol-4-yl)-3-(5-(pyridin-3-yl)thiophen-2-yl)imidazo[1,2-a]pyridine (5i) The title compound was synthesized from 4a and pyridin-3-ylboronic acid following general procedure D. Yellow solid(18 mg, 54%).¹H NMR (400 MHz, DMSO-d₆) δ 8.98 (s, 1H), 8.66 (d, J = 7.2 Hz, 1H), 8.53 (d, J = 4.6 Hz, 1H), 8.38 (s, 1H), 8.10 (s, 1H), 7.89 (d, J = 10.2 Hz, 2H), 7.80 (d, J = 3.8 Hz, 1H), 7.60 (d, J = 3.9 Hz, 2H), 7.48 (dd, J = 7.9, 4.7 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 3.89 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 148.64, 146.11, 138.45, 136.74, 133.97, 132.57, 131.55, 131.46, 130.29, 129.82, 128.93, 128.85, 128.74, 126.19, 124.90, 124.17, 119.95, 112.05, 110.75, 38.84. HRMS (ESI) m/z: [M+H]+ calcd for C₂₀H₁₅N₅S, 358.1048; found 358.1131.

7-(1-methyl-1H-pyrazol-4-yl)-3-(5-(2-(trifluoromethyl)phenyl) thiophen-2-yl)imidazo[1,2-a]pyridine (5j) The title compound was synthesized from 4a and (2-(trifluoromethyl)phenyl)boronic acid following general procedure D. yellow solid (24 mg, 76%).¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (d, J = 6.7 Hz, 1H), 8.58 (s, 1H), 8.34 (s, 1H), 8.30–8.14 (m, 1H), 8.08 (s, 1H), 7.91 (d, J = 6.9 Hz, 1H), 7.88 (s, 1H), 7.69 (s, 2H), 7.60 (s, 1H), 7.49 (d, J = 20.0 Hz, 1H), 7.33 (d, J = 6.3 Hz, 1H), 3.88 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 150.55, 140.26, 138.90, 136.86, 134.29, 131.77, 130.18, 129.10, 127.80, 126.24, 125.08, 120.03, 119.49, 112.36, 110.84, 109.78, 38.96. HRMS (ESI) m/z: [M+H]+ calcd for C₂₂H₁₅F₃N₄S, 425.0970; found 425.1040.

3-(5-(1-methyl-1H-pyrazol-4-yl)thiophen-2-yl)-7-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridine (5k) The title compound was synthesized from 4b and 1-Methyl-1H-pyrazole-4-boronic acid following general procedure D. Yellow solid(35 mg, 64%). Yellow solid (0.035 g, 63.63%); ¹H NMR (400 MHz, CDCl₃): δ 8.51 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 3H), 7.91 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.70 (s, 1H), 7.59 (s, 1H), 7.20 (d, J = 4.0 Hz, 1H), 7.15 (d, J = 4.0 Hz, 1H), 7.12 (d, J = 4.0 Hz, 1H), 3.94 (s, 3H), 3.09 (s, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 146.23, 143.92, 139.97, 136.86, 136.07, 135.00, 134.77, 128.34, 128.26, 127.66, 127.08, 126.68, 126.62, 124.24, 123.00, 116.40, 116.09, 112.23, 44.57, 39.15; HRMS (ESI) m/z: [M+H]+ calcd for C₂₂H₁₈N₄O₂S₂, 435.0871; found 435.0942.

3-(5-(4-fluorophenyl)thiophen-2-yl)-7-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridine (5l) The title compound was synthesized from 4b and (4-fluorophenyl)boronic acid following general procedure D. Yellow solid (21 mg, 67%).¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (d, J = 6.9 Hz, 1H), 8.31 (d, J = 12.6 Hz, 2H), 8.20 (d, J = 8.2 Hz, 2H), 8.09 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 4.8 Hz, 1H), 7.72 (s, 1H), 7.68 (d, J = 3.7 Hz, 1H), 7.37–7.24 (m, 3H), 3.17 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 163.21, 160.76, 141.33, 141.08, 128.05, 127.87, 127.69, 127.60, 127.44, 127.35, 126.33, 125.03, 119.83, 116.37, 116.16, 43.45. HRMS (ESI) m/z: [M+H]+ calcd for C₂₄H₁₇FN₂O₂S₂, 449.0716; found 449.0783.

7-(4-(methylsulfonyl)phenyl)-3-(5-(2-(trifluoromethyl)phenyl)thiophen-2-yl)imidazo[1,2-a]pyridine (5m) The title compound was synthesized from 4b and (2-(trifluoromethyl)phenyl)boronic acid following general procedure D. Yellow solid (39 mg, 61%).¹H NMR(400 MHz, DMSO-d₆) δ 8.79 (d, J = 7.3 Hz, 1H), 8.20 (s, 1H), 8.16 (d, J = 7.6 Hz, 2H), 8.04 (d, J = 8.5 Hz, 3H), 7.92 (d, J = 7.9 Hz, 1H), 7.82–7.76 (m, 1H), 7.73–7.67 (m, 2H), 7.63 (d, J = 3.7 Hz, 1H), 7.54 (d, J = 7.4 Hz, 1H), 7.31 (d, J = 3.5 Hz, 1H), 3.28 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 142.36, 140.28, 138.45, 134.80, 134.50, 133.27, 132.68, 130.49, 129.15, 127.75, 127.56, 126.67, 125.77, 125.05, 115.24, 112.50, 43.52.HRMS (ESI) m/z: [M+H]+ calcd for C₂₅H₁₇F₃N₂O₂S₂, 499.0684; found 499.0749.

7-(4-(methylsulfonyl)phenyl)-3-(5-(pyridin-4-yl)thiophen-2-yl)imidazo[1,2-a]pyridine (5n) The title compound was synthesized from 4b and pyridin-4-ylboronic acid following general procedure D. Yellow solid (37 mg, 67%).¹H NMR (400 MHz, CDCl₃) δ 8.62 (d, J = 8.0 Hz, 1H), 8.53 (dd, J = 20.0, 8.0 Hz, 1H), 8.05 (dd, J = 8.0, 4.0 Hz, 2H), 7.96-7.72 (m, 4H), 7.58 (dd, J = 12.0, 8.0 Hz, 1H), 7.55-7.40 (m, 3H), 7.40-7.28 (m, 1H), 7.24-7.16 (m, 1H), 3.09 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 150.24, 143.64, 140.18, 135.16, 132.49, 132.00, 131.10, 131.62, 128.73, 128.60, 128.31, 127.66, 126.80, 126.28, 124.25, 119.68, 116.16, 112.74, 44.56; HRMS (ESI) m/z: [M+H]+ calcd for C₂₃H₁₇N₃O₂S₂, 432.0762; found 432.0835.

7-(4-(methylsulfonyl)phenyl)-3-(5-(pyridin-3-yl)thiophen-2-yl)imidazo[1,2-a]pyridine (5o) The title compound was synthesized from 4b and pyridin-3-ylboronic acid following general procedure D. Yellow solid (38 mg, 70%). ¹H NMR (400 MHz, CDCl₃) δ 8.93 (s, 1H), 8.57 (d, J = 6.6 Hz, 2H), 8.07 (d, J = 8.1 Hz, 2H), 7.90 (dd, J = 24.9, 13.9 Hz, 5H), 7.47 (d, J = 3.4 Hz, 1H), 7.39–7.30 (m, 2H), 7.21 (d, J = 7.2 Hz, 1H), 3.11 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆): δ148.93, 146.82, 143.79, 140.81, 140.08, 135.31, 135.24, 132.84, 130.10, 129.71, 128.29, 127.63, 126.90, 124.96, 124.24, 123.76, 119.35, 116.18, 112.50, 44.56. HRMS (ESI) m/z: [M+H]+ calcd for C₂₃H₁₇N₃O₂S₂, 432.0762; found 432.0838.

3-(5-(4-(tert-butyl)phenyl)thiophen-2-yl)-7-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridine (5p) The title compound was synthesized from 4b and (4-(tert-butyl)phenyl)boronic acid following general procedure D. Yellow solid (38 mg, 61%). Yellow solid (0.038 g, 61.29%); ¹H NMR (400 MHz, CDCl₃): δ 8.55 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.92 (s, 1H), 7.84 (d, J = 8.0 Hz, 3H), 7.57 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 4.0 Hz, 1H), 7.24 (s, 1H), 7.17 (d, J = 7.0 Hz, 1H), 3.09 (s, 3H), 1.34 (s, 9H); ¹³C NMR(101 MHz, DMSO-d₆): δ 151.27, 143.92, 139.95, 134.98, 134.82, 130.83, 128.35, 128.13, 127.88, 127.59, 126.81, 126.25, 125.98, 125.56, 124.33, 123.33, 116.09, 112.25, 44.57, 34.66, 31.22; HRMS (ESI) m/z: [M+H]+ calcd for C₂₈H₂₆N₂O₂S₂, 487.1436; found 487.1510.

3-(5-(furan-3-yl)thiophen-2-yl)-7-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridine (5q) The title compound was synthesized from 4b and furan-3-ylboronic acid following general procedure D. Yellow solid (23 mg 61%). Yellow solid (0.033 g, 61.11%); ¹H NMR (400 MHz, CDCl₃) δ 8.50 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.92-7.89 (m, 1H), 7.83 (d, J = 8.0 Hz, 3H), 7.71 (d, J = 2.0 Hz, 1H), 7.47 (dd, J = 4.0, 2.0 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 7.15 (dd, J = 8.0, 4.0 Hz, 2H), 6.64 (dd, J = 4.0, 2.0 Hz, 1H), 3.09 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 146.27, 143.89, 143.85, 139.97, 138.45, 135.67, 135.05, 134.82, 128.24, 127.59, 127.38, 126.49, 124.23, 123.04, 120.00, 116.06, 112.29, 109.09, 44.55; HRMS (ESI) m/z: [M+H]+ calcd for C₂₂H₁₆N₂O₃S₂, 421.0602; found 421.0666.

6-(1-methyl-1H-pyrazol-4-yl)-3-(5-(1-methyl-1H-pyrazol-4-yl)thiophen-2-yl)imidazo[1,2-a]pyridine (6a) The title compound was synthesized from 4c and 1-Methyl-1H-pyrazole-4-boronic acid pinacol ester following general procedure D. Brown solid(23 mg, 76%).¹H NMR(400 MHz, CDCl₃) δ 8.47 (s, 1H), 7.72 (d, J = 3.9 Hz, 3H), 7.66 (d, J = 9.3 Hz, 1H), 7.60 (d, J = 4.7 Hz, 2H), 7.33 (dd, J = 9.3, 0.7 Hz, 1H), 7.19 (d, J = 3.7 Hz, 1H), 7.12 (d, J = 3.6 Hz, 1H), 3.95 (d, J = 3.5 Hz, 6H).¹³C NMR (101 MHz, CDCl₃) δ 145.15, 136.68, 136.47, 135.69, 133.67, 126.98, 126.91, 126.53, 124.38, 122.80, 119.15, 118.98, 117.98, 116.33, 39.03, 38.97.HRMS (ESI) m/z: [M+H]+ calcd for C₁₉H₁₆N₆S, 361.1157; found 361.1229.

3-(5-(4-fluorophenyl)thiophen-2-yl)-6-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (6b) The title compound was synthesized from 4c and (4-fluorophenyl)boronic acid following general procedure D. Brownish green solid(17 mg, 65%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (s, 1H), 8.28 (s, 1H), 7.98 (s, 1H), 7.84 (s, 1H), 7.81–7.76 (m, 2H), 7.71 (d, J = 9.3 Hz, 1H), 7.64 (d, J = 3.8 Hz, 1H), 7.62–7.59 (m, 2H), 7.33–7.26 (m, 2H), 3.88 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 162.97, 160.52, 144.74, 141.58, 136.33, 133.59, 129.89, 129.86, 128.81, 128.33, 127.49, 127.41, 126.85, 124.91, 124.52, 119.39, 118.93, 118.72, 118.01, 117.73, 116.20, 115.98, 38.69.HRMS (ESI) m/z: [M+H]+ calcd for C₂₁H₁₅FN₄S, 375.1002; found 375.1079.

3-(5-(4-chlorophenyl)thiophen-2-yl)-6-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (6c) The title compound was synthesized from 4c and (4-chlorophenyl)boronic acid following general procedure D. Yellow solid (21 mg, 61%).¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (s, 1H), 8.28 (s, 1H), 7.98 (s, 1H), 7.85 (s, 1H), 7.75 (d, J = 7.8 Hz, 2H), 7.71 (d, J = 9.5 Hz, 2H), 7.60 (d, J = 17.2 Hz, 2H), 7.49 (d, J = 7.7 Hz, 2H), 3.88 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 144.79, 141.15, 136.32, 133.68, 132.22, 132.12, 129.34, 129.10, 128.32, 126.96, 126.76, 125.42, 124.54, 119.41, 118.89, 118.73, 118.00, 117.71, 38.69. HRMS (ESI) m/z: [M+H]+ calcd for C₂₁H₁₅ClN₄S, 391.0706; found 391.0779.

3-(5-(furan-3-yl)thiophen-2-yl)-6-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (6d) The title compound was synthesized from 4c and furan-3-ylboronic acid following general procedure D. Light brown solid (14 mg, 63%).¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (s, 1H), 8.27 (s, 1H), 8.17 (s, 1H), 7.97 (s, 1H), 7.79 (d, J = 4.0 Hz, 2H), 7.70 (d, J = 9.3 Hz, 1H), 7.57 (dd, J = 12.0, 6.4 Hz, 2H), 7.43 (d, J = 3.4 Hz, 1H), 6.93 (s, 1H), 3.88 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 144.59, 139.05, 136.37, 133.98, 133.43, 128.39, 127.30, 126.35, 125.06, 124.55, 119.83, 119.39, 119.00, 118.71, 118.07, 117.75, 109.17, 38.75.HRMS (ESI) m/z: [M+H]+ calcd for C₁₉H₁₄N₄OS, 347.0888; found 347.0968.

3-(5-(1-methyl-1H-pyrazol-4-yl)thiophen-2-yl)-6-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridine (6e) The title compound was synthesized from 4d and 1-Methyl-1H-pyrazole-4-boronic acid pinacol ester following general procedure D. Yellow solid (22 mg, 54%).¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (s, 1H), 8.10 (s, 1H), 8.03 (s, 4H), 7.86 (s, 1H), 7.81 (d, J = 11.0 Hz, 2H), 7.72 (d, J = 9.3 Hz, 1H), 7.57 (d, J = 3.7 Hz, 1H), 7.32 (d, J = 3.7 Hz, 1H), 3.86 (s, 3H), 3.26 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 144.99, 141.83, 139.98, 136.18, 135.28, 133.85, 128.00, 127.95, 127.78, 126.92, 125.74, 124.92, 124.81, 123.45, 122.62, 119.80, 117.86, 115.53, 43.63, 38.71. HRMS (ESI) m/z: [M+H]+ calcd for C₂₂H₁₈N₄O₂S₂, 435.0871; found 435.0944.

3-(5-(4-fluorophenyl)thiophen-2-yl)-6-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridine (6f) The title compound was synthesized from 4d and (4-fluorophenyl)boronic acid following general procedure D. Yellow solid(24 mg, 53%). ¹H NMR (400 MHz, CDCl₃) δ 8.66 (s, 1H), 8.05 (d, J = 8.3 Hz, 2H), 7.85 (s, 1H), 7.80 (d, J = 9.9 Hz, 2H), 7.77 (s, 1H), 7.61 (dd, J = 8.6, 5.3 Hz, 2H), 7.50 (dd, J = 9.2, 1.4 Hz, 1H), 7.34 (d, J = 3.7 Hz, 1H), 7.29 (d, J = 3.7 Hz, 1H), 7.12 (t, J = 8.6 Hz, 2H), 3.10 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 163.67, 161.20, 144.03, 142.77, 139.75, 134.50, 129.73, 128.38, 128.16, 127.75, 127.45, 127.37, 127.21, 125.68, 125.56, 124.38, 123.67, 121.88, 118.49, 116.05, 115.83, 44.41.HRMS (ESI) m/z: [M+H]+ calcd for C₂₄H₁₇FN₂O₂S₂, 449.0716; found 449.07825.

3-(5-(3-chlorophenyl)thiophen-2-yl)-6-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridine (6g) The title compound was synthesized from 4d and (3-chlorophenyl)boronic acid following general procedure D. Yellow solid (26 mg, 55%).¹H NMR (400 MHz, DMSO-d₆) δ 8.87 (s, 1H), 8.07 (d, J = 8.7 Hz, 2H), 8.04 (d, J = 6.5 Hz, 2H), 7.97 (s, 1H), 7.85 (d, J = 9.6 Hz, 2H), 7.80 (d, J = 2.5 Hz, 1H), 7.76 (d, J = 9.3 Hz, 1H), 7.73 (d, J = 3.7 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 3.27 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 141.71, 141.05, 139.98, 135.22, 134.36, 133.97, 131.03. 129.41, 127.95, 127.66, 127.55, 127.20, 126.07, 125.00, 124.74, 124.05, 122.77, 117.82, 43.54. HRMS (ESI) m/z: [M+H]+ calcd for C₂₄H₁₇ClN₂O₂S₂ 465.0420; found 465.0493.

3-(5-(4-fluorophenyl)thiophen-2-yl)imidazo[1,2-a]pyridine (9a) The title compound was synthesized from 8a and (4-fluorophenyl)boronic acid following general procedure D. Yellow solid (0.018 g, 45%) ¹H NMR (400 MHz, CDCl₃) δ 8.42 (d, J = 6.5 Hz, 1H), 7.77 (s, 1H), 7.66 (d, J = 8.9 Hz, 1H), 7.60–7.52 (m, 2H), 7.22 (d, J = 8.9 Hz, 3H), 7.07 (t, J = 8.5 Hz, 2H), 6.87 (t, J = 6.6 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 162.63, 143.64, 133.79, 130.18, 129.35, 127.61, 126.83, 124.76, 124.03, 123.80, 118.38, 116.16, 113.16. HRMS (ESI) m/z: [M+H]+ calcd for C₁₇H₁₁FN₂S 295.06270; found 295.0698.

3-(5-(1-methyl-1H-pyrazol-4-yl)thiophen-2-yl)imidazo[1,2-a]pyridine (9b) The title compound was synthesized from 8a and 1-Methyl-1H-pyrazole-4-boronic acid pinacol ester following general procedure D. Yellow solid (0.021 g, 43%)¹H NMR (400 MHz, CDCl₃) δ 8.42 (d, J = 4.0 Hz, 1H), 7.75 (s, 1H), 7.70 (d, J = 2.3 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.22 (s, 1H), 7.14 (dd, J = 26.1, 3.5 Hz, 2H), 6.87 (dd, J = 6.3, 3.9 Hz, 1H), 3.94 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 136.99, 135.83, 133.65, 128.68, 128.57, 127.19, 126.62, 124.62, 124.02, 123.06, 118.33, 116.68, 113.04, 39.28.HRMS (ESI) m/z: [M+H]+ calcd for C₁₅H₁₂N₄S 281.0783; found 281.0855.

3-(5-(4-fluorophenyl)thiophen-2-yl)-8-methylimidazo[1,2-a] pyridine (9c) The title compound was synthesized from 8b and (4-fluorophenyl)boronic acid following general procedure D. Yellow solid (0.019 g, 67%)¹H NMR (400 MHz, DMSO-d₆) δ 8.55 (d, J = 6.8 Hz, 1H), 7.87 (d, J = 3.2 Hz, 1H), 7.80–7.74 (m, 2H), 7.63 (d, J = 3.4 Hz,1H), 7.52 (d, J = 3.6 Hz, 1H), 7.30 (t, J = 8.6 Hz, 2H), 7.19 (d, J = 6.0 Hz, 1H), 6.99 (t, J = 6.8 Hz, 1H), 2.54 (s, 3H).¹³C NMR (101 MHz, DMSO-d₆) δ 161.53, 146.04, 141.46, 132.52, 127.41, 126.98, 126.51, 124.86, 123.75, 122.51, 119.17, 116.14, 113.48, 39.52.HRMS (ESI) m/z: [M+H]+ calcd for C₁₈H₁₃FN₂S 309.0784; found 309.0856.

8-methyl-3-(5-(1-methyl-1H-pyrazol-4-yl)thiophen-2-yl)imidazo[1,2-a]pyridine (9d) The title compound was synthesized from 8b and 1-Methyl-1H-pyrazole-4-boronic acid pinacol ester following general procedure D. Light yellow solid (0.015 g, 65%)¹H NMR (400 MHz, CDCl₃) δ 8.33 (d, J = 6.9 Hz, 1H), 7.81 (s, 1H), 7.71 (s, 1H), 7.60 (s, 1H), 7.26 (d, J = 0.8 Hz, 1H), 7.19 (d, J = 3.6 Hz, 1H), 7.12 (d, J = 3.7 Hz, 1H), 6.92 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H), 2.73 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 137.03, 127.75, 127.52, 127.32, 126.65, 123.16, 122.16, 119.99, 116.51, 114.26, 39.32, 17.32. HRMS (ESI) m/z: [M+H]+ calcd for C₁₆H₁₄N₄S, 295.0939; found 295.1012.

Biochemical Enzymatic Inhibition Assay.

Kinase activity was measured in a microfluidics assay that monitors the separation of a phosphorylated product from substrate [28,29]. The assay was run using a 12-sipper chip on a Caliper EZ Reader II (PerkinElmer, Walthman, USA) with separation buffer (100 mM HEPES, 10 mM EDTA, 0.015% Brij-35, 0.1% CR-3 (PerkinElmer, Walthman, USA)). In 96-well plates, compound stocks (20 mM in DMSO) were diluted with kinase buffer (50 mM HEPES, 0.075% Brij-35, 0.1% Tween 20, 4 mM DTT, 30 mM MgCl₂ and 1 mg/ml BSA) in 12-point ½log dilutions (2mM–6.32 nM). Then, 1 μL of compound solution was transferred into a 384-well assay plate. The FLT3 enzyme (Life Technologies, Grand Island, USA) was diluted in kinase buffer and 4 μL of the enzyme solution was transferred to the 384-well plate. The inhibitors with FLT3 enzyme were incubated for 60 min with minor shaking. Further, 5 μL of substrate mix containing 10 μM ATP (Ambresco, Solon, USA) and 3 μM 5FAM tagged FLT3 peptide (peptide #22, 5′ FAM-EPLYWSFPA, PerkinElmer, Walthman, USA) in kinase buffer was added to the assay plate. The final reaction concentrations were as follows: ATP (5 μM), peptide (1.5 μM), compound 12-point ½log dilutions (0.2 mM–0.632 nM). For positive control, no inhibitor was added. For negative control, no enzyme was added. The plate was run until 10–20% conversion of the substrate based on the positive control wells. The following separation conditions were utilized as follow: upstream voltage −500 V; downstream voltage, −1900V; chip pressure −0.8. Percent inhibition was measured for each well comparing starting peptide to phosphorylated product peaks relative to the baseline. Dose-response curves, spanning the IC₅₀ dose, were generated in GraphPad Prisim 8. IC₅₀ values were generated from two to four measurements and error was calculated from the standard deviation between values. For NEK2 inhibition assay, all the procedure are the same, but with different peptide, which was 5FAM tagged NEK2 peptide (peptide #11, 5′-FAM-KKLNRTLSVA-COOH, PerkinElmer, Walthman, USA). For RET inhibition assay, it was exact same as FLT3 assay. For EGFR inhibition assay, the same procedure was performed with different buffer, which was 20 mM MOPS, 0.1% Brij-35, 0.1% Tween 20, 5% glycerol, 4 mM DTT, 30 mM MgCl₂, and 1 mg/ml BSA. For CSF-1R inhibition assay, the same procedure was performed and the same kinase buffer for EGFR screening was used, but the peptide used in here is peptide#30 (5′-FAM-KKKKEEIYFFF-CONH₂, PerkinElmer, Walthman, USA). For the Aurora A inhibition assay, the same procedure was utilized, but with different kinase buffer (20 mM MOPS, 0.01% Brij-35, 5% glycerol, 2 mM DTT, 30 mM MgCl₂, and 1 mg/ml BSA) and different peptide (peptide #21, 5′-FAM-LRRASLG-CONH₂, PerkinElmer, Walthman, USA).

Computational Modeling

FLT3 homology model was generated from Swiss-model web server (https://swissmodel.expasy.org/). The DFG-in structure of PDGFRA (PDB ID: 6JOI) was used as template. The molecular docking studies were completed using AutoDock Vina, AutoDock Tools and Maestro [28–30]. The protein preparation was done in Maestro using the protein preparation wizard tool. All the hydrogens were added and bond orders assigned. A grid box around the ATP binding site was created. The ligands were docked in this grid using AutoDock Vina. The results were visualized and analyzed with the Maestro suite.

Inhibition Kinetic Study

Compounds 5e and 5g was prepared in a serial indicated concentrations using the same kinase buffer as FLT3 inhibition assay. And a serial of substrate solution with different concentrations of ATP were also prepared using FLT3 kinase buffer. Same as previously described, FLT3 solution, compounds solution and substrate solution were added to assay plate orderly. Then recorded the reaction time and followed the biochemical enzymatic assay procedure. Collected the activity data for each compound concentration with different ATP concentrations. Calculate the reaction rate based on the converted amount of the peptide and the recoded reaction time. The Lineweaver-Burk plot were generated by GraphPad Prisim 8.

Anti-proliferation Assay

The FLT3 mutants driven MOLM14 cell lines and Ba/F3 cell line were obtained from the laboratory of Dr. Neil Shah (University of California, San Francisco). All the cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C. RPMI-1640 (Gibco, Carlsbad, USA) medium with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, USA) were used to culture MOLM14 cell lines. While Ba/F3 cell line used the same growth medium as MOLM14 but with additional 2 ng/ml murine IL3. Cells were treated with compounds (200μM–0.0002 μM) and proliferation was assessed using resazurin assay (Biotium, Fremont, USA) following the manufacturer’s instruction. The plates were read at 540 nm (excitation)/590 nm (emission) using a Synergy Neo2 microplate reader (Biotek, Winooski, VT). Then, IC₅₀ values were calculated from the dose-response curves which were generated in GraphPad Prism 8 (GraphPad Software, San Diego, USA). Error was determined as the standard deviation between three independent IC₅₀ measurements.

Detection of Apoptosis

Three FLT3 mutants fused MOLM14 cell lines were cultured in 2 ml of growth medium in 6-well plate for 24 h. Then the cells were treated with DMSO or 1 μM of compound 5o. After 24 h or 48 h exposure to the compound, collected the cells and centrifuged for 5 min at 1200 rpm. Then re-suspended the cells with PBS and counted using Countess II Automated Cell Counter (Applied Biosystems, Foster City, USA). After that, centrifuged the cells again and removed the PBS. Further, the cells were washed with cold PBS for twice and resuspended in binding buffer (BD Bioscience, San Jose, USA) to a concentration of 1 × 10⁶ cells/ml. Then, transferred 100 μl of the cell suspension solution to FACS tubes, followed by adding 5 μl of FITCA Annexin V (BD Bioscience, San Jose, USA) and 5 μl PI (BD Bioscience, San Jose, USA) as the manufacture’s instruction stated. 400 μl of binding buffer was added to each sample after 15 min incubation at room temperature in the dark. Lastly, all the samples were analyzed by flow cytometry using BD Accuri C6 Plus flow cytometer (BD Bioscience, San Jose, USA).