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Article

Design, Synthesis and Biological Evaluation of 6-(Imidazo[1,2-a]pyridin-6-yl)quinazoline Derivatives as Anticancer Agents via PI3Kα Inhibition

1
State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, China
2
The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550014, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(7), 6851; https://doi.org/10.3390/ijms24076851
Submission received: 15 February 2023 / Revised: 30 March 2023 / Accepted: 3 April 2023 / Published: 6 April 2023
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

Abstract

:
Aberrant expression of the phosphatidylinositol 3-kinase (PI3K) signalling pathway is often associated with tumourigenesis, progression and poor prognosis. Hence, PI3K inhibitors have attracted significant interest for the treatment of cancer. In this study, a series of new 6-(imidazo[1,2-a]pyridin-6-yl)quinazoline derivatives were designed, synthesized and characterized by 1H NMR, 13C NMR and HRMS spectra analyses. In the in vitro anticancer assay, most of the synthetic compounds showed submicromolar inhibitory activity against various tumour cell lines, among which 13k is the most potent compound with IC50 values ranging from 0.09 μΜ to 0.43 μΜ against all the tested cell lines. Moreover, 13k induced cell cycle arrest at G2/M phase and cell apoptosis of HCC827 cells by inhibition of PI3Kα with an IC50 value of 1.94 nM. These results suggested that compound 13k might serve as a lead compound for the development of PI3Kα inhibitor.

1. Introduction

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that plays a key regulatory role in various cellular physiological processes including cell growth, proliferation, survival and metabolism [1,2]. Akt (protein kinase B, PKB) is a serine/threonine kinase and participates in the key role of the PI3K signalling pathway. Research shows that mutations and abnormal activation of the PI3K-AKT pathway are often identified as one of the major factors resulted in tumourigenesis, progression and poor prognosis [3,4,5]. PI3K is usually divided into three categories (classes I, II and III) [6]. PI3Kα belongs to class I, which mainly consists of a regulatory subunit (p85) and a catalytic subunit (p110) [7]. The mutation of PIK3CA, the encoding gene of PI3Kα, is one of the most common mutations in tumours and would result in the under-expression or absence of PTEN (phosphatase and tensin homolog) and hyperactivation of PI3K downstream signalling pathways [8,9]. Due to the critical roles of PI3K signalling pathway in tumour occurrence, development and drug resistance, inhibitors targeting PI3K have attracted widespread attention [10,11]. Currently, dozens of subtype-selective and pan-PI3K inhibitors are in various stages of clinical studies for the treatment of human malignancies, yet the discovery of additional lead compounds for novel PI3Kα inhibitors with better efficacy and less toxic side effects remains an urgent therapeutic need [12,13,14].
Quinazolines are the major compounds in the aromatic backbone of nitrogen-containing heterocyclic compounds with a wide range of biological activities such as anti-inflammatory, antimicrobial, antimalarial and antitumour [15,16,17]. In particular, many drugs containing 4-aminoquinazoline structures have been reported to exhibit prominent antitumour activity through various mechanisms [18,19,20,21]. In recent years, it has been shown that 4-aminoquinazoline derivatives show good antitumour activity by inhibiting PI3Kα [22]. This shows that 4-aminoquinazolines are an important class of molecular scaffold that can be used for the development of antitumour drugs.
In a previous study, we designed and synthesised a series of 4-aminoquinazoline derivatives and obtained a compound 6b as a PI3Kα inhibitor [23]. Based on the previous structure activity relationships (SAR) analysis and pharmacophore fusion strategy, structure modification of 6b was performed to further improve the activity. According to the SAR analysis, 4-aminoquinazoline derivative moiety is the main critical pharmacophore of 6b for its PI3Kα inhibitory activity. Therefore, this moiety was retained as the basic scaffold for our target compound. Since imidazo[1,2-a]pyridine, the key pharmacodynamic group of PI3Kα inhibitors TAK-117 and HS-173, is an important class of nitrogen-containing fused heterocyclics compounds that can effectively inhibit the growth of cancer cells, it was introduced to the position 6 of 4-aminoquinazoline [24,25,26,27,28]. Herein, a series of 6-(imidazo[1,2-a]pyridin-6-yl)quinazoline derivatives were designed and synthesized (Figure 1), and biological evaluation was performed to verify their PI3Kα inhibitory activities and antitumour effects.

2. Results and Discussion

2.1. Chemistry

The synthetic route for intermediates 7ao of target products 10au is shown in Scheme 1. A purchased raw material, 6-iodoquinazoline 4-3(H)-one was chlorinated in POCl3 in the presence of DIPEA to give intermediate 2. Intermediates 5aq were obtained by nucleophilic substitution reaction with primary or secondary amines, which subsequently reacted with 2-aminopyridine-5-boronic pinacol ester acid by Suzuki–Miyaura cross-coupling reaction to give intermediates 7ao. Intermediates 7ao were cyclized with methyl bromopyruvate or ethyl bromopyruvate to give the target products 10au, as shown in Scheme 2. To improve the inhibitory activities of the target compounds, we performed further optimization of the substituents. Unfortunately, when the ester side chain was replaced with a cycloalkane, we failed to yield our target products by Scheme 2, so we opted for an alternative synthetic route. As shown in Scheme 3, intermediate 6 reacted with compound 11 to afford compound 12, which was coupled with intermediates 5 to give our target products 13ak by Suzuki–Miyaura cross-coupling reaction. In this thesis, we introduced different substituents at the C6 and C4 positions of the 4-aminoquinazoline backbone and synthesised various ester and amines to further explore their possible structure–activity relationship (SAR), and all compounds are shown in Table 1.

2.2. Biological Evaluation

2.2.1. Antiproliferation Activity Assay

To test the antiproliferative activity of all target compounds, IC50 values were measured by MTT assay on various cancer cell lines including HCC827 (human non-small cell lung cancer cells), A549 (human non-small cell lung cancer cells), SH-SY5Y (human neuroblastoma cells), HEL (human erythroid and leukocyte leukaemia cells) and MCF-7 (human breast cancer cells). As shown in Table 1, most of the compounds showed significant antiproliferative activity in all the test cancer cells. Notably, most of the active compounds were more sensitive to HCC827 cells. In addition to HCC827 cells, PI3K was also overexpressed in other tested cells [29,30,31,32]. As to the reasons for the different sensitivity of the compounds to these tested cells, we hypothesized it might be because the PI3K pathway is not as equally important in the survival and proliferation of these cells as it is in HCC827 cells. For example, when PI3K signalling pathway is inhibited in A549 cells, cells can still maintain cell survival and proliferation through Ras/MERK/ERK pathway [33], which hence leads to different inhibitory activities of PI3K inhibitors in these two cells. According to the data of the antiproliferative assay, we conclude the following structure activity relationship. (I) In general, the antiproliferative activity of the compounds significantly decreased when R1 substituent group was an alkyl, suggesting that simultaneous alkylation of NH2 at the 4-position of quinazoline would impair the antiproliferative activity of the target compounds. (II) When R3 = COOCH3, most of the compounds are more active than R3 = COOC2H5, such as compounds 10q and 10h, 10r and 10i, and when R3 = COOC2H5 and R2 is pyridine, the ortho-nitrogen is more active than meta-nitrogen. (III) The activity of the compounds was generally increased when benzene was introduced into the R3, as in 13c and 10l, 13a and 10r, but a decrease in activity was found with the introduction of the electron withdrawing group F on the R3-substituted benzene, as in compounds 13a and 13b, 13c and 13d. Overall, compound 13k showed the best antiproliferative activity against HCC827 cells with an IC50 value of 0.09 μM, which could be attributed to the conventional hydrogen bond formed between the R2-substituted tetrahydropyran and residue Gln859 in the active site of the target proprotein. To evaluate the selectivity of 13k on cancer cells, the cytotoxicity of 13k on human normal cell MRC-5 (human embryonic lung fibroblasts) was determined. Compound 13k showed much less antiproliferative activity against MRC-5 with an IC50 value of 1.98 μM, which is more than 20-fold different from HCC827 cells (Table 2). Moreover, as shown in Figure 2, 13k treatment time-dependently inhibited the proliferation of HCC827 cells. Taken together, we chose HCC827 cells to further explore the anticancer effects and mechanisms of 13k.

2.2.2. Compound 13k Inhibits PI3Kα and Blocks the PI3K Pathway in HCC827 Cells

To evaluate the in vitro kinase inhibitory activity of 13k against PI3Kα, the kinase activity of PI3Kα was tested using the ADP-GloTM Max Assay method. HS-173, a known PI3Kα inhibitor, was used as a positive control. As shown in Table 3, 13k significantly inhibited the kinase activity of PI3Kα with an IC50 value of 1.94 nM. This suggests that compound 13k is a potential PI3Kα inhibitor.
Aberrant expression of PI3K signalling pathway is closely related to the process of tumourigenesis [34]. Lung cancer is the most lethal malignancy in the world, with non-small cell lung cancer (NSCLC) being the most commonly reported histological subtype [35]. According to reports, new oncogene changes have been discovered in NSCLC, including genetic changes in the PI3K pathway, and PIK3CA mutations in NSCLC may co-occur with epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homologue (KRAS) and anaplastic lymphoma kinase (ALK) mutations [36,37]. Therefore, we chose compound 13k to investigate the mechanism of this compound in HCC827 cells. Since 13k significantly inhibited PI3Kα activity, we further verified the effects of 13k on the PI3K/AKT pathway by Western blot. As shown in Figure 3, the phosphorylation level of PI3K was significantly reduced after 13k treatment in a dose-dependent manner. The phosphorylation levels of its downstream proteins, AKT, mTOR and GSK3β, were correspondingly reduced. The results confirmed the inhibitory effect of 13k on PI3K pathway. The AKT/MAPK signalling pathway, downstream of PI3K, is considered a classical cancer signalling pathway and is involved in the development of many cancers [38,39,40]. Hence, PI3K inhibitors usually also affect the activation of three major categories of MAPK including ERK, JNK and p38 [41]. As shown in Figure 4, the p-JNK/JNK and p-p38/p38 values of HCC827 cells after 13k treatment were significantly higher than those of the control group, indicating that 13k can regulate the MAPK pathway through AKT.

2.2.3. Molecular Docking Study of Compound 13k

Molecular docking simulations were performed to investigate the binding mode between 13k and its target protein PI3Kα (PDB code: 4ZOP). Similar to the binding mode of PI3Kα inhibitor previously discovered, 13k formed two conventional hydrogen bonds with the residues Lys802 and Gln859 as well as hydrophobic interactions including van der Waals, pi–pi T-shaped and pi–sulfur interactions in the active site of PI3Kα. As shown in Figure 5, the benzene ring of compound 13k also formed a pi–alkyl interaction with Leu807 disability. The results indicated that 13k could engage the ATP-binding pocket of PI3Kα. In addition, 13k also formed similar hydrophobic interactions with residues in the acetyl–lysine binding sites.

2.2.4. Compound 13k Induced G2/M Phase Block in HCC827 Cells

It has shown that the anti-proliferative activity of PI3Kα inhibitors was associated with cell cycle arrest [42]. Therefore, we examined the effects of 13k on cell cycle distribution. As shown in Figure 6, 13k treatment for 48 h resulted in a significant G2/M phase block of HCC827 cells (52.21%), when compared to the control group (20.84%). In order to elucidate the potential regulatory mechanism of 13k on cell cycle, proteins associated with cell cycle regulation were detected using Western blot. As described in Figure 6C–G, the protein levels of cyclin B1, c-Myc and CDK1 were dose-dependently decreased by 13k treatment. Additionally, both the total and phosphorylated proteins of CHK1 and CDC25A were also reduced by compound 13k.

2.2.5. Compound 13k Induced Cell Apoptosis

To further investigate the effects of 13k on apoptosis, cells were treated with various doses of 13k ranging from 0 to 0.32 μM. The percentage of apoptotic HCC827 cells was detected using Annexin V-FITC /PI double staining. The results showed that 13k dose-dependently induced cellular apoptosis from 1.73–37.61%. In addition, Hoechst 33342 staining analysis indicated 13k treatment caused cell shrinkage and DNA fragmentation, which resulted in an enhanced absorption and intensity of Hoechst staining. To further elucidate the mechanism of 13k-induced apoptosis, the apoptosis-related protein levels was examined by Western blot. We found that compound 13k increased the protein levels of cleaved caspase-9 and cleaved PARP in a concentration-dependent manner, while the ratios of Bax/Bcl-2 were upregulated, further indicating that compound 13k promotes cell apoptosis (Figure 7).

2.2.6. In 3D Spheroid Cell Inhibition Assay

The 3D cell culture has been proved to more realistically reproduce the interactions between cell–cell and cell–extracellular matrix interactions and more accurately simulate the actual microenvironment of cells in tissues [43,44,45]. These allow the cell behaviour characteristics of cells in 3D cell culture to be closer to the survival state in living organisms. Hence, it was widely applied in research fields including new drug screening, tumour cell system biology, stem cell research and functional tissue implantation [46,47,48]. Additionally, previous findings indicated that the phenotype of the 3D lung cancer tumour sphere in vitro is closer to that of real cancer tissue in vivo [49,50]. Thus, it is considered a reasonable method to evaluate the in vivo efficacy of active compounds in the early stages of new drug development [51]. To gain insight into the effects of long-term 13k treatment, we used a 3D spheroid tumour growth model that was built using HCC827 cancer cells. After the 3D tumour spheres had been formed, they were treated with different concentrations of 13k for 12 days, changing the drug-containing culture medium every 3 days. As shown in Figure 8, the tumour spheres were slightly contracted and flattened after treatment with 0.4 μM 13k for 12 days. However, the spheres were gradually split and became loose and eventually collapsed when treated with increased concentration of 13k (0.8 μM and 1.6 μM), indicating that 13k could effectively inhibit the tumour sphere formation and has potential for further preclinical studies.

3. Conclusions

In summary, a series of new 6-(imidazo[1,2-a]pyridin-6-yl)quinazoline derivatives (10au and 13ak) were designed, synthesized and evaluated for their in vitro anti-proliferative activities against five cancer cell lines (HCC827, A549, SH-SY5Y, HEL and MCF-7). As a result, most of the synthetic compounds showed submicromolar inhibitory activity against various tumour cell lines. Among them, 13k is the most potent compound with IC50 values ranging from 0.09 μΜ to 0.43 μΜ against all the test cell lines. Moreover, compound 13k showed strong inhibitory activity against PI3Kα, and 13k induced cell cycle arrest at G2/M phase and cell apoptosis of HCC827 cells by inhibition of PI3Kα with an IC50 value of 1.94 nM. Compound 13k showed better antitumour activity and PI3Kα kinase activity compared to the lead compound 6b. Therefore, compound 13k could be a promising PI3Kα inhibitor for the development of novel targeted antitumour drugs.

4. Experimental Procedure

4.1. Chemistry

4.1.1. Instruments and Materials

All reagents and solvents were commercially available and used without further purification. 1H NMR, 13C NMR and 19F NMR spectra were recorded with a 600, 150 and 565MHz NMR spectrometer (Bruker AVANCE NEO), respectively. The NMR spectra were generated by using Mestrenova 12.0 as processing software, deuterated chloroform (CDCl3) and dimethyl sulfoxide-d6 (DMSO-d6) as solvents, and tetramethylsilane (TMS) as an internal standard. All chemical shifts are expressed in ppm (δ), and the coupling constants (J) are expressed in hertz (Hz). The melting points of the compounds were determined using a Beijing micro melting point apparatus. High-resolution accurate mass measurements were performed on a quadrupole time-of-flight (QTOF) mass spectrometer (micro TOF-Q, Bruker Inc., Billerica, MA, USA) using electrospray ionisation (positive mode).

4.1.2. General Experimental Protocol for Preparation of Compounds 10au

Preparation of 4-Chloro-6-iodoquinazoline (2)

A mixture of 6-iodoquinazolin-4(3H)-one (2.45 g, 9 mmol), N, N-diisopropylethylamine (2.33 g, 18 mmol), phosphorus oxychloride (2.76 g, 18 mmol) and anhydrous toluene (50 mL) was reacted at 80 °C for 4 h under argon atmosphere. After completion of the reaction (monitored by TLC), the crude reaction mixture was cooled, and the solvent was removed under reduced pressure. The mixture was extracted 2–3 times with ethyl acetate and saturated sodium bicarbonate solution. The organic phase was dried with anhydrous Na2SO4 and rotary dried under vacuum. The residue was purified through a column chromatography on silica with EtOAc/PE to afford 4-chloro-6-iodoquinazoline 2 as white flocculent (2.27 g, 7.81 mmol, 86.83% yield), ESI-MS: m/z 291.5 [M + H]+.

Steps for the Preparation of 6-Iodo-N-(4-methoxybenzyl)quinazolin-4-amine (5a)

A mixture of 4-chloro-6-iodoquinazoline (0.58 g, 2 mmol) and 4-Methoxybenzylamine (0.33 g, 2.4 mmol) was added to isopropanol (10 mL) and refluxed at 60 °C for 2 h under argon protection. After completion of the reaction (monitored by TLC), the solvent of the reaction mixture was removed under reduced pressure and extracted 2–3 times with ethyl acetate and saturated Na2CO3 solution. The organic phase was dried over anhydrous Na2SO4 and rotary dried under vacuum to form 6-iodo-N-(4-methoxybenzyl)quinazolin-4-amine 5a as white solid (0.60 g, 1.54 mmol, 77.0% yield), ESI-MS: m/z 392.1 [M + H]+.
Compounds 5bq were synthesized according to the procedure described in 5a. The ESI-MS information of compounds 5bq is listed as below:

N-(cyclopropylmethyl)-6-iodoquinazolin-4-amine (5b)

Off-white solid, 91.2% yield, ESI-MS: m/z 326.0 [M + H]+.

N-(4-fluorobenzyl)-6-iodoquinazolin-4-amine (5c)

Off-white solid, 77.8% yield, ESI-MS: m/z 380.0 [M + H]+.

6-iodo-N-(4-(trifluoromethyl)benzyl)quinazolin-4-amine (5d)

Off-white solid, 86.1% yield, ESI-MS: m/z 430.2 [M + H]+.

6-iodo-N-(3-methylbenzyl)quinazolin-4-amine (5e)

Pale yellow solid, 94.5% yield, ESI-MS: m/z 376.2 [M + H]+.

N1,N1-diethyl-N2-(6-iodoquinazolin-4-yl)ethane-1,2-diamine (5f)

Pale yellow oily substance, 91.0% yield, ESI-MS: m/z 371.0 [M + H]+.

6-iodo-N-(2-methylbenzyl)quinazolin-4-amine (5g)

Pale yellow solid, 95.3% yield, ESI-MS: m/z 376.0 [M + H]+.

6-iodo-N-(pyridin-2-ylmethyl)quinazolin-4-amine (5h)

Pale yellow solid, 92.1% yield, ESI-MS: m/z 385.2 [M + Na]+.

N-(2-fluorobenzyl)-6-iodoquinazolin-4-amine (5i)

Off-white solid, 61.9% yield, ESI-MS: m/z 380.0 [M + H]+.

N-(3-fluorophenyl)-6-iodoquinazolin-4-amine (5j)

Pale yellow solid, 85.3% yield, ESI-MS: m/z 366.1 [M + H]+.

N-(3,5-dimethoxyphenyl)-6-iodoquinazolin-4-amine (5k)

Pale yellow solid, 90.7% yield, ESI-MS: m/z 408.2 [M + H]+.

6-iodo-N-(pyridin-3-ylmethyl)quinazolin-4-amine (5l)

Pink solid, 94.2% yield, ESI-MS: m/z 385.2 [M + H]+.

N-(2,3-difluorophenyl)-6-iodoquinazolin-4-amine (5m)

Off-white solid, 93.6% yield, ESI-MS: m/z 384.1 [M + H]+.

6-iodo-N-methyl-N-(p-tolyl)quinazolin-4-amine (5n)

Pale yellow solid, 94.0% yield, ESI-MS: m/z 376.0 [M + H]+.

N-ethyl-6-iodo-N-phenylquinazolin-4-amine (5o)

Pale yellow solid, 95.7% yield, ESI-MS: m/z 376.0 [M + H]+.

6-iodo-N-(1H-pyrazol-3-yl)quinazolin-4-amine (5p)

White solid, 91.5% yield, ESI-MS: m/z 338.1 [M + H]+.

6-iodo-N-((tetrahydro-2H-pyran-4-yl)methyl)quinazolin-4-amine (5q)

White solid, 90.1% yield, ESI-MS: m/z 392.0 [M + Na]+.

Procedure for the Preparation of 6-(6-Aminopyridin-3-yl)-N-(4-methoxybenzyl)quinazolin-4-amine (7a)

The 6-iodo-N-(4-methoxybenzyl)quinazolin-4-amine 5a (0.6 g, 1.5 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (0.34 g, 1.5 mmol) and K2CO3 (0.64 g, 4.6 mmol) were added to 15 mL of solvent [V(1,4-dioxane):V(water) = 4:1]. The mixture was heated to 100 °C under a protective atmosphere of argon followed by the addition of Pd(dppf)Cl2. The mixture continues to be stirred under these conditions for a further 4–6 h. After completion of the reaction (monitored by TLC), 1,4-dioxane and water were removed under reduced pressure, and the residue was purified through a column chromatography on silica with dichloromethane/methanol to afford white solid 6-(6-aminopyridin-3-yl)-N-(4-methoxybenzyl)quinazolin-4-amine 7a (0.36 g, 0.99 mmol, 66.6% yield), ESI-MS: m/z 358.1 [M + H]+.
Compounds 7bo were synthesized according to the procedure described in 7a. The ESI-MS information of compounds 7bo is listed as below:

6-(6-Aminopyridin-3-yl)-N-(cyclopropylmethyl)quinazolin-4-amine (7b)

Off-white solid, 71.2% yield, ESI-MS: m/z 291.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-(4-fluorobenzyl)quinazolin-4-amine (7c)

Off-white solid, 92.2% yield, ESI-MS: m/z 246.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-(4-(trifluoromethyl)benzyl)quinazolin-4-amine (7d)

Off-white solid, 85.7% yield, ESI-MS: m/z 396.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-(3-methylbenzyl)quinazolin-4-amine (7e)

Off-white solid, 73.4% yield, ESI-MS: m/z 342.1 [M + H]+.

N1-(6-(6-aminopyridin-3-yl)quinazolin-4-yl)-N2,N2-diethylethane-1,2-diamine (7f)

Brown solid, 82.9% yield, ESI-MS: m/z 359.1 [M + Na]+.

6-(6-Aminopyridin-3-yl)-N-(2-methylbenzyl)quinazolin-4-amine (7g)

Off-white solid, 76.3% yield, ESI-MS: m/z 342.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-(pyridin-2-ylmethyl)quinazolin-4-amine (7h)

Yellow solid, 70.6% yield, ESI-MS: m/z 328.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-(2-fluorobenzyl)quinazolin-4-amine (7i)

Off-white solid, 86.3% yield, ESI-MS: m/z 346.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-(3-fluorophenyl)quinazolin-4-amine (7j)

Pale yellow solid, 77.6% yield, ESI-MS: m/z 332.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-(3,5-dimethoxyphenyl)quinazolin-4-amine (7k)

Yellow solid, 81.3% yield, ESI-MS: m/z 396.1 [M + Na]+.

6-(6-Aminopyridin-3-yl)-N-(pyridin-3-ylmethyl)quinazolin-4-amine (7l)

Off-white solid, 67.6% yield, ESI-MS: m/z 329.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-(2,3-difluorophenyl)quinazolin-4-amine (7m)

White solid, 88.7% yield, ESI-MS: m/z 352.1 [M + Na]+.

6-(6-Aminopyridin-3-yl)-N-methyl-N-(p-tolyl)quinazolin-4-amine (7n)

Pale yellow solid, 79.8% yield, ESI-MS: m/z 342.1 [M + H]+.

6-(6-Aminopyridin-3-yl)-N-ethyl-N-phenylquinazolin-4-amine (7o)

Yellow solid, 75.7% yield, ESI-MS: m/z 342.1 [M + H]+.

Procedure for the Preparation of Ethyl 6-(4-((4-Methoxybenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10a) or Methyl 6-(4-((4-methoxybenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10o)

A mixture of 6-(6-aminopyridin-3-yl)-N-(4-methoxybenzyl)quinazolin-4-amine 7a (0.18 g, 0.5 mmol), ethyl bromopyruvate (0.29 g, 1.5 mmol) or methyl bromopyruvate (0.27 g, 1.5 mmol) and NaHCO3 (0.13 g, 1.5 mmol) was added to EtOH (5 mL), and the mixture was warmed to 80 °C and refluxed by condensation under argon for 4 h. After completion of the reaction (monitored by TLC), the solvents were removed under reduced pressure and the residue was purified through a column chromatography on silica with dichloromethane/methanol to obtain ethyl 6-(4-((4-methoxybenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate 10a or methyl 6-(4-((4-methoxybenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate 10o; 10a as white solid (0.142 g, 0.31 mmol, 62.0% yield), m.p. 131.2–133.6 °C. 1H NMR (600 MHz, Chloroform-d) δ 8.69 (s, 1H), 8.22 (s, 1H), 8.09 (s, 1H), 8.05 (s, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 9.4 Hz, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.24–7.17 (m, 1H), 6.83 (d, J = 8.0 Hz, 2H), 4.84 (s, 2H), 4.37 (q, J = 7.1 Hz, 2H), 3.76 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 163.1, 159.5, 159.2, 156.0, 149.1, 144.3, 137.3, 134.1, 131.2, 130.2, 129.6 (2C), 129.2, 127.5, 126.9, 123.6, 120.0, 118.7, 117.3, 115.4, 114.1 (2C), 61.3, 55.3, 44.9, 14.4. HRMS (ESI): calcd for C26H24O3N5 [M + H]+ m/z 454.1874, found 454.1866; C26H23O3N5Na [M + Na]+ m/z 476.1693, found 476.1687; 10o as white solid (0.096 g, 0.218 mmol, 43.7% yield), m.p. 138.6–140.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.25 (s, 1H), 9.04 (d, J = 0.8 Hz, 1H), 8.73 (s, 1H), 8.54 (s, 2H), 8.13 (d, J = 8.7 Hz, 1H), 7.86 (d, J = 9.6 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 9.7 Hz, 1H), 7.34 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 4.77 (d, J = 5.7 Hz, 2H), 3.85 (s, 3H), 3.71 (s, 3H). 13C NMR (150 MHz, DMSO) δ 163.0, 159.5, 158.4, 154.9, 148.5, 143.9, 135.9, 133.6, 131.2, 130.9, 130.9, 128.9 (2C), 126.8, 125.5, 125.1, 120.8, 118.5, 118.0, 115.0, 113.8 (2C), 55.1, 51.7, 43.4. HRMS (ESI): calcd for C25H22O3N5 [M + H]+ m/z 440.1717, found 440.1711.
Compounds 10bn and 10pu were synthesized according to the procedure described in 10a or 10o. The information of compounds 10bn and 10pu is listed as below:

Ethyl 6-(4-((Cyclopropylmethyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10b)

White solid, 49.8% yield, m.p. 128.5–130.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.64 (d, J = 2.1 Hz, 1H), 8.57 (d, J = 4.9 Hz, 2H), 8.47 (s, 1H), 8.09 (dd, J = 8.7, 2.0 Hz, 1H), 7.87 (dd, J = 9.6, 1.9 Hz, 1H), 7.79 (dd, J = 9.1, 6.6 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.48–3.42 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H), 1.22 (ddd, J = 11.6, 7.3, 5.3 Hz, 1H), 0.53–0.46 (m, 2H), 0.34–0.29 (m, 2H). 13C NMR (150 MHz, DMSO) δ 162.6, 159.5, 155.5, 148.9, 143.8, 136.2, 133.2, 130.7, 128.4, 126.9, 125.8, 125.0, 120.7, 118.4, 118.0, 115.2, 60.3, 45.1, 14.3, 10.6, 3.6 (2C). HRMS (ESI): calcd for C22H22O2N5 [M + H]+ m/z 388.1768, found 388.1760; C22H21O2N5Na [M + Na]+ m/z 410.1588, found 410.1580.

Ethyl 6-(4-((4-Fluorobenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10c)

White solid, 55.1% yield, m.p. 127.8–129.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.04 (t, J = 1.3 Hz, 1H), 9.01 (t, J = 5.9 Hz, 1H), 8.68 (d, J = 2.2 Hz, 1H), 8.56 (s, 1H), 8.49 (s, 1H), 8.12 (dd, J = 8.7, 2.0 Hz, 1H), 7.87 (dd, J = 9.6, 1.9 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 9.5 Hz, 1H), 7.45 (dd, J = 8.5, 5.7 Hz, 2H), 7.16 (t, J = 8.9 Hz, 2H), 4.81 (d, J = 5.7 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.6, 162.1, 160.5, 159.4, 155.4, 148.9, 143.9, 136.2, 135.5, 133.4, 130.9, 129.4, 129.4, 128.5, 126.8, 125.7, 125.0, 120.7, 118.4, 118.0, 115.2, 115.0, 60.3, 43.0, 14.3. 19F NMR (565 MHz, DMSO) δ −115.99. HRMS (ESI): calcd for C25H21O2N5F [M + H]+ m/z 442.1674, found 442.1667; C25H20O2N5FNa [M + Na]+ m/z 464.1493, found 464.1489.

Ethyl 6-(4-((4-(Trifluoromethyl)benzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10d)

White solid, 56,8% yield, m.p. 129.8–131.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.14 (t, J = 6.0 Hz, 1H), 9.05 (s, 1H), 8.70 (s, 1H), 8.56 (s, 1H), 8.49 (s, 1H), 8.14 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 9.4 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 9.4 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.1 Hz, 2H), 4.92 (d, J = 5.6 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.6, 159.5, 155.3, 148.7, 144.3, 143.8, 136.2, 133.5, 131.0, 128.4, 128.0 (2C), 127.7, 126.7, 125.6, 125.3 (2C), 125.0, 123.5, 120.7, 118.4, 118.0, 115.1, 60.3, 43.4, 14.3. 19F NMR (565 MHz, DMSO) δ −60.78. HRMS (ESI): calcd for C26H21O2N5F3 [M + H]+ m/z 492.1642, found 492.1632; C26H20O2N5F3Na [M + Na]+ m/z 514.1461, found 514.1452.

Ethyl 6-(4-((3-Methylbenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10e)

Off-white solid 53.0% yield, m.p. 130.1–132.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.03 (s, 1H), 9.01 (t, J = 5.9 Hz, 1H), 8.70 (s, 1H), 8.55 (s, 1H), 8.50 (s, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.87 (d, J = 9.4 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.79 (d, J = 9.5 Hz, 1H), 7.20 (dd, J = 12.5, 7.3 Hz, 3H), 7.06 (d, J = 7.1 Hz, 1H), 4.80 (d, J = 5.7 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 2.28 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.6, 159.5, 155.4, 148.7, 143.9, 139.2, 137.5, 136.2, 133.4, 130.9, 128.3, 128.3, 128.0, 127.6, 126.8, 125.7, 125.0, 124.5, 120.7, 118.5, 118.0, 115.2, 60.4, 43.7, 21.1, 14.3. HRMS (ESI): calcd for C26H24O2N5 [M + H]+ m/z 438.1925, found 438.1918; C26H23O2N5Na [M + Na]+ m/z 460.1744, found 460.1737.

Ethyl 6-(4-((2-(Diethylamino)ethyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10f)

Brown solid, 42.1% yield, m.p. 235.7–237.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.28 (s, 1H), 9.24 (s, 1H), 8.98 (s, 1H), 8.51 (d, J = 11.6 Hz, 2H), 8.16 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 9.6 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 9.4 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 3.94 (s, 2H), 3.17 (s, 6H), 1.33 (t, J = 7.1 Hz, 3H), 1.22 (s, 6H). 13C NMR (150 MHz, DMSO) δ 162.6, 159.6, 155.2, 148.8, 143.9, 136.1, 133.1, 130.6, 128.3, 126.7, 125.3, 125.2, 121.1, 118.3, 117.9, 115.4, 60.4 (2C), 46.6 (3C), 14.3 (3C). HRMS (ESI): calcd for C24H29O2N6 [M + H]+ m/z 433.2347, found 433.2341.

Ethyl 6-(4-((2-Methylbenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10g)

Pink solid, 45.8% yield, m.p. 149.6–151.7 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.94 (s, 1H), 8.76 (s, 1H), 8.56 (s, 1H), 8.51 (s, 1H), 8.15 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 9.4 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.30 (d, J = 7.3 Hz, 1H), 7.22–7.14 (m, 3H), 4.81 (d, J = 5.4 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 2.37 (s, 3H), 1.33 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.6, 159.5, 155.2, 143.8, 136.5, 136.2, 135.9, 133.5, 133.5, 131.0, 130.0, 128.0, 127.4, 127.0, 126.8, 125.8, 125.7, 125.0, 120.8, 118.4, 118.0, 115.1, 60.3, 42.1, 18.8, 14.3. HRMS (ESI): calcd for C26H24O2N5 [M + H]+ m/z 438.1925, found 438.1917; C26H23O2N5Na [M + Na]+ m/z 460.1744, found 460.1735.

Ethyl 6-(4-((Pyridin-2-ylmethyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10h)

Yellow solid, 57.3% yield, m.p. 129.6–131.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.20–9.14 (m, 1H), 9.05 (s, 1H), 8.73 (s, 1H), 8.58–8.51 (m, 2H), 8.47 (s, 1H), 8.14 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 9.5 Hz, 1H), 7.84–7.78 (m, 2H), 7.73 (t, J = 7.6 Hz, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.29–7.24 (m, 1H), 4.92 (d, J = 5.7 Hz, 2H), 4.32 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.6, 159.6, 158.5, 155.3, 149.0, 148.6, 143.8, 136.8, 136.2, 133.4, 130.9, 128.3, 126.7, 125.6, 125.0, 122.2, 121.2, 120.7, 118.4, 118.0, 115.2, 60.3, 45.7, 14.3. HRMS (ESI): calcd for C24H21O2N6 [M + H]+ m/z 425.1721, found 425.1712; C24H20O2N6Na [M + Na]+ m/z 447.1540, found 447.1532.

Ethyl 6-(4-((2-Fluorobenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10i)

Off-white solid, 59.2% yield, m.p. 142.3–144.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.48 (s, 1H), 9.08 (s, 1H), 8.81 (s, 1H), 8.60 (s, 1H), 8.55(s, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 9.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 9.4 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.33 (q, J = 6.9 Hz, 1H), 7.22 (t, J = 9.3 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H), 4.90 (d, J = 5.2 Hz, 2H), 4.33 (q, J = 7.0 Hz, 2H), 1.33 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.5, 161.1, 159.8, 159.5, 154.3, 143.8, 136.2, 134.1, 131.6, 129.6, 129.1, 126.7, 126.5, 125.3, 125.2, 124.4, 121.0, 118.4, 118.0, 115.3, 115.2, 114.7, 60.3, 38.0, 14.3. 19F NMR (565 MHz, DMSO) δ −118.51. HRMS (ESI): calcd for C25H21O2N5F [M + H]+ m/z 442.1674, found 442.1664; C25H20O2N5FNa [M + Na]+ m/z 464.1493, found 464.1486.

Ethyl 6-(4-((3-Fluorophenyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10j)

Yellow solid, 46.7% yield, m.p. 148.3–150.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.08 (s, 1H), 8.86 (s, 1H), 8.68 (s, 1H), 8.56 (s, 1H), 8.19 (d, J = 8.7 Hz, 1H), 7.92 (t, J = 8.0 Hz, 3H), 7.82 (d, J = 9.4 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.45 (q, J = 7.8 Hz, 1H), 6.98 (td, J = 8.5, 2.6 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.9, 162.6, 161.3, 157.7, 154.6, 149.2, 143.9, 136.3, 134.3, 131.6, 130.1, 128.7, 126.9, 125.6, 125.4, 120.8, 118.5, 118.1, 117.9, 115.4, 110.4, 109.1, 60.4, 14.3. 19F NMR (565 MHz, DMSO) δ −112.46. HRMS (ESI): calcd for C24H18O2N5FNa [M + Na]+ m/z 450.1337, found 450.1328.

Ethyl 6-(4-((3,5-dimethoxyphenyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10k)

Yellow solid, 50.2% yield, m.p. 156.2–158.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.90 (s, 1H), 9.09 (s, 1H), 8.87 (s, 1H), 8.66 (s, 1H), 8.57 (s, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 9.3 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.82 (d, J = 9.4 Hz, 1H), 7.19 (s, 2H), 6.34 (s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 3.78 (s, 6H), 1.34 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.6, 160.4 (2C), 157.8, 154.6, 148.9, 143.9, 140.6, 136.2, 134.2, 131.5, 128.4, 126.9, 125.6, 125.3, 120.8, 118.5, 118.0, 115.4, 100.8 (2C), 95.8, 60.3, 55.3 (2C), 14.3. HRMS (ESI): calcd for C26H24O4N5 [M + H]+ m/z 470.1823, found 470.1809; C26H23O4N5Na [M + Na]+ m/z 492.1642, found 492.1633.

Ethyl 6-(4-((2,3-difluorophenyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10l)

White solid, 55.3% yield, m.p. 131.2–133.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.21 (s, 1H), 9.10 (s, 1H), 8.83 (s, 1H), 8.57 (d, J = 7.1 Hz, 2H), 8.23 (d, J = 8.7 Hz, 1H), 8.02–7.86 (m, 2H), 7.82 (d, J = 9.5 Hz, 1H), 7.39 (s, 2H), 7.30 (s, 1H), 4.33 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.6, 158.4, 154.9, 151.3, 151.2, 149.6, 149.6, 143.9, 136.3, 134.2, 131.6, 128.6, 126.7, 125.5, 125.3, 124.3, 123.2, 121.0, 118.5, 118.1, 115.1, 114.5, 60.4, 14.3. 19F NMR (565 MHz, DMSO) δ −138.40, −142.37. HRMS (ESI): calcd for C24H18O2N5F2 [M + H]+ m/z 446.1423, found 446.1411; C24H17O2N5F2Na [M + Na]+ m/z 468.1243, found 468.1231.

Ethyl 6-(4-(Ethyl(phenyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10m)

Pink flocculent, 63.1% yield, m.p. 176.7–177.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.74 (s, 1H), 8.50 (s, 1H), 8.45 (s, 1H), 7.97 (dd, J = 8.7, 2.1 Hz, 1H), 7.85 (d, J = 8.6 Hz, 1H), 7.60–7.55 (m, 3H), 7.50 (t, J = 7.4 Hz, 1H), 7.38 (d, J = 7.6 Hz, 2H), 7.10 (d, J = 2.1 Hz, 1H), 6.86 (dd, J = 9.5, 1.8 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.20 (q, J = 6.9 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H), 1.25 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.5, 160.0, 154.5, 151.1, 145.8, 143.6, 136.2, 131.9, 130.5 (2C), 130.4, 129.1, 127.3, 127.2 (2C), 125.8, 125.4, 124.7, 124.0, 118.4, 117.9, 115.8, 60.4, 48.2, 14.3, 11.7. HRMS (ESI): calcd for C26H24O2N5 [M + H]+ m/z 438.1925, found 438.1916; C26H23O2N5Na [M + Na]+ m/z 460.1744, found 460.1735.

Ethyl 6-(4-((Pyridin-3-ylmethyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10n)

White solid, 47.3% yield, m.p. 120.3–122.4 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.08 (t, J = 5.9 Hz, 1H), 9.04 (s, 1H), 8.66 (d, J = 7.1 Hz, 2H), 8.55 (s, 1H), 8.51 (s, 1H), 8.47 (d, J = 4.8 Hz, 1H), 8.13 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 9.5 Hz, 1H), 7.84–7.77 (m, 3H), 7.36 (dd, J = 7.9, 4.7 Hz, 1H), 4.85 (d, J = 5.7 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.6, 159.5, 155.3, 149.0, 148.7, 148.2, 143.8, 136.2, 135.3, 134.8, 133.5, 131.0, 128.4, 126.8, 125.7, 125.0, 123.6, 120.7, 118.4, 118.0, 115.1, 60.4, 41.5, 14.3. HRMS (ESI): calcd for C24H21O2N6 [M + H]+ m/z 425.1721, found 425.1711; C24H20O2N6Na [M + Na]+ m/z 447.1540, found 447.1534.

Methyl 6-(4-((2-Methylbenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10p)

Off-white solid, 36.7% yield, m.p. 150.3–152.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.05 (s, 2H), 8.77 (s, 1H), 8.57 (s, 1H), 8.53 (s, 1H), 8.23–8.08 (m, 1H), 7.96–7.70 (m, 3H), 7.30 (d, J = 7.2 Hz, 1H), 7.24–7.11 (m, 3H), 4.81 (d, J = 4.7 Hz, 2H), 3.86 (s, 3H), 2.37 (s, 3H). 13C NMR (150 MHz, DMSO) δ 163.0, 159.6, 155.0, 147.7, 143.9, 136.4, 135.9, 133.7, 131.7, 131.2, 130.1, 127.6, 127.5, 127.0, 126.9, 125.8, 125.6, 125.1, 120.9, 118.5, 118.0, 115.0, 51.7, 42.2, 18.9. HRMS (ESI): calcd for C25H22O2N5 [M + H]+ m/z 424.1768, found 424.1760; C25H21O2N5Na [M + Na]+ m/z 446.1588, found 446.1580.

Methyl 6-(4-((Pyridin-2-ylmethyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10q)

Yellow solid, 53.8% yield, m.p. 143.7–145.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.23 (s, 1H), 9.06 (s, 1H), 8.75 (s, 1H), 8.57 (s, 1H), 8.53 (d, J = 4.8 Hz, 1H), 8.48 (s, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 9.5 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 9.5 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.29–7.25 (m, 1H), 4.92 (d, J = 5.5 Hz, 2H), 3.86 (s, 3H). 13C NMR (150 MHz, DMSO) δ 163.0, 159.7, 158.4, 155.2, 149.0, 148.2, 143.9, 136.8, 135.9, 133.5, 131.0, 128.0, 126.7, 125.6, 125.1, 122.3, 121.2, 120.8, 118.5, 118.0, 115.1, 51.7, 45.8. HRMS (ESI): calcd for C23H19O2N6 [M + H]+ m/z 411.1564, found 411.1556; C23H18O2N6Na [M + Na]+ m/z 433.1384, found 433.1372.

Methyl 6-(4-((2-Fluorobenzyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10r)

Off-white solid, 52.1% yield, m.p. 174.0–176.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.72 (s, 1H), 9.09 (s, 1H), 8.85 (s, 1H), 8.64 (s, 1H), 8.55 (s, 1H), 8.21 (s, 1H), 7.87 (t, J = 11.8 Hz, 2H), 7.76 (d, J = 9.2 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.33 (s, 1H), 7.22 (t, J = 9.2 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H), 4.92 (s, 2H), 3.85 (s, 3H). 13C NMR (150 MHz, DMSO) δ 162.9, 161.1, 159.9, 159.4, 153.9, 143.8, 135.9, 134.3, 131.9, 129.7, 129.2, 126.6, 125.6, 125.3, 125.2, 124.4, 121.1, 118.5, 118.0, 115.3, 115.2, 114.5, 51.7, 38.1. 19F NMR (565 MHz, DMSO) δ −118.44. HRMS (ESI): calcd for C24H19O2N5F [M + H]+ m/z 428.1517, found 428.1509; C24H18O2N5FNa [M + Na]+ m/z 450.1337, found 450.1330.

Methyl 6-(4-((3-Fluorophenyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10s)

White solid, 48.6% yield, m.p. 179.3–181.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.22–10.05 (m, 1H), 9.10 (s, 1H), 8.88 (s, 1H), 8.69 (s, 1H), 8.57 (s, 1H), 8.21 (dd, J = 8.7, 2.0 Hz, 1H), 7.97–7.88 (m, 3H), 7.81 (d, J = 9.5 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.49–7.41 (m, 1H), 6.98 (td, J = 8.5, 2.6 Hz, 1H), 3.86 (s, 3H). 13C NMR (150 MHz, DMSO) δ 163.0, 162.8, 161.2, 157.7, 154.4, 148.8, 143.9, 140.8, 135.9, 134.3, 131.6, 130.1, 128.4, 126.9, 125.6, 125.4, 120.8, 118.5, 118.0, 115.3, 110.4, 109.2, 51.7. 19F NMR (565 MHz, DMSO) δ −112.46. HRMS (ESI): calcd for C23H17O2N5F [M + H]+ m/z 414.1361, found 414.1347; C23H16O2N5FNa [M + Na]+ m/z 436.1180, found 436.1172.

Methyl 6-(4-(Methyl(p-tolyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10t)

Pink flocculent, 39.5% yield, m.p. 172.8–174.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.58 (s, 1H), 8.41 (s, 1H), 7.95 (dd, J = 8.7, 2.1 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 9.5 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 7.06 (d, J = 2.1 Hz, 1H), 6.77 (dd, J = 9.5, 1.9 Hz, 1H), 3.85 (s, 3H), 3.57 (s, 3H), 2.41 (s, 3H). 13C NMR (150 MHz, DMSO) δ 162.9, 160.4, 154.4, 150.8, 145.1, 143.6, 137.0, 135.9, 131.8, 130.9 (2C), 130.2, 128.9, 126.4 (2C), 125.6, 125.3, 124.9, 124.0, 118.4, 117.8, 115.7, 51.7, 42.0, 20.6. HRMS (ESI): calcd for C25H22O2N5 [M + H]+ m/z 424.1768, found 424.1757; C25H21O2N5Na [M + Na]+ m/z 446.1588, found 446.1580.

Methyl 6-(4-(Ethyl(phenyl)amino)quinazolin-6-yl)imidazo[1,2-a]pyridine-2-carboxylate (10u)

Orange flocculent, 51.4% yield, m.p. 286.5–287.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.38 (d, J = 9.2 Hz, 2H), 7.94 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 8.1 Hz, 3H), 7.46 (t, J = 7.4 Hz, 1H), 7.33 (d, J = 7.7 Hz, 2H), 7.20 (s, 1H), 6.98 (d, J = 8.7 Hz, 1H), 4.24 (q, J = 7.0 Hz, 2H), 3.88 (s, 3H), 1.31 (t, J = 6.9 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.3, 159.8, 153.7 (2C), 150.7, 145.6, 143.2, 135.8, 131.5, 129.7 (2C), 129.6, 128.4, 126.4 (2C), 126.3, 125.2, 123.8, 123.4, 117.4, 117.2, 115.7, 50.7, 47.4, 11.4. HRMS (ESI): calcd for C25H22O2N5 [M + H]+ m/z 424.1768, found 424.1762; C25H21O2N5Na [M + Na]+ m/z 446.1588, found 446.1581.

4.1.3. General Experimental Protocol for Preparation of Compounds 13ak

Procedure for the Preparation of 2-Phenyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-a]pyridine (12a)

The components 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (1.2 g, 5.4 mmol), 2-bromoacetophenone 11a (1.3 g, 6.5 mmol) and NaHCO3 (1.4 g, 16 mmol) were added to EtOH (10 mL), and the mixture was heated to 80 °C and refluxed by condensation under argon for 4 h. After completion of the reaction (monitored by TLC), the solvent of the reaction mixture was removed under reduced pressure, and the mixture was extracted 2–3 times with ethyl acetate and saturated Na2CO3 solution. The organic phase was dried over anhydrous Na2SO4 and rotary dried under vacuum to form 2-phenyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-a]pyridine 12a as pale-yellow oil substance (1.5 g, 4.7 mmol, 86.8% yield), ESI-MS: m/z 321.1 [M + H]+.
Compounds 12bc were synthesized according to the procedure described in 12a. The ESI-MS information of compounds 12bc is listed as below:

Compound 2-(4-Fluorophenyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-a]pyridine (12b)

Yellow solid, 81.5% yield, ESI-MS: m/z 337.2 [M + H]+.

Compound 2-Cyclopropyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-a]pyridine (12c)

Yellow solid, 81.5% yield, ESI-MS: m/z 283.2 [M + H]+.

Procedure for the Preparation of N-(2-fluorobenzyl)-6-(2-phenylimidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine (13a)

N-(2-fluorobenzyl)-6-iodoquinazolin-4-amine 5i (0.19 g, 0.5mmol), 2-phenyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[1,2-a]pyridine 12a (0.16 g, 0.5 mmol) and K2CO3 (0.21 g, 1.5 mmol) were added to 1,4-dioxane/water 10 mL [V(1,4-dioxane):V(water) = 4:1], and the mixture was heated to 100 °C under a protective atmosphere of argon followed by the addition of Pd(dppf)Cl2. The mixture continues to be stirred under these conditions for a further 4–5 h. After completion of the reaction (monitored by TLC), the 1, 4-dioxane and water were removed under reduced pressure, and the residue was purified through a column chromatography on silica with dichloromethane/methanol to afford N-(2-fluorobenzyl)-6-(2-phenylimidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine 13a as pink flocculent, 67.3% yield, m.p. 130.8–132.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.98 (t, J = 5.8 Hz, 1H), 8.72 (s, 1H), 8.50 (s, 1H), 8.44 (s, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.01 (d, J = 7.9 Hz, 2H), 7.82 (d, J = 8.6 Hz, 1H), 7.77 (q, J = 9.4 Hz, 2H), 7.46 (t, J = 7.6 Hz, 3H), 7.36–7.30 (m, 2H), 7.25–7.20 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 4.88 (d, J = 5.5 Hz, 2H). 13C NMR (150 MHz, DMSO) δ 161.2, 159.6, 159.5, 155.2, 148.7, 145.2, 144.2, 134.0, 133.8, 131.0, 129.6, 129.1, 128.8 (2C), 128.4, 127.9, 125.9, 125.8, 125.7 (2C), 125.1, 124.4, 120.3, 116.8, 115.3, 115.2, 109.7, 37.8. 19F NMR (565 MHz, DMSO) δ −118.62. HRMS (ESI): calcd for C28H21N5F [M + H]+ m/z 446.1776, found 446.1768.
Compounds 13b13k were synthesized according to the procedure described in 13a. The information of compounds 13b13k is listed as below:

N-(2-fluorobenzyl)-6-(2-(4-fluorophenyl)imidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine (13b)

Pink flocculent, 70.7% yield, m.p. 133.7–135.6 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.96 (t, J = 5.4 Hz, 1H), 8.72 (s, 1H), 8.50 (s, 1H), 8.42 (s, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.04 (dd, J = 8.5, 5.5 Hz, 2H), 7.82 (d, J = 8.6 Hz, 1H), 7.79 (d, J = 9.4 Hz, 1H), 7.75 (d, J = 9.4 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.35–7.31 (m, 1H) 7.29 (t, J = 8.8 Hz, 2H), 7.24–7.20 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 4.88 (d, J = 5.5 Hz, 2H). 13C NMR (150 MHz, DMSO) δ 162.8, 161.1, 159.5, 155.2, 148.6, 144.2, 134.0, 131.0, 130.3, 129.6, 129.0, 128.3, 127.6, 125.9, 125.8, 125.1, 124.5, 124.4 (2C), 120.3, 116.7 (2C), 115.7, 115.6, 115.3, 115.2, 109.5, 37.7. 19F NMR (565 MHz, DMSO) δ −113.19, −117.60. HRMS (ESI): calcd for C28H20N5F2 [M + H]+ m/z 464.1681, found 464.1677.

N-(2,3-difluorophenyl)-6-(2-phenylimidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine (13c)

Pink flocculent, 73.2% yield, m.p. 142.0–144.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.22 (s, 1H), 9.08 (s, 1H), 8.86 (s, 1H), 8.56 (s, 1H), 8.46 (s, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 7.3 Hz, 2H), 7.93 (d, J = 8.7 Hz, 1H), 7.87–7.76 (m, 2H), 7.47 (t, J = 7.7 Hz, 2H), 7.40 (q, J = 8.6 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H) 7.30 (q, J = 7.2 Hz, 1H). 13C NMR (150 MHz, DMSO) δ 158.5, 154.7, 151.3, 149.7, 149.6, 145.1, 144.2, 134.7, 133.6, 131.6, 129.7, 128.8 (2C), 128.4, 128.0, 125.7 (2C), 125.0, 124.7, 124.3, 124.3, 123.3, 120.7, 116.8, 115.1, 114.6, 109.7. 19F NMR (565 MHz, DMSO) δ −137.37, −141.29. HRMS (ESI): calcd for C27H18N5F2 [M + H]+ m/z 450.1525, found 450.1520; C27H17N5F2Na [M + Na]+ m/z 472.1344, found 472.1337.

N-(2,3-difluorophenyl)-6-(2-(4-fluorophenyl)imidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine (13d)

Pink flocculent, 72.5% yield, m.p. 136.1–138.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.21 (s, 1H), 9.07 (s, 1H), 8.85 (s, 1H), 8.56 (s, 1H), 8.43 (s, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.05 (dd, J = 8.5, 5.5 Hz, 2H), 7.92 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 9.4 Hz, 1H), 7.78 (d, J = 9.4 Hz, 1H), 7.40 (q, J = 8.4 Hz, 2H), 7.29 (t, J = 8.7 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 162.8, 161.2, 158.4, 154.7, 151.2, 149.7, 144.2, 134.7, 131.6, 130.2, 128.4, 127.7 (2C), 127.6, 125.1, 124.7, 124.3, 124.2, 123.3, 120.6, 116.8, 115.7 (2C), 115.6, 115.1, 114.6, 109.6. 19F NMR (565 MHz, DMSO) δ −113.15, −137.34, −141.32. HRMS (ESI): calcd for C27H17N5F3 [M + H]+ m/z 468.1431, found 468.1426; C27H16N5F3Na [M + Na]+ m/z 490.1250, found 490.1239.

N-(cyclopropylmethyl)-6-(2-(4-fluorophenyl)imidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine (13e)

Pink flocculent, 56.1% yield, m.p. 227.5–229.7 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.64 (d, J = 2.2 Hz, 1H), 8.56 (t, J = 5.6 Hz, 1H), 8.48 (s, 1H), 8.43 (s, 1H), 8.18–8.11 (m, 1H), 8.05 (dd, J = 8.6, 5.6 Hz, 2H), 7.87–7.65 (m, 3H), 7.29 (t, J = 8.8 Hz, 2H), 3.46 (t, J = 6.1 Hz, 2H), 1.22 (td, J = 11.8, 4.7 Hz, 1H), 0.57–0.45 (m, 2H), 0.32 (q, J = 5.0 Hz, 2H). 13C NMR (150 MHz, DMSO) δ 162.8, 161.1, 159.5, 155.4, 148.7, 144.2, 133.7, 130.7, 130.3, 128.3, 127.6 (2C), 125.2, 124.6, 124.3, 120.3, 116.7, 115.7 (2C), 115.2, 109.5, 45.2, 10.6, 3.6 (2C). 19F NMR (565 MHz, DMSO) δ −114.24. HRMS (ESI): calcd for C25H21N5F [M + H]+ m/z 410.1776, found 410.1773.

Compound 6-(2-phenylimidazo[1,2-a]pyridin-6-yl)-N-(1H-pyrazol-3-yl)quinazolin-4-amine (13f)

Pink solid, 54.6% yield, m.p. 289.1–290.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 12.61 (s, 1H), 10.86 (s, 1H), 9.09 (s, 1H), 9.06 (s, 1H), 8.66 (s, 1H), 8.46 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H), 8.03 (d, J = 7.6 Hz, 2H), 7.93 (d, J = 9.4 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H),7.83–7.68 (m, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.36 (t, J = 7.3 Hz, 1H), 6.91 (s, 1H). 13C NMR (150 MHz, DMSO) δ 154.4, 149.4, 148.1, 145.0, 144.2, 134.4, 133.6, 131.2, 129.0, 128.8 (2C), 128.0, 127.8, 125.7 (2C), 125.2, 124.6, 124.3, 120.6, 116.6, 116.6, 115.2, 109.7, 98.4. HRMS (ESI): calcd for C24H18N7 [M + H]+ m/z 404.1618, found 404.1613; C24H17N7Na [M + Na]+ m/z 426.1438, found 426.1426.

Compound 6-(2-cyclopropylimidazo[1,2-a]pyridin-6-yl)-N-(cyclopropylmethyl)quinazolin-4-amine (13g)

Yellow solid, 60.6% yield, m.p. 118.6–120.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.89 (s, 1H), 8.60 (d, J = 2.1 Hz, 1H), 8.55 (t, J = 5.6 Hz, 1H), 8.46 (s, 1H), 8.09 (dd, J = 8.6, 2.0 Hz, 1H), 7.79 (s, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.68 (dd, J = 9.4, 1.9 Hz, 1H), 7.57 (d, J = 9.3 Hz, 1H), 3.45 (t, J = 6.2 Hz, 2H), 2.05 (ddd, J = 13.2, 8.3, 4.9 Hz, 1H), 1.25–1.18 (m, 1H), 0.92 (dt, J = 8.2, 2.9 Hz, 2H), 0.88–0.84 (m, 2H), 0.52–0.45 (m, 2H), 0.34–0.27 (m, 2H). 13C NMR (150 MHz, DMSO) δ 159.4, 155.2, 149.3, 148.5, 143.4, 134.0, 130.7, 128.2, 124.0, 123.8, 123.7, 120.1, 115.9, 115.2, 109.2, 45.2, 10.6, 9.5, 8.3 (2C), 3.6 (2C). HRMS (ESI): calcd for C22H22N5 [M + H]+ m/z 356.1870, found 356.1863; C22H21N5Na [M + Na]+ m/z 378.1690, found 378.1682.

Compound 6-(2-cyclopropylimidazo[1,2-a]pyridin-6-yl)-N-ethyl-N-phenylquinazolin-4-amine (13h)

Yellow solid, 58.3% yield, m.p. 166.8–168.7 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.15 (s, 1H), 7.94 (dd, J = 8.7, 2.1 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.67 (s, 1H), 7.55 (t, J =7.3 Hz, 2H), 7.53–7.50 (m, 1H), 7.34 (d, J = 7.5 Hz, 3H), 7.04 (d, J = 2.1 Hz, 1H), 6.76 (dd, J = 9.3, 1.9 Hz, 1H), 4.18 (q, J = 7.0 Hz, 2H), 1.99–2.05 (m, 1H), 1.23 (t, J = 7.0 Hz, 3H), 0.90 (dt, J = 8.2, 2.9 Hz, 2H), 0.85–0.81 (m, 2H). 13C NMR (150 MHz, DMSO) δ 160.0, 154.2, 150.8, 149.4, 145.9, 143.2, 132.6, 130.4 (2C), 130.4, 128.8, 127.2 (2C), 127.1, 123.5, 123.4, 123.3, 122.9, 115.8, 115.8, 109.1, 48.1, 11.7, 9.4, 8.2 (2C). HRMS (ESI): calcd for C26H24N5 [M + H]+ m/z 406.2026, found 406.2019; C26H23N5Na [M + Na]+ m/z 428.1846, found 428.1840.

N-ethyl-N-phenyl-6-(2-phenylimidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine (13i)

Pink solid, 67.4% yield, m.p. 203.0–205.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.74 (s, 1H), 8.32 (s, 1H), 8.26 (s, 1H), 8.03–7.97 (m, 3H), 7.83 (d, J = 8.6 Hz, 1H), 7.61–7.57 (m, 3H), 7.54 (d, J = 9.3 Hz, 1H), 7.45 (t, J = 7.6 Hz, 2H), 7.38 (dd, J = 6.8, 2.8 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 6.87 (dd, J = 9.4, 1.9 Hz, 1H), 4.21 (q, J = 7.0 Hz, 2H), 1.25 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 160.0, 154.1, 150.5, 145.8, 145.2, 143.9, 133.6, 132.4, 130.5 (2C), 128.7 (2C), 128.6, 127.9, 127.4, 127.2 (2C), 125.7 (2C), 124.3, 124.1, 123.9, 123.7, 116.6, 115.7, 109.5 (2C), 48.2, 11.7. HRMS (ESI): calcd for C29H24N5 [M + H]+ m/z 442.2026, found 442.2021.

Compound 6-(2-phenylimidazo[1,2-a]pyridin-6-yl)-N-(pyridin-2-ylmethyl)quinazolin-4-amine (13j)

Pink solid, 70.2% yield, m.p. 141.9–143.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 9.13 (t, J = 5.9 Hz, 1H), 9.02 (s, 1H), 8.75 (s, 1H), 8.54 (d, J = 4.3 Hz, 1H), 8.46 (s, 1H), 8.44 (s, 1H), 8.19 (dd, J = 8.7, 2.0 Hz, 1H), 8.01 (d, J = 7.5 Hz, 2H), 7.84–7.78 (m, 2H), 7.78–7.70 (m, 2H), 7.46 (t, J = 7.6 Hz, 2H), 7.39 (d, J = 7.9 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 7.29–7.25 (m, 1H), 4.93 (d, J = 5.8 Hz, 2H). 13C NMR (150 MHz, DMSO) δ 159.6, 158.6, 155.2, 149.0, 148.6, 145.2, 144.2, 136.8, 133.9, 133.7, 130.9, 128.8 (2C), 128.3, 127.9, 125.7 (2C), 125.0, 124.4, 124.4, 122.2, 121.2, 120.3, 116.8, 115.2, 109.7, 45.7. HRMS (ESI): calcd for C27H21N6 [M + H]+ m/z 429.1822, found 429.1817.

Compound 6-(2-phenylimidazo[1,2-a]pyridin-6-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)quinazolin-4-amine (13k)

Pink solid, 69.8% yield, m.p. 127.0–129.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.64 (s, 1H), 8.51 (t, J = 5.8 Hz, 1H), 8.49 (s, 1H), 8.44 (s, 1H), 8.14 (dd, J = 8.6, 2.0 Hz, 1H), 8.01 (d, J = 7.5 Hz, 2H), 7.80–7.74 (m, 3H), 7.46 (t, J = 7.6 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 3.86 (d, J = 11.2 Hz, 2H), 3.49 (t, J = 6.4 Hz, 2H), 3.28 (t, J = 11.0 Hz, 2H), 2.06–1.97 (m, 1H), 1.67 (d, J = 10.4 Hz, 2H), 1.32–1.24 (m, 2H). 13C NMR (150 MHz, DMSO) δ 159.7, 155.3, 148.5, 145.2, 144.2, 133.8, 133.8, 130.8, 128.8 (2C), 128.2, 127.9, 125.7 (2C), 125.1, 124.5, 124.3, 120.3, 116.8, 115.2, 109.7, 66.8 (2C), 46.3, 34.2, 30.7 (2C). HRMS (ESI): calcd for C27H26ON5 [M + H]+ m/z 436.2132, found 436.2126.

4.2. Biological (Pharmacological) Research

4.2.1. Cell Culture

Human cell lines HCC827, A549, SH-SY5Y, HEL, MCF-7 and MRC-5 obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) were treated with 10% foetal bovine serum (FBS, Biological Industries, Cromwell, CT, USA) and 1% antibiotics-antimycotics (100 units/mL penicillin G sodium, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B) added to RPMI-1640 (HCC827, SH-SY5Y, HEL) or DMEM (A549, MCF-7, MRC-5) in culture. Cells were grown at 37 °C in an incubator containing water and 5% CO2.

4.2.2. Antiproliferative Activity Assay

Cells were seeded in 96-well plates at 3000–5000 cells/well and treated with different concentrations of compounds for 72 h. After treatment, 20 μL MTT (Sigma-Aldrich, St. Louis, MO, USA) was added to each well, and incubation was continued in the incubator for 4 h. Purple formazan crystals were formed, the medium was discarded, 150 μL DMSO was added to dissolve the formazan, and the absorbance at 490 nm was measured by a multi-well spectrophotometer (Thermo Scientific, VARIOSKAN LUX, Waltham, MA, USA) to measure absorbance at 490 nm and to measure viability. IC50 values were calculated based on the inhibition rate using GraphPad Prism software.

4.2.3. Molecular Modelling

Molecular docking simulations were performed using Molecular Operating Environment (MOE, Version 2020) [52]. PI3Kα (PDB code: 4ZOP) is selected for docking studies. Protein optimisation was performed by quickprep of the MOE. Docking sites were defined by the Site Finder program and Accelrys Discovery Studio Visualizer 4.5 was used for graphical display.

4.2.4. Kinase Assay

The inhibitory activity of compound 13k against PI3Kα was determined using the ADP-GloTM Max Assay, with HS-173 as a positive control, according to the kit instructions. Chemiluminescence values were measured by multi-well spectrophotometer (Thermo Scientific, VARIOSKAN LUX, USA).

4.2.5. Cell Cycle Assays

HCC827 cells were incubated in 6-well plates and treated with specific concentrations of 13k for 48 h. Cells were collected and washed with PBS buffered solution, fixed overnight at −20 °C with pre-cooled 70% ethanol, supernatant discarded, washed with PBS buffered solution, stained by a mixture of propidium iodide (PI) and RNase, incubated for 30 min at room temperature protected from light and then detected using flow cytometry.

4.2.6. Hochest 33342 Staining Assay

A portion of HCC827 cells were taken and inoculated overnight in 6-well plates and treated with different concentrations of compound 13k for 48 h. Subsequent steps were carried out according to the instructions of the Hochest 33342 staining kit (Beyotime, Shanghai, China). Final pictures were taken with a microscope (DMi8, Leica, Wetzlar, Germany).

4.2.7. Apoptosis Assay

Apoptosis was detected by flow cytometry after staining with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s protocol (BD Biosciences). HCC827 cells were inoculated overnight in 6-well plates, treated with specific concentrations of compound 13k for 48 h. Cells were collected and incubated with 5 μL of membrane linked protein V-FITC and 5 μL of PI for 15–20 min protected from light, followed by flow cytometry analysis.

4.2.8. Western Blot Assay

Cells were treated with different concentrations of compound 13k and then subjected to immunoblot analysis as described in a previous study. Blots were imaged by a ChemiDocTM MP imaging system (Bio-Rad, Hercules, CA, USA). All bands were analysed using Image J software. Antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

4.2.9. 3D Spheroid Cell Inhibition Assay

To culture HCC827 cancer cells into three-dimensional spheroids, we used PerkinElmer’s CellCarrier Spheroid ULA 96-well microtiter plates (PerkinElmer, Waltham, MA, USA). In all experiments, cells were seeded at 40,000 cells per well. After spheroid formation, the spheroids were treated with 13k at the indicated concentrations every 3 days. When significant changes in tumour spheroids were observed, photographs were taken using a ZEISS LSM 900 Airyscan 2 confocal laser scanning microscopy (ZEISS, Jena, Germany).

4.2.10. Statistical Analysis

All experimental data were replicated three times, and experimental results are expressed as mean ± standard deviation (SD). Statistical analyses were manipulated and plotted using Photoshop, ImageJ, Graph Pad, etc., and tests were performed to assess statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001 or n.s. (not significant)).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24076851/s1.

Author Contributions

Conceptualization, M.L. and D.W.; Data curation, M.L. and D.W.; Formal analysis, Q.L., F.L. and T.Z.; Funding acquisition, Y.F.; Investigation, M.S.; Methodology, M.L. and D.W.; Project administration, Y.F.; Resources, Y.F.; Software, Q.L., F.L. and T.Z.; Supervision, Y.F.; Validation, H.W., L.X. and M.Y.; Visualization, M.L. and D.W.; Writing—original draft, M.L.; Writing—review and editing, M.L., D.W. and Y.F. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grants 21907020 and 22167010), Merit-based Funding Program for Innovation and Entrepreneurship of High-level Overseas Talents of Guizhou Province (grant No. 202001), Natural Science Foundation of Guizhou Provincial Science and Technology Projects (QKH-zk [2022]030) and the Science and Technology Plan Project of Guizhou Province, China (QKHPTRC[2018]5779-58).

Institutional Review Board Statement

Not applicable for studies not involving humans or animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are included within the article and Supplementary Materials.

Acknowledgments

The authors wish to express their gratitude to their Analytical Testing Center for testing the NMR and HR-MS (ESI) data and to Ailing Linghu, Xinran Zhao of the Guizhou Medical University for helpful discussions on topics related to this work.

Conflicts of Interest

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

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Figure 1. Design strategies for target compounds.
Figure 1. Design strategies for target compounds.
Ijms 24 06851 g001
Scheme 1. Preparation of 7a7p reagents and conditions: (i) DIPEA, POCl3, Toluene, 80 °C, 4 h; (ii) isopropanol, 60 °C, 2 h; (iii) K2CO3, Pd(dppf)Cl2, 1,4-dioxacyclohexane/H2O, 100 °C, 5 h.
Scheme 1. Preparation of 7a7p reagents and conditions: (i) DIPEA, POCl3, Toluene, 80 °C, 4 h; (ii) isopropanol, 60 °C, 2 h; (iii) K2CO3, Pd(dppf)Cl2, 1,4-dioxacyclohexane/H2O, 100 °C, 5 h.
Ijms 24 06851 sch001
Scheme 2. Preparation of 10a10u reagents and conditions: (iv) NaHCO3, EtOH, 80 °C, 4 h.
Scheme 2. Preparation of 10a10u reagents and conditions: (iv) NaHCO3, EtOH, 80 °C, 4 h.
Ijms 24 06851 sch002
Scheme 3. Preparation of 13a13k reagents and conditions: (i) NaHCO3, EtOH, 80 °C, 4 h; (ii) K2CO3, Pd(dppf)Cl2, 1,4-dioxacyclohexane/H2O, 100 °C, 5 h.
Scheme 3. Preparation of 13a13k reagents and conditions: (i) NaHCO3, EtOH, 80 °C, 4 h; (ii) K2CO3, Pd(dppf)Cl2, 1,4-dioxacyclohexane/H2O, 100 °C, 5 h.
Ijms 24 06851 sch003
Figure 2. The time-dependent activity of 13k on HCC827 cells. Cells were treated with 13k (0.03 to 0.50 µM) for 24 h to 72 h, and the survival rates were detected via MTT assay.
Figure 2. The time-dependent activity of 13k on HCC827 cells. Cells were treated with 13k (0.03 to 0.50 µM) for 24 h to 72 h, and the survival rates were detected via MTT assay.
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Figure 3. Compound 13k inhibited the expression of PI3K and its downstream related proteins (AE). Expression of PI3K-related proteins was analysed by immunoblotting after treatment of cells with different concentrations of compound 13k (0, 0.08, 0.16 and 0.32 μM) for 48 h. Expression of the associated proteins was analysed using Image J. Each bar data are expressed as mean ± SD from three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control).
Figure 3. Compound 13k inhibited the expression of PI3K and its downstream related proteins (AE). Expression of PI3K-related proteins was analysed by immunoblotting after treatment of cells with different concentrations of compound 13k (0, 0.08, 0.16 and 0.32 μM) for 48 h. Expression of the associated proteins was analysed using Image J. Each bar data are expressed as mean ± SD from three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control).
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Figure 4. Effect of compound 13k on the MAPK signalling pathway (AD). Expression of related proteins was analysed by immunoblotting after treatment of cells with different concentrations of compound 13k (0, 0.08, 0.16 and 0.32 μM) for 48 h. Expression of the associated proteins was analysed using Image J. Each bar Data are expressed as mean ± SD from three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control).
Figure 4. Effect of compound 13k on the MAPK signalling pathway (AD). Expression of related proteins was analysed by immunoblotting after treatment of cells with different concentrations of compound 13k (0, 0.08, 0.16 and 0.32 μM) for 48 h. Expression of the associated proteins was analysed using Image J. Each bar Data are expressed as mean ± SD from three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control).
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Figure 5. Molecular docking model of compound 13k with PI3Kα. (A) Docking of 13k to the active site of PI3Kα (PDB code: 4ZOP); (B) 13k docked in the ATP-binding pocket of PI3Kα; (C) 2D binding model of 13k and PI3Kα. The image was observed with BIOVIA Discovery Studio Visualizer 4.5.
Figure 5. Molecular docking model of compound 13k with PI3Kα. (A) Docking of 13k to the active site of PI3Kα (PDB code: 4ZOP); (B) 13k docked in the ATP-binding pocket of PI3Kα; (C) 2D binding model of 13k and PI3Kα. The image was observed with BIOVIA Discovery Studio Visualizer 4.5.
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Figure 6. Effect of 13k on the cell cycle of HCC827. (A) Compound 13k alters the distribution of the cell cycle. Cells were treated with compound 13k for 48 h, stained with propidium iodide mixed with RNase, incubated for 30 min at room temperature and protected from light and analysed by flow cytometry. ‘Ctrl’ refers to the control without the addition of compound 13k. (B) Quantitative histograms of the different phases of the cell cycle. (CG) Western blot analysis of protein expression associated with G2/M phase. Changes in the corresponding proteins were quantified using Image j. Each bar represents the mean ± SD (n = 3) and was considered statistically significant when compared to the corresponding control values at * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 6. Effect of 13k on the cell cycle of HCC827. (A) Compound 13k alters the distribution of the cell cycle. Cells were treated with compound 13k for 48 h, stained with propidium iodide mixed with RNase, incubated for 30 min at room temperature and protected from light and analysed by flow cytometry. ‘Ctrl’ refers to the control without the addition of compound 13k. (B) Quantitative histograms of the different phases of the cell cycle. (CG) Western blot analysis of protein expression associated with G2/M phase. Changes in the corresponding proteins were quantified using Image j. Each bar represents the mean ± SD (n = 3) and was considered statistically significant when compared to the corresponding control values at * p < 0.05, ** p < 0.01 and *** p < 0.001.
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Figure 7. Compound 13k induces apoptosis in HCC827 cells. (A) Apoptosis as well as nuclear morphology was measured by Hoechst 33342 staining after treatment of cells with compound 13k, scale bar = 250 μM. (B) Apoptosis was quantified by flow cytometry using Annexin V-FITC/PI double staining. ‘Ctrl’ refers to the control without the addition of compound 13k. (CF) Western blot analysis was used to measure the regulation of apoptosis-associated proteins, using Image J for analysis. Each data is expressed as the mean ± SD of three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control).
Figure 7. Compound 13k induces apoptosis in HCC827 cells. (A) Apoptosis as well as nuclear morphology was measured by Hoechst 33342 staining after treatment of cells with compound 13k, scale bar = 250 μM. (B) Apoptosis was quantified by flow cytometry using Annexin V-FITC/PI double staining. ‘Ctrl’ refers to the control without the addition of compound 13k. (CF) Western blot analysis was used to measure the regulation of apoptosis-associated proteins, using Image J for analysis. Each data is expressed as the mean ± SD of three parallel experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. control).
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Figure 8. Effect of 13k on HCC827 spheroid formation. HCC827 cells were seeded in ultralow attachment 96-well U bottom plates (40,000 cells/well) to generate tumour spheroids and treated with 5 fold of IC50 concentrations of 13k for the spheroid assay. After initiation, the spheroids were treated with 13k at the indicated concentrations every 3 days. After 12 days, pictures were taken with a ZEISS LSM 900 Airyscan 2 confocal laser scanning microscopy. ‘Ctrl’ refers to the control without the addition of compound 13k.
Figure 8. Effect of 13k on HCC827 spheroid formation. HCC827 cells were seeded in ultralow attachment 96-well U bottom plates (40,000 cells/well) to generate tumour spheroids and treated with 5 fold of IC50 concentrations of 13k for the spheroid assay. After initiation, the spheroids were treated with 13k at the indicated concentrations every 3 days. After 12 days, pictures were taken with a ZEISS LSM 900 Airyscan 2 confocal laser scanning microscopy. ‘Ctrl’ refers to the control without the addition of compound 13k.
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Table 1. Anti-tumour activity of different cell lines (IC50, μM) a.
Table 1. Anti-tumour activity of different cell lines (IC50, μM) a.
Ijms 24 06851 i001
Comp.R1R2R3HCC827A549SH-SY5YHELMCF-7
10aHIjms 24 06851 i002Ijms 24 06851 i003>10>10>10>10>10
10bHIjms 24 06851 i004Ijms 24 06851 i0030.59 ± 0.181.16 ± 0.271.89 ± 0.600.82 ± 0.252.86 ± 0.80
10cHIjms 24 06851 i005Ijms 24 06851 i003>10>10>10>10>10
10dHIjms 24 06851 i006Ijms 24 06851 i003>10>10>10>10>10
10eHIjms 24 06851 i007Ijms 24 06851 i003>10>10>10>10>10
10fHIjms 24 06851 i008Ijms 24 06851 i003>10>10>10>10>10
10gHIjms 24 06851 i009Ijms 24 06851 i003>10>10>10>10>10
10hHIjms 24 06851 i010Ijms 24 06851 i0037.26 ± 1.357.30 ± 1.977.06 ± 0.165.37 ± 1.07>10
10iHIjms 24 06851 i011Ijms 24 06851 i003>10>10>109.51 ± 1.60>10
10jHIjms 24 06851 i012Ijms 24 06851 i0031.76 ± 0.778.49 ± 1.97>106.87 ± 3.80>10
10kHIjms 24 06851 i013Ijms 24 06851 i003>10>10>10>10>10
10lHIjms 24 06851 i014Ijms 24 06851 i0031.04 ± 0.792.01 ± 0.881.20 ± 0.352.90 ± 0.517.47 ± 1.31
10mIjms 24 06851 i015Ijms 24 06851 i016Ijms 24 06851 i003>10>109.26 ± 1.54>10>10
10nHIjms 24 06851 i017Ijms 24 06851 i003>10>10>10>10>10
10oHIjms 24 06851 i018Ijms 24 06851 i019>10>10>10>10>10
10pHIjms 24 06851 i020Ijms 24 06851 i019>10>10>10>10>10
10qHIjms 24 06851 i021Ijms 24 06851 i0191.83 ± 0.704.22 ± 0.234.27 ± 0.662.10 ± 0.664.94 ± 0.54
10rHIjms 24 06851 i022Ijms 24 06851 i0195.21 ± 2.403.31 ± 2.134.64 ± 0.715.49 ± 0.948.15 ± 1.10
10sHIjms 24 06851 i023Ijms 24 06851 i0190.91 ± 0.215.71 ± 3.99>102.91 ± 1.025.52 ± 1.02
10tIjms 24 06851 i024Ijms 24 06851 i025Ijms 24 06851 i019>10>10>107.22 ± 2.44>10
10uIjms 24 06851 i015Ijms 24 06851 i026Ijms 24 06851 i019>10>10>10>10>10
13aHIjms 24 06851 i022Ijms 24 06851 i0270.94 ± 0.141.18 ± 0.453.24 ± 1.760.55 ± 0.252.54 ± 0.57
13bHIjms 24 06851 i022Ijms 24 06851 i0284.14 ± 0.653.11 ± 0.204.38 ± 1.112.10 ± 0.414.47 ± 0.39
13cHIjms 24 06851 i029Ijms 24 06851 i0270.99 ± 0.231.78 ± 0.961.91 ± 0.611.45 ± 0.734.72 ± 0.72
13dHIjms 24 06851 i029Ijms 24 06851 i0287.45 ± 1.36>107.91 ± 1.847.11 ± 3.30>10
13eHIjms 24 06851 i030Ijms 24 06851 i0288.47 ± 0.764.60 ± 0.387.54 ± 1.067.58 ± 1.546.11 ± 1.90
13fHIjms 24 06851 i031Ijms 24 06851 i0270.83 ± 0.132.08 ± 0.305.63 ± 1.501.65 ± 0.235.66 ± 2.64
13gHIjms 24 06851 i030Ijms 24 06851 i0320.50 ± 0.271.18 ± 0.694.62 ± 1.231.40 ± 0.845.39 ± 1.76
13hIjms 24 06851 i015Ijms 24 06851 i026Ijms 24 06851 i032>10>107.99 ± 2.545.08 ± 0.21>10
13iIjms 24 06851 i015Ijms 24 06851 i026Ijms 24 06851 i027>10>106.62 ± 1.05>10>10
13jHIjms 24 06851 i033Ijms 24 06851 i0277.10 ± 1.831.23 ± 0.342.94 ± 0.770.83 ± 0.22>10
13kHIjms 24 06851 i034Ijms 24 06851 i0270.09 ± 0.010.18 ± 0.010.37 ± 0.080.19 ± 0.010.43 ± 0.04
HS-173 3.90 ± 0.345.91 ± 0.1910.71 ± 1.925.24 ± 1.284.36 ± 0.90
a IC50 values are the mean of triplicate measurements.
Table 2. Cytotoxicity of 13k to normal human cells (IC50 μM) a.
Table 2. Cytotoxicity of 13k to normal human cells (IC50 μM) a.
CellMRC-5
13k1.98 ± 0.89
a IC50 value is the mean of triplicate measurements.
Table 3. PI3Kα kinase inhibition by 13k (IC50 nM) a.
Table 3. PI3Kα kinase inhibition by 13k (IC50 nM) a.
CompoundsPI3Kα (IC50)
13k1.94 ± 0.66
HS-1733.72 ± 0.93
a IC50 values are the mean of triplicate measurements.
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Li, M.; Wang, D.; Li, Q.; Luo, F.; Zhong, T.; Wu, H.; Xiong, L.; Yuan, M.; Su, M.; Fan, Y. Design, Synthesis and Biological Evaluation of 6-(Imidazo[1,2-a]pyridin-6-yl)quinazoline Derivatives as Anticancer Agents via PI3Kα Inhibition. Int. J. Mol. Sci. 2023, 24, 6851. https://doi.org/10.3390/ijms24076851

AMA Style

Li M, Wang D, Li Q, Luo F, Zhong T, Wu H, Xiong L, Yuan M, Su M, Fan Y. Design, Synthesis and Biological Evaluation of 6-(Imidazo[1,2-a]pyridin-6-yl)quinazoline Derivatives as Anticancer Agents via PI3Kα Inhibition. International Journal of Molecular Sciences. 2023; 24(7):6851. https://doi.org/10.3390/ijms24076851

Chicago/Turabian Style

Li, Mei, Daoping Wang, Qing Li, Fang Luo, Ting Zhong, Hongshan Wu, Liang Xiong, Meitao Yuan, Mingzhi Su, and Yanhua Fan. 2023. "Design, Synthesis and Biological Evaluation of 6-(Imidazo[1,2-a]pyridin-6-yl)quinazoline Derivatives as Anticancer Agents via PI3Kα Inhibition" International Journal of Molecular Sciences 24, no. 7: 6851. https://doi.org/10.3390/ijms24076851

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