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

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.


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.
Quinazolines are the major compounds in the aromatic backbone of nitrogen-containing heterocyclic compounds with a wide range of biological activities such as antiinflammatory, 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 nitrogencontaining 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.

Chemistry
The synthetic route for intermediates 7a-o of target products 10a-u 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 5a-q 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 7a-o. Intermediates 7a-o were cyclized with methyl bromopyruvate or ethyl bromopyruvate to give the target products 10a-u, 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

Chemistry
The synthetic route for intermediates 7a-o of target products 10a-u is shown in Scheme 1. A purchased raw material, 6-iodoquinazoline 4-3(H)-one was chlorinated in POCl 3 in the presence of DIPEA to give intermediate 2.
Intermediates 5a-q 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 7a-o. Intermediates 7a-o were cyclized with methyl bromopyruvate or ethyl bromopyruvate to give the target products 10a-u, 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 13a-k by Suzuki-Miyaura cross-coupling reaction. In this thesis, we introduced different substituents at the C 6 and C 4 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. give our target products 13a-k by Suzuki-Miyaura cross-coupling reaction. In this thesis, we introduced different substituents at the C 6 and C 4 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 give our target products 13a-k by Suzuki-Miyaura cross-coupling reaction. In this thesis, we introduced different substituents at the C 6 and C 4 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

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 overex-

Antiproliferation Activity Assay
To test the antiproliferative activity of all target compounds, IC 50 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 R 1 substituent group was an alkyl, suggesting that simultaneous alkylation of NH 2 at the 4-position of quinazoline would impair the antiproliferative activity of the target compounds. (II) When R 3 = COOCH 3 , most of the compounds are more active than R 3 = COOC 2 H 5 , such as compounds 10q and 10h, 10r and 10i, and when R 3 = COOC 2 H 5 and R 2 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 R 3 , 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 R 3 -substituted benzene, as in compounds 13a and 13b, 13c and 13d. Overall, compound 13k showed the best antiproliferative activity against HCC827 cells with an IC 50 value of 0.09 µM, which could be attributed to the conventional hydrogen bond formed between the R 2 -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 IC 50 value of 1.98 µM, which is more than 20-fold different from HCC827 cells (Table 2). Moreover, as shown in Figure 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-Glo TM 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 nonsmall 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 cooccur 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

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-Glo TM 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 IC 50 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 nonsmall 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. 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.

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 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.

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

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.

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.

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. 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.

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 dosedependently 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). 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.

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 dosedependently 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). (C-F) 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).

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. (C-F) 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).

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. 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.

Conclusions
In summary, a series of new 6-(imidazo[1,2-a]pyridin-6-yl)quinazoline derivatives (10a-u and 13a-k) 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.

Instruments and Materials
All reagents and solvents were commercially available and used without further purification. 1 H NMR, 13 C NMR and 19 F 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).

General Experimental Protocol for Preparation of Compounds 10a-u
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 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 IC 50 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.

Conclusions
In summary, a series of new 6-(imidazo[1,2-a]pyridin-6-yl)quinazoline derivatives (10a-u and 13a-k) 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 IC 50 values ranging from 0.09 µM to 0.43 µM 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 IC 50 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.

Instruments and Materials
All reagents and solvents were commercially available and used without further purification. 1 H NMR, 13 C NMR and 19 F NMR spectra were recorded with a 600, 150 and 565 MHz NMR spectrometer (Bruker AVANCE NEO), respectively. The NMR spectra were generated by using Mestrenova 12.0 as processing software, deuterated chloroform (CDCl 3 ) and dimethyl sulfoxide-d 6 (DMSO-d 6 ) 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).

General Experimental Protocol for Preparation of Compounds 10a-u 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 Na 2 SO 4 and rotary dried under vacuum. The residue was purified through a column chromatography