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. 2025 Feb 10;10(6):5686–5698. doi: 10.1021/acsomega.4c08645

Synthesis and Antitumor Activity of 6-(2-Aminobenzo[d]thiazol-5-yl) quinazolin-4(3H)-one Derivatives

Ailing Linghu †,‡, Lei Tang †,‡, Qing Li †,‡, Ting Zhong †,‡, Fang Luo †,‡, Xinran Zhao †,‡, Feng Zhang †,‡, Mingzhi Su †,‡, Yanhua Fan †,‡,*, Linzhen Li †,‡,*
PMCID: PMC11840607  PMID: 39989817

Abstract

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Quinazolinones are key scaffolds in anticancer drug development. We previously identified the lead compound 16h from a series of 6-(1H-benzo[d]imidazol-6-yl) quinazolin-4(3H)-one derivatives. In this study, we optimized 16h to develop new 6-(2-aminobenzo[d]thiazol-5-yl) quinazolin-4(3H)-one derivatives, with compound 45 showing the best antiproliferative activity against A549 lung cancer cells (IC50: 0.44 μM) and good selectivity. Mechanistic studies revealed that compound 45 induced G1-phase arrest, inhibited ALK/PI3K/AKT signaling, disrupted mitochondrial membrane potential, and promoted apoptosis. It also significantly inhibited spheroid formation in a 3D cell culture model. In summary, the results suggest that compound 45 might have potential for the development of anticancer drugs.

1. Introduction

Lung cancer remains one of the most prevalent and deadly malignancies worldwide, accounting for approximately 1.8 million deaths in 2022.1 Nonsmall cell lung cancer (NSCLC), which constitutes about 85% of all lung cancer cases, is often diagnosed at locally advanced or metastatic stages.2 As a result of this late-stage diagnosis, the median survival time following diagnosis is typically less than one year.3

Several oncogenic driver mutations and gene fusions have been identified in NSCLC, including mutations in EGFR and KRAS, as well as ALK rearrangements. In the context of precision medicine, these genetic abnormalities have become key therapeutic targets, with the development of selective inhibitors offering improved clinical outcomes.4

Receptor tyrosine kinases (RTKs) make up a class of transmembrane receptors that play a pivotal role in extracellular signal transduction, regulating critical cellular processes such as proliferation and survival. Dysregulation of RTKs or their downstream signaling pathways is frequently observed in NSCLC and other malignancies.5−9

Anaplastic lymphoma kinase (ALK), a member of the receptor tyrosine kinase family and structurally related to the insulin receptor family,10−12 is implicated in 3–7% of NSCLC cases, predominantly of the adenocarcinoma subtype.13 ALK rearrangements occur in a mutually exclusive manner with KRAS and EGFR mutations.14 The most prevalent ALK fusion partner in NSCLC is the echinoderm microtubule-associated protein-like 4 (EML4) gene.3 This fusion leads to constitutive activation of ALK, resulting in aberrant signaling that drives tumorigenesis.15 Importantly, ALK-positive NSCLC tends to occur in younger patients and those with minimal or no history of smoking,16 suggesting that ALK fusion variants may have unique biological and clinical implications.17

Crizotinib, a small-molecule multitarget tyrosine kinase inhibitor, was approved by the U.S. Food and Drug Administration (FDA) in 2011 for the treatment of ALK-positive NSCLC.18 While initial response rates to crizotinib are promising, resistance to the therapy inevitably develops.19 In response to this challenge, second- and third-generation ALK inhibitors, such as ceritinib and lorlatinib, have been developed and subsequently approved to overcome crizotinib resistance.20,21 However, the emergence of resistance to these newer agents remains a concern, underscoring the need for the concurrent development of more effective next-generation ALK inhibitors for sustained clinical benefit.

Quinazolinone is a nitrogen-containing heterocyclic compound with many biological activities and is a vital drug scaffold in drug discovery.22−24 Many marketed drugs or experimental drug candidates contain quinazolinone fragments, and they demonstrate different biological activities such as antidiabetic, antibacterial, anticonvulsant, and antitumor effects.25−27 In recent years, a large number of quinazolin-4(3H)-one derivatives have been investigated for their anticancer activity. Moreover, some derivatives containing quinazolin-4(3H)-one have been used in clinical anticancer therapy.28 Studies have demonstrated that quinazolinone derivatives are able to exert antitumor effects by affecting RTK family members.29

Quinazolinone derivatives exert antitumor effects through various mechanisms, including inhibition of EGFR-TK, CDK4,30 microtubule polymerization,31 HER2,32 and c-Met.33 Additionally, compounds such as CM9, a quinazolinone-based multikinase inhibitor, show activity not only against targets like FGFR1, FLT1 (VEGFR1), and FLT4 (VEGFR3), but also exhibit notable efficacy against ALK,34 highlighting their potential as ALK inhibitors.

Overall, quinazolinones make up an essential class of molecular parent structures in the field of drug research and have the potential to be developed as antitumor agents.

Based on the binding mode of Aurora A kinase and its inhibitors, our group selected quinazoline-4-(3H)-one as a scaffold, designing selective Aurora A kinase inhibitors. Compound 16h emerged as a lead, inhibiting A549 cells (IC50 = 8.27 ± 0.52 μM) and showing strong selectivity for Aurora A kinase (IC50 = 21.94 nM), inducing G2/M phase arrest and apoptosis.35 To improve compound 16h, we synthesized and evaluated new quinazolinone derivatives for antiproliferative activity. Compound 45 displayed the best anticancer activity and was further studied for its mechanism of action, including effects on the cell cycle, mitochondrial membrane potential, apoptosis, and 3D spheroid culture. Results suggest compound 45 might have potential for the development of anticancer drugs.

2. Materials and Methods

2.1. Chemistry

2.1.1. Synthesis of Intermediates 4a–j

2.1.1.1. Preparation of 3-Benzyl-6-bromoquinazolin-4(3H)-one (4a)

Under the protection of argon, 2-amino-5-bromobenzoic acid (4.32 g, 20 mmol), triethyl orthoformate (3.86 g, 26 mmol), phenylmethanamine (2.79 g, 26 mmol), and iodine (0.05 g, 0.2 mmol) were reacted at reflux in anhydrous ethanol (60 mL) for 6 h. Upon completion of the reaction (monitored by TLC), the residue was concentrated in a vacuum to give the residue, which was dissolved in ethyl acetate (100 mL). The ethyl acetate solution was washed with 1 N aqueous sodium hydroxide (50 mL × 3), dried over anhydrous magnesium sulfate, and concentrated to give 3-benzyl-6-bromoquinazolin-4(3H)-one (4a) as a light yellow solid (5.35 g, 17 mmol, 85% yield). ESI-MS: m/z = 316.0 [M + H]+. Compounds 4b–i were synthesized according to the method described for 4a.

A mixture of 2-amino-5-bromobenzoic acid (2.16 g, 10 mmol) and formamide (1.80 g, 40 mmol) was stirred at 130 °C for 4 h before adding water (30 mL). The reaction mixture was cooled to 60 °C, and 20 mL of water was added. After stirring for an additional 30 min, the precipitated product was isolated by vacuum filtration. The crude product was washed with anhydrous ethanol to afford 6-bromoquinazolin-4(3H)-one 3 in 91% yield. Intermediate 3 (1.125 g, 5 mmol) was added to 15 mL anhydrous DMF, and NaH (60% dispersed in mineral oil, 0.24 g, 6 mmol) was added under argon protection. The reaction mixture was stirred at room temperature for 3 min, and then 2-chloro-1-morpholinoethan-1-one (1.145 g, 7 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight and then extracted with ethyl acetate (100 mL) and brine (100 mL). The organic layer was washed several times with brine (100 mL), dried with anhydrous MgSO4 and finally concentrated under vacuum. Purification by EtOAc/PE (100:1) column chromatography afforded 6-bromo-3-(2-morpholino-2-oxoethyl)quinazolin-4(3H)-one (4j) as a white solid (1.21 g, 3.44 mmol, 69% yield). ESI-MS: m/z 374.0, 376.0 [M + Na]+.

2.1.2. Synthesis of Intermediate 5

Using the “one-pot method”, a mixture of 2-amino-5-bromobenzothiazole (2.29 g, 10 mmol), bis(pinacolato)diboron (5.08 g, 20 mmol), potassium acetate (2.94 g, 29.96 mmol), and the catalyst [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (0.4 g, 0.5 mmol) was added to a solvent of 1,4-dioxane (60 mL) and allowed to react overnight at 100 °C. Upon completion of the reaction (monitored by TLC), 1,4-dioxane was removed under reduced pressure, and the residue was purified by column chromatography with dichloromethane/methanol (50:1) on silica to afford the yellow solid 5-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazol-2-amine (2.23 g, 8 mmol, 80% yield). ESI-MS: m/z 277.1 [M + H]+.

2.1.3. Synthesis of Intermediates 6a–j

Intermediates 4a (1.26 g, 4 mmol) and 5 (2.21 g, 8 mmol) were added to 1,4-dioxane (20 mL) with stirring. Then, K2CO3 (1.66 g, 12 mmol) and Pd(dppf)Cl2 (0.15 g, 0.2 mmol) were added, and the reaction was heated up to 100 °C for 1 h (the color of the reaction solution changed from red to black). The reaction was continued by adding water ([V(1,4-dioxane):V(water) = 4:1]) for 4 h. When the reaction was complete (monitored by TLC), the solvent was removed under vacuum, and the residue was purified by column chromatography with dichloromethane/methanol (50:1) on silica to give a yellowish solid, 6-(2-aminobenzo[d]thiazol-5-yl)-3-benzylquinazolin-4(3H)-one (6a) (1.19 g, 3.16 mmol, 79% yield). ESI-MS: m/z 385.1 [M + H]+. Compounds 6b–j were synthesized according to the method described for 6a.

2.1.4. Synthesis of Compounds 7–11

Intermediate 6a (100 mg, 0.26 mmol) and triethylamine (61.63 mg, 0.78 mmol) were added to THF (3 mL) with stirring at 0 °C. Then, acyl chloride (0.34 mmol) was slowly dripped in using a dropping funnel, and after the completion of the dropwise addition, the temperature was raised to room temperature to continue the reaction for 5 h. After completion of the reaction (monitored by TLC), THF was removed in a vacuum. The residue was purified using dichloromethane/methanol (80:1) by column chromatography on silica to afford compound N-(5-(3-benzyl-4-oxo-3,4-dihydroquinazolin-6-yl)benzo[d]thiazol-2-yl)acetamide (7) as a white solid (60.17 g, 0.14 mmol, 53% yield); mp 276.7–278.5 °C.

Compounds 8–11 were synthesized according to the methods described for compound 7. Information about compounds 8–11 is provided in Supporting Information S3.

2.1.5. Synthesis of Intermediates 12–47

Intermediate 6a (100 mg, 0.26 mmol) and tolyl isocyanate (45.08 mg, 0.34 mmol) were added to 1,4-dioxane (2 mL), and the reaction was stirred at 80 °C for 4 h. After completion of the reaction (TLC monitoring of the reaction), the dioxane solvent was removed in a vacuum, and the residue was purified by column chromatography on silica with dichloromethane/methanol (50:1) to give the compound 1-(5-(3-benzyl-4-oxo-3,4-dihydroquinazolin-6-yl)benzo[d]thiazol-2-yl)-3-(p-tolyl)urea (12) as a white solid (128 mg, 0.25 mmol, 95% yield); mp 304.8–306.5 °C. Compounds 13–47 were synthesized according to the methods described for intermediate 12. Information about compounds 13–47 is given in Supporting Information S4.

2.2. Biological Evaluation

2.2.1. Cell Culture

Human nonsmall cell lung cancer cells (A549, PC9, and H1975) and human normal lung epithelial cells (BEAS-2B) were obtained from the cell bank of the Chinese Academy of Sciences. A549, PC9, and BEAS-2B were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antifungal drugs (100 units/mL penicillin G sodium, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B). H1975 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% antibiotic-antimicrobial drugs. All cells were cultured in a cell culture incubator at 37 °C with 5% CO2.

2.2.2. Antiproliferative Assay

Cells in the logarithmic growth phase were inoculated into 96-well plates at 3 × 103 cells per well and cultured overnight at 37 °C and 5% CO2 in a cell culture incubator. Cells were treated with different concentrations of compounds or DMSO for 72 h. Then, 20 μL of 5 mg/mL MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide) was added to each well and incubated for 4 h in a cell culture incubator under light-avoiding conditions, which allows the formamide crystals to completely dissolve in DMSO. Cell viability was determined by a multiwell spectrophotometer (Thermo Scientific, VARIOSKAN LUX, USA) at a wavelength of 490 nm. In each well, 20 μL of MTT was added, and after 4 h, the culture solution was aspirated, 150 μL of DMSO solution was added, and shaken on a shaker to completely dissolve formamide crystals in DMSO. Finally, the absorbance at 490 nm was measured using an enzyme meter, and three parallel experiments were set up to calculate the cell viability.

2.2.3. Western Blotting

Cells treated with different concentrations of compound 45 were collected, and the cells were lysed by adding lysis solution and heat denatured at 104 °C for 12 min. The protein content of the cells was calculated using the bicinchoninic acid (BCA) method. An equal amount of protein was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane in the correct order. The membrane was blocked with 5% bovine serum albumin or skimmed milk for 1 h. The membrane was incubated with a specific primary antibody at 4 °C overnight. On the next day, the membrane was washed three times with TBST solution for 10 min each time. The secondary antibody was incubated at room temperature for 2 h. The secondary antibody was removed and washed rapidly with TBST solution 3 times for 5 min each. Finally, the blots were imaged by the ChemiDoc MP Imaging System (Bio-Rad, Hercules, California, USA) using ECL luminescent solution (A:B = 1:1), and the results were processed using Image Lab software.

2.2.4. Cell Cycle Analysis

Cells treated with different concentrations of compound 45 were collected, washed once with cold PBS, fixed by slowly adding precooled 70% ethanol, and stored at −20 °C overnight. The fixed cells were removed, and the supernatant was discarded. The cells were washed once with PBS, stained with PI (50 μg/mL PI and 100 μg/mL RNase A) for 30 min at room temperature with light protection, and then analyzed by flow cytometry.

2.2.5. Determination of Mitochondrial Membrane Potential

Mitochondrial membrane potential was determined using the assay kit with JC-1 (cat. no. C2006, Beyotime), following the instructions strictly. Cells in six-well plates were stained with JC-1 working solution, configured according to the kit instructions, for 20 min at 37 °C. Subsequently, cells were washed with PBS and then observed under a fluorescence microscope or detected by flow cytometry.

2.2.6. Cell Apoptosis Analysis

Apoptosis was detected by flow cytometry using an Annexin V-FITC/PI dual labeling assay. A549 cells were inoculated in 6-well plates at a density of 1 × 105 cells per well overnight. Cells were treated with compound 45 for 48 h, then treated cells were collected by rinsing with PBS and incubated with Annexin V and propidium iodide (PI) staining buffer for 15 min at room temperature, protected from light. Finally, the cells were analyzed by flow cytometry.

2.2.7. RTK Array Kit Assay

The Proteome Profiler Human Phospho-RTK Array Kit (R and D Systems, Minneapolis, MN) was used, and operations were performed in strict accordance with the kit instructions. The array allowed the simultaneous screening of 49 different phospho-RTKs. Briefly, cells in the blank group and the dosed (2.0 μM) group were collected separately and lysed with lysis buffer containing a mixture of protease and phosphatase inhibitors. Protein quantification was performed using the Bradford Protein Concentration Assay Kit (Cat: PC0015, Solarbio). After blocking the arrays with Array Buffer 1 for 1 h, 450 μg of protein lysates were incubated with the arrays overnight at 4 °C, which allowed for phosphorylated and unphosphorylated RTK binding. The array was washed 3 times with wash buffer and then incubated for 2 h with the Anti-Phospho-Tyrosine-HRP Detection Antibody. After 3 washes, the membrane with the identification number was developed face-up using Chemical Reagent Mix.

2.2.8. 3D Spheroid Cell Inhibition Assay

To culture A549 lung cancer cells into three-dimensional spheres, we used PerkinElmer’s Cell Carrier Spheroid ULA 96-well microtiter plates (PerkinElmer, Waltham, MA, USA). Cells were seeded at a density of 20 000 cells per well. After the formation of the tumor microspheres, they were treated with the indicated concentrations of compound 45 every 3 days, and the tumor spheres were photographed and documented using a microscope.

3. Results

3.1. Chemistry

To explore the relationship between the structure and biological activity and to identify additional compounds with antitumor potential, we performed structural modifications on compound 16h. The N atom on benzimidazole in the structure of lead compound 16h is prone to isomerization, which makes the synthesis and purification of the product difficult and makes it challenging to achieve industrial production. To retain the biological activity of quinazolinone as much as possible, the N atom on the benzimidazole was replaced by an S atom using bioisosterism. In addition, after analyzing the structure–activity relationship of some synthetic derivatives, we accessed the acylurea group on the amino group of benzothiazole, which improved its activity (Figure 1).

Figure 1.

Figure 1

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Design strategies for the target compound.

As shown in Scheme 1, the amino group of 2-amino-5-bromobenzoic acid and the added primary amine are excellent nucleophilic reagents. They attacked the carbonyl carbon of 2-amino-5-bromobenzoic acid and triethyl orthoformate. Eventually, a stable structure was formed, which is what we needed for the quinazolinone fragment (intermediates 4a–i).

Scheme 1. Synthetic Routes of Intermediates 6a–j.

Scheme 1

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Then, intermediates 6a–j were prepared by the Suzuki-Miyaura cross-coupling reaction. 2-Amino-5-bromothiazole and bis(pinacol)diboron produced intermediate 5 by the Miyaura coupling reaction in the presence of potassium acetate. In this step, we found that replacing the commonly used catalyst Pd(dppf)Cl2 with the catalyst 1,1′-bis(diphenylphosphino)ferrocene-palladium(II) dichloride dichloromethane complex increased the reaction yield from 40% to 80%. In the presence of a base, the Pd(0) catalysts underwent oxidative addition reactions with intermediates 4a–j to generate strongly electrophilic organopalladium intermediates. Meanwhile, intermediate 5 interacted with the base to generate borate intermediates, which underwent metal transfer to obtain Pd(II) intermediates with bis-alkyl coordination, and finally, the target products (intermediates 6a–j) were obtained by a reductive elimination reaction (Supporting Information S12).

End products 7–11 were prepared by the substitution of different chlorides for the H atom of the amino group on intermediate 6a. Finally, intermediates 6a–j as nucleophilic reagents attacked the carbonyl carbon of different isocyanates. An addition reaction was then carried out to prepare the remaining final products (Scheme 2).

Scheme 2. Synthetic Routes of the Target Compounds.

Scheme 2

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3.2. Biological Evaluation

3.2.1. Antiproliferation Activity Assay

To investigate the antiproliferative activity of the synthesized compounds, the cell viability of three human nonsmall cell lung cancer cell lines (A549, H1975, and PC9) treated with these synthesized compounds was measured by MTT assay. As shown in Table 1, the evaluated compounds exhibited different antiproliferative effects on three different lung cancer cell lines, and most of the compounds were more effective against A549 cells than against the other two lung cancer cell lines. In our previous studies, we demonstrated that the introduction of benzyl to N3 of quinazolin-4(3H)-one enhanced the antitumor activity of the target compound (16h vs 9a).36 In this study, we introduce benzyl at the N3 position of quinazolin-4(3H)-one. Following the replacement of the 1H-benzo[d]imidazole moiety at the 6-position of quinazolin-4(3H)-one with a benzo[d]thiazole scaffold, a significant reduction in antitumor activity was observed. This finding suggests that the replacement of bioisosterism altered the manner in which the compound interacts with the target, thereby reducing its antitumor activity (16h vs 8). It was unexpected that replacing the propionamide at the 2-position of the 1H-benzo[d]imidazole with butyramide resulted in the restoration of antitumor activity (8 vs 9). We also introduced benzyl at the 3-position of quinazolin-4(3H)-one, but it did not improve the activity well (18 vs 32). This finding suggests that the replacement of bioisosterism altered the manner in which the compound interacts with the target, thereby reducing its antitumor activity. It is postulated that this region may be situated within a narrow pocket of the target, which can accommodate only small volume groups such as alkane chains. Therefore, we introduced an alkane group with a small volume. It turned out that the compounds with cyclopropane introduced at the 3-position of the quinazolinone had better activity than phenylmethyl (18 vs 42). Furthermore, there is no increase in activity after splitting cyclopropane into chain alkanes (42 vs 43 and 44). However, the activity of the compounds increased significantly after we shortened the length of the carbon chain (43 vs 45). Meanwhile, compounds 29, 32, and 36 showed superior antiproliferative activity compared to compounds 46, 47, and 41. These results suggest that the introduction of a carbonyl group at the 3-position of quinazolin-4(3H)-one led to a decrease in activity. Furthermore, in consideration of the poor solubility of these derivatives, we introduced a urea moiety into benzothiazole to enhance their water solubility. To our surprise, both the antitumor activity and hydrophilicity of the majority of derivatives were improved to varying degrees (8 vs 24 and 11 vs 15). In addition, activity is increased when F atoms or methoxy are accessed on acylurea (12–18 vs 24–27).

Table 1. Antitumor Activity of Different Cell Lines (IC50, μM)a.

3.2.1.

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a

Data are expressed as mean ± SD from three different experiments.

Among the tested compounds, 38 and 45 showed significantly higher antitumor activity against A549, H1975, and PC9 cell lines. Consequently, we focused on these two compounds for further investigation. To determine the optimal candidate for subsequent studies, we assessed the toxicity of compounds 38 and 45 in NSCLC cells (A549 and H1975) and normal human bronchial epithelial cells (BEAS-2B). Compound 38 had IC50 values of 0.98 ± 0.08 μM for A549 and 1.39 ± 0.55 μM for H1975, but it also demonstrated considerable cytotoxicity with an IC50 of 1.28 ± 0.15 μM in BEAS-2B cells. In contrast, compound 45 showed IC50 values of 0.44 ± 0.03 μM for A549 and 0.49 ± 0.09 μM for H1975, with a higher IC50 of 2.9 ± 0.55 μM for BEAS-2B cells, suggesting that compound 45 may be a safer option.

To further evaluate compound 45 as an antitumor compound, we compared its cytotoxicity with clinically used chemotherapeutics, doxorubicin and paclitaxel, in A549, H1975, and BEAS-2B cells. As shown in Figure 2C, paclitaxel had IC50 values of 0.64 ± 0.035 and 0.072 ± 0.008 μM for A549 and H1975, respectively, and 0.25 μM for BEAS-2B cells. Doxorubicin had IC50 values of 0.52 ± 0.2 and 0.067 ± 0.032 μM for A549 and H1975, respectively, with an IC50 of 0.22 μM for BEAS-2B cells (Figure 2D). Compound 45 demonstrated comparable antitumor activity to paclitaxel and doxorubicin, while showing superior selectivity toward tumor cells (Table 2). Thus, compound 45 was chosen for further mechanistic studies.

Figure 2.

Figure 2

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Antiproliferative activity of compounds on cancer and normal cells. (A, B) BEAS-2B, A549, and H1795 cells were subjected to treatment with compounds 38 and 45 at various concentrations for 72 h. Cell viability was assessed using an MTT assay. (C, D) BEAS-2B, A549, and H1795 cells were subjected to treatment with doxorubicin and paclitaxel at various concentrations for 72 h. Cell viability was assessed using the MTT assay. n = 3.

Table 2. Selectivity of Compounds on Cancer and Normal Cells.
  selectivity (fold)
compounds A549 vs BEAS-2B H1975 vs BEAS-2B
compound 38 1.30 0.92
compound 45 7.10 5.80
doxorubicin 0.39 3.47
paclitaxel 0.42 3.28
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3.2.2. Compound 45 Induced Cell Cycle Arrest

The cell cycle is closely related to cell growth. Subsequently, to evaluate the effects of compound 45 on the cell cycle distribution, the cells were treated with compound 45 for 48 h, and the cell cycle distribution was detected by flow cytometry. Treatment with compound 45 induced cell cycle arrest at the G1 phase in a concentration-dependent manner compared to that of the control groups of A549 cells (Figure 3A). The effects of compound 45 on the cell cycle distribution were further confirmed by Western blot analysis. As illustrated in Figure 3C–G, with increasing concentrations of compound 45 from 0.5 to 2.0 μM, the expression levels of p27 and p21 were markedly upregulated in a dose-dependent manner, but the stabilization of CDK2 and p-Rb expression levels was reduced in a concentration-dependent manner. This demonstrated that compound 45 inhibited tumor cell proliferation by affecting the cell cycle distribution.

Figure 3.

Figure 3

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Compound 45 induced cell cycle arrest at the G1 phase in A549 cells. (A) The effect of compound 45 on the cell cycle distribution in A549 cells was performed by the PI-staining assay. Cells were treated with compound 45 for 48 h. (B) The quantitative histograms of different cell cycle phases by treatment of 45 in A549 cells. (C–G) Cells were treated with the indicated concentrations of 45 for 48 h and then the protein expressions associated with the G1 phase were analyzed by Western blot. The changes of corresponding proteins were quantified using ImageJ. Each bar represents mean ± SD (n = 3); *p < 0.05, **p < 0.01, or ***p < 0.001 was considered statistically significant compared with corresponding control values.

3.2.3. Mechanisms of Action Studies of Compound 45

To further explore the antitumor action of compound 45, we investigated its mechanism of action. Aurora A is activated at the onset of the S phase, and its activity peaks at the G2/M phase transition, stimulating replicating centrosomes to segregate and initiate mitotic entry.37 The lead compound 16h targets Aurora A and induces G2/M phase arrest. The cell cycle distribution results in this study showed that compound 45 induced cellular G1 phase arrest, so we speculated that the mechanism of action of compound 45 might be altered. According to the group’s previous study, compounds that act on PI3K may induce G1-phase arrest,38 so we verified the inhibitory effect of compound 45 on PI3K and its downstream protein AKT by Western blotting. The results demonstrated that compound 45 inhibited the protein expression of p-PI3K and the downstream p-AKT (Figure 4A).

Figure 4.

Figure 4

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Signaling pathway studies of compound 45. (A) Cells were treated with the indicated concentrations of compound 45 for 48 h and then the protein expressions of PI3K and AKT were analyzed by Western blot. (B) Relative levels of tyrosine phosphorylation of human receptor tyrosine kinases with treatment of compound 45. A549 cells were treated with compound 45 (2.0 μM) for 24 h and then human Phospho-RTK array analyses were performed. (C–G) Cells were treated with the indicated concentrations of 45 for 48 h, and then the protein expressions of ALK and p-ALK were analyzed by Western blot. The changes of corresponding proteins were quantified using ImageJ. Each bar represents mean ± SD (n = 3); *p < 0.05, **p < 0.01, or ***p < 0.001 was considered statistically significant compared with corresponding control values.

Studies have shown that PI3K is involved in the signaling pathways of the RTK family.39 RTKs are not only important regulators of normal cellular physiological activities but also play a key role in the development of many types of tumors, indicating that hyperactivation of human RTKs contributes significantly to cancer progression.40 Therefore, to better understand the mechanisms of compound 45 action in A549 cells, we determined the phosphorylation status of RTKs following treatment with compound 45 using a Proteome Profiler Human Phospho-RTK Array Kit, which showed that the presence of compound 45 reduced the phosphorylation levels of ALK in this study (Figure 4B,C). Additionally, we found that compound 45 not only reduces the phosphorylation levels of ALK but also decreases the total protein levels of ALK (Figure 4C), suggesting its potential role as an ALK degrader. However, since the cellular model (A549) is not ALK mutant and is instead dependent on the KRAS mutation and the MAPK pathway, future studies should focus on evaluating the effects of compound 45 in ALK mutant cell lines, such as H3122.

3.2.4. Compounds 45 Induced Cell Apoptosis

Mitochondria are prominent organelles in eukaryotic cells and are a major source of reactive oxygen species (ROS), the abnormal production of which leads to oxidative stress.41 Oxidative stress affects gene expression, cell proliferation, and apoptosis, which are important in tumorigenesis, tumor progression, metastasis, and recurrence.42,43 The mitochondrial membrane potential reflects the functional state of mitochondria and is related to cell differentiation, tumorigenesis, and malignancy.43 To determine whether the changes in the mitochondrial membrane potential (Δψm) were related to compound 45, the effects of compound 45 on the mitochondrial membrane potential were analyzed using JC-1 dye. When the Δψm was lower in the control group (19.25%), JC-1 aggregated in the mitochondrial matrix to form polymers, producing red fluorescence. The fluorescence in the administered group changed from red to green (Figure 5B), indicating that compound 45 treatment induced an increase in the Δψm in a dose-dependent manner (47.72%, 51.16%, and 60.52%, respectively) (Figure 5A).

Figure 5.

Figure 5

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Effect of compound 45 on mitochondria. After compound 45 treatments, the cells were stained with JC-1 dye. (A) Changes in the mitochondrial membrane potential were examined via flow cytometry. (B) Treated cells were examined using an inverted fluorescence microscope; size: 200 μm.

Disruption of the mitochondrial transmembrane potential is widely recognized as one of the earliest events that occur during apoptosis. To verify whether the antiproliferative activity of compound 45 is accompanied by apoptosis in cancer cells, the percentage of apoptosis in A549 cells was assayed by flow cytometry after double staining with Annexin V-FITC/PI. The results showed a significant increase in the percentage of annexin V positive cells among the cells treated with 2.0 μM and 4.0 μM of compound 45, from 2.64% in the control group to 7.13% in the 2.0 μM treatment group and 20.36% in the 4.0 μM treatment group (Figure 6A). To further elucidate the molecular mechanism by which compound 45 is involved in apoptosis, we examined the effect of compound 45 on the expression levels of apoptosis-related proteins using Western blotting. Compared with those in the control group, the levels of proapoptotic proteins, cleaved-caspase 9 and Bax, were increased in a concentration-dependent manner after treatment with compound 45, whereas the level of the antiapoptotic protein Bcl-2 was decreased (Figure 6C–F). In summary, these results suggest that compound 45 induces apoptosis in A549 cells via a mitochondrial pathway.

Figure 6.

Figure 6

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Compound 45 facilitated cell apoptosis in A549 cells. (A) Annexin V-FITC/PI double staining assay was performed to evaluate the apoptotic effect of 45 on A549 cells after treatment with the indicated concentrations of compound 45 for 48 h. (B) The quantitative histograms of the apoptosis percentage in A549 cells. (C–F) Cells were treated with the indicated concentrations of compound 45 for 48 h and then analyzed for apoptosis-related protein expression by Western blotting. Each bar represents the mean ± SD (n = 3); *p < 0.05, **p < 0.01, or ***p < 0.001 was considered statistically significant compared with corresponding control values.

3.2.5. In Vitro 3D Spheroid Cell Inhibition Assay

Traditional two-dimensional (2D) in vitro cell cultures do not mimic the natural tumor microenvironment and do not ideally represent physiological conditions.44 To ameliorate these limitations, three-dimensional (3D) models of cells show characteristics that more closely resemble complex conditions in vivo, enabling more accurate observation of growth phenotypes similar to those observed in tumors.45,46 As shown in Figure 7, the crude tumor spheres of the untreated control group gradually grew; the growth rate of the tumor spheres in the low concentration group decreased compared to that in the control group; the tumor spheres in the medium concentration group gradually became loose from solid; and the tumor spheres in the high concentration group became fragmented. This demonstrates that compound 45 can inhibit the growth of tumor spheres and has the potential for further research.

Figure 7.

Figure 7

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Effect of compound 45 on A549 spheroid formation. A549 cells were seeded in ultralow attachment 96-well U bottom plates (20 000 cells/well) to generate tumor spheroids and treated with 5-fold IC50 concentrations of compound 45 for the spheroid assay. After initiation, the spheroids were treated with compound 45 at the indicated concentrations every 3 days. “Control” refers to the control without the addition of compound 45.

4. Discussion

Cancer remains one of the hotspots of research in today’s medicine, and lung cancer is also widely noted for its high mortality rate. In the present study, a series of derivatives were synthesized by using 16h obtained from the group’s previous research as the lead compound, which was modified to obtain the parent structure of the target compounds and produce a series of derivatives. To preserve the biological activity of the quinazolinone fragment as much as possible, we optimized only the benzimidazole ring in the lead compound 16h. We introduced the carbamide structure with a view to improve the antitumor activity of the compounds. In fact, most of the compounds we obtained showed significantly enhanced antitumor activity compared with the lead compound, with compounds 38 and 45 demonstrating the most notable improvements. However, considering safety, we found that compound 45 exhibited greater selectivity toward tumor cells over normal cells. Additionally, its inhibitory activity against A549 was comparable to that of the clinical lung cancer chemotherapeutic agents, paclitaxel and doxorubicin, while also showing superior selective inhibition of tumor cells. Of course, relying solely on IC50 determinations for normal and tumor cells may not be sufficient to fully establish the safety profile of compound 45. Therefore, in subsequent research, we will further investigate the structure–activity relationship (SAR) of compounds 38 and 45. Furthermore, we will conduct in vivo studies to evaluate the broader safety and antitumor efficacy of compound 45 in animal models.

Evidence demonstrated a more effective strategy based on the combination of ALK and PI3K inhibitors in the treatment of NSCLC. Studies have reported that quinazolinone derivatives can inhibit the PI3K/AKT signaling pathway. We therefore hypothesize that compound 45 retains the inhibitory effect of quinazolinone derivatives on the PI3K/AKT pathway. Unfortunately, we have not yet found a reason why compound 45 is able to act on ALK. This may be the key to the ability of compound 45 to inhibit the ALK/PI3K/AKT signaling pathway. In future studies, we will also continue to investigate the manner in which compound 45 interacts with ALK and explore evidence that compound 45 inhibits the ALK/PI3K/AKT signaling pathway.

Compound 45 is only a simple structural optimization of the lead compound 16h. However, we unexpectedly found that the results of the bioactivity studies of compound 45 were completely different from those of 16h. The lead compound 16h was found to be an excellent inhibitor of Aurora A kinase and had a good antiproliferative effect on breast cancer cells MDA-MB-231. However, most of the target compounds in this study were more sensitive to nonsmall cell lung cancer cells A549, with compound 45 performing more prominently. Moreover, compound 45 induced cellular G1 phase arrest and was not an Aurora A kinase inhibitor. Although it is a simple structural optimization, it is easy to see that the mechanism of action of compound 45 is dramatically changed from that of the lead compound 16h. We will continue to explore the reasons why compounds are differentially sensitive to different cell lines. Furthermore, the lead compound 16h was not found to possess an antioxidative stress effect at the present time. Compound 45 significantly altered the mitochondrial membrane potential and affected the oxidative stress response of the cells. This suggests that simple changes in the structures of compounds may also bring new discoveries.

5. Conclusions

In summary, we synthesized 6-(2-aminobenzo[d]thiazol-5-yl) quinazolin-4(3H)-one derivatives and evaluated their antitumor activity in vitro. Most compounds inhibited A549 lung cancer cells, with compound 45 showing the best activity (IC50: 0.44 μM). Compound 45 altered mitochondrial membrane potential, promoted apoptosis, and induced G1 phase arrest, differing from lead compound 16h’s Aurora A kinase targeting. Further studies confirmed that compound 45 inhibited the ALK/PI3K/AKT signaling pathway. Additionally, the 3D tumor microsphere culture demonstrated that compound 45 inhibited spheroid growth. These findings suggest that compound 45 is a promising lead compound for antitumor drugs.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 82360674), the Natural Science Foundation of Guizhou Provincial Science and Technology Projects (QKH-zk [2022]030), and the National Natural Science Foundation of China (Grant No. 22167010).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c08645.

  • Information for compounds; picture of the synthesis mechanism; HPLC analysis/copies of 1H and 13C NMR spectra (PDF)

Author Contributions

# These authors contributed equally to this work. The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Special Issue

Published as part of the ACS Omegaspecial issue “Chemistry in Brazil: Advancing through Open Science”.

Supplementary Material

ao4c08645_si_001.pdf (6.6MB, pdf)

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