Synthesis, evaluation, and mechanism study of novel benzimidazole acylhydrazone derivatives for antitumor activity

doi:10.21203/rs.3.rs-5257593/v1

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Synthesis, evaluation, and mechanism study of novel benzimidazole acylhydrazone derivatives for antitumor activity

https://doi.org/10.21203/rs.3.rs-5257593/v1

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This study focuses on the design, synthesis, and evaluation of benzimidazole derivatives for their anti-tumor activity against A549 and PC-3 cells. Initial screening using the MTT assay identified compound 5m as the most potent inhibitor of A549 cells with an IC₅₀ of 7.19 μM, which was superior to the positive agents 5-Fluorouracil and Gefitinib. Cellular mechanism studies elucidated 5m arrests cell cycle at G2/M phase, induces apoptosis along with the decrease of mitochondrial membrane potential and increased reactive oxygen species. Colony formation and wound healing assays demonstrated that 5m markedly inhibited the clonogenic and migratory abilities of A549 cells. Western blot analysis showed an upregulation of pro-apoptotic protein Bax, downregulation of anti-apoptotic protein Bcl-2, and significant downregulation of cell cycle proteins CyclinB1 and CDK-1. These findings suggest that compound 5m effectively suppresses A549 cell proliferation and migration through multiple mechanisms, highlighting its potential as a novel anti-lung cancer agent.

benzimidazole derivative

A549

apoptosis

cell cycle

Benzimidazoles and their derivatives have garnered significant attention in medicinal chemistry due to their broad range of biological activities, particularly their antiviral, antibacterial, and anticancer properties, through different mechanisms such as protein kinase inhibition, apoptosis, and microtubule inhibition [1–3]. Escaping cell apoptosis and cycle are significant features of cancer cells, causing uncontrolled tumor growth, treatment resistance, cell proliferation, and accumulation of different oncogenic changes [4–6]. In recent years, significant progress has been made in cancer treatment, with the in-depth study of apoptosis and cycle-related mechanisms and the further understanding of the relationship between tumors and changed in apoptosis and cycle-regulating protein expression [7–10]. Due to its excellent biological activities and ability to bind to intracellular target molecules, benzimidazole has attracted great interest in recent years in targeting members of the apoptosis and cycle-related protein family (Fig. 1).

Lung cancer is the primary cause of cancer-related mortality (18% of all cancer-related deaths) throughout the world. Lung cancer is the most frequently occurring cancer in men, whereas it is the 3rd most commonly diagnosed cancer in women. Especially non-small cell lung cancer (NSCLC), remains one of the leading causes of cancer-related mortality worldwide [11–13]. The A549 cell line, a model for NSCLC, is widely used to evaluate the efficacy and mechanisms of new anticancer agents. Despite advancements in surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, the prognosis for NSCLC patients remains poor due to issues like drug resistance and tumor recurrence [14–16]. Therefore, based on our research, we introduced an active fragment of hydrazone into the parent nucleus of benzimidazole to evaluate its effectiveness against A549 cell line.

This study aims to design, synthesize, and evaluate a series of benzimidazole derivatives for their antitumor activity against A549 cells. Initial screening using MTT assays identified compound 5m as the most potent inhibitor of A549 cells, with an IC₅₀ of 7.19 µM. To further explore the mechanism of action, we conducted in-depth cellular and molecular analyses, focusing on apoptosis induction, mitochondrial membrane potential disruption, reactive oxygen species generation, and the modulation of cell cycle and apoptosis-related proteins. Our findings provide a theoretical basis for developing 5m as a potential anti-lung cancer agent and open new avenues for the application of benzimidazole derivatives in cancer therapy.

2.1 Chemistry

To a stirred solution of 3-aminoacetophenone 1 (0.01 mol) and anhydrous potassium carbonate (0.01 mol) in dry dimethylformamide was added dropwise chloroacetyl chloride 2 (0.8 mL, 0.01 mol) at room temperature for 24 h. After completion of reaction, the solution was poured into ice-cold water and the product was crystallized from benzene to give 3. To a solution of N-(4-acetylphenyl)-2-chloroacetamide 3 (0.01 mol), 2-mercapto benzothiazole (0.01 mol) and K₂CO₃ (0.02mol) in acetone (20 mL) was stirred for 6 h at room temperature. The precipitate obtained was collected and dried, the solid obtained was recrystallized from ethanol to give 4. Intermediate compound 4 (0.001 mol) was treated with the appropriate benzohydrazide derivative (0.001 mol) and catalytic amount of glacial acetic acid in ethanol (15 mL) was stirred at room temperature for 8 h. The resulting solids were filtered and recrystallized from ethanol to afford the compounds 5 [17, 27]. The structures of the obtained compounds were established by means of analysis using techniques such as ¹H NMR, ¹³C NMR, and HRMS spectra. The analysis confirmed that assumed structure of these derivatives was correct.

2.2 Biological evaluation

2.2.1 Activity evaluation and structure-activity relationship

As indicated in Tables 1 and 2, the antitumor effects of the target compounds were greatly affected by structural variations. Some structure–activity relationship (SAR) analyses are discussed below. Many compounds exhibited potent antibacterial activities. For example, compounds 5d, 5g and 5m had the inhibition rates of 51.78, 50.29, 71.4 and 66.10% against A549 at 10 µM, which was better than the 5-Fluorouracil (49.86%) and the inhibition rates of all compounds were higher than Gefitinib (23.79%).

Table 1

The inhibition rate of the compound on A549 and PC-3 cells
Compound	Inhibitory activity/ % at 10 µM
Compound	PC-3	A549
5a	49.25 ± 4.42	45.29 ± 2.73
5b	38.78 ± 3.80	43.44 ± 1.70
5c	40.60 ± 3.50	30.29 ± 1.95
5d	49.02 ± 2.57	51.78 ± 1.02
5e	28.04 ± 5.45	47.27 ± 3.66
5f	53.85 ± 1.57	48.48 ± 1.11
5g	50.51 ± 2.33	50.29 ± 2.73
5h	46.04 ± 1.44	41.27 ± 3.66
5i	20.15 ± 4.79	48.79 ± 0.94
5j	3.95 ± 3.30	39.10 ± 1.17
5k	45.04 ± 0.91	36.81 ± 0.78
5l	43.23 ± 4.33	30.44 ± 1.70
5m	46.59 ± 6.45	66.10 ± 1.17
5n	46.37 ± 5.20	43.48 ± 1.11
50	53.11 ± 2.48	34.85 ± 1.88
5p	46.37 ± 5.20	43.29 ± 1.95
5q	53.11 ± 2.48	47.85 ± 1.88
5-Fluorouracil	22.70 ± 0.43	49.86 ± 1.38
Gefitinib	30.12 ± 2.95	23.79 ± 2.15

Table 2

IC₅₀ values of target compounds 5m against A549
Compd.	IC₅₀ (µM)
Compd.	A549
5m	7.19 ± 0.29
5-Fluorouracil	10.56 ± 0.63
Gefitinib	27.07 ± 4.50
Data are expressed as mean ± SD

The activity of benzene 4-site electron-donating substituents is better than that of electron-absorbing substituents, such as 5d (4-OCH₃), 5e (4-OH), 5f (4-C(CH₃)₃), 5i (4-OCH₃) > 5a (4-Br), 5b (4-Cl), 5c (4-F), 5h (4-CF₃). The activity of benzene 3-substituted groups is generally poor. The 2-site electron-donating substituents of benzene ring are more active than electron-absorbing substituents, such as 5m (2-OCH₃) > 5n (2-Cl), 5o (2-Br). In order to further expand the antitumor spectrum of the compound, we tested the bioactivity of the compound PC-3. The inhibitory activity of the compound on PC-3 cells was generally better than that of the positive control drugs 5-Fluorouracil and Gefitinib.

The screening tests demonstrated that 5m presented excellent antitumor activities against A549 with IC₅₀ value was 7.19 µM, which was superior to the positive agents 5-Fluorouracil (10.56 µM) and Gefitinib (27.07 µM).

2.2.2 Compound 5m induced apoptosis of A549 cells

In order to further explore the antitumor mechanism of compound 5m on A549 cell line, the effect of compound 5m on apoptosis of A549 cells was tested with 5-Fluorouracil and Gefitinib as positive control. As shown in Fig. 2, after co-culturing compound 5m (0, 5, 10, 20 µM) and A549 cells for 24 h, DAPI staining experiment showed that compound 5m caused shrinkage and nuclear chromatin condensation in A549 cells, suggesting apoptosis, and the number of apoptotic cells is concentration-dependent. And the cell apoptosis of A549 cells induced by different concentrations of compound 5m (0, 5, 10, 20 µM) for 48 h was evaluated using Annexin V-FITC/PI double-staining technique, and the apoptosis ratio of A549 cells was detected by flow cytometry. According to the results, it can be clearly seen that the ratio of apoptotic cells induced by compound 5m obviously increased in a dose-dependent manner compared to control group. When the concentration of compound 5m was 0, 5, 10 and 20 µM, the apoptosis rates are as follows: 6.31%, 18.34%, 23.78%, and 26.37%. The results showed that compound 5m could induce apoptosis in A549 cell.

2.2.3 Compound 5m inhibit A549 cell proliferation and migration

In our study, after treating A549 cell with different concentrations (0, 5, 10, 20 µM) of 5m for 0, 12 and 24 h. Wound healing assay suggested that 5m weakened the migration capability of A549 cell (Fig. 2A). Colony formation tests showed that treatment of A549 cells with different concentrations of 5m significantly weakened their proliferative activity (Fig. 2B).

2.2.4 Compound 5m induced ROS accumulation in A549 cells

Tumor cells produce reactive oxygen species (ROS), which are essential for the progression of the cancer cell cycle. However, excessive ROS production by oxidants or drugs may lead to toxicity in cancer cells, ultimately inducing apoptosis. The changes of reactive oxygen species (ROS) in A549 cells were detected by fluorescence probe DCFH-DA. As shown in Fig. 4, flow cytometry results showed that compound 5m significantly increased ROS levels in A549 cells.

2.2.5 Compound 5m induced a decrease in mitochondrial membrane potential in A549 cells

It has been reported that the damage of mitochondrial membrane integrity and the loss or collapse of mitochondrial membrane potential are the early process of inducing apoptosis. Therefore, we tested this possibility by using a JC-1 fluorescent probe to detect the effect of compound 5m on mitochondrial membrane potential in A549 cancer cells. Mitochondria with normal membrane potential retain JC-1 probes, but destruction of membrane potential leads to loss of mitochondrial JC-1 probes, decreased intracellular red fluorescence intensity (JC-1 aggregates), and increased green fluorescence intensity (JC-1 monomers). The change of green fluorescence intensity was observed by flow cytometry. After the intervention of compound 5m with different concentrations in A549 cells for 24 h, the results of Fig. 5 showed that its red fluorescence intensity decreased from 95.5–53.7%, and its green fluorescence intensity increased from 4.40–46.2%. These results suggested that compound 5m could induce mitochondrial membrane potential collapse and mitochondrial dysfunction. Then the expression of apoptosis-related proteins was changed, and finally apoptotic cell death was induced in A549 cells.

2.2.6 Compound 5m induced A549 cell cycle arrest in G2\M phase

Cell cycle is an important regulator of cell proliferation. Most chemotherapy drugs exert their cytotoxic effects by inducing apoptosis or by blocking the cell cycle at specific checkpoints. Inappropriate regulation of the cell cycle by anticancer drugs has been recognized as an effective strategy for developing new tumor therapies. Therefore, the effect of compound 5m on cell cycle progression of A549 by flow cytometry is the focus of our study. A549 cells were treated with different concentrations of compound 5m for 24 h, It can be clearly seen from Fig. 6 that after 5m treatment, the proportion of G2/M phase cells increased significantly, from 2.95% (0 µM) to 11.72% (5 µM), 12.50% (10 µM) and14.38% (20 µM) in the control group.

2.2.7 Compound 5m significantly affected the expression of apoptosis and G2\M phase regulatory proteins

Bcl-2 family proteins are crucial components of mitochondrial stress-induced cellular apoptosis. Therefore, the expression of apoptosis-related proteins was also determined. Further evidence from the Western blot assay confirmed that 5m up-regulated the expression of pro-apoptotic proteins (e.g., Bax) and correspondingly down-regulated the expression of antiapoptotic proteins (e.g., Bcl-2) in a dose dependent manner (Fig. 7).

Cell cycle experiments showed that 5m may promote cell cycle arrest in G2/M phase. Cyclin B1 and CDK-1 are all involved in cell cycle regulation and serve as key checkpoint proteins during G2/M phase progression. Therefore, the expression of cell division regulated proteins was investigated. As shown in Fig. 8, 5m caused a dose-dependent decrease in cyclin B1and CDK-1. These results, which were consistent with the cell cycle analysis, further illustrate the mechanism of the cell cycle arrest effect.

2.2.8 ADMET test

To further evaluate the drug similarity and non-target toxicity of the lead compound, we evaluated the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of compound 5m using ADMET lab 2.0 software to obtain pharmacokinetic informatics. The predicted results of compound 5m are shown in Fig. 9 and Table 3. The radar map shows that the physical and chemical properties of compound 5m are in the appropriate range: molecular weight = 473.15, logS = -5.496, logP = 4.274, logD = 3.556. In addition, the ADMET and drug properties of compound 5m are as follows: (1) In the absorption part, compound 5m has almost no inhibitory activity against P-glycoprotein (Pgp). It has good absorption effect in MDCK permeability, human intestinal absorption (HIA), 20% bioavailability and so on. (2) In terms of distribution, compound 5m has good volume distribution and blood-brain barrier penetration ability. (3) In terms of metabolism, compound 5m has no inhibitory effect on CYP1A2 and CYP2DA6, but has inhibitory effect on CYP2C19. (4) In terms of excretion, compound 5m has a medium clearance rate and half-life. (5) In terms of toxicology, compound 5m has better safety in terms of human hepatotoxicity, eye corrosion and eye irritation, etc. (6) Compound 5m conforms to Lipinski and the Golden Triangle (compounds satisfying the golden triangle rule may have a more favorable ADMET profile) rules. In summary, compound 5m showed good pharmacokinetic properties and could be further explored and developed.

Table 3

ADMET properties and drug properties of compound 5m were predicted by ADMETab. 2.0
Category	Model	Value	Decision
Medicinal chemistry	QED	0.573	Medium
	SA score	2.24	Excellent
	NP score	-2.101	Excellent
	Lipinski Rule	-	Excellent
	Golden T riangle	-	Excellent
Absorption	Caco-2 Permeability	-6.056	Medium
	MDCK Permeability	5 x 10^− 5	Excellent
	Pg p-inhibitor	0.026	Medium
	Pg p-substrate	0.235	Excellent
	HIA	0.017	Excellent
	F20%	0.004	Excellent
	F30%	0.966	Medium
Distribution	PPB	100.2%	Medium
	VD	0.31	Excellent
	BBB Penetration	0.037	Excellent
	Fu	0.746%	Bad
Metabolism	CYP1A2 inhibitor	0.543	Medium
	CYP1A2 substrate	0.745	Excellent
	CYP2C19 inhibitor	0.868	Bad
	CYP2C19 substrate	0.065	Excellent
	CYP2DA6 inhibitor	0.551	Medium
	CYP2DA6 substrate	0.687	Excellent
Excretion	CL	3.21	Medium
	T_1/2	0.451	Medium
Toxicity	hERG Blockers	0.397	Medium
	H-HT	0.274	Excellent
	AMES Toxicity	0.854	Bad
	Rat Oral Acute Toxicity	0.037	Excellent
	FDAMDD	0.376	Medium
	Skin Sensiti zation	0.925	Bad
	Eye Corrosion	0.003	Excellent
	Eye Irritation	0.107	Excellent
	Respiratory Toxicity	0.881	Bad

In our study, 17 new benzimidazole derivatives were designed and synthesized. The compound 5m has good inhibitory activity on A549 cells. The compound 5m was selected for further study, including its mechanism of growth inhibition on the A549 cell line. DAPI staining showed that A549 cells showed contraction and nuclear chromatin agglutination after 5m treatment. Flow cytometry results further confirmed that compound 5m could cause mitochondrial membrane potential collapse and induce apoptosis of A549 cells. In addition, flow cytometry was used to detect reactive oxygen species in A549 cells, and the results showed that compound 5m could increase the reactive oxygen species in A549 cells. The results of cell cycle experiment showed that compound 5m could put A549 cells in G2/M phase. Taken together, these results provide a basis for further optimization of the structures of benzimidazole antitumor compounds. Further verification showed that compound 5m significantly decreased the expression of anti-apoptotic protein Bcl-2 and up-regulated the expression of pro-apoptotic protein Bax. In addition, the key proteins CyclinB1 and CDK-1 involved in the regulation of G2/M phase of cell growth were significantly down-regulated. Throughout the data, 5m halted cell cycle progression at the G2/M phase and altered the expression of cell cycle-related proteins. Moreover, 5m effectively induced cell apoptosis, altered the expression of apoptosis-related proteins, decreased MMP and increased reactive oxygen species. All together, these results demonstrate the promising worth of 5m as a potential chemotherapeutic agent.

4.1 Methods and materials

The solvents and chemicals employed were of analytical purity. The melting points of the compounds were determined using an X-4D melting point apparatus, which was not calibrated before use. ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy was conducted in a Bruker Avance III, 400 MHz nuclear magnetic resonance (NMR) instrument. The target compounds were analyzed by HRMS-ESI spectra with the use of Thermo Scientific Q Exactive series chromatography. All reagents and solvents were sourced commercially and used without further purification.

Fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI 1640) medium were obtained from Basalmedia Technologies (Shanghai, China). Penicillin-Streptomycin Solution was purchased form Biosharp. MTT (3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide) was received from Leyan (Shanghai, China). DAPI, Annexin V-FITC Apoptosis Detection Kit, DNA Content Quantitation Assay (Cell Cycle) Kit, Mitochondrial Membrane Potential Assay Kit with JC-1, Reactive Oxygen Species Assay Kit, protease and phosphatase inhibitor cocktail were purchased from Solarbio Science & Technology (Beijing, China). Cell lysis buffer was purchased from Beytime (Shanghai China). The antibodies against Cyclin B1 (AF6188, 1:500), CDK1 (DF8024, 1:500), β-actin (AF7018, 1:3000) were purchased from Affinity (JiangSu, China); P21 (10355-1-AP,1:1000), Bax (5099-2-Ig, 1:2000) were purchased from Proteintech (WuHan, China); Bcl-2 (H651066060,1:2000) was purchased from Huabio (ZheJiang, China). The human NSCLC cell lines A549 and PC-3 were kindly provided by the Third Military Medical University.

4.2 Biological evaluation

4.2.1 Cell culture

All cancer cells were cultured in RPMI-1640 (EXCEL, USA) with Gibco at 37℃ in5% CO₂ and 95% air. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in an incubator at 37°C with 5% CO₂.

4.2.2 MTT assay

The in vitro inhibitory activity of the target compounds against A549 and PC-3 tumor cell lines were tested by MTT method. The logarithmic cells were cultured in a 96-well plate and pre-cultured for 24 h before adding the test compound. Then different concentrations of 20 µL compound solution were added to the 96-well plate. After the cells were cultured at 37°C and 5% carbon dioxide for 48 hours, 20 µL tetramethylazolium salt solution with a concentration of 5 µg/mL was added and reacted in the same environment for 4 h, and the absorbance was measured at 490 nm by microplate reader (Tecan, Infinite M200 Pro).

4.2.3 Colony-forming assay

A549 (2 × 10⁵ cells/well) were seeded in 6-well plate and allowed to attach overnight. Subsequently, the cells were treated with 5m for a duration of 24 h. Drug-treated cells were seeded into 6-well plates at 1000 cells/well and continue to incubate for 14 days. Then cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min, washed sometimes with ddH₂O. After airdrying, the colonies were photographed and counted using Image J software [18].

4.2.4 Wound-healing assay

A549 was seeded at a density of 1 × 10⁶ cells per well in 6-well culture plates and cultured overnight. Once the cell confluence reached over 90%, the monolayer was scratched with a sterile pipette tip (10 µL) and floating cells were removed by rinsing with PBS three times. The cells were then incubated in 1% serum medium containing varying concentrations of 5m (0, 5, 10, 20 µM). The wound healing process was monitored using microscopy (NIKON, Japan), and images of the scratches were captured at 0, 12, and 24 hours. Finally, Image J software was utilized to accurately delineate the wound areas for subsequent analysis [19].

4.2.5 DAPI staining assay

Cells were spread in 12-well plates at a density of 1 × 10⁵ cells per well. After incubated for 24 h, cells were treated with different concentrations of 5m (0, 5, 10, 20 µM) and 5-fluorouracil (20 µM ) for 24 h. At the end of the incubation period, the medium was removed, and the cells were washed with PBS for several times and then fixed by adding 4% paraformaldehyde for 20 min, after which the fixing solution was removed, the cells were washed by adding PBS. Then added with DAPI staining solution and stained for 20 min in the dark. Finally, the staining solution was removed, and anti-fluorescent attenuating sealer was dropped into each well, and the pictures were taken by inverted fluorescence microscope (NIKON, Japan) [20].

4.2.6 Cell apoptosis analysis

Annexin V-FITC/PI kit was utilized to evaluate the apoptosis induction. Briefly, Cells were seeded in 6-well plates (2 × 10⁵ cells/well) and incubated for 24 hours. Then, the exponential cells were treated with different concentrations of 5m (0, 5, 10, 20 µM) for a duration of 24 hours. Cells were harvested, washed twice with PBS, and suspended in 100 µL of binding buffer. Approximately 5 µL each of Annexin V and PI were added to the solution, followed by gentle vortexing then a 15-minutes and 5-minutes incubation at room temperature in the dark respectively. After adding 400 µL of 1 × binding buffer, apoptotic cells were measured using flow cytometry (FACS Calibur, Becton Dickenson). Data analysis was performed using Flow jo [21, 22].

4.2.7 Cell cycle assay

DNA Content Quantitation Assay (Cell Cycle) kit was used to detect cell cycle. Briefly, A549 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells/well and incubated for 24 h. The exponential cells were subsequently exposed to varying concentrations of 5m (0, 5, 10, 20 µM) for a duration of 24 hours. Following treatment, according to the manufacture’s instruction, the cells were harvested and fixed with 75% cold ethanol overnight at a temperature of 4 ℃. Following centrifugation, the cells were washed with PBS and subsequently treated with 100 µL RNase A at 37 ℃ for 30 min. The fixed cells were then stained with 400 µL propidium iodide (PI) at 4 ℃ for a duration of 30 min. Flow cytometry analysis was performed using FACS Calibur (Becton Dickenson) to determine cell cycle distribution. Data analysis was performed using Modifit LT5.0 [23].

4.2.8 Evaluation of mitochondrial membrane potential (Δѱm)

The effect of 5m on Δѱm was determined using a JC-1 Assay Kit. JC-1 is a fluorescence probe, and the change of the fluorescence from red to green suggested that JC-1 changed from polymer to monomer, and the decrease of the ratio of JC-1 polymer/monomer (JC-1 red/green rate) indicated a loss of Δѱm. Cells were seeded evenly at the density of 2 × 10⁵ cells/well in 6- well plates and allowed to attach for 24 h. After treated with 5m for 48 h, the cells were collected and resuspended in incubating buffer containing JC-1 for 30 min at 37 ℃. The cells were centrifugated and washed twice using ice-cold JC-1 buffer for later flow cytometry analysis. Data analysis was performed using Flow jo [24, 25].

4.2.9 Intracellular ROS Assay

Cells were seed evenly in 6-well plates (2×10⁵) for 24 h and treated with 5m. DCFH-DA was a cell-permeable probe and could be oxidized by ROS in cells. The ROS level could be detected by measuring fluorescence of the oxidized form of DCFH-DA. The cells were stained with of DCFH-DA at 37℃ for 20 min, and the labeled cells were washed with FBS free medium for three times and analyzed using flow cytometry (FACS Calibur, Becton Dickenson). Data analysis was performed using Flow jo [26].

4.2.10 Western blotting

Protein levels were analyzed by Western blotting. After treatment, A549 cells was harvested and lysed with 25–40 µL lysis buffer on ice for 40 min to extract intracellular proteins. The lysate was then centrifuged at 12000 rpm for 15 min at 4°C to isolate the supernatant, which was then quantified using a BCA assay kit (Solarbio Science & Technology, Beijing, China) according to the manufacturer’s insructions. Equal amounts (30 µg) protein samples were processed through 10%, 12.5% or 15%SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% non-fat milk for 2 h, the membrane was washed three times with TBST and followed by incubation with primary antibody at 4 ℃ overnight. The next day, the membrane was incubated for 1 hour at room temperature, then washed 3 times with TBST and incubated with the respective secondary horseradish peroxidase (HRP)-conjugated antibodies for 1 hour at room temperature. Finally, the protein bands were visualized by chemiluminescence system [27–30].

4.2.11 Statistical analysis

All statistical were analyzed by GraphPad Prism 9.0 software (GraphPad Inc., San Diego, CA, USA) and data were presented as mean ± standard deviation (SD). Statistical differences between groups were evaluated using One-way analysis of variance (ANOVA) tests, when P < 0.05 was considered a significant difference.

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.

Acknowledgement

This study was supported by the National Natural Science Foundation of China (32360689, 22364008, 32260694, 21867004), Guizhou Provincial Foundation for Excellent Scholars Program (GCC[2023]072), Guizhou Provincial Natural Science Foundation (ZZK[2021]034, ZK[2022]073), Guizhou Provincial Young Science and Technology Talents Development Project (KY[2022]146), and the National Key R&D Program of China (2023YFD1400400).

Author Contribution

Lihui shao wrote the main manuscript text; Nianlin Feng prepared figures; Yue Zhou, Chengpeng Li, Danping Chen, Chenchen Li, and Xiang Zhou helped with the data analysis； Zhurui Li and Zhenchao Wang assisted in the revision of the article；All authors reviewed the manuscript.

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

GraphicalAbstract.png
Graphical Abstract
Scheme1.png
Scheme 1 Synthesis of target compounds 5.
Surpportinginformation202410092.docx
Supplementary Information The ¹H NMR and ¹³C NMR spectra and data of target compounds.

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Synthesis, evaluation, and mechanism study of novel benzimidazole acylhydrazone derivatives for antitumor activity

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Version 1

Abstract

Figures

1 Introduction

2 Results and discussion

2.1 Chemistry

2.2 Biological evaluation

2.2.1 Activity evaluation and structure-activity relationship

2.2.2 Compound 5m induced apoptosis of A549 cells

2.2.3 Compound 5m inhibit A549 cell proliferation and migration

2.2.4 Compound 5m induced ROS accumulation in A549 cells

2.2.5 Compound 5m induced a decrease in mitochondrial membrane potential in A549 cells

2.2.6 Compound 5m induced A549 cell cycle arrest in G2\M phase

2.2.7 Compound 5m significantly affected the expression of apoptosis and G2\M phase regulatory proteins

2.2.8 ADMET test

3 Conclusion

4 Experimental

4.1 Methods and materials

4.2 Biological evaluation

4.2.1 Cell culture

4.2.2 MTT assay

4.2.3 Colony-forming assay

4.2.4 Wound-healing assay

4.2.5 DAPI staining assay

4.2.6 Cell apoptosis analysis

4.2.7 Cell cycle assay

4.2.8 Evaluation of mitochondrial membrane potential (Δѱm)

4.2.9 Intracellular ROS Assay

4.2.10 Western blotting

4.2.11 Statistical analysis

Declarations

Author Contribution

References

Scheme 1

Additional Declarations

Supplementary Files

Status:

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