Design, Synthesis, and Antitumor Activity of Isoliquiritigenin Amino Acid Ester Derivatives

Isoliquiritigenin (ISL) is a chalcone that has shown great potential in the treatment of cancer. However, its relatively weak activity and low water solubility limit its clinical application. In this study, we designed and synthesized 21 amino acid ester derivatives of ISL and characterized the compounds using 1H NMR and 13C NMR. Among them, compound 9 (IC50 = 14.36 μM) had a better inhibitory effect on human cervical cancer (Hela) than ISL (IC50 = 126.5 μM), and it was superior to the positive drug 5-FU (IC50 = 33.59 μM). The mechanism of the action experiment showed that compound 9 could induce Hela cell apoptosis and autophagy through the PI3K/Akt/mTOR pathway.


Introduction
Cancer is a major disease that threatens human health.According to the 2020 study report published by the Agency for Cancer study of the World Health Organization, there are roughly 19.2928 million new cases of cancer worldwide, with 9.9581 million new cancer deaths [1].The report also projects a modest annual increase in the number of new cases.Owing to the intricate etiology and challenging aspects of cancer, the three primary therapeutic modalities still used in cancer care are surgery, radiation therapy, and chemical medication therapy [2].Despite the fact that all three treatments can increase patient lifespan, some cancers cannot be surgically removed, radiation and chemotherapy can cause severe side effects, and chemotherapy can cause drug resistance [3,4].A medication created in accordance with the principles of traditional Chinese medicine is known as traditional Chinese medicine.Because of its distinct anti-tumor action and minimally hazardous side effects, it is frequently employed in the treatment of malignancies.About half of the 80 different types of anti-tumor medications currently in clinical use worldwide are derived either directly or indirectly from plants.Commonly used plant-based medications include vinblastine [5], paclitaxel [6], artemisinin [7], betulinic acid [8], berberine [9], ginsenosides [10], etc.
One chalcone component that can be separated from the leguminous plant Glycyrrhiza uralensis was isoliquiritigenin [11].It has a wide range of pharmacological properties, including anti-inflammatory [12], anti-tumor [12], anti-angiogenesis [13], antibacterial [14], and anti-diabetic activities [15].For instance, it possesses many modes of action and exhibits some inhibitory effect on human cancer cells, including those of the liver [16], breast [17], prostate [18], and cervical regions [19].By preventing the cell cycle, triggering tumor autophagy, preventing tumor cell spread, and inducing apoptosis, among other mechanisms, it can cause tumor cells to die [20].However, the application of isoliquiritigenin is still limited by its poor solubility and low anti-tumor activity.
Amino acids are the fundamental units of proteins and the main substances that maintain biological life activities.The chemical structure of an amino acid includes an amino group (-NH 2 ) a carboxyl group (-COOH), and a unique carbon side chain connecting the amino and carboxyl groups.Amino acid structures are simple and diverse, with significant anti-tumor activity, making them easy to use in drug synthesis and structural modification.According to reports, after combination with an amino acid, the solubility of the compound is enhanced, the selectivity towards tumor cells is improved, and the toxicity to normal cells is reduced [21].In the preliminary research results of the research group, the anti-tumor activity was significantly enhanced by the introduction of amino acids separately, such as Arctigenin [22], α-mangostin [23], and podophyllotoxin [24].A common chalcone structure, isoliquiritigenin, is joined to the A and B rings by cross-conjugation with propylene ketone.Three atoms with large rotational degrees of freedom are present in the associated bridge bonds.Consequently, isoliquiritigenin's binding to receptors has a wide geographical distribution.Ref. [25] verified that isoliquiritigenin's anti-tumor action requires the presence of hydroxyl groups as functional groups.According to research [19], the best locations for chalcone compounds to substitute active groups are at positions 2 ′ and 4 ′ of the A ring and 4 of the B ring.Structure-activity relationship analysis demonstrated that, following chlorination modification at positions 2 ′ and 4 ′ of the A-ring, the modified chlorination at these locations had considerably greater activity on Hela and SiHa cells and less toxicity on normal CHO cells than at positions 3 ′ , 4 ′ , 2 ′ , and 5 ′ .
Therefore, this study designed and synthesized a series of amino acid derivatives of isoliquiritigenin, screened out isoliquiritigenin derivatives with stronger anti-tumor activity, and conducted preliminary discussions on the structure-activity relationship and apoptosis mechanism, providing theoretical reference for the further development and utilization of isoliquiritigenin.

Chemistry
The structures of all synthesized target derivatives were characterized by 1 H NMR, 13 C NMR, and HRMS.The characterization data were consistent with the predicted structures.

Toxicity of Compounds on Different Human Tumor Cell Lines
The anti-tumor activity of all target compounds 1-23 was evaluated in vitro by an MTT assay against Bel-7402, A549, Hela, MCF-7 and PC-3M cell lines, with 5-FU as the positive control.Their inhibition rates and IC 50 values are listed in Table 1.
The following structure-activity relationship pattern was obtained by assessing the anti-tumor activity of the aforementioned compounds: (1) The structurally modified derivatives had a lower overall inhibitory action on tumor cells than the mother nucleus isoliquiritigenin when the C4 ′ -OH of the A ring and the C4-OH of the B ring of isoliquiritigenin were esterified concurrently; and (2) the structurally modified derivatives exhibited a greater overall inhibitory action on tumor cells than isoliquiritigenin, the parent nucleus, when C4-OH on the B ring was esterified alone.Four of them had far greater inhibitory effects against tumor cells when modified with amino acids that had cycloalkyl groups or halogen atoms.Specifically, compound 21 had an IC 50 value of 66.5 for Hela cells and 87.5 µM for PC-3M cells.This is because compound 21 esterifies phenylalanine on the B-ring alone by C4-OH without adding halogen atoms.However, the IC 50 values of compound 15 and compound 17 on Hela dropped to 27.36 and 30.4 µM, respectively, upon the introduction of halogen atoms Br and Cl.The PC-3M's IC 50 values were lowered by compound 15 and compound 17 to 30.2 µM and 37.4 µM, respectively.Furthermore, the inhibitory activity against PC-3M cells was significantly increased by adding cyclic alkyl amino acids, specifically compound 7 and compound 9, with compound 9 exhibiting the largest change in activity.

Toxicity of Compounds on Different Human Tumor Cell Lines
The anti-tumor activity of all target compounds 1-23 was evaluated in vitro by an MTT assay against Bel-7402, A549, Hela, MCF-7 and PC-3M cell lines, with 5-FU as the positive control.Their inhibition rates and IC50 values are listed in Table 1.To further confirm the toxicity of compound 9 to normal cells, we used the MTT method to determine the cytotoxicity of human normal cervical epithelial cells (HUCEC), and the results showed that compound 9 had cytotoxicity (IC 50 > 200 µM).Therefore, compound 9 can serve as a specific mechanism for further research.

Compound 9 Inhibits Hela Cell Migration
Inhibiting tumor cell migration is a therapeutic approach for malignancies, as it is a necessary condition for invasion and metastasis.In order to verify the effects of ISL and compound 9 on the in vitro migration ability of Hela cells, the scratch test results showed in Figure 1, compared with the control group, compounds ISL and 9 could inhibit the migration of Hela cells.Compared with the ISL group, the scratch healing degree of the compound 9 group was relatively more significant, and the scratch healing degree significantly decreased with the increase in drug concentration.

Compound 9 Inhibits Hela Cell Proliferation
In order to verify the proliferative and killing effect of compound 9 on cervical cancer cells, this study used plate clone formation experiments to detect the effects of compound 9 and ISL on Hela cell clone proliferation ability.The results showed in Figure 2, compared with the control group, both compound ISL and compound 9 could inhibit Hela cell proliferation.Compared with the ISL group, compound 9 group had relatively fewer colonies, and the number of colonies significantly decreased with increasing drug concentration.

Compound 9 Affects Hela Cell Morphology
As shown in Figure 3, compared with the control group, compounds ISL and 9 could both inhibit the apoptosis of Hela cells.The control group cells showed a good growth status, with spherical and intact nuclei.However, as the drug concentration increased, the number of cells in the treatment group decreased, and apoptosis began to occur, resulting in fragmented nucleus.The number of cells in compound 9 of 50 µM decreased sharply, and there were basically no complete nuclei present.Therefore, it can be concluded that this derivative can induce apoptosis of Hela cells and exhibits a certain dose-dependent effect.

Compound 9 Inhibits Hela Cell Proliferation
In order to verify the proliferative and killing effect of compound 9 on cervical cancer cells, this study used plate clone formation experiments to detect the effects of compound 9 and ISL on Hela cell clone proliferation ability.The results showed in Figure 2, compared with the control group, both compound ISL and compound 9 could inhibit Hela cell proliferation.Compared with the ISL group, compound 9 group had relatively fewer colonies, and the number of colonies significantly decreased with increasing drug concentration.

Compound 9 Affects Hela Cell Morphology
As shown in Figure 3, compared with the control group, compounds ISL and 9 could both inhibit the apoptosis of Hela cells.The control group cells showed a good growth status, with spherical and intact nuclei.However, as the drug concentration increased, the number of cells in the treatment group decreased, and apoptosis began to occur, resulting in fragmented nucleus.The number of cells in compound 9 of 50 µM decreased sharply,

Compound 9 Affects Hela Cell Apoptosis
Quantitative analysis of apoptosis by Figure 4 showed that the apoptosis ratio of Hela cells in the control group was 6.41%, while the apoptosis ratios of Hela cells in the 12.5 µM, 25 µM, and 50 µM groups after 48 h of treatment were 13.57%, 22.88%, and 30.2%, respectively.The apoptotic proportions of Hela cells were 9.75%, 16.43%, and 20.93% after 48 h of treatment in the ISL group, which confirmed that compound 9 could induce apoptosis of Hela cells.

Compound 9 Affects Hela Cell Apoptosis
Quantitative analysis of apoptosis by Figure 4 showed that the apoptosis ratio of Hela cells in the control group was 6.41%, while the apoptosis ratios of Hela cells in the 12.5 µM, 25 µM, and 50 µM groups after 48 h of treatment were 13.57%, 22.88%, and 30.2%, respectively.The apoptotic proportions of Hela cells were 9.75%, 16.43%, and 20.93% after 48 h of treatment in the ISL group, which confirmed that compound 9 could induce apoptosis of Hela cells.

Anti-Tumor PI3K/AKT/mTOR Pathway Analysis of Compound 9 Based on Molecular Docking Technology
Molecular docking is a widely used computational tool in molecular recognition research whose purpose is to predict the binding pattern and binding affinity of a complex composed of two or more molecules of known structure [26].At present, molecular docking has been widely used in the efficacy evaluation and mechanism of action of TCM, such as preliminary verification of targets, interpretation of the TCM mechanism of action, drug function localization, and identification of toxic components of TCM [27].
At present, studies have confirmed that the occurrence and development of cervical cancer are closely related to apoptosis and autophagy [28].Autophagy is a catabolic pathway through which some cellular components are transported to lysosomes by autophagosomes [29].Autophagy-related factors Beclin1, LC3, and p62, as means to assist in the diagnosis of cervical cancer lesions, are also the targets of a variety of anti-tumor drugs.The PI3K/AKT/mTOR signaling pathway related to autophagy also regulates a variety of cellular processes in the process of tumor occurrence, including tumor growth, tumor survival, tumor cell proliferation, tumor immunity, tumor metabolism, and tumor angiogenesis [30].The activation of the PI3K/AKT/mTOR signaling pathway begins with the activation of growth factor receptors, cytokines, hormones, etc., on phosphatidylinoinosidine-3-kinase.PI3K enables PIP2 on the cell membrane to produce a second messenger, PIP3, which binds to the signaling proteins AKT and PDK1 containing the PH domain in the cell.AKT is translocated in the cell membrane and achieves catalytic activity, catalyzing its own phosphorylation at Serl24 and Thr450.PDKl can catalyze phosphorylation of the AKT protein at Thr308.AKT may also be fully activated by the phosphorylation of Ser473 by mTORC2 [31].Activated AKT activates or inhibits a series of downstream substrates such as mTOR, Bad, Bcl-2, Caspase-3 and P70S6K through phosphorylation targeting, thereby regulating cell proliferation, differentiation, apoptosis, and migration.

Compound 9 Affects Hela Cell Apoptosis
Quantitative analysis of apoptosis by Figure 4 showed that the apoptosis ratio of Hela cells in the control group was 6.41%, while the apoptosis ratios of Hela cells in the 12.5 µM, 25 µM, and 50 µM groups after 48 h of treatment were 13.57%, 22.88%, and 30.2%, respectively.The apoptotic proportions of Hela cells were 9.75%, 16.43%, and 20.93% after 48 h of treatment in the ISL group, which confirmed that compound 9 could induce apoptosis of Hela cells.

Anti-Tumor PI3K/AKT/mTOR Pathway Analysis of Compound 9 Based on Molecular Docking Technology
Molecular docking is a widely used computational tool in molecular recognition research whose purpose is to predict the binding pattern and binding affinity of a complex composed of two or more molecules of known structure [26].At present, molecular docking has been widely used in the efficacy evaluation and mechanism of action of TCM, such as preliminary verification of targets, interpretation of the TCM mechanism of action, drug function localization, and identification of toxic components of TCM [27].
At present, studies have confirmed that the occurrence and development of cervical cancer are closely related to apoptosis and autophagy [28].Autophagy is a catabolic pathway through which some cellular components are transported to lysosomes by autophagosomes [29].Autophagy-related factors Beclin1, LC3, and p62, as means to assist in the diagnosis of cervical cancer lesions, are also the targets of a variety of anti-tumor drugs.The PI3K/AKT/mTOR signaling pathway related to autophagy also regulates a variety of cellular processes in the process of tumor occurrence, including tumor growth, tumor sur- In order to explore the binding ability of ISL and compound 9 to PI3K/Akt/mTOR pathway proteins, PI3K and mTOR were selected for docking analysis.As showed Figure 5, Auto Dock data showed that ISL formed hydrophobic interactions with the residues THR 369, ILE 381, LYS 382, and PHE 394 in the PI3K protein.Meanwhile, hydrogen bonding with the residue SER361 was measured to be 3.20 ngstrom.And the binding affinity was 8.1 kcal/mol.However, compound 9 formed hydrophobic interactions with the residues ILE381, PHE 384, PHE 392, and TYR 416 in the PI3K protein.Hydrogen bonding with the residue THR 369 was measured to be 2.54 ngstrom, and the residue ILE 381 was measured to be 4.06 ngstrom.And the binding affinity was 8.9 kcal/mol.Furthermore, ISL formed hydrophobic interactions with the residues TYR 337, PHE 348, and PHE 367 in the mTOR protein.Hydrogen bonding with the residue ASN 342 was measured to be 2.50 ngstrom.And the binding affinity was 6.9 kcal/mol.Nevertheless, compound 9 formed hydrophobic interactions with the residues TYR 336, TYR 337, PHE 348, and PHE 367 in the mTOR protein.Hydrogen bonding with the residue ASN 342 was measured to be 3.16 ngstrom.And the binding affinity was 7.6 kcal/mol.Thus, compound 9 can be further studied on the PI3K/Akt/mTOR pathway.

Real-Time Fluorescence Quantitative PCR Was Used to Detect PI3K/AKT/mTOR Pathway-Related Gene Expression
As shown in Figure 6, based on the prediction results of molecular docking, the star genes PI3K, AKT, and mTOR; the downstream genes P70S6K, Bad, Bcl-2, and Caspase-3; and autophagy-related genes Maplc3A and Beclin1 in the PI3K/AKT/mTOR pathway were detected in this experiment, and the results showed that, compared with the control group, PI3K, AKT, mTOR, and P70S6K, the mRNA levels of five genes of Bcl-2 showed a downward trend, and the mRNA levels of three genes, Bad, Maplc3A, and Beclin1, showed upward trends.In addition, compared with ISL, the changes in compound 9 groups were more significant.In order to explore the binding ability of ISL and compound 9 to PI3K/Akt/mTOR pathway proteins, PI3K and mTOR were selected for docking analysis.As showed Figure 5, Auto Dock data showed that ISL formed hydrophobic interactions with the residues THR 369, ILE 381, LYS 382, and PHE 394 in the PI3K protein.Meanwhile, hydrogen bonding with the residue SER361 was measured to be 3.20 ngstrom.And the binding affinity was 8.1 kcal/mol.However, compound 9 formed hydrophobic interactions with the residues ILE381, PHE 384, PHE 392, and TYR 416 in the PI3K protein.Hydrogen bonding with the residue THR 369 was measured to be 2.54 ngstrom, and the residue ILE 381 was measured to be 4.06 ngstrom.And the binding affinity was 8.9 kcal/mol.Furthermore, ISL formed hydrophobic interactions with the residues TYR 337, PHE 348, and PHE 367 in the mTOR protein.Hydrogen bonding with the residue ASN 342 was measured to be 2.50 ngstrom.And the binding affinity was 6.9 kcal/mol.Nevertheless, compound 9 formed hydrophobic interactions with the residues TYR 336, TYR 337, PHE 348, and PHE 367 in the mTOR protein.Hydrogen bonding with the residue ASN 342 was measured to be 3.16 ngstrom.And the binding affinity was 7.6 kcal/mol.Thus, compound 9 can be further studied on the PI3K/Akt/mTOR pathway.

Real-Time Fluorescence Quantitative PCR Was Used to Detect PI3K/AKT/mTOR Pathway-Related Gene Expression
As shown in Figure 6, based on the prediction results of molecular docking, the star genes PI3K, AKT, and mTOR; the downstream genes P70S6K, Bad, Bcl-2, and Caspase-3; and autophagy-related genes Maplc3A and Beclin1 in the PI3K/AKT/mTOR pathway were detected in this experiment, and the results showed that, compared with the control group, PI3K, AKT, mTOR, and P70S6K, the mRNA levels of five genes of Bcl-2 showed a downward trend, and the mRNA levels of three genes, Bad, Maplc3A, and Beclin1, showed upward trends.In addition, compared with ISL, the changes in compound 9 groups were more significant.

Chemistry
Every chemical and solvent was above analytical purity.Purchased from commercial vendors, all reagents and solvents were utilized without additional purification.A QSTAR spectrometer was used to collect HR-ESI-MS data (FL, USA).Utilizing an American Varian NMR System 300 MHz, NMR spectroscopy was conducted in CDCl3 to determine chemical shift (δ) values, with solvent peaks or tetramethylsilane (0.00 ppm) serving as the internal reference.All compounds were separated using silica gel columns (Qingdao Ocean Chemical Group Co., Ltd., Qingdao, China).

Chemistry
Every chemical and solvent was above analytical purity.Purchased from commercial vendors, all reagents and solvents were utilized without additional purification.A QSTAR spectrometer was used to collect HR-ESI-MS data (FL, USA).Utilizing an American Varian NMR System 300 MHz, NMR spectroscopy was conducted in CDCl 3 to determine chemical shift (δ) values, with solvent peaks or tetramethylsilane (0.00 ppm) serving as the internal reference.All compounds were separated using silica gel columns (Qingdao Ocean Chemical Group Co., Ltd., Qingdao, China).

Preparation of Isoliquiritigenin Derivatives
Isoliquiritigenin with a purity of 98% was used as a raw material (Chengdu Na Lithium Biotechnology Co., Ltd., Chengdu, China).As shown in the procedure in Scheme 1, isoliquiritigenin (1.0 mmol), different kinds of amino acids with Boc protection (3.0 mmol, 3.0 eq), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (3.0 mmol, 3.0 eq), and 4-Dimethylaminopyridine (1.0 mmol, 1.0 eq) were added to 20 mL of CH 2 Cl 2 and reacted in an ice bath for 12-24 h.The reaction was detected by TLC.After the reaction was complete, the crude product was obtained by reducing the pressure concentration.Finally, target products were purified using petroleum ether (PE): acetone (AC).

Cell Scratch Assay
Hela cells were inoculated into the 6-well plate at a density of 1 × 10 4 cells/well.When the cell growth reached 80-90% density, the medium was discarded, and a straight line was drawn with the gun head perpendicular to the horizontal line behind the 6-well plate.PBS was used to wash off the delimit cells twice; then, different concentrations of drugs were added to the PBS, and cell migration images of 0 h were recorded under the microscope.It was put into the incubator for 48 h and then photographed again, and the cell migration images were recorded at the same position for 48 h.

Cell Clonal Formation Assay
Hela cells were inoculated into the 6-well plate at a density of 1 × 10 3 cells/well.After the cells were attached to the wall and treated with drugs for 48 h, the medium was discarded and the complete medium was added, and the liquid was changed every 4 days.After 8-9 days of culture, visible cell masses in the culture plate were observed, that is, cell cloning occurred.After clone formation, we discarded the medium, added PBS to clean it 3 times, and then added 4% paraformaldehyde along the hole to fix for 15 min.After discarding the paraformaldehyde, it was washed with PBS for 3 times, and then we added 0.1% crystal violet dye along the hole for 15 min.After dyeing, the crystal violet was recovered, washed with PBS 3-4 times, dried at room temperature, and photographed.

Cell DAPI Staining Assay
Hela cells were seeded in 6-well plates at a density of 1 × 10 4 /well, and the cells were treated with drugs for 48 h after the cells grew to 80% density, the medium was discarded, PBS was added to wash 3 times, and 4% paraformaldehyde was added along the wells for 15 min.The paraformaldehyde was discarded, PBS was added for washing 3 times, DAPI staining solution was added along the well to protect from light for 15 min.Then, the staining solution was washed off with PBS, and we immediately took pictures under the microscope to observe the morphological changes.

Cell Apoptosis Assay
Hela cells were seeded in 6-well plates at a density of 1 × 10 5 /well and cultured in a cell culture incubator for 24 h.The medium was discarded, and PBS was added for 3 washes.Drug treatment lasted 48 h, the medium was discarded, PBS was added for 3 washes, trypsin digestion without EDTA was added, 500 µL of binding buffer was added to each group and gently blown into a single-cell suspension, 5 µL of Annexin V-FITC was mixed, and 5 µL of propidium was added to iodide.We set up a single positive tube and a blank tube at the same time, mixed the sample well at room temperature, protected it from light, reacted it for 10 min, and then observed and detected the flow cytometer within 1 h.

Molecular Docking
The X-ray crystal structures of PI3K (PDB ID: 5GJI) and mTOR (PDB ID: 7SOQ) were obtained from the Protein Data Bank (https://www.rcsb.org/accessed on 2 March 2024).The structure of isoliquiritigenin was downloaded from the PubChem database (https://www.pubchem.ncbi.nlm.nih.gov/accessed on 2 March 2024), and the structure of compound 9 was drawn from Chemdraw V20.0.We used ChemBio3D Ultra 14.0 software (PerkinElmer Informatics) for optimization.Auto Dock Vina 1.2.0 software (Center for Computational Structural Biology) was used to dock conformation.PyMOL 2.5.2 was used to visualize the conformation.

RT-qPCR
After treating Hela cells with different concentrations of drugs for 48 h, the total RNA was extracted according to the instructions of the RNA extraction kit, and the RNA concentration and purity were determined using an ultra-micro spectrophotometer.The A260-A280 ratio was between 1.8 and 2.1 for reverse transcription to synthesize the cDNA template.The primer sequences (designed and synthesized by Sangon Biotech) are shown in Table 2, and the expression levels of the target gene in the blank control group were used as the reference factor.The 2 −△△Ct method was used for analysis, and the result was calculated as follows: relative expression amount = 2 −(Ct target gene-Ct internal reference gene) .

Conclusions
In this study, a series of isoliquiritigenin derivatives was designed and synthesized.The anti-tumor activity of Bel-7402, A549, Hela, MCF-7, and PC-3M was evaluated via the MTT method using 5-FU and Isoliquiritigenin as positive control drugs.The results showed that compound 9, containing the macromolecular amino acid cyclohexyl glycine, had strong inhibitory effects on all five types of tumor cells.The experiments showed that compound 9 induced Hela cell apoptosis and autophagy through the PI3K/Akt/mTOR pathway in a concentration-dependent manner.

Figure 2 .
Figure 2. ISL and compound 9 inhibit the proliferation of Hela cells.

Figure 2 .
Figure 2. ISL and compound 9 inhibit the proliferation of Hela cells.

Figure 3 .
Figure 3. DAPI staining was used to detect the effects of ISL and compound 9 on the morphology of Hela cells.

Figure 3 .
Figure 3. DAPI staining was used to detect the effects of ISL and compound 9 on the morphology of Hela cells.

Figure 4 .
Figure 4. Annexin V-FITC/PI double-staining assay was used to investigate the effect of compound 9 on the apoptosis of Hela cells; compared to the control group, * p < 0.05, ** p < 0.01.

Figure 4 .
Figure 4. Annexin V-FITC/PI double-staining assay was used to investigate the effect of compound 9 on the apoptosis of Hela cells; compared to the control group, * p < 0.05, ** p < 0.01.

Figure 6 .
Figure 6.Effects of ISL and compound 9 on mRNA expression in Hela cells; compared to the control group, * p < 0.05, ** p < 0.01.

Figure 6 .
Figure 6.Effects of ISL and compound 9 on mRNA expression in Hela compared to the control group, * p < 0.05, ** p < 0.01.

Scheme 1. Synthetic route of target compounds 1-23.Table 1 .
Inhibition rates of target compounds on proliferation of different tumor cells.