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Ginsengenin derivatives synthesized from 20(R)-panaxotriol: Synthesis, characterization, and antitumor activity targeting HIF-1 pathway

Background Ginseng possesses antitumor effects, and ginsenosides are considered to be one of its main active chemical components. Ginsenosides can further be hydrolyzed to generate secondary saponins, and 20(R)-panaxotriol is an important sapogenin of ginsenosides. We aimed to synthesize a new ginsengenin derivative from 20(R)-panaxotriol and investigate its antitumor activity in vivo and in vitro. Methods Here, 20(R)-panaxotriol was selected as a precursor and was modified into its derivatives. The new products were characterized by 1H-NMR, 13C-NMR and HR-MS and evaluated by molecular docking, MTT, luciferase reporter assay, western blotting, immunofluorescent staining, colony formation assay, EdU labeling and immunofluorescence, apoptosis assay, cells migration assay, transwell assay and in vivo antitumor activity assay. Results The derivative with the best antitumor activity was identified as 6,12-dihydroxy-4,4,8,10,14-pentamethyl-17-(2,6,6-trimethyltetrahydro-2H-pyran-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl(tert-butoxycarbonyl)glycinate (A11). The focus of this research was on the antitumor activity of the derivatives. The efficacy of the derivative A11 (IC50 < 0.3 μM) was more than 100 times higher than that of 20(R)- panaxotriol (IC50 > 30 μM). In addition, A11 inhibited the protein expression and nuclear accumulation of the hypoxia-inducible factor HIF-1α in HeLa cells under hypoxic conditions in a dose-dependent manner. Moreover, A11 dose-dependently inhibited the proliferation, migration, and invasion of HeLa cells, while promoting their apoptosis. Notably, the inhibition by A11 was more significant than that by 20(R)-panaxotriol (p < 0.01) in vivo. Conclusion To our knowledge, this is the first study to report the production of derivative A11 from 20(R)-panaxotriol and its superior antitumor activity compared to its precursor. Moreover, derivative A11 can be used to further study and develop novel antitumor drugs.


Introduction
Cancer can be described as the uncontrolled growth and spread of abnormal cells, which can also affect other body parts and may spread to other organs. Etiology and pathogenesis of tumor are not fully understood. Tumor may act as a leading trigger for death worldwide [1], with its incidence increasing every year [2]. The discovery of cancer drugs has presented a significant challenge for the scientific community due to the challenges associated with evaluating and verifying various possible targets [3]. Globally, cervical cancer is the fourth most common cancer amongst women, ranking only after breast cancer (2.1 million cases), colorectal cancer (0.8 million) and lung cancer (0.7 million). Globally, the average age at diagnosis of cervical cancer was 53 years, and the global average age at death was 59 years [4].
A lack of oxygen is the central feature of many solid tumors. Tumor cells grow rapidly in the body, compress blood vessels, and cause hypoxia in body tissues [5,6]. In the hypoxic microenvironment, hypoxia-inducible factor-1 (HIF-1) acts as a regulatory factor in the transcription process and promotes the occurrence and development of tumors. In consequence, HIF-1 proved to be pivotal anti-cancer drug mark [7e9]. HIF-1a and HIF-1b constitute the heterodimeric transcription factor HIF-1 [10]. The level of HIF-1a is strictly controlled by the concentration of oxygen in the cell, but the level of HIF-1b is not influenced by the oxygen content [11]. HIF-1a regulates the spread, migration and production of tumor cells [12], and there is a positive correlation between the cellular concentration of HIF-1a and the severity of cancer [13]. In addition, cancer recurrence after resection is related to overexpression of HIF-1a [14]. Therefore, HIF-1a has been used as an effective target for working on new antitumor medicine [15e17].
Ginseng, a herbal supplement, has cardioprotective, antitumor, anti-ischemic shock, anti-arrhythmia, anti-myocardial ischemia, anti-inflammatory, anti-apoptotic and antioxidant effects [18e20]. Among its chemical components, ginsenosides are considered one of the main active components [21,22]. Ginsenosides can be hydrolyzed to generate secondary sapogenins. Among them, 20(R)panaxotriol ( Fig. 1) is an important sapogenin of ginsenosides [23]. Several studies have shown that the potential antitumor effect of panaxotriol on DU-15 cells is adjusted by sub-G1 cell cycle arrest, cell migration repression, and mitochondrial-mediated apoptosis [24]. In addition, Li et al. evaluated the inhibitory effects of panaxotriol derivatives on K562/ADR, Du-145, HeLa, MCF-7, and HepG2 cell lines. It has been proven that panaxotriol can selectively inhibit the human leukemia progenitor K562/ADR by arresting the cell cycle [25].
Based on the principle of drug combination and the above findings, we designed and synthesized four new 20(R)-panaxotriol derivatives by attaching these pharmacophores to the C-3 or C-6 position of panaxotriol. The anti-proliferative activity of the target compound against human hepatoma cancer (Hep3B) was evaluated. In addition, we selected the derivative A11 ( Fig. 1) with the strongest anti-proliferative activity, studied its possible mechanism of action of HIF-1, and particularly focused on the activity of this panaxotriol derivative on cell apoptosis and cell migration. Finally, we conducted in vivo experiments to verify its antitumor properties.

Procedure for the preparation of compounds A13-A15
The mixture of compounds A10-A12 (50 mg) and 200e300 mesh silicone (150 mg) was stirred in 2 mL of toluene at 120 C for 6 h. The crude compounds were obtained by evaporating the solvent after TLC, confirming the completion of the reaction. The desired compounds were then purified by silica gel column chromatography (dichloromethane/methanol, 100:1-30:1 v/v).

Analysis of 20(R)-panaxatriol derivatives
Silica gel plate GF254 was used for TLC analysis to detect the reaction. The instrument used for the 1 H-NMR and 13 C-NMR spectroscopic analyses of all compounds was: AV-300 (Bruker BioSpin, Switzerland), with CDCl 3 as the solvent and trimethylsilane as the interior label. The coupling constant is expressed in Hz, and the chemical shift is indicated in ppm. The high-resolution mass spectra (HR-MS) were analyzed using electrospray ionization (ESI) on a Thermo Scientific LTQ Orbitrap XL spectrometer.

Cytotoxic (MTT assay)
Hep3B cells were cultured in a 96-well plate at a density of 5 Â 10 4 /well, in 200 mL medium, and cultured for 12 h to allow them to adhere. Then, 20(R)-panaxotriol and its derivatives were processed according to a concentration gradient (final concentrations of 0.3, 1, 3, 10, and 30 mM), each concentration was repeated three times, and the cells were cultured for 24 h. The incubation was continued for 4 h after the addition of the thiazole blue reagent in the dark. After removing the thiazole blue reagent, 150 mL of dimethyl sulfoxide was added for coloring and dissolved by shaking for 10 min. The absorbance (optical density, OD) was measured at a wavelength of 492 nm on a microplate reader, and the data were analyzed.

Luciferase reporter assay
When the Hep3B cells reached 60%e80% confluence, they were used for plasmid transfection. The medium was changed, Lipofectamine 2000 was used to transfect the cells with pGL-HRE-Luc plasmid, and the cells were cultured for 24 h. After digesting the cells with trypsin, they were seeded into a 96-well plate (5 Â 10 4 / well, 200 mL) and incubated for 24 h to allow adherence. The cells were treated with a concentration gradient of 20(R)-panaxotriol and its derivatives (final concentrations of 0.3, 1, 3, 10, and 30 mM).
Three replicate wells were prepared for each concentration, and cells were cultured for 12e16 h. The medium was aspirated, 80 mL of cell lysate was added to each well (5 Â cell lysate was diluted with 3:1 distilled water), and the plate was shaken on a shaker for 1 h to fully lyse the cells. Then, 30 mL of lysed cells per well was added to the white plate. The OD was measured using Luminoskan, and the data were analyzed.

Molecular docking
A molecular docking study was performed to investigate the binding mode of the compound A11 to HIF-1a protein using the Discovery Studio 2017/CDOCKER protocol (Accelrys, San Diego, USA), according to a previously described method [46]. HIF-1a structure was obtained from the Protein Data Bank (https://www. rcsb.org, PDB ID: 4ZPR). Compound A11 was treated with the ligand preparation and minimization models in Discovery Studio 2017 to investigate the binding patterns of compound A11 and HIF-1a LBD.

Western blotting
Cell extracts were analyzed using western blotting [47,48]. HeLa cells were incubated with 3, 10, and 30 mM of compound A11 for 12 h. Cells were lyse in an ice-cold lysis buffer to obtain the nuclear extract protein. A bicinchoninic acid protein quantitative detection kit was used to quantify the protein at 570 nm. Sodium dodecyl sulfate-polyacrylamide gel was used to separate 30 mg of nuclear extract protein per lane, and then the protein bands were transferred to polyvinylidene fluoride membranes (Hybond-P). Membranes were blocked with 5% nonfat milk and then incubated with the corresponding antibodies at 4 C overnight, followed by incubation with secondary antibodies for 2 h, and detection with a highefficiency chemiluminescence kit.

Immunofluorescence staining
HeLa cells were cultured for 24 h on a 24-well plate (1 Â 10 4 / well) to facilitate adherence, and treated with the derivative A11 (final concentration 30 mM) for 12h. The cells were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde at room temperature for half an hour, and then permeabilized in 0.2% Triton X-100. They were blocked in 5% bovine serum albumin for half an hour, incubated with the HIF-1a antibody at 4 C overnight, followed by incubation with a secondary antibody at room temperature for half an hour. They were finally stained with 4 0 , 6diamidino-2-phenylindole for half an hour, and observed under a fluorescence microscope. Blue color represent the nucleus and green represents the HIF-1a protein. The green and blue-colored images were merged using NIS-Elements software.

Colony formation assay
HeLa cells were incubated for 12 h in a 6-well plate (1 Â 10 3 / well). After changing to a new medium, the cells were treated with the derivative A11 (final concentrations of 3, 10, and 30 mM) and incubated for 10 days until the colonies were clearly visible. Next, the cells were rinsed in 1 mL of PBS, fixed in 700 mL of 10% formaldehyde, stained in 700 mL of 1% crystal violet, washed with 1 mL of PBS three times, and naturally air-dried.

5-ethynyl-2 0 -deoxyuridine (EdU) labeling and immunofluorescence
HeLa cells were incubated for 24 h in a 96-well plate (5 Â 10 4 / well). After treatment with the derivative A11 (final concentrations of 3, 10, and 30 mM), the cells were incubated for 12 h, followed by incubation with EdU (RiboBio, Guangzhou, China) for 1 h. The cells were then fixed with 4% paraformaldehyde for 30 min, treated with 0.5% Triton X-100 for 10 min, and washed in PBS. They were then incubated in a 1 Â Apollo® reaction cocktail for half an hour and stained with Hoechst 33,342. Finally, the stained cells were visualized using an Olympus IX83 inverted fluorescence microscope.

Apoptosis assay
Apoptosis was analyzed using flow cytometry, as previously described [49]. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). Briefly, the HeLa cells were cultured for 24 h in a 6-well plate (5 Â 10 5 /well) and incubated for 24 h after treatment with derivative A11 (final concentrations of 3, 10, and 30 mM). Then, the cells were collected, washed twice with PBS, and mixed with the binding buffer (10 mM 4-(2hydroxyethyl)-1-piperazine€ ethanesulfonic acid (HEPES), pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 ). The cells were then stained with Annexin V-FITC and 2 mg/mL propidium iodide in a dark environment at 37 C for 15 min. Analysis was performed by flow cytometry after the addition of 400 mL binding buffer to the samples. Cell Quest software (Becton-Dickinson, Franklin Lakes, NJ, USA) was used to analyze the data.

Cell migration assay
Scratch experiments were performed to detect the migration of HeLa cells. The cells were inoculated in a 24-well plate (1 Â 10 5 / well) and incubated for 24 h tofacilitate adherence. A sterile pipette tip was used to make vertical scratches in each hole, and the plates were washed thrice with PBS to remove the scratched cells. The medium was then replaced with a serum-free medium. After treatment with the derivative A11 (final concentrations of 3, 10, and 30 mM), the cells were incubated for 12 h. Images were taken with a fluorescence microscope to observe the degree of migration of the cells to the scratched area.

Transwell assay
The invasion assay was performed using a Transwell chamber (Costar, MA, USA). The Transwell membrane was coated with Matrigel (BD Biocoat, Bedford, MA, USA) for the invasion assay and the cells pretreated with or without A11 (3, 10, and 30 mM) in serum-free DMEM were added to the upper compartment of the chamber. The lower compartment contained DMEM supplemented with 10% FBS. After 24 h of invasion at 37 C, the cells migrated to the bottom chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Then, the cells were photographed under a microscope.

In vivo antitumor activity
Male athymic Nu/Nu nude mice (4e5 weeks of age, 24 ± 2 g; Vital River Laboratory Animal, Beijing, China) were reared under septic conditions as per the Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).
Normal saline (200 mL) was injected subcutaneously into the mice with HeLa cells. Two weeks later, the mice were randomly divided into five groups (n ¼ 4), 50 mg/kg A11, 30 mg/kg A11, 30 mg/kg 20(R)-panaxotriol, 30 mg/kg 5-fluorouracil, and a negative control group. An intraperitoneal injection was administered every two days, and the body weights and tumor volumes of mice were simultaneously measured for 20 days. On the 21st day, the mice were sacrificed, and the tumors were excised and weighed. The following standard formula was used to calculate the tumor volume (mm 3 ): (length Â (width) 2 )/2. Ratio of inhibition of tumor (%) ¼ (1average tumor weight of treated group/average tumor weight of control group) Â 100%.

Statistical analysis
All data are presented as the mean ± standard deviation (SD), and all experiments were independently performed in triplicate. A comparison of the results was performed using one-way analysis of variance and Tukey's multiple comparison tests (GraphPad Software, Inc. San Diego, CA, USA). Statistically significant differences between the groups were defined as p < 0.05.

In vitro HIF-1a inhibitory activity and SAR study
MTT assay determined the cytotoxicity of 20(R)-panaxotriol and its derivatives in Hep3B cells. The results in Figure 2A show that, except for A13 (IC 50 ¼ 6.99 mM), all other compounds exhibited no cytotoxicity (IC 50 > 30 mM). To detect the effect of 20(R)-panaxotriol and its derivatives on the transcriptional activity of HIF-1, a luciferase reporter gene experiment was performed on human liver cancer cells Hep3B ( Fig. 2A), with 20(R)-panaxotriol as a positive control. The great majority of the derivatives had inhibitory effects on HIF-1a and were stronger than the positive control 20(R)-panaxotriol. Among them, the compound A11 with t-butyloxycarbonylglycine was the most effective derivative, and its IC 50 for inhibiting HIF-1a transcription activity in the Hep3B cell line was <0.3 mM. Its efficacy was over 100 times higher than that of 20(R)-panaxotriol. The derivatives C1-C12 inhibited transcription of HIF-1a weaker than those of derivatives A1-A12 and B1-B12, especially aminotriazoles and cinnamic acids (derivatives C4-C9: IC 50 > 30 mM), indicating that the double-modified derivatives at the C-3 and C-6 positions of 20(R)-panaxotriol have weaker activity than the C-3 or C-6 single-modified derivatives. Among 20(R)-panaxotriol isocyanate derivatives, the C-3 monosubstituted derivatives had the best activity with a benzene ring A2 (IC 50 ¼ 1.58 mM), and the C-6 monosubstituted derivatives with cycloalkyl B3 had the best activity (IC 50 ¼ 1.08 mM), the activity of the cycloalkyl group in the C-3 and C-6 disubstituted derivatives C3 was the highest (IC 50 ¼ 14.85 mM). This shows that the aromatic ring and cycloalkyl are equally important in 20(R)-panaxotriol isocyanate derivatives. For the 20(R)-panaxotriol derivatives with phenyl-1,2,3 triazole mono-substituted at the C-3 position, the electron donating group showed better effects than the electron withdrawing group (p-OCH 3 > p-H > p-Cl) (IC 50 are 0.33 mM, 0.43 mM, and >30 mM, respectively). Unfortunately, no clear structure-activity relationship has been found in cinnamic acid-substituted 20(R)-panaxotriol derivatives. It can be seen from the derivatives A10-A15 that the influence of the protection of the tert-butoxycarbonyl group in amino acids on the inhibition of HIF-1a transcription activity is not obvious (Fig. 2B). These findings indicate that 20(R)-panaxotriol derivatives effectively reduced the level of HIF-1a without significant cytotoxicity in Hep3B cells.
Further activity analysis was conducted on derivative A11 with the best HIF-1a inhibitory activity. The results in Fig. 2C and Fig. 2D show that, compared with 20% oxygen situation, under 1% oxygen situation, the transcription activity of HIF-1a was significantly increased (p < 0.001). After adding 0.3 mM of derivative A11, the transcription activity of HIF-1a was reduced. As the concentration of the derivative A11 drug increased, the transcriptional activation of HIF-1a decreased. The results indicated that derivative A11 dosedependent inhibited the transcriptional activity of HIF-1a (Fig. 2C).
Derivative A11 at concentrations as high as 30 mM had no adverse effects on cell viability (Fig. 2D).

Compound A11 inhibits HIF-1a protein expression in HeLa cells
To explore the interaction between compound A11 and HIF-1a, we investigated its binding mode through a molecular docking study. As shown in Fig. 3A, the central structure of compound A11 was considered to form conventional hydrogen bond interactions with surrounding residues (Cys351, Gln359, and His286), and the tbutoxycarbonyl part of compound A11 was considered to form a mixed pi/alkyl hydrophobic interaction with the surrounding residues (Arg156, Pro154 and Met354). Moreover, the nitrogen atom of the amino acids of compound A11 formed a hydrogen bond with Ser355. We also found that the compound A11 showed good binding interactions with the related protein (4ZPR) in the binding pocket. Western blotting determined the effect of compound A11 on the expression of HIF-1a protein in HeLa cells. As shown in Fig. 3B, under normoxic conditions, HIF-1a was not expressed. Under 1% O 2 conditions, HIF-1a was shown to accumulate in large amounts. The addition of different concentrations of A11 dosedependently reduced the HIF-1a protein content in HeLa cells. However, A11 had almost no influence on the expression level of HIF-1b. Next, we performed an immunofluorescence experiment to determine whether compound A11 inhibited the increase of HIF-1a in the nucleus of HeLa cells. The results in Fig. 3C show that, in the nucleus, the HIF-1a protein was not expressed under normoxic conditions. Under 1% O 2 conditions, in the nucleus, the HIF-1a protein was largely amassed; however, after adding 30 mM of compound A11, HIF-1a expression in the nucleus was not detected. These results indicate that compound A11 inhibited nuclear accumulation of HIF-1a.
HIF-1a is known to regulate the expression of VEGF, a crucial growth factor involved in tumor cell proliferation, angiogenesis, invasion, and metastasis [9,54]. Therefore, in this study, we tested the effect of A11 on VEGF protein expression by western blotting, We found that hypoxic conditions significantly increased the expression levels of VEGF, while treatment with A11 resulted in a dose-dependent decrease in its levels (Fig. 3D), and the effective concentrations were comparable to those inhibiting HIF-1a protein expression.

Compound A11 inhibits HeLa cell proliferation and promotes apoptosis
Malignant tumor cells can grow without restriction. Colony forming test can determine the size of cell colony formation [55]; thus, the colony formation test can evaluate the inhibitory ability of A11 on cell multiplication more directly. The results in Fig. 4A show that, compound A11 apparently inhibited the colony formation of cells, and the inhibitory effect became more obvious when the concentration was increased. The EdU experiment yielded similar results. As shown in Fig. 4B, compared with the control group, compound A11 concentration-dependently suppressed the number of EdU-positive cells. In addition, we wanted to confirm whether compound A11 could induce cell apoptosis in addition to inhibiting cell multiplication. Therefore, Annexin V-FITC/PI double staining was performed. As shown in Fig. 4C, compared with the control group, incubation with compound A11 (3, 10, and 30 mM) resulted in a concentration-dependent increase in the percentage of total apoptotic (early and late) cells (5.2%, 14.0%, and 24.2%, respectively). These results indicated that compound A11 could effectively induce cell apoptosis. To further understand the anti-apoptotic effects, western blotting analysis was used to determine the expression levels of cleaved-caspase 3, Bcl-2, and Bax proteins in vivo. As shown in Fig. 4D, the expression levels of Bax were increased in the compound A11-treated HeLa cells compared to those in the control group, whereas the levels of Bcl-2 were decreased after treatment with compound A11. Therefore, the ratio of Bax/Bcl-2 increased in a dose-dependent manner. In addition, it was observed that with the treatment of compound A11 (0, 3, 10, and 30 mM), the expression levels of cleaved caspase-3 in HeLa cells significantly increased in a concentration-dependent manner. Taken together, these data further confirmed that compound A11 could induce apoptosis in HeLa cells.

Compound A11 inhibits HeLa cell migration and invasion
Because HIF-1a is a key regulator of tumor invasion [56,57], we performed cell scratch assay and transwell assay to study the effect of compound A11 on HeLa cell motility. The results showed that the migration of HeLa cells in the cells treated with compound A11 decreased compared with the control group, and the degree of reduction in the migration of HeLa cells increased as the concentration of compound A11 increased. Treatment with compound A11 significantly reduced the number of tumor cells that crossed the transwell chamber compared to the control. Moreover, the invasion and migration of HeLa cells decreased to a greater extent at an increased dose of A11, suggesting that A11 inhibited the invasion and migration of HeLa cells in a concentration-dependent manner ( Fig. 5A and Fig. 5B). Next, the effect of A11 on the expression of the invasion and migration-associated protein, MMP-9, was investigated. Western blotting experiments showed that treatment with A11 (10 mM) significantly reduced the expression levels of MMP-9 ( Fig. 5C), further confirming that A11 plays an inhibitory role in the migration and invasion of HeLa cells.

In vivo antitumor activity of compound A11
Antitumor activity of compound A11 was remarkable in vitro, prompting us to verify its antitumor effect in vivo. The measured nude mouse body weight and nude mouse tumor volume results are shown in Fig. 6A and 6B. In the control group, the average tumor volume was obviously larger than that in the 20(R)-panaxotriol, A11, and 5-fluorouracil treatment groups. A11 significantly inhibit tumor volume (p < 0.001). But, the mice in the control group and other groups had very little difference in body weight, and this difference was not statistically significant. This indicates that the treatment could not produce toxicity in vivo. After administration of the treatment to nude mice, the nude mice were died by spinal dislocation, and the solid tumor was removed and photographed. The experimental results are shown in Fig. 6C. It can be seen more clearly from the figure that compared with the control group, the A11 group was associated with inhibition of cancer cell growth. The tumor growth inhibition rates in mice treated with 30 mg/kg 20(R)panaxotriol, 30 mg/kg A11, 50 mg/kg A11, and 30 mg/kg 5fluorouracil were 40.15%, 70.33%, 76.21%, and 61.89%, respectively (Fig. 6D). According to the above pharmacological data, in vivo, the antitumor ability of derivative A11 was remarkably stronger than that of 20(R)-panaxotriol (p < 0.01). In addition, the antitumor activity of A11 increased as its concentration increased. Whole-cell lysates for HIF-1b and nuclear extract for HIF-1a were analyzed using western blotting. Data are respresented as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, significant with respect to the hypoxia control. (C) HeLa cells were incubated with or without A11 (30 mM) under normoxia or hypoxia for 12 h and then analyzed for the intracellular distribution of HIF-1a by immunofluorescence assay (magnification, 400Â). (D) HeLa cells were incubated under normoxia or hypoxia for 12 h in the absence or presence of indicated concentrations of A11. VEGF were analyzed using western blotting. Data are respresented as the mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, significant with respect to the hypoxia control.

Conclusion
For the study, 39 new C-3 and C-6 derivatives of 20(R)-panaxotriol were designed and synthesized, and their HIF-1a transcription inhibitory activities were studied. Most of the derivatives showed strong HIF-1a transcription inhibitory activity. Among them, the derivative A11 with t-butyloxycarbonyl-glycine showed the strongest HIF-1a transcription inhibitory activity, and its IC 50 for inhibiting HIF-1a transcription activity in the Hep3B cell line was <0.3 mM. Its efficacy was shown to be over 100 times higher Bcl-2 and Cleaved Caspase-3. Data represented as mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different when compared with control group. than that of 20(R)-panaxotriol. The results of molecular docking highlight that glycine with t-butyloxycarbonyl on C-3 of 20(R)panaxotriol may be important for good HIF-1a inhibition. In addition, derivative A11 dose-dependently inhibited the transcriptional ability of HIF-1a, dose-dependently inhibited HIF-1a protein content in HeLa tumor cells, and inhibited the nuclear accumulation of HIF-1a. A11 was also observed to dose-dependently inhibit HeLa cell proliferation and promote apoptosis, as well as inhibit migration of HeLa cells. Furthermore, in the HeLa cervical cancer xenograft model, compound A11 significantly inhibited tumor volume by 76.21% (50 mg/kg) and 70.33% (30 mg/kg) (ip). It was better than 20(R)-panaxotriol (40.15%) and 5-fluorouracil (61.89%). In short, derivative A11 shows excellent antitumor activity in vivo and in vitro, and may be used as an effective antitumor agent in the future after more in-depth research. So far, only a few types of 20(R)-panaxotriol derivatives with amino acids have been synthesized, and there may be certain compounds that have better activity than A11, Therefore, more follow-up studies are needed to explore and analyze such compounds in the future. represented as mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different when compared with control group.

Declaration of competing interest
The authors have no conflicts of interest to report.