Design and Synthesis of Novel Betulin Derivatives Containing Thio-/Semicarbazone Moieties as Apoptotic Inducers through Mitochindria-Related Pathways

Two new series of betulin derivatives with semicarbazone (7a–g) or thiosemicarbazone (8a–g) groups at the C-28 position were synthesized. All compounds were evaluated for their in vitro cytotoxicities in human hepatocellular carcinoma cells (HepG2), human breast carcinoma cells (MCF-7), human lung carcinoma cells (A549), human colorectal cells (HCT-116) and normal human gastric epithelial cells (GES-1). Among these compounds, 8f displayed the most potent cytotoxicity with an IC50 value of 5.86 ± 0.61 μM against MCF-7 cells. Furthermore, the preliminary mechanism studies in MCF-7 cells showed that compound 8f could trigger the intracellular mitochondrial-mediated apoptosis pathway by losing MMP level, which was related with the upregulation of Bax, P53 and cytochrome c expression; the downregulation of Bcl-2 expression; activation of the expression levels of caspase-3, caspase-9, cleaved caspase-3 and cleaved caspase-9; and an increase in the amounts of intracellular reactive oxygen species. These results indicated that compound 8f may be used as a valuable skeleton structure for developing novel antitumor agents.

Thus, inspired by the good biological property of thio-/semicarbazones, in view o the potential medicinal research value of betulin and in continuation of an ongoing pro gram aiming at developing more potential anticancer drugs, in the present study, 14 bet ulin derivatives modified at the C-28 position with thio-/semicarbazide groups were de signed and synthesized ( Figure 3). We hope to obtain valuable information about the im pact of thio/semicarbazone moiety at the C-28 position on cytotoxic activity and their un derlying mechanisms of antitumor effect Thiosemicarbazones and semicarbazones are classes of Schiff bases, which are highly regarded by medicinal chemists due to their extensive pharmacological properties or bioactivities, such as antifungal [30], anti-chlamydial [31], antibacterial [32][33][34] and antimalarial activities [35][36][37][38]. Notably, thio-/semicarbazone compounds have a wide antitumor spectrum against various tumor types, such as breast cancer [39][40][41], non-small-cell lung cancer [42], pancreatic cancer [43], leukemia [44], bladder cancer [45] and prostate cancer [46]. Recently, several representative compounds containing thio-/semicarbazone groups in the clinical trial have been reported, such as DcP [47], 3-AP [48] and COTI-2 [49]. 3-AP was initially designed as a potent ribonucleotide reductase (RR) inhibitor for cancer treatment and has since entered Phase II clinical trials [50]. Representative compounds containing thiosemicarbazone groups are shown in Figure 2. Thiosemicarbazones and semicarbazones are classes of Schiff bases, which are highly regarded by medicinal chemists due to their extensive pharmacological properties or bioactivities, such as antifungal [30], anti-chlamydial [31], antibacterial [32][33][34] and antimalarial activities [35][36][37][38]. Notably, thio-/semicarbazone compounds have a wide antitumor spectrum against various tumor types, such as breast cancer [39][40][41], non-small-cell lung cancer [42], pancreatic cancer [43], leukemia [44], bladder cancer [45] and prostate cancer [46]. Recently, several representative compounds containing thio-/semicarbazone groups in the clinical trial have been reported, such as DcP [47], 3-AP [48] and COTI-2 [49]. 3-AP was initially designed as a potent ribonucleotide reductase (RR) inhibitor for cancer treatment and has since entered Phase II clinical trials [50]. Representative compounds containing thiosemicarbazone groups are shown in Figure 2. On the one hand, our previous work mainly focused on the structural modifications of natural compounds [51][52][53]. We successfully synthesized a series of endoperoxide steroidal derivatives containing semicarbazone or thiosemicarbazone groups and obtained several new structures with significant antitumor activity [54]. On the other hand, there are three independent active positions in betulin, namely, the isopropenyl side chain at C-19 and two hydroxyl groups at C-3 and C-28. It is quite feasible to make a chemical modification of different sites to obtain novel betulin derivatives with desired biological properties. According to the structure-activity relationship of betulin, introducing a hydrogen donor group at the C-28 position may improve cytotoxic activity significantly.
Thus, inspired by the good biological property of thio-/semicarbazones, in view of the potential medicinal research value of betulin and in continuation of an ongoing program aiming at developing more potential anticancer drugs, in the present study, 14 betulin derivatives modified at the C-28 position with thio-/semicarbazide groups were designed and synthesized ( Figure 3). We hope to obtain valuable information about the impact of thio/semicarbazone moiety at the C-28 position on cytotoxic activity and their underlying mechanisms of antitumor effect On the one hand, our previous work mainly focused on the structural modifications of natural compounds [51][52][53]. We successfully synthesized a series of endoperoxide steroidal derivatives containing semicarbazone or thiosemicarbazone groups and obtained several new structures with significant antitumor activity [54]. On the other hand, there are three independent active positions in betulin, namely, the isopropenyl side chain at C-19 and two hydroxyl groups at C-3 and C-28. It is quite feasible to make a chemical modification of different sites to obtain novel betulin derivatives with desired biological properties. According to the structure-activity relationship of betulin, introducing a hydrogen donor group at the C-28 position may improve cytotoxic activity significantly.
Thus, inspired by the good biological property of thio-/semicarbazones, in view of the potential medicinal research value of betulin and in continuation of an ongoing program aiming at developing more potential anticancer drugs, in the present study, 14 betulin derivatives modified at the C-28 position with thio-/semicarbazide groups were designed and synthesized ( Figure 3). We hope to obtain valuable information about the impact of thio/semicarbazone moiety at the C-28 position on cytotoxic activity and their underlying mechanisms of antitumor effect.

Synthesis of Botulin Derivatives
The general synthesis route of betulin derivatives is shown in Scheme 1. Two hidroxyls of betulin (1) were acetylated with acetic anhydride in dry pyridine to synthesis compound 2. Compound 2 further reacted with Ti(i-PrOH) 4 in dry isopropyl alcohol for selective deacetylation at C-28 to give compound 3. Then, the 28-OH of betulin was oxidized to a carbonyl group in the presence of pyridinium chlorochromate in dry dichloromethane to give compound 4. Subsequently, compound 4 reacted with sodium hydroxide for deacetylation at C-3 to give compound 5. Compound 5 was further reacted with hydrazine hydrate in ethanol to get hydrazine 6. At last, hydrazine 6 reacted with phenyl isothiocyanate or isocyanate substituents in the presence of acetic acid in ethanol to obtain target novel betulin-(thio-)semicarbazone derivatives 7a-g and 8a-g. The chemical structures of all new synthesized compounds were characterized by HRMS and NMR spectrum methods (Supplementary Materials).

Betulin Derivatives Inhibited Proliferation of Various Human Cancer Cells
The in vitro cytotoxicities of all betulin derivatives 7a-g and 8a-g were evaluated using MTT assays against human hepatocellular carcinoma cells (HepG2), human breast carcinoma cells (MCF-7), human lung carcinoma cells (A549), human colorectal cells (HCT-116) and normal gastric cells (GES-1). Mitomycin C was tested as a positive drug control. The cytotoxicities of all compounds are summarized as IC 50 values in Table 1. The results showed that most of compounds have remarkable cytotoxicities toward all four tested human tumor cell lines and were more potent than betulin parent.  50 : Concentration of the tested compound that inhibits 50% of cell growth. All data are recorded as means ± SD of three experiments. b X means oxygen atom (O) when the substituent is semicarbazone; X means sulphur atom (S) when the substituent is thiosemicarbazone. "-" not active.
For the HepG2 cell line, the semicarbazone series of compounds 7a, 7b, 7d, 7e and 7g (X = O) displayed lower cytotoxic activities than betulin (IC 50 = 20.42 µM). The substitutions (methyl, methoxy) on the semicarbazone moiety led to compounds 7c and 7f without improvement in cytotocxicity. Compound 7e possessing 3-trifluoromethyl group displayed significant cytotoxic activity with IC 50 value of 8.93 µM. It is about 2.28-fold higher than botulin. The thiosemicarbazone series of compounds 8a, 8b, 8f and 8g also displayed lower cytotoxic activities than betulin. For compound 8f possessing 3-chloro group (IC 50 = 6.87 µM), the cytotoxicity was 2.97-fold higher than betulin. Furthermore, compounds 7e and 8f were less toxic to GES-1 cells with IC 50 values of 196.18 and 214.60 µM, respectively. The data showed that the incorporation of a 3-chloro or 3-trifluoromethyl group with semicarbazone or thiosemicarbazone moieties at the C28 of betulin led to significant improvement in cytotoxic activity than tertiary butyl or methyl.
Taken together, the two series of betulin derivatives generally exhibited potent cytotoxicity compared with betulin against MCF-7 and HepG2 cell lines. The results suggest that both the conjugated N,N,S-and N,N,O-tridentate donor sets are essential for the cytotoxicities of the novel betulin derivatives.
Among these compounds under biological study, the thiosemicarbazone derivative 8f and semicarbazone derivative 7e were the most potent compounds against MCF-7 cell lines, with IC 50 values of 5.96 and 5.86 µM, respectively). One of the major indices of a potent effective anti-cancer drug lies in that it can inhibit cancer cell growth and subsequently induce apoptosis. Here, compound 8f was chosen for subsequent biological functions experiments in MCF-7 cells.

Betulin Derivatives Induced Apoptosis in Various Human Cancer Cells
Firstly, the Acridine Orange/Ethidium Bromide (AO/EB) staining of MCF-7 cells treated with compound 8f was observed under a fluorescence microscope. A large number of normal cells in the control group were stained green and their nuclei were intact. As the concentration of compound 8f increased (0, 3, 6 and 12 µM), some cells showed apoptotic characteristics such as chromosome pyknosis, fragmentation and sparse cytoplasm, and the number of cells gradually increased. Furthermore, the number of early apoptotic cells and late apoptotic cells also increased, the latter was characterized by the nucleus with EB staining, orange red, concentration or bias. The necrotic cells showed uneven orange-red fluorescence and were not clearly defined and disintegrated or nearly disintegrated. The results were shown in Figure 4. thiosemicarbazone moiety at the C28 of betulin was beneficial for compounds to displayed remarkable cytotoxicity against MCF-7 cells. Taken together, the two series of betulin derivatives generally exhibited potent cytotoxicity compared with betulin against MCF-7 and HepG2 cell lines. The results suggest that both the conjugated N,N,S-and N,N,O-tridentate donor sets are essential for the cytotoxicities of the novel betulin derivatives.
Among these compounds under biological study, the thiosemicarbazone derivative 8f and semicarbazone derivative 7e were the most potent compounds against MCF-7 cell lines, with IC50 values of 5.96 and 5.86 μM, respectively). One of the major indices of a potent effective anti-cancer drug lies in that it can inhibit cancer cell growth and subsequently induce apoptosis. Here, compound 8f was chosen for subsequent biological functions experiments in MCF-7 cells.

Betulin Derivatives Induced Apoptosis in Various Human Cancer Cells
Firstly, the Acridine Orange/Ethidium Bromide (AO/EB) staining of MCF-7 cells treated with compound 8f was observed under a fluorescence microscope. A large number of normal cells in the control group were stained green and their nuclei were intact. As the concentration of compound 8f increased (0, 3, 6 and 12 μM), some cells showed apoptotic characteristics such as chromosome pyknosis, fragmentation and sparse cytoplasm, and the number of cells gradually increased. Furthermore, the number of early apoptotic cells and late apoptotic cells also increased, the latter was characterized by the nucleus with EB staining, orange red, concentration or bias. The necrotic cells showed uneven orange-red fluorescence and were not clearly defined and disintegrated or nearly disintegrated. The results were shown in Figure 4. In order to confirm whether tumor cell apoptosis was induced by compound 8f in tumor cells, the MCF-7 cells were stained in sequence with Annexin V-FITC (AV) and propidium iodide (PI). MCF-7 cells deal with compound 8f by gradient concentration (0, 3, 6 and 12 μM) for 24 h, and the rates of apoptotic cells were detected by flow cytometry. As shown in Figure 5, after treatment with 3, 6 or 12 μM of 8f for 24 h, the percentage of apoptosis cells was increased from 13.20% to 48.66%, while the control group's percentage In order to confirm whether tumor cell apoptosis was induced by compound 8f in tumor cells, the MCF-7 cells were stained in sequence with Annexin V-FITC (AV) and propidium iodide (PI). MCF-7 cells deal with compound 8f by gradient concentration (0, 3, 6 and 12 µM) for 24 h, and the rates of apoptotic cells were detected by flow cytometry. As shown in Figure 5, after treatment with 3, 6 or 12 µM of 8f for 24 h, the percentage of apoptosis cells was increased from 13.20% to 48.66%, while the control group's percentage of apoptosis cells was only 10.54%. Notably, the apoptosis of the MCF-7 cells treatment with compound 8f increased in a dose-dependent manner. The above results suggested that compound 8f could induce apoptosis in MCF-7 cells significantly. of apoptosis cells was only 10.54%. Notably, the apoptosis of the MCF-7 cells treatment with compound 8f increased in a dose-dependent manner. The above results suggested that compound 8f could induce apoptosis in MCF-7 cells significantly.

Compound 8f Induced MMP Loss in MCF-7 Cells
Mitochondria dysfunction has been proven to be an important role in inducing apoptosis in tumor cells. The loss of mitochondrial membrane potential (MMP) has been considered to be an early manifestation of mitochondrial dysfunction in apoptotic process. Thus, we tried to explore the contribution of mitochondria in compound-8f-induced apoptosis in MCF-7 cells. JC-1 cationic dye is an ideal MMP-sensitive probe, which can measure the MMP lost by flow cytometry. As shown in Figure 6, after exposed to compound 8f from 3.0 to 12.0 μM, the fluorescence intensity decreased from 79.00% to 47.06%, respectively, compared to control group. Notably, the loss of MMP in the MCF-7 cell treatment with compound 8f occurred in a dose-dependent manner. The results clearly proved that compound 8f caused MMP to collapse significantly and induced cell apoptosis in MCF-7 cells through the intrinsic mitochondrial-mediated pathways.

Compound 8f Induced MMP Loss in MCF-7 Cells
Mitochondria dysfunction has been proven to be an important role in inducing apoptosis in tumor cells. The loss of mitochondrial membrane potential (MMP) has been considered to be an early manifestation of mitochondrial dysfunction in apoptotic process. Thus, we tried to explore the contribution of mitochondria in compound-8f-induced apoptosis in MCF-7 cells. JC-1 cationic dye is an ideal MMP-sensitive probe, which can measure the MMP lost by flow cytometry. As shown in Figure 6, after exposed to compound 8f from 3.0 to 12.0 µM, the fluorescence intensity decreased from 79.00% to 47.06%, respectively, compared to control group. Notably, the loss of MMP in the MCF-7 cell treatment with compound 8f occurred in a dose-dependent manner. The results clearly proved that compound 8f caused MMP to collapse significantly and induced cell apoptosis in MCF-7 cells through the intrinsic mitochondrial-mediated pathways.

Compound 8f Induced MMP Loss in MCF-7 Cells
Mitochondria dysfunction has been proven to be an important role in inducing ap tosis in tumor cells. The loss of mitochondrial membrane potential (MMP) has been c sidered to be an early manifestation of mitochondrial dysfunction in apoptotic proce Thus, we tried to explore the contribution of mitochondria in compound-8f-indu apoptosis in MCF-7 cells. JC-1 cationic dye is an ideal MMP-sensitive probe, which measure the MMP lost by flow cytometry. As shown in Figure 6, after exposed to co pound 8f from 3.0 to 12.0 μM, the fluorescence intensity decreased from 79.00% to 47.06 respectively, compared to control group. Notably, the loss of MMP in the MCF-7 cell tre ment with compound 8f occurred in a dose-dependent manner. The results clearly prov that compound 8f caused MMP to collapse significantly and induced cell apoptosis MCF-7 cells through the intrinsic mitochondrial-mediated pathways.  (B) Data were revealed as the means ± SD for independent tests in triplicate. * p < 0.05, ** p < 0.01 vs. control (8f 0 µM) group.

Compound 8f Triggered ROS Generation
In the past years, many studies reported that intracellular amounts of reactive oxygen species (ROS) play an important role in some kinds of biological processes in tumor cells. In addition, it has been demonstrated that the death-inducing capacity of many chemotherapeutic drugs could be associated with the generation of ROS. Therefore, we explored if ROS stimulated by compound 8f induced apoptosis in MCF-7 cells. Cells were treated with compound 8f by gradient concentration (0, 3, 6 and 12 µM) for 24 h and then using H 2 DCFDA staining analysis by flow cytometry. As shown in Figure 7, after exposure to 12 µM of compound 8f for 24 h, the generation of ROS level was increased to 75.22% compared to the control group (2.61%). Notably, the ROS level of MCF-7 cells treated with compound 8f increased in a dose-dependent manner. The results demonstrated that compound 8f significantly induced ROS generation in MCF-7 cells, which could in turn lead to MCF-7 cell apoptosis.

Compound 8f Triggered ROS Generation
In the past years, many studies reported that intracellular amounts of reactive oxygen species (ROS) play an important role in some kinds of biological processes in tumor cells. In addition, it has been demonstrated that the death-inducing capacity of many chemotherapeutic drugs could be associated with the generation of ROS. Therefore, we explored if ROS stimulated by compound 8f induced apoptosis in MCF-7 cells. Cells were treated with compound 8f by gradient concentration (0, 3, 6 and 12 μM) for 24 h and then using H2DCFDA staining analysis by flow cytometry. As shown in Figure 7, after exposure to 12 μM of compound 8f for 24 h, the generation of ROS level was increased to 75.22% compared to the control group (2.61%). Notably, the ROS level of MCF-7 cells treated with compound 8f increased in a dose-dependent manner. The results demonstrated that compound 8f significantly induced ROS generation in MCF-7 cells, which could in turn lead to MCF-7 cell apoptosis. Data were revealed as the means ± SD for independent tests in triplicate. * p < 0.05, ** p < 0.01 vs. control (8f 0 μM) group.

Compound 8f Regulated Apoptosis-Related Protein Expression
To further explore the molecular mechanism of compound-8f-induced apoptosis in MCF-7 cells, the expression levels of the related apoptotic proteins Bcl-2, Bax, P53, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and cytochrome c under treatment of compound 8f were carried out. MCF-7 cells were co-cultured with compound 8f at the gradient concentrations of 3, 6 and 12 μM for 24 h, and the related apoptotic proteins expression levels were tested by means of Western blotting. The GAPDH expression level was used as an internal control group. As shown in Figure 8, compound 8f could remarkably suppressed the expression levels of Bcl-2 but increased the expression levels of cytochrome c (cyt-c), P53 and Bax in a dose-dependent manner. As shown in Figure 9, the results indicated that compound 8f remarkably activated the expression levels of both caspase-3 and caspase-9 in a dose-dependent manner. Meanwhile, the cleavage forms of the caspase proteins were evaluated ( Figure 10). Compound 8f could also activated the expression levels of both cleaved caspase-3 and cleaved caspase-9 in a dose-dependent manner. The above results have further demonstrated that compound 8f could regulated apoptosis-related protein expression in MCF-7 cells.

Compound 8f Regulated Apoptosis-Related Protein Expression
To further explore the molecular mechanism of compound-8f-induced apoptosis in MCF-7 cells, the expression levels of the related apoptotic proteins Bcl-2, Bax, P53, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and cytochrome c under treatment of compound 8f were carried out. MCF-7 cells were co-cultured with compound 8f at the gradient concentrations of 3, 6 and 12 µM for 24 h, and the related apoptotic proteins expression levels were tested by means of Western blotting. The GAPDH expression level was used as an internal control group. As shown in Figure 8, compound 8f could remarkably suppressed the expression levels of Bcl-2 but increased the expression levels of cytochrome c (cyt-c), P53 and Bax in a dose-dependent manner. As shown in Figure 9, the results indicated that compound 8f remarkably activated the expression levels of both caspase-3 and caspase-9 in a dose-dependent manner. Meanwhile, the cleavage forms of the caspase proteins were evaluated ( Figure 10). Compound 8f could also activated the expression levels of both cleaved caspase-3 and cleaved caspase-9 in a dose-dependent manner. The above results have further demonstrated that compound 8f could regulated apoptosis-related protein expression in MCF-7 cells.
x FOR PEER REVIEW 8 of 18

Chemistry
All materials and reagents were purchased from commercial suppliers (Energy, Shanghai, China). All unit reaction progress were real-time monitored by TLC using F254 silica gel plates (Biohonor, Guangzhou, China). The intermediates and target derivatives were purified by flash column chromatography (300 mesh silica gel, Yinlong, Qingdao, China). Melting points (mp) of all new derivatives were tested using a MP120 melting point apparatus (Haineng, Fujian, China). NMR spectra were tested using BrukerAvance DRX400 spectrometers 600 MHz for 1 H NMR and 150 MHz for 13 C NMR (Bruker, Berlin, Germany). The chemical shifts were expressed in ppm using tetramethylsilane as an internal standard. Low resolution mass spectra were recorded on Esquire 6000 mass spectrometer (Bruker, Berlin, Germany). HRMS spectra were obtained using an Agilent 6250 mass spectrometer (Agilient, San Francisco, CA, USA). The values of MS were recorded in a positive ion mode with ESI source.

Chemistry
All materials and reagents were purchased from commercial suppliers (Energy, Shanghai, China). All unit reaction progress were real-time monitored by TLC using F254 silica gel plates (Biohonor, Guangzhou, China). The intermediates and target derivatives were purified by flash column chromatography (300 mesh silica gel, Yinlong, Qingdao, China). Melting points (mp) of all new derivatives were tested using a MP120 melting point apparatus (Haineng, Fujian, China). NMR spectra were tested using BrukerAvance DRX400 spectrometers 600 MHz for 1 H NMR and 150 MHz for 13 C NMR (Bruker, Berlin, Germany). The chemical shifts were expressed in ppm using tetramethylsilane as an internal standard. Low resolution mass spectra were recorded on Esquire 6000 mass spectrometer (Bruker, Berlin, Germany). HRMS spectra were obtained using an Agilent 6250 mass spectrometer (Agilient, San Francisco, CA, USA). The values of MS were recorded in a positive ion mode with ESI source.

Synthesis of 3-O-Acetyl-betulin (3)
To a solution of 3-O,28-O-acetyl-betulin (2, 3.41 g, 6.4 mmol) in isopropyl alcohol (i-PrOH, 160 mL) was added titanium propoxide (Ti(i-PrOH) 4 , 10 mL, 35 mmol). The reaction temperature was increased to 85 • C and stirred for 5 h. The solution was evaporated and then CH 2 Cl 2 (50 mL) and water (50 mL) were added. The filtration was washed with brine twice and dried over anhydrous Na 2 SO 4 . The organic solvent was evaporated and then purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/7) to give 3 (2. To a solution of betulinicaldehyde (5, 1.55 g, 3.5 mmol) in ethanol (80 mL) was added hydrazine hydrate (85%, 2 mL). The reaction mixture was continuously stirred at 40 • C for 5 h. The solvent was evaporated, and crude product was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/1) to give 6 (1.  To a solution of 28-hydrazonomethyl-betulin (6, 1 mmol) in ethanol (20 mL) was added phenyl isocyanate or phenyl isothiocyanate substituent (2 mmol) and five drops of acetic acid. The reaction mixture was continuously stirred at 30 • C for 5~10 h until no material. The solvent was evaporated, and crude product was purified by silica gel column chromatography (dichloromethane/methanol) to obtain compounds 7a-g and 8a-g.

Cell Apoptosis Analysis
Apoptosis was determined by staining cells with an Annexin VFITC/PI detection kit (BD Biosciences, San Jose, CA, USA). Briefly, MCF-7 cells were seeded on six-well plates (6 × 10 4 cells/mL) and incubated with compound 8f (0, 3, 6 and 12 µM) for 24 h. Cells were washed twice with cold PBS and resuspended in 500 µL of 1 × binding buffer, and then 10 µL of Annexin V-FITC and 5 µL of PI were applied to stain the cells for 15 min at room temperature in the dark. The status of stained cells was analyzed using a flow cytometer (BD, FACSCalibur, San Jose, CA, USA) [60,61].

Mitochondrial Membrane Potential (MMP) Assay
MCF-7 cells were cultured into 12-well plates at a concentration of 1 × 10 6 cells/well of the RPMI-1640 medium. Then, cells were co-cultured with compound 8f (0, 3, 6 and 12 µM) for 24 h. After treatment, cells were stained with 3 µM cationic dye JC-1 at room temperature in the dark for 30 min according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA). Finally, cells were harvested and washed with PBS twice, and then the results were recorded by flow cytometry analysis [62].

ROS Level Assay
MCF-7 cells were cultured into 12-well plate at a concentration of 1 × 10 6 cells/well for 24 h of the RPMI-1640 medium with 10% FBS. Then, cells were co-cultured with compound 8f (0, 3, 6 and 12 µM) for 24 h. Then, the MCF-7 cells were stained with 5 µM H 2 DCFDA solution (400 µL/well) at room temperature in the dark for 30 min. The cells were harvested and washed with PBS twice according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). The results were obtained by flow cytometry [63].

Conclusions
In summary, according to the special structural features of betulin and thio-/semicarbazone groups, 14 new betulin derivatives with thiocarbazone or semicarbazone sidechains on the C-28 position were synthesized. All new compounds were evaluated for their in vitro cytotoxicities in human carcinoma cells (HepG, MCF-7, A549, HCT-116) and normal human gastric epithelial cells (GES-1). Among them, compound 8f displayed the most potent cytotoxicity, with an IC 50 value of 5.86 ± 0.61 µM against MCF-7 cells. Furthermore, the intracellular mechanism studies proved that compound 8f could trigger the mitochondrial-mediated apoptosis pathway by losing MMP, which was associated with the downregulation of Bcl-2 and P53, the upregulation of Bax expression, the activation of the levels of caspase-3 and caspase-9, and the formation of ROS. The above results indicated that compound 8f could be used as a valuable skeleton for developing novel antitumor agents.