Novel Steroidal 5α,8α-Endoperoxide Derivatives with Semicarbazone/Thiosemicarbazone Side-chain as Apoptotic Inducers through an Intrinsic Apoptosis Pathway: Design, Synthesis and Biological Studies.

A series of novel steroidal 5α,8α-endoperoxide derivatives bearing semicarbazone (7a–g) or thiosemicarbazone (7h–k) side chain were designed, synthesized and evaluated for their cytotoxicities in four human cancer cell lines (HepG2, HCT-116, MCF-7, and A549) using the MTT assay in vitro. The results showed that compound 7j exhibited significant cytotoxic activity against HepG2 cells (IC50 = 3.52 μM), being more potent than ergosterol peroxide. Further cellular mechanism studies in HepG2 cells indicated that compound 7j triggered the mitochondrial-mediated apoptosis by decreasing mitochondrial membrane potential (MMP), which was associated with up-regulation of Bax, down-regulation of Bcl-2, activation levels of the caspase cascade, and formation of reactive oxygen species (ROS). The above findings indicated that compound 7j may be used as a promising skeleton for antitumor agents with improved efficacy.


Introduction
Cancer is a serious disease that poses a risk people's health. The discovery of potent anticancer drugs from natural products is one of the important directions in drug research. Nowadays, it's still more than 50% of drugs used in the areas of cancer and infectious diseases are based on natural origins [1,2]. Natural endoperoxides are widely found in nature, which all have a distinct peroxide bond (-O-O-) in their structures as cyclic organic compounds, such as artemisinin, schinalactone A, talaperoxides B and gracilioethers A, yingzaosu, etc. (Figure 1) [3][4][5]. Most of natural endoperoxides have been proved with antiviral, anticancer or antifungal activity [6][7][8]. The most representative discovery of endoperoxide from natural products is artemisinin by Youyou Tu (2015 Nobel Award winner) [9]. Artemisinin was extracted from a traditional Chinese medicine (Artemisia annua L.), and many derivatives of it have been synthesized and used for treatment of malaria [10,11].
Steroidal compounds have drawn attention not only due to their unusual and interesting chemical structures, but also due to their widespread application as anti-inflammatory, diuretic, anabolic, contraceptive, and anticancer agents [12]. Most steroidal drugs in use today are semisynthetic compounds widely used in traditional medicines by modification of the steroid ring system and side chains [13]. The interesting structural and stereochemical features of the steroid nucleus provide Ergosterol peroxide (5α,8α-epidioxiergosta-6,22-dien-3β-ol, EP) is a member of a class of fungal secondary metaboliters of sterol 5α,8α-endoperoxidederivatives ( Figure 2). A number of biological activities have been attributed to EP, including antitumor, anti-inflammatory, antiviral and immunomodulatory activities and antioxidant activities [16][17][18][19][20][21]. As an important active lead compound in drug discovery, EP is well known for its 5α,8α-peroxy moiety. During the last years, our research group has been working on the modifications of steroids to obtained more active compounds as potential antitumor agents. In our previous studies, we synthesized a series of steroidal 5α,8α-endoperoxide derivatives with aliphatic side-chain. We found that most of these derivatives exhibited significant anticancer activities compared to ergosterol, which has no peroxidic bridge at C-5 and C-8 positions with barely inhibition activity against all the tested cells. The studies showed that the 5α,8α-peroxygroup is requisite pharmacophore for these derivatives [22]. Then we synthesized several series of steroidal 5α,8α-endoperoxide derivatives with 17-(Ocarboxymethyl)oxime side-chain and hydrazone hybrid side-chain. The MTT assay indicated that some derivatives possessing the 5α,8α-peroxy group and different side chains displayed distinct cytotoxic activity against some cancer cell lines [23][24][25][26][27]. Based on the above results, we speculate that the difference between side chains at C-17 position and 5α,8α-peroxy group would provide a synergistic effect for the bioactivity. Semicarbazones and thiosemicarbazones are a class of Schiff bases that have been gaining considerable attention due to their wide array of pharmacological effects or biological activities [28,29]. In a literature review, we found that semicarbazone based derivatives have shown anticancer, anti-protozoal, antimicrobial, pesticidal activity, etc. [30][31][32][33]. In addition, thiosemicarbazones (TSCs) have a wide clinical antitumor spectrum with efficacy in various tumor types such as bladder cancer, pancreatic cancer, cervical cancer, non-small cell lung cancer, breast cancer, prostate cancer and leukemia [34][35][36][37]. Figure 3 shows some representative compounds with thiosemicarbazones structure in the clinical trial. In addition, it is reported that combining steroids (progesterone and testosterone) with semicarbazide or thiosemicarbazide moieties could be a good anticancer drug [38]. Ergosterol peroxide (5α,8α-epidioxiergosta-6,22-dien-3β-ol, EP) is a member of a class of fungal secondary metaboliters of sterol 5α,8α-endoperoxidederivatives ( Figure 2). A number of biological activities have been attributed to EP, including antitumor, anti-inflammatory, antiviral and immunomodulatory activities and antioxidant activities [16][17][18][19][20][21]. As an important active lead compound in drug discovery, EP is well known for its 5α,8α-peroxy moiety. During the last years, our research group has been working on the modifications of steroids to obtained more active compounds as potential antitumor agents. In our previous studies, we synthesized a series of steroidal 5α,8α-endoperoxide derivatives with aliphatic side-chain. We found that most of these derivatives exhibited significant anticancer activities compared to ergosterol, which has no peroxidic bridge at C-5 and C-8 positions with barely inhibition activity against all the tested cells. The studies showed that the 5α,8α-peroxygroup is requisite pharmacophore for these derivatives [22]. Then we synthesized several series of steroidal 5α,8α-endoperoxide derivatives with 17-(O-carboxymethyl)oxime side-chain and hydrazone hybrid side-chain. The MTT assay indicated that some derivatives possessing the 5α,8α-peroxy group and different side chains displayed distinct cytotoxic activity against some cancer cell lines [23][24][25][26][27]. Based on the above results, we speculate that the difference between side chains at C-17 position and 5α,8α-peroxy group would provide a synergistic effect for the bioactivity. nucleus provide additional fascination for researchers, and the introduction of heteroatoms, heterocycles, or amides, or replacement of one or more carbon atoms in the steroidal skeleton has been envisaged to discover new chemical entities with the potential to become promising future drugs, resulting in notable modifications in biological activity [14][15][16]. Ergosterol peroxide (5α,8α-epidioxiergosta-6,22-dien-3β-ol, EP) is a member of a class of fungal secondary metaboliters of sterol 5α,8α-endoperoxidederivatives ( Figure 2). A number of biological activities have been attributed to EP, including antitumor, anti-inflammatory, antiviral and immunomodulatory activities and antioxidant activities [16][17][18][19][20][21]. As an important active lead compound in drug discovery, EP is well known for its 5α,8α-peroxy moiety. During the last years, our research group has been working on the modifications of steroids to obtained more active compounds as potential antitumor agents. In our previous studies, we synthesized a series of steroidal 5α,8α-endoperoxide derivatives with aliphatic side-chain. We found that most of these derivatives exhibited significant anticancer activities compared to ergosterol, which has no peroxidic bridge at C-5 and C-8 positions with barely inhibition activity against all the tested cells. The studies showed that the 5α,8α-peroxygroup is requisite pharmacophore for these derivatives [22]. Then we synthesized several series of steroidal 5α,8α-endoperoxide derivatives with 17-(Ocarboxymethyl)oxime side-chain and hydrazone hybrid side-chain. The MTT assay indicated that some derivatives possessing the 5α,8α-peroxy group and different side chains displayed distinct cytotoxic activity against some cancer cell lines [23][24][25][26][27]. Based on the above results, we speculate that the difference between side chains at C-17 position and 5α,8α-peroxy group would provide a synergistic effect for the bioactivity. Semicarbazones and thiosemicarbazones are a class of Schiff bases that have been gaining considerable attention due to their wide array of pharmacological effects or biological activities [28,29]. In a literature review, we found that semicarbazone based derivatives have shown anticancer, anti-protozoal, antimicrobial, pesticidal activity, etc. [30][31][32][33]. In addition, thiosemicarbazones (TSCs) have a wide clinical antitumor spectrum with efficacy in various tumor types such as bladder cancer, pancreatic cancer, cervical cancer, non-small cell lung cancer, breast cancer, prostate cancer and leukemia [34][35][36][37]. Figure 3 shows some representative compounds with thiosemicarbazones structure in the clinical trial. In addition, it is reported that combining steroids (progesterone and testosterone) with semicarbazide or thiosemicarbazide moieties could be a good anticancer drug [38]. Semicarbazones and thiosemicarbazones are a class of Schiff bases that have been gaining considerable attention due to their wide array of pharmacological effects or biological activities [28,29]. In a literature review, we found that semicarbazone based derivatives have shown anticancer, anti-protozoal, antimicrobial, pesticidal activity, etc. [30][31][32][33]. In addition, thiosemicarbazones (TSCs) have a wide clinical antitumor spectrum with efficacy in various tumor types such as bladder cancer, pancreatic cancer, cervical cancer, non-small cell lung cancer, breast cancer, prostate cancer and leukemia [34][35][36][37]. Figure 3 shows some representative compounds with thiosemicarbazones structure in the clinical trial. In addition, it is reported that combining steroids (progesterone and testosterone) with semicarbazide or thiosemicarbazide moieties could be a good anticancer drug [38]. Molecules 2020, 25, x FOR PEER REVIEW 3 of 15 Inspired by the good biological properties of semicarbazones and thiosemicarbazones, and in continuation with our previous work on the modifications of steroids, we here report the design and synthesis of a series of novel steroidal endoperoxide derivatives with semicarbazone/thiosemicarbazone side-chain ( Figure 4) and their underlying action mechanisms of anticancer activity, wishing to find new effective antitumor agents.

Chemistry
The general procedure for the synthesis of steroidal derivatives is shown in Scheme 1. Dehydroepiandrosterone (DHEA, 1) was reacted with acetic anhydride in the presence of pyridine and dichloromethane to give compound 2. Compound 2 further underwent bromination with N-Bromosuccinimide (NBS) and debromination with 2,4,6-collidineto afford compound 3. Compound 3 was reacted with potassium hydroxide for deacetylation to give compound 4. Subsequently, compound 4 was reacted with oxygen to give endoperoxide 5, using phloxine B as photosensitizer. Then, compound 5 was reacted with hydrazine hydrate to obtain the corresponding steroidal hydrazine 6. At last, the resulting hydrazine 6was reacted with different phenyl isocyanate or isothiocyanate substituents to obtain target novel 5α,8α-endoperoxide steroidal conjugation semicarbazone/thiosemicarbazone derivatives 7a-k. The structures of all compounds were characterized by MS, 1 H-NMR and 13 C-NMR spectrum.

In Vitro Cytotoxic Activity
All of the newly synthesized steroidal endoperoxide derivatives 7a-k were investigated for their anti-proliferative activities against human hepatocellular carcinomacells (HepG2), human breast carcinoma cells (MCF-7), human colorectal cells (HCT-116), and human lung carcinoma cells (A549) by the MTT assay in vitro. Mitomycin was used as positive reference drug. The results of the MTT assay were summarized as IC50 values in Table 1.
The results showed that most of the synthesized derivatives displayed significant cytotoxic activities against all the four tested tumor cell lines, and were more potent than ergosterol peroxide. In addition, it seems that most compounds exhibited significant anti-proliferative activities against HepG2 and MCF-7 cell lines than HCT-116 and A549 cell lines. The results suggested the following rough structure-activity relationships considerations. Inspired by the good biological properties of semicarbazones and thiosemicarbazones, and in continuation with our previous work on the modifications of steroids, we here report the design and synthesis of a series of novel steroidal endoperoxide derivatives with semicarbazone/thiosemicarbazone side-chain ( Figure 4) and their underlying action mechanisms of anticancer activity, wishing to find new effective antitumor agents. Inspired by the good biological properties of semicarbazones and thiosemicarbazones, and in continuation with our previous work on the modifications of steroids, we here report the design and synthesis of a series of novel steroidal endoperoxide derivatives with semicarbazone/thiosemicarbazone side-chain ( Figure 4) and their underlying action mechanisms of anticancer activity, wishing to find new effective antitumor agents.

Chemistry
The general procedure for the synthesis of steroidal derivatives is shown in Scheme 1. Dehydroepiandrosterone (DHEA, 1) was reacted with acetic anhydride in the presence of pyridine and dichloromethane to give compound 2. Compound 2 further underwent bromination with N-Bromosuccinimide (NBS) and debromination with 2,4,6-collidineto afford compound 3. Compound 3 was reacted with potassium hydroxide for deacetylation to give compound 4. Subsequently, compound 4 was reacted with oxygen to give endoperoxide 5, using phloxine B as photosensitizer. Then, compound 5 was reacted with hydrazine hydrate to obtain the corresponding steroidal hydrazine 6. At last, the resulting hydrazine 6was reacted with different phenyl isocyanate or isothiocyanate substituents to obtain target novel 5α,8α-endoperoxide steroidal conjugation semicarbazone/thiosemicarbazone derivatives 7a-k. The structures of all compounds were characterized by MS, 1 H-NMR and 13 C-NMR spectrum.

In Vitro Cytotoxic Activity
All of the newly synthesized steroidal endoperoxide derivatives 7a-k were investigated for their anti-proliferative activities against human hepatocellular carcinomacells (HepG2), human breast carcinoma cells (MCF-7), human colorectal cells (HCT-116), and human lung carcinoma cells (A549) by the MTT assay in vitro. Mitomycin was used as positive reference drug. The results of the MTT assay were summarized as IC50 values in Table 1.
The results showed that most of the synthesized derivatives displayed significant cytotoxic activities against all the four tested tumor cell lines, and were more potent than ergosterol peroxide. In addition, it seems that most compounds exhibited significant anti-proliferative activities against HepG2 and MCF-7 cell lines than HCT-116 and A549 cell lines. The results suggested the following rough structure-activity relationships considerations.

Chemistry
The general procedure for the synthesis of steroidal derivatives is shown in Scheme 1. Dehydroepiandrosterone (DHEA, 1) was reacted with acetic anhydride in the presence of pyridine and dichloromethane to give compound 2. Compound 2 further underwent bromination with N-Bromosuccinimide (NBS) and debromination with 2,4,6-collidineto afford compound 3. Compound 3 was reacted with potassium hydroxide for deacetylation to give compound 4. Subsequently, compound 4 was reacted with oxygen to give endoperoxide 5, using phloxine B as photosensitizer. Then, compound 5 was reacted with hydrazine hydrate to obtain the corresponding steroidal hydrazine 6. At last, the resulting hydrazine 6 was reacted with different phenyl isocyanate or isothiocyanate substituents to obtain target novel 5α,8α-endoperoxide steroidal conjugation semicarbazone/thiosemicarbazone derivatives 7a-k. The structures of all compounds were characterized by MS, 1 H-NMR and 13 C-NMR spectrum.
Taken together, it is notable that compounds 7b and 7f with semicarbazone side-chain, 7h and 7j with thiosemicarbazone side-chain exhibited more obvious cytotoxic activity to all tested cells as compared to ergosterol peroxide, which suggests that both conjugated N,N,S-tridentate donor set and N,N,O-tridentate donor set are essential for the biological activities of these novel derivatives. In addition, the activity of the compounds was greatly influenced by the change in substitutions at different positions of phenyl ring.  The results showed that most of the synthesized derivatives displayed significant cytotoxic activities against all the four tested tumor cell lines, and were more potent than ergosterol peroxide. In addition, it seems that most compounds exhibited significant anti-proliferative activities against HepG2 and MCF-7 cell lines than HCT-116 and A549 cell lines. The results suggested the following rough structure-activity relationships considerations.
Taken together, it is notable that compounds 7b and 7f with semicarbazone side-chain, 7h and 7j with thiosemicarbazone side-chain exhibited more obvious cytotoxic activity to all tested cells as compared to ergosterol peroxide, which suggests that both conjugated N,N,S-tridentate donor set and N,N,O-tridentate donor set are essential for the biological activities of these novel derivatives. In addition, the activity of the compounds was greatly influenced by the change in substitutions at different positions of phenyl ring.
Among the compounds under study, the semicarbazonederivative (7f) and thiosemicarbazone derivative (7j) were the most potent compounds against HepG2 cell lines (IC 50 < 5 µM). To further investigate the cellular mechanism of these new compounds, compound 7j was chosen for subsequent biological functions experiments in HepG2 cells.

Compound 7j Induces Apoptosis in HepG2 Cells
Apoptosis is a major type of cell death. In our previous studies, we have reported that ergosterol peroxide exhibited cytotoxic activity against HepG2 and MCF-7 cells by inducing apoptosis [20,21]. In this work, the changes of the morphological characters in HepG2 cells were studies by Hoechst33342/PI staining, which to prove that the inhibitory activity of compound 7j was related to the inducing of cell apoptosis. As shown in Figure 5A, staining the cells with Hoechst 33342 showed the typical features of apoptosis such as chromatin condensation, and formation of apoptotic bodies. To further demonstrate whether 7j could induce apoptosis in HepG2 cells, 7j-treated HepG2 cells were doubly stained with Annexin V-FITC and propidium iodide (PI). As shown in Figure 5B, the percentage of total apoptotic and necrotic cells increased to 16.22% and 41.32% in a dose-dependent manner after treatment with 7j at the concentrations of 4 and 8 µM, respectively. The results suggested that 7j exerted its inhibitory effects on proliferation by inducing HepG2 cells apoptosis.

Compound 7j Induces Oxidative Stress in HepG2 Cells
Intracellular reactive oxygen species (ROS) plays a vital role in many kinds of biological processes. At the proper concentrations, ROS generation is considered as a major factor in mitochondrial-dependent apoptosis [39,40]. Hence, we explored the ROS inducing capability of 7j at the cellular levels using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining analysis by flow cytometry. As shown in Figure 7, we found a doses-dependent increase in the ROS generation was observed for HepG2 cells after treated with 7j (2, 4 and 8 μM) for 24 h. The relative ROS level in HepG2 cells that were treated with 7j (8 μM) was about six-fold higher than that of the control group, which was also apparently higher than the Rosup group. The results demonstrate that 7j significantly increases ROS generation, which could be accountable for inducing apoptosis.

Compound 7j Induces Mitochondrial Membrane Potential (MMP) Loss in HepG2 Cells
Mitochondria dysfunction plays an important role in inducing apoptosis in cancer cells. The loss of MMP (∆Ψ m ) has been indicated as an early hallmark of mitochondrial dysfunction in apoptotic cells [38]. The cationic dye JC-1 is an ideal MMP-sensitive probe, which can be used to detect the changes of MMP by flow cytometry. As shown in Figure 6, 7j induced a doses-dependent increase in depolarized cell population from 3.24% of control to 15.21%, 25.60% and 59.96% at the concentrations of 2, 4 and 8 µM, respectively. The results indicated that the apoptosis of HepG2 cells induced by 7j was associated with the intrinsic mitochondrial-mediated pathways.

Compound 7j Induces Apoptosis Via the Activation of Caspases and Regulated Apoptosis Releated Protein Expression
To further provide the molecular mechanistic insight into how compound 7j induces apoptosis in HepG2 cells, we examined the expression of apoptotic proteins Bcl-2, Bax, caspase-3 and caspase-9 in response to compound 7j treatment. First, HepG2 cells were treated with compound 7j at the concentrations of 2, 4 and 8 μM for 24 h, and the expression levels of pro-apoptosis proteins Bax and pro-survival protein Bcl-2 were investigated by western blotting. The GAPDH expression was served as an internal control groups. As shown in Figure 8, the immunoblot analysis indicated that compound 7j significantly suppressed the levels of Bcl-2 expression, but increased the expression levels of Bax in a dose-dependent manner.

Compound 7j Induces Oxidative Stress in HepG2 Cells
Intracellular reactive oxygen species (ROS) plays a vital role in many kinds of biological processes. At the proper concentrations, ROS generation is considered as a major factor in mitochondrial-dependent apoptosis [39,40]. Hence, we explored the ROS inducing capability of 7j at the cellular levels using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining analysis by flow cytometry. As shown in Figure 7, we found a doses-dependent increase in the ROS generation was observed for HepG2 cells after treated with 7j (2, 4 and 8 µM) for 24 h. The relative ROS level in HepG2 cells that were treated with 7j (8 µM) was about six-fold higher than that of the control group, which was also apparently higher than the Rosup group. The results demonstrate that 7j significantly increases ROS generation, which could be accountable for inducing apoptosis.  To further provide the molecular mechanistic insight into how compound 7j induces apoptosis in HepG2 cells, we examined the expression of apoptotic proteins Bcl-2, Bax, caspase-3 and caspase-9 in response to compound 7j treatment. First, HepG2 cells were treated with compound 7j at the

Compound 7j Induces Apoptosis Via the Activation of Caspases and Regulated Apoptosis Releated Protein Expression
To further provide the molecular mechanistic insight into how compound 7j induces apoptosis in HepG2 cells, we examined the expression of apoptotic proteins Bcl-2, Bax, caspase-3 and caspase-9 in response to compound 7j treatment. First, HepG2 cells were treated with compound 7j at the concentrations of 2, 4 and 8 µM for 24 h, and the expression levels of pro-apoptosis proteins Bax and pro-survival protein Bcl-2 were investigated by western blotting. The GAPDH expression was served as an internal control groups. As shown in Figure 8, the immunoblot analysis indicated that compound 7j significantly suppressed the levels of Bcl-2 expression, but increased the expression levels of Bax in a dose-dependent manner. Moreover, the expression levels of cleaved caspase-3 and caspase-9 were evaluated in HepG2 cells by spectrophotometry using caspase-3 and caspase-9 activity assay kits. As shown in Figure 9, the expression levels of cleaved caspase-3 and caspase-9 were significantly increased after being treated with 7j (2, 4 and 8 μM), in a dose-dependent manner. The above data proved that compound 7j induces HepG2 cells apoptosis via the intrinsic mitochondrial-mediated pathway.

Conclusions
In summary, we have successfully prepared a series of novel 5α,8α-endoperoxidesteroidal derivatives with semicarbazone/thiosemicarbazone side-chain on the C-17 position. The antiproliferative activity of the compounds against HepG2, HCT-116, MCF-7 and A549cell lines were evaluated using the MTT assay in vitro. The results showed that compound 7j exhibited significant cytotoxic activity against HepG2 cells (IC50 = 3.52 μM), and was more potent than ergosterol peroxide. Further cellular mechanism studies in HepG2 cells indicated that compound 7j triggered the Moreover, the expression levels of cleaved caspase-3 and caspase-9 were evaluated in HepG2 cells by spectrophotometry using caspase-3 and caspase-9 activity assay kits. As shown in Figure 9, the expression levels of cleaved caspase-3 and caspase-9 were significantly increased after being treated with 7j (2, 4 and 8 µM), in a dose-dependent manner. The above data proved that compound 7j induces HepG2 cells apoptosis via the intrinsic mitochondrial-mediated pathway. Moreover, the expression levels of cleaved caspase-3 and caspase-9 were evaluated in HepG2 cells by spectrophotometry using caspase-3 and caspase-9 activity assay kits. As shown in Figure 9, the expression levels of cleaved caspase-3 and caspase-9 were significantly increased after being treated with 7j (2, 4 and 8 μM), in a dose-dependent manner. The above data proved that compound 7j induces HepG2 cells apoptosis via the intrinsic mitochondrial-mediated pathway.

Conclusions
In summary, we have successfully prepared a series of novel 5α,8α-endoperoxidesteroidal derivatives with semicarbazone/thiosemicarbazone side-chain on the C-17 position. The anti-

Conclusions
In summary, we have successfully prepared a series of novel 5α,8α-endoperoxidesteroidal derivatives with semicarbazone/thiosemicarbazone side-chain on the C-17 position. The anti-proliferative activity of the compounds against HepG2, HCT-116, MCF-7 and A549cell lines were evaluated using the MTT assay in vitro. The results showed that compound 7j exhibited significant cytotoxic activity against HepG2 cells (IC 50 = 3.52 µM), and was more potent than ergosterol peroxide. Further cellular mechanism studies in HepG2 cells indicated that compound 7j triggered the mitochondrial-mediated apoptosis by decreasing mitochondrial membrane potential (MMP), which was associated with up-regulation of Bax, down-regulation of Bcl-2, activation levels of the caspase cascade, and formation of reactive oxygen species (ROS). The above findings indicated that compound 7j may be used as a promising skeleton for antitumor agents with improved efficacy.

Chemistry
Dehydroepiandrosterone, NBS, hydrazine hydrate, phloxine B, isocyanate, isothiocyanate substituents and other regents were purchased from Energy Chemical (Shanghai) Co. Ltd. China. All regents and solvent (analytical grade) used without further purification. 1 H-NMR (600 MHz) and 13 C-NMR (150 MHz) were recorded on a Bruker Avance DRX400 instrument (Bruker, Karlsruhe, Germany), using tetramethylsilane (TMS) as internal standards. Melting points (mp) were determined on an MP120 auto melting point apparatus (Haineng, Jinan, China). Mass spectra (ESI) of all compounds were recorded on Esquire 6000 mass spectrometer (Bruker, Karlsruhe, Germany). Flash chromatography was performed using 400 mesh silica gel. 4.1.1. Synthesis of 3β-Acetoxyandrosta-5-en-17-one (2) DHEA (1) (0.05 mol, 14.4 g) reacted with acetic anhydride (0.07 mol) in dichloromethane-pyridine (4:1, 100 mL). The reaction mixture was stirred at room temperature for 10 h. After completion of reaction, water (50 mL) was added into the mixture, and then extracted with dichloromethane (100 mL). The combined organic phase was washed with saturated NaHCO 3 aqueous (2 × 60 mL), brine (2 × 60 mL) and dried over anhydrous Na 2 SO 4 . The solvent was evaporated and the crude product was purified by flash chromatography to obtain compound 2 as white solid (16. Intermediate 2 (0.05 mol, 16 g) reacted with NBS in cyclohexane (100 mL). The mixture was heated to 70 • C and refluxed for 1 h. The solids were removed by filtration, then the solvent was collected and evaporated to get a brown solid (17.0 g, 83%). Subsequently, the solid prepared above was dissolved in xylene (150 mL) and 2,4,6-collidine (25 mL). The mixture was heated to 135 • C and refluxed for 3~4 h. After completion of reaction, the solids were removed by filtration. The solvent was collected and washed with water (2 ×80 mL), brine (2 ×80 mL) and dried over anhydrous Na 2 SO 4 . The solvent was evaporated and recrystallized in cooled methanol overnight, and then filtered to obtain pale yellow solid as compound 3 (5.6 g). Yield: 33%, mp: 111. 8 (4) Intermediate 3 (0.034 mol, 10.8 g) reacted with potassium hydroxide (4.5 g) in methanol (80 mL). The mixture was heated to 80 • C for 1 h. After completion of reaction, the mixture was cooled for crystallization overnight. The precipitate was washed with cooled methanol (20 mL) to get a brown solid crude product 4 (9.1 g). Yield: 93%, mp: 156.8-157. Intermediate 4 (3.24 mmol, 1.0 g) in methanol (150 mL) was added phloxine B (5 mg) in a 250 mL round-bottom flask. The mixture kept in a water-cooled bath and stirred by bubbling into high purity oxygen. While, the mixture was lighted with a 500 W iodine tungsten lamp (220 V) for 3 h. After completion of reaction, the solids were removed by filtration. The methanol was evaporated, and then the crude product was purified by chromatographic column ( (6) Intermediate 5 (10.0 mmol, 3.0 g) reacted with 85% hydrazine hydrate (4 mL) in ethanol (100 mL). The mixture was heated to 40 • C and stirred for 1~2 h. After completion of reaction, the solvent was evaporated, and then the crude product was purified by chromatographic column (ethyl acetate/petroleum ether, 1:1) to give 6 as white needles (2.

Western Blot Analysis
After treatment with compound 7j (0, 2, 4 and 8 µM), HepG2 cells were harvested with trypsin and lysed in RIPA buffer and boiled at 100 • C for 10 min. Protein was separated on a 15% SDS-PAGE gel, and transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA for 1~2 h, and incubated with a 1:1000 dilution of primary antibody overnight. Then the membranes were incubated with a 1:5000 dilution of secondary antibody for 2 h. Positive bands were visualized by using an enhanced chemiluminescence system Kit [44].