Synthesis of Ergosterol Peroxide Conjugates as Mitochondria Targeting Probes for Enhanced Anticancer Activity

Inspired by the significant bioactivity of ergosterol peroxide, we designed and synthesized four fluorescent coumarin and ergosterol peroxide conjugates 8a–d through the combination of ergosterol peroxide with 7-N,N-diethylamino coumarins fluorophore. The cytotoxicity of synthesized conjugates against three human cancer cells (HepG2, SK-Hep1, and MCF-7) was evaluated. The results of fluorescent imaging showed that the synthesized conjugates 8a–d localized and enriched mainly in mitochondria, leading to significantly enhanced cytotoxicity over ergosterol peroxide. Furthermore, the results of biological functions of 8d showed that it could suppress cell colony formation, invasion, and migration; induce G2/M phase arrest of HepG2 cells, and increase the intracellular ROS level.

In our previous study, we obtained pure ergosterol peroxide by chemical extraction and separation from Ganoderma lucidum. As the isolated amount of ergosterol peroxide from natural sources is not sufficient for an in-depth study on it, we got an effective chemical method to synthesize ergosterol peroxide (Figure 1). The synthesis of ergosterol peroxide was achieved by the treatment of natural ergosterol with oxygen in the presence of visible light and photosensitive catalysis [20][21][22]. Also, we have proved that ergosterol peroxide could inhibit forkhead-box O3 transcription factor (Foxo3a) functions by the inhibition of phosphorylate protein kinase (pAKT) to induce tumor cell death [23].
Herein, we designed four fluorescent coumarin and ergosterol peroxide conjugates 8a-d through the combination of ergosterol peroxide with different targeting coumarin-3-carboxamide analogs. We postulated that the mitochondria-targeting coumarin analogs could efficiently take ergosterol peroxide selective accumulated in mitochondria, in which subsequent production of ROS from the hemolytic cleavage of the endoperoxide bond in ergosterol peroxide would induce mitochondrial dysfunction and kill tumor cells ( Figure 1B).

Chemistry
First, using ergosterol (EG) as material, we achieved a chemical synthesis of ergosterol peroxide by the treatment of ergosterol in the presence of eosin Y, oxygen, and visible light (Scheme 1) [22]. Natural ergosterol, which is lacking a peroxidic bond (O-O), has been proved with no significant activity against most of the cancer cells [24]. Hence, it is generally accepted that the peroxide bridge is a crucial functional group to the biological activities [25,26]. The hemolytic cleavage of the peroxide bridge in a reducing environment induces reactive oxygen species (ROS), which could be cytotoxic to cancer cells [27][28][29][30].
Selective delivery of drug probes to subcellular organelles in tumor cells has emerged as an effective and attractive strategy for cancer therapy. It exerts synchronous tumor targeting and molecular imaging of drug delivery. Therefore, various theranostic probes that target different subcellular organelles in cancer cells, including the endoplasmic reticulum, lysosomes, and mitochondria, have been continuously reported in recent years [31][32][33]. Among them, mitochondria represent the most attractive target for effective theranostic probe development due to their key role in controlling many essential functions in cancer cells. The specific motivation of inducing mitochondrial dysfunctions has been acknowledged as an effective method in cancer therapy [34][35][36][37].
Herein, we designed four fluorescent coumarin and ergosterol peroxide conjugates 8a-d through the combination of ergosterol peroxide with different targeting coumarin-3-carboxamide analogs. We postulated that the mitochondria-targeting coumarin analogs could efficiently take ergosterol peroxide selective accumulated in mitochondria, in which subsequent production of ROS from the hemolytic cleavage of the endoperoxide bond in ergosterol peroxide would induce mitochondrial dysfunction and kill tumor cells ( Figure 1B).

Chemistry
First, using ergosterol (EG) as material, we achieved a chemical synthesis of ergosterol peroxide by the treatment of ergosterol in the presence of eosin Y, oxygen, and visible light (Scheme 1) [22]. 2.7-(N,N-Diethylamino)coumarin-3-carboxylic acid (2) was generated according to known procedures [38]. Three different amino acid esters were introduced to the carboxyl group of compound 2 for amide bond formation by the same acylation reaction to give compounds 3a-c. Then, the ester protecting a group of compounds 3a-c could be hydrolyzed with an aqueous solution of hydrochloric acid to give three coumarin fluorophore carboxylic acid analogs 4a-c. Besides, as a comparison, we also synthesized an ergosterol peroxide and fluorophore conjugate 6 with piperazine as the linker. Finally, conjugates 8a-c were obtained by introducing ergosterol peroxide directly to the carboxylic group of 4a-c, using dicyclohexylcarbodi imide (DCC) as the coupling reagent (Scheme 2). Also, ergosterol peroxide was reacted with 4-nitrophenyl chloroformate using pyridine as the base in dichloromethane to get intermediate 7. Then, 7 reacted with intermediate 6 to obtain the desired conjugate 8d. There is a hydroxyl group at the C-3 position of ergosterol peroxide that is also a feasible functional site. We designed and synthesized four ergosterol peroxide-coumarin conjugates 8a-d to evaluate the biological activities of various groups of linker at the C-3 position. The reason for the use of amino acids as spacers is that the amide bond is fairly stable, and the synthesis of compounds in which the amide bond is formed by coupling reagents is simple. Thus, three different amino acids (glycine, β-alanine, or γ-aminobutyric acid) were selected as the linker, to increase the space distance between ergosterol peroxide and coumarin fluorophore.
The synthetic routes of different coumarin fluorophore analogs are presented in Scheme 2. 2.7-(N,N-Diethylamino)coumarin-3-carboxylic acid (2) was generated according to known procedures [38]. Three different amino acid esters were introduced to the carboxyl group of compound 2 for amide bond formation by the same acylation reaction to give compounds 3a-c. Then, the ester protecting a group of compounds 3a-c could be hydrolyzed with an aqueous solution of hydrochloric acid to give three coumarin fluorophore carboxylic acid analogs 4a-c. Besides, as a comparison, we also synthesized an ergosterol peroxide and fluorophore conjugate 6 with piperazine as the linker.

Optical Properties and Subcellular Localization
First, the optical properties of four ergosterol peroxide conjugates 8a-d were investigated (Table 1). In general, all four conjugates possess typical optical properties [39]. Especially, probe 8d with a maximum excitation wavelength (λex) of about 469.5 nm and an emission wavelength (λem) of Finally, conjugates 8a-c were obtained by introducing ergosterol peroxide directly to the carboxylic group of 4a-c, using dicyclohexylcarbodi imide (DCC) as the coupling reagent (Scheme 2). Also, ergosterol peroxide was reacted with 4-nitrophenyl chloroformate using pyridine as the base in dichloromethane to get intermediate 7. Then, 7 reacted with intermediate 6 to obtain the desired conjugate 8d.

Optical Properties and Subcellular Localization
First, the optical properties of four ergosterol peroxide conjugates 8a-d were investigated (Table 1). In general, all four conjugates possess typical optical properties [39]. Especially, probe 8d with a maximum excitation wavelength (λ ex ) of about 469.5 nm and an emission wavelength (λ em ) of about 404 nm. The large Stokes shift (65.5 nm) of probe 8d ensured ideal photophysical properties for living cells fluorescence imaging studies. Thus, probe 8d was chosen for further subcellular localization study. The subcellular localization of probe 8d in living HepG2 cells was carried with the green mitochondria-specific dye Rhodamine 123 (Rh123) as a comparison (λ ex = 488 nm, λ em = 515-530 nm) [40]. The intracellular microscopic fluoroscope imaging was captured by Carl Zeiss 710M confocal laser scanning microscopy. As shown in Figure 2A,B, HepG2 cells were universality stained by 8d (5 µM and 10 µM) after 2 h of incubation. Bright blue fluorescence was captured successfully in HepG2 cells (λ ex = 430-500 nm), which demonstrated both satisfactory permeability into the cell membrane and high cellular uptake of 8d. What's more, the extensive merged blue-green color fluorescence images illustrated that probe 8d could strongly co-localize with specific dye Rh123 in mitochondria. Also, as shown in Figure 2C,D, MCF-7 cells were successfully stained by 8c or 8d (10 µM). The results suggested that the fluorescent coumarin-3-carboxamide could successfully deliver ergosterol peroxide to mitochondria.

Cytotoxic Activity
The newly synthesized conjugates 8a-d and ergosterol peroxide were evaluated for their cytotoxicities against human hepatic carcinoma cells (HepG2, Sk-Hep2) and human breast cancer cells (MCF-7) by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cisplatin was chosen as a positive control drug. The results in Table 2 show that all conjugates have ideal cytotoxicity to these three tumor cell lines, with half maximal inhibitory concentration (IC50) values at the sub-micromolar levels. Among them, probes 8c and 8d were the most potent to the tested cancer cells. Probe 8c with a longer linker (γ-aminobutyric acid) had around twofold improved potency against three tested cancer cells relative to that of 8a. Besides, probe 8d with a special pyridine moiety as linker exhibited significant cytotoxicity against human liver cancer cells (HepG2, SK-Hep1), with IC50 values of 6.60 μM for HepG2, which is nearly equivalent to 8c for Sk-Hep1. Moreover, much weaker cytotoxicities were measured for either 1 or 2, which suggested that the cytotoxicities of probes 8a-d were associated with the synergistic effect of these two components. Overall, three probes 8b, 8c, and 8d exhibited significant cytotoxicities against HepG2

Cytotoxic Activity
The newly synthesized conjugates 8a-d and ergosterol peroxide were evaluated for their cytotoxicities against human hepatic carcinoma cells (HepG2, Sk-Hep2) and human breast cancer cells (MCF-7) by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cisplatin was chosen as a positive control drug. The results in Table 2 show that all conjugates have ideal cytotoxicity to these three tumor cell lines, with half maximal inhibitory concentration (IC 50 ) values at the sub-micromolar levels. Among them, probes 8c and 8d were the most potent to the tested cancer cells. Probe 8c with a longer linker (γ-aminobutyric acid) had around twofold improved potency against three tested cancer cells relative to that of 8a. Besides, probe 8d with a special pyridine moiety as linker exhibited significant cytotoxicity against human liver cancer cells (HepG2, SK-Hep1), with IC 50 values of 6.60 µM for HepG2, which is nearly equivalent to 8c for Sk-Hep1. Moreover, much weaker cytotoxicities were measured for either 1 or 2, which suggested that the cytotoxicities of probes 8a-d were associated with the synergistic effect of these two components. Overall, three probes 8b, 8c, and 8d exhibited significant cytotoxicities against HepG2 cell lines, with IC 50 values lower than 10 µM. To understand the mechanism of cancer cells death caused by treatment with coumarin-1 conjugates, we chose probe 8d for the series of biological functions experiments. The effect of probe 8d on tumor cell cycle distribution was carried by flow cytometry. We treated HepG2 cells with probe 8d (3 µM) for 24 h. The low concentration of 8d was used to avoid induction of HepG2 cell death. As shown in Figure 3A, in HepG2 cells, the cell phase distribution was 49.30 ± 1.03% vs. 53.12 ± 1.12% in G0/G1 phase, 25.09 ± 1.18% vs. 33.49 ± 1.05% in S phase, and 22.89 ± 1.17% vs. 12.31 ± 2.05% in G2/M phase before and after the effect of 8d. The G0/G1 and S phases were increased significantly before and after the effect of 8d, suggesting a chemical effect of 8d on cell cycle progression. Also, the number of cells decreased in G2/M phases (Figure 3).

Effect of Probe 8d on the Cell Cycle Distribution
The effect of probe 8d on tumor cell cycle distribution was carried by flow cytometry. We treated HepG2 cells with probe 8d (3 μM) for 24 h. The low concentration of 8d was used to avoid induction of HepG2 cell death. As shown in Figure 3A, in HepG2 cells, the cell phase distribution was 49.30 ± 1.03% vs. 53.12 ± 1.12% in G0/G1 phase, 25.09 ± 1.18% vs. 33.49 ± 1.05% in S phase, and 22.89 ± 1.17% vs. 12.31 ± 2.05% in G2/M phase before and after the effect of 8d. The G0/G1 and S phases were increased significantly before and after the effect of 8d, suggesting a chemical effect of 8d on cell cycle progression. Also, the number of cells decreased in G2/M phases (Figure 3).

Effect of Probe 8d on the Cell Colony, Migration, and Invasion
The clonogenic assay is an effective method to evaluate the neoplastic transformation indirectly. Hence, we tested the effect of probe 8d on HepG2 cells colony formation. The cells were treated with 7 μM probe 8d for 20 days. The colony formation was observed under light microscopy. The colony numbers were 61.3 ± 3.1 vs. 9.1 ± 1.4 in HepG2 cells before and after the effect of 8d (Figure 4). The results suggested that probe 8d had a significant inhibitory activity to HepG2 cells growth.

Effect of Probe 8d on the Cell Colony, Migration, and Invasion
The clonogenic assay is an effective method to evaluate the neoplastic transformation indirectly. Hence, we tested the effect of probe 8d on HepG2 cells colony formation. The cells were treated with 7 µM probe 8d for 20 days. The colony formation was observed under light microscopy. The colony numbers were 61.3 ± 3.1 vs. 9.1 ± 1.4 in HepG2 cells before and after the effect of 8d (Figure 4). The results suggested that probe 8d had a significant inhibitory activity to HepG2 cells growth.  To determine if 8d can prevent cancer cell invasion and migration, transwell assays were performed. Under the transwell assay, the number of migratory cells was 120.4 ± 4.2 vs. 63.2 ± 3.5 for HepG2 cells before and after the effect of 8d (6 μM). The HepG2 cell number was reduced significantly after the effect of 8d. The results suggested that 8d suppressed HepG2 cancer cells migration ( Figure 5A,B). The number of invasive cells was 107.1 ± 5.2 vs. 52.7 ± 4.6 for HepG2 cells before and after the effect of 8d (6 μM). The HepG2 cell number was reduced significantly after the effect of 8d. The results suggested that 8d suppressed HepG2 cancer cell migration and invasion ( Figure 5C,D). To determine if 8d can prevent cancer cell invasion and migration, transwell assays were performed. Under the transwell assay, the number of migratory cells was 120.4 ± 4.2 vs. 63.2 ± 3.5 for HepG2 cells before and after the effect of 8d (6 µM). The HepG2 cell number was reduced significantly after the effect of 8d. The results suggested that 8d suppressed HepG2 cancer cells migration ( Figure 5A,B). The number of invasive cells was 107.1 ± 5.2 vs. 52.7 ± 4.6 for HepG2 cells before and after the effect of 8d (6 µM). The HepG2 cell number was reduced significantly after the effect of 8d. The results suggested that 8d suppressed HepG2 cancer cell migration and invasion ( Figure 5C,D).
for HepG2 cells before and after the effect of 8d (6 μM). The HepG2 cell number was reduced significantly after the effect of 8d. The results suggested that 8d suppressed HepG2 cancer cells migration ( Figure 5A,B). The number of invasive cells was 107.1 ± 5.2 vs. 52.7 ± 4.6 for HepG2 cells before and after the effect of 8d (6 μM). The HepG2 cell number was reduced significantly after the effect of 8d. The results suggested that 8d suppressed HepG2 cancer cell migration and invasion ( Figure 5C,D).

Effect of 8d on the Level of ROS
To comprehend the mechanism of tumor cells death caused by treatment with probe 8d, the level of intracellular ROS in HepG2 cells was monitored by using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) as an indicator and detected by flow cytometry [41]. Figure 6 shows the effect of probe 8d on ROS generation in HepG2 cells. A concentration-dependent increase in the ROS level was observed for HepG2 cell lines after incubation with probe 8d (5 and 20 μM) for 24 h. The relative ROS level in HepG2 cancer cells, resulting from incubation with 20 μM probe 8d, was about twofold higher than that of control cells without 8d. This suggested that the intracellularly generated ROS was responsible for cell death.

Effect of 8d on the Level of ROS
To comprehend the mechanism of tumor cells death caused by treatment with probe 8d, the level of intracellular ROS in HepG2 cells was monitored by using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) as an indicator and detected by flow cytometry [41]. Figure 6 shows the effect of probe 8d on ROS generation in HepG2 cells. A concentration-dependent increase in the ROS level was observed for HepG2 cell lines after incubation with probe 8d (5 and 20 µM) for 24 h. The relative ROS level in HepG2 cancer cells, resulting from incubation with 20 µM probe 8d, was about twofold higher than that of control cells without 8d. This suggested that the intracellularly generated ROS was responsible for cell death.

Chemistry
Reagents were commercially available and used without purification. The 1 H-and 13 C-NMR spectra were recorded using the Bruker Avance DRX400 spectrometer (400 MHz and 100 MHz) in CDCl3 or DMSO-d6. Tetramethylsilane (TMS) was used as internal standards. Melting points were measured by an MP120 point apparatus. Mass spectra were measured in electrospray (ESI) mode on a mass spectrometer (Esquire 6000). Flash chromatography was performed using 400 mesh silica gel. 1 H-and 13 C-NMR spectra of new compounds are available online (See supplementary materials).

Chemistry
Reagents were commercially available and used without purification. The 1 H-and 13 C-NMR spectra were recorded using the Bruker Avance DRX400 spectrometer (400 MHz and 100 MHz) in CDCl 3 or DMSO-d 6 . Tetramethylsilane (TMS) was used as internal standards. Melting points were measured by an MP120 point apparatus. Mass spectra were measured in electrospray (ESI) mode on a mass spectrometer (Esquire 6000). Flash chromatography was performed using 400 mesh silica gel. 1 H-and 13 C-NMR spectra of new compounds are available online (See Supplementary Materials). (1) Ergosterol (150 mg), eosin (1 mg), and pyridine (20 mL) were added into a quartz tube. The mixture was kept in a water-cooled bath and vigorously stirred by the gas bubbling (O 2 ), radiating with an iodine tungsten lamp (500 W, 220 V) for 0.5 h. After the reaction, the mixture was poured into ice-water (20 mL) and then extracted with 50 mL ethyl acetate twice. The ethyl acetate phase was washed with 50 mL saturated brine twice and then dried with anhydrous Na 2 SO 4 . The crude product was purified by chromatographic column (ethyl acetate/petroleumether = 1/5) to get pure ergosterol peroxide as white solid [22].

Synthesis of Amino-Acid Ester Derivatives
Under N 2 , dry methanol or ethanol (100 mL) was cooled down to −5 • C, and SOCl 2 (21 mL, 0.3 mmol) was added dropwise. Glycine, β-alanine, or γ-aminobutyric acid (0.1 mmol) was added to this solution, and stirring was continued at room temperature for 3 h. The solvent was removed under reduced pressure to obtain the correspondent pure product as colorless crystals.

Synthesis of Intermediate 7
To a stirred solution of ergosterol peroxide (200 mg, 0.56 mmol) and 4-nitrophenyl chloroformate (258 mg, 1.28 mmol) in DCM (5 mL) under N 2 gas, pyridine (100 mg, 1.28 mmol) was added, and stirring continued at room temperature for 3 h. The reaction course was followed on TLC. Then, DCM (20 mL) and H 2 O (20 mL) were added into the mixture. The DCM phase was combined, washed with brine, and then dried with anhydrous Na 2 SO 4 . The solution was concentrated to give yellow residue 7 for the next step.