Potent Cytotoxicity of Novel L-Shaped Ortho-Quinone Analogs through Inducing Apoptosis

Twenty-seven L-shaped ortho-quinone analogs were designed and synthesized using a one pot double-radical synthetic strategy followed by removing methyl at C-3 of the furan ring and introducing a diverse side chain at C-2 of the furan ring. The synthetic derivatives were investigated for their cytotoxicity activities against human leukemia cells K562, prostate cancer cells PC3, and melanoma cells WM9. Compounds TB1, TB3, TB4, TB6, TC1, TC3, TC5, TC9, TC11, TC12, TC14, TC15, TC16, and TC17 exhibited a better broad-spectrum cytotoxicity on three cancer cells. TB7 and TC7 selectively displayed potent inhibitory activities on leukemia cells K562 and prostate cancer cells PC3, respectively. Further studies indicated that TB3, TC1, TC3, TC7, and TC17 could significantly induce the apoptosis of PC3 cells. TC1 and TC17 significantly induced apoptosis of K562 cells. TC1, TC11, and TC14 induced significant apoptosis of WM9 cells. The structure-activity relationships evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped ortho-quinone analogs.


Introduction
Over several decades, cancer continues to be the most awful disease due to its uncontrolled cell growth and the fact that it is a dominate killer of human beings worldwide [1]. Especially in China, millions of deaths have been caused by tumor. The common cancer types in Chinese male, in 2018, were lung, stomach, colorectum, liver, and esophageal cancer. Additionally, breast, lung, colorectum, thyroid, and stomach cancer were the common types in Chinese female [2]. The incidence of colorectal cancer in males and females has increased, however, the incidence of esophageal, stomach, and liver cancer has decreased between 2000 and 2011 [3]. Meanwhile, the incidence and mortality of prostate cancer and bladder cancer in males, together with obesity and hormonal exposure-related cancers, some nitrogen, oxygen-substituted, and amino acid substrates can improve aqueous solubility and antitumor activities [21][22][23][24][25], we have attempted to introduce a great diversity of oxygen-substituted, nitrogen-containing groups and amino acids. Thus, in this work, we developed quinone-directed agents by removing methyl at C-3 of the furan ring and introducing a diverse side chain at C-2 of the furan ring, culminating in the discovery of a promising scaffold. The inhibitory activity was assessed in vitro using three cell lines including K562, PC3, and WM9.

In Vitro Cytotoxicity Assay
The cytotoxic activities of 5 µmol/L of the synthesized L-shaped ortho-quinone analogs were determined by using three cancer cell lines ( Table 1). The results revealed that compounds TB1, TB3,  TB4, TB6, TC1, TC3, TC5, TC9, TC11, TC12, TC15, TC16, and TC17 showed a broad-spectrum potent inhibitory activity on the proliferation of the cancer cell lines, with a more than 70% inhibition rate, and TB7 showed better inhibitory activity on K562 cells as compared with other cells. Moreover, we observed that TC7 inhibited the growth of PC3 cells more efficiently than other cells.
The concentration inhibition curves ( Figure 2) were analyzed to calculate the IC50 values of the selected active compounds. The results indicated there was a dose-dependent trend of the inhibitory response of all active compounds on three cancer cells for treating 48 h. The IC50 values were summarized in Table 2 and show that the cytotoxicity of compounds TB3, TC1, TC3, TC7, TC9, and TC17 on PC3 were better (P < 0.05) than that of the positive control (tanshinone IIA and paclitaxel), and another active compound exhibited similar activity to that of the positive control. The inhibition activity of TC1 against the growth of K562 was better than that of the positive control, paclitaxel, and tanshinone IIA. Compounds TB6, TC1, TC11, TC14, and TC15 inhibited the growth of WM9 better (P < 0.05) than that of tanshinone IIA and paclitaxel. In summary, most of novel L-shaped orthoquinone analogs exhibited relatively better cytotoxicity activity as compared with the two positive controls, which indicated that the analogs containing L-shaped ortho-quinone as the core structure, Initially, dealing 8 with N-bromosuccinimide (NBS) and 2,2 -azobis(2-methylpropionitrile) (AIBN) afforded only trace amounts of 8a. Another intermediate, 8b, was obtained by Nelson's method [27] as shown in Scheme 1. Then, compound 9 was obtained from 8b through a second radical reaction. Considering the same reaction condition, we successfully got 9 from 8 through a bis-radical reaction. The brominated intermediate 9 was reacted with substituted phenol or amine to provide ortho-quinone derivatives TB1-TB9 and TC1-TC18, respectively. All the structures of ortho-quinone derivatives were identified through 1 H, 13 C, and HRMS.
In summary, we successfully established an effective synthetic strategy, which removed the methyl at C-3 of the furan ring and introduced diverse side chains at C-2 of the furan ring. In addition, we replaced the bromide of 9 with a variety of oxygen-substituted, nitrogen-containing group, and amino acid by a nucleophilic substitution.

In Vitro Cytotoxicity Assay
The cytotoxic activities of 5 µmol/L of the synthesized l-shaped ortho-quinone analogs were determined by using three cancer cell lines ( Table 1). The results revealed that compounds TB1, TB3,  TB4, TB6, TC1, TC3, TC5, TC9, TC11, TC12, TC15, TC16, and TC17 showed a broad-spectrum potent inhibitory activity on the proliferation of the cancer cell lines, with a more than 70% inhibition rate, and TB7 showed better inhibitory activity on K562 cells as compared with other cells. Moreover, we observed that TC7 inhibited the growth of PC3 cells more efficiently than other cells.  Figure 2. Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562, and WM9 cells by MTT assay. The IC50 values (µM) of the compounds were determined according to these curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments.  Figure 2. Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562, and WM9 cells by MTT assay. The IC50 values (µM) of the compounds were determined according to these curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments.  Figure 2. Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562, and WM9 cells by MTT assay. The IC50 values (µM) of the compounds were determined according to these curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments.  Figure 2. Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562, and WM9 cells by MTT assay. The IC50 values (µM) of the compounds were determined according to these curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments.  Figure 2. Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562, and WM9 cells by MTT assay. The IC50 values (µM) of the compounds were determined according to these curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments.  Figure 2. Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562, and WM9 cells by MTT assay. The IC50 values (µM) of the compounds were determined according to these curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments.  Figure 2. Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562, and WM9 cells by MTT assay. The IC50 values (µM) of the compounds were determined according to these curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments. WM9 cells by MTT assay. The IC50 values (µM) of the compounds were determined according to these curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments. curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments. curves at different incubation times. The 100 µL tested compounds were added to 96-well microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments. microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments. microculture plates and 100 µL cells (a final concentration of 5 × 10 4 /well) were incubated for 48 h at 37 °C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in the Methods section. Data was presented as mean ± SD of three independent experiments.           The concentration inhibition curves ( Figure 2) were analyzed to calculate the IC 50 values of the selected active compounds. The results indicated there was a dose-dependent trend of the inhibitory response of all active compounds on three cancer cells for treating 48 h. The IC 50 values were summarized in Table 2 and show that the cytotoxicity of compounds TB3, TC1, TC3, TC7, TC9, and TC17 on PC3 were better (P < 0.05) than that of the positive control (tanshinone IIA and paclitaxel), and another active compound exhibited similar activity to that of the positive control. The inhibition activity of TC1 against the growth of K562 was better than that of the positive control, paclitaxel, and tanshinone IIA. Compounds TB6, TC1, TC11, TC14, and TC15 inhibited the growth of WM9 better (P < 0.05) than that of tanshinone IIA and paclitaxel. In summary, most of novel L-shaped ortho-quinone analogs exhibited relatively better cytotoxicity activity as compared with the two positive controls, which indicated that the analogs containing L-shaped ortho-quinone as the core structure, possessed stronger anticancer activity. This result provided a preliminary biological activity basis for the investigation of anticancer candidate agents.

Structure-Activity Relationships Study
To obtain two series of analogs, we successfully built an effective synthetic strategy by removing the methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring. On the basis of the cytotoxicity results (Tables 1 and 2), a preliminary structure-activity relationships could be established. The TB series molecules bearing electron-withdrawing groups or multi-substituted groups such as compounds TB3, TB4, and TB6 showed a better inhibitory effect on PC3 cell lines, K562 cell lines, and WM9 cell lines, whereas the TB series molecules bearing alkane groups at the 2-position showed decreased cytotoxicity in PC3 cell lines, K562 cell lines, and WM9 cell lines, such as compounds TB9. The TC series molecules with electron-withdrawing groups, saturate six-membered rings, or multi-substituted groups emerged greater inhibitory effects on three cancer cell lines, such as TC11, TC12, TC15, and TC16, whereas the TC series molecules bearing donating groups or alkane groups at the 2-position showed reduced cytotoxicity in three cancer cell lines. The structure-activity relationships evaluation also showed that removing methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring were good strategies for improving the anticancer activity of L-shaped ortho-quinone analogs.

Effects of Active Compounds on Cell Apoptosis
According to the above IC 50 values of all active compounds, we selected six active compounds (TB3, TC1, TC3, TC7, TC9, and TC17) for PC3, three active compounds (TB6, TC1, and TC17) for K562, and four active compounds (TB6, TC1, TC11, and TC14) for WM9, based on their higher activities than that of the positive control and better selectivity and, then, studied their effects on cell apoptosis by microscope observation (Figure 3) and flow cytometry (Figure 4). The microscopic observations ( Figure 3A) showed that the number of PC3 cells was significantly reduced by treatments with 2.5 µmol/L of TB3, TC1, TC3, and TC7; the apoptotic bodies and cell fragments were significantly observed as compared with the control group. The PC3 cells treated with TC9 showed that the number of cells were significantly reduced, while fewer cells died and there were no significant apoptotic bodies. The PC3 cells treated with TC7 showed a significant decrease in the number of cells, meanwhile, some cells died, apoptotic bodies appeared obviously, and the morphology of some cells became an irregular shape of spindle length. Above all, the inhibitory activity of TB3, TC1, TC3, and TC7 may be through inducing apoptosis, another two compounds may be through different types. that the number of cells were significantly reduced, while fewer cells died and there were no significant apoptotic bodies. The PC3 cells treated with TC7 showed a significant decrease in the number of cells, meanwhile, some cells died, apoptotic bodies appeared obviously, and the morphology of some cells became an irregular shape of spindle length. Above all, the inhibitory activity of TB3, TC1, TC3, and TC7 may be through inducing apoptosis, another two compounds may be through different types.
The K562 cells treated with TC1 were obviously dead and dispersed, with the appearance of apoptotic bodies as comparing with the control group ( Figure 3B). The cells treated with TB6 had a significantly reduced number of cells and most cells clumped growth similar to the control cells. For the cells treated with TC17 we observed both dead cells and fewer clumps of cells. Furthermore, for the WM9 cell lines treated with TB6, TC1, TC11, and TC14 ( Figure 3C), we observed that the cells treated with TC1 and TC11 were obviously dead with a large number of apoptotic bodies and dispersed cells; the cells treated with TB6 showed a significant decrease in the number of cells and fewer dead cells. Observation of the cells treated with TC14 showed that the number of cells was significantly reduced, while some cells were obviously dead with apoptotic bodies appearing, and the morphology of some cells also became an irregular shape of spindle length. The above results indicated that TC1 can induce apoptosis for K562 and WM9 cells to inhibit the growth; TB6, TC11, TC14, and TC17 can jointly inhibit the proliferation of cell through a variety of mechanisms.

Instruments and Materials
High-resolution mass spectra (HRMS) were obtained on an electrospray ionization (ESI) mode on a Bruker ESI-QTOF mass spectrometry. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance NEO ( 1 H NMR, 600 MHz; 13 C NMR, 150 MHz, Bruker, Switzerland) with TMS as an internal standard. The IR spectra were recorded by using a FTIR Spectrometer (IR 200 Fourier Energy Spectrum Technology Co., Ltd., TianJin, China) and the KBr disk method was adopted. The melting points (mp) were determined on an WRX-4 microscope melting point apparatus. The column chromatography was performed on silica gel (Qingdao, 200-300 mesh) and the thin-layer (0.25 mm) chromatography (TLC) analysis was carried out on silica gel plates The K562 cells treated with TC1 were obviously dead and dispersed, with the appearance of apoptotic bodies as comparing with the control group ( Figure 3B). The cells treated with TB6 had a significantly reduced number of cells and most cells clumped growth similar to the control cells. For the cells treated with TC17 we observed both dead cells and fewer clumps of cells. Furthermore, for the WM9 cell lines treated with TB6, TC1, TC11, and TC14 ( Figure 3C), we observed that the cells treated with TC1 and TC11 were obviously dead with a large number of apoptotic bodies and dispersed cells; the cells treated with TB6 showed a significant decrease in the number of cells and fewer dead cells. Observation of the cells treated with TC14 showed that the number of cells was significantly reduced, while some cells were obviously dead with apoptotic bodies appearing, and the morphology of some cells also became an irregular shape of spindle length. The above results indicated that TC1 can induce apoptosis for K562 and WM9 cells to inhibit the growth; TB6, TC11, TC14, and TC17 can jointly inhibit the proliferation of cell through a variety of mechanisms.

Instruments and Materials
High-resolution mass spectra (HRMS) were obtained on an electrospray ionization (ESI) mode on a Bruker ESI-QTOF mass spectrometry. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance NEO ( 1 H NMR, 600 MHz; 13 C NMR, 150 MHz, Bruker, Switzerland) with TMS as an internal standard. The IR spectra were recorded by using a FTIR Spectrometer (IR 200 Fourier Energy Spectrum Technology Co., Ltd., TianJin, China) and the KBr disk method was adopted. The melting points (mp) were determined on an WRX-4 microscope melting point apparatus. The column chromatography was performed on silica gel (Qingdao, 200-300 mesh) and the thin-layer (0.25 mm) chromatography (TLC) analysis was carried out on silica gel plates (Qingdao, China). Other reagents were analytical grade or guaranteed reagent commercial product and used without further purification, unless otherwise noted.

Synthesis of 2-Allyl-3-hydroxy-1,4-naphthoquinone (7)
A mixture of lawsone 6 (10.0 g, 57.42 mmol) and anhydrous K 2 CO 3 (7.94 g, 57.42 mmol) in anhydrous DMF (100 mL) were stirred for 15 min at room temperature. Allyl bromide (17.37 g, 143.55 mmol) in DMF (5 mL) was added dropwise and stirred for 15 min at 0 • C. The mixture was refluxed at 120 • C for 3 h and then cooled to room temperature before it was poured into water and extracted with EA. The organic phase was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (eluent: petroleum ether/EtOAc 15:1) to afford 7 (7.8 g, 63% yield) as a light yellow solid. Other data was found in reference [26].

In Vitro Cytotoxicity Assay
The human cancer cell lines, including prostate cancer cells PC3, leukemia cells K562, and melanoma cells WM9, were stored in the biology laboratory of the Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences (Guiyang, China). All cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Sijiqing, Hangzhou, China) and incubated at 37 • C under 5% CO 2 , 95% air, and 95% humidity. Cytotoxicity was evaluated by performing the MTT assay [24]. Briefly, the cells were seeded in 96-well microculture plates at a density from 4 × 10 3 to 8 × 10 3 cells/well. Cells were then exposed to different concentrations of the assayed compounds for 48 h. Then, 20 µL of MTT solution (5 mg/mL) was added to each well and incubated at 37 • C for an additional 4 h. The medium was then removed and 200 µL Tris-DMSO solution was added. Plates were lightly shaking up to dissolve the dark blue formazan crystals and the absorbance was measured in an ELISA plate reader at 570 nm.

Flow Cytometry Assay
Cell apoptosis was determined by an inverted fluorescence microscope observation and flow cytometry as describe in our previous study [28]. Briefly, the cancer cells treated with compounds were harvested for centrifugation at 1000 rpm for 5 min at room temperature, washed twice with PBS and resuspended with binding buffer, and then PI (Sigma, St. Louis, MO, USA) was added to a final concentration of 20 mg/mL. The cell lines were analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Statistical Analysis
The IC 50 values were calculated from the semilogarithmic dose-response curves. The data were analyzed using SPSS 18.0 and reported as mean ± SD of the number of experiments indicated. For all measurements, one-way ANOVA followed by Student's t-test was used to assess the statistical significance of the difference between each group. The LSD method was used to assess the statistical significance of the difference between the two groups. A statistically significant difference was considered at the level of P < 0.05. The data are presented as the mean ± SEM of three assays.

Conclusions
In this study, 27 novel L-shaped ortho-quinone analogs were synthesized and evaluated for their anti-cancer activities. Compounds TB1, TB3, TB4, TB6, TC1, TC3, TC5, TC9, TC11, TC12, TC14,  TC15, TC16, and TC17 possessed broad-spectrum potent cytotoxicity against PC3, K562, and WM9 cells. With more than a 70% inhibitory rate, TB7 showed better inhibitory activity of K562 cells as compared with other cells. Moreover, we observed that TC7 inhibited the growth of PC3 cells more efficiently than other cells. Some of the active compounds such as TB3, TC1, TC3, and TC7 inhibited cell proliferation mainly through inducing apoptosis. The structure-activity relationships evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped ortho-quinone analogs.