Design, synthesis and mechanism of novel shikonin derivatives as potent anticancer agents

Abstract

In this study, a series of novel shikonin derivatives (30–49) were designed and synthesized and their anti-proliferative activities were evaluated against five different cancer cell lines, including HeLa, HepG2, MCF-7, BGC and A549. Some of the compounds show strong anti-proliferative effects against HeLa, HepG2 and MCF-7 with IC₅₀ values ranging from 1.26 to 18.50 μM and show lower side effects towards normal cell lines as compared to shikonin. Compared to other compounds and shikonin itself, compound 40 displayed much stronger anti-proliferative effects against various cancer cell lines. Furthermore, the flow cytometry results demonstrated that compound 40 could obviously induce apoptosis in a dose- and time-dependent manner and also cause cell cycle arrest at the G2/M phase. For further investigation of the aforementioned mechanisms, we performed Western blot experiments and found that the cleavage of PARP and upstream caspase-3 increased; moreover, caspase-9 was activated by cleavage but not caspase-8. These aforementioned results also indicate that compound 40 could induce caspase-9 involved apoptosis and G2/M phase cell cycle arrest via the P21, p-CDC2 (Tyr15) pathway independent of P53.

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1. Introduction

Cancer is one of the major health issues throughout the world. It has been ranked first in carnage the peoples and needs special attention for its eradication. Recently, some advancement has been achieved with effectively increased anticancer activities, yet the continued promise to the tedious task of discovering new anticancer agents remains critically important.^1–6 A variety of anti-cancer drugs have been developed and applied to cure cancer patients, but numerous drugs have not been able to show desirable results due to the problem of drug tolerance by cancer cells. Conventional anti-cancer drugs frequently result in apoptosis, although the cancer cells were sensitive to apoptotic initiation at an early stage, sooner or later they exhibit resistance to it because of the deregulation of the apoptotic machinery, which is indicated by the overexpression of anti-apoptosis proteins and also signaling defects in apoptosis.^7,8

Moreover, due to the high impact of cancer on human health, apoptosis plays a significant role in cell death mechanisms and largely takes place in various cancer cells as well.^9–11 In contrast, the mitochondrial apoptotic pathway contains caspase-9, which is an essential member of the caspase family of proteins,¹² and once the mitochondrial apoptotic pathway is activated, cytochrome C is released from the mitochondrial inter-membrane space and interacts with dATP and APAF1 to develop a composite receptor.^12,13 Moreover, the receptor also helps to recruit and activate caspase-9 to induce the activation of downstream proteases (for example, caspase-3 and caspase-7).¹⁴ PARP and caspase-3 are the vital agents of apoptosis that restrictively cleave almost all or part of the key proteins; thereafter cleaved caspase-3 and cleaved PARP exist in their activated forms. Therefore, after the increased level of all cleaved caspases and PARP, they induce apoptosis of cancer cells.^15,16

Several new drugs have been obtained from natural products and have been active against a wide variety of diseases, including cancer. Numerous plants from the Boraginaceae family have been used as anti-inflammatory, anti-arthritic and anti-microbial agents in Eurasia.^17–21 Shikonin and its derivatives, which primarily occur in Lithospermum erythrorhizon, have aroused great interest as hallmark molecules responsible for significant and fascinating anti-tumor activities by different mechanisms.^22,23 The specific protocols of the cancer research that relates to shikonin and alkannin have been focused on the induction of cell apoptosis^24,25 and necroptosis,^26,27 DNA topoisomerases, inhibition of angiogenesis,^28,29 and protein tyrosine kinases.^30,31 However, as a potential anti-cancer drug, shikonin itself is poorly soluble and believed to exert strong cytotoxic effects on normal cells.³² Hence, large numbers of researchers are dedicated to synthesizing new and effective shikonin derivatives.

During the past decades, a number of shikonin derivatives have been synthesized and studied for their anticancer activities. Many shikonin derivatives with side-chain hydroxyl group modifications have been synthesized and evaluated for their anti-tumor effects on various cancer cell lines. Most of the derivatives showed better cytotoxicity than the parent compound shikonin and the mechanisms of action were studied.^33,34

Moreover, acetylshikonin, isovalerylshikonin and SH-7 exhibited obvious inhibitory actions on topoisomerase I, stronger than the parent compound shikonin.^35–37 Shikonin glycosyl derivatives were also reported to show similar or stronger cytotoxicity than the parent shikonin.³⁸ To reduce the toxicity and side effects of shikonin, we synthesized some related derivatives of shikonin in our previous studies by the modification of its structure and found that the toxicity of shikonin was greatly reduced after ester modification.^39–44 In this study, we also decided to augment shikonin with some anticancer components to make it more effective against different cancer cell lines. Shikonin and augmenting parts were conjugated by introducing amino acids as bridges. Compounds of different steric configuration can be synthesized using different chiral amino acids as bridges and these new compounds could also act with different mechanisms against cancer cells. Based on the results of previous experiments, a series of novel shikonin derivatives were synthesized as potent anticancer agents using alanine and phenylalanine as bridges.

2. Results and discussion

2.1. Chemistry

With the aim of obtaining a series of shikonin derivatives containing two functional structures, we designed some twin medicines that introduced cantharidin, norcantharidin and their analogs into the shikonin skeleton. We finally conjugated norcantharidin or an adjacent dicarboxylic acid to the skeleton of shikonin using alanine, phenylalanine or norcantharidin as bridges (Schemes 1–4). However, after several attempts we could not link cantharidin to shikonin; this was probably due to cantharidin's amino acid conformation or the electron donating effect of the methyl that prevented the linkage. Among the compounds obtained, the yield of compounds 34, 35, 36 was >97% (highest), the yield of compounds 44, 45, 46 was <65% (lower), and the compounds 41, 42, 43 had the lowest yield of <30% due to their amino acid conformation. It was found that all compounds displayed different dimensional conformations. The carboxyl of 11 is completely exposed after derivatization, which is favorable for the next esterification reaction. However, the esterification reaction of 12 is blocked owing to its carboxyl group being partially covered by phthalic anhydride. No significant effect was found on the anti-proliferative activity due to the chiral configuration of amino acids in the bridges; however, derivatives in which phenylalanine was used as a bridge moiety showed better anti-proliferative activity than those bridging alanine.

Scheme 1 Synthesis of phenylalanine derivatives. ^aReagents and conditions: acetic acid, reflux, 12 h.

Scheme 2 Synthesis of alanine derivatives. ^aReagents and conditions: acetic acid, reflux, 12 h.

Scheme 3 Synthesis of phenylalanine shikonin esters. ^bReagents and conditions: DCC, DMAP, CH₂Cl₂ as solvent, ice-bath, overnight.

Scheme 4 Synthesis of alanine shikonin esters. ^bReagents and conditions: DCC, DMAP, CH₂Cl₂ as solvent, ice-bath, overnight.

2.2. Biological activities

2.2.1 In vitro antitumor and cytotoxicity evaluation. Antitumor activities of all the compounds against different cancer cell lines, i.e., HeLa (human cervix cell line), HepG2 (human liver cell line), MCF-7 (human breast cell line), A549 (human lungs cell line) and BGC (human gastric cell line) were determined using the MTT assay. Each of these cell lines were incubated with five different concentrations (0 μM, 0.3 μM, 1 μM, 3 μM, 10 μM) of all synthesized compounds for 24 hours and subsequently the IC₅₀ (half maximal inhibitory concentration) values were calculated as shown in Table 1.

Table 1 In vitro anticancer activity of shikonin derivatives against five cancer cell lines

Entry	Compd	IC50 ± SD^a (μM)
		HeLa	HepG2	MCF-7	BGC	A549	L02
a SD: standard deviation. All experiments were independently performed at least three times.
1	31	4.88 ± 0.87	2.33 ± 0.43	6.06 ± 1.49	20.53 ± 3.76	>100	>100
2	32	4.51 ± 0.66	2.48 ± 0.38	4.58 ± 0.34	>100	>100	>100
3	33	5.68 ± 1.34	2.98 ± 0.61	7.59 ± 1.15	52.70 ± 4.12	>100	>100
4	34	3.21 ± 0.45	2.96 ± 0.88	6.12 ± 0.84	>100	>100	>100
5	35	2.92 ± 0.67	1.69 ± 0.25	3.05 ± 0.21	>100	>100	>100
6	36	2.92 ± 0.34	5.18 ± 0.87	3.51 ± 0.33	36.12 ± 3.89	>100	>100
7	37	9.99 ± 1.85	2.77 ± 1.05	5.88 ± 0.65	>100	>100	>100
8	38	3.75 ± 0.44	3.65 ± 1.30	8.47 ± 0.53	7.63 ± 1.63	>100	>100
9	39	5.91 ± 0.98	2.21 ± 0.32	3.43 ± 0.21	>100	>100	>100
10	40	1.26 ± 0.25	1.92 ± 0.19	3.55 ± 0.34	11.56 ± 2.81	>100	>100
11	41	5.54 ± 1.24	2.33 ± 0.54	5.69 ± 0.88	>100	>100	>100
12	42	7.38 ± 2.08	2.67 ± 0.29	9.06 ± 1.54	>100	>100	>100
13	43	3.28 ± 0.71	2.15 ± 0.14	3.82 ± 0.58	>100	>100	>100
14	44	5.54 ± 1.90	5.14 ± 0.91	4.24 ± 0.82	92.85 ± 6.73	>100	>100
15	45	1.93 ± 1.36	5.35 ± 0.28	2.55 ± 0.35	13.34 ± 1.78	>100	>100
16	46	2.96 ± 0.98	5.11 ± 0.47	4.30 ± 0.51	14.67 ± 2.05	>100	>100
17	47	8.53 ± 0.67	2.10 ± 0.13	4.19 ± 0.32	14.94 ± 2.41	>100	>100
18	48	18.50 ± 2.71	3.16 ± 0.45	6.27 ± 0.69	57.54 ± 4.67	>100	>100
19	49	11.39 ± 2.28	3.57 ± 0.19	16.08 ± 2.09	61.25 ± 3.65	>100	>100
20	Shikonin	3.11 ± 0.82	0.92 ± 0.12	1.03 ± 0.21	2.20 ± 0.19	2.51 ± 0.35	65.34 ± 3.18

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From IC₅₀ values it was found that after modification not all the obtained compounds showed higher IC₅₀ against the five cell lines compared with shikonin. Interestingly, all the compounds lost their anti-proliferative activities against A549 and the anti-proliferative activities were much lower than that of shikonin against BGC. This is probably due to our modification that improved the selectivity and reduced the cytotoxicity. To determine the reduced cytotoxicity and clinically safe use of the compounds, MTT assay against L02 (human normal liver cell line) was performed and the results (Table 1) showed that all the compounds have no effects against L02. Some of the compounds showed strong effects against HeLa, HepG2 and MCF-7 cell lines with IC₅₀ values ranging from 1.26 μM to 18.50 μM. Furthermore, compound 40 showed best anti-proliferative activities against HeLa cells with the lowest IC₅₀ value (1.26 μM) compared to shikonin itself (3.11 μM) and this was selected for further experiments.

2.2.2 Apoptosis is induced in HeLa cells in dose- and time-dependent manner. Annexin V and PI staining can distinguish the living cells (annexin V−/PI−), as early apoptotic cells (annexin V+/PI−) and late apoptotic cells (annexin V+/PI+). To validate whether compound 40 could cause the growth inhibition of HeLa cells by in vitro apoptosis, annexin V-FITC/PI double staining assay was performed. After treating HeLa cells with different concentrations (0 μM, 1 μM, 3 μM, 10 μM) of compound 40 for 24 hours, HeLa cells showed considerable sensitivity to compounds 40 in a dose-dependent manner. For the highest concentration (10 μM), apoptotic rates reached up to 43.95% (Fig. 1A and B), thus suggesting that these compounds can induce apoptosis in vitro by targeting the cancer cells. Moreover, the time-dependent assay results also indicated that when HeLa cells were treated with compound 40 in a time-dependent manner, the percentage of apoptotic cells increased compared with the mock group, as shown in (Fig. 2A and B). In conclusion, it is obvious from the aforementioned results that compound 40 could induce apoptosis in HeLa cells in a dose- and time-dependent manner.

Fig. 1 (A and B) Cellular apoptosis study of compound 40 tested on HeLa cells in a dose dependent manner (0 μM, 1 μM, 3 μM, 10 μM). The percentage of early apoptotic cells in the lower right quadrant (annexin V-FITC-positive/PI-negative cells), as well as late apoptotic cells located in the upper right quadrant (annexin V-FITC-positive/PI-positive cells).

Fig. 2 (A and B) Cellular apoptosis study of compound 40 tested on HeLa cells in a time dependent manner compared (0 h, 8 h, 16 h and 24 h) with the mock group.

2.2.3 Cell cycle arrest in HeLa cells in dose-dependent manner. To gain better understanding of the potency of compound 40, we further explored the effect of compound 40 on the cell cycle to ascertain whether the cells are blocked in mitosis. HeLa cells were treated with different concentrations (0 μM, 1 μM, 3 μM, 10 μM) of compound 40 for 12 hours. The results demonstrated that the treatment of HeLa cells with compound 40 led to an obvious G2/M arrest in a concentration-dependent manner as shown in Fig. 3. The incubation of the cells with 3 μM of compound 40 caused 39.1% of cells to be arrested at the G2/M phase compared to the control. When the concentration of compound 40 increased to 10 μM, 47.9% of cells were arrested in the G2/M phase. In summary, effective doses of compound 40 seem to cause an arrest of cells in the G2/M phase, which ultimately leads to a significant increase in the number of apoptotic cells.

Fig. 3 Effect of compound 40 on the cell cycle distribution of HeLa cells in a dose dependent manner (0 μM, 1 μM, 3 μM, 10 μM).

2.2.4. Western blot analysis. In order to investigate the process of apoptosis, we further performed the Western blot analysis to detect the expression of some related proteins in the apoptosis-related pathway. From the Western blot results, we found that the cleavage of PARP and upstream caspase-3 increased, indicating that compound 40 could induce caspase activation in apoptosis. Furthermore, determining the protein levels of caspase-8 and caspase-9, we found that caspase-9 rather than caspase-8 was activated by cleavage. Thus, caspase-9 but not caspase-8 was involved in the apoptosis induced by compound 40. We also found the level of P21, which is the downstream target of P53, was upregulated, and enhanced the phosphorylation of its downstream target, CDC2. However, no obvious change was found for P53 as shown in Fig. 4. Based on the abovementioned cell cycle results, we concluded that P21 was activated independent of P53 and that the downstream CDC2 was phosphorylated, which contributed to the G2/M arrest in HeLa cells as described in the pathway (Fig. 5).

Fig. 4 Immunodetection of apoptosis related proteins of HeLa cells treated with different concentrations (0 μM, 1 μM, 3 μM, 10 μM) of compound 40.

Fig. 5 Compound 40 induced caspase-9 involved apoptosis and G2/M phase cell cycle arrest via P21, p-CDC2 (Tyr15) pathway independent of P53.

3. Conclusion

In our present study, we synthesized a series of novel shikonin derivatives (30–49). Selectivity and cytotoxicity assays were performed against five cancer cell lines along with one normal cell line. Some of the compounds showed strong effects against HeLa, HepG2 and MCF-7 with IC₅₀ values ranging from 1.26 μM to 18.50 μM. Among them, compound 40 displayed much stronger anti-proliferative effects against various cancer cell lines. Detailed apoptotic mechanistic studies with compound 40 suggested that through the cell cycle and apoptosis analysis, compound 40 showed the best anti-proliferation activities and exhibited strong ability to inhibit the proliferation of HeLa cancer cells by inducing high levels of apoptosis in a dose- and time-dependent manner, and also causes HeLa cells to be arrested in the G2/M phase. Western blot results also indicated that compound 40 could induce caspase-9 involved apoptosis and G2/M phase cell cycle arrest via P21, p-CDC2 (Tyr15) pathway independent of P53.

4. Materials and methods

4.1. Chemicals

All chemicals (reagent grade) were purchased from J&K Chemical Ltd. and Nanjing Chemical Reagent Co. Ltd. (China). All the ¹H NMR spectra were recorded on a Bruker DRX 500 spectrometer in CDCl₃. TLC was carried out on glass-backed silica gel sheets (silica gel 60 Å GF254). The ESI-MS spectra were obtained on a Mariner Biospectrometry Workstation (ESI-TOF) mass spectrometer. Chemical shifts (δ) for ¹H NMR spectra were reported in ppm (δ). Melting points (uncorrected) were measured on a XT4 MP Apparatus (Taike Corp., Beijing, China).

4.1.1 General synthesis procedure of compounds 8–17 and 21–29. A mixture of compounds 4–7 (50 mmol) and amino acid (50 mmol) were dissolved in acetic acid and stirred at 120 °C overnight. The reaction mixture was poured into ice water and a white precipitate was filtered and dried under vacuum to obtain the compounds 8–17 and 21–29 (Schemes 1 and 2).

4.1.2 General synthesis procedure of compounds 31–49. Compounds 8–17 and 21–29 were dissolved in 16 mL of dichloromethane and 0.072 g (0.354 mmol) of N,N′-dicyclohexylcarbodiimide (DCC) was added into the reaction system. The reaction mixture was stirred under a nitrogen atmosphere in an ice bath for 15 min. 0.004 g (0.044 mmol) of 4-dimethylaminopyridine (DMAP) was added and stirred in the ice bath for further 15 min. Then, 0.050 g of shikonin (0.175 mmol) was added to the reaction mixture and stirred in the ice bath for 12 hours to afford the target compounds 31–49 (Schemes 3 and 4).
4.1.2.1. (2R)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-((4R,7S)-1,3-dioxohexahydro-1H-4,7-epoxyisoindol-2(3H)-yl)-3-phenylpropanoate (31). Red powder, 76% yield, Mp: 69.5–71.4 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.592 (s, 1H); 12.477 (s, 1H); 7.286–7.136 (m, 8H); 7.033 (s, 1H); 6.133 (t, 1H, J₁ = 6.5 Hz, J₂ = 5 Hz); 5.148–5.066 (m, 1H); 5.024–4.991 (m, 2H); 4.898–4.843 (m, 1H); 3.504–3.308 (m, 3H); 2.833–2.715 (m, 2H); 2.607–2.560 (m, 1H); 2.501–2.440 (m, 1H); 1.975–1.518 (m, 4H); 1.369–1.095 (m, 4H). ESI-MS: calcd for C₃₃H₃₁NO₉ ([M − H]⁻) 584.20, found 584.1025. Anal. calcd for C₃₃H₃₁NO₉: C, 67.68; H, 5.34; N, 2.39; O, 24.59. Found: C, 67.71; H, 5.38; N, 2.41; O, 24.61.
4.1.2.2. (2S)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-((4R,7S)-1,3-dioxohexahydro-1H-4,7-epoxyisoindol-2(3H)-yl)-3-phenylpropanoate (32). Red powder, 70% yield, Mp: 77.3–79.2 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.591 (s, 1H); 12.476 (s, 1H); 7.286–7.136 (m, 8H); 7.034 (s, 1H); 6.135 (t, 1H, J₁ = 4.5 Hz, J₂ = 7.5 Hz); 5.104 (t, 1H, J₁ = 6.5 Hz, J₂ = 6.5 Hz); 5.025–4.991 (m, 2H); 4.909–4.851 (m, 1H); 3.504–3.456 (m, 2H); 3.360–3.309 (m, 1H); 2.834–2.793 (m, 1H); 2.731–2.702 (m, 1H); 2.611–2.537 (m, 1H); 2.502–2.430 (m, 1H); 1.899–1.772 (m, 2H); 1.975–1.518 (m, 3H); 1.369–1.095 (m, 3H). ESI-MS: calcd for C₃₃H₃₁NO₉ ([M − H]⁻) 584.20, found 584.2288. Anal. calcd for C₃₃H₃₁NO₉: C, 67.68; H, 5.34; N, 2.39; O, 24.59. Found: C, 67.70; H, 5.36; N, 2.40; O, 24.62.
4.1.2.3. 1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-((4R,7S)-1,3-dioxohexahydro-1H-4,7-epoxyisoindol-2(3H)-yl)-3-phenylpropanoate (33). Red powder, 70% yield, Mp: 68.7–70.5 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.591 (s, 1H); 12.476 (s, 1H); 7.286–7.136 (m, 8H); 7.034 (s, 1H); 6.135 (t, 1H, J₁ = 4.5 Hz, J₂ = 7.5 Hz); 5.104 (t, 1H, J₁ = 6.5 Hz, J₂ = 6.5 Hz); 5.025–4.991 (m, 2H); 4.909–4.851 (m, 1H); 3.504–3.456 (m, 2H); 3.360–3.309 (m, 1H); 2.834–2.793 (m, 1H); 2.731–2.702 (m, 1H); 2.611–2.537 (m, 1H); 2.502–2.430 (m, 1H); 1.899–1.772 (m, 2H); 1.975–1.518 (m, 3H); 1.369–1.095 (m, 3H). ESI-MS: calcd for C₃₃H₃₁NO₉ ([M − H]⁻) 584.20, found 584.2055. Anal. calcd for C₃₃H₃₁NO₉: C, 67.68; H, 5.34; N, 2.39; O, 24.59. Found: C, 67.69; H, 5.40; N, 2.43; O, 24.64.
4.1.2.4. (2R)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxoisoindolin-2-yl)-3-phenylpropanoate (34). Red powder, 98% yield, Mp: 70.5–71.8 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.566 (s, 1H); 12.443 (s, 1H); 7.849–7.736 (m, 4H); 7.224–7.196 (m, 7H); 6.948 (s, 1H, naphthoquinone–H); 6.108 (t, 1H, J₁ = 5 Hz, J₂ = 6 Hz); 5.266–5.233 (m, 1H); 5.019 (t, 1H, J₁ = 7.5 Hz, J₂ = 5.5 Hz); 3.640–3.572 (m, 2H); 2.640–2.610 (m, 1H); 2.499–2.440 (m, 1H); 1.615–1.585 (m, 3H); 1.483–1.465 (m, 3H). ESI-MS: calcd for C₃₃H₂₇NO₈ ([M − H]⁻) 564.17, found 564.2332. Anal. calcd for C₃₃H₂₇NO₈: C, 70.08; H, 4.81; N, 2.48; O, 22.63. Found: C, 70.13; H, 4.95; N, 2.71; O, 22.72.
4.1.2.5. (2S)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxoisoindolin-2-yl)-3-phenylpropanoate (35). Red powder, 97% yield, Mp: 60.9–62.6 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.565 (s, 1H); 12.443 (s, 1H); 7.849–7.833 (m, 2H); 7.751–7.735 (m, 2H); 7.240–7.168 (m, 7H); 6.949 (s, 1H); 6.109 (t, 1H, J₁ = 5.5 Hz, J₂ = 5.5 Hz); 5.268–5.236 (m, 1H); 5.021 (t, 1H, J₁ = 7 Hz, J₂ = 6 Hz); 3.653–3.546 (m, 2H); 2.641–2.611 (m, 1H); 2.499–2.441 (m, 1H); 1.615–1.585 (m, 3H); 1.484–1.465 (m, 3H). ESI-MS: calcd for C₃₃H₂₇NO₈ ([M − H]⁻) 564.17, found 564.2804. Anal. calcd for C₃₃H₂₇NO₈: C, 70.08; H, 4.81; N, 2.48; O, 22.63. Found: C, 70.21; H, 4.86; N, 2.51; O, 22.73.
4.1.2.6. 1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxoisoindolin-2-yl)-3-phenylpropanoate (36). Red powder, 98% yield, Mp: 61.6–63.8 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.565 (s, 1H); 12.442 (s, 1H); 7.839–7.810 (m, 2H); 7.745–7.713 (m, 2H); 7.210–7.194 (m, 7H); 6.949 (s, 1H); 6.108 (t, 1H, J₁ = 5.5 Hz, J₂ = 5.5 Hz); 5.266–5.237 (m, 1H); 5.022 (t, 1H, J₁ = 7 Hz, J₂ = 6 Hz); 3.641–3.573 (m, 2H); 2.640–2.612 (m, 1H); 2.497–2.442 (m, 1H); 1.614–1.586 (m, 3H); 1.483–1.465 (m, 3H). ESI-MS: calcd for C₃₃H₂₇NO₈ ([M − H]⁻) 564.17, found 564.2018. Anal. calcd for C₃₃H₂₇NO₈: C, 70.08; H, 4.81; N, 2.48; O, 22.63. Found: C, 70.23; H, 4.91; N, 2.62; O, 22.69.
4.1.2.7. (2R)-(R)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxohexahydro-1H-isoindol-2(3H)-yl)-3-phenylpropanoate (37). Red powder, 64% yield, Mp: 72.1–73.6 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.601 (s, 1H); 12.455 (s, 1H); 7.315–7.168 (m, 6H); 7.022 (s, 1H); 6.104 (t, 1H, J₁ = 5 Hz, J₂ = 7 Hz); 5.160–5.091 (m, 3H); 3.903 (d, 2H, J = 15 Hz); 3.778 (t, 3H, J₁ = 7 Hz, J₂ = 8.5 Hz); 3.532–3.509 (m, 3H); 2.943 (t, 3H, J₁ = 8 Hz, J₂ = 7 Hz); 2.782 (t, 3H, J₁ = 4 Hz, J₂ = 3.5 Hz); 1.715 (s, 3H); 1.586 (s, 3H). ESI-MS: calcd for C₃₃H₃₃NO₈ ([M − H]⁻) 570.62, found 570.631. Anal. calcd for C₃₃H₃₃NO₈: C, 69.34; H, 5.82; N, 2.45; O, 22.39. Found: C, 69.47; H, 5.96; N, 2.51; O, 22.42.
4.1.2.8. (R)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxohexahydro-1H-isoindol-2(3H)-yl)-3-phenylpropanoate (38). Red powder, 64% yield, Mp: 72.1–73.6 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.596 (s, 1H); 12.452 (s, 1H); 7.286–7.180 (m, 6H); 7.020 (s, 1H); 6.103 (t, 1H, J₁ = 5 Hz, J₂ = 6.5 Hz); 5.161–5.091 (m, 3H); 3.903 (d, 2H, J = 15 Hz); 3.778 (t, 3H, J₁ = 7 Hz, J₂ = 8.5 Hz); 3.568–3.508 (m, 3H); 2.943 (t, 3H, J₁ = 8 Hz, J₂ = 7 Hz); 2.782 (t, 3H, J₁ = 4 Hz, J₂ = 3.5 Hz); 1.716 (s, 3H); 1.585 (s, 3H). ESI-MS: calcd for C₃₃H₃₃NO₈ ([M − H]⁻) 570.62, found 570.631. Anal. calcd for C₃₃H₃₃NO₈: C, 69.34; H, 5.82; N, 2.45; O, 22.39. Found: C, 69.45; H, 5.93; N, 2.58; O, 22.46.
4.1.2.9. (2R)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-((3aR,4S,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindol-2(3H)-yl)-3-phenylpropanoate (39). Red powder, 64% yield, Mp: 60.5–62.6 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.594 (s, 1H); 12.448 (s, 1H); 7.286–7.256 (m, 2H); 7.223–7.173 (m, 5H); 6.960 (s, 1H); 6.286–6.220 (m, 2H); 6.121 (t, 1H, J₁ = 6 Hz, J₂ = 5.5 Hz); 5.140–5.077 (m, 2H); 3.554–3.438 (m, 2H); 3.232 (s, 1H); 3.168 (s, 1H); 2.676–2.592 (m, 2H); 2.536–2.466 (m, 2H); 1.715–1.582 (m, 8H). ESI-MS: calcd for C₃₄H₃₁NO₈ ([M − H]⁻) 580.20, found 580.2168. Anal. calcd for C₃₄H₃₁NO₈: C, 70.21; H, 5.37; N, 2.41; O, 22.01. Found: C, 70.34; H, 5.51; N, 2.55; O, 22.31.
4.1.2.10. 1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-((3aR,4S,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindol-2(3H)-yl)-3-phenylpropanoate (40). Red powder, 67% yield, Mp: 51.6–53.7 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.598 (s, 1H); 12.451 (s, 1H); 7.287–7.176 (m, 7H); 6.963 (s, 1H); 6.289–6.223 (m, 2H); 6.125 (t, 1H, J₁ = 4.5 Hz, J₂ = 6 Hz); 5.140–5.079 (m, 2H); 3.556–3.439 (m, 2H); 3.253 (s, 1H); 3.173 (s, 1H); 2.689–2.595 (m, 2H); 2.538–2.470 (m, 2H); 1.718–1.584 (m, 8H). ¹³C NMR (300 MHz, CDCl₃) δ: 185.37, 183.12, 175.23, 172.57, 161.21, 160.14, 150.24, 149.22, 139.93, 139.56, 139.15, 135.68, 134.33, 133.64, 132.26, 125.28, 118.37, 74.38, 62.86, 55.34, 48.80, 47.65, 16.89, 36.97, 31.23, 20.21, 19.87. ESI-MS: calcd for C₃₄H₃₁NO₈ ([M − H]⁻) 580.20, found 580.2368. Anal. calcd for C₃₄H₃₁NO₈: C, 70.21; H, 5.37; N, 2.41; O, 22.01. Found: C, 70.32; H, 5.41; N, 2.51; O, 22.31.
4.1.2.11. (2R)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-((4R,7S)-1,3-dioxohexahydro-1H-4,7-epoxyisoindol-2(3H)-yl)propanoate (41). Red powder, 27% yield, Mp: 63.2–64.8 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.601 (s, 1H); 12.484 (s, 1H); 7.028 (s, 2H); 7.049 (s, 1H); 6.151–6.063 (m, 1H); 6.035 (t, 1H, J₁ = 7.5 Hz, J₂ = 11 Hz); 5.168–5.063 (m, 1H); 5.024–4.991 (m, 2H); 4.898–4.843 (m, 1H); 3.504–3.308 (m, 3H); 2.833–2.715 (m, 2H); 2.607–2.560 (m, 1H); 2.501–2.440 (m, 1H); 1.975–1.518 (m, 3H); 1.568–1.412 (m, 3H); 1.387–1.196 (m, 3H). ESI-MS: calcd for C₂₇H₂₇NO₉ ([M − H]⁻) 508.50, found 508.5245. Anal. calcd for C₂₇H₂₇NO₉: C, 63.65; H, 5.34; N, 2.75; O, 28.26. Found: C, 63.72; H, 5.43; N, 2.81; O, 28.37.
4.1.2.12. (2S)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-((4R,7S)-1,3-dioxohexahydro-1H-4,7-epoxyisoindol-2(3H)-yl)propanoate (42). Red powder, 25% yield, Mp: 62.7–63.8 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.601 (s, 1H); 12.484 (s, 1H); 7.028 (s, 2H); 7.049 (s, 1H); 6.151–6.063 (m, 1H); 6.035 (t, 1H, J₁ = 7.5 Hz, J₂ = 11 Hz); 5.168–5.063 (m, 1H); 5.024–4.991 (m, 2H); 4.898–4.843 (m, 1H); 3.504–3.308 (m, 3H); 2.833–2.715 (m, 2H); 2.607–2.560 (m, 1H); 2.501–2.440 (m, 1H); 1.975–1.518 (m, 3H); 1.568–1.412 (m, 3H); 1.387–1.196 (m, 3H). ESI-MS: calcd for C₂₇H₂₇NO₉ ([M − H]⁻) 508.50, found 508.5245. Anal. calcd for C₂₇H₂₇NO₉: C, 63.65; H, 5.34; N, 2.75; O, 28.26. Found: C, 63.72; H, 5.43; N, 2.81; O, 28.37.
4.1.2.13. 1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-((4R,7S)-1,3-dioxohexahydro-1H-4,7-epoxyisoindol-2(3H)-yl)propanoate (43). Red powder, 30% yield, Mp: 71.8–72.7 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.601 (s, 1H); 12.484 (s, 1H); 7.028 (s, 2H); 7.049 (s, 1H); 6.151–6.063 (m, 1H); 6.035 (t, 1H, J₁ = 7.5 Hz, J₂ = 11 Hz); 5.168–5.063 (m, 1H); 5.024–4.991 (m, 2H); 4.898–4.843 (m, 1H); 3.504–3.308 (m, 3H); 2.833–2.715 (m, 2H); 2.607–2.560 (m, 1H); 2.501–2.440 (m, 1H); 1.975–1.518 (m, 3H); 1.568–1.412 (m, 3H); 1.387–1.196 (m, 3H). ESI-MS: calcd for C₂₇H₂₇NO₉ ([M − H]⁻) 508.50, found 508.5245. Anal. calcd for C₂₇H₂₇NO₉: C, 63.65; H, 5.34; N, 2.75; O, 28.26. Found: C, 63.72; H, 5.43; N, 2.81; O, 28.37.
4.1.2.14. (R)-(R)-2-(3-Hydroxybenzoyl)-6-methylhepta-1,5-dien-3-yl2-(1,3-dioxoisoindolin-2-yl)propanoate (44). Red powder, 60% yield, Mp: 76.5–77.3 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.569 (s, 1H); 12.444 (s, 1H); 7.952–7.923 (m, 2H); 7.821–7.794 (m, 2H); 7.204 (s, 2H); 6.950 (s, 1H); 6.080 (t, 1H, J₁ = 9 Hz, J₂ = 11 Hz); 5.117–4.978 (m, 2H); 2.677–2.585 (m, 1H); 2.518–2.419 (m, 1H); 1.795–1.755 (m, 3H); 1.629 (s, 3H); 1.485 (s, 3H). ESI-MS: calcd for C₂₆H₂₅NO₆ ([M − H]⁻) 446.17, found 446.1760. Anal. calcd for C₂₆H₂₅NO₆: C, 69.79; H, 5.63; N, 3.13; O, 21.45. Found: C, 69.36; H, 5.88; N, 3.09; O, 21.32.
4.1.2.15. (S)-(R)-2-(3-Hydroxybenzoyl)-6-methylhepta-1,5-dien-3-yl2-(1,3-dioxoisoindolin-2-yl)propanoate (45). Red powder, 55% yield. Mp: 55.7–57.5 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.581 (s, 1H); 12.476 (s, 1H); 7.929–7.901 (m, 2H); 7.822–7.770 (m, 2H); 7.208 (s, 2H); 7.013 (s, 1H); 6.078 (t, 1H, J₁ = 9 Hz, J₂ = 10.5 Hz); 5.117–4.975 (m, 2H); 2.634–2.568 (m, 1H); 2.516–2.441 (m, 1H); 1.795–1.741 (m, 3H); 1.648–1.565 (m, 3H); 1.491 (s, 3H). ¹³C NMR (300 MHz, CDCl₃) δ: 186.3, 184.8, 173.9, 171.5, 166.5, 165.8, 151.8, 140.2, 130.8, 130.1, 125.1, 122.5, 110.6, 73.8, 60.5, 31.9, 25.8, 19.1, 12.4. ESI-MS: calcd for C₂₆H₂₅NO₆ ([M − H]⁻) 446.17, found 446.1728. Anal. calcd for C₂₆H₂₅NO₆: C, 69.79; H, 5.63; N, 3.13; O, 21.45. Found: C, 69.76; H, 5.52; N, 3.09; O, 21.57.
4.1.2.16. (R)-2-(3-Hydroxybenzoyl)-6-methylhepta-1,5-dien-3-yl2-(1,3-dioxoisoindolin-2-yl)propanoate1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxoisoindolin-2-yl)propanoate (46). Red powder, 65% yield, Mp: 43.2–44.8 °C. ¹H NMR (500 MHz, CDCl₃) δ: 12.572 (s, 1H); 12.466 (s, 1H); 7.954–7.952 (m, 2H); 7.824–7.795 (m, 2H); 7.207 (s, 2H); 6.950 (s, 1H); 6.081 (t, 1H, J₁ = 7.5 Hz, J₂ = 11 Hz); 5.118–5.069 (m, 2H); 2.652–2.609 (m, 1H); 2.510–2.445 (m, 1H); 1.796–1.631 (m, 9H). ESI-MS: calcd for C₂₆H₂₅NO₆ ([M − H]⁻) 446.17, found 446.1728. Anal. calcd for C₂₆H₂₅NO₆: C, 69.79; H, 5.63; N, 3.13; O, 21.45. Found: C, 69.66; H, 5.52; N, 3.24; O, 21.40.
4.1.2.17. (2R)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxohexahydro-1H-isoindol-2(3H)-yl)propanoate (47). Red oil, 90% yield. ¹H NMR (500 MHz, CDCl₃) δ: 12.588 (s, 1H); 12.455 (s, 1H); 7.207 (s, 2H); 7.027 (s, 1H); 6.070 (t, 1H, J₁ = 11.5 Hz, J₂ = 6.5 Hz); 5.094 (t, 1H, J₁ = 11.5 Hz, J₂ = 12 Hz); 4.919–4.846 (m, 1H); 2.962–2.905 (m, 3H); 2.694–2.607 (m, 1H); 2.570–2.472 (m, 1H); 1.861–1.481 (m, 16H). ESI-MS: calcd for C₂₇H₂₉NO₈ ([M − H]⁻) 494.19, found 494.2081. Anal. calcd for C₂₇H₂₉NO₈: C, 65.44; H, 5.90; N, 2.83; O, 25.83. Found: C, 65.56; H, 6.08; N, 2.99; O, 25.97.
4.1.2.18. (2S)-1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxohexahydro-1H-isoindol-2(3H)-yl)propanoate (48). Red oil, 87% yield. ¹H NMR (500 MHz, CDCl₃) δ: 12.594 (s, 1H); 12.447 (s, 1H); 7.203 (s, 2H); 7.006 (s, 1H); 6.067 (t, 1H, J₁ = 6.5 Hz, J₂ = 13.5 Hz); 5.099 (t, 1H, J₁ = 12 Hz, J₂ = 12.5 Hz); 4.921–4.848 (m, 1H); 2.961–2.900 (m, 3H); 2.715–2.625 (m, 1H); 0.559–2.461 (m, 1H); 1.876–1.580 (m, 16H). ESI-MS: calcd for C₂₇H₂₉NO₈ ([M − H]⁻) 494.19, found 494.1879. Anal. calcd for C₂₇H₂₉NO₈: C, 65.44; H, 5.90; N, 2.83; O, 25.83. Found: C, 65.59; H, 6.67; N, 2.97; O, 25.90.
4.1.2.19. 1-(5,8-Dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-3-yl)-4-methylpent-3-en-1-yl-2-(1,3-dioxohexahydro-1H-isoindol-2(3H)-yl)propanoate (49). Red oil, 93% yield. ¹H NMR (500 MHz, CDCl₃) δ: 12.599 (s, 1H); 12.453 (s, 1H); 7.206 (s, 2H); 7.012 (s, 1H); 6.071 (t, 1H, J₁ = 6.5 Hz, J₂ = 4 Hz); 5.102 (t, 1H, J₁ = 11.5 Hz, J₂ = 12.5 Hz); 4.923–4.850 (m, 1H); 2.964–2.908 (m, 3H); 2.718–2.610 (m, 1H); 2.566–2.467 (m, 1H); 1.880–1.547 (m, 16H). ESI-MS: calcd for C₂₇H₂₉NO₈ ([M − H]⁻) 494.19, found 494.2054. Anal. calcd for C₂₇H₂₉NO₈: C, 65.44; H, 5.90; N, 2.83; O, 25.83. Found: C, 65.58; H, 6.01; N, 2.94; O, 25.90.

4.2. Cell culture

All cell lines were obtained from the State Key Laboratory of Pharmaceutical Biotechnology (Nanjing University) and maintained in Dulbecco's modified Eagle's medium (DMEM) with L-glutamine, supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO₂.

4.3. Cell viability assay (MTT assay)

Cells were planted in 96-well plates at appropriate densities to ensure exponential growth throughout the experimental period (2.0 × 10³ cells per well), and then allowed to adhere for 6 hours. Cells were then treated for 20 hours with four serial concentrations (0 μM, 1 μM, 3 μM, 10 μM) of each compound. Shikonin and norcantharidin were used as positive controls. After 20 hours incubation, 20 μL MTT solution was added to each well to a final concentration of 4 mg mL⁻¹. Plates were then incubated for a further 4 hours, after incubation all the plates were centrifuged (1500 rpm, 10 min) and then the entire medium was removed. 150 μL of DMSO was added to each well for coloration. The plates were shaken vigorously to ensure complete solubilization for 10 min at room temperature. Optometric density (OD) was read on a microplate reader (ELx800, BioTek, Highland Park, Winooski, VT, USA) at a wavelength of 570 nm and the data were subsequently analyzed using Origin7.5.

4.4. Analysis for apoptosis by flow cytometry

Apoptosis was detected using an Apoptosis Detection Kit (Invitrogen, Eugene, Oregon, USA). Briefly, cells were plated in 6 well plates (5.0 × 10⁴ cells per well) and incubated at 37 °C for 12 hours. Exponentially growing cells were then incubated with compound 40 at different concentrations (0 μM, 1 μM, 3 μM, 10 μM). Following 24 hours treatment, cells were collected and washed twice with PBS, once with 1× binding buffer and then stained with 5 μL of annexin V-FITC and 2.5 μL of PI (5 μg mL⁻¹) in 1× binding buffer for 30 min at room temperature in the dark. Apoptotic cells were quantified using a FACScan cytofluorometer (PT. MadagasiBrosa Inc. Jl. BatangHari No. 73, Propinsi Sumatera Utara, Indonesia). Statistical analysis was performed using WINMDI software version 2.8 (The Scripps Research Institute (TSRI), San Diego, CA, USA).

4.5. Analysis for cell cycle by flow cytometry

HepG2 cells were plated in 6-well plates (5.0 × 10³ cells per well) and incubated at 37 °C for 24 hours. Exponentially growing cells were then incubated with compound 40 at different concentrations (0 mM, 1 mM, 3 mM, and 10 mM). After 12 hours, untreated cells (control) or cells treated with compound 40 were centrifuged at 1500 rpm at 4 °C for 10 min, and then fixed in 70% ethanol at 4 °C for at least 12 hours and subsequently re-suspended in phosphate buffered saline (PBS) containing 0.1 mg mL⁻¹ RNase A and 5 μg mL⁻¹ propidium iodide (PI). The cellular DNA content for cell cycle distribution analysis was measured by flow cytometry using a Becton-Dickinson FACScan cytoflouorometer, plotting at least 10 000 events per sample. The percentage of cells in the G0/G1, S and G2/M phases of the cell cycle were determined using the Verity Software BD Accuri C6 software.

4.6. Western blot analysis

Cells were rinsed with PBS and lysed in cold RIPA buffer (10 mM Tris-HCl, 1 mM EDTA, 1% SDS, 1 mM DTT, 0.1 mM PMSF, protease inhibitors, 1% Nonidet P-40, pH 8.0). Lysates were centrifuged at 12 000 g for 10 min at 4 °C to remove cell debris and the protein content was then analyzed by a Micro BCA Protein Assay kit (Pierce). Aliquots of proteins (40–60 μg) were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to polyvinylidenedifluoride (PVDF) membranes. Membranes were blocked with 5% non-fat dry milk or BSA in TBST (TBS plus 0.1% Tween 20) for 1 hour. Blots were then probed with primary antibodies against PARP, caspase-3, caspase-8, caspase-9, P53, P21, CDC2, phosphor-CDC2 (Tyr 15) and α-tubulin, incubated at 4 °C overnight, followed by HRP-conjugated secondary antibodies and protein expression was detected with an enhanced chemiluminescent reagent (Cell Signaling Technology). The PARP antibody was purchased from Oncogene Company and other antibodies were purchased from Cell Signalling Technology Company. The autoradiographic intensity of each band was scanned.

Acknowledgements

The authors are grateful to the National Natural Science Foundation of China (NSFC) (nos 31171161, 31170275, 31470384), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R27), the Project of New Century Excellent Talents in University (NECT-11-0234), the fund for University Ph.D. Program from the Ministry of Education of China (20120091110037), and the Natural Science Foundation of the Jiangsu (BK2011414).

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Footnote

† These two authors equally contribute to this paper.

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