Nitric oxide-releasing derivatives of brefeldin A as potent and highly selective anticancer agents

European Journal of Medicinal Chemistry 136 (2017) 131e143

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Nitric oxide-releasing derivatives of brefeldin A as potent and highly selective anticancer agents Kangtao Tian a, 1, Fanxing Xu b, 1, Xiang Gao a, Tong Han a, Jia Li a, Huaqi Pan c, **, Linghe Zang d, Dahong Li a, ***, Zhanlin Li a, Takahiro Uchita e, Ming Gao e, Huiming Hua a, * a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, and School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China b Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China c Shenyang Institute of Applied Ecology, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang 110016, PR China d School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China e School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien, Nishinomiya 663-8179, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 February 2017 Received in revised form 1 May 2017 Accepted 4 May 2017 Available online 5 May 2017

A series of NO-donating mono- or diester derivatives of brefeldin A were designed, synthesized and biologically evaluated. Some derivatives exhibited potent antiproliferative activity with low IC50 values. The most potent NO-donating hybrid 13b exhibited stronger cytotoxicity against human prostate cancer PC-3 cells, human colon carcinoma HT-29 cells and human liver cancer HepG-2 cells than BFA with IC50 values of 25 nM, 160 nM and 180 nM, respectively. More importantly, compound 13b showed good selectivity between human normal and tumor liver cells with selectivity index of 33. Additionally, 13b released higher levels of NO in HepG-2 cells than L-02 cells. Further mechanism concerning cellular apoptosis showed that 13b induced apoptosis and S phase cell cycle arrest in HepG-2 cells. Incubation with 13b increased the number of HepG-2 cells with collapsed mitochondrial membrane at low concentrations in dose-dependent manner. In addition, by using the Human Apoptosis Protein Array kit, several apoptosis-related proteins, including HO-1, HO-2 and survivin, were found to be markedly downregulated by 13b in HepG-2 cells. Furthermore, in western blot assay, 13b increased the expression of Bax, Cyt c and caspase 3, and reduced the relative levels of Bcl-2, Bcl-xl and pro-caspase 3 in HepG2 cells. © 2017 Published by Elsevier Masson SAS.

Keywords: Brefeldin A Antitumor Nitric oxide Apoptosis

1. Introduction Brefeldin A (BFA) (1, Fig. 1) was first isolated from Penicillium decumbens by Singleton et al. [1]. It is a macrolide antibiotic and secondary metabolite of Ascomycetes species. Most importantly, BFA exhibited a variety of biological activities including antitumor [2], antifungal [3], antiviral [4], antimitotic [5] and so on. The potent antiproliferative activity of BFA was tested by the National Cancer Institute's 60 cancer cell line assay (NCI-60) with the mean graph midpoint (MGM) GI50 value of 40 nM [6]. Furthermore, the

* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (H. Pan), [email protected] (D. Li), [email protected] (H. Hua). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2017.05.018 0223-5234/© 2017 Published by Elsevier Masson SAS.

mechanism was widely investigated, that BFA could induce apoptosis in numerous human cancer cell lines [7,8] and disrupt the cis Golgi apparatus [9]. Treatment of eukaryotic cells with BFA could induce the breakdown of vesicle-mediated protein transport, which resulted in the Golgi complex redistributing into the endoplasmic reticulum (ER) [10]. Moreover, it has been reported that BFA would cause apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways [11]. In addition, BFA also effectively inhibited clonogenic activity and the migration and matrix metalloproteinases-9 activity of MDA-MB-231 cells. Western blot analysis indicated that BFA could mediate the down-regulation of breast cancer stem cells marker CD 44 and anti-apoptotic proteins Bcl-2 and Mcl-1, as well as the reversal of epithelial-mesenchymal transition [12]. These biological results and mechanism investigation prompted BFA to be a potential therapeutic lead for future investigation [13,14].

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2. Results and discussion 2.1. Chemistry

Fig. 1. Chemical structure and key numbering of brefeldin A.

Although the cytostatic and apoptotic effects of BFA have been well studied, it is unsuitable to subject BFA to direct clinical development mainly because of its undesirable drug-like properties [15,16], including low selectivity between tumor and normal cells. In this study, the antiproliferative activity, especially the selectivity between cancerous and normal cells was improved, which was probably contributed to the introducing of nitric oxide (NO) donor moieties. NO is known as a notable mediator in biological system and a wide range of physiological processes [17,18]. Active macrophages produce NO, which induce cytostasis and generate cytotoxity on tumor cells, and the mechanism seems to be the inhibition of ribonucleotide reductase [19]. Recently, NO donor hybrid compounds have become a focus in the field of oncotherapy since NO influences the apoptosis process of tumor cells [18]. It is reported that OA/furoxan hybrid compounds exhibited remarkably improved cytotoxicity because of the cytotoxic effects of NO released from the furoxan moieties [20]. In addition, nonsteroidal anti-inflammatory drugs/nitrate hybrids have been identified as the prototypical chemopreventive agents against many forms of cancers [21]. We have also synthesized a variety of furoxan-based NO donating compounds, such as evodiamine/furoxan [22] and spirolactone-type diterpenoid/furoxan hybrids [23] with potent antitumor activities and/or selectivity between cancerous and normal cells. Therefore, generation of furoxan-based hybrids is a promising strategy to develop potential antitumor drug candidates. On the basis of above, the present article outlined the synthesis of 18 mono- or diester derivatives of BFA, which were obtained by introducing furoxan-type NO donors into the two hydroxyl (at C4 and C7 position) groups. The cytotoxicity, NO releasing ability and cellular mechanism of these derivatives were investigated. The research herein aimed to obtain a befitting dual BFA/NO releasing agent that still maintained the antiproliferative activity and showed selectivity between cancerous and normal cells.

The derivatives 11a
f, 12a
f and 13a
f were synthesized from thiophenol and BFA. The BFA was isolated from the fermentation liquor and mycelium of Eupenicillium brefeldianum, and characterized by 1H NMR, 13C NMR, high-resolution mass spectra, X-ray crystal structure analysis and optical rotation [24]. Thiophenol 2 was treated with chloroacetic acid in sodium hydroxide yielding the (phenylthio)acetic acid 3. A one-pot reaction of (phenylthio) acetic acid by oxidization with 30% H2O2 produced compound 4, which was further reacted with fuming HNO3 at 90  C to yield 5. Reacting 5 with ethanediol or propanediol in the presence of 30% NaOH in THF afforded 6a
b, which were further treated with anhydride, triethylamine (Et3N) and 4-dimethylaminopyridine (DMAP) to obtain intermediates 7a
f (Scheme 1). In order to get the ester derivatives at 4-position, the 4,7-OH groups of BFA were protected with tert-butyldimethylsilyl (TBS) groups using tertbutyldimethylsilyltriflate (TBSOTf) and 2,6-lutidine in dichloromethane (DCM). Then the TBS group on 4-OH of compound 8 was removed by tert-butylammonium fluoride (TBAF) in THF to obtain the intermediate 9. Reacting compound 9 with intermediates 7a
f in the presence of DMAP and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) afforded the intermediates 10a
f. The derivatives 11a
f were got from 10a
f by the reaction with TBAF in THF (Scheme 2). Target compounds 12a
f and 13a
f were prepared from the combination of 7a
f and BFA under the condition of DMAP and EDCI in DCM, followed by column chromatography on silica gel using petroleum ether-ethyl acetate (V:V 2:1) (Scheme 3). Intermediate 7a was further treated with TMSCHN2 to offer compound 14. 15 was prepared by the reaction of BFA with succinic anhydride in 2,6-lutidine (Scheme 4).

2.2. Pharmacology 2.2.1. The antiproliferative activities of BFA derivatives Applying the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay, the antiproliferative activities of BFA and the derivatives against one normal (L-02) and three tumor (PC-3, HT-29 and HepG-2) cell lines were evaluated and the results were summarized in Table 1. 5-FU was served as positive control. Comparing the antiproliferative activities of NO-donating BFA derivatives (11a
f, 12a
f and 13a
f) with those of NO donors 7a
f and BFA derivative 15, it could be found that the hybrids showed better antiproliferative profiles. It

Scheme 1. Synthesis of NO donors 7a
f. Reagents and conditions: (a) ClCH2COOH, NaOH (aq), 140  C, 2 h; (b) 30% H2O2, AcOH, rt, 3 h; (c) fuming HNO3, AcOH, 90  C, 4 h; (d) (1) HOCH2CH2OH, THF, 30% NaOH, 0  C, 4e8 h; (2) HOCH2CH2CH2OH, THF, 30% NaOH, 0  C, 72 h; (e) (1) succinic anhydride, Et3N, DMAP, DCM, rt, 2e3 h; (2) glutaric anhydride, Et3N, DMAP, DCM, rt, 6e24 h; (3) phthalic anhydride, Et3N, DMAP, DCM, rt, 12e15 h.

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Scheme 2. Synthesis of BFA derivatives 11a
f. Reagents and conditions: (a) TBSOTf, 2,6-lutidine, DCM, 0e25  C, 3 h; (b) TBAF, THF, 0e25  C, 24 h; (c) 7a¡f, EDCI, DMAP, DCM, rt, 1e2 h; (d) TBAF, THF, 0e25  C, 20 h.

Scheme 3. Synthesis of BFA derivatives 12a
f and 13a
f. Reagents and conditions: (a) 7a¡f, EDCI, DMAP, DCM, rt, 4e72 h.

was obvious that most of diester derivatives of BFA (except 13f) exhibited more potent cytotoxic activities against three human

cancer cell lines than the monoester derivatives. Moreover, the IC50 values of BFA derivatives against three cancer cell lines were lower

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Scheme 4. Synthesis of NO donor and BFA derivatives 14 and 15. Reagents and conditions: (a) TMSCHN2, CH3OH, 0  C, 10 min; (b) succinic anhydride, 2,6-lutidine, 110  C, 12 h.

Table 1 Antiproliferative activity (IC50, mM) of BFA derivatives and NO donors against three cancer and one normal cell lines. Compounds

BFA 11a 12a 13a 11b 12b 13b 11e 12e 13e 11c 12c 13c 11d 12d 13d 11f 12f 13f 15 7a 7b 7e 7c 7d 7f 5-FU

IC50 (mM)a

SIc

PC-3

HT-29

HepG-2

L-02

0.068 ± 0.003 0.12 ± 0.006 0.14 ± 0.004 0.056 ± 0.002 0.046 ± 0.004 0.059 ± 0.002 0.025 ± 0.001 0.11 ± 0.005 0.064 ± 0.003 0.13 ± 0.007 0.25 ± 0.009 0.73 ± 0.05 0.12 ± 0.008 0.095 ± 0.006 0.16 ± 0.01 0.083 ± 0.008 0.15 ± 0.01 0.095 ± 0.004 1.3 ± 0.09 1.6 ± 0.07 3.8 ± 0.3 5.1 ± 0.3 9.9 ± 0.4 >10 3.3 ± 0.2 4.9 ± 0.3 24.3 ± 0.4

0.16 ± 0.007 0.23 ± 0.008 0.46 ± 0.02 0.22 ± 0.01 0.28 ± 0.02 0.18 ± 0.01 0.16 ± 0.01 0.4 ± 0.02 0.28 ± 0.01 0.23 ± 0.01 0.79 ± 0.03 0.91 ± 0.04 1.2 ± 0.05 5.7 ± 0.4 1.6 ± 0.09 0.62 ± 0.03 0.86 ± 0.05 0.3 ± 0.02 4.4 ± 0.3 6.3 ± 0.2 >10 >10 >10 >10 >10 >10 4.9 ± 0.3

0.35 ± 0.01 0.51 ± 0.01 0.91 ± 0.02 0.51 ± 0.02 0.41 ± 0.02 0.82 ± 0.03 0.18 ± 0.01 0.82 ± 0.04 0.54 ± 0.04 0.55 ± 0.03 0.73 ± 0.03 1.3 ± 0.08 0.84 ± 0.04 1.8 ± 0.09 0.75 ± 0.06 0.38 ± 0.02 0.62 ± 0.04 0.62 ± 0.04 4.0 ± 0.3 1.6 ± 0.1 >10 4.0 ± 0.2 >10 6.4 ± 0.3 >10 >10 29.1 ± 0.9

<0.0004 5.0 ± 0.4 5.2 ± 0.2 5.9 ± 0.3 5.1 ± 0.2 5.1 ± 0.2 6.0 ± 0.1 5.7 ± 0.4 5.9 ± 0.4 6.2 ± 0.3 6.9 ± 0.5 7.8 ± 0.4 7.2 ± 0.4 6.7 ± 0.2 6.7 ± 0.3 5.1 ± 0.4 5.9 ± 0.4 5.4 ± 0.2 9.8 ± 0.5 9.5 ± 0.4 36 ± 0.9 32 ± 1 37 ± 0.8 29 ± 2 35 ± 1 40 ± 0.8 NTb

NCd 10 6 12 12 6 33 7 11 11 9 6 9 4 9 13 10 9 2 6 NC 8 NC 5 NC NC NC

a

IC50: concentration of the tested compound that inhibits 50% of cell growth. Not tested. c Selectivity index expressed as the ratio of IC50(L-02)/IC50(HepG-2) and rounded to the nearest integer. d NC: not calculated. Results are expressed as the means ± SD of three independent experiments. b

than those against human normal liver cell line L-02. Among these hybrids, compound 13b exhibited the most potent activities against PC-3, HT-29 and HepG-2 cells with IC50 values of 25 nM, 162 nM and 178 nM, respectively and also exhibited 15-fold less inhibitory activity against human normal liver L-02 cells than BFA. The selectivity indexes (SI) between HepG-2 and L-02 cells were calculated and compound 13b showed the highest SI of above 30 which deserved further investigation. 2.2.2. The ability of NO-releasing The NO-releasing ability of some synthesized derivatives was investigated by Griess assay. HepG-2 and L-02 cells were exposed to

Table 2 NO releasing ability of the BFA derivatives (mM/L) in vitro.a Compound

HepG-2

11b 12b 13a 13b 13d 13e

68.53 65.19 98.37 98.47 92.30 89.28

a

± ± ± ± ± ±

L-02 2.84 3.08 1.54 3.25 3.62 2.51

13.90 14.57 19.54 22.36 23.42 20.60

± ± ± ± ± ±

1.04 0.63 1.23 0.98 0.75 1.47

Results are expressed as mean ± SD of three independent experiments.

100 mM of 11b, 12b, 13a, 13b, 13d or 13e, respectively. The levels of NO generated in the cell lysates were determined and presented as those of nitrite. As shown in Table 2, the tested compounds could produce various levels of nitrite at the time point of 1 h in human liver cancer HepG-2 cells with the concentrations ranging from 65.19 mM to 98.47 mM, among which, the most active compound 13b released the highest concentration of nitrite. As expected, the treatment with selected compounds resulted in less nitrite in human normal liver L-02 cells which was consistent with our previous study [22]. It was also observed that diester derivatives 13a, 13b, 13d and 13e released higher levels of NO and exhibited stronger inhibitory effects than monoester derivatives 11b and 12b against HepG-2 cells, indicating that the amounts of NO released by these compounds were positively correlated with their antiproliferative activity against HepG-2 cells. 2.2.3. Apoptosis and cell cycle effects of 13b on HepG-2 cells Cell cycle arrest is an important sign for the inhibition of proliferation and the series of events take place in cells, leading to cell division and duplication (replication). Some NO-donating hybrids were reported to exhibit cell cycle arrest properties [25]. Based on the antiproliferative data, the most potent compound 13b was selected for further mechanistic study and the effects of 13b on HepG-2 cell cycle were examined. HepG-2 cells were treated with different concentrations of 13b (0, 0.0625 mM, 0.125 mM and 0.25 mM) for 48 h and stained with propidium iodide (PI), followed by flow cytometry analysis. As shown in Fig. 2A, compared with the control cells treated with 0.1% DMSO, when HepG-2 cells were treated with increasing concentrations of 13b, the percentage of cells in the S phase increased from 43.88% to 48.20%, 51.22% and 72.16%, respectively. The results indicated that compound 13b suppressed HepG-2 cell proliferation through inducing cell cycle arrest at S phase. Cancer cells arrested at S phase are connected with high sensitivity to apoptotic triggers [26]. Changes of the morphological

K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143 Fig. 2. (A) Effects of 13b on HepG-2 cell cycle. Cells were treated with the indicated concentrations of 13b for 48 h and stained with PI, followed by flow cytometry analysis. (B) Effects of 13b on cell cycle of hemoglobin pretreated HepG2 cells. HepG-2 cells were pretreated with 10 mM of hemoglobin for 1 h and then treated with different concentrations of 13b for 48 h.

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characters in HepG-2 cells were studied by Hoechst 33258 staining under fluorescence microscopy to estimate whether the growth inhibitory activities of 13b were related to the inducing of apoptosis. As shown in Fig. 3, the vehicle-treated HepG-2 cells (0.1% DMSO) displayed weak homogeneous blue in the nuclei while the 13b (0.0625 mM, 0.125 mM and 0.25 mM) treated HepG-2 cells exhibited bright chromatin condensation and nuclear fragmentation, a hallmark of apoptosis. These data indicated that the treatment of 13b induced HepG-2 cells apoptosis. Apoptosis is a process of programmed cell death and cancer cells usually have an abnormal ability of proliferation mainly due to the lack of apoptosis. Thus, activation of apoptosis could reduce the accumulation of cancer cells. Furthermore, high levels of NO could act as a tumor cell apoptosis inducer [27,28]. So, we investigated whether the antiproliferative activity of 13b against HepG-2 cells was attributed to apoptosis. HepG-2 cells were treated with variable concentrations of hybrid 13b (0.0625 mM, 0.125 mM and 0.25 mM). The cells were harvested and stained with 7aminoactinomycin D (7-AAD) and annexin-V, then the percentage of apoptotic cells was determined by flow cytometry analysis. Significantly, it was found that hybrid 13b induced apoptosis of HepG-2 cells in a dose-dependent manner (Fig. 4A). After 48 h treatment, the observed apoptotic cells were 14.86%, 34.65% and 55.24% (early and late apoptosis) at the indicated concentrations, indicating the remarkable apoptosis inducing effects of hybrid 13b, while the vehicle-treated HepG-2 cells displayed 6.54% of apoptotic cells. To verify the contribution of NO to apoptosis and cell cycle arrest, NO scavenger hemoglobin was explored in the tests of apoptosis and cell cycle arrest. HepG-2 cells were pretreated with 10 mM of hemoglobin for 1 h and then treated with 13b (0, 0.0625 mM, 0.125 mM and 0.25 mM) for 48 h. The apoptotic and cell cycle effects were reduced (Figs. 2B and 4B). These results suggested that NO produced by 13b would contribute to the apoptosis and cell cycle arrest of HepG-2 cells.

2.2.4. Mitochondrial membrane potential assay The mitochondrion is an organelle that plays an essential role in bio-energetic function. Its dysfunction, including the loss of mitochondrial membrane potential, is a key event that takes place during drug-induced apoptosis [29]. JC-1 is a commercially available florescent probe. In healthy cells, JC-1 aggregates in the mitochondria and spontaneously forms complexes which fluoresce red. In necrotic or apoptosis cells, JC-1 exists in the monomeric form and stains the cytosol green. HepG-2 cells were incubated with different concentrations (0.0625 mM, 0.125 mM and 0.25 mM) of hybrid 13b for 48 h prior to staining with JC-1. As depicted in Fig. 5, the percentage of apoptotic cells increased in a dosedependent fashion (16.58%, 35.45% and 60.14%, respectively). These results showed that incubation with 13b increased the number of HepG-2 cells with collapsed mitochondrial membrane at low concentrations and in dose-dependent manner. So compound 13b would induce apoptosis in HepG-2 cells via mitochondriarelated pathways. 2.2.5. The effects on apoptosis-related proteins of 13b-treated HepG-2 cells Heme oxygenase (HO) is a cytoprotective enzyme that can be overexpressed in some pathological conditions, including certain cancers [30]. Recent work reported significant tumor regression mediated by the downregulation of both HO-1 and HO-2 systems [31]. Survivin is a member of the inhibitor in apoptosis family that has been found to abundantly express in fetal tissues and the most common human tumors, and is considered absent in normal tissues in initial studies [32]. At the same time, survivin, a member of the inhibitor of apoptosis protein family that inhibits caspases and blocks cell death, is highly expressed in most cancers and associated with poor clinical outcome [33]. In order to identify the contribution of cellular proteins involved in apoptosis, we detected the relative expression of 35 key apoptosis associated proteins in HepG-2 cells by using the Human Apoptosis Protein Array kit

Fig. 3. Hoechst staining of 13b treated HepG-2 cells. Cells were treated with the indicated concentrations of 13b or vehicle (control) for 48 h, stained with Hoechst 33258 and examined with a fluorescent microscope.

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Fig. 4. Flow cytometry analysis of 13b induced HepG-2 cell apoptosis. (A) Cells were incubated with the indicated concentrations of 13b for 48 h, and the apoptotic cells were determined by flow cytometry after staining with 7-aminoactinomycin D (7-AAD) and annexin-V. (B) Effects of 13b on apoptosis of hemoglobin pretreated HepG-2 cells. HepG2 cells were pretreated with 10 mM of hemoglobin for 1 h and then treated with different concentrations of 13b for 48 h. The apoptotic cells were determined by flow cytometry after staining with PI and annexin-V.

Fig. 5. The effects of 13b on mitochondrial membrane potential (MMP) of HepG-2 cells.

[34,35]. Cell lysates were collected after the treatment of 13b for the analysis of apoptosis-related proteins. The images of the most

significantly changed proteins in the Human Apoptosis Protein Array were showed in Fig. 6. It is notable that 13b could

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Fig. 6. The effects of 13b on the expression of apoptosis-related proteins of HepG2 cells. Cells were treated with 13b or control for 24 h and the total cell lysates were collected for the analysis by Human Apoptosis Protein Array kit.

downregulate the expression of HO-1, HO-2 and survivin. Besides, the whole photographic films of the Human Apoptosis Protein Array assay were shown in supplementary materials. NO is known to induce apoptosis by activating the intrinsic mitochondrionmediated pathway, which results from a variety of key events, including the loss of mitochondrial membrane potential, the regulation of both pro- and anti-apoptotic Bcl-2 family proteins, the release of cytochrome c (Cyt c) into the cytoplasm, and finally the activation of caspase 9 and caspase 3. We therefore conducted western blot analysis of some apoptosis related proteins. And the results revealed that 13b could dramatically increase the relative expression of pro-apoptotic Bax, total Cyt c and caspase 3, but reduced the expression of anti-apoptotic Bcl-2, Bcl-xl and procaspase 3 levels in HepG-2 cells (Fig. 7). 3. Conclusions In summary, a novel series of BFA derivatives capable of releasing NO were designed and synthesized for the treatment of cancer by the multifunctional drug approach. All the synthesized compounds were evaluated for their antiproliferative effects against PC-3, HT-29, HepG-2 and L-02 cell lines and the NOreleasing ability was also measured. Compound 13b exhibited the strongest cytotoxicity against three selected tumor cell lines with IC50 values of 25 nM, 160 nM and 180 nM, respectively. Compound 13b also displayed 15-fold less cytotoxicity than BFA against L02 cells. So NO-donor/BFA hybrid 13b was highly selective between tumor and normal liver cells and NO donating groups might be effective to increase antiproliferative selectivity. Additionally, 13b released higher levels of NO than other tested derivatives in HepG2 cells. And all the selected derivatives released lower levels of NO in normal L-02 liver cells than tumorous HepG-2 cells. Further mechanistic studies revealed that 13b induced apoptosis and S phase arrest in HepG-2 cells. Incubation with 13b increased the number of cells with collapsed mitochondrial membrane potentials at low concentrations in dose-dependent manner. So the mitochondrial pathway would be involved in BFA derivatives-mediated apoptosis. Results of the Human Apoptosis Protein Array assay disclosed that the apoptosis related proteins survivin, HO-1 and HO-2 were downregulated. These proteins played a critical role in the apoptotic process of HepG-2 cells induced by compound 13b. In addition, western blot analysis revealed that treatment with 13b increased the expression of Bax, Cyt c and caspase 3 and reduced the relative levels of Bcl-2, Bcl-xl and pro-caspase 3 in HepG-2 cells. Taken together, compound 13b, a potent compound with NO releasing ability, was discovered and deserved further investigation as a potential chemotherapeutic agent for colon and liver cancer. 4. Experimental 4.1. General methods Most chemicals and solvents were purchased from commercial

Fig. 7. Western blot of apoptosis-related proteins in HepG-2 cells after exposure to different concentrations (0, 0.0625 mM, 0.125 mM and 0.25 mM) of 13b for 48 h.

sources. Further purification by standard methods were employed when necessary. Melting points were determined on an XT-4 micro melting point apparatus and uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker ARX-400 NMR spectrometer in the indicated solvents (TMS as internal standard): the values of the chemical shifts were expressed in d values (ppm) and the coupling constants (J) in Hz. Mass spectra were obtained on Agilent 1100 Ion trap mass spectrometer. HR-MS were carried out on Agilent Q-TOF B.05.01 (B5125.2).

4.2. General procedure to synthesize compounds 11a
f, 12a
f and 13a
f 6a
b was obtained from thiophenol (2) in a four-step sequence according to the literature [26,27]. Then 6a
b (0.5 mmol) in 10 mL of anhydrous DCM was mixed with corresponding anhydride (1 mmol), Et3N (2.5 mmol) and catalytic amount of DMAP by stirring at room temperature for 2e24 h. The mixture was dissolved in 15 mL of H2O and extracted with DCM (15 mL  3). The organic layer was combined, washed with water and saturated NaCl solution sequentially, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography (petroleum ether-ethyl acetate 1:1) to afford compounds

K. Tian et al. / European Journal of Medicinal Chemistry 136 (2017) 131e143

7a
f. 9 was obtained from 1 in a two-step sequence according to the literature [6]. To a solution of 9 (32 mg, 0.08 mmol) in 2.5 mL of anhydrous DCM, 7a
f (0.08 mmol), EDCI (29 mg, 0.15 mmol) and catalytic amount of DMAP were added. The reaction solution was stirred for 1e2 h at room temperature. The mixture was poured into 1 mL of H2O, and extracted with DCM (5 mL  3). The organic layer was combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and concentrated in vacuo. The residues were purified by chromatography on silica gel (petroleum etherethyl acetate 5:1) to afford compounds 10a
f. Then 10a
f (0.06 mmol) in 2 mL of THF was mixed with TBAF (0.19 mL, 0.19 mmol) by stirring at room temperature for 20 h. The mixture was dissolved in 1 mL of H2O and extracted with ethyl acetate (5 mL  3). The organic layer was combined, washed with water and saturated NaCl solution sequentially, dried over anhydrous Na2SO4, and concentrated in vacuo. The residues were purified by column chromatography (petroleum ether-ethyl acetate 2:1) to yield compounds 11a
f. To a solution of 7a
f (0.5 mmol) in 10 mL of anhydrous DCM, BFA (140 mg, 0.5 mmol), EDCI (191.7 mg, 1 mmol) and catalytic amount of DMAP were added. The reaction solution was stirred for 4e24 h at room temperature and quenched with 10 mL of H2O, extracted with DCM (10 mL  3), dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate 2:1) to afford the title compounds 12a
f and 13a
f. 4.2.1. 4-(2-((4-(((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-1-yl)oxy)-4-oxobutanoyl)oxy)ethoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide(11a) White solid, 21% yield: mp. 148e151  C; 1H NMR (400 MHz, DMSO-d6): d 7.72e8.09 (5H, m), 7.23 (1H, dt, J ¼ 15.8, 3.1 Hz), 5.71 (1H, m), 5.62 (1H, dt, J ¼ 15.7, 1.6 Hz), 5.22 (2H, m), 4.72 (1H, m), 4.36e4.60 (5H, m), 4.06 (1H, m), 2.59e2.73(4H, m), 0.72e1.99 (15H, m); 13C NMR (400 MHz, DMSO-d6): d 171.86, 171.00, 164.98, 158.69, 148.43, 136.70, 136.17, 131.59, 130.16, 130.00, 129.81, 128.74, 128.36, 117.00, 110.48, 76.38, 71.26, 70.35, 69.23, 61.70, 54.92, 49.03, 42.93, 42.84, 33.38, 31.38, 28.51, 27.22, 26.39, 20.61; HRMS (ESI) m/z calcd for C30H36N2O12S [MþNa]þ 671.1881, found 671.1881.

139

(1H, dd, J ¼ 15.0, 9.6 Hz), 5.00 (1H, m), 4.72 (1H, m), 4.37e4.60 (8H, t, J ¼ 4.4 Hz), 2.55e2.76 (8H, m), 0.72e2.54 (15H, m); 13C NMR (400 MHz, DMSO-d6): d 171.87, 171.83, 171.39, 170.93, 164.87, 158.68, 158.68, 148.02, 137.22, 137.20, 136.11, 136.09, 135.76, 130.66, 129.97, 129.97, 129.97, 129.97, 128.33, 128.33, 128.33, 128.33, 117.24, 110.46, 110.44, 76.09, 75.06, 71.25, 69.21, 69.21, 61.59, 61.46, 49.14, 49.14, 42.43, 40.06, 37.38, 33.38, 31.29, 28.78, 28.57, 28.47, 26.26, 20.56; HRMS (ESI) m/z calcd for C44H48N4O20S2 [MþNa]þ 1039.2196, found 1039.2197. 4.2.4. 4-(2-((5-(((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-1-yl)oxy)-5-oxopentanoyl)oxy)ethoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11b) Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6): d 7.72e8.01 (5H, m), 7.24 (1H, dd, J ¼ 15.8, 3.2 Hz), 5.70 (1H, m), 5.58 (1H, dd, J ¼ 15.9, 1.7 Hz), 5.19e5.23 (2H, m), 4.72 (1H, m), 4.41e4.62 (4H, t, J ¼ 3.9 Hz), 4.54 (1H, d, J ¼ 3.4 Hz), 4.04 (1H, m), 2.50 (4H, m), 0.73e2.40 (17H, m); 13C NMR (400 MHz, DMSO-d6): d 172.64, 171.99, 165.29, 159.07, 149.03, 137.60, 137.08, 136.48, 130.34, 130.34, 130.13, 128.69, 128.69, 117.27, 110.84, 76.44, 71.62, 70.73, 69.72, 61.67, 49.48, 43.32, 43.20, 40.68, 33.76, 32.78, 32.73, 31.75, 26.74, 20.96, 20.00; HRMS (ESI) m/z calcd for C31H38N2O12S [MþNa]þ 685.2038, found 685.2046. 4.2.5. 4-(2-((5-(((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6methyl-4-oxo-4,6,7,8, 9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-13-yl)oxy)-5-oxopentanoyl)oxy)ethoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12b) Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6): d 7.73e7.99 (5H, m), 7.32 (1H, dd, J ¼ 15.6, 3.0 Hz), 5.70 (2H, m), 5.19 (1H, d, J ¼ 5.6 Hz), 5.14 (1H, dd, J ¼ 15.1, 9.6 Hz), 5.00 (1H, m), 4.70 (1H, m), 4.39e4.60 (4H, t, J ¼ 4.2 Hz), 4.00 (1H, m), 2.32e2.37 (4H, m), 0.73e2.46 (17H, m); 13C NMR (400 MHz, DMSO-d6): d 172.30, 172.09, 165.60, 158.69, 154.03, 137.20, 136.27, 136.14, 130.12, 129.98, 129.98, 128.31, 128.31, 116.47, 110.46, 74.99, 74.00, 70.89, 69.33, 61.25, 51.88, 42.69, 38.16, 38.07, 33.43, 32.71, 32.38, 31.37, 26.38, 20.69, 19.79; HRMS (ESI) m/z calcd for C31H38N2O12S [MþNa]þ 685.2038, found 685.2039.

4.2.2. 4-(2-((4-(((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6methyl-4-oxo-4,6,7,8, 9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-13-yl)oxy)-4-oxobutanoyl)oxy)ethoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12a) Yellow oil, 7% yield: 1H NMR (400 MHz, DMSO-d6): d 7.74e8.01 (5H, m), 7.32 (1H, dd, J ¼ 15.5, 3.0 Hz), 5.70e5.75 (2H, m), 5.21 (1H, d, J ¼ 5.7 Hz), 5.16 (1H, dd, J ¼ 15.3, 9.7 Hz), 4.98 (1H, m), 4.70 (1H, m), 4.39e4.59 (4H, t, J ¼ 4.2 Hz), 4.00 (1H, m), 2.54e2.60 (4H, m), 0.73e2.46 (15H, m); 13C NMR (400 MHz, DMSO-d6): d 171.86, 171.42, 165.60, 158.68, 154.03, 137.21, 136.14, 130.10, 129.99, 129.99, 128.34, 128.34, 116.46, 110.47, 75.29, 73.99, 70.89, 69.23, 61.47, 51.89, 42.67, 40.05, 38.17, 33.43, 31.38, 28.81, 28.58, 26.37, 20.69, 20.69; HRMS (ESI) m/z calcd for C30H36N2O12S [MþNa]þ 671.1881, found 671.1864.

4.2.6. 4,4'-((((5,5'-(((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1] oxacyclotridecine-1,13-diyl)bis(oxy))bis(5-oxopentanoyl))bis(oxy)) bis(ethane-2,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5oxadiazole 2-oxide) (13b) Yellow oil, 13% yield: 1H NMR (400 MHz, DMSO-d6): d 7.72e8.02 (10H, m), 7.23 (1H, dd, J ¼ 15.7, 3.3 Hz), 5.73 (1H, m), 5.60 (1H, dd, J ¼ 15.8, 1.7 Hz), 5.28 (1H, m), 5.16 (1H, dd, J ¼ 15.1, 9.8 Hz), 5.01 (1H, m), 4.73 (1H, m), 4.43e4.62 (8H, m), 2.39e2.63 (8H, m), 0.73e2.34 (19H, m); 13C NMR (400 MHz, DMSO-d6): d 172.83, 172.29, 172.05, 171.56, 164.86, 158.69, 158.69, 148.34, 137.21, 136.13, 136.13, 136.10, 135.78, 130.68, 129.97, 129.97, 129.97, 129.97, 128.31, 128.31, 128.31, 128.31, 117.13, 110.46, 110.46, 75.76, 74.74, 71.26, 69.33, 69.33, 61.28, 61.24, 51.28, 42.43, 42.40, 37.43, 33.40, 32.71, 32.66, 32.40, 32.28, 31.27, 26.26, 20.57, 19.82, 19.77; HRMS (ESI) m/z calcd for C46H52N4O20S2 [MþNa]þ 1067.2509, found 1067.2510.

4.2.3. 4,4'-((((4,4'-(((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1] oxacyclotridecine-1,13-diyl)bis(oxy))bis(4-oxobutanoyl))bis(oxy)) bis(ethane-2,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5oxadiazole 2-oxide) (13a) White solid, 11% yield: mp. 60e65  C; 1H NMR (400 MHz, DMSO-d6): d 7.72e8.02 (10H, m), 7.22 (1H, dd, J ¼ 15.8, 3.1 Hz), 5.75 (1H, m), 5.64 (1H, dd, J ¼ 15.7, 1.9 Hz), 5.27 (1H, d, J ¼ 9.6 Hz), 5.18

4.2.7. 4-(2-((2-((((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-1-yl)oxy)carbonyl)benzoyl)oxy)ethoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11e) Yellow oil, 5% yield: 1H NMR (400 MHz, DMSO-d6): d 7.51e7.95 (9H, m), 7.26 (1H, dd, J ¼ 15.9, 3.4 Hz), 5.71 (1H, m), 5.55 (1H, dd, J ¼ 15.8, 1.7 Hz), 5.42 (1H, dq, J ¼ 10.6, 1.78 Hz), 5.24 (1H, dd, J ¼ 15.1, 9.76 Hz), 4.59e4.73 (5H, m), 4.55 (1H, d, J ¼ 3.6 Hz), 4.08 (1H, m),

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0.76e2.56 (15H, m); 13C NMR (400 MHz, DMSO-d6): d 167.16, 165.73, 165.16, 159.06, 148.26, 137.56, 137.06, 136.32, 132.51, 132.27, 132.03, 130.96, 130.39, 130.12, 130.12, 128.66, 128.66, 128.66, 128.66, 117.61, 110.76, 78.08, 71.69, 70.78, 69.36, 63.05, 49.70, 43.36, 43.16, 40.73, 33.80, 31.78, 26.65, 20.91; HRMS (ESI) m/z calcd for C34H36N2O12S [MþNa]þ 719.1881, found 719.1862. 4.2.8. 4-(2-((2-((((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6methyl-4-oxo-4,6,7,8, 9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-13-yl)oxy)carbonyl)benzoyl)oxy)ethoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12e) Yellow oil, 6% yield: 1H NMR (400 MHz, DMSO-d6): d 7.53e7.95 (9H, m), 7.33 (1H, dd, J ¼ 15.7, 3.04 Hz), 5.72 (2H, m), 5.22 (1H, d, J ¼ 5.7 Hz), 5.19 (1H, m), 5.13 (1H, dd, J ¼ 15.2, 9.58 Hz), 4.64e4.71 (5H, m), 4.03 (1H, m), 0.73e2.26 (15H, m); 13C NMR (400 MHz, DMSO-d6): d 166.77, 166.10, 165.57, 158.71, 153.94, 137.17, 136.08, 136.00, 131.74, 131.69, 131.43, 131.39, 130.25, 129.81, 129.81, 128.87, 128.70, 128.19, 128.19, 116.50, 110.45, 76.65, 73.97, 70.86, 69.09, 62.59, 51.87, 42.69, 40.04, 38.05, 33.41, 31.33, 26.34, 20.66; HRMS (ESI) m/z calcd for C34H36N2O12S [MþNa]þ 719.1881, found 719.1884. 4.2.9. 4,4'-((((2,2'-((((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1] oxacyclotridecine-1,13-diyl)bis(oxy))bis(carbonyl))bis(benzoyl)) bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5oxadiazole 2-oxide) (13e) White solid, 27% yield: mp. 50e54  C; 1H NMR (400 MHz, DMSO-d6): d 7.51e7.92 (18H, m), 7.25 (1H, dd, J ¼ 15.8, 3.5 Hz), 5.75 (1H, m), 5.58 (1H, dd, J ¼ 15.8, 1.6 Hz), 5.51 (1H, m), 5.24 (1H, m), 5.16 (1H, dd, J ¼ 15.0, 9.8 Hz), 4.71 (1H, m), 4.63 (8H, m), 0.73e2.32 (15H, m); 13C NMR (400 MHz, DMSO-d6): d 167.05, 167.02, 166.54, 165.79, 165.10, 159.06, 159.02, 147.84, 137.54, 137.54, 136.36, 136.29, 135.90, 132.45, 132.32, 132.20, 132.01, 131.92, 131.81, 131.54, 131.26, 131.06, 130.17 130.17, 130.09, 130.09, 129.37, 129.33, 129.29, 129.15, 128.62, 128.62, 128.56, 128.56, 117.96, 110.82, 110.74, 77.83, 76.91, 71.68, 69.41, 69.31, 62.99, 62.92, 49.73, 42.99, 40.43, 37.77, 33.81, 31.67, 26.49, 20.87; HRMS (ESI) m/z calcd for C52H48N4O20S2 [MþNa]þ 1135.2196, found 1135.2176. 4.2.10. 4-(3-((4-(((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-1-yl)oxy)-4-oxobutanoyl)oxy)propoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11c) Yellow oil, 5% yield: 1H NMR (400 MHz, DMSO-d6): d 7.72e8.02 (5H, m), 7.23 (1H, dd, J ¼ 15.8, 3.1 Hz), 5.71 (1H, m), 5.61 (1H, dd, J ¼ 15.8, 1.6 Hz), 5.18e5.24 (2H, m), 4.71 (1H, m), 4.57 (1H, s), 4.15e4.45 (4H, t, J ¼ 6.1 Hz), 4.03 (1H, m), 2.59e2.73 (4H, m), 0.72e2.48 (17H, m); 13C NMR (400 MHz, DMSO-d6): d 172.38, 171.45, 165.33, 159.14, 148.79, 137.54, 137.09, 136.51, 130.38, 130.38, 130.15, 128.72, 128.72, 117.35, 110.89, 76.72, 71.60, 70.72, 68.62, 60.89, 49.43, 43.31, 43.20, 40.63, 33.75, 31.75, 28.93, 28.93, 27.76, 26.76, 20.96; HRMS (ESI) m/z calcd for C31H38N2O12S [MþNa]þ 685.2038, found 685.2039. 4.2.11. 4-(3-((4-(((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6methyl-4-oxo-4,6,7,8, 9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-13-yl)oxy)-4-oxobutanoyl)oxy)propoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12c) Yellow oil, 8% yield: 1H NMR (400 MHz, CDCl3): d 7.63e8.10 (5H, m), 7.34 (1H, dd, J ¼ 15.7, 3.1 Hz), 5.91 (1H, dd, J ¼ 15.6, 1.9 Hz), 5.71 (1H, m), 5.21 (1H, dd, J ¼ 15.2, 9.7 Hz), 5.08 (1H, m), 4.86 (1H, m), 4.30e4.51 (4H, t, J ¼ 6.2 Hz), 4.12 (1H, m), 2.59e2.64 (4H, m), 0.71e2.38 (17H, m); 13C NMR (400 MHz, CDCl3): d 172.37 171.88, 165.98, 159.16, 154.42, 137.51, 136.68, 136.53, 130.46, 130.38, 138.38,

128.73, 128.73, 116.83, 110.91, 75.64, 74.37, 71.27, 68.55, 60.71, 52.28, 43.05, 38.56, 38.45, 33.81, 31.77, 30.17, 29.25, 29.00, 27.75, 21.07; HRMS (ESI) m/z calcd for C31H38N2O12S [MþNa]þ 685.2038, found 685.2035. 4.2.12. 4,4'-((((4,4'-(((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1] oxacyclotridecine-1,13-diyl)bis(oxy))bis(4-oxobutanoyl))bis(oxy)) bis(propane-3,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5oxadiazole 2-oxide) (13c) Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6): d 7.71e8.01 (10H, m), 7.22 (1H, dd, J ¼ 15.9, 3.3 Hz), 5.74 (1H, m), 5.63 (1H, dd, J ¼ 15.8, 1.7 Hz), 5.25 (1H, m), 5.18 (1H, dd, J ¼ 15.0, 9.7 Hz), 4.94 (1H, m), 4.71 (1H, m), 4.15e4.45 (8H, t, J ¼ 6.1 Hz), 2.52e2.70 (8H, m), 0.72e2.52 (19H, m); 13C NMR (400 MHz, DMSO-d6): d 172.36, 172.36, 171.83, 171.40, 165.26, 159.15, 159.15, 148.45, 137.53, 137.50, 136.50, 136.50, 136.16, 131.03, 130.37, 130.37, 130.37, 130.37, 128.72, 128.72, 128.72, 128.72, 117.60, 110.88, 110.88, 76.43, 75.42, 71.62, 68.62, 68.56, 60.90, 60.73, 49.57, 42.80, 40.42, 37.78, 33.76, 31.67, 29.22, 28.98, 28.90, 28.90, 27.75, 27.75, 26.64, 20.93; HRMS (ESI) m/ z calcd for C46H52N4O20S2 [MþNa]þ 1067.2509, found 1067.2456. 4.2.13. 4-(3-((5-(((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-1-yl)oxy)-5-oxopentanoyl)oxy)propoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11d) White solid, 23% yield: mp. 55e60  C; 1H NMR (400 MHz, DMSO-d6): d 7.70e8.00 (5H, m), 7.22 (1H, dd, J ¼ 15.8, 3.3 Hz), 5.68 (1H, m), 5.55 (1H, dd, J ¼ 15.8, 1.6 Hz), 5.19 (2H, m), 4.69 (1H, m), 4.52 (1H, d, J ¼ 3.7 Hz), 4.13e4.44 (4H, t, J ¼ 6.1 Hz), 4.03 (1H, m), 2.32e2.45 (4H, m), 0.68e2.10 (19H, m); 13C NMR (400 MHz, DMSOd6): d 172.78, 171.95, 165.29, 159.16, 149.05, 137.56, 137.09, 136.51, 130.38, 130.38, 130.14, 128.72, 128.72, 117.25, 110.88, 76.42, 71.62, 70.73, 68.62, 60.62, 49.50, 43.32, 43.20, 40.69, 33.76, 32.83, 32.79, 31.75, 27.76, 26.74, 20.97, 20.28; HRMS (ESI) m/z calcd for C32H40N2O12S [MþNa]þ 699.2194, found 699.2196. 4.2.14. 4-(3-((5-(((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-13-yl)oxy)-5-oxopentanoyl)oxy)propoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12d) Yellow oil, 11% yield: 1H NMR (400 MHz, DMSO-d6): d 7.73e8.02 (5H, m), 7.33 (1H, dd, J ¼ 15.5, 3.0 Hz), 5.72 (2H, m), 5.20 (1H, d, J ¼ 5.7 Hz), 5.16 (1H, dd, J ¼ 15.1, 9.7 Hz), 5.00 (1H, m), 4.71 (1H, m), 4.14e4.45 (4H, t, J ¼ 6.1 Hz), 4.00 (1H, m), 2.28e2.36 (4H, m), 0.73e2.46 (19H, m); 13C NMR (400 MHz, DMSO-d6): d 172.82, 172.44, 165.99, 159.16, 154.43, 137.55, 136.68, 136.53, 130.51,130.40, 130.40, 128.71, 128.71, 116.84, 110.88, 75.40, 74.38, 71.28, 68.63, 60.59, 52.31, 43.06, 40.44, 38.57, 33.81, 33.16, 32.82, 31.77, 27.75, 26.77, 21.07, 20.26; HRMS (ESI) m/z calcd for C32H40N2O12S [MþNa]þ 699.2194, found 699.2193. 4.2.15. 4,4'-((((5,5'-(((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4-oxo4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1] oxacyclotridecine-1,13-diyl)bis(oxy))bis(5-oxopentanoyl))bis(oxy)) bis(propane-3,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)-1,2,5oxadiazole 2-oxide) (13d) Yellow oil, 19% yield: 1H NMR (400 MHz, DMSO-d6): d 7.70e7.99 (10H, m), 7.21 (1H, dd, J ¼ 15.8, 3.3 Hz), 5.72 (1H, m), 5.56 (1H, dd, J ¼ 15.9, 1.8 Hz), 5.26 (1H, m), 5.15 (1H, dd, J ¼ 15.2, 9.8 Hz), 4.99 (1H, m), 4.69 (1H, m), 4.10e4.44 (8H, m), 2.30e2.48 (8H, m), 0.70e2.65 (23H, m); 13C NMR (400 MHz, DMSO-d6): d 172.79, 172.76, 172.37, 171.92, 165.21, 159.15, 159.15, 148.68, 137.56, 137.56, 136.49, 136.49, 136.13, 131.07, 130.36, 130.36, 130.36, 130.36, 128.70, 128.70, 128.70, 128.70, 117.52, 110.86, 110.86, 76.15, 75.13, 71.62,

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68.60, 68.60, 60.60, 60.57, 55.27, 49.60, 42.82, 37.80, 33.77, 33.10, 32.81, 32.81, 32.70, 31.67, 27.75, 27.75, 26.63, 20.93, 20.21, 20.21; HRMS (ESI) m/z calcd for C48H56N4O20S2 [MþNa]þ 1095.2822, found 1095.2771.

DMSO and 5-FU was used as the positive reference. The IC50 values were calculated according to the inhibition ratios.

4.2.16. 4-(3-((2-((((1R,2E,6S,10E,11aS,13S,14aR)-13-hydroxy-6methyl-4-oxo-4,6, 7,8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-1-yl)oxy)carbonyl)benzoyl)oxy)propoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11f) Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6): d 7.70e8.02 (9H, m), 7.27 (1H, dd, J ¼ 15.8, 3.44 Hz), 5.71 (1H, m), 5.66 (1H, dd, J ¼ 15.8, 1.7 Hz), 5.38 (1H, m), 5.25 (1H, dd, J ¼ 15.2, 9.8 Hz), 4.74 (1H, m), 4.55 (1H, d, J ¼ 3.5 Hz), 4.37e4.49 (4H, t, J ¼ 6.2 Hz), 4.09 (1H, m), 0.75e2.57 (17H, m); 13C NMR (400 MHz, DMSO-d6): d 167.48, 165.72, 165.24, 159.20, 148.26, 137.51, 137.06, 136.51, 132.57, 131.97, 130.58, 130.36, 130.36, 130.20, 129.32, 129.18, 129.04, 128.73, 128.73, 117.75, 110.92, 78.02, 71.71, 70.77, 68.54, 62.09, 49.71, 43.41, 43.16, 40.76, 38.46, 33.80, 28.74, 27.58, 20.93; HRMS (ESI) m/z calcd for C35H38N2O12S [MþNa]þ 733.2038, found 733.2048.

NO-releasing data were acquired for tested compounds using the Griess reaction in HepG-2 and L-02 cells according to the manufacturer's instructions (Beyotime, China). Briefly, cells were treated with 100 mM of each compound for 60 min. Subsequently, the cells were harvested and their cell lysates were prepared and then mixed with Griess reagent for 10 min at 37  C, followed by measurement at 550 nm on a microplate reader. The cells treated with 0.4% of DMSO in medium were used as negative controls for the background levels of nitrite production, while sodium nitrite at different concentrations was prepared as the positive control for the establishment of a standard curve [22].

4.2.17. 4-(3-((2-((((1R,2E,6S,10E,11aS,13S,14aR)-1-hydroxy-6methyl-4-oxo-4,6,7, 8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta [f][1]oxacyclotridecin-13-yl)oxy)carbonyl)benzoyl)oxy)propoxy)-3(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12f) Yellow oil, 9% yield: 1H NMR (400 MHz, DMSO-d6): d 7.66e8.02 (9H, m), 7.33 (1H, dd, J ¼ 15.6, 3.0 Hz), 5.71 (2H, m), 5.23 (1H, d, J ¼ 5.7 Hz), 5.14 (2H, m), 4.70 (1H, m), 4.38e4.50 (4H, t, J ¼ 6.2 Hz), 4.03 (1H, m), 0.73e2.46 (17H, m); 13C NMR (400 MHz, DMSO-d6): d 167.41, 166.57, 165.97, 159.20, 154.36, 137.50, 136.52, 136.52, 132.20, 132.08, 131.90, 131.67, 130.64, 130.36, 130.36,129.17, 129.06, 128.75, 128.75, 116.90, 110.94, 77.03, 74.37, 71.26, 68.50, 61.93, 52.37, 43.09, 40.44, 38.55, 33.81, 31.74, 27.63, 26.73, 21.06; HRMS (ESI) m/z calcd for C35H38N2O12S [MþNa]þ 733.2038, found 733.2045. 4.2.18. 4,4'-((((2,2'-((((1R,2E,6S,10E,11aS,13S,14aR)-6-methyl-4oxo-4,6,7,8,9,11a, 12,13,14,14a-decahydro-1H-cyclopenta[f][1] oxacyclotridecine-1,13-diyl)bis(oxy))bis(carbonyl))bis(benzoyl)) bis(oxy))bis(propane-3,1-diyl))bis(oxy))bis(3-(phenylsulfonyl)1,2,5-oxadiazole2-oxide) (13f) Yellow oil, 8% yield: 1H NMR (400 MHz, DMSO-d6): d 7.69e7.99 (18H, m), 7.28 (1H, dd, J ¼ 15.8, 3.6 Hz), 5.77 (1H, m), 5.69 (1H, dd, J ¼ 15.8, 1.7 Hz), 5.50 (1H, m), 5.21 (2H, m), 4.74 (1H, m), 4.34e4.44 (8H, m), 0.74e2.32 (19H, m); 13C NMR (400 MHz, DMSO-d6): d 167.36, 167.25, 166.63, 165.77, 165.16, 159.16, 159.14, 147.91, 137.50, 137.49, 136.48, 136.48, 135.95, 132.49, 132.34, 132.09, 131.97, 131.97, 131.91, 131.79, 131.26, 130.66, 130.33, 130.33, 130.33, 130.33, 129.34, 129.20, 129.11, 129.04, 128.70, 128.70, 128.70, 128.70, 118.06, 110.89, 110.89, 76.90, 71.71, 71.52, 68.53, 67.78, 62.11, 61.87, 49.83, 43.01, 40.44, 40.44, 38.45, 30.17, 28.74, 27.58, 23.62, 22.77; HRMS (ESI) m/z calcd for C54H52N4O20S2 [MþNa]þ 1163.2509, found1163.2464. 4.3. MTT assay The antiproliferative activities were determined by the MTT method as previously described [36]. The assay was performed in 96-well plates. Cells were added to each well and incubated for 24 h at 37  C in a humidified atmosphere of 5% CO2. Then cells were incubated in the presence or absence of test compounds. After 72 h, 20 mL of MTT solution (5 mg/mL) per well was added to each cultured medium, which was incubated for another 4 h. Then, DMSO (150 mL) was added to each well and the plates were shaken for 10 min at room temperature. After 10 min, the OD of each well was measured on a Microplate Reader (BIO-RAD) at the wavelength of 570 nm. In these experiments, the negative reference was 0.1%

4.4. Griess assay

4.5. Cell cycle assay Cell cycle effects were tested by flow cytometry with PI (KGA511, KeyGEN Biotech, Nanjing, China). HepG-2 cells were plated in 6well plates and incubated at 37  C for 24 h. Cells were then incubated with 13 b at certain concentrations for 48 h. Besides, HepG2 cells were pretreated with 10 mM of hemoglobin for 1 h and then treated with different concentrations (0, 0.0625 mM, 0.125 mM and 0.25 mM) of 13b for 48 h. Then, cells were centrifuged and fixed in 70% ethanol at 4  C overnight and resuspended in PBS containing 100 mL RNase A and 400 mL PI. Cellular DNA content, for cell cycle distribution analysis, was measured using a flow cytometer (FACS Calibur BectoneDickinson) [37]. 4.6. Cell apoptosis assay Apoptosis was analyzed using Annexin-V and 7aminoactinomycin D (7-AAD) double staining by flow cytometry according to the manufacturer's instructions (KGA1024, KeyGEN Biotech, Nanjing, China) in order to detect apoptotic cells [38]. The HepG-2 cells were seeded in 6-well plates to grow overnight, and then treated with or without 13 b at indicated concentrations for 48 h. Cells were then washed twice in PBS and resuspended in Annexin V binding buffer. Annexin V-FITC was then added and the mixture was incubated for 15 min under dark conditions at 25  C. 7AAD was added just prior to acquisition. The percentage of cells positive for 7-AAD and/or Annexin V-FITC was reported inside the quadrants. In addition, HepG-2 cells were pretreated with 10 mM of hemoglobin for 1 h and then treated with different concentrations (0, 0.0625 mM, 0.125 mM and 0.25 mM) of 13b for 48 h. The percentages of apoptotic HepG-2 cells were determined by PI and annexin-V staining and flow cytometry. 4.7. Mitochondrial membrane potential assay Briefly, HepG-2 cells were incubated with 13b or vehicle for 48 h, and then washed with PBS and stained with JC-1 dye under dark conditions according to the manufacturer's instruction (KGA601, KeyGEN Biotech, Nanjing, China). The percentage of cells with healthy or collapsed mitochondrial membrane potentials was monitored by flow cytometry analysis [39]. 4.8. Human Apoptosis Protein Array assay The Human Apoptosis Protein Array (Proteome Profiler™ Ary009; R&D Systems) was used to analyze the relative expression levels of 35 apoptosis-related proteins according to manufacturer instructions in HepG-2 cells. Proteins were extracted from cells

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treated for 24 h with compound 13b which were incubated with the array membrane overnight at 4  C, followed by incubation with a biotinylated detection antibody cocktail at room temperature for 1 h. A digital imaging system (Bio Pioneer Tech) was used to detect the chemiluminescent signals which were further analyzed using the Image J program [40]. 4.9. Western blot assay HepG2 cells were extracted with a cell lysis buffer (Beyotime). Then, cells were centrifuged and washed twice. The pellet was then resuspended in lysis buffer. After the cells were lysed for 20 min on ice, lysates were centrifuged for 15 min 4  C [25,41]. The total protein concentration was measured and adjusted to equal concentrations across different samples. The protein was separated on a 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membranes were incubated with the indicated primary antibodies overnight at 4  C and then incubated with the secondary antibodies conjugated to horseradish peroxidase. The proteins were visualized by a Keygen ECL system (KeyGEN Biotech, Nanjing, China) and scanned with a Clinx ChemiScope chemiluminescence imaging system. The relative optical densities of the specific proteins were determined with a ChemiScope analysis program. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (41576136). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2017.05.018. References [1] V.L. Singleton, N. Bohonos, A.J. Ullstrup, Decumbin, a new compound from a species of Penicillium, Nature 181 (1958) 1072e1073. [2] B.M. Fox, J.A. Vroman, P.E. Fanwick, M. Cushman, Preparation and evaluation of sulfide derivatives of the antibiotic brefeldin A as potential prodrug candidates with enhanced aqueous solubilities, J. Med. Chem. 44 (2001) 3915e3924. [3] A. Argade, R.D. Haugwitz, R. Devraj, M. Cushman, Highly efficient diastereoselective Michael addition of various thiols to (þ)-brefeldin A, J. Org. Chem. 63 (1998) 273e278. [4] A.B. Argade, R. Devraj, J.A. Vroman, R.D. Haugwitz, M. Hollingshead, M. Cushman, Design and synthesis of brefeldin A sulfide derivatives as prodrug candidates with enhanced aqueous solubilities, J. Med. Chem. 41 (1998) 3337e3346. [5] A. Takatsuki, I. Yamaguchi, G. Tamura, T. Misato, K. Arima, Correlation between the anti-animal and anti-plant-virus activities of several antibiotics. (Studies on antiviral and antitumor antibiotics. XIX), J. Antibiot. 22 (1969) 442e445. [6] N.O. Anadu, V.J. Davisson, M. Cushman, Synthesis and anticancer activity of brefeldin A ester derivatives, J. Med. Chem. 49 (2006) 3897e3905. [7] J.L. Moon, S.Y. Kim, S.W. Shin, J.W. Park, Regulation of brefeldin A-induced ER stress and apoptosis by mitochondrial NADPþ-dependent isocitrate dehydrogenase, Biochem. Biophys. Res. Commun. 417 (2012) 760e764. [8] D.E. Larsson, M. Wickstrom, S. Hassan, K. Oberg, D. Granberg, The cytotoxic agents NSC-95397, brefeldin A, bortezomib and sanguinarine induce apoptosis in neuroendocrine tumors in vitro, Anticancer Res. 30 (2010) 149e156. [9] A. Dinter, E.G. Berger, Golgi-disturbing agents, Histochem. Cell Biol. 109 (1998) 571e590. [10] J. Lippincott-Schwartz, L.C. Yuan, J.S. Bonifacino, R.D. Klausner, Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER, Cell 56 (1989) 801e813. [11] S.A. Lee, Y.J. Kim, C.S. Lee, Brefeldin A induces apoptosis by activating the mitochondrial and death receptor pathways and inhibits focal adhesion kinase-mediated cell invasion, Basic Clin. Pharmacol. Toxicol. 113 (2013) 329e338.

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