Scutellarin derivatives as apoptosis inducers: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2017.03.020

To explore novel antitumor agents with high efficiency and low toxicity, a series of NO-donating scutellarin derivatives (14–17) were synthesized and the antiproliferative activities against MCF-7, HCT-116, PC-3 and HepG2 cancer cell lines were assessed. Among them, compound 14b was the strongest with IC₅₀ values of 2.96?μM, 7.25?μM, 0.09?μM and 0.50?μM, respectively, and displayed low toxicity against normal human liver L-O2 cells with an IC₅₀ of 47.96?μM, showing good selectivity between normal and malignant liver cells. Moreover, NO releasing ability of the derivatives has been studied. Mechanism studies of the most promising compounds 14b and 15a were carried out. The results indicated that 14b and 15a could induce apoptosis, cell cycle arrest at the S phase and led to mitochondrial dysfunction in the HepG2 and PC-3?cell lines, respectively. Furthermore, Human Apoptosis Protein Array kit assay demonstrated that 14b could induce apoptosis through down-regulating the levels of procaspase-3 and inhibiting the expression of survivin, c-IAP1, HSP27, HSP60, HSP70, HO-1/HMOX1/HSP32 and HO-2/HMOX2 in HepG2 cell line. These results guaranteed compound 14b to be a drug candidate against liver cancer for further investigation.

Full text of DOI:10.1016/j.ejmech.2017.03.020

Accepted Manuscript
Scutellarin derivatives as apoptosis inducers: Design, synthesis and biological
evaluation
Tong Han, Jia Li, Jingjing Xue, He Li, Fanxing Xu, Keguang Cheng, Dahong Li,
Zhanlin Li, Ming Gao, Huiming Hua
PII:
S0223-5234(17)30177-0
10.1016/j.ejmech.2017.03.020
EJMECH 9280
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 January 2017
Revised Date: 10 March 2017
Accepted Date: 11 March 2017
Please cite this article as: T. Han, J. Li, J. Xue, H. Li, F. Xu, K. Cheng, D. Li, Z. Li, M. Gao, H. Hua,
Scutellarin derivatives as apoptosis inducers: Design, synthesis and biological evaluation, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.020.
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Scutellarin derivatives as apoptosis inducers: design, synthesis and
biological evaluation
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Tong Han , Jia Li , Jingjing Xue , He Li , Fanxing Xu , Keguang Cheng , Dahong Li , Zhanlin
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Li , Ming Gao and Huiming Hua
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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, China
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Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China
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State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, and School of
Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Road, Guilin 541004, China
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School of Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Koshien, Nishinomiya 663-8179,
Japan
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Corresponding authors. Tel.: +86-24-23986465; Fax: +86-24-23986465.
E-mail address: huimhua@163.com (H. Hua); lidahong0203@163.com (D. Li); fanxing0011@163.com (F. Xu).
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Abstract:
To explore novel antitumor agents with high efficiency and low toxicity, a series of
NO-donating scutellarin derivatives (14−17) were synthesized and the antiproliferative activities
against MCF-7, HCT-116, PC-3 and HepG2 cancer cell lines were assessed. Among them,
compound 14b was the strongest with IC50 values of 2.96 µM, 7.25 µM, 0.09 µM and 0.50 µM,
respectively, and displayed low toxicity against normal human liver L-O2 cells with an IC50 of
47.96 µM, showing good selectivity between normal and malignant liver cells. Moreover, NO
releasing ability of the derivatives has been studied. Mechanism studies of the most promising
compounds 14b and 15a were carried out. The results indicated that 14b and could induce
apoptosis, cell cycle arrest at the S phase and led to mitochondrial dysfunction in the HepG2 and
PC-3 cell lines, respectively. Furthermore, Human Apoptosis Protein Array kit assay demonstrated
that 14b could induce apoptosis through down-regulating the levels of procaspase-3 and inhibiting
the expression of survivin, c-IAP1, HSP27, HSP60, HSP70, HO-1/HMOX1/HSP32 and
HO-2/HMOX2 in HepG2 cell line. These results guaranteed compound 14b to be a drug candidate
against liver cancer for further investigation.
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Key words: scutellarin, antiproliferative activity, selectivity, NO, apoptosis.
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1. Introduction
Natural products have been the major sources of chemical diversity for lead compounds,
inspiring for the research and development of new drugs [1,2]. According to the statistics, there
are nearly 51% of currently marketed drugs owing their origins to natural compounds [3].
Flavonoids, as a large class of natural products, are widely distributed in the plant kingdom as
secondary metabolites [4]. They show various biological activities, especially for the treatment of
cancer [5], cardiovascular diseases [6] and neurodegenerative disorders [7]. Moreover, flavonoids
are considered to exhibit positive impact on human health without significant side effects, since
they are also existed in fruits and vegetables which we consume inadvertently in our daily diet
[8,9].
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Scutellarin, an extract from traditional Chinese herb Erigeron breviscapus (vant.) Hand-Mazz,
is the main effective flavonoid component of breviscapine [10]. It has been widely reported for the
treatment of cardiovascular diseases, such as hypertension [11], myocardial fibrosis [12,13] and
cardiomyocyte ischemia/reperfusion injury [14,15]. Recently, scutellarin was found to show
anticancer activity [16,17], which could suppress proliferation, migration and invasion of human
hepatocellular carcinoma cells via inhibiting the STAT3/Girdin/Akt activity [18,19]. There were
also accumulating evidences suggesting that scutellarin could significantly inhibit the growth,
adhesion and migration of human tongue squamous carcinoma cells by reducing the expression of
MMP-2, MMP-9 and integrin α β [20,21,22]. All these results demonstrated that scutellarin
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possessed potent antitumor activities with great therapeutic potentials. However, some perceived
disadvantages of scutellarin limited its further application, mainly including low stability and poor
oral bioavailability [23]. In order to overcome these disadvantages, methyl and benzyl as
protective groups were introduced to 6-OH and 4′-OH in order to avoid oxidation, and the
antitumor activities of the modified derivatives could also be further improved since methyl and
benzyl groups were benefit to the antitumor activity of flavonoid derivatives according to the
literatures [24,25].
Nitric oxide (NO) is a low molecular weight signaling molecule and plays pivotal roles in
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diverse physiological and pathophysiological processes [26]. Over the past decade, NO has
aroused great interest as a potential antitumor agent [27]. The molecular mechanisms involved in
NO-mediated cell death are multiple. In general, high concentration of NO can induce apoptosis,
inhibit metastasis of tumor cells, and sensitize tumor cells to chemotherapy, radiation and
immunotherapy [28,29]. Some kinds of NO donors can produce high levels of NO with a wide
range of half-lives in vitro and in vivo, and have been widely used in drug research, especially the
types of furoxan and nitrate [29-31]. Furoxans are an important class of thermodynamically stable
NO donors. A variety of active compounds or natural products including bifendate, coumarin and
oleanolic acid have been hybrided with furoxan (Figure 1) and displayed potent antitumor
activities [32-34]. Another kind of important NO donors with a long history, nitrates, can also
exert great influence in the treatment of tumor, such as some oleanolic acid derivatives (Figure 1)
[35]. Hence, design and synthesis of NO-releasing based hybrids is an effective and promising
strategy for cancer treatment [36]. As an important class of natural products with a variety of
biological activities, flavonoids are attractive to many researchers. Some NO-donating derivatives
of flavonoids were reported as well, for instance, the nitrate derivatives of apigenin, chrysin and
luteolin were found to prevent the development of diabetic complications [37,38]. However, there
were only a few reports concerning the antitumor activity of NO-donating flavoid derivatives [39].
On the basis of these reasons, we synthesized 12 new scutellarin derivatives 14−17 with
furoxan or nitrate moiety through different linkers and evaluated their antiproliferative activities
by MTT assay against human tumor MCF-7, HCT-116, PC-3, HepG2 cells and normal liver L-O2
cells. Furthermore, the NO-releasing ability and the mechanisms concerning cell cycle progression,
induction of apoptosis and effects on apoptosis-related proteins by representative derivatives were
investigated.
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2. Results and discussion
2.1. Chemistry
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The synthetic routine of target compounds is illustrated in Scheme 1. 5 was synthesized in a
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three-step sequence according to the literature [40], and then converted to various
monophenylsulfonylfuroxans (6a−d) by treatment with corresponding amino-substituted alcohol
(ethanolamine, 3-aminopropanol, 1-aminopropan-2-ol and N-(2-hydroxyethyl)piperazine)
(Scheme 1). 2-Bromoethanol and 3-bromo-1-propanol were treated with HNO and H SO in
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CH Cl to give its mononitrates 9a and 9b (Scheme 2).
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Scutellarin was used as the raw material, and compound 10 and 11 were prepared by reacted
with benzyl bromide and methyl iodide. Then they were hydrolyzed by KOH to generate the
intermediates 12 and 13. Finally, the furoxan (6a−d) and nitrate (9a and 9b) intermediates were
treated with the derivatives (12 and 13) of scutellarin to give the target compounds 14a−d, 15a−d,
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16a, 16b, 17a and 17b (Scheme 3). The structures of all the derivatives were confirmed by H
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NMR, C NMR and high resolution mass spectrum (HR-MS).
2.2. Biological evaluation
2.2.1 Antiproliferative activity
Twelve new scutellarin/furoxan or nitrate hybrids (14a−d, 15a−d, 16a, 16b, 17a and 17b)
were evaluated for their inhibitory effects against four different human cancer cell lines (human
breast carcinoma cell line MCF-7, human colorectal carcinoma cell line HCT-116, human
prostatic cancer cell line PC-3 and human liver carcinoma cell line HepG2), in comparison with
the reference 5-FU. Meanwhile, the activities against the human normal hepatic cell line (L-O2)
were also evaluated since a potential anticancer drug candidate would be better to show selective
cytotoxicity between malignant and normal cells.
As shown in Table 1, most target compounds displayed more potent inhibitory activities than
scutellarin, and some of them showed superior cytotoxic activities to 5-FU. The furoxan hybrids
14a−d and 15a−d showed significant antiproliferative activities against four cancer cell lines with
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IC values ranging from 0.09 µM to 39.97 µM, especially against PC-3 (IC 0.09 µM−3.65 µM) ,
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HCT-116 (IC50 1.27 µM−13.58 µM) and HepG2 (IC50 0.50 µM−12.92 µM) cells. Compounds
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14a−d with benzyl groups at the 6- and 4′-hydroxyl of scutellarin displayed more potential
activities against MCF-7, PC-3 and HepG2 cells than corresponding ones with methyl groups. The
results were in accord with previous literatures that bulky benzyl group was preferred for
antiproliferative activity [24,25]. Interestingly, the antiproliferative activities showed almost no
differences among compounds 14a, 14b, 14c and 14d (15a, 15b, 15c and 15d) against most
cancer cell lines when the linkers were changed. Notably, compound 14b displayed the most
potent antiproliferative activity among compounds 14a−d, with IC values of 0.09 µM−7.25 µM,
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especially against PC-3 (IC50 0.09 µM) and HepG2 cells (IC50 0.50 µM). Compounds 15a−d
showed comparable antitumor potency to 5-FU against HCT-116 cell line and more potent
activities against PC-3 and HepG2 cell lines. On the other hand, nitrate hybrids 16a, 16b, 17a and
17b exhibited no significant activity against any of the selected cancer cell lines, except for
compound 16b showing moderate inhibitory activity against HCT-116 cells with IC50 of 17.40
µM.
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In addition, to investigate whether the hybrids exhibited selective antiproliferative activities
between normal and malignant liver cells, 14a−d and 15a−d were screened against human normal
liver L-O2 cells with scutellarin as control. The results were listed in Table 1. All the tested
compounds showed low antiproliferative activities against L-O2 cells with IC values from 19.31
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µM to 47.96 µM. In particular, compound 14b displayed 96-fold less antiproliferative activity
against nontumor L-O2 cells than HepG2 tumor cells.
2.2.2. NO-releasing ability in vitro
The levels of NO released by the target compounds were tested by Griess assay [41]. As
shown in Table 2, generally, the series of furoxan-based NO donor hybrids (14a−d and 15a−d)
displayed more potent NO-releasing ability than the series bearing nitrate moiety (16a, 16b, 17a
and 17b). This indicated that the antiproliferative activities would be partly attributed to the NO
releasing ability. Meanwhile, the derivatives 15a−d with 6- and 4'-hydroxyl substitutions of
methyl groups were found to release more NO than 14a−d with benzyl groups. These results
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suggested that the benzyl groups might hinder the metabolism of furoxans. Compound 14b which
showed the strongest growth inhibitory activity also produced a sustained release of more than 20
µM/L of NO at the time point of 3 h. The maximum amount of NO was produced by compound
15a with above 59 µM/L at the time point of 3 h. In addition, we determined the intracellular NO
levels produced by compounds14a−d and 15a−d in HepG2 and L-O2 cells. As shown in Figure 2,
all the derivatives released more than 26.3 µM/L of NO at the time point of 1 h in HepG2 cell, and
less than 15.46 µM/L of NO in L-O2 cells. These results indicated that relatively high levels of
NO produced by the scutellarin derivatives were benifit to their strong antiproliferative activities.
In order to investigate the mechanism of action, the functions of compound 14b in HepG2 cells
and 15a in PC-3 cells were chosen for further study.
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2.2.3. Cell cycle analysis
The effects on the cell cycle distribution in HepG2 and PC-3 cells were first evaluated. HepG2
and PC-3 cells were treated with compound 14b (0, 0.3, 0.6 and 1.2 µM) and compound 15a (0,
0.16, 0.31 and 1.25 µM) for 72 h, respectively, then stained with propidium iodide (PI) and
analyzed by flow cytometry. Non-treated cells were used as control. As shown in Figure 3, cells in
the S phase increased from 44.60% in control group to 47.14%, 50.45% and 69.63% in a
concentration-dependent manner in HepG2 cell lines. These results revealed that compound 14b
caused S phase arrest in a concentration-dependent manner. At the same time, the similar results
were observed by compound 15a in PC-3 cell line that the cell cycle was significantly arrested in
the S phase.
2.2.4. The morphological analysis by Hoechst 33258 staining
The changes of morphological features, such as cell shrinkage, chromatin condensation,
nuclear membrane blebbing are the characteristics of apoptotic cells [42]. Hoechst 33258, which
stains the cell nuclei and emits fluorescence allowing the visualization of nuclear morphological
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changes, is a membrane permeable dye. Hence, we used fluorescence microscopy to observe the
morphology of HepG2 and PC-3 cells stained with Hoechst, after treatment with different
concentrations (same as the cell cycle test) of compounds 14b and 15a, respectively, for 48 h.
The morphological analysis, depicted in Figure 4, showed that control cells were uniformly
stained with Hoechst 33258 and presented round homogeneous nuclei, without morphological
changes. HepG2 cells were exposed to 0.3 µM and 0.6 µM of compound 14b for 48 h, which
presented remarkable morphological changes, such as cell shrinkage, chromatin condensation and
evident reduction in the number of adherent cells. The rupture of cell membrane and the nuclear
fragmentation were evident after the treatment of HepG2 cells with 1.2 µM of compound 14b.
These typical apoptotic morphology changes were also observed in different concentrations of 15a
treated PC-3 cells. These results strongly supported the pro-apoptotic effects of compounds 14b
and 15a.
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2.2.5. Cell apoptosis assay
Since high levels of NO induce cellular apoptosis [43], the cellular apoptosis ability of 14b
and 15a were tested. The cells were treated with different concentrations (same as the cell cycle
test) of compounds 14b or 15a for 72 h. Then, the cells were harvested and stained with
7-aminoactinomycin D (7-AAD) and annexin-V, and the percentages of apoptotic cells were
determined by flow cytometry analysis. As shown in Figure 5 (A, B), the treatment of HepG2
cells with compound 14b at concentrations of 0.3, 0.6 and 1.2 µM for 72 h resulted in cell
apoptosis at the ratios of 16.54%, 33.1% and 52.62%, respectively, as compared with 8.05% in the
control group. And the treatment of PC-3 cells with 1.25 µM of compound 15a decreased the
percentage of live cells from 88.74% to 36.34% and increased the apoptotic cells from 8.83% to
58.45% (14.91% of earlier apoptotic cells and 43.54% of late apoptotic cells). These results
showed that compounds 14b and 15a caused a marked increase of the cellular apoptosis in a
concentration-dependent manner in HepG2 and PC-3 cells, respectively.
Morover, the effects of apoptosis in normal human liver cells L-O2 induced by 14b or 15a
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were also tested. As shown in Figure 5 (C), when L-O2 cells were treated with compounds 14b
and 15a at the concentrations of 1.2 µM and 1.25 µM, respectively, almost no cellular apoptosis
was observed. However, at the same concentrations, they can induce apoptosis obviously in
HepG2 and PC-3 cells. These data comfirmed the good selectivity of compounds 14b and 15a
between normal and malignant liver cells.
2.2.6. Mitochondria membrane potential (∆ψm)
Mitochondria play a crucial role in the induction and control of apoptosis, which results in a
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variety of key events, including the loss of mitochondrial membrane potential (∆ψ ) and release
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of pro-apoptotic factors such as cytochrome c and other apoptosis-inducing factors [44]. NO is
also known to induce apoptosis by activating the intrinsic mitochondrion-mediated pathways [45].
We therefore studied the effects on mitochondrial membrane potential of target compounds 14b
and 15a in HepG2 and PC-3 cell lines, respectively. The cells were treated with different
concentrations (same as the cell cycle test) of 14b and 15a for 48 h, respectively, and the changes
of mitochondrial membrane potential (∆ψ ) were monitored by flow cytometry using the dye
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5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-caebocyanine (JC-1). As shown in Figure 6
in HepG2 and PC-3 cells, compounds 14b and 15a induced a concentration-dependent increase in
the proportion of cells with depolarized mitochondria. This illustrated that the induction of
apoptosis by hybrids 14b and 15a was associated with the mitochondrial (intrinsic) pathways.
2.2.7. Quantitation of cellular proteins involved in apoptosis
Apoptosis is an essential physiological cell death process in development, homeostasis, and
immune defense of multicellular animals [46]. To better understand the mechanism of scutellarin
derivatives, we performed a protein array assay using the Human Apoptosis Protein Array kit.
Compound 14b was chosen for this study in HepG2 cells. The visible changes of some apoptotic
proteins were observed (Figure 7) and the whole experimental results were presented in the
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Caspases are key factors in apoptotic cell death. Caspase-3 in particular, can be activated by
the proteolytic processing of procaspase-3 in response to exogenous apoptosis inducers [47]. The
inhibitors of apoptosis proteins (IAP), a family of anti-apoptotic regulators, express at high levels
and prevent caspase-mediated apoptosis in cancer cells. The human IAP family contains eight
proteins: c-IAP1, c-IAP2, NAIP, survivin, XIAP, bruce, ILP-2 and livin [48,49]. As shown in
Figure 7, the results indicated that caspase-3 was activated because of decreased expression of
procaspase-3 after the treatment of 14b for 24 h. Moreover, in comparison with the control cells,
14b induced a significant reduction in the levels of survivin and c-IAP1.
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Heat shock proteins (HSPs), especially Hsp 27, Hsp 60, Hsp 70 and Hsp 90, are not only
involved in normal physiology but also in cancer [50-52]. They can inhibit the activity of caspases
directly or indirectly, thereby blocking the intrinsic and extrinsic apoptotic pathways through
interaction with key apoptotic proteins [53,54]. Heme oxygenase (HO) is a cytoprotective enzyme
that can be overexpressed in cancer cells and promotes proliferation and survival. Two
isoenzymes of HO, the inducible form HO-1 and the constitutive form HO-2, have been well
characterized [55-57]. The effects of compound 14b on the constitutive levels of HSPs and HO in
HepG2 cells are shown in Figure 7. Exposure of HepG2 cells to compound 14b caused a dramatic
decrease in the levels of Hsp27, Hsp 60, Hsp 70, HO-1 and HO-2, as compared with control.
These observations suggested that compound 14b might induce HepG2 cells apoptosis through
caspase cascade and inhibit the expression of anti-apoptotic proteins.
3. Conclusion
In summary, a series of hybrids of scutellarin and NO donors (furoxans or nitrate) (14a−d,
15a−d, 16a,b and 17a,b) were synthesized and evaluated. Most of them showed strong
antiproliferative activities against four cancer cell lines (MCF-7, HCT-116, PC-3 and HepG2) and
weak cytotoxic activities against human normal liver cell line L-O2. Particularly, for compound
14b, exhibited the best selectivity with the selective index of 96. In addition, 14b showed
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sustained NO releasing ability in the Griess assay and compound 15a produced the greatest
amount of NO of above 59 µM/L by the time point of 3 h. Hence, compounds 14b and 15a were
chosen for further investigation in order to reveal the cellular mechanisms in different cell lines.
The apoptosis-inducing activity of representative compounds 14b in HepG2 cells and 15a in PC-3
cells were tested. The results demonstrated that hybrids 14b and 15a showed similar effects which
could induce cell apoptosis and arrest cell cycle at the S stage. Human Apoptosis Protein Array kit
was further applied to disclose the effects of apoptosis-related proteins by compound 14b in
HepG-2 cells. The results suggested that compound 14b induced HepG-2 cells apoptosis through
down-regulation of the level of pro-caspase-3 and inhibited the expression of anti-apoptotic
proteins (c-IAP1, survivin, HSP27, HSP60, HSP70, HO/HMOX1/HSP32 and HO-2/HMOX2).
Consequently, the rational design of scutellarin derivatives containing NO-donating moiety
offered a good strategy for the discovery of antitumor agents with high efficiency and low
toxicity.
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4. Experimental
4.1. Chemistry
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Melting points (mp) were determined on an X-4 melting point apparatus and uncorrected. H
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NMR and C NMR spectra were measured on Bruker ARX-300, AV400 MHz or ARX-600
spectrometers with tetramethylsilane (TMS) as the internal standard. Chemical shifts were
reported in δ (ppm). Mass spectra (MS) were determined on Finnigan MAT/USA spectrometer
(LC-MS). High-resolution mass spectra were obtained on Bruker microOTOF-Q in the ESI mode
(HR-ESI-MS). Scutellarin was purchased from Jiangsu Zelang Bio-pharm. Pty. Ltd. China, with
the purity over 95%. The other reagents were obtained from commercial suppliers and used
without purification. TLC analysis was carried out on silica gel plates GF254 (Qindao Haiyang
Chemical, China). Column chromatography was performed on silica gel (200−300 mesh).
Compounds 6a−d, 9a,b and intermediates 10−13 were prepared as described previously [37], and
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their chemical characterizations were shown in the Supplementary materials.
4.1.1. General procedure for the synthesis of compounds 14a-d and 15a-d
A mixture of 12 or 13 (0.5 mmol) and HOBt (0.6 mmol) in anhydrous DMF (5 mL) was
stirred
at
room
temperature
for
0.5
h.
After
the
addition
of
(6a),
(6b),
or
4-(2-aminoethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
4-(3-aminopropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
4-((1-aminopropan-2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
(6c)
3-(phenylsulfonyl)-4-(2-(piperazin-1-yl)ethoxy)-1,2,5-oxadiazole-2-oxide (6d) (0.75 mmol) and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
EDCI (0.75 mmol) to the solution, the mixture was further stirred at room temperature for 3 h.
Then, the mixture was poured into 20 mL of H O, and extracted with EtOAc (3 × 20 mL). The
2
organic layer was combined, washed with brine, dried over anhydrous Na SO , and concentrated
2
4
in vacuo. The crude product was purified on silica gel column chromatography eluting with
dichloromethane/methanol system.
4.1.1.1.
4-(2-(-6-((6-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-5-hydroxy-4-oxo-4H-
chromen-7-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxamido)ethoxy)-3-(phenylsulfony
l)-1,2,5-oxadiazole-2-oxide (14a).
o
+
Yellow power, 241 mg; yield: 53.1%, mp: 179−181
C; MS(ESI) m/z: 910.2 [M + H] , 932.2
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H N NaO S [M + Na] 932.1943, found 932.1943; H
4
5
39
3
16
NMR (DMSO-d , 400 MHz) δ (ppm): 12.95 (s, 1H, 5-OH), 8.23 (t, 1H, J = 5.6 Hz, -NH-), 8.02 (d,
6
2H, J = 8.9 Hz, H-2′,6′), 7.95 (d, 2H, J = 7.4 Hz, Ar-H), 7.75 (t, 1H, Ar-H), 7.65 (d, 2H, J = 7.4
Hz, Ar-H), 7.56 (d, 2H, J = 6.6 Hz, Ar-H), 7.47 (d, 2H, J = 7.4 Hz, Ar-H), 7.43−7.31 (m, 6H,
Ar-H), 7.17 (d, 2H, J = 8.9 Hz, H-3′,5′), 7.12 (s, 1H, H-8), 6.95 (s, 1H, H-3), 5.65 (brs, 1H, H-1′′),
5.37−5.31 (m, 3H, sugar hydroxyl), 5.21 (s, 2H, -CH -), 5.09 (d, 1H, J = 10.9 Hz, -CH -), 4.98 (d,
25
26
27
28
2
2
1H, J = 10.9 Hz, -CH -), 4.43 (t, 2H, -CH -), 4.06 (d, 1H, J = 9.7 Hz, H-5′′), 3.62−3.52 (m, 3H,
2
2
1
3
H-2′′, 3′′, 4′′), 3.44 (t, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.79, 169.19,
2
6
164.28, 161.94, 159.18, 156.75, 153.30, 152.72, 138.05, 137.35, 136.91, 136.36, 131.82, 130.76,
1
2

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
130.42, 130.35, 130.35, 129.21, 128.97, 128.97, 128.87, 128.87, 128.57, 128.57, 128.51, 128.51,
128.30, 128.30, 123.37, 115.79, 115.79, 111.06, 111.06, 106.39, 103.87, 100.21, 94.51, 76.64,
76.27, 74.66, 73.36, 71.70, 70.03, 69.93, 67.87.
4.1.1.2.
4-(3-(6-((6-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-5-hydroxy-4-oxo-4H-
chromen-7-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxamido)propoxy)-3-(phenylsulfo
nyl)-1,2,5-oxadiazole-2-oxide (14b).
o
+
Yellow power, 210 mg; yield: 45.5%, mp: 127−129
C; MS(ESI) m/z: 924.2 [M + H] , 946.2
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H N O S [M + H] 946.2100, found 946.2089; H
4
6
42
3
16
1
1
1
1
1
NMR (DMSO-d , 400 MHz) δ (ppm): 12.97 (s, 1H, 5-OH), 8.19 (t, 1H, J = 5.7 Hz, -NH-), 8.05 (d,
6
2H, J = 8.9 Hz, H-2′,6′), 7.98 (d, 2H, J = 7.4 Hz, Ar-H), 7.81 (t, 1H, Ar-H), 7.69 (d, 2H, J = 7.9
Hz, Ar-H), 7.56 (d, 2H, J = 6.8 Hz, Ar-H), 7.47 (d, 2H, J = 6.8 Hz, Ar-H), 7.40−7.35 (m, 6H,
Ar-H), 7.18 (d, 2H, J = 8.9 Hz, H-3′,5′), 7.07 (s, 1H, H-8), 6.95 (s, 1H, H-3), 5.64 (brs, 1H, H-1′′),
5.44−5.27 (m, 3H, sugar hydroxyl), 5.21 (s, 2H, -CH -), 5.08 (d, 1H, J = 10.8 Hz, -CH -), 4.96 (d,
3
4
2
2
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1H, J = 10.8 Hz, -CH -), 4.34 (t, 2H, -CH -), 3.96 (d, 1H, J = 9.6 Hz, H-5′′), 3.53−3.45 (m, 3H,
2 2
1
3
H-2′′,3′′,4′′), 3.24 (t, 2H, -CH -), 1.90 (m, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm):
2
2
6
182.82, 168.55, 164.27, 162.00, 159.21, 156.73, 153.28, 152.72, 138.02, 137.55, 136.90, 136.53,
132.06, 131.80, 130.45, 130.45, 129.12, 128.96, 128.96, 128.87, 128.87, 128.80, 128.57, 128.57,
128.50, 128.50, 128.29, 128.29, 123.35 , 115.80, 115.80, 110.85, 110.85, 106.39, 103.86, 100.39,
94.54, 76.81, 76.52, 74.64, 73.41, 71.40, 70.04, 69.42, 67,86, 52.47.
4.1.1.3.
4-((1-(6-((6-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-5-hydroxy-4-oxo-4H-
chromen-7-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxamido)propan-2-yl)oxy)-3-(phe
nylsulfonyl)-1,2,5-oxadiazole-2-oxide (14c).
o
+
Yellow power, 306 mg; yield: 66.3%, mp: 135−137
C; MS(ESI) m/z: 924.1 [M + H] , 946.2
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H N NaO S [M + Na] 946.2100, found 946.2097; H
4
6
41
3
16
NMR (DMSO-d , 400 MHz) δ (ppm): 12.96 (s, 1H, 5-OH), 8.23 (t, 1H, -NH-), 8.05 (d, 2H, J =
6
8.9 Hz, H-2′,6′), 7.97 (d, 2H, J = 7.3 Hz, Ar-H), 7.77 (t, 1H, Ar-H), 7.68 (d, 2H, J = 7.4 Hz, Ar-H),
1
3

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
7.57 (d, 2H, J = 7.0Hz, Ar-H), 7.48 (brs, 2H, Ar-H), 7.43−7.33 (m, 6H, Ar-H), 7.18 (d, 2H, J = 8.9
Hz, H-3′,5′), 7.06 (s, 1H, H-8), 6.94 (s, 1H, H-3), 5.65 (d, 1H, J = 5.2 Hz, H-1′′), 5.42−5.27 (m,
3H, sugar hydroxyl), 5.21 (s, 2H, -CH -), 4.96 (dd, 2H, J = 10.6 Hz, -CH -), 4.23 (m, 1H, -CH-),
2
2
4.03 (d, 1H, J = 9.0 Hz, H-5′′), 3.61−3.50 (m, 3H, H-2′′,3′′,4′′), 3.43 (d, 2H, -CH -), 1.27 (t, 2H,
2
13
-CH ). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.82, 168.92, 164.28, 161.96, 158.66, 156.73,
3
6
153.29, 152.70, 138.04, 137.40, 136.90, 136.37, 131.85, 131.81, 130.44, 130.44, 129.06, 128.97,
128.97, 128.88, 128.88, 128.83, 128.57, 128.57, 128.51, 128.51, 128.30, 128.30, 123.33, 115.79,
115.79, 111.14, 111.14, 106.40, 103.87, 100.33, 94.72, 76.83, 76.27, 74.66, 73.46, 73.37, 71.57,
71.35, 70.03, 17.27.
1
1
1
1
1
1
1
1
1
4.1.1.4.
4-(2-(4-(6-((6-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-5-hydroxy-4-oxo-4H-
chromen-7-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboyl)piperazin-1-yl)ethoxy)-3-(phe
nylsulfonyl)-1,2,5-oxadiazole-2-oxide (14d).
o
+
Yellow power, 150 mg; yield: 51.1%, mp: 123−125
C
; MS(ESI) m/z: 979.1 [M + H] , 1001.1
+
+
[M + Na] ; HRMS (ESI) m/z calcd for C H N NaO S [M + Na] 1001.2522, found 1001.2512;
4
9
46
4
16
1
H NMR (DMSO-d , 400 MHz) δ (ppm): 12.92 (s, 1H, 5-OH), 8.02 (d, 2H, J = 8.9 Hz, H-2′,6′),
6
7.94 (d, 2H, J = 7.3 Hz, Ar-H), 7.82 (t, 1H, Ar-H), 7.68 (d, 2H, J = 7.9 Hz, Ar-H), 7.54 (d, 2H, J =
7.1 Hz, Ar-H), 7.45 (d, 2H, J = 7.1 Hz, Ar-H), 7.39−7.34 (m, 6H, Ar-H), 7.21 (s, 1H, H-8), 7.17 (d,
2H, J = 8.9 Hz, H-3′,5′), 6.93 (s, 1H, H-3), 5.67 (brs, 1H, H-1′′), 5.53 (d, 1H, sugar hydroxyl),
19
20
21
5.28 (brs, 1H, sugar hydroxyl), 5.24 (s, 1H, sugar hydroxyl), 5.17 (s, 2H, -CH -), 5.07 (d, 1H, J =
2
11.0 Hz, -CH -), 4.96 (d, 1H, J = 11.0 Hz, -CH -), 4.48 (d, 1H, J = 9.3 Hz, H-5′′), 4.24 (m, 2H,
2
2
22
23
24
25
26
27
28
-CH -), 3.73−3.51 (m, 4H, -CH - × 2), 3.45−3.42 (m, 3H, H-2′′,3′′,4′′), 3.21 (t, 2H, -CH -), 2.47 (t,
2 2 2
1
3
2H, -CH -), 2.19 (m, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.78, 165.80,
2
2
6
164.12, 162.02, 159.16, 156.41, 153.30, 152.60, 137.93, 137.71, 136.87, 136.52, 131.81, 130.42,
130.42, 128.93, 128.93, 128.85, 128.85, 128.62, 128.57, 128.57, 128.50, 128.50, 128.34, 128.34,
128.24, 127.78, 124.97, 123.31, 119.60, 115.81, 115.81, 110.07, 110.07, 106.26, 103.87, 99.33,
94.55, 76.39, 74.62, 73.18, 71.48, 70.49, 70.04, 68.79, 55.83, 53.35, 52.80, 45.43, 41.91.
1
4

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
4.1.1.5.
3-(Phenylsulfonyl)-4-(2-(3,4,5-trihydroxy-6-((5-hydroxy-6-methoxy-2-(4-
methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)tetrahydro-2H-pyran-2-carboxamido)ethoxy)-1,2,5-
oxadiazole-2-oxide (15a).
o
+
Yellow power, 49.9 mg; yield: 13.2%, mp: 175−177
C; MS(ESI) m/z: 758.1 [M + H] , 780.1
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H N NaO S [M + Na] 780.1317, found 780.1315; H
3
3
31
3
16
NMR (DMSO-d , 400 MHz) δ (ppm): 12.92 (s, 1H, 5-OH), 8.24 (t, 1H, J = 5.4 Hz, -NH-), 8.02 (d,
6
2H, J = 9.0 Hz, H-2′,6′), 7.95 (d, 2H, J = 7.5 Hz, Ar-H), 7.82 (t, 1H, Ar-H), 7.69 (t, 2H, J = 7.5 Hz,
Ar-H), 7.09 (d, 2H, J = 9.0 Hz, H-3′,5′), 7.04 (s, 1H, H-8), 6.93 (s, 1H, H-3), 5.59 (d, 1H, J =5.3
Hz, H-1′′), 5.35 (d, 1H, J = 4.5 Hz, sugar hydroxyl), 5.30 (d, 1H, J = 4.5 Hz, sugar hydroxyl), 5.28
(brs, 1H, sugar hydroxyl), 4.43 (t, 2H, -CH -), 4.02 (d, 1H, J = 9.5 Hz, H-5′′), 3.85 (s, 3H, -OCH ),
8
9
0
1
2
3
1
3
1
1
1
1
1
1
1
1
1
2
2
2
1
2
3
4
5
6
7
8
9
0
1
2
3.76 (s, 3H, -OCH ), 3.57−3.49 (m, 3H, H-2′′,3′′,4′′), 3.40 (t, 2H, -CH -). C NMR (DMSO-d6,
3 2
100 MHz) δ (ppm): 182.81, 169.15, 164.36, 162.88, 159.19, 156.61, 153.07, 152.61, 137.36,
136.40, 133.05, 130.37, 130.37, 128.92, 128.92, 128.86, 128.86, 123.18, 115.00, 115.00, 111.07,
106.42, 103.82, 100.35, 94.66, 76.54, 76.27, 73.23, 71.67, 69.95, 60.78, 56.04, 37.81.
4.1.1.6.
3-(Phenylsulfonyl)-4-(3-(3,4,5-trihydroxy-6-((5-hydroxy-6-methoxy-2-(4-
methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)tetrahydro-2H-pyran-2-carboxamido)propoxy)-1,2,5
-oxadiazole-2-oxide (15b).
o
+
Yellow power, 60.1 mg; yield: 15.6%, mp: 126−128
C; MS(ESI) m/z: 772.1 [M + H] , 794.1
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H N NaO S [M + Na] 794.1474, found 794.1457; H
3
4
33
3
16
NMR (DMSO-d , 400 MHz) δ (ppm): 12.96 (s, 1H, 5-OH), 8.36 (t, 1H, J = 5.2 Hz, -NH-), 8.05 (d,
6
2H, J = 8.9 Hz, H-2′,6′), 7.98 (d, 2H, J = 7.3 Hz, Ar-H), 7.84 (t, 1H, Ar-H), 7.69 (d, 2H, J = 7.5
Hz, Ar-H), 7.10 (d, 2H, J = 8.9 Hz, H-3′,5′), 7.06 (s, 1H, H-8), 6.95 (s, 1H, H-3), 5.70−5.51 (m,
4H, sugar hydroxyl), 4.35 (t, 2H, -CH -), 3.94 (d, 1H, J = 9.5 Hz, H-5′′), 3.84 (s, 3H, -OCH ),
23
24
25
26
27
2
3
1
3
3.73 (s, 3H, -OCH ), 3.52−3.44 (m, 3H, H-2′′,3′′,4′′), 3.26 (t, 2H, -CH -), 1.90 (t, 2H, -CH -). C
3
2
2
NMR (DMSO-d , 100 MHz) δ (ppm): 182.83, 169.15, 163.93, 162.76, 159.24, 155.59, 153.85,
6
152.72, 137.62, 136.55, 133.37, 130.48, 130.48, 128.82, 128.82, 128.06, 128.06, 122.14, 118.06,
1
5

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
0
1
118.06, 112.62, 106.18, 103.24, 100.53, 95.08, 76.75, 76.49, 74.39, 71.43, 69.45, 60.81, 56.05,
40.90.
4.1.1.7.
3-(Phenylsulfonyl)-4-((1-(3,4,5-trihydroxy-6-((5-hydroxy-6-methoxy-2-(4-
methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)tetrahydro-2H-pyran-2-carboxamido)propan-2-yl)ox
y)-1,2,5-oxadiazole-2-oxide (15c).
o
+
Yellow power, 195.1 mg; yield: 50.6%, mp: 130−132
C; MS(ESI) m/z: 772.1 [M + H] , 794.1
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H N NaO S [M + Na] 794.1474, found 794.1458; H
3
4
33
3
16
NMR (DMSO-d , 400 MHz) δ (ppm): 12.92 (s, 1H, 5-OH), 8.23 (t, 1H, J = 5.6 Hz, -NH-), 8.05 (d,
6
1
1
2H, J = 8.9 Hz, H-2′,6′), 7.97 (d, 2H, J = 7.5 Hz, Ar-H), 7.83 (t, 1H, Ar-H), 7.69 (d, 2H, J = 7.5
Hz, Ar-H), 7.09 (d, 2H, J = 8.9 Hz, H-3′,5′), 7.03 (s, 1H, H-8), 6.95 (s, 1H, H-3), 5.60 (brs, 1H,
H-1′′), 5.35−5.51 (m, 3H, sugar hydroxyl), 4.97 (m, 1H, -CH-), 4.01 (d, 1H, J = 9.3 Hz, H-5′′),
3.86 (s, 3H, -OCH ), 3.75 (s, 3H, -OCH ), 3.59−3.50 (m, 3H, H-2′′,3′′,4′′), 3.42(d, 2H, -CH -),
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
3
3
2
1
3
1.28 (d, 3H, -CH3). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.83, 168.99, 164.32, 162.90,
6
158.67, 156.61, 153.06, 152.59, 137.41, 136.41, 133.05, 130.31, 130.31, 129.06, 129.06, 128.84,
128.84, 123.14, 119.55, 119.55, 111.13, 106.45, 103.80, 100.47, 94.65, 78.03, 76.72, 73.24, 71.54,
71.34, 60.79, 56.04, 17.26.
4.1.1.8.
3-(Phenylsulfonyl)-4-(2-(4-(3,4,5-trihydroxy-6-((5-hydroxy-6-methoxy-2-(4-
methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)tetrahydro-2H-pyran-2-carbonyl)piperazin-1-yl)etho
xy)-1,2,5-oxadiazole-2-oxide. (15d).
o
+
Yellow power, yield: 10.9%, mp: 119−121
C; MS(ESI) m/z: 827.1 [M + H] , 849.1 [M +
+
+
1
Na] ; HRMS (ESI) m/z calcd for C H N NaO S [M + Na] 849.1896, found 849.1879; H
3
7
38
4
16
NMR (DMSO-d , 400 MHz) δ (ppm): 12.90 (s, 1H, 5-OH), 8.01 (d, 2H, J = 8.9 Hz, H-2′,6′), 7.95
6
(d, 2H, J = 8.1 Hz, Ar-H), 7.85 (t, 1H, Ar-H), 7.70 (d, 2H, J = 8.1 Hz, Ar-H), 7.17 (s, 1H, H-8),
7.06 (d, 2H, J = 8.9 Hz, H-3′,5′), 6.94 (s, 1H, H-3), 5.61 (d, 1H, J = 4.0 Hz, H-1′′), 5.45 (d, 1H, J
= 7.2 Hz, sugar hydroxyl), 5.22−5.17 (m, 3H, sugar hydroxyl), 4.48 (d, 1H, J = 9.4 Hz, H-5′′),
4.24 (m, 2H, -CH -), 3.82 (s, 3H, -OCH ), 3.75 (s, 3H, -OCH ), 3.68−3.53 (m, 4H, -CH - × 2),
2
7
8
2
2
3
3
2
1
6

ACCEPTED MANUSCRIPT
1
3
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
3.43−3.37 (m, 3H, H-2′′,3′′,4′′), 3.22 (t, 2H, -CH -), 2.47 (m, 2H, -CH -), 2.23 (m, 2H, -CH -). C
2 2 2
NMR (DMSO-d , 100 MHz) δ (ppm): 182.80, 165.81, 164.17, 162.93, 159.16, 156.28, 153.05,
6
152.51, 137.71, 136.54, 133.01, 130.44, 130.44, 128.63, 128.63, 127.78, 127.78, 124.96, 119.60,
119.60, 110.08, 106.30, 103.80, 99.40, 94.60, 76.32, 73.05, 71.37, 70.50, 68.82, 60.77, 56.04,
55.08, 53.38, 52.87, 45.46, 41.93.
4.1.2. General procedure for the synthesis of compounds 16a, 16b, 17a and 17b
To a solution of 12 or 13 (0.5 mmol) and DBU (0.6 mmol) in DMF (5 mL) 9a or 9b (0.6
mmol) was added. The mixture was stirred at room temperature for 3 h. Then, the mixture was
1
1
1
1
1
1
1
1
1
1
poured into 20 mL of H O, and extracted with EtOAc (3 × 20 mL). The organic layer was
2
combined, washed with brine, dried over anhydrous Na SO , and concentrated in vacuo. The
2
4
crude product was purified by silica gel column chromatography eluting with
dichloromethane/methanol system.
4.1.2.1.
2-(Nitrate)ethyl-6-((6-(benzyloxy)-2-(4-(benzyloxy)phenyl)-5-hydroxy-4-oxo
-4H-chromen-7-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate (16a).
o
+
Yellow power, 142 mg; yield: 38.8%, mp: 156−158
C; MS(ESI) m/z: 732.2 [M + H] , 754.1
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H NNaO [M + Na] 754.1742, found 754.1739; H
3
7
33
15
NMR (DMSO-d , 400 MHz) δ (ppm): 12.97 (s, 1H, 5-OH), 8.05 (d, 2H, J = 8.9 Hz, H-2′,6′), 7.55
6
20
21
22
23
24
25
26
27
28
(d, 2H, J = 7.4 Hz, Ar-H), 7.48 (d, 2H, J = 7.4 Hz, Ar-H), 7.42−7.31 (m, 6H, Ar-H), 7.21 (d, 2H, J
= 8.9 Hz, H-3′,5′), 7.12 (s, 1H, H-8), 6.95 (s, 1H, H-3), 5.71 (d, 1H, J =5.1 Hz, H-1′′), 5.54 (d, 1H,
J =5.8 Hz, sugar hydroxyl), 5.42 (d, 1H, J =7.8 Hz, sugar hydroxyl), 5.41 (brs, 1H, sugar
hydroxyl), 5.22 (s, 2H, -CH -), 5.09 (d, 1H, J =10.8 Hz, -CH -), 4.96 (d, 1H, J =10.8 Hz, -CH -),
2
2
2
4.22 (d, 1H, J = 9.6 Hz, H-5′′), 4.14−4.09 (m, 2H, -CH -), 3.50−3.47 (m, 3H, H-2′′,3′′,4′′), 3.44 (t,
2
1
3
2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.84, 169.00, 164.32, 162.00, 156.58,
2
6
153.31, 152.70, 137.98, 136.91, 131.78, 128.98, 128.98, 128.86, 128.86, 128.58, 128.58, 128.52,
128.52, 128.34, 128.34, 123.40, 115.87, 115.87, 106.40, 103.94, 99.80, 94.42, 76.14, 75.61, 74.66,
73.38, 71.72, 71.72, 70.05, 61.38.
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4.1.2.2.
3-(Nitrate)propyl-6-((6-(benzyloxy)-2-(4-(benzyloxy)phenyl)-5-hydroxy-4-
oxo-4H-chromen-7-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate (16b).
o
+
Yellow power, 295 mg; yield: 79.2%, mp: 106−108
C; MS(ESI) m/z: 746.1 [M + H] , 768.1
+
1
[M + Na] ; H NMR (DMSO-d , 400 MHz) δ (ppm): 12.97 (s, 1H, 5-OH), 8.03 (d, 2H, J = 8.6 Hz,
6
H-2′,6′), 7.56 (d, 2H, J = 7.3 Hz, Ar-H), 7.47 (d, 2H, J = 7.3 Hz, Ar-H), 7.42−7.32 (m, 6H, Ar-H),
7.20 (d, 2H, J = 8.6 Hz, H-3′,5′), 7.12 (s, 1H, H-8), 6.93 (s, 1H, H-3), 5.71 (d, 1H, J = 5.4 Hz,
H-1′′), 5.59 (d, 1H, J = 5.8 Hz, sugar hydroxyl), 5.43 (d, 2H, J = 7.6 Hz, sugar hydroxyl), 5.21 (s,
2H, -CH -), 5.09 (d, 1H, J =10.9 Hz, -CH -), 4.96 (d, 1H, J =10.9 Hz, -CH -), 4.54 (t, 2H, -CH -),
8
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0
1
2
3
4
5
6
7
8
9
0
1
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
4.25 (d, 1H, J = 9.7 Hz, H-5′′), 4.18 (t, 2H, -CH -), 3.51−3.47 (m, 3H, H-2′′, 3′′,4′′), 2.02−1.98 (m,
2
1
3
2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.80, 169.08, 164.30, 162.00, 156.57,
2
6
153.33, 152.69, 138.01, 136.91, 131.85, 128.95, 128.95, 128.84, 128.84, 128.56, 128.56, 128.49,
128.49, 128.28, 128.28, 123.39, 115.84, 115.84, 106.42, 103.91, 99.95, 94.47, 76.28, 75.73, 74.67,
+
73.44, 71.77, 70.07, 66.72, 61.47, 26.00. HRMS (ESI) m/z calcd for C H NNaO [M + Na]
38
35
15
768.1899, found 768.1853.
4.1.2.3.
2-(Nitrate)ethyl-3,4,5-trihydroxy-6-((5-hydroxy-6-methoxy-2-(4-
methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)tetrahydro-2H-pyran-2-carboxylate (17a).
o
+
Yellow power, 202 mg; yield: 69.8%, mp: 149−151
C; MS(ESI) m/z: 580.1 [M + H] , 602.1
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H NO [M + H] 602.1116, found 602.1119; H NMR
2
5
26
15
(DMSO-d , 400 MHz) δ (ppm): 12.94 (s, 1H, 5-OH), 8.06 (d, 2H, J = 8.9 Hz, H-2′,6′), 7.12 (d, 2H,
6
J = 8.9 Hz, H-3′,5′), 7.08 (s, 1H, H-8), 6.97 (s, 1H, H-3), 5.65 (d, 1H, J =5.0 Hz, H-1′′), 5.56 (d,
23
24
25
26
27
1H, J =5.6 Hz, sugar hydroxyl), 5.39−5.36 (m, 2H, sugar hydroxyl), 4.75 (t, 2H, -CH -), 4.41 (m,
2
2H, -CH -), 4.25 (d, 1H, J = 9.4 Hz, H-5′′), 3.86 (s, 3H, -OCH ), 3.76 (s, 3H, -OCH ), 3.47−3.40
2
3
3
1
3
(m, 3H, H-2′′,3′′,4′′). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.85, 169.00, 164.39, 162.94,
6
156.47, 153.09, 152.61, 133.03, 128.85, 128.85, 123.20, 115.07, 115.07, 106.45, 103.88, 99.96,
94.57, 76.06, 75.58, 73.26, 71.74, 71.74, 61.38, 60.79, 56.07.
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4.1.2.4.
3-(Nitrate)propyl-3,4,5-trihydroxy-6-((5-hydroxy-6-methoxy-2-(4-
methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)tetrahydro-2H-pyran-2-carboxylate (17b).
o
+
Yellow power, 50.1 mg; yield: 16.9%, mp: 105−107
C; MS(ESI) m/z: 594.0 [M + H] , 615.9
+
+
1
[M + Na] ; HRMS (ESI) m/z calcd for C H NO [M + H] 594.1453, found 594.1483; H NMR
2
6
28
15
(DMSO-d , 400 MHz) δ (ppm): 12.93 (s, 1H, 5-OH), 8.04 (d, 2H, J = 8.9 Hz, H-2′,6′), 7.13 (d, 2H,
6
J = 8.9 Hz, H-3′,5′), 7.07 (s, 1H, H-8), 6.94 (s, 1H, H-3), 5.62 (d, 1H, J =5.1 Hz, H-1′′), 5.54 (d,
8
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0
1
2
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7
8
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0
1
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3
4
5
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7
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1H, J =5.7 Hz, sugar hydroxyl), 5.36−5.34 (m, 2H, sugar hydroxyl), 4.55 (t, 2H, -CH -), 4.21 (d,
2
1H, J = 9.7 Hz, H-5′′), 4.17 (t, 2H, -CH -), 3.86 (s, 3H, -OCH ), 3.77 (s, 3H, -OCH ), 3.47−3.38
2
3
3
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
(m, 3H, H-2′′,3′′,4′′), 2.01 (m, 2H, -CH -). C NMR (DMSO-d , 100 MHz) δ (ppm): 182.83,
2 6
169.07, 164.38, 162.94, 156.44, 153.09, 152.60, 133.06, 128.82, 128.82, 123.21, 115.07, 115.07,
106.45, 103.87, 100.03, 94.56, 76.18, 75.68, 73.30, 71.72, 70.87, 61.45, 60.77, 56.06, 26.00.
4.2. MTT assay
Cytotoxicity of all the tested compounds against MCF-7, HCT-116, PC-3, and HepG2 cells
was determined by MTT assay. Cells were planted into 96-well micro-plates at a density of
4
o
2.0-4.0 × 10 cells/mL (100 µL per well). After overnight incubation at 37
C, 5-9 different
concentrations of test compounds were added to the wells. Compounds were dissolved in DMSO
before being diluted in the growth medium. The concentration of DMSO in the wells did not
exceed 0.5%. Cells were further incubated for 72 h, at the end of the incubation time, fresh
medium containing 0.5 mg/mL of MTT were added. Plates were incubated for another 4 h at 37
o
C, the media was removed and formazan crystals formed in the cells were dissolved in 200 µL of
DMSO. Optical density was measured at 570 nm with DMSO as blank using a Bio-Rad
microplate reader (Model 680). In these experiments, the negative reference was 0.1% DMSO,
scutellarin and 5-FU were used as the positive references.
4.3. NO releasing test
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The levels of NO generated by individual compounds were determined by the colorimetric
assay using the nitrite colorimetric assay kit (Beyotime, China), according to the manufacturer’s
instructions. Incubation in phosphate buffer solution (pH 7.4) containing 2% dimethyl sulfoxide
−5
and 10 M of the test compound (1 mL of 0.2 mM solution in 0.1 M phosphate buffer, pH 7.4)
was mixed with a freshly prepared solution of L-cysteine (1 mL of a 3.6 mM solution in 0.1 M
o
phosphate buffer, pH 7.4) at 37 C for 1 h, 2h and 3h in the absence of air. After exposure to air
o
for 10 min at 25 C, an aliquot of the Griess reagent I (50 µL) and Griess reagent II (50 µL) was
added to an equal volume (50 µL) of each test compound’s incubation solution. After 10 min, the
absorbance was measured at 540 nm. Solutions of 1 M sodium nitrite were used to prepare a
nitrite absorbance versus concentration curve under the same experimental conditions. The
different concentrations of nitrite were used as standards to calculate the concentrations of NO
formed by individual test compound.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
Intracellular NO release data were acquired using the Griess reaction in HepG2 and L-O2 cells
according to the manufacturer’s instructions (S0024, Beyotime, China). Briefly, cells were treated
with 100 µM of each compound for 2.5 h. Subsequently, the cells were harvested and their cell
o
lysates were prepared and then mixed with Griess reagent for 10 min at 37 C, followed by
measurement at 540 nm by a microplate reader. The cells treated with 0.4% DMSO in medium
were used as negative control 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.
4.4. Cell cycle study
The cell were incubated with different concentrations of the test compounds 14b and 15a or
vehicle as described above. After incubation for 72 h, the treated cells were trypsinized, washed
with PBS and centrifuged at 2000 rpm/min. The collected cells were fixed by adding cold ethanol
o
(4 C overnight) and incubated for 30 min in PBS containing 100 µL RNase A of RNase and 400
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8
µL of PI. Cell DNA content was measured using flow cytometry (FACS Calibur
Bectone-Dickinson) for cell cycle distribution analysis.
4.5. Hoechst 333258 staining
Cells grown on a sterile cover slip in 6-well plates were treated with test compounds 14b and
15a or vehicle for a certain range of time. The culture medium containing compounds was
removed, and the cells were fixed in 4% paraformaldehyde for 10 min. After being washed twice
with PBS, the cells were stained with 0.5 mL of Hoechst 33258 (Keygen, Nanjing, China) for 5
min and then again washed twice with PBS. The stained nuclei were observed under a flow
cytometry (FACS Calibur Bectone-Dickinson) using 350 nm excitation and 460 nm emission.
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4.6. Analysis of cellular apoptosis
o
Cells were seed into 6-well plates and incubated for 24 h at 37 C under an atomosphere of 5%
CO . Then test compounds 14b and 15a were added in a certain concentration and the negative
2
control which was treated with DMSO were included. After incubation for 72 h, the treated cells
were trypsinized, then washed with PBS twice and centrifuged at 2000 rpm to collect the cells (5
5
× 10 ). After 500 µL of binding buffer was added to suspend the cells, 5 µL of Annexin V-APC
and 7-AAD or PI were added successively and mixed well. Followed by 5−15 min incubation at
room temperature in dark, cell apoptosis was analyzed with flow cytometry (FACS Calibur
Bectone-Dickinson).
4.7. Cell mitochondrial membrane potential assay
Cells were cultured overnight and incubated in triplicate with the test compounds 14b and 15a
or vehicle for 48 h. The cells were stained with the lipophilic cationic dye JC-1, according to the
manufacturer’s instruction (Keygen, KGA601). The percentage of cells with healthy or collapsed
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mitochondrial membrane potentials was monitored by flow cytometry analysis.
4.8. Quantitation of cellular proteins involved in apoptosis
The relative expression levels of 35 apoptosis-related proteins were evaluated using Human
Apoptosis Array kit (R&D Systems, Abingdon, UK) in HepG2 cells. Proteins were extracted
according to the manufacturer’s protocol from cells treated for 24 h with compound 14b (2 µM).
Acknowledgment
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1
This work was financially supported by the Key Laboratory for the Chemistry and Molecular
Engineering of Medicinal Resources (Guangxi Normal University), the Ministry of the Education
of China (CMEMR2015-B07).
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Table 1
Effects of the compounds on cell viability against different cell lines.
a
Compound
IC (µM)
50
b
MCF-7
HCT-116
PC-3
HepG-2
L-O2
SI
1
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
7a
7b
Scutellarin
-FU
2.51 ± 0.34 13.58 ± 0.47
0.36 ± 0.03
0.09 ± 0.01
0.74 ± 0.31
0.67 ± 0.12
1.48 ± 0.15 19.31 ± 0.86 13.0
0.50 ± 0.07 47.96 ± 1.24 95.9
1.83 ± 0.59 32.46 ± 1.10 17.7
6.40 ± 0.66 39.87 ± 1.75 6.2
1
2.96 ± 0.21
1.99 ± 0.99
1.65 ± 0.78
39.97 ± 1.89
21.71 ± 0.93
8.25 ± 0.77
3.64 ± 0.86
> 50
7.25 ± 1.38
2.59 ± 0.82
5.70 ± 3.63
1.74 ± 0.38
2.46 ± 0.57
2.16 ± 1.26
1.27 ± 0.32
> 50
1
1
1
1.45 ± 0.53 10.88 ± 1.32 34.49 ± 1.22 3.2
3.10 ± 0.46 12.35 ± 0.83 45.90 ± 2.37 3.7
3.01 ± 0.89 12.92 ± 1.41 41.58 ± 1.84 3.2
1
1
1
3.65 ± 0.81
> 50
9.53 ± 0.72 22.94 ± 0.56 2.4
c
1
> 50
NT
NT
1
30.83 ± 4.79 17.40 ± 1.7
18.79 ± 1.34 36.75 ± 2.23
1
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
NT
1
NT
> 100
77 ± 2.35
72.90 ± 1.37 56.09 ± 1.23
> 100
NT
5
28.73 ± 3.94 4.16 ± 0.23 24.33 ± 0.36 29.07 ± 0.93
a
IC : Half inhibitory concentrations measured by the MTT assay. The values are expressed as
50
b
average ± standard deviations of three independent experiments. SI: selectivity index. It was
calculated as: SI = IC50, L-O2/HepG-2. NT: not tested.
c

ACCEPTED MANUSCRIPT
Table 2
The amount of NO released by the tested compounds
Compound The amounts of released-NO (µmol/L)
0
.25 h
0.5 h
14.27
10.11
13.91
11.74
33.26
15.54
20.78
20.78
4.51
0.75 h
17.53
11.92
15.72
12.64
41.94
20.42
24.94
26.03
4.69
1 h
2 h
3 h
1
4a
4b
11.20
7.58
19.52
13.55
17.71
14.27
49.35
24.22
34.16
33.80
6.13
26.39
16.26
19.33
21.87
58.03
30.55
46.46
37.96
4.87
39.95
20.06
18.97
23.86
59.12
30.37
44.29
39.41
5.05
1
1
4c
11.92
7.94
14d
15a
15b
26.93
11.74
12.46
13.73
3.96
1
5c
15d
16a
16b
17a
17b
4.14
4.33
4.69
5.05
3.78
3.24
1.07
1.97
2.34
2.34
1.97
1.79
0.53
1.25
1.61
1.25
1.43
1.25

ACCEPTED MANUSCRIPT
1
. Legends for Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 , Fig. 7, Scheme 1 and Scheme 2
Fig. 1. The chemical structures of reported natural product/nitric oxide donor hybrids (18−21).
Fig. 2. The NO level in HepG2 and L-O2 cells.
Fig. 3. Cell cycle analysis of compounds 14b (A) in HepG2 and 15a (B) in PC-3 cells by flow cytometry.
Fig. 4. Hoechst staining of compounds 14b (A) in HepG2 and 15a (B) in PC-3 cells.
Fig. 5. Flow cytometry analysis of compounds 14b (A) induced apoptosis in HepG2, 15a (B) in PC-3 cells and the
apoptosis of nontumor LO-2 cells induced by 14b and 15a (C).
Fig. 6. 14b (A) induced mitochondrial depolarization in HepG2 and 15a (B) in PC-3 cells.
Fig. 7. The effects exerted by compound 14b on the expression of apoptosis-related proteins using the Human
Apoptosis Array kit in HepG2 cells.
Scheme 1. Synthesis of compounds (6a-d) and (9a, b). Reagents and conditions: (a) ClCH COOH, NaOH (aq),
2
o
reflux, 2 h; (b) 30% H O , AcOH, rt, 3 h; (c) fuming HNO , 90 C, 4 h; (d) aminoalcohol, THF, NaH,0 °C, 4 h; (e)
2
2
3
o
fuming HNO , H SO , 0 C, 3 h.
3
2
4
Scheme 2. Synthesis of compounds (14a-d), (15a-d), (16a, b) and (17a, b). Reagents and conditions: (a) BnBr or
CH I, K CO , DMF, rt, 24 h; (b) KOH, MeOH/DCM, rt, 2-3 h; (c) 6a-d, HOBt, EDCI, DMF, rt, 3 h; (d) 9a, b,
3
2
3
DBU, DMF, rt, 2 h.

ACCEPTED MANUSCRIPT
2
. Graphics for Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 , Fig. 7, Scheme 1 and Scheme 2
Figure 1