Discovery of novel antitumor nitric oxide-donating β-elemene hybrids through inhibiting the PI3K/Akt pathway

Article abstract of DOI:10.1016/j.ejmech.2017.04.045

A series of novel furoxan-based NO-donating β-elemene hybrids were designed and synthesized to improve the anticancer efficacy of natural β-elemene. The bioassay results indicated that all of the target compounds exhibited significantly improved antiproliferative activities against three cancer cell lines (SGC-7901, HeLa and U87) compared to parent compound β-elemene. Interestingly, these compounds displayed excellent sensitivity to U87?cells with IC₅₀ values ranging from 173 to 2?nM. Moreover, most compounds produced high levels of NO in?vitro, and the antitumor activity of 11a in U87?cells was markedly attenuated by an NO scavenger (hemoglobin or carboxy-PTIO). Further mechanism studies revealed that 11a caused the G2 phase arrest of the cell cycle and induced apoptosis of U87?cells by preventing the activation of the PI3K/Akt pathway. Moreover, 11a significantly suppressed the tumor growth in H22 liver cancer xenograft mouse model with a tumor inhibitory ratio (TIR) of 64.8%, which was superior to that of β-elemene (TIR, 49.6%) at the same dose of 60?mg/kg. Together, the remarkable biological profiles of these novel NO-donating β-elemene derivatives may make them promising candidates for the intervention of human cancers.

Full text of DOI:10.1016/j.ejmech.2017.04.045

Accepted Manuscript
Discovery of novel antitumor nitric oxide-donating β-elemene hybrids through
inhibiting the PI3K/Akt pathway
Jichao Chen, Tianyu Wang, Shengtao Xu, Pengfei Zhang, Aijun Lin, Liang Wu,
Hequan Yao, Weijia Xie, Zheying Zhu, Jinyi Xu
PII:
S0223-5234(17)30311-2
10.1016/j.ejmech.2017.04.045
EJMECH 9396
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2017
Revised Date: 16 April 2017
Accepted Date: 19 April 2017
Please cite this article as: J. Chen, T. Wang, S. Xu, P. Zhang, A. Lin, L. Wu, H. Yao, W. Xie, Z. Zhu, J.
Xu, Discovery of novel antitumor nitric oxide-donating β-elemene hybrids through inhibiting the PI3K/Akt
pathway, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.045.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of novel antitumor nitric oxide-donating
β-elemene hybrids through inhibiting the PI3K/Akt pathway
a
a
a
a
a
Jichao Chen , Tianyu Wang , Shengtao Xu , Pengfei Zhang , Aijun Lin , Liang
Wu b,*, Hequan Yao a, Weijia Xie a, Zheying Zhu c,*, Jinyi Xu a,*
a State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical
University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China.
b
Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
210009, P. R. China.
c
Division of Molecular Therapeutics & Formulation, School of Pharmacy, the University of Nottingham,
University Park Campus, Nottingham NG7 2RD, U.K.
*Corresponding authors: E-Mail: wul2004@hotmail.com (L. Wu);
E-Mail: Zheying.Zhu@nottingham.ac.uk (Z. Zhu);
E-Mail: jinyixu@china.com; Tel.: +86-25-8327-1299 (J. Xu).
Abstract: A series of novel furoxan-based NO-donating β-elemene hybrids were designed and
synthesized to improve the anticancer efficacy of natural β-elemene. The bioassay results
indicated that all of the target compounds exhibited significantly improved antiproliferative
activities against three cancer cell lines (SGC-7901, HeLa and U87) compared to parent
compound β-elemene. Interestingly, these compounds displayed excellent sensitivity to U87 cells
with IC50 values ranging from 173 to 2 nM. Moreover, most compounds produced high levels of
NO in vitro, and the antitumor activity of 11a in U87 cells was markedly attenuated by an NO
scavenger (hemoglobin or carboxy-PTIO). Further mechanism studies revealed that 11a caused
the G2 phase arrest of the cell cycle and induced apoptosis of U87 cells by preventing the
activation of the PI3K/Akt pathway. Moreover, 11a significantly suppressed the tumor growth in
H22 liver cancer xenograft mouse model with a tumor inhibitory ratio (TIR) of 64.8%, which was
superior to that of β-elemene (TIR, 49.6%) at the same dose of 60 mg/kg. Together, the
remarkable biological profiles of these novel NO-donating β-elemene derivatives may make them
promising candidates for the intervention of human cancers.
Key words: β-elemene; furoxan; antitumor activity; apoptosis; PI3K/Akt pathway
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1. Introduction
Over past centuries, natural products have played a key role in drug discovery and development.
They provided the basis and inspiration for developing effective therapeutics for human diseases
[1,2]. Review of natural products (NPs) over the 34 years from 1981 to 2014 revealed that
approximately 51% of the developed therapeutic agents approved by the Food and Drug
Administration were NPs, their derivatives, and synthetic mimetics related to NPs. Especially in
the antitumor area, the percentage has risen to ca. 65% [3].
β-Elemene (7, Scheme 1), a sesquiterpene compound isolated from the traditional Chinese
medicinal herb Curcuma aromatica Salisb. [4], has been used as an anticancer drug in China for
many years [5]. Numerous reports have demonstrated that β-elemene exhibited its antineoplastic
activity in vivo against various human tumors such as glioblastoma [6], liver [7] and breast [8]
cancers. It inhibited tumor cell growth via diverse mechanisms including induction of apoptosis,
autophagy and cell cycle arrest, and inhibition of cell proliferation and migration [6-9]. No severe
side effects were observed in the clinical studies, and on the contrary, patient immunity was
improved during the therapy with β-elemene [10,11]. Despite these attractive anticancer properties,
the moderate antitumor potency and poor bioavailability of β-elemene hampers its wide
application in clinic. In particular, high concentrations of β-elemene were frequently required in
order to reach the therapeutic effects [11,12].
Nitric oxide (NO) is an important signaling and/or effector molecule involved in various
physiological and pathophysiological processes [13]. High levels of NO exert its antitumor
activity by inducing cell apoptosis, inhibiting tumor metastasis as well as sensitizing drug-resistant
tumor cells to chemotherapy, radiation and immunotherapy in vitro and in vivo [14-16]. However,
due to its short half-life and chemical instability, NO donors are usually employed as surrogates
for NO in anticancer studies [17]. As an important class of NO donors, furoxans are able to
produce high levels of NO in vitro and inhibit the growth of tumors in vivo [18]. In recent decade,
numerous efforts were directed towards the construction of hybrids incorporating furoxan subunits
to advance the pharmacological profile of the parent drug. Many studies have shown that
(phenylsulfonyl)furoxan-based NO-releasing natural products possessed greater antitumor activity
than corresponding parent compounds, furoxan precursors and/or their combinations in vitro and
in vivo [19-22].
In order to improve the anticancer efficacy of natural β-elemene, based on the above findings,
we hypothesized that hybridization of (phenylsulfonyl)furoxan and β-elemene might release high
levels of NO to exert synergistic antitumor effects with β-elemene. Therefore, a series of hybrids
(11a-f, 14, 18a-f) from such furoxan and β-elemene were designed and synthesized. Their
antitumor activity in vitro and in vivo, NO-releasing ability and mechanism of anticancer action
were biologically evaluated.
2. Results and discussions
2.1. Chemistry
The synthesis of compounds 11a-f was illustrated in Scheme 1. The substituted furoxans 6a-f
were synthesized in a five-step sequence. The starting material benzenethiol 1 was treated with
NaOH and chloroacetic acid under reflux to give 2-(phenylthio)acetic acid 2, which was oxidized
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with 30% H2O2 to generate 2-(phenylsulfonyl)acetic acid 3, followed by treatment with fuming
HNO3 to produce di(phenylsulfonyl)furoxan 4. Then, reaction of 4 with different diols gave
mono(phenylsulfonyl)furoxans 5a-e, which were converted to 6a-f by treatment with
corresponding anhydrides in the presence of DMAP/Et3N. Chlorination of β-elemene (7) with
NaClO produced the chlorinated mixture of 8a and 8b, followed by treatment with NaOAc to give
the acylated compounds 9a and 9b. The resulting products were subjected to alkaline hydrolysis to
give a mixture of 13-β-elemol (10a) and 14-β-elemol (10b), which was separated by HPLC to
provide the main component 10a. Subsequent condensation of 10a and 6a-f afforded the target
compounds 11a-f.
The preparation of compounds 14 and 18a-f was depicted in Scheme 2. Reaction of 10a with
Boc-L-Asp in the presence of DCC and DMAP using dry DMF as the solvent generated
intermediate 12 [23], followed by esterification with 5b to give intermediate 13, which was
deprotected using trifluoroacetic acid (TFA) to provide compound 14. Similarly, condensation of
10a and varying Boc-amino acids in dichloromethane produced intermediates 15a-f, followed by
N-Boc deprotection to give intermediates 16a-f. Treatment of 6b with oxalyl chloride formed
intermediate 17, which reacted with 16a-f in the presence of DMAP/Et3N to yield the title
compounds 18a-f.
Scheme 1. Synthetic routes of the title compounds 11a-f. Reaction conditions and reagents: (a)
chloroacetic acid, 5M NaOH, reflux, 2 h, 91%; (b) 30% H2O2, AcOH, rt, 2 h; (c) fuming HNO3,
AcOH, 100 °C, 3 h, 75%; (d) diols, 5M NaOH, 0.5 h, 78-86%; (e) anhydride, DMAP, Et3N,
CH2Cl2, rt, 2 h, 84-93%; (f) NaClO, HOAc/CH2Cl2, 0-5 oC, 6 h, 55%; (g) NaOAc, DMF, 120 oC, 8
h, 75%; (h) MeOH/CHCl3, KOH, reflux, 2 h, 85%; (i) Separated by HPLC, Hexane/EtOH = 98/2
(V/V), UV = 214 nm; (j) 6a-f, EDCI, DMAP, rt, 4-8 h, 82-88%.
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Scheme 2. Synthetic routes of the title compounds 14 and 18a-f. Reaction conditions and reagents:
(a) Boc-L-Asp, DCC, DMAP, dry DMF, rt, 16 h, 62%; (b) 5b, DCC, DMAP, rt, 8 h, 85%; (c) TFA,
CH2Cl2, rt, 1 h, 78%; (d) Boc-amino acids, DCC, DMAP, rt, 4-10 h, 82-91%; (e) TFA, CH2Cl2, rt,
0.5-1.5 h, 77-84%; (f) oxalyl chloride, Cat. DMF, CH2Cl2, rt, 1.5 h; (g) DMAP, Et3N, CH2Cl2, rt,
1-2 h, 45-67%.
2.2. In vitro antiproliferative activity
Initially, compounds 11a-f were first prepared and tested for their antiproliferative effects on
three cancer cell lines (SGC-7901: human gastric carcinoma; HeLa: human cervical
adenocarcinoma; U87: human glioblastoma). The results were presented in Table 1. As expected,
all of the synthesized compounds showed much more potent antiproliferative activities than
β-elemene, and most of them were comparable or even superior to positive control cisplatin.
Interestingly, these compounds exhibited excellent sensitivity to U87 cells with IC50 values
ranging from 0.154 to 0.002 µM. It was found that improvement of the activity was acquired by
decreasing the length of the diol chain from C6 to C2 (e.g. 11c < 11b < 11a). Replacement of
butane-1,4-diol (11b) with 2-butine-1,4-diol (11d) or diethylene glycol (11e) resulted in slightly
increased activity in SGC-7901 and HeLa cells, whereas the activity markedly decreased in U87
cells. Surprisingly, change of succinic anhydride (11b) to o-phthalic anhydride (11f) led to
dramatically decreased activity in all the three cell lines.
Considering that the poor oral bioavailability of β-elemene is one of the main factors that lead
to its moderate antitumor activity, some amino acids were introduced into the linker to improve
the druggability of β-elemene [23-26] and further investigated for the structure-activity
relationships. As a result, compounds 14 and 18a-f were synthesized and evaluated for their
antiproliferative activities against SGC-7901, HeLa and U87 cells. As depicted in Table 1, these
compounds exhibited significantly improved activities compared to β-elemene, which were
comparable or even superior to positive control cisplatin. Replacement of succinic anhydride (11b)
with L-aspartic acid (14) strengthened the activity against SGC-7901 and HeLa cells, whereas the
activity in U87 cells sharply decreased, suggesting that introduction of a polar amino group (-NH2)
into the linker was much detrimental for the cytotoxicity against U87 cells. Incorporating several
other amino acids, such as glycine (18a), β-alanine (18b), L-alanine (18c), etc. into 11b had no
significant effects on SGC-7901 and HeLa cells compared to 11b, while the activity in U87 cells
remarkably diminished except 18d containing L-isoleucine. Extending the chain length from
glycine (18a) to β-alanine (18b) resulted in 3 times increased activity in U87 cells. It was
observed that substitution of varying lipophilic groups at α-position of glycine (e.g. 18c-f)
enhanced the activity at different levels, in particular, 18d showed over 17 times more potent
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activity than 18a in U87 cells, which was comparable to 11b, but still 5 times less than 11a.
Among all target compounds, 11a exhibited the strongest antiproliferative activity against
SGC-7901, HeLa, and U87 cells with IC50 values of 3.18, 4.94, and 0.002 µM, respectively, which
were significantly superior to that of positive control cisplatin (IC50, 9.09, 16.28, and 21.39 µM,
respectively). Moreover, 11a was further assayed with lower cytotoxicity in normal liver LO-2
cells (IC50, 30.42 µM). Thus, compound 11a was selected for subsequent anticancer mechanism
and in vivo cancer xenograft mouse model studies.
Table 1
Antiproliferative activity (IC50, µM) of β-elemene derivatives against three cancer cell lines
a
Cell lines (IC50 , µM)
Compd.
SGC-7901
236.27 ± 18.41
3.18 ± 0.22
8.76 ± 0.53
22.86 ± 1.64
4.97 ± 0.72
4.66 ± 0.25
27.81 ± 2.08
2.92 ± 0.21
15.31 ± 1.36
7.78 ± 0.49
11.81 ± 0.97
3.66 ± 0.26
9.97 ± 0.85
9.49 ± 0.77
9.09 ± 0.83
HeLa
U87
β-Elemene
11a
213.51 ± 15.23
4.94 ± 0.34
10.92 ± 0.79
23.16 ± 0.93
3.78 ± 0.16
5.78 ± 0.55
21.86 ± 1.91
3.64 ± 0.27
10.20 ± 0.58
8.86 ± 0.74
10.92 ± 0.66
4.25 ± 0.55
10.76 ± 0.88
11.24 ± 0.95
16.28 ± 1.06
179.72 ± 15.37
0.002 ± 0.0005
0.007 ± 0.0013
0.154 ± 0.0172
0.085 ± 0.0057
0.024 ± 0.0046
0.081± 0.0062
0.124 ± 0.0089
0.173 ± 0.0203
0.058 ± 0.0038
0.114 ± 0.0092
0.010 ± 0.0018
0.061 ± 0.0071
0.055 ± 0.0034
21.39 ± 1.91
11b
11c
11d
11e
11f
14
18a
18b
18c
18d
18e
18f
Cisplatin
a IC50: concentration of the test compound that inhibits 50% of cell growth. Data are expressed
as the mean ± SD (n = 3).
2.3. Effects of NO on the antitumor activity
To examine whether NO contributes to the antitumor activity, the levels of NO released by the
test compounds in vitro were first detected and presented as that of nitrite by Griess assay. As
shown in Figure 1A, all of the compounds produced high levels of NO with time. It was observed
that the concentrations of released NO gradually decreased with the extension of the diol chain
length (e.g. 11a > 11b > 11c), which was consist with the antiproliferative activity. Among these
compounds, 11a, 14, 18d released higher NO levels than other compounds, while 11c and 11f
exhibited the lowest NO-releasing ability, which might, at least in part, result in their relatively
poor antiproliferative activities.
Subsequently, 11a was investigated for its inhibitory activity against U87 cells in the presence
or absence of an NO scavenger, hemoglobin (Hb) or carboxy-PTIO (PTIO). U87 cells were
pretreated with varying concentrations of Hb or PTIO for 2 h and then treated with 0.01 µM 11a
for another 72 h. The effects of different treatments on the growth of U87 cells were determined
by the MTT assay (Figure 1B). Treatment with 11a alone significantly inhibited the proliferation
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of U87 cells and this inhibitory effect was reduced dose-dependently by pretreatment with Hb or
PTIO. These results demonstrated that NO generated by 11a enhanced its inhibition effect on U87
cell proliferation in vitro.
Furthermore, 10a, 6a as the two moieties of 11a and their equimolar combination were also
examined for their antiproliferative activitities against U87 cells. As shown in Figure 2, the
inhibitory effects of 10a (IC50 = 197.65 µM), 6a (IC50 = 32.24 µM) and their combination (IC50
=
22.53 µM) were much less potent than 11a (IC50 = 0.002 µM), respectively. These results
suggested that the excellent antitumor activity of 11a resulted from a synergic effect of β-elemene
and NO donor moieties.
Figure 1. Effects of NO produced by the title compounds on the antitumor activity. (A) The
levels of released NO for the test compounds were measured at 100 µM over duration of 120 min
by Griess assay. (B) Effect of hemoglobin (Hb) or carboxy-PTIO (PTIO) on the antiproliferative
activity of 11a. U87 cells were pretreated with the indicated concentrations of Hb or PTIO for 2 h
and then treated with 0.01 µM of 11a for another 72 h. Data are expressed as the mean ± SD
(n=3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the group without Hb or PTIO.
Figure 2. Antiproliferative activity of 11a in comparison with that of 10a, 6a and their equimolar
combination in U87 cells.
2.4. Effect of 11a on cell cycle
To investigate whether 11a suppressed the cell growth by a cell-cycle arrest, the cell cycle
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distribution was analyzed by flow cytometry after staining of the DNA with propidium iodide (PI).
As shown in Figure 3, 11a influenced cell cycle progression at low nanomolar concentrations.
Treatment of U87 cells with 11a at concentrations of 0.5-2.0 nM increased the percentage of cells
at the G2 phase from 18.86% to 47.99% accompanying by a decrease in G1-phase cells from
54.11% to 26.96% in a dose-dependent manner, respectively. The results indicated that 11a caused
the G2 phase arrest of the cell cycle in 11a-induced cell growth inhibition.
Figure 3. Effect of 11a on cell cycle progression of U87 cells. Treatment of U87 cells with 11a (0,
0.5, 1.0, 2.0 nM) for 72 h, intracellular DNA was stained with propidium iodide (PI). Cell cycle
distribution was analyzed by flow cytometry.
2.5. Effect of 11a on cell apoptosis
To determine the possible role of 11a in cell apoptosis, U87 cells were treated with different
concentrations of 11a for 72 h and stained with Annexin V-APC/7-AAD. The percentages of
apoptotic U87 cells were measured by flow cytometry. As depicted in Figure 4, 11a significantly
induced apoptosis of U87 cells in a concentration-dependent manner. Treatment of U87 cells with
11a at concentrations of 0.5, 1.0 and 2.0 nM for 72 h resulted in 18.32%, 34.94% and 53.70%
apoptotic cells (Q2 + Q4), respectively, as compared with 6.84% in an untreated group. The
results indicated that 11a exerted its antitumor activity potentially via the induction of cell
apoptosis.
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Figure 4. Effect of 11a on apoptosis of U87 cells. Treatment with 11a at the indicated
concentrations for 72 h, U87 cells were collected and stained with Annexin V-APC/7-AAD,
followed by flow cytometric analysis.
2.6. Effect of 11a on apoptosis-related proteins
Akt plays a key role in regulating a diversity of cellular functions, such as protein synthesis, cell
cycle, cell survival and apoptosis [27,28]. It has been reported that apoptosis might be prevented
through the activation of the PI3K/Akt pathway in several cancer cells [29-32]. Activated Akt
phosphorylates the intracellular protein Bad, resulting in the release of the anti-apoptotic protein
Bcl-2, which contributes to cancer cell survival. Bax, a dominant-negative inhibitor of Bcl-2,
induces a mitochondrial permeability transition and promotes apoptosis [33]. Caspase-3 acts as a
final executor in apoptosis and can be irreversibly activated through cleavage of pro-caspase-3
[34]. As shown in Figure 5, 11a significantly inhibited p-Akt activation, Bcl-2 expression and
promoted Bax expression, caspase-3 activation in concentration-dependent manners, while the
total protein level of Akt was not altered. The results indicated that 11a induced apoptosis of U87
cells possibly through suppressing the activation of the PI3K/Akt pathway.
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Figure 5. Effects of 11a on apoptosis-related proteins in U87 cells. U87 cells were treated with
varying concentrations of 11a for 72 h. (A) The expressions of p-Akt, Akt, Bcl-2, Bax,
pro-caspase-3 and caspase-3 were determined by Western Blotting using specific antibodies.
GAPDH was used as internal control; (B) The density ratios of proteins to GAPDH are shown as
relative expression. Data are expressed as the mean ± SD (n = 3). **P < 0.01 vs. control group.
2.7. In vivo antitumor activity of 11a
To further evaluate the in vivo antitumor activity of 11a, human liver cancer xenograft was
established by subcutaneous inoculation of H22 cells into the right flank of mice. 32 mice were
then divided into four groups at random and administered intravenously with 60 mg/kg β-elemene,
30 or 60 mg/kg 11a in a vehicle of 10% DMF/2% Tween 80/88% saline once a day, respectively.
All the mice were sacrificed after three weeks, and the tumors were excised and weighed. As
presented in Table 2, 11a markedly inhibited the tumor growth in a dose-dependent manner. At a
dose of 60 mg/kg, 11a possessed a tumor inhibitory rate (TIR) of 64.8%, which was significantly
superior to β-elemene with a TIR of 49.6%. Thus, 11a may deserve further investigation as a
potential anti-cancer drug candidate.
Table 2. In vivo antitumor activity of 11a in H22 liver cancer xenograft mouse model
Number of
mice
Weight of
tumor X ± SD
(g)
Ratio of
inhibition
(%)
Weight of mice (g)
Dose
Drugs
mg/kg
Start
End
Start
End
Controla
-
8
8
18.0 ± 0.2
18.1 ± 0.1
18.2 ± 0.2
18.0 ± 0.2
22.9 ± 0.4
20.0 ± 0.3
21.6 ± 0.4
20.9 ± 0.6
5.68 ± 0.49
2.86 ± 0.27**
2.83 ± 0.19**
2.00 ± 0.16**
β-Elemene
60
30
60
8
8
8
8
8
8
49.6%
50.2%
64.8%
11a
a Control: vehicle of 10% DMF/2% Tween 80/88% saline. **P < 0.01 vs. control group.
3. Conclusion
In summary, thirteen novel furoxan-based NO-donating β-elemene derivatives were synthesized
and displayed much more potent antiproliferative activities than parent compound β-elemene
against SGC-7901, HeLa and U87 cell lines, which are comparable or even superior to that of
positive control cisplatin. Moreover, most compounds released high levels of NO in vitro, and the
antitumor activity of 11a in U87 cells was remarkably diminished by an NO scavenger in a
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dose-dependent manner. Further mechanism studies showed that 11a blocked the G2 phase of the
cell cycle and induced apoptosis through inhibiting the activation of the PI3K/Akt pathway.
Finally, the in vivo antitumor activity of 11a was verified in H22 liver cancer xenograft mouse
model. Collectively, the current finding may provide a new insight for the design of natural
product-based drugs to enhance the efficacy of chemotherapy.
4. Experimental section
4.1. Chemistry
4.1.1. General
Most chemicals and solvents were purchased from commercial sources. Further purification and
drying by standard methods were employed when necessary. 1H NMR and 13C NMR spectra were
recorded on Bruker-300 spectrometers in the indicated solvents (TMS as internal standard). Data
are reported as follows: chemical shift in ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, brs = broad singlet, m = multiplet), coupling constant (Hz), and integration. High
Resolution Mass measurement was performed on Agilent QTOF 6520 mass spectrometer with
electron spray ionization (ESI) as the ion source. Purity of all tested compounds was ≥ 95%, as
estimated by HPLC analysis. Flash column chromatography was carried out using commercially
available silica gel (200-300 mesh) under pressure. Intermediates 6a-f [35] and 10a [36] were
synthesized according to our reported procedures.
4.1.2. General procedure for synthesis of the title compound 11a-f
To a solution of 10a (44 mg, 0.2 mmol) in CH2Cl2 (5 mL) was added 6 (0.24 mmol), EDCI (58
mg, 0.3 mmol), DMAP (3 mg, 0.02 mmol), the mixture was stirred for 4-8 h at room temperature.
The reaction mixture was then diluted with CH2Cl2, washed with 10% HCl, water, and brine
successively, dried over anhydrous Na2SO4, and concentrated. The residue was purified by flash
column chromatography using petroleum ether/ethyl acetate (4/1, V/V) as an eluent to afford the
title compounds.
4.1.2.1. Compound 11a
Colorless liquid, yield 84%. 1H NMR (300 MHz, CDCl3) δ 8.13 – 8.01 (m, 2H), 7.77 (t, J = 7.5
Hz, 1H), 7.64 (t, J = 7.7 Hz, 2H), 5.81 (dd, J = 17.8, 10.5 Hz, 1H), 5.05 (s, 1H), 5.01 (s, 1H), 4.93
(d, J = 3.3 Hz, 1H), 4.88 (d, J = 1.2 Hz, 1H), 4.82 (s, 1H), 4.66 – 4.57 (m, 5H), 4.56 – 4.50 (m,
2H), 2.72 (s, 4H), 2.10 – 1.95 (m, 2H), 1.71 (s, 3H), 1.69 – 1.55 (m, 3H), 1.53 – 1.40 (m, 3H),
1.00 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 171.53, 171.38, 158.18, 149.50, 147.70, 146.88,
137.51, 135.19, 129.20, 128.14, 111.77, 110.43, 109.56, 68.35, 65.93, 60.92, 52.10, 41.21,
39.28(2), 32.48, 28.49, 28.40, 26.51, 24.33, 16.07; HRMS (ESI) calculated for C29H36N2NaO9S
[M + Na]+ 611.2034, found 611.2038.
4.1.2.2. Compound 11b
Colorless liquid, yield 87%. 1H NMR (300 MHz, CDCl3) δ 8.12 – 8.01 (m, 2H), 7.77 (dd, J =
10.7, 4.3 Hz, 1H), 7.63 (t, J = 7.7 Hz, 2H), 5.81 (dd, J = 17.8, 10.5 Hz, 1H), 5.05 (s, 1H), 5.01 (s,
1H), 4.93 (d, J = 3.4 Hz, 1H), 4.88 (d, J = 1.0 Hz, 1H), 4.82 (s, 1H), 4.61 (s, 2H), 4.58 (s, 1H),
4.46 (t, J = 6.2 Hz, 2H), 4.20 (t, J = 6.2 Hz, 2H), 2.76 – 2.62 (m, 4H), 2.08 – 1.91 (m, 4H), 1.90 –
1.75 (m, 2H), 1.71 (s, 3H), 1.69 – 1.55 (m, 3H), 1.53 – 1.41 (m, 3H), 1.00 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 171.77, 171.52, 158.42, 149.50, 147.72, 146.88, 137.52, 135.16, 129.20, 128.05,
111.77, 110.41, 109.56, 70.44, 65.88, 63.41, 52.10, 41.21, 39.28(2), 32.50, 28.61, 28.53, 26.52,
24.69, 24.49, 24.34, 16.07; HRMS (ESI) calculated for C31H40N2NaO9S [M + Na]+ 639.2347,
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found 639.2347.
4.1.2.3. Compound 11c
Colorless liquid, yield 85%. 1H NMR (300 MHz, CDCl3) δ 8.06 (d, J = 7.5 Hz, 2H), 7.76 (t, J =
7.5 Hz, 1H), 7.62 (t, J = 7.9 Hz, 2H), 5.81 (dd, J = 17.7, 10.5 Hz, 1H), 5.05 (s, 1H), 5.01 (s, 1H),
4.93 (d, J = 4.5 Hz, 1H), 4.88 (s, 1H), 4.82 (s, 1H), 4.60 (s, 2H), 4.58 (s, 1H), 4.42 (t, J = 6.5 Hz,
2H), 4.12 (t, J = 6.5 Hz, 2H), 2.72 – 2.61 (m, 4H), 2.09 – 1.96 (m, 2H), 1.94 – 1.82 (m, 2H), 1.71
(s, 3H), 1.69 – 1.57 (m, 6H), 1.51 – 1.42 (m, 6H), 1.00 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
172.19, 171.92, 158.93, 149.90, 148.17, 147.26, 138.07, 135.50, 129.55, 128.42, 112.17, 110.86,
109.94, 71.33, 66.26, 64.45, 52.57, 41.67, 39.71, 39.64, 32.94, 29.09, 29.01, 28.37, 28.23, 26.95,
25.36, 25.18, 24.70, 16.51; HRMS (ESI) calculated for C33H44N2NaO9S [M + Na]+ 667.2660,
found 667.2676.
4.1.2.4. Compound 11d
Colorless liquid, yield 78%. 1H NMR (300 MHz, CDCl3) δ 8.08 (d, J = 7.9 Hz, 2H), 7.77 (t, J =
6.8 Hz, 1H), 7.64 (t, J = 7.1 Hz, 2H), 5.82 (dd, J = 17.2, 10.7 Hz, 1H), 5.10 (s, 2H), 5.05 (s, 1H),
5.02 (s, 1H), 4.93 (d, J = 3.8 Hz, 1H), 4.88 (s, 1H), 4.83 (s, 1H), 4.77 (s, 2H), 4.62 (s, 2H), 4.59 (s,
1H), 2.71 (s, 4H), 2.09 – 1.95 (m, 2H), 1.71 (s, 3H), 1.64 – 1.54 (m, 3H), 1.53 – 1.41 (m, 3H),
1.01 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 171.65, 171.35, 157.86, 149.91, 148.14, 147.27,
137.81, 135.64, 129.64, 128.57, 112.20, 110.95, 109.97, 83.74, 78.61, 66.39, 58.52, 52.59, 52.04,
41.70, 39.73, 39.66, 32.96, 28.90, 28.74, 26.97, 24.70, 16.53; HRMS (ESI) calculated for
C31H36N2NaO9S [M + Na]+ 635.2034, found 635.2049.
4.1.2.5. Compound 11e
Colorless liquid, yield 80%. 1H NMR (300 MHz, CDCl3) δ 8.14 – 8.03 (m, 2H), 7.77 (t, J = 7.5
Hz, 1H), 7.63 (t, J = 7.7 Hz, 2H), 5.81 (dd, J = 17.8, 10.5 Hz, 1H), 5.04 (s, 1H), 5.00 (s, 1H), 4.93
(d, J = 3.6 Hz, 1H), 4.88 (s, 1H), 4.82 (s, 1H), 4.65 – 4.52 (m, 5H), 4.30 (t, J = 4.7 Hz, 2H), 3.92 (t,
J = 4.4 Hz, 2H), 3.80 (t, J = 4.7 Hz, 2H), 2.69 (s, 4H), 2.08 – 1.96 (m, 2H), 1.71 (s, 3H), 1.69 –
1.56 (m, 3H), 1.54 – 1.41 (m, 3H), 1.00 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 171.79, 171.48,
158.41, 149.51, 147.72, 146.87, 137.51, 135.18, 129.19, 128.08, 111.76, 110.38, 109.55, 70.03,
68.86, 67.84, 65.83, 63.19, 52.08, 41.17, 39.27(2), 32.48, 28.55, 28.43, 26.50, 24.35, 16.06;
HRMS (ESI) calculated for C31H40N2NaO10S [M + Na]+ 655.2296, found 655.2299.
4.1.2.6. Compound 11f
Colorless liquid, yield 81%. 1H NMR (300 MHz, CDCl3) δ 8.16 – 7.99 (m, 2H), 7.86 – 7.77 (m,
1H), 7.77 – 7.65 (m, 2H), 7.67 – 7.53 (m, 4H), 5.82 (dd, J = 17.8, 10.5 Hz, 1H), 5.15 (s, 1H), 5.07
(s, 1H), 4.94 (d, J = 3.4 Hz, 1H), 4.89 (d, J = 1.0 Hz, 1H), 4.84 (s, 3H), 4.59 (s, 1H), 4.48 (t, J =
5.9 Hz, 2H), 4.41 (t, J = 5.9 Hz, 2H), 2.16 – 1.88 (m, 6H), 1.71 (s, 3H), 1.70 – 1.56 (m, 3H), 1.55
– 1.42 (m, 3H), 1.01 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 167.23, 166.53, 158.42, 149.47,
147.52, 146.87, 137.53, 135.15, 131.96, 131.10, 130.87, 130.58, 129.20, 128.55, 128.28, 128.03,
111.78, 110.79, 109.59, 70.43, 66.77, 64.39, 52.10, 41.22, 39.26(2), 32.53, 26.52, 24.80, 24.43,
24.35, 16.07; HRMS (ESI) calculated for C35H40N2NaO9S [M + Na]+ 687.2347, found 687.2345.
4.1.3. Synthesis of the title compound 14
To a solution of 10a (88 mg, 0.4 mmol) in anhydrous DMF (8 mL) was added Boc-L-aspartic
acid (280 mg, 1.2 mmol), DCC (91 mg, 0.44 mmol), DMAP (0.04 mmol, 5 mg), the mixture was
stirred for 16 h at room temperature. The reaction mixture was added 8 mL H2O and extracted
with ethyl acetate (15 mL× 3). The combined organic extracts were washed with water (10 mL ×
2), dried over anhydrous Na2SO4 and concentrated to give the crude intermediate 12 in 62% yield
11

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without further purification [23].
12 (87 mg, 0.2 mmol), 5b (76 mg, 0.24 mmol), DCC (46 mg, 0.22 mmol) and DMAP (3 mg,
0.02 mmol) were added in CH2Cl2 (5 mL) and stirred for 6 h at room temperature. The reaction
mixture was then diluted with CH2Cl2, washed with 10% HCl, water, dried over anhydrous
Na2SO4, and concentrated. The residue was purified by flash column chromatography using
petroleum ether/ethyl acetate (10/1, V/V) as an eluent to obtain intermediate 13 as yellowish
liquid, yield 85%. 1H NMR (300 MHz, CDCl3) δ 8.07 (d, J = 7.4 Hz, 2H), 7.77 (t, J = 7.5 Hz, 1H),
7.63 (t, J = 7.7 Hz, 2H), 5.81 (dd, J = 17.8, 10.5 Hz, 1H), 5.52 (d, J = 8.6 Hz, 1H), 5.05 (s, 1H),
5.02 (s, 1H), 4.93 (d, J = 2.7 Hz, 1H), 4.88 (d, J = 1.9 Hz, 1H), 4.82 (s, 1H), 4.73 – 4.54 (m, 4H),
4.45 (t, J = 6.1 Hz, 2H), 4.19 (t, J = 6.2 Hz, 2H), 3.10 – 2.80 (m, 4H), 2.10 – 1.91 (m, 2H), 1.90 –
1.75 (m, 2H), 1.70 (s, 3H), 1.68 – 1.52 (m, 3H), 1.45 (s, 12H), 1.00 (s, 3H); MS (ESI) m/z: 732.0
[M + H]+; 749.2 [M + NH4]+; 766.0 [M + Cl]-.
To a solution of 13 (125 mg, 0.17 mmol) in 5 mL CH2Cl2, 0.5 mL trifluoroacetic acid was
dropwise added and stirred at room temperature for 1 h. The reaction solution was concentrated,
and the residue was purified by flash column chromatography using CH2Cl2/MeOH (50/1, V/V) as
an eluent to afford the title compound 14 as yellowish liquid, yield 78%. 1H NMR (300 MHz,
CDCl3) δ 8.12 – 8.00 (m, 2H), 7.77 (t, J = 7.5 Hz, 1H), 7.63 (t, J = 7.7 Hz, 2H), 5.81 (dd, J = 17.8,
10.5 Hz, 1H), 5.05 (s, 1H), 5.03 (s, 1H), 4.93 (d, J = 3.1 Hz, 1H), 4.88 (d, J = 1.4 Hz, 1H), 4.83 (s,
1H), 4.64 (s, 2H), 4.58 (s, 1H), 4.46 (t, J = 6.1 Hz, 2H), 4.21 (t, J = 6.2 Hz, 2H), 3.95 – 3.85 (m,
1H), 2.92 – 2.71 (m, 2H), 2.05 (s, 2H), 2.03 – 1.90 (m, 4H), 1.89 – 1.77 (m, 2H), 1.71 (s, 3H),
1.68 – 1.56 (m, 3H), 1.54 – 1.42 (m, 3H), 1.00 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 173.28,
170.63, 158.42, 149.41, 147.46, 146.83, 137.50, 135.16, 129.20, 128.04, 111.79, 110.77, 109.61,
70.41, 66.43, 63.55, 52.10, 50.77, 41.25, 39.24(2), 38.25, 32.50, 26.51, 24.69, 24.47, 24.34, 16.07;
HRMS (ESI) calculated for C31H42N3NaO9S [M + H]+ 632.2636, found 632.2639.
4.1.4. General procedure for synthesis of the title compounds 18a-f
4.1.4.1. Synthesis of intermediates 15a-f
To a solution of 10a (88 mg, 0.4 mmol) in CH2Cl2 (5 mL) was added corresponding Boc-amino
acid (0.48 mmol), DCC (58 mg, 0.44 mmol), DMAP (5 mg, 0.04 mmol), the mixture was stirred
for 4-10 h at room temperature. The reaction mixture was then diluted with CH2Cl2, washed with
10% HCl, water, dried over anhydrous Na2SO4, and concentrated. The residue was purified by
flash column chromatography using petroleum ether/ethyl acetate (10/1, V/V) as an eluent to give
intermediates 15a-f as yellowish liquid, yield 82-91%.
4.1.4.2. Synthesis of intermediates 16a-f
To a solution of 15 (0.3 mmol) in CH2Cl2 (5 mL), 0.5 mL trifluoroacetic acid was dropwise
added and stirred at room temperature for 0.5-1.5 h. The reaction solution was concentrated, and
the residue was purified by flash column chromatography using CH2Cl2/MeOH (40/1, V/V) as an
eluent to give intermediates 16a-f as yellowish liquid, yield 77-84%.
4.1.4.3. Synthesis of intermediate 17
To a solution of 6b (124 mg, 0.3 mmol) in CH2Cl2 (5 mL) were added oxalyl chloride (37 µL,
0.45 mmol) and a catalytic amount of DMF, the mixture was stirred at room temperature for 1.5 h
and then concentrated to yield intermediate 17 as yellowish liquid without further purification.
4.1.4.4. Synthesis of compounds 18a-f
A mixture of 16 (0.2 mmol), DMAP (3 mg, 0.02 mmol), triethylamine (83 µL, 0.6 mmol) in
CH2Cl2 (5 mL) were dropwise added a 2 mL CH2Cl2 solution of 17 (0.3 mmol) and stirred at room
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temperature for 1-2 h. The reaction mixture was then diluted with CH2Cl2, washed with 10% HCl,
water, dried over anhydrous Na2SO4, and concentrated. The residue was purified by flash column
chromatography using petroleum ether/ethyl acetate (2/1, V/V) as an eluent to afford the title
compounds.
4.1.4.4.1. Compound 18a
Yellowish liquid, yield 49%. 1H NMR (300 MHz, CDCl3) δ 8.06 (d, J = 7.3 Hz, 2H), 7.76 (t, J =
7.4 Hz, 1H), 7.63 (t, J = 7.6 Hz, 2H), 6.19 (s, 1H), 5.81 (dd, J = 17.8, 10.6 Hz, 1H), 5.05 (s, 1H),
5.03 (s, 1H), 4.93 (d, J = 3.3 Hz, 1H), 4.88 (s, 1H), 4.83 (s, 1H), 4.65 (s, 2H), 4.58 (s, 1H), 4.46 (t,
J = 6.1 Hz, 2H), 4.19 (t, J = 6.2 Hz, 2H), 4.08 (d, J = 5.0 Hz, 2H), 2.70 (t, J = 6.4 Hz, 2H), 2.58 (t,
J = 6.5 Hz, 2H), 2.06 – 1.91 (m, 4H), 1.87 – 1.81 (m, 2H), 1.71 (s, 3H), 1.68 – 1.55 (m, 3H), 1.53
– 1.40 (s, 3H), 1.00 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 172.68, 171.46, 169.60, 158.86, 149.84,
147.69, 147.22, 138.01, 135.55, 129.60, 128.45, 112.22, 111.54, 110.00, 70.91, 66.95, 63.81,
52.56, 41.61, 41.37, 39.69, 39.63, 32.96, 30.51, 29.26, 26.97, 25.14, 24.89, 24.71, 16.52; HRMS
(ESI) calculated for C33H43N3NaO10S [M + Na]+ 696.2561, found 696.2576.
4.1.4.4.2. Compound 18b
Yellowish liquid, yield 53%. 1H NMR (300 MHz, CDCl3) δ 8.14 – 7.99 (m, 2H), 7.77 (t, J = 7.5
Hz, 1H), 7.64 (t, J = 7.7 Hz, 2H), 6.24 (s, 1H), 5.81 (dd, J = 17.8, 10.5 Hz, 1H), 5.04 (s, 1H), 5.02
(s, 1H), 4.93 (d, J = 3.1 Hz, 1H), 4.88 (d, J = 1.4 Hz, 1H), 4.83 (s, 1H), 4.61 (s, 2H), 4.58 (s, 1H),
4.46 (t, J = 6.2 Hz, 2H), 4.18 (t, J = 6.2 Hz, 2H), 3.54 (dd, J = 11.9, 6.0 Hz, 2H), 2.68 (t, J = 6.7
Hz, 2H), 2.59 (t, J = 5.9 Hz, 2H), 2.48 (t, J = 6.7 Hz, 2H), 2.06 – 1.91 (m, 4H), 1.90 – 1.78 (m,
2H), 1.71 (s, 3H), 1.70 – 1.54 (m, 3H), 1.53 – 1.40 (m, 3H), 1.01 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 172.33, 171.85, 170.76, 158.43, 149.43, 147.62, 146.85, 137.52, 135.16, 129.20, 128.04,
111.79, 110.63, 109.60, 70.46, 65.87, 63.32, 52.11, 41.23, 39.26(2), 34.39, 33.52, 32.51, 30.34,
28.87, 26.52, 24.72, 24.47, 24.33, 16.07; HRMS (ESI) calculated for C34H46N3NaO10S [M + H]+
688.2898, found 688.2908.
4.1.4.4.3. Compound 18c
Yellowish liquid, yield 64%. 1H NMR (300 MHz, CDCl3) δ 8.06 (d, J = 7.6 Hz, 2H), 7.76 (t, J =
7.4 Hz, 1H), 7.63 (t, J = 7.7 Hz, 2H), 6.28 (d, J = 6.9 Hz, 1H), 5.81 (dd, J = 17.7, 10.6 Hz, 1H),
5.05 (s, 1H), 5.02 (s, 1H), 4.93 (d, J = 3.6 Hz, 1H), 4.88 (s, 1H), 4.82 (s, 1H), 4.70 – 4.57 (m, 4H),
4.46 (t, J = 6.1 Hz, 2H), 4.19 (t, J = 6.0 Hz, 2H), 2.73 – 2.63 (m, 2H), 2.55 (t, J = 6.5 Hz, 2H),
2.03 – 1.93 (m, 4H), 1.86 – 1.80 (m, 2H), 1.70 (s, 3H), 1.67 – 1.52 (m, 3H), 1.49 – 1.41 (m, 6H),
1.00 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 172.59, 172.53, 170.85, 158.74, 149.72, 147.67,
147.09, 137.88, 135.47, 129.51, 128.31, 112.10, 111.16, 109.88, 70.81, 66.76, 63.67, 52.47, 48.03,
41.55, 39.58, 39.51, 32.84, 30.47, 29.16, 26.87, 25.01, 24.77, 24.60, 18.15, 16.42; HRMS (ESI)
calculated for C34H45N3NaO10S [M + Na]+ 710.2718, found 710.2734.
4.1.4.4.4. Compound 18d
Yellowish liquid, yield 67%. 1H NMR (300 MHz, CDCl3) δ 8.13 – 7.99 (m, 2H), 7.77 (t, J = 7.5
Hz, 1H), 7.64 (t, J = 7.7 Hz, 2H), 6.25 (d, J = 8.6 Hz, 1H), 5.81 (dd, J = 17.8, 10.5 Hz, 1H), 5.07
(s, 1H), 5.03 (s, 1H), 4.93 (d, J = 3.4 Hz, 1H), 4.88 (d, J = 0.9 Hz, 1H), 4.83 (s, 1H), 4.72 – 4.61
(m, 3H), 4.58 (s, 1H), 4.45 (t, J = 6.2 Hz, 2H), 4.19 (t, J = 6.2 Hz, 2H), 2.81 – 2.64 (m, 2H), 2.64 –
2.54 (m, 2H), 2.11 – 1.78 (m, 7H), 1.71 (s, 3H), 1.67 – 1.53 (m, 3H), 1.53 – 1.37 (m, 4H), 1.28 –
1.11 (m, 1H), 1.01 (s, 3H), 0.96 – 0.88 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 172.31, 171.26,
170.62, 158.42, 149.43, 147.27, 146.81, 137.51, 135.17, 129.20, 128.03, 111.79, 111.27, 109.60,
70.46, 66.45, 63.37, 56.01, 52.15, 41.00, 39.26(2), 37.51, 32.45, 30.34, 28.90, 26.46, 24.70, 24.57,
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24.46, 24.34, 16.06, 14.98, 11.18; HRMS (ESI) calculated for C37H52N3O10S [M + H]+ 730.3368,
found 730.3371.
4.1.4.4.5. Compound 18e
Yellowish liquid, yield 61%. 1H NMR (300 MHz, CDCl3) δ 8.09 – 7.98 (m, 2H), 7.76 (t, J = 7.5
Hz, 1H), 7.62 (t, J = 7.7 Hz, 2H), 7.35 – 7.18 (m, 3H), 7.16 – 7.07 (m, 2H), 6.17 (d, J = 7.7 Hz,
1H), 5.81 (dd, J = 17.8, 10.5 Hz, 1H), 5.03 (s, 2H), 4.96 – 4.87 (m, 3H), 4.84 (s, 1H), 4.71 – 4.52
(m, 3H), 4.45 (t, J = 6.2 Hz, 2H), 4.17 (t, J = 6.2 Hz, 2H), 3.22 – 3.06 (m, 2H), 2.70 – 2.60 (m,
2H), 2.51 (t, J = 6.6 Hz, 2H), 2.05 – 1.91 (m, 4H), 1.88 – 1.72 (m, 2H), 1.71 (s, 3H), 1.69 – 1.55
(m, 3H), 1.54 – 1.39 (m, 3H), 1.00 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 172.21, 170.78, 170.35,
158.43, 149.44, 147.09, 146.83, 137.53, 135.29, 135.17, 129.21, 128.82, 128.08, 128.04, 126.64,
111.84, 111.42, 109.62, 70.47, 66.65, 63.38, 52.75, 52.06, 41.02, 39.23(2), 37.41, 32.47, 30.18,
28.73, 26.47, 24.71, 24.46, 24.36, 16.07; HRMS (ESI) calculated for C40H50N3O10S [M + H]+
764.3211, found 764.3207.
4.1.4.4.6. Compound 18f
Yellowish liquid, yield 45%. 1H NMR (300 MHz, CDCl3) δ 8.06 (d, J = 7.4 Hz, 2H), 7.76 (t, J =
7.2 Hz, 1H), 7.63 (t, J = 7.7 Hz, 2H), 5.81 (dd, J = 17.7, 10.5 Hz, 1H), 5.04 (s, 1H), 4.99 (s, 1H),
4.92 (d, J = 4.0 Hz, 1H), 4.88 (s, 1H), 4.82 (s, 1H), 4.69 – 4.63 (m, 1H), 4.61 – 4.52 (m, 3H), 4.45
(t, J = 6.1 Hz, 2H), 4.18 (t, J = 6.0 Hz, 2H), 3.68 – 3.54 (m, 2H), 2.83 – 2.54 (m, 4H), 2.26 – 2.14
(s, 1H), 2.07 – 1.90 (m, 7H), 1.85 – 1.79 (m, 2H), 1.70 (s, 3H), 1.67 – 1.53 (m, 3H), 1.52 – 1.39
(m, 3H), 1.00 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 172.85, 171.84, 169.95, 158.82, 149.90,
148.05, 147.25, 138.03, 135.53, 129.59, 128.41, 112.15, 110.80, 109.90, 70.92, 66.52, 63.60,
58.72, 52.60, 46.78, 41.66, 39.71, 39.66, 32.96, 29.13, 29.05, 28.76, 26.99, 25.11, 24.87, 24.66,
24.58, 16.51; HRMS (ESI) calculated for C36H47N3NaO10S [M + Na]+ 736.2874, found 736.2868.
4.2. Pharmacology
4.2.1. In vitro antiproliferative assay
SGC-7901, HeLa and U87 cells were purchased from Nanjing Key Gen Biotech Co. Ltd.
(Nanjing, China). The cytotoxicity of the compounds was determined using MTT assay. Briefly,
test cell lines were plated on 96-well plates at the density of 5×104/well and incubated for 24 h at
o
37 C under an atomosphere of 5% CO2. The test compounds were dissolved in the culture
medium with 0.5% DMSO at different concentrations and treated to the cells for another 72 h. The
MTT (5 mg/mL in PBS) was added and incubated for another 4 h, the optical density was detected
with a microplate reader at 490 nm. The IC50 values were calculated according to the
dose-dependent curves. All the tests were repeated in at least three independent experiments
[38-40].
4.2.2. Griess assay
The levels of NO generated by individual compounds in the lysates are presented as that of
nitrite. Briefly, a mixture of 100 µM test compound and 5.0 mM L-cysteine was incubated at
37 °C for 120 min, and sampled every 15 min. The collected samples (2 mL) were mixed with 0.5
mL of Griess reagent and incubated at 37 °C for another 15 min, followed to be determined at 540
nm using a Microplate Reader (Tecan, Männedorf, Switzerland). Each compound was measured in
triplicate. Standard sodium nitrite solutions at different concentrations were used to construct the
calibration curve, from which the amount of NO release was calculated [35,37].
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4.2.3. Cell cycle analysis
U87 cells were treated with 11a at concentrations of 0, 0.5, 1.0 and 2.0 nM for 72 h,
respectively. The cells were trypsinized, washed with PBS and centrifuged. The collected cells
were fixed by adding 70% ethanol at 4 oC overnight and incubated for 30 min in PBS containing
100 µL RNase A and 400 µL of propidium iodide. Analysis of the cell DNA content was
performed with the system software (Cell Quest, BD Biosciences, USA) [38-40].
4.2.4. Cellular apoptosis analysis
o
U87 cells were seeded into 6-well plates and incubated at 37 C for 24 h, and then treated
different concentrations of 11a (0, 0.5, 1.0 and 2.0 nM) for another 72 h. Cells were washed twice
in PBS and resuspended in 500 µL Annexin V binding buffer. Then 5 µL Annexin V-APC and 5
µL 7-AAD were added successively and the mixture was incubated for 15 min under dark
conditions at 25 °C. Apoptosis was analyzed using a FACS Calibur flow cytometer
(BectoneDickinson, San Jose, CA, USA) [38-40].
4.2.5. Western blotting analysis
U87 cells were incubated with 11a at the indicated concentrations for 72 h. The cells were
harvested and lysed using lysis buffer, and the solution was centrifuged at 14,000 g for 10 min at 4
°C. Then the protein concentrations were determined, and individual cell lysates (25 µg per lane)
were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% gel,
SDS-PAGE) and transferred onto nitrocellulose membranes. After being blocked with 5% fat-free
milk, the target proteins in the membranes were probed with monoclonal antibodies against p-Akt,
Akt, Bcl-2, Bax, caspase-3, pro-caspase-3, GAPDH (KeyGEN Biotech, Nanjing, China) at 4 °C
overnight, respectively. The bound antibodies were detected by horseradish peroxidase (HRP)
conjugated second antibodies and visualized using an enhanced chemiluminescent reagent. The
relative levels of each signaling event to control GAPDH were determined by densimetric
scanning [20,29,40].
4.2.6. In vivo antitumor assay
Five-week-old male Institute of Cancer Research (ICR) mice were purchased from Shanghai
SLAC Laboratory Animals Co. Ltd. A total of 1×106 H22 cells were subcutaneously inoculated
into the right flank of ICR mice according to protocols of tumor transplant research, to initiate
tumor growth. Incubated after one day, mice were weighted and at random divided into 4 groups
(8 mice/group). The groups were treated with β-elemene (60 mg/kg) and 11a (30, 60 mg/kg) in a
vehicle of 10% DMF/2% Tween 80/88% saline, respectively. Vehicle was used as negative control.
Treatments were done at a frequency of intravenous injection one dose per day for three weeks.
The mice were sacrificed, and the tumors were excised and weighed. The inhibition rate was
calculated as follows: Tumor inhibitory ratio (%) = (1-average tumor weight of treated
group/average tumor weight of control group) × 100% [40,41].
Acknowledgments
This study was financially supported by the National Natural Science Foundation of China (No.
81373280, 81673306), the Project Program of the State Key Laboratory of Natural Medicines,
China Pharmaceutical University (No. SKLNMKF201710), and the Innovation project of Jiangsu
Province (No. KYLX15_0633).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
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1. Thirteen novel furoxan-based NO-donating β-elemene hybrids were synthesized.
2. All of the compounds exhibited significantly improved antitumor activity in vitro.
3. The activity of 11a in U87 cells was markedly diminished by an NO scavenger.
4. 11a induced cell apoptosis by preventing the activation of the PI3K/Akt pathway.
5. 11a effectively inhibited liver tumor growth in an in vivo mouse model.