Conjugates of podophyllotoxin and norcantharidin as dual inhibitors of topoisomeraseⅡ and protein phosphatase 2A

Article abstract of DOI:10.1016/j.ejmech.2016.07.031

A series of novel conjugates of podophyllotoxin and norcantharidin was designed using association strategy, and synthesized by coupling 4′-demethylepipodophyllotoxin with N-amino acid norcantharimides, and their cytotoxicitiy was evaluated against four hu

Full text of DOI:10.1016/j.ejmech.2016.07.031

Accepted Manuscript
Conjugates of podophyllotoxin and norcantharidin as dual inhibitors of
topoisomeraseⅡ and protein phosphatase 2A
Zhen-Bo Tang, Yao-Zhang Chen, Jie Zhao, Xiao-Wen Guan, Yong-Xin Bo, Shi-Wu
Chen, Ling Hui
PII:
S0223-5234(16)30579-7
10.1016/j.ejmech.2016.07.031
EJMECH 8749
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 January 2016
Revised Date: 15 July 2016
Accepted Date: 16 July 2016
Please cite this article as: Z.-B. Tang, Y.-Z. Chen, J. Zhao, X.-W. Guan, Y.-X. Bo, S.-W. Chen, L. Hui,
Conjugates of podophyllotoxin and norcantharidin as dual inhibitors of topoisomeraseⅡ and protein
phosphatase 2A, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.031.
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ACCEPTED MANUSCRIPT
Conjugates of podophyllotoxin and norcantharidin as dual inhibitors of
topoisomerase and protein phosphatase 2A
Zhen-Bo Tang, Yao-Zhang Chen, Jie Zhao, Xiao-Wen Guan, Yong-Xin Bo, Shi-Wu Chen*,
Ling Hui*

ACCEPTED MANUSCRIPT
Conjugates of podophyllotoxin and norcantharidin as dual
inhibitors of topoisomerase and protein phosphatase 2A
Zhen-Bo Tang a, Yao-Zhang Chen c, Jie Zhao a, Xiao-Wen Guan a, Yong-Xin Bo a,
Shi-Wu Chen a,*, Ling Hui b,d,*
a School of Pharmacy, Lanzhou University, Lanzhou 730000, China
b Experimental Center of Medicine, General Hospital of Lanzhou Military Command, Lanzhou 730050,
China
c Department of Parmaceutics, Gansu Provincial Hospital of TCM, Lanzhou 730050, China
d
Key Laboratory of Stem Cells and Gene Drug of Gansu Province, General Hospital of Lanzhou
Military Command, Lanzhou 730050, China
Abstract: A series of novel conjugates of podophyllotoxin and norcantharidin was
designed using association strategy, and synthesized by coupling 4′-demethylepi-
podophyllotoxin with N-amino acid norcantharimides, and their cytotoxicitiy was
evaluated against four human tumor cell lines (A-549, HepG2, HeLa and HCT-8) and
normal human diploid fibroblast line WI-38. These compounds exhibited potent
cytotoxic effects on tumor cell lines, whereas it was less toxic to WI-38 cells than
anticancer drug VP-16 or its parent compound norcantharidin. Furthermore,
conjugates 7a, 7c, 7f, 7j, 7k and 7l displayed excellent PP2A inhibition activity with
IC50 values of 0.49–9.52 ꢀM. The most potent compound 7l also exhibited
topoisomeraseꢀinhibition activity. In addition, compound 7l induced cell-cycle arrest
in the G2/M phase in HepG2 by regulating levels of cyclinB1 and cdc2.
Key words: podophyllotoxin, cantharidin, TOP-Ⅱ, PP2A, cell-cycle arrest
* Corresponding author.
For Prof. Shi-Wu Chen: e-mail, chenshw@lzu.edu.cn; Tel./fax, 86-931-8915686.
For Prof. Ling Hui: e-mail, zyhuil@hotmail.com; Tel./fax, 86-931-8994582.
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1. Introduction
In tumor therapy, anticancer drugs based on a single biological target or pathway
show good efficacy, and reduce the risk of side effects. However, cancer is a complex
disease with multiple causes. In many cases, when a particular target or pathway is
completely blocked, the tumor cells will compensate for this change by another
potential pathway, and the therapeutic effect disappears [1]. For each specific tumour
type, effective chemotherapy treatment depends on the use of a suitable drug
combination. Such drug combinations consist of two or more single agents or drugs
joined together either directly or indirectly via a chemical linker [2].
Podophyllin, an ethanolic extract of Podophyllum peltatum L. or P. emodi Wall,
is a good source of the aryltetralin-type lignan, podophyllotoxin (PPT, 1). The latter
compound, as well as its congeners and derivatives exhibit pronounced biological
activity mainly as strong antiviral agents and as antineoplastic drugs [3]. The
podophyllotoxin derivatives etoposide (VP-16, 3), etopophos (etoposide phosphate),
and teniposide (VM26, 4) are thus successfully utilized in the treatment of a variety of
malignant conditions [4]. The cytotoxic mechanism of VP-16 is the inhibition of
topoisomeraseꢀ (TOPꢀ), unlike the parent compound which inhibits mitosis by
interfering with tubulin. VP-16 induces cell death by enhancing the TOP II-mediated
DNA cleavage through the stabilization of the transient DNA/TOP II cleavage
complex [5]. The clinical success of VP-16 and VM26 has triggered the search for
compounds with a similar mechanism of action but without their shortcoming such as
poor water-solubility and acquired drug-resistance. Continuing research on PPT is
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currently focused on structure optimization to generate derivatives with superior
pharmacological profiles and broader therapeutic scope. Tafluposide (F11782) and
F14512 are two potential drug candidates based on PPT recently applied in clinical
trials. F11782 is a dual inhibitor of TOP I andꢀ which impairs the binding of the
enzyme to DNA, but does not stabilize the cleavage complex [6]. The
spermine-conjugated 4'-demethy-4-epipodophyllotoxin (DMEP, 2) derivative F14512
is a TOP ꢀ poison that inhibits the polyamines transport system to target
preferentially tumor cells [7].
Cantharidin (CTD, 5), the principle active ingredients of Epicanta gorhami or
Mylabris spp (blister beetles), is active in vitro against several tumor cell lines,
including cervical, hepatoma, ovarian, laryngocarcinoma, colon, osteocarcinoma, and
leukamia cell lines. However its applications are limited by its severe nephrotoxic and
inflammatory side effects, and it has mainly been used to treat the gastrointestinal
tract, the ureter, and the kidney [8]. Norcantharidin (NCTD, 6, Fig. 1), the
demethylated analogue of cantharidin, is active in vitro against several tumor cell
lines and appears to cause less nephrotoxic and inflammatory side effects [9].
Recently, a series of bioactive analogues of CTD or NCTD have been synthesized in
an attempt to reduce its toxicity and thereby increase its potential use [10–14]. The
anticancer activity of CTD and NCTD is thought to come from the inhibition of
serine/threonine protein phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A)
[15–17]. These two phosphatases are known to be involved in many different cellular
processes, including repairing DNA damage, cell cycle progression, and apoptosis
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[18,19].
Recently, we have reported some conjugates of podophyllotoxin together with
5-FU [20 22], d4T [23,24] and dichloroplatinum [25], which show better antitumor or
–
antiviral activities than the parent compound. In our continuing efforts to find new
compounds with potent activities and low toxicity based on a natural product, we
report herein a series of conjugates of DMEP together with NCTD, and their
cytotoxicities in vitro against four human tumor cell lines, together with a human lung
fibroblast cell line. The most potent compound 7l was further evaluated for its effect
on inhibition of PP2A and TOPꢀ, cell cycle progression, and interaction with CT
DNA.
2. Results and discussion
2.1 Chemistry
In a previous publication [26], amino acid substituted norcantharimides were
synthesized by a modified Gabriel synthesis, by reaction of NCTD and amino acids in
the presence of equivalents anhydrous Et3N in anhydrous toluene in a thick-walled
glass pressure vessel for 24 h. However, this reaction was time consuming and
dangerous. We synthesized cantharimides 6a-l with high yield via a melting method
as the procedure outlined in Scheme 1. Briefly, the mixture of NCTD [27] and
appropriate α-amino acids, or β-alanine, or γ-aminobutyric acid, or 4-aminobenzoic
acid were heated to melting and reacted for another 40–60 mins under N2. The above
mixture was then cooled to room temperature and recrystallized to provide the
corresponding intermediate 6a-l.
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The synthesis of conjugates 7a-l was carried out according to the procedure
outlined in Scheme 2. Firstly, the intermediate 4β-amino-4′-demethyl-
4-deoxylpodophyllotoxin was obtained from DMEP as described in previous
publication [24]. Compounds 7a-l were obtained through reaction of the intermediate
4β-amino-4′-demethyl-4-deoxypodophyllotoxin with the corresponding 6a-l under
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) in the
presence of N-hydroxybenzotriazole (HOBT) in dichloromethane. The structures of
target compounds 7a-l were identified by IR, 1H NMR spectra, 13C NMR spectra and
high resolution mass spectrometry (HRMS).
Furthermore, the octanol-water partition coefficients of 7a-l were also
determined to evaluate their water-solubility [28]. The results are shown in Table 1.
As expected, compounds 7a-l were more soluble in water than VP-16.
2.2 Biological activity
2.2.1 Cytotoxicities of compounds 7a-7l
The in vitro cytotoxicity of compounds 7a-7l were evaluated against a panel of
four human cancer cell lines (lung carcinoma A-549, hepatic carcinoma HepG2,
cervical carcinoma HeLa and human colorectal adenocarcinoma HCT-8 cells)
together with the human lung fibroblast cell line WI-38, using VP-16 and NCTD as
reference compounds. The screening procedure was based on the standard MTT
method [29], and the IC50 values are summarized in Table 1.
The conjugates 7a-7l were generally more potent than VP-16 and NCTD in their
cytotoxicity to these four cell lines (Table 1). In the group of compounds, these
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compounds were most effective in HepG2 cells, and had lowest potency in HCT-8
cells, although the order of potency varied in each cell line. The IC50 values for
compound 7l, which is the most potent conjugate in the group compounds, were
4.57±0.21, <0.01, 4.33±0.12, and 3.48±0.45 ꢀM for the A-549, HepG2, HeLa, and
HCT-8 tumor cells, respectively. Whereas compound 7l was less toxic than VP16 and
NCTD to the normal human lung fibroblast cell WI-38, the IC50 values for compound
7l, VP16 and NCTD were >100, 17.6±3.29 and 26.73±2.28 ꢀM for WI-38,
respectively.
2.2.2 PP2A inhibition activities
Serine/threonine protein phosphatases (S/T-PPs) are thought to be cancer
suppressive target, since the inhibition of the S/T-PPs can lead to an increased
phosphorylation and hence the activation of substrate kinases [18]. Cantharidin and its
analogues have been of considerable interest as potent inhibitors of the PP1 and PP2A
S/T-PPs. However, cantharimides with non-polar or acidic amino acid residues are
only poor inhibitors of PP1 and PP2A [26], or even the norcantharimides have shown
to be devoid of protein phosphatase inhibition [30,31]. To determine whether
conjugates 7a–7l inhibit PP1 and PP2A, we evaluated their ability to directly inhibit
PP1 and PP2A in vitro assay, NCTD was included as internal standards to ensure the
relative validity of our protocol [32].
All conjugates were initially screened at a dose of 100 ꢀM. In the case of PP1
inhibition, none of the conjugates reported herein displayed any noteworthy activity
(data not shown). Otherwise, conjugates 7a, 7c, 7f, 7j, 7k and 7l display excellent
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potency on inhibition of PP2A, their IC50 value were shown in Table 2. Compound 7l
possess the most potent PP2A inhibition with IC50 values of 0.49 ꢀM. These results
indicate that compound 7l shows the selectivity for PP2A over PP1, which is
consistent with previous results with the cantharimides [15,26].
To further confirm and analyze the cellular effects related to the alteration of
PP2A activity by evaluating the effect of compound 7l. The HepG2 cells were
incubated with different concentration 7l for 24 h, and a phosphopeptide substrate was
measured [33]. PP2A activity was reduced to approximately 73%, 32% and 27% of
the control activity at 1 ꢀM, 2 ꢀM and 5 ꢀM, respectively (Figure 2). PP2A activity
was decreased by 7l exposure in a concentration-dependent manner.
In view of the differential cytotoxicity, inhibition of PP2A and solubility,
compound 7l has the most potential as an anticancer drug, and we choose the HepG2
cell line to further investigate the mechanism of its cytotoxic effect.
2.2.3 Compound 7l inhibits Topo
ꢀ
In a previous paper it was demonstrated the ability of DMEP derivates can
inhibit the relaxation activity of TOPꢀ [34]. Thus, we investigate the effect of 7l on
the catalytic activity of TOP ꢀ by evaluated the effect of compound 7l on
topoisomerase II-mediated DNA relaxation [35]. The supercoiled DNA was converted
into relaxed forms by the enzyme (TOPꢀ) as shown in Figure 3 (lane 2). At 50 ꢀM
concentration 7l exerts a partial inhibitory effect (lane 3), as demonstrated by the
increase of supercoiled DNA and by the concurrent decrease in relaxed products.
Nevertheless, by increasing the concentration up to 100 ꢀM, a complete inhibition is
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observed for 7l (lane 4), but only a partial effect on TOPꢀ relaxation for VP-16 (lane
5). Taking into account that VP-16 exerts the lower antiproliferative capacity, these
data could suggest the contribution of the inhibition on TOPꢀ to the cytotoxicity.
2.2.4 Compound 7l induces cell cycle arrest in G2/M phase by effect on cyclin B1 and
cdc2
Podophyllum derivates including VP-16 [29,34], and cantharidin [36] inhibit
cancer cells though G2/M cell cycle arrest. Thus, we investigated the effect of 7l on
cell cycle progression by means of fluorescence-activated cell sorting analysis of
HepG2 cells stained with propidium iodide. Treatment with 7l led to a dose-dependent
accumulation of cells in the G2/M phase with a concomitant decrease in the
population of G1 phase cells (Figure 4). After exposure to 0.5 ꢀM (Fig. 4B) and 1 ꢀM
(Fig. 4C) 7l for 24 h, 27.6% and 58.6%, respectively, of the cells were in G2/M phase,
compared with 9.4% in untreated cultures (Fig. 4A). These results demonstrated that
7l also induces cell cycle arrest in G2/M phase.
During normal cell cycle, the progression of cells from the G2 to M phase is
triggered by activation of the cyclin B1-dependent cdc2 kinase [37]. We therefore
investigated the expression levels of cyclin B1 and cdc2 in HepG2 cells following
treatment with 7l (0, 0.5, 1 ꢀM) for 24 h. Western blot analysis revealed a marked
increase in cyclin B1 and cdc2 protein levels in 7l-treated HepG2 cells compared with
the control (Figure 5). Furthermore, the effect of 7l treatment on these cell-cycle
regulators was also time-dependant. These results indicate that the 7l-induced G2/M
cell cycle arrest involves cell-cycle regulators cdc2 and cyclin B1 in a dose-dependent
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manner.
2.2.5 Interaction of 7l with CT DNA
DNA is an important pharmacological target of antitumor drugs. It is therefore
essential to the development of effective chemotherapeutic agents to explore their
interaction with DNA. A binding assay of compound 7l with CT DNA was performed
by monitoring changes in the fluorescence spectroscopy emission pattern of
compound 7l (1 ꢀM, excited at 264 nm) with increasing concentrations of CT DNA (0,
1, 2, 3, 4, and 5 ꢀM ) in pH 7.2 PBS [38]. Even though no appreciable change in the
position of the charge transfer band of the complex was observed upon addition of
DNA, the fluorescence intensity of the complex decreases progressively with
increasing concentration of DNA, suggesting that compound 7l-based emission is
quenched when it is bound to DNA (Figure 6).
3. Conclusion
In summary, a series of novel conjugates of PPT and NCTD show promising in
vitro cytotoxicities against a panel of human cancer cell lines and less toxicities on
human normal cell line WI-38. Especially, the most potential conjugate 7l which
inhibits TOPꢀ and PP2A, interacts with CT DNA, and induces cell cycle arrest in
the G2/M phase involving cell-cycle regulators cyclin B1 and cdc2. These results
suggest that conjugate 7l is a dual inhibitor of TOPꢀ and PP2A with potential for
further development as anticancer agent.
4. Experimental Methods
4.1. Chemistry
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All starting materials and regents were purchased commercially and used without
further purification, unless otherwise stated. All reactions were monitored by thin
layer chromatograph (TLC) on silica gel GF254 (0.25 mm thick). Column
chromatography (CC) was performed on Silica Gel 60 (230-400 mesh, Qingdao
Ocean Chemical Ltd., China). Melting points were determined in Kofler apparatus
and were uncorrected. IR spectra were measured on a NicoLET iS5 spectrometer on
1
neat samples placed between KBr plates. H NMR and 13C NMR spectra were
recorded with a Mecury-600BB spectrometer with TMS as an internal standard, all
chemical shift values are reported as ppm. Mass spectra were recorded on a Bruker
Dalton APEXꢀ 49e and Esquire 6000 (ESI-ION TRAP) spectrometer with ESI
source as ionization, respectively.
4.1.1. General procedure of synthesis of 6a-l
A mixture of NCTD (336 mg, 2.0 mmol), appropriate α-amino acids, or β-alanine,
or γ-aminobutyric acid, or 4-aminobenzoic acid (2.0 mmol) were heated to melt and
further reacted for 40–60 mins under N2. The above mixture was then cooled to room
temperature and recrystallized to provide the corresponding intermediate 6a-l.
4.1.1.1 (2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)acetic acid (6a)
Yield: 76%; 1H NMR (600 MHz, DMSO-d6) δ 4.63 (s, 2 H), 4.00 (s, 2 H), 3.10 (s,
2 H), 1.60–1.58 (m, 4 H).
4.1.1.2 (2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)propanoic acid (6b)
Yield: 72%; 1H NMR (600 MHz, DMSO-d6) δ 5.43–5.42 (m, 1 H), 4.76–4.32 (m,
2 H), 2.96–2.92 (s, 2 H), 1.87–1.84 (m, 2 H), 1.63–1.60 (m, 2 H), 1.58 (d, J = 7.2 Hz,
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3 H).
4.1.1.3 (2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-3-methylbutanoic acid
(6c)
Yield: 70%; 1H NMR (600 MHz, DMSO-d6) δ 4.85 (d, J = 4.2 Hz, 1 H), 4.78 (d,
J = 3.6 Hz, 1 H), 4.25 (d, J = 10.2 Hz, 1 H), 2.95–2.90 (m, 3 H), 1.88–1.86 (m, 2 H),
1.63–1.60 (m, 2 H), 1.10 (d, J = 6.6 Hz, 3 H), 0.78 (d, J = 6.6 Hz, 3 H).
4.1.1.4
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-4-methylpentanoic
acid (6d)
1
Yield: 51%; H NMR (600 MHz, DMSO-d6) δ 4.75 (d, J = 4.2 Hz, 2 H),
4.73–4.71 (m, 1 H), 2.95−1.92 (m, 2 H), 1.98−1.93 (m, 1 H), 1.88−1.83 (m, 2 H),
1.64−1.61 (m, 3 H), 1.37−1.36 (m, 1 H), 0.92 (d, J = 6.6 Hz, 3 H), 0.90 (d, J = 6.6 Hz,
3 H).
4.1.1.5 (2S,3S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-3-methylpentanoic
acid (6e)
Yield: 65%; 1H NMR (600 MHz, DMSO-d6) δ 4.87 (d, J = 4.8 Hz, 1 H), 4.79 (d,
J = 4.8 Hz, 1 H), 4.34 (d, J = 11.4 Hz, 1 H), 2.95−2.88 (m, 2 H), 1.90−1.87 (m, 2 H),
1.63−1.58 (m, 4 H), 1.28−1.24 (m, 1 H), 1.06 (d, J = 6.0 Hz, 3 H), 0.83 (t, J = 7.2 Hz,
3 H).
4.1.1.6
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-4-(methylthio)
butanoic acid (6f)
1
Yield: 68%; H NMR (600 MHz, DMSO-d6) δ 4.85–4.84 (m, 1 H), 4.76 (t, J =
5.4 Hz, 1 H), 2.98−2.93 (m, 2 H), 2.58–2.33 (m, 4 H), 2.08 (s, 3 H), 1.88−1.82 (m, 2
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H), 1.65−1.59 (m, 2 H).
4.1.1.7 (2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-2-phenylacetic acid
(6g)
Yield: 70%; 1H NMR (600 MHz, DMSO-d6) δ 7.46–7.43 (m, 2 H), 7.38–7.36 (m,
3 H), 5.76 (s, 1 H), 4.88 (d, J = 5.4 Hz, 1 H), 4.67 (d, J = 4.8 Hz, 1 H), 2.98−2.95 (m,
2 H), 1.86−1.80 (m, 2 H), 1.64−1.60 (m, 2 H).
4.1.1.8
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-3-phenylpropanoic
acid (6h)
Yield: 64%; 1H NMR (600 MHz, DMSO-d6) δ 7.25–7.19 (m, 3 H), 7.12–7.10 (m,
2 H), 4.97–4.92 (m, 1 H), 4.71 (d, J = 7.2 Hz, 1 H), 4.67 (d, J = 6.6 Hz, 1 H),
3.61–3.56 (m, 1 H), 3.44–3.37 (m, 1 H), 2.80 (d, J = 10.8 Hz, 1 H), 2.68 (d, J = 10.2
Hz, 1 H), 1.86−1.75 (m, 2 H), 1.62−1.51 (m, 2 H).
4.1.1.9
(2S)-3-(1H-benzo[d]imidazol-2-yl)-2-(1,3-dioxooctahydro-2H-4,7-
epoxyisoindol-2-yl)propanamide (6i)
Yield: 56%; 1H NMR (600 MHz, DMSO-d6) δ 8.12 (s, 1 H), 7.48 (d, J = 7.8 Hz,
1 H), 7.32 (d, J = 7.8 Hz, 1 H), 7.18 (t, J = 7.2 Hz, 1 H), 7.06 (d, J = 7.8 Hz, 1 H),
7.02 (d, J = 1.8 Hz, 1 H), 5.04–5.01 (m, 1 H), 4.74 (d, J = 4.8 Hz, 1 H), 4.68 (d, J =
4.8 Hz, 1 H), 3.72–3.68 (m, 1 H), 3.57–3.52 (m, 1 H), 2.76 (d, J = 6.6 Hz, 1 H), 2.61
(d, J = 6.6 Hz, 1 H), 1.84−1.77 (m, 2 H), 1.57−1.48 (m, 2 H).
4.1.1.10 4-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)benzoic acid (6j)
Yield: 66%; 1H NMR (600 MHz, DMSO-d6) δ 7.80 (d, J = 9.0 Hz, 2 H), 7.31 (d,
J = 9.6 Hz, 2 H), 4.98 (s, 2 H), 4.67 (d, J = 4.8 Hz, 1 H), 3.04−3.01 (m, 2 H),
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1.94−1.92 (m, 2 H), 1.69−1.68 (m, 2 H).
4.1.1.11 3-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)propanoic acid (6k)
Yield: 74%; 1H NMR (600 MHz, DMSO-d6) δ 4.82 (d, J = 4.2 Hz, 1 H), 4.71 (d,
J = 4.2 Hz, 1 H), 3.75−3.72 (m, 2 H), 2.86 (t, J = 7.2 Hz, 2 H), 2.56 (t, J = 6.6 Hz, 2
H), 1.85−1.82 (m, 2 H), 1.60−1.58 (m, 2 H).
4.1.1.12 4-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)butanoic acid (6l)
Yield: 70%; 1H NMR (600 MHz, DMSO-d6) δ 4.82 (d, J = 4.8 Hz, 1 H), 4.77 (d,
J = 4.2 Hz, 1 H), 3.53−3.50 (m, 2 H), 2.84−2.81 (m, 2 H), 2.17–2.11 (m, 2 H),
1.98−1.92 (m, 2 H), 1.85−1.82 (m, 2 H), 1.61−1.60 (m, 2 H).
4.1.2. General procedure of synthesis of 7a-l
A mixture of 4β-amino-4-deoxylpodophyllotoxin (210 mg , 0.53 mmol), 6a-l (0.5
mmol), EDC·HCl (115 mg, 0.6 mmol) and HOBt (81 mg, 0.6 mmol)) was stirred in
dried CH2Cl2 (5 ml) for 1 h at 0ꢁ, then stirred for overnight at room temperature.
After complete reaction, the mixture was washed by H2O and then the extract was
dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by
silica gel column chromatography, using CH2Cl2/acetone 20:1 as the eluent to afford
pure compounds 7a-l.
4.1.2.1 2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,9R)-9-(4-
hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho
[2,3-d][1,3]dioxol-5-yl)acetamide (7a)
23
Yield: 68%; mp: 201−203 ; [α]D –78° (c 0.5, CHCl3); IR (KBr cm–1) 3348,
℃
1
2953, 1775, 1705, 1613, 1516, 1484, 1393, 1229, 1112, 1036, 999, 938; H NMR
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(600 MHz, CDCl3) δ 6.75 (s, 1 H), 6.50 (s, 1 H), 6.40 (d, J = 7.2 Hz, 1 H), 6.27 (s, 1
H), 5.97 (d, J = 5.4 Hz, 2 H), 5.24−5.21 (m, 1 H), 4.79 (d, J = 4.8 Hz, 1 H), 4.61 (d, J
= 4.8 Hz, 1 H), 4.53 (d, J = 4.8 Hz, 1 H), 4.33 (t, J = 8.4 Hz, 1 H), 4.25 (d, J = 16.2
Hz, 1H), 4.16 (d, J = 16.2 Hz, 1 H), 3.82 (t, J = 9.6 Hz, 1 H), 3.75 (s, 6 H), 2.99−2.96
(m, 2 H), 2.92–2.90 (m, 1 H), 2.80 (dd, J = 14.4, 4.8 Hz, 1 H), 1.84−1.79 (m, 2 H),
1.63−1.61 (m, 2 H); 13C NMR (150 MHz, CDCl3) δ 176.3, 176.2, 174.5, 165.5, 148.2,
147.4, 146.4 (2 C), 133.9, 132.4, 130.2, 128.5, 109.9, 109.1, 107.7 (2 C), 101.5, 79.8
(2 C), 68.6, 56.3 (2 C), 49.9 (2 C), 49.8, 48.0, 43.5, 41.6, 36.8, 28.4, 28.2; LRMS(ESI)
m/z 629.1861 ([M+Na]+, 100%); HRMS (ESI) 629.1731 for [M+Na]+ (calcd 629.1742
for C31H30N2O11Na).
4.1.2.2
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,
9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]
naphtho[2,3-d][1,3]dioxol-5-yl)propanamide (7b)
23
D
Yield: 52%; mp: 169−170 ; [α] –92° (c 0.5, CHCl3); IR (KBr cm–1) 3379,
℃
1
2943, 1774, 1704, 1614, 1515, 1484, 1391, 1228, 1114, 1035, 998, 931; H NMR
(600 MHz, CDCl3) δ 6.71 (s, 1 H), 6.48 (s, 1 H), 6.27 (s, 2 H), 5.99−5.98 (m, 2 H),
5.91 (d, J = 7.8 Hz, 1 H), 5.43 (s, 1 H), 5.31−5.27 (m, 1 H), 4.76−4.74 (m, 3 H), 4.54
(d, J = 4.8 Hz, 1 H), 4.34 (t, J = 8.4 Hz, 1 H), 3.94 (t, J = 9.9 Hz, 1 H), 3.76 (s, 6 H),
2.96−2.90 (m, 3 H), 2.81 (dd, J = 14.4, 4.8 Hz, 1 H), 1.87−1.85 (m, 2 H), 1.63−1.61
(m, 2 H), 1.59 (d, J = 7.2 Hz, 3 H); 13C NMR (150 MHz, CDCl3) δ 176.6, 176.3,
174.6, 168.1, 148.3, 147.5, 146.4 (2 C), 133.9, 132.3, 130.3, 128.8, 110.0, 109.0,
107.7 (2 C), 101.5, 80.2, 80.0, 68.5, 56.4 (2 C), 49.6 (2 C), 49.5, 49.4, 48.0, 43.5, 41.4,
14

ACCEPTED MANUSCRIPT
36.6, 30.9, 28.4, 28.1, 13.7; MS-ESI: 643.2007 ([M+Na]+, 100%); HRMS (ESI)
643.1887 for [M+Na]+ (calcd 643.1898 for C32H32N2O11Na).
4.1.2.3
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,
9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]
naphtho[2,3-d][1,3]dioxol-5-yl)-3-methylbutanamide (7c)
23
D
Yield: 57%; mp: 163−165 ; [α] –78° (c 0.5, CHCl3); IR (KBr cm–1) 3368,
℃
1
2966, 1775, 1705, 1613, 1505, 1484, 1386, 1228, 1114, 1037, 999, 930; H NMR
(600 MHz, CDCl3) δ 6.68 (s, 1 H), 6.53 (d, J = 7.2 Hz, 1 H), 6.50 (s, 1 H), 6.28 (s, 2
H), 5.98 (d, J = 3.6 Hz, 2 H), 5.30 (s, 1 H), 5.18−5.15 (m, 1 H), 4.86 (d, J = 4.2 Hz, 1
H), 4.78 (d, J = 3.6 Hz, 1 H), 4.56 (d, J = 4.8 Hz, 1 H), 4.36 (t, J = 8.4 Hz, 1 H), 4.25
(d, J = 10.2 Hz, 1 H), 3.85 (t, J = 9.9 Hz, 1 H), 3.76 (s, 6 H), 2.96−2.90 (m, 3 H), 2.84
(dd, J = 14.4, 4.8 Hz, 1 H), 2.77–2.74 (m, 1 H), 1.89−1.86 (m, 2 H), 1.62-1.60 (m, 2
13
H), 1.11 (m, 3 H), 0.79−0.77 (m, 3 H); C NMR (150 MHz, CDCl3) δ 177.4, 177.2,
174.5, 168.0, 148.3, 147.4, 146.4 (2 C), 134.0, 132.5, 130.3, 128.7, 110.1, 108.9,
107.7 (2 C), 101.5, 79.6 (2 C), 68.7, 62.0, 56.4 (2 C), 49.6, 49.3, 48.2, 43.5, 41.6, 37.0,
28.5, 28.4, 26.3, 20.6, 19.0; MS-ESI: 671.2226 ([M+Na]+, 100%); HRMS (ESI)
649.2382 for [M+H]+ (calcd 649.2392 for C34H37N2O11).
4.1.2.4 (2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,9R)-
9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naph
tho[2,3-d][1,3]dioxol-5-yl)-4-methylpentanamide (7d)
23
D
Yield: 52%; mp: 171−173 ; [α] –96° (c 0.5, CHCl3); IR (KBr cm–1) 3378,
℃
1
2959, 1775, 1700, 1614, 1506, 1484, 1388, 1228, 1114, 1037, 999, 933; H NMR
15

ACCEPTED MANUSCRIPT
(600 MHz, CDCl3) δ 6.71 (s, 1 H), 6.48 (s, 1 H), 6.27 (s, 2 H), 6.04 (d, J = 8.4 Hz, 1
H), 5.98 (s, 2 H), 5.43 (s, 1 H), 5.30−5.25 (m, 1 H), 4.76−4.71 (m, 3 H), 4.53 (d, J =
4.8 Hz , 1 H), 4.34 (t, J = 4.4 Hz, 1 H), 3.89 (t, J = 9.9 Hz, 1 H), 3.76 (s, 6 H),
2.96−2.88 (m, 3 H), 2.82 (dd, J = 14.4, 5.4 Hz, 1 H), 1.98−1.93 (m, 1 H), 1.88−1.83
(m, 2 H), 1.64−1.61 (m, 3 H), 1.37−1.36 (m, 1 H), 0.93−0.89 (m, 6 H); 13C NMR (150
MHz, CDCl3) δ 176.8, 176.7, 174.6, 168.2, 148.3, 147.5, 146.3 (2 C), 133.9, 132.3,
130.3, 128.8, 110.0, 109.0, 107.6 (2 C), 101.5, 80.2, 80.0, 68.5, 56.3 (2 C), 52.8, 49.5
(2 C), 48.0, 43.5, 41.5, 36.7, 35.6, 28.5, 28.1, 24.8, 23.1, 21.1; MS-ESI: 685.1986
([M+Na]+, 100%); HRMS (ESI) 685.2354 for [M+Na]+ (calcd 685.2368 for
C35H38N2O11 Na).
4.1.2.5
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,
9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]
naphtho[2,3-d][1,3]dioxol-5-yl)-3-methylpentanamide (7e)
23
D
Yield: 53%; mp: 177−179 ; [α] –74° (c 0.5, CHCl3); IR (KBr cm–1) 3463,
℃
1
2966, 1775, 1704, 1615, 1506, 1484, 1385, 1227, 1114, 1037, 999, 931; H NMR
(600 MHz, CDCl3) δ 6.68 (s, 1 H), 6.60 (d, J = 7.2 Hz, 1 H), 6.51 (s, 1 H), 6.28 (s, 2
H), 5.98 (d, J = 5.4 Hz, 2 H), 5.43 (s, 1 H), 5.16−5.13 (m, 1 H), 4.87 (d, J = 4.8 Hz, 1
H), 4.79 (d, J = 4.8 Hz, 1 H), 4.58−4.56 (m, 1 H), 4.38 (t, J = 4.5 Hz, 1 H), 4.33 (d, J
= 11.4 Hz, 1 H), 3.83 (t, J = 9.9 Hz, 1 H), 3.77 (s, 6 H), 2.95−2.88 (m, 3 H), 2.84 (dd,
J = 14.4, 4.8 Hz, 1 H), 2.61−2.59 (m, 1 H), 1.90−1.87 (m, 2 H), 1.63−1.59 (m, 3 H),
1.28−1.24 (m, 1 H), 1.06 (d, J = 6.0 Hz, 3 H), 0.83 (t, J = 7.2 Hz, 3 H); 13C NMR (150
MHz, CDCl3) δ 177.7, 177.2, 174.6, 168.3, 148.3, 147.4, 146.4 (2 C), 133.9, 132.5,
16

ACCEPTED MANUSCRIPT
130.3, 128.6, 110.1, 108.9, 107.7 (2 C), 101.5, 79.6 (2 C), 68.8, 61.2, 56.4 (2 C), 49.6,
49.3, 48.3, 43.5, 41.6, 37.1, 31.6, 28.5, 28.4, 24.9, 16.5, 10.1; MS-ESI: 685.1986
([M+Na]+, 100%); HRMS (ESI) 685.2362 for [M+Na]+ (calcd 685.2368 for
C35H38N2O11 Na).
4.1.2.6
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,
9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]
naphtho[2,3-d][1,3]dioxol-5-yl)-4-(methylthio)butanamide (7f)
23
D
–1
℃
Yield: 67%; mp: 158−160 ; [α] –88° (c 0.5, CHCl3); IR (KBr cm ) 3371,
1
2917, 1775, 1705, 1613, 1505, 1484, 1386, 1227, 1113, 1037, 999, 931; H NMR
(600 MHz, CDCl3) δ 6.71 (s, 1 H), 6.48 (s, 1 H), 6.27 (s, 2 H), 5.98 (d, J = 3.6 Hz, 2
H), 5.95 (d, J = 8.4 Hz, 1 H), 5.28−5.26 (m, 1 H), 4.87−4.84 (m, 1 H), 4.76 (t, J = 5.4
Hz, 1 H), 4.54 (d, J = 5.4 Hz, 1 H), 4.34 (t, J = 8.4 Hz, 1 H), 3.91 (t, J = 10.2 Hz, 1 H),
3.75 (s, 6 H), 2.98−2.87 (m, 3 H), 2.81 (dd, J = 14.4, 5.4 Hz, 1 H), 2.59−2.33 (m, 4 H),
2.08 (s, 3 H), 1.88−1.82 (m, 2 H), 1.65−1.59 (m, 3 H); 13C NMR (150 MHz, CDCl3) δ
176.8, 176.7, 174.6, 167.4, 148.3, 147.5, 146.4 (2 C), 133.9, 132.3, 130.3, 128.7,
110.0, 109.0, 107.7 (2 C), 101.6, 80.2, 80.1, 68.4, 56.4 (2 C), 53.1, 49.6, 49.5, 48.0,
43.5, 41.4, 36.6, 28.4, 28.1, 25.9 (2 C), 15.4; MS-ESI: 703.1952 ([M+Na]+, 100%);
HRMS (ESI) 703.1923 for [M+ Na]+ (calcd 703.1932 for C34H36N2O11 SNa).
4.1.2.7
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,
9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]
naphtho[2,3-d][1,3]dioxol-5-yl)-2-phenylacetamide (7g)
23
D
Yield: 63%; mp: 184−185 ; [α] –28° (c 0.5, CHCl3); IR (KBr cm–1) 3416,
℃
17

ACCEPTED MANUSCRIPT
1
2912, 1775, 1707, 1613, 1504, 1484, 1387, 1228, 1113, 1037, 1000, 941; H NMR
(600 MHz, CDCl3) δ 7.46−7.40 (m, 2 H), 7.38−7.34 (m, 3 H), 6.72 (s, 1 H), 6.45 (s, 1
H), 6.26 (s, 2 H), 5.97 (d, J = 6.0 Hz, 2 H), 5.76 (s, 1 H), 5.24−5.22 (m, 1 H), 4.89 (d,
J = 5.4 Hz, 1 H), 4.67 (d, J = 4.8 Hz, 1 H), 4.49 (d, J = 4.2 Hz, 1 H), 4.38 (t, J = 8.7
Hz, 1 H), 4.10 (t, J = 10.2 Hz, 1 H), 3.76 (s, 6 H), 2.98−2.92 (m, 3 H), 2.65 (dd, J =
13
13.8, 4.8 Hz, 1 H), 1.86−1.80 (m, 2 H), 1.64−1.60 (m, 2 H); C NMR (150 MHz,
CDCl3) δ 176.5, 176.2, 174.4, 166.7, 148.3, 147.5, 146.4 (2 C), 134.0, 133.1, 132.6,
130.2, 129.3, 129.2 (2 C), 129.1 (2 C), 128.2, 110.1, 108.9, 107.8, 107.7, 101.6, 79.6,
79.4, 68.9, 58.3, 56.4 (2 C), 49.9 (2 C), 48.9, 43.5, 41.8, 37.2, 28.5, 28.4; MS-ESI:
705.1467 ([M+Na]+, 100%); HRMS (ESI) 705.2040 for [M+ Na]+ (calcd 705.2055 for
C37H34N2O11Na).
4.1.2.8
(2S)-2-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,
9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]
naphtho[2,3-d][1,3]dioxol-5-yl)-3-phenylpropanamide (7h)
23
D
Yield: 56%; mp: 175−177 ; [α] –124° (c 0.5, CHCl3); IR (KBr cm–1) 3387,
℃
1
2955, 1775, 1704, 1613, 1505, 1484, 1387, 1228, 1113, 1037, 999, 931; H NMR
(600 MHz, CDCl3) δ 7.24 (d, J = 11.4 Hz, 2 H), 7.20 (d, J = 10.2 Hz, 1 H), 7.12 (d, J
= 10.2 Hz, 2 H), 6.71 (s, 1 H), 6.48 (s, 1 H), 6.27 (s, 2 H), 5.98 (d, J = 4.2 Hz, 2 H),
5.95 (d, J = 8.4 Hz, 1 H), 5.43 (s, 1 H), 5.32−5.29 (m, 1 H), 4.96−4.93 (m, 1 H), 4.71
(d, J = 4.8 Hz, 1 H), 4.68 (d, J = 4.8 Hz, 1 H), 4.54 (d, J = 4.8 Hz, 1 H), 4.35 (t, J =
8.4 Hz, 1 H), 3.91 (t, J = 9.9 Hz, 1 H), 3.77 (s, 6 H), 3.58 (dd, J = 14.4, 5.4 Hz, 1 H),
3.43−3.38 (m, 1 H), 2.92−2.90 (m, 1 H), 2.80−2.67 (m, 3 H), 1.85−1.78 (m, 2 H),
18

ACCEPTED MANUSCRIPT
1.56−1.25 (m, 2 H); 13C NMR (150 MHz, CDCl3) δ 176.9, 176.1, 174.6, 167.2, 148.3,
147.5, 146.4 (2 C), 136.4, 134.0, 132.2, 130.3, 128.8, 128.7 (2 C), 128.6 (2 C), 127.0,
110.0, 109.0, 107.7 (2 C), 101.5, 80.1, 79.9, 68.4, 56.4 (2 C), 55.1, 49.3 (2 C), 47.9,
43.5, 41.4, 36.6, 33.0, 28.3, 28.1; MS-ESI: 719.2307 ([M+Na]+, 100%); HRMS (ESI)
697.2389 for [M+ H]+ (calcd 697.2392 for C38H37N2O11).
4.1.2.9
(2S)-3-(1H-benzo[d]imidazol-2-yl)-2-(1,3-dioxooctahydro-2H-4,7-
epoxyisoindol-2-yl)-N-((5S,5aS,8aR,9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo- 5,
5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)propanamide (7i)
23
D
Yield: 58%; mp: 195−196 ; [α] –106° (c 0.5, CHCl3); IR (KBr cm–1) 3384,
℃
1
2910, 1773, 1701, 1616, 1506, 1483, 1389, 1227, 1112, 1036, 998, 932; H NMR
(600 MHz, CDCl3) δ 8.14 (s, 1 H), 7.50 (d, J = 7.8 Hz, 1 H), 7.34 (d, J = 7.8 Hz, 1 H),
7.9 (t, J = 7.2 Hz, 1 H), 7.08 (t, J = 7.2 Hz, 1 H), 7.04 (d, J = 1.8 Hz, 1 H), 6.72 (s, 1
H), 6.46 (s, 1 H), 6.26 (s, 2 H), 6.00 (d, J = 7.8 Hz, 1 H), 5.99 (s, 2 H), 5.43 (s, 1 H),
5.30−5.26 (m, 1 H), 5.04−5.01 (m, 1 H), 4.74 (d, J = 4.8 Hz, 1 H), 4.69 (d, J = 4.8 Hz,
1 H), 4.46 (d, J = 5.4 Hz, 1 H), 4.30 (t, J = 8.4 Hz, 1 H),, 3.81−3.77 (m, 1 H), 3.76 (s,
6 H), 3.70 (dd, J = 15.0, 6.0 Hz, 1 H), 3.57−3.52 (m, 1 H), 2.88−2.84 (m, 1 H), 2.76
(d, J = 6.6 Hz, 1 H), 2.62 (d, J = 6.6 Hz, 1 H), 2.50 (dd, J = 14.4, 4.8 Hz, 1 H),
2.18−1.77 (m, 2 H), 1.63 (s, 1 H), 1.57−1.48 (m, 2 H); 13C NMR (150 MHz, CDCl3) δ
177.0, 176.4, 174.5, 167.4, 148.3, 147.5, 146.4 (2 C), 136.0, 133.9, 132.2, 130.3,
128.7, 127.0, 122.6, 122.4, 119.8, 118.2, 111.4, 110.7, 110.0, 109.0, 107.6 (2 C),
101.5, 79.9, 79.8, 68.4, 56.4 (2 C), 54.9, 49.5, 49.4, 47.8, 43.5, 41.3, 36.6, 28.4, 28.1,
23.1; MS-ESI: 758.2207 ([M+Na]+, 100%); HRMS (ESI) 758.2310 for [M+ Na]+
19

ACCEPTED MANUSCRIPT
(calcd 758.2320 for C40H37N3O11Na).
4.1.2.10 4-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,9R)-9-(4-
hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho
[2,3-d][1,3]dioxol-5-yl)benzamide (7j)
23
D
Yield: 72%; mp: 220−221 ; [α] –64° (c 0.5, CHCl3); IR (KBr cm–1) 3469,
℃
1
2961, 1774, 1712, 1609, 1503, 1483, 1383, 1228, 1113, 1036, 999, 929; H NMR
(600 MHz, CDCl3) δ 7.82 (d, J = 9.0 Hz, 2 H), 7.32 (d, J = 8.4 Hz, 2 H), 6.79 (s, 1 H),
6.64 (d, J = 7.8 Hz, 1 H), 6.53 (s, 2 H), 6.30 (s, 2 H), 5.98 (d, J = 7.8 Hz, 1 H), 5.50 (s,
1 H), 5.43–5.40 (m, 1 H), 4.98 (s, 2 H), 4.58 (d, J = 4.8 Hz, 1 H), 4.45 (t, J = 8.1 Hz, 1
H), 3.83 (t, J = 9.6 Hz, 1 H), 3.77 (s, 6 H), 3.05−2.97 (m, 4 H), 1.93−1.91 (m, 2 H),
1.69−1.67 (m, 2 H); 13C NMR (150 MHz, CDCl3) δ 176.0, 175.9, 174.4, 166.4, 148.4,
147.6, 146.4 (2 C), 134.9, 134.0, 133.1, 132.7, 130.1, 128.6, 127.9 (2 C), 126.5 (2 C),
110.0, 109.1, 107.7 (2 C), 101.7, 79.5 (2 C), 69.1, 56.4 (2 C), 50.0 (2 C), 48.6, 43.6,
41.8, 37.3, 28.6, 28.5; MS-ESI: 691.1542 ([M+Na]+, 100%); HRMS (ESI) 691.1887
for [M+Na]+ (calcd 691.1898 for C36H32N2O11 Na).
4.1.2.11
4-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,9R)-9-
(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphth
o[2,3-d][1,3]dioxol-5-yl)butanamide (7k)
23
D
Yield: 65%; mp: 238−240 ; [α] –85° (c 0.5, CHCl3); IR (KBr cm–1) 3344,
℃
1
2955, 1770, 1691, 1612, 1524, 1480, 1396, 1224, 1116, 1032, 996, 920; H NMR
(600 MHz, CDCl3) δ 6.81 (s, 1 H), 6.51 (s, 1 H), 6.27 (s, 2 H), 6.20 (d, J = 7.2 Hz, 1
H), 5.98 (d, J = 7.2 Hz, 2 H), 5.21−5.18 (m, 1 H), 4.82 (d, J = 4.2 Hz, 1 H), 4.72 (d, J
20

ACCEPTED MANUSCRIPT
= 4.2 Hz, 1 H), 4.56 (d, J = 4.2 Hz, 1 H), 4.38−4.35 (m, 1 H), 3.85−3.82 (m, 1 H),
3.81−3.73 (m, 8 H), 2.91−2.85 (m, 4 H), 2.58−2.55 (m, 2 H), 1.86−1.82 (m, 2 H),
1.60−1.58 (m, 2 H); 13C NMR (150 MHz, CDCl3) δ 177.1, 177.0, 174.6, 169.8, 148.3,
147.5, 146.4 (2 C), 133.9, 132.5, 130.3, 128.7, 110.0, 109.2, 107.6 (2 C), 101.6, 79.1,
79.0, 68.9, 56.3 (2 C), 49.8 (2 C), 48.2, 43.5, 41.6, 37.0, 35.2, 34.1, 28.5 (2 C);
MS-ESI: 643.1505 ([M+Na]+, 100%); HRMS (ESI) 643.1895 for [M+ Na]+ (calcd
643.1898 for C32H32N2O11 Na).
4.1.2.12 5-(1,3-dioxooctahydro-2H-4,7-epoxyisoindol-2-yl)-N-((5S,5aS,8aR,9R)-9-(4-
hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho
[2,3-d][1,3]dioxol-5-yl)pentanamide (7l)
23
D
Yield: 68%; mp: 178−179 ; [α] –82° (c 0.5, CHCl3); IR (KBr cm–1) 3350,
℃
1
2952, 1773, 1698, 1613, 1516, 1484, 1404, 1228, 1113, 1036, 999, 940; H NMR
(600 MHz, CDCl3) δ 6.81 (s, 1 H), 6.52 (s, 1 H), 6.31−6.29 (m, 3 H), 5.99−5.97 (m, 2
H), 5.26−5.23 (m, 1 H), 4.83 (d, J = 4.8 Hz, 1 H), 4.78 (d, J = 4.2 Hz, 1 H), 4.59 (d, J
= 4.2 Hz, 1 H), 4.42−4.38 (m, 1 H), 3.93−3.89 (m, 1 H), 3.78 (s, 6 H), 3.53−3.50 (m,
2 H), 2.98−2.95 (m, 2 H), 2.89−2.87 (m, 2 H), 2.17−2.12 (m, 2 H), 1.98−1.92 (m, 2
13
H), 1.85−1.82 (m, 2 H), 1.62−1.60 (m, 2 H); C NMR (150 MHz, CDCl3) δ 177.7,
177.5, 174.6, 171.9, 148.2, 147.5, 146.4 (2 C), 133.9, 132.4, 130.3, 129.1, 110.0,
109.0, 107.7 (2 C), 101.5, 79.2 (2 C), 69.0, 56.4 (2 C), 49.8 (2 C), 48.0, 43.6, 41.7,
38.0, 37.1, 32.8, 28.5 (2 C), 23.7; MS-ESI: 657.1684 ([M+Na]+, 100%); HRMS (ESI)
657.2043 for [M+ Na]+ (calcd 657.2055 for C33H34N2O11Na).
4.1.3. logP of 7a-l determination
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The partition coefficient P was determined according to the standard method [36]
and calculated with the following equation: P = (compound in 1-octanol)/(compound
in water).
4.2 Biological evaluation
4.2.1 Cytotoxicity assays
Cells were incubated at 37 in a 5% CO2 atmosphere. The MTT assay was used
to determined growth inhibition. The synthetic compounds 7a-l and reference
compounds VP16 and norcantharidin were dissolved in saline for five concentrations
(0.01–100 ꢀM). The A-549, HepG2, HeLa, HCT-8 and WI-38 cells were seeded in
96-well plates at a density of 5×103 cells/well in 150 ꢀL of the RPMI1640 medium
with 10% heat-inactivated FBS and allowed to attach for 4–6 h, and then incubated
with the indicated concentrations of compound for 48 h. The media was aspirated, and
10 ꢀL of 5 mg/mL MTT solution (dilute in sterile PBS) diluted in 100 ꢀL serum-free
media was added to each well. After 4 h of incubation, the solution was discarded and
then added 100 ꢀL DMSO, 96-well plates were shaken on an oscillator for 10 min.
The absorbance at 490 nm was determined on a plate reader. IC50 values were
determined from a log plot of percent of control versus concentration.
4.2.2 Protein phosphatase inhibition
Inhibition of protein phosphatase 1 and 2A activity was performed according to
previously reported assay [12,27]. Protein phosphatase PP1 was purchased from New
England Biolabs (Beijing) LTD., and PP2A was purchased from Merck Millipore
Corporation (USA). A serine/threonine PP assay was used in which free phosphate ion
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released from a substrate phosphopeptide (Lys-Arg-pThr-Ile-Arg) was quantified by
colorimetric analysis using the Malachite Green method. PP1, PP2 and substrate used
in the assay were 30 mU/well, 0.6 mU/well and 200 ꢀM, respectively. The reaction
was initiated by addition of substrate (5 ꢀL) to a mixture containing enzyme (5 ꢀL),
reaction buffer (10 ꢀL; 50 mM Tris-HCl, pH 7.0, 100 ꢀM CaCl2) and test compound
(10 ꢀL, 100 ꢀM), producing a total reaction volume of 30 ꢀL/well and incubated at
room temperature for 60 min. Reactions were halted via addition of a malachite green
solution (50 ꢀL), and the absorbance readings were taken at 650 nm after 10 min
development time. Enzyme dilutions were made with buffer containing 20 mM
MOPS, pH 7.5, 0.15 M NaCl, 60 mM 2-mercaptoethanol, 1 mM MgCl2, 2 mM EGTA,
0.1 mM MnCl2, 1mM DTT, 10% glycerol and 0.1 mg/mL serum albumin. For each of
the enzymatic assays, the enzyme and the inhibitor were incubated at 37 ˚C for 10 min
prior to the addition of the substrate. Samples were blanked against wells containing
enzyme and buffer only. Initial inhibitor dilutions were made in DMSO and
subsequent dilutions were made in distilled deionised H2O. A dose-response curve of
percentage enzyme activity versus drug concentration was produced from which an
IC50 value was calculated indicating the concentration of drug required to inhibit
enzyme activity by 50%. Data represents the mean IC50 of three independent
experiments performed in duplicate.
4.2.3 PP2A inhibition of 7l in HepG2 cells
PP2A activity was assayed according to the PP2A phosphatase-activity assay kit
obtained from Promega Corporation (V2460, Madison, WI) protocol [33]. The PP2A
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phosphatase-activity assay kit contains the chemically synthesized phosphopeptide,
RRAT (pT) VA, a peptide substrate that is compatible with PP2A. PP2A
dephosphorylates the phosphopeptide substrate, allowing the free phosphate to react
with the molybdate dye mixture and show the green color.
The HepG2 cells were incubated in six-well plates. After treatment with
compound 7l for 24 h, the cells were collected and lysed, and endogenous phosphates
were removed. The prepared enzyme samples were added to premixes containing 5
ꢀL of 1 mM phosphopeptide substrate, after 30 min of incubation at 37°C, 50 ꢀL of
molybdate dye-additive mixture was added to stop the reaction. The absorbance was
measured at 600 nm after 15 min, and PP2A activity was obtained using the
phosphate standard curve.
4.2.4 DNA topoisomerase-ꢀ inhibition assay
Topoisomerase-ꢀ activity was determined using a kit (Topogen Inc., USA, Cat
No. 2000H). The reaction was run in microcentrifuge tube that contained supercoiled
pBR322 plasmid DNA 250 ng/ꢀL and topoisomerase-ꢀ (4 units) in assay buffer (A
0.1 volume and B 1volume). In each reaction 2 ꢀL sample was added and the volume
was made up to 20 ꢀL with water and then incubated for 60 mins at 37ꢁ. The
reaction was terminated by the addition of 2 ꢀL of 10% SDS. Each sample tube was
treated with proteinase K and extracted once with chloroform/isoamyl alcohol (24:1).
Products were resolved by 1% agarose gel electrophoresis in TAE buffer (40 mM
tris-acetate, pH8.0, and 1 mM EDTA) and stained with 0.5 mg/mL ethidium bromide
[35].
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4.2.5 Analysis of cell cycle by flow cytometry
The cell cycle was analyzed by flow cytometry. Firstly, HepG2 cells were treated
with different concentrations of compound 7l (0, 0.5, and 1 ꢀM) for 24 h. After
incubation, a total of (1–5)×105 cells were harvested from the treated and untreated
samples. The cells were washed twice with PBS and fixed in 70% ice-cold ethanol for
at least overnight. The sample was concentrated by removing the ethanol and then
cells were then washed three times with PBS, staining the cellular DNA with
fluorescent solution (1% (v/v) Triton X-100, 0.01% RNase, 0.05% PI) for 15 min in
darkness. The cell cycle distribution was then detected by flow cytometry (COULTER
EPICS XL, USA).
4.2.6 Western blotting anaylsis
HepG2 cells were seeded into culture flasks. After 24 h incubation, cells were
treated with 7l (0, 0.5, and 1 ꢀM), respectively. For total cell protein extracts, cells
were washed and lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 1% NP-40, 2 mM
EDTA, 10 mM NaCl, 10 ꢀg/ml aprotinin, 10 ꢀg/ml leupeptin, 1 mM DTT, 0.1% SDS
and 1 mM phenyl methyl sulfonyl fluoride). Total proteins were obtained by
centrifuging (12,000 g for 20 min at 4 ). The protein concentrations were
determined by using Bradford method. The Western blotting assay was performed as
described previously [29]. For Western blot analysis, equal amounts of proteins (30 ꢀg)
were separated on 10% or 12% SDS-PADE gels and transferred to polyvinylidine
difluoride (PVDF) membranes (Millipore Corporation, USA). The blot was blocked
in blocking buffer (5% non-fat dry milk in TBST) for 2 h at room temperature, and
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then incubated with dilute solution (1:500~1:1000) of cdc2 or CyclinB1 antibodies
(BioLegend, US) in blocking buffer overnight at 4 . The blot was then incubated
with appropriate secondary antibody (ZSGB-BIO, Beijing, China) (1:5000~1:10000
dilution), β-Actin (ZSGB-BIO, Beijing, China) was used as a loading control. The
protein bands were visualized using the Gel Imaging System (ChemDoc-It610, UVP,
USA).
4.2.7 Fluorescence titrations
The binding of the compound 7l to CT DNA was performed by monitoring the
changes in the emission spectral pattern of compound 7l in pH 7.2 PBS by increasing
the concentrations of CT DNA. After addition of indicated amount of DNA (0–5 ꢀM)
to the compound 7l (1 ꢀM), the resulting solution was allowed to equilibrate for 5 min
at 25 , excited at 318 nm followed by recording the emission spectral changes in the
range of 280–400 nm. The appropriate concentration samples of CT DNA in PBS
were also determined as blank.
Acknowledgments
We gratefully acknowledge the financial support from the National Natural
Science Foundation of China (Grant No. 21372110, 81372177 and 21302079).
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