Design, synthesis and anticancer activities of novel otobain derivatives

Article abstract of DOI:10.1039/c5ob02176f

A series of novel racemic otobain derivatives was designed and synthesised using 2-piperonyl-1,3-dithianes in the conjugate addition-alkylation to 5H-furan-2-one, followed by cationic cyclisation. All the synthesised compounds were consequently evaluated for their anticancer activity against several human cancers in vitro. The efficacy of the most active compound 27g was comparable with etoposide, with IC₅₀ values ranging from 1.06 μM to 4.16 μM in different cancer cell lines. Notably, compound 27g strongly induced cell cycle arrest and increased the expression of mitosis-specific markers MPM-2 and phosphorylated histone H3, but it did not trigger cell apoptosis. Further a colony formation assay showed that compound 27g effectively inhibited the anchor growth of lung cancer cells in a dose-dependent manner. More importantly, compound 27g at 40 mg kg^-1 significantly suppressed tumour volume (P < 0.01) and tumour weight (P < 0.05) in a human lung cancer cell xenograft mouse model without causing systematic toxicity in mice. Our findings indicated that compound 27g has significant potential for further drug development.

Full text of DOI:10.1039/c5ob02176f

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Design, synthesis and anticancer activities of novel
otobain derivatives†
Cite this: DOI: 10.1039/c5ob02176f
Zhongzhou Li,‡a Hui Su,‡b Weiwei Yu,b Xinjun Li,a Hao Cheng,a Mingyao Liu,b
Xiufeng Pang*b and Xinzhuo Zou*a
A series of novel racemic otobain derivatives was designed and synthesised using 2-piperonyl-1,3-
dithianes in the conjugate addition–alkylation to 5H-furan-2-one, followed by cationic cyclisation. All the
synthesised compounds were consequently evaluated for their anticancer activity against several human
cancers in vitro. The efficacy of the most active compound 27g was comparable with etoposide, with IC50
values ranging from 1.06 µM to 4.16 μM in different cancer cell lines. Notably, compound 27g strongly
induced cell cycle arrest and increased the expression of mitosis-specific markers MPM-2 and phos-
phorylated histone H3, but it did not trigger cell apoptosis. Further a colony formation assay showed that
compound 27g effectively inhibited the anchor growth of lung cancer cells in a dose-dependent manner.
More importantly, compound 27g at 40 mg kg−1 significantly suppressed tumour volume (P < 0.01) and
tumour weight (P < 0.05) in a human lung cancer cell xenograft mouse model without causing systematic
toxicity in mice. Our findings indicated that compound 27g has significant potential for further drug
development.
Received 22nd October 2015,
Accepted 17th November 2015
DOI: 10.1039/c5ob02176f
www.rsc.org/obc
Podophyllum peltatum and Podophyllum hexandrum.16 Its semi-
synthetic derivatives, including etoposide (VP-16, ETO, 2), teni-
Introduction
Cancer is a serious threat to human health. Approximately poside (3) and etopophos (4) (Fig. 1), have been developed as
14.1 million new cases of cancer were diagnosed worldwide in clinically useful medicines against various cancers.17–19
2012. The number of new cases could further rise to However, their high toxicity, low water solubility and drug
19.3 million by 2025.1 Although current therapies are effective resistance have limited their application in cancer chemo-
in treating early stages of cancer, the survival rates of cancer therapy.20 To obtain better therapeutic agents, a great number
remain limited.2 Therefore, the search for promising new of podophyllotoxin analogues have been synthesized. Among
approaches to treat this deadly disease is an urgent need. them, NK611 (5), NPF (6), GL-311 (7) and TOP53 (8) (Fig. 1) are
Among these approaches, natural compounds have been con- presently under clinical trials. The clinical success of podo-
sidered as a good source of novel anticancer drugs.
Lignans, a class of phenylpropanoid compounds widely dis- exploration of new lignans with improved antitumor activity.
tributed in the plant kingdom, have generated particular inter- In our investigation on the bioactive components from the
phyllotoxin derivatives has stimulated great interest in the
est because of their broad range of biological activities, stem barks of Myristica fragrans Houtt, we isolated one lignan,
including antitumor,3–6 anti-inflammatory,6,7 cardiovascular,8 hydroxyotobain (9) (Fig. 2) in 0.50% yield from ethanolic
antioxidant,9–11 neuroprotective effects,11 antiviral actions12–14 extracts.21 Preliminary structural modification of hydroxy-
and anti-liver fibrotic activities.15 The best known antitumor otobain obtained methoxyotobain (10), benzyloxyotobain (11),
lignan is podophyllotoxin (PPT, 1), a bioactive component of allyloxyotobain (12) and otobaene (13) (Fig. 2).22 These com-
pounds showed some anticancer activity, but their levels were
still not ideal. On the one hand, considering that the hydroxy
of hydroxyotobain was prone to an elimination reaction,
otobain (14) was then designed. In addition, studies have
shown that trans-fused γ-lactone is essential for the anticancer
activity of podophyllotoxin derivatives.23,24 Therefore, the
trans-fused γ-lactone was introduced to otobain. On the other
hand, a large number of previous studies on podophyllotoxin
derivatives focused on C-4 position modification,25 but very
little work has been directed to the biological activity of
aDepartment of Chemistry, School of Chemistry and Molecular Engineering,
East China Normal University, 500 Dongchuan Road, Shanghai 200241, China.
E-mail: xzzou@chem.ecnu.edu.cn
bShanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and
School of Life Sciences, East China Normal University, Shanghai 200241, China.
E-mail: xfpang@bio.ecnu.edu.cn
†Electronic supplementary information (ESI) available: NMR spectra of all new
compounds. CCDC 1430247 and 1430248. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c5ob02176f
‡These authors contributed equally to this work.
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Fig. 3 Structures of synthesized otobain derivatives.
Results and discussion
Chemistry
The total synthesis of otobain 22 was carried out by the syn-
thetic sequence illustrated in Scheme 1. Compound 16 was
prepared from piperonal by employing p-TsOH and 1,3-
propanedithiol in excellent yield. Treatment of compound 16
with n-butyllithium by the addition of 2(5H)-furanone gave rise
to compound 17 with good yield. The enolate derived from
compound 17 by treating 17 with lithium diisopropylamide
was captured by piperonal, which led to a mixture of diastereo-
mers of compound 18 in a 3 : 2 ratio. Erythro 18a and threo 18b
Fig. 1 Structures of PPT and related compounds.
1
are distinguished based on the H NMR chemical shift of H-1
and JH1–H2 coupling constants (erythro: δH1 > 5.12 ppm, JH1–H2
(2–4 Hz); threo: δH1 ≤ 4.95 ppm, JH1–H2 (6–9 Hz)).31–33 Cyclisa-
tion of compound 18 under TFA in DCM yielded a mixture of
compound 19 (cis–trans, podophyllotoxin-type; minor) and
compound 20 (trans–trans, isopodophyllotoxin-type; major;
Fig. 2 Structures of hydroxyotobain and related compounds.
isopodophyllotoxin (15) with the relative stereochemistry at JH1–H2 and JH2–H3 >10 Hz) in a 1 : 15 ratio. These results were
positions 1, 2 and 3 in (1,2–2,3)-trans–trans. Recently, Thanh consistent with those usually described for such a cyclisation
et al. reported that a novel aryl-tetralin lignan, cleistantoxin, reaction.34 The stereochemically preferential compound 20
which was isolated from the fruits of Cleistanthus indochinen- was selected for use in subsequent syntheses. Deprotection of
sis, shows potent activities against several human cancers compound 20 with HgO and BF3·Et2O gave the ketoderivative
in vitro.3,26 The structure of cleistantoxin is interesting as the 21 with no change in the relative stereochemistry at positions
relative stereochemistry of cleistantoxin at positions 1, 2 and 3 1, 2 and 3 (JH1–H2 and JH2–H3 >10 Hz).34 Reduction of ketone 21
is similar to that of podophyllotoxin, whereas the benzodioxole with NaBH4 afforded alcohol 22 in 70% yield. The assignment
structure of cleistantoxin at position 1 is similar to that of of relative stereochemistry for C-4 on compound 22 was based
otobain. According to some literature reports on podophyllo- on JH3–H4 coupling constants: JH3–H4 ≥10 Hz indicates that H-3
toxin derivatives, we find that some podophyllotoxin and H-4 are in a trans relationship,35 which was further con-
ester, amide and amine derivatives show good anticancer firmed by the X-ray crystal of ester 23a (Fig. 4).36 The synthesis
activity.27–30 From these studies, we recently synthesised 20 of otobain analogues 23a–c, g–h, 26b–h and 27g–m is
novel racemic otobain ester, amide and amine derivatives described in Scheme 2. Compounds 23a–c and g–h were
(Fig. 3), which were stereochemically preferential compounds obtained in good yields by the reaction of compound 22 with
with a known synthetic route (Scheme 1). These compounds the corresponding carboxylic acids in the presence of N,N′-
were evaluated for their cytotoxicities against a panel of dicyclohexylcarbodiimide and 4-(N,N-dimethylamino)pyridine.
human cancer cell lines and human immortal keratinocyte Intermediate 24 was prepared from compound 22 by employ-
cells.
ing the TFA/NaN3 reagent system. The assignment of relative
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Scheme 1 Synthesis of 22. Reagents and conditions: (i) SH (CH2)3SH, p-TsOH, DCM, rt; (ii) n-BuLi, furan-2(5H)-one, THF, −78 °C; (iii) LDA, piperonal,
THF, −78 °C; (iv) TFA, DCM, rt; (v) HgO, BF3·Et2O, THF/H2O (85/15), 0 °C–rt; (vi) NaBH4, THF, 0 °C.
Fig. 4 X-ray crystal structure of compound 23a.
Fig. 5 X-ray crystal structure of compound 24.
lyst to yield the key intermediate 25. Compound 25 was
coupled with various chlorides or anhydrides in dichloro-
methane to obtain compounds 26b–h in good yields. Com-
pounds 27g–m were obtained in 39%–48% yields by the
coupling compound 25 with the corresponding benzyl iodide.
The structures of the target compounds were characterised by
1H NMR and 13C NMR spectral analyses.
In vitro cytotoxicity assay
Target compounds 23a–c, g–h, 26b–h and 27g–m were evalu-
ated for in vitro cytotoxicity against five human tumour cell
lines and one human normal cell line using the SRB assay.
Etoposide was used as a reference. The results showed that
most of the compounds possessed considerable anticancer
activity (Table 1). Lead compound 22 and ester derivatives
Scheme 2 Synthesis of 23a–c, g–h, 26b–h and 27g–m. Reagents and
conditions: (i) DCC, DMAP, RCOOH, DCM, 28 °C; (ii) TFA, NaN3, CH3Cl,
rt; (iii) H2/Pd–C, ethyl acetate, rt; (iv) RCOCl or (RO)2CO, DMAP, DCM, rt;
activity than acetates (23a) and aromatic esters (23g and 23h),
(v) RCH2I (from RCH2Br + NaI), 1,2-dichloroethane, 75–80 °C.
(23a–c and g–h) showed poor activities. Aromatic heterocyclic
esters (23b and 23c) possessed more potent antiproliferative
which implied that aromatic heterocyclic esters were better
substituents in the C-4 position of otobain by the ester bond.
Except for compounds 26g and 26h, most of the amide deriva-
stereochemistry for C-4 on compound 24 was based on JH3–H4
coupling constants: JH3–H4 ≈ 4 Hz indicates that H-3 and H-4
are in a cis relationship.35 The precise 3D structural infor-
mation on compound 24 was determined by single-crystal
X-ray diffraction as illustrated in Fig. 5.36 Compound 24 was
then reduced under a hydrogen atmosphere with a Pd/C cata-
tives (26b–f) showed promising activity compared with ester
derivatives (23a–c and g–h) and lead compound 22. The amine
derivatives (27g–m) showed good activity against the HL-60
cancer cell line with IC50 values ranging from 1.06 µM to
15.47 μM, but only moderate activity against other cell lines.
Notably, 27g showed potent actions against HL-60 (IC50
=
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Table 1 Cytotoxicity of compounds 23a–c, g–h, 26b–h and 27g–m
IC50 (μmol L−1
)
Compounds
HL60a
SGC-7901b
MCF-7c
HCT116d
A549e
HaCatf
23a
23b
23c
23g
23h
26b
26c
26d
26e
26f
26g
26h
27g
27h
27i
>100
80.91 2.3
28.95 1.6
21.35 1.7
—
61.11 0.7
14.53 0.4
17.08 0.2
—
>100
>100
87.75 1.9
66.55 1.8
16.33 0.4
—
53.52 2.7
35.57 2.9
>100
41.22 1.5
29.41 0.5
—
71.00 1.3
24.50 4.3
>100
>100
—
—
—
>100
—
3.83 0.3
6.71 1.6
10.71 0.9
7.22 1.4
8.09 0.1
>100
15.22 1.4
12.72 0.3
36.13 0.3
>100
73.40 9.2
—
14.03 1.5
16.06 0.5
32.13 2.8
47.15 3.5
19.66 1.4
—
11.52 0.8
16.82 0.4
15.24 0.2
31.86 1.2
14.79 0.7
—
9.80 0.5
11.15 0.4
21.23 0.6
22.48 0.9
17.61 0.7
>100
6.81 0.1
7.72 0.2
12.39 0.3
7.84 0.2
11.49 0.5
—
>100
—
—
—
>100
—
1.06 0.1
8.08 0.2
4.65 0.1
4.51 0.4
4.90 0.01
15.47 0.6
7.16 0.8
>100
4.16 0.4
26.19 2.1
19.81 0.5
18.48 1.0
43.92 3.4
>100
2.25 0.2
27.89 2.0
7.91 0.3
5.67 0.1
61.44 1.1
>100
1.65 0.1
12.64 0.6
10.02 0.3
7.73 0.2
49.08 5.7
>100
2.32 0.2
17.94 2.0
10.21 0.7
7.53 0.5
20.52 4.3
98.60 11.0
67.99 2.6
>100
1.16 0.1
6.42 0.3
4.40 0.2
3.94 0.2
7.47 0.3
26.38 0.1
8.80 0.2
—
27j
27k
27l
27m
22
47.16 4.2
—
41.55 5.6
—
32.68 2.2
—
Etoposide
0.38 0.01
3.00 0.3
1.68 0.1
2.64 0.1
2.90 0.4
0.58 0.02
a Human promyelocytic leukemia cells. b Human gastric cancer cells. c Human breast cancer cells. d Human colon carcinoma cell. e Human non-
small cell lung cancer cells. f Human skin cell line/keratinocyte. IC50 is shown as mean standard deviation. The assays were performed in three
independent experiments in triplicate.
1.06 μM), SGC-901 (IC50 = 4.16 μM), MCF-7 (IC50 = 2.25 μM),
HCT-116 (IC50 = 1.65 μM) and A549 (IC50 = 2.32 μM) cancer cell
lines, whereas etoposide showed IC50 values of 0.38, 3.00, 1.68,
2.64 and 2.90 μM in the respective cell lines. Moreover, the
cytotoxicity of 27g against HaCat (i.e., IC50 = 1.16 μM) was two-
fold higher than that of etoposide (i.e., IC50 = 0.58 μM). These
results suggested that the target compounds showed promis-
ing anticancer effects and structure–activity relationships.
Compound 27g arrests cell cycle
We found that cells treated with 27g decreased in size and
became detached and round, a phenomenon that usually
occurred during mitosis (Fig. 6A). Cell cycle analysis was
further applied to examine how 27g inhibited cell growth. As
shown in Fig. 6B, compound 27g dose-dependently arrested
Fig. 6 27g dramatically blocked cell cycle, but had little effects on cell
the cell cycle at the G2/M phase with a concomitant decrease
apoptosis. (A) A549 cells were incubated with different dilutions of 27g
in the G1 phase as compared with the DMSO control. Treat-
ment of 27g with the indicated concentrations (1, 3 and
10 μM) for 12 h led to a marked increase in cell population in
the G2/M phase from 13.09% to 63.80%, and a further increase
to 90.45% after 24 h of treatment in A549 cancer cells.
for 24 h. Morphological observation was visualized under a microscope
at a magnification of 100. (B) Cells were incubated with the indicated
concentrations of 27g for 12 h and 24 h, followed by being stained with
propidium iodide (PI). The DNA contents were analyzed by using a flow
cytometer. (C) Annexin V-FITC apoptosis was examined by using a flow
cytometer in treated cells.
Apoptosis is a kind of programmed cell death, and an
annexin V-FITC/propidium iodide binding assay was further
carried out to test whether compound 27g induces cell apopto-
sis. Our results in Fig. 6C showed that compound 27g did not
apparently induce cell apoptosis at the drug concentrations of the results of apoptosis detection. Therefore, these results
1–10 μM. Furthermore, there was no population of sub-G1 indicated that 27g inhibited cell proliferation by arresting the
phase cells in the cell cycle assay, which was consistent with cell cycle in the G2/M phase without inducing apoptosis.
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Fig. 8 27g inhibited colony formation of lung cancer cells. (A) A-549
cells were incubated with the indicated concentrations of 27g for 10
days and stained with 0.2% crystal violet. The colonies of the local
region were visualized under a microscope (magnification; 40×). (B) The
Fig. 7 Compound 27g increased the expression of MPM-2 and phos-
relative colony number is shown as mean
SD. Statistical differences
phorylated histone H3.
are noted; ***P < 0.001.
Compound 27g increases the expression of mitotic markers
with the DMSO control group (P < 0.001). 27g at 1 μM appar-
ently suppressed both the number and size of colonies (Fig. 8A
and B) and almost blocked the anchor growth of lung cancer
cells at 3 μM.
To further validate the inhibitory function of 27g on cell
mitosis, we carried out a western blot assay to examine the
expression levels of MPM-2 and phosphorylated histone H3
(p-HH3), which are two mitosis-specific markers. MPM-2
reflected the phosphorylation level of mitosis-specific pro-
teins,37 and the expression of phosphorylation of histone H3
at Ser 10 peaked in the M phase.38 As shown in Fig. 7, MPM-2
expression was slightly increased when the cells were treated
with 3 μM 27g, but it significantly increased under 10 μM 27g.
Consistent with this result, p-HH3 remarkably increased in
27g-treated cells, which was consistent with the cell cycle
arrest in mitosis mediated by 27g (Fig. 6B).
In vivo antitumor efficacy of compound 27g
Based on the above in vitro cytotoxic activities, compound 27g
was then chosen to investigate its anticancer properties in vivo.
We established a xenograft mouse model of human lung
cancer using A549 lung cancer cells. Compound 27g (20 and
40 mg per kg body weight) and etoposide (20 mg kg−1) were
administered intraperitoneally for four weeks. The therapeutic
efficacy of 27g is shown in Fig. 9. Compound 27g at 20
mg kg−1 marginally delayed tumour growth, but 27g at
40 mg kg−1 significantly suppressed tumour volume (P < 0.01)
and tumour weight (P < 0.05). The inhibitory action of 27g was
Compound 27g inhibits colony formation of cancer cells
Colony formation assay is an in vitro cell survival assay based durable. Although etoposide blocked solid tumour growth
on the ability of a single cell to grow into a colony. This assay more potently as compared with 27g, it was much more toxic
was carried out to test the inhibitory effect of compound 27g than 27g because the body weights in the etoposide-treated
on the “unlimited” division of cancer cells. The results showed group were reduced by 16.75% at the end of the experiments
that compound 27g could block the colony formation of A-549 (P < 0.001). However, tumour-bearing mice were much more
cells in a dose-dependent manner (Fig. 8). The number of colo- tolerant to 27g as the body weight of mice was not obviously
nies was significantly reduced by 27g even at 0.3 μM compared affected after treatment with 27g. These data suggested that
Fig. 9 27g suppressed tumor growth of xenograft lung tumors in vivo (n = 6 per group). (A) Tumor size was calculated by using the formula (major
circumference × minor circumference2 × 0.5). Data are shown as mean SEM. (B) Tumor weight was measured when the nude mice were sacrificed.
Each dot indicates the tumor weight of an individual mouse. (C) Body weight was measured every three days. Statistical differences among different
groups are noted; *P < 0.05, **P < 0.01, ***P < 0.001.
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compound 27g might be an effective antitumor candidate with acetate to give the products. The characterization and spectral
low toxicity.
data of the final compounds 24a–e are given below.
9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-
[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl acetate (23a). White
solid, yield: 85%, m.p. 237–239 °C; 1H NMR (500 MHz, CDCl3):
δ 2.21(s, 3H), 2.66–2.74(m, 2H), 4.07(d, J = 10.5 Hz, 1H),
4.20–4.24(m, 1H), 4.42(dd, J1 = 9.3 Hz, J2 = 6.1 Hz, 1H), 5.92(s,
2H), 5.94(dd, J1 = 3.4 Hz, J2 = 1.4 Hz, 2H), 6.07(d, J = 9.6 Hz,
1H), 6.31(s, 1H), 6.55(s, 1H), 6.70(s, 1H), 6.72(d, J = 7.9 Hz,
1H), 6.78(d, J = 7.9 Hz, 1H); 13C NMR (125 MHz, CDCl3):
δ 21.07, 45.42, 46.05, 46.25, 70.01, 72.84, 101.11, 101.51,
105.67, 108.27, 109.01, 109.59, 123.09, 127.95, 133.97, 135.89,
146.80, 147.03, 147.91, 147.96, 171.39, 174.04; HRMS (ESI):
calc. for C22H18O8Na (M + Na)+ 433.0894, found: 433.0899.
9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-
Conclusions
In this study, a series of novel racemic otobain derivatives with
ester, amide or amine linkages at the C-4 position was syn-
thesised. These derivatives were examined for anti-proliferative
activity against several cancer cell lines. Most of the com-
pounds showed improved activity compared with lead com-
pound 22. Compound 27g showed the most promising
anticancer action with IC50 values ranging from 1.06 µM to
4.16 μM in various human cancer cells. Compound 27g was
found to cause G2/M phase arrest in A-549 cells, but it did not
induce apoptosis. Colony formation results showed that com-
pound 27g effectively blocked the anchor growth of lung
cancer cells. Compound 27g was a promising agent that
significantly inhibited the growth of human lung cancer
(P < 0.01). Additionally, 27g was relatively safe and tolerant
compared with etoposide because it did not cause gross
toxicity in mice at the tested dosages in vivo. Additional work
on the enantioselective synthesis of compound 27g is currently
underway in our laboratory.
[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl
furan-2-carboxylate
(23b). White solid, yield: 81%, m.p. 236–239 °C; 1H NMR
(500 MHz, CDCl3): δ 2.75–2.82(m, 2H), 4.10–4.12(m, 1H), 4.31
(t, J = 9.6 Hz, 1H), 4.47–4.50(m, 1H), 5.92(d, J = 2.2 Hz, 2H),
5.95(s, 2H), 6.29(d, J = 8.0 Hz, 1H), 6.34(s, 1H), 6.58(s, 2H),
6.74(d, J = 7.7 Hz, 1H), 6.79–6.80(m, 2H), 7.29(d, J = 3.2 Hz,
1H), 7.66(s, 1H); 13C NMR (125 MHz, CDCl3): δ 45.44, 46.08,
46.30, 70.01, 73.33, 101.12, 101.52, 105.79, 108.30, 109.05,
109.63, 112.25, 119.31, 123.11, 127.76, 134.11, 135.85, 143.66,
146.83, 147.15, 147.28, 147.99, 148.05, 158.88, 173.99; HRMS
(ESI): calc. for C25H18O9Na (M + Na)+ 485.0843, found:
485.0850.
Experimental section
9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-
[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl pyrazine-2-carboxylate
(23c). White solid, yield: 77%, m.p. 235–236 °C; 1H NMR
(500 MHz, CDCl3): δ 2.82–2.96(m, 2H), 4.14(d, J = 10.6 Hz, 1H),
4.38(t, J = 9.7 Hz, 1H), 4.49–4.52(m, 1H), 5.94(s, 2H), 5.97(s,
2H), 6.37(s, 1H), 6.46(d, J = 9.4 Hz, 1H), 6.60(s, 1H), 6.77(d, J =
7.2 Hz, 1H), 6.82(d, J = 7.5 Hz, 2H), 8.80(s, 1H), 8.85(s, 1H),
9.40(s, 1H); 13C NMR (125 MHz, CDCl3): δ 45.32, 46.07, 46.24,
69.90, 74.86, 101.14, 101.59, 105.75, 108.31, 109.02, 109.70,
123.14, 127.17, 134.33, 135.71, 142.64, 144.80, 146.51, 146.87,
147.29, 148.01, 148.24, 148.32, 164.42, 173.75; HRMS (ESI):
calc. for C25H18O8N2Na (M + Na)+ 497.0955, found: 497.0961.
9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-
[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl benzoate (23g). White
solid, yield: 80%, m.p. 247–249 °C; 1H NMR (500 MHz, CDCl3):
δ 2.76–2.84(m, 2H), 4.13(d, 1H, J = 9.3 Hz), 4.32–4.36(m, 1H),
4.46(dd, J1 = 9.3 Hz, J2 = 5.8 Hz, 1H), 5.92–5.95(m, 4H), 6.32(d,
J = 8.1 Hz, 1H), 6.36(s, 1H), 6.59(s, 1H), 6.76(d, J = 7.8 Hz, 1H),
6.80(d, J = 9.0 Hz, 2H), 7.49(t, J = 7.8 Hz, 2H), 7.63(t, J = 7.4 Hz,
1H), 8.10(d, J = 7.4 Hz, 2H); 13C NMR (125 MHz, CDCl3):
δ 45.62, 46.13, 46.34, 70.15, 73.30, 101.12, 101.51, 105.85,
108.30, 109.06, 109.65, 123.12, 128.18, 128.71, 129.15, 129.89,
Chemistry
Materials and methods. All reagents and chemicals were
purchased from commercial suppliers and used without
further purification unless otherwise stated. 1H (500 MHz) and
13C (125 MHz) NMR spectra were recorded on a Varian INOVA
spectrometer with CDCl3 or DMSO-d6 and tetramethylsilane
(TMS) as the internal standard. All chemical shift values were
reported in units of δ (ppm). The following abbreviations were
used to indicate the peak multiplicity: s = singlet; d = doublet;
t = triplet; m = multiplet; br = broad. Analytical thin-layer
chromatography (TLC) was carried out on precoated plates
(silica gel 60 F254), and the spots were visualized with ultra-
violet (UV) light. Flash column chromatography was carried
out with silica gel (300–350 mesh). Melting points were deter-
mined on a Yanano MP 500. High-resolution mass data were
obtained on a Bruker microOTOFQ II spectrometer.
The preparation and characterization details of intermedi-
ate compounds 16–22, 24, 25 are included within the ESI.†
General procedure for the synthesis of compounds 23a–c, g–h
A mixture of 23 (0.40 mmol), the corresponding acids RCO2H 133.78, 134.06, 135.93, 146.82, 147.12, 147.96, 147.99, 166.84,
(0.48 mmol), N,N′-dicyclohexylcarbodiimide (DCC, 0.48 mmol) 174.08; HRMS (ESI): calc. for C27H20O8Na (M + Na)+ 495.1050,
and 4-(N,N-dimethylamino)pyridine (DMAP, 0.04 mmol) in dry found: 490.1052.
CH2Cl2 (25 mL) was stirred at 28 °C. Upon completion, 0.1 N
9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro-
HCl (25 mL) was added. The organic layer was washed with [3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl 4-fluorobenzoate (23h).
1
saturated brine (3 × 15 mL), dried over anhydrous Na2SO4, fil- White solid, yield: 75%, m.p. 262–264 °C; H NMR (500 MHz,
tered, concentrated in vacuo, and recrystallized from ethyl CDCl3): δ 2.78–2.83(m, 2H), 4.13(d, J = 9.3 Hz, 1H), 4.31–4.35
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(m, 1H), 4.44–4.47(m, 1H), 5.92(s, 1H), 5.94(s, 1H), 5.95(d, J = 1.5 Hz, 1H), 6.77(d, J = 7.9 Hz, 1H), 6.89(d, J = 7.0 Hz, 1H); 13C
1.9 Hz, 2H), 6.31(d, J = 8.1 Hz, 1H), 6.36(s, 1H), 6.59(s, 1H), NMR (125 MHz, CDCl3): 41.98, 43.41, 46.17, 48.85, 67.80,
6.75–6.81(m, 3H), 7.17(t, J = 8.6 Hz, 2H), 8.12(dd, J1 = 8.6 Hz, 101.17, 101.75, 108.27, 108.76, 108.87, 109.85, 116.72(q, 1JC–F
=
J2 = 5.5 Hz, 2H); 13C NMR (125 MHz, CDCl3): δ 45.58, 46.11, 286.2 Hz), 123.30, 125.95, 134.53, 135.08, 146.90, 147.36,
2
46.30, 70.09, 73.47, 101.13, 101.54, 105.76, 108.30, 109.05, 148.00, 148.91, 157.41(q, JC–F = 37.5 Hz), 174.37; HRMS (ESI):
109.68, 115.86(2JC–F = 21.3 Hz), 123.13, 125.41, 128.02, 132.48 calc. for C22H16NO7F3Na (M + Na)+ 486.0771, found: 486.0893.
3
(d, JC–F = 8.7 Hz), 134.13, 135.87, 146.84, 147.14, 148.00,
N-(9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydro-
1
165.24(d, JC–F = 253.7 Hz), 165.87, 174.01; HRMS (ESI): calc. furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)thiophene-2-carbox-
for C27H19O8FNa (M + Na)+ 513.0956, found: 513.0961.
amide (26e). White solid, yield: 68%, m.p. 261–263 °C; 1H
NMR (500 MHz, CDCl3) δ 2.73(dd, J1 = 14.2 Hz, J2 = 11.3 Hz,
1H), 2.79–2.86(m, 1H), 3.97(t, J = 10.4 Hz, 2H), 4.56(dd, J1 = 9.2
General procedure for the synthesis of compounds 26b–h
A
mixture of 25 (0.70 mmol), chloride or anhydride Hz, J2 = 6.8 Hz, 1H), 5.30(dd, J1 = 6.4, J2 = 3.8 Hz, 1H), 5.92(d,
(0.85 mmol), and 4-(N,N-dimethylamino)pyridine (DMAP, J = 4.3 Hz, 2H), 5.96(d, J = 4.6 Hz, 2H), 6.20(d, J = 6.4 Hz, 1H),
1.12 mmol) in dry CH2Cl2 (25 mL) was stirred at room temp- 6.37(s, 1H), 6.61(s, 1H), 6.73(s, 1H), 6.77(d, J = 7.9 Hz, 1H),
erature under argon. Upon completion, the organic layer was 6.81(d, J = 7.9 Hz, 1H), 7.11(t, J = 4.3 Hz, 1H), 7.55(d, J = 4.4
washed with saturated brine (3 × 15 mL), dried over anhydrous Hz, 2H); 13C NMR (125 MHz, CDCl3): 42.25, 44.28, 46.19,
Na2SO4, filtered, concentrated in vacuo, and purified by silica 48.73, 68.73, 101.15, 101.60, 108.37, 108.91, 109.12, 109.80,
column chromatography (ethyl acetate/dichloromethane, 123.14, 127.84, 127.90, 128.89, 131.07, 134.39, 135.76, 137.43,
1 : 30–1 : 10) to give the products. The characterization and 146.81, 147.22, 147.99, 148.55, 162.09, 174.87; HRMS (ESI):
spectral data of the final compounds 26b–f are given below.
N-(9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydro-
calc. for C25H19NO7SNa (M + Na)+ 500.0774, found: 500.0870.
N-(9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydro-
furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)furan-2-carboxamide furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)quinoline-2-carbox-
(26b). White solid, yield: 74%, m.p. 262–265 °C; 1H NMR amide (26f). White solid, yield: 64%, m.p. 179–181 °C; 1H
(500 MHz, CDCl3) δ 2.76(dd, J1 = 14.2, J2 = 11.4 Hz, 1H), NMR (500 MHz, CDCl3) δ 2.85–2.94(m, 2H), 3.98(dd, J1 = 10.8,
2.82–2.89(m, 1H), 3.97(d, J = 9.7 Hz, 1H), 4.01(d, J = 11.9 Hz, J2 = 9.3 Hz, 1H), 4.03–4.05(m, 1H), 4.57–4.60(m, 1H), 5.49–5.41
1H), 4.58(dd, J1 = 9.2, J2 = 6.9 Hz, 1H), 5.33(dd, J1 = 6.7, J2 = 3.8 (m, 1H), 5.91(d, J = 3.9 Hz, 2H), 5.98(d, J = 2.3 Hz, 2H), 6.42(s,
Hz, 1H), 5.94–5.99(m, 4H), 6.39(s, 1H), 6.53(d, J = 6.7 Hz, 1H), 1H), 6.75(s, 1H), 6.77(s, 1H), 6.86(s, 2H), 7.63(t, J = 7.5 Hz, 1H),
6.58(dd, J1 = 3.3, J2 = 1.6 Hz, 1H), 6.65(s, 1H), 6.75(s, 1H), 7.77(t, J = 7.2 Hz, 1H), 7.89(d, J = 8.1 Hz, 1H), 8.10(d, J = 8.5
6.80–6.85(m, 2H), 7.23(d, J = 3.4 Hz, 1H), 7.50(s, 1H); 13C NMR Hz, 1H), 8.31(d, J = 8.5 Hz, 1H), 8.36(d, J = 8.5 Hz, 1H), 8.50(d,
(125 MHz, CDCl3): 42.24, 44.25, 46.17, 47.95, 68.60, 101.14, J = 7.6 Hz, 1H); 13C NMR(125 MHz, CDCl3): 42.51, 44.40, 46.14,
101.58, 108.37, 108.97, 109.15, 109.79, 112.59, 115.58, 123.19, 48.26, 68.57, 101.16, 101.56, 108.40, 109.11, 109.33, 109.76,
127.85, 134.30, 135.82, 144.53, 146.80, 146.82, 147.18, 147.98, 118.77, 123.08, 127.81, 128.35, 129.57, 129.83, 130.41, 134.12,
148.54, 158.35, 174.83; HRMS (ESI): calc. for C25H19NO8Na 136.25, 137.89, 146.45, 146.78, 147.13, 148.05, 148.49, 148.60,
(M + Na)+ 484.1003, found: 484.1045.
164.62, 174.99; HRMS (ESI): calc. for C30H22N2O7Na (M + Na)+
N-(9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydro- 545.1319, found: 545.1456.
furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)pyrazine-2-carbox-
N-(9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydro-
amide (26c). White solid, yield: 66%, m.p. 263–265 °C; 1H NMR furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)benzamide (26g).
(500 MHz, CDCl3) δ 2.72–2.77(m, 1H), 2.85–2.92(m, 1H), 3.93(t, White solid, yield: 78%, white solid, m.p. 280–281 °C; 1H NMR
J = 10.1 Hz, 1H), 4.02(d, J = 11.3 Hz, 1H), 4.55–4.58(m, 1H), (500 MHz, CDCl3) δ 2.72(dd, J1 = 14.2 Hz, J2 = 11.4 Hz, 1H),
5.37(dd, J1 = 7.1 Hz, J2 = 3.7 Hz, 1H), 5.91(d, J = 4.2 Hz, 2H), 2.80–2.88(m, 1H), 3.92(dd, J1 = 10.7 Hz, J2 = 9.5 Hz, 1H), 3.97
5.96(d, J = 3.3 Hz, 2H), 6.39(s, 1H), 6.66(s, 1H), 6.72(s, 1H), (d, J = 11.3 Hz, 1H), 4.57(dd, J1 = 9.3 Hz, J2 = 6.9 Hz, 1H), 5.33
6.79–6.83(m, 2H), 7.98(d, J = 7.1 Hz, 1H), 8.54(s, 1H), 8.80(s, (dd, J1 = 6.5 Hz, J2 = 3.9 Hz, 1H), 5.92(dd, J1 = 8.3 Hz, J2 = 1.1
1H), 9.44(s, 1H); 13C NMR (125 MHz, CDCl3): 42.41, 44.20, Hz, 1H), 5.95(dd, J1 = 6.6 Hz, J2 = 1.4 Hz, 1H), 6.36(d, J = 6.7
46.24, 48.27, 68.31, 101.10, 101.54, 108.35, 109.02, 109.09, Hz, 1H), 6.37(s, 1H), 6.60(s, 1H), 6.73(s, 1H), 6.77(dd, J1 = 8.0
109.82, 123.17, 127.91, 134.22, 135.91, 142.67, 143.52, 144.61, Hz, J2 = 1.3 Hz, 1H), 6.80(d, J = 7.9 Hz, 1H), 7.45–7.48(m, 2H),
146.84, 147.23, 147.90, 148.02, 148.61, 163.18, 174.51; HRMS 7.58(t, J = 7.4 Hz, 1H), 7.79–7.81(m, 2H); 13C NMR (125 MHz,
(ESI): calc. for C25H19N3O7Na (M + Na)+ 496.1115, found: CDCl3): 42.30, 44.29, 46.20, 48.72, 68.75, 101.14, 101.57,
496.1229.
108.35, 108.91, 109.09, 109.81, 123.19, 127.06, 128.03, 128.88,
N-(9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydro- 132.30, 133.28, 134.37, 135.79, 146.80, 147.21, 147.98, 148.50,
furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,2,2-trifluoroacet- 167.73, 174.85; HRMS (ESI): calc. for C27H21NO7Na (M + Na)+
amide (26d). White solid, yield: 81%, m.p. 228–230 °C; 1H 494.1210, found: 494.1342.
NMR (500 MHz, CDCl3) δ 2.64(dd, J1 = 14.3 Hz, J2 = 11.5 Hz,
N-(9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexahydro-
1H), 2.77–2.85(m, 1H), 3.78(dd, J1 = 11.0 Hz, J2 = 9.3 Hz, 1H), furo[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-4-fluorobenzamide
3.93(d, J = 11.4 Hz, 1H), 4.48(dd, J1 = 9.2 Hz, J2 = 7.0 Hz, 1H), (26h). White solid, yield: 70%, m.p. 256–259 °C; 1H NMR
5.17(dd, J = 7.2 Hz, J2 = 4.1 Hz, 1H), 5.90–5.93(m, 4H), 6.34(s, (500 MHz, CDCl3) δ 2.70(dd, J1 = 14.1 Hz, J2 = 11.5 Hz, 1H),
1H), 6.54(d, J = 1.2 Hz, 1H), 6.66(s, 1H), 6.72(dd, J1 = 8.0, J2 = 2.80–2.87(m, 1H), 3.90(t, J = 10.1 Hz, 1H), 3.98(d, J = 11.3 Hz,
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1H), 4.55(dd, J1 = 9.0 Hz, J2 = 7.1 Hz, 1H), 5.33(dd, J1 = 6.1 Hz, J2 = 11.5 Hz, 1H), 3.90(d, J = 2.9 Hz, 1H), 3.94(d, J = 2.9 Hz,
J2 = 4.2 Hz, 1H), 5.91(d, J = 6.0 Hz, 2H), 5.94(d, J = 5.1 Hz, 2H), 1H), 3.97(d, J = 6.1 Hz, 1H), 4.16(d, J = 14.1 Hz, 1H), 4.28(dd,
6.27(d, J = 6.4 Hz, 1H), 6.38(s, 1H), 6.61(s, 1H), 6.73(s, 1H), J1 = 10.9 Hz, J2 = 8.3 Hz, 1H), 4.40(t, J = 7.4 Hz, 1H), 5.89–5.93
6.77(d, J = 7.9 Hz, 1H), 6.80(d, J = 7.9 Hz, 1H), 7.13(t, J = 8.4 (m, 4H), 6.36(s, 1H), 6.58(d, J = 7.9 Hz, 2H), 6.78(s, 2H), 7.56(d,
Hz, 2H), 7.80(dd, J1 = 8.4 Hz, J2 = 5.3 Hz, 2H); 13C NMR J = 8.4 Hz, 2H), 8.23(d, J = 8.4 Hz, 2H); 13C NMR (125 MHz,
(125 MHz, CDCl3): 42.34, 44.28, 46.25, 48.83, 68.63, 101.10, CDCl3): δ 42.13, 45.01, 45.38, 53.38, 56.06, 67.73, 101.07,
2
101.54, 108.32, 108.89, 109.02, 109.82, 115.83(d, JC–F = 21.3 101.39, 108.23, 108.40, 108.98, 110.20, 123.02, 123.92, 128.80,
3
Hz), 123.17, 128.05, 129.43(d, JC–F = 8.7 Hz), 129.54, 134.39, 131.09, 133.25, 136.78, 146.62, 146.65, 147.19, 147.39, 147.89,
1
135.79, 146.85, 147.27, 148.01, 148.57, 164.17(d, JC–F = 251.3 147.92, 175.90; HRMS (ESI): calc. for C27H22O8N2Na (M + Na)+
Hz), 166.60, 174.65; HRMS (ESI): calc. for C27H20NO7FNa 525.1268, found: 525.1271.
(M + Na)+ 512.1116, found: 512.1188.
5-(Benzo[d][1,3]dioxol-5-yl)-9-((2-nitrobenzyl)amino)-5,5a,8a,9-
tetrahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-6(8H)-one
(27j). Light yellow solid, yield: 39%, m.p. 163–165 °C; 1H NMR
General procedure for the synthesis of compounds 27g–m
To a solution of substituted benzyl bromide (0.79 mmol) in (500 MHz, CDCl3) δ 2.65–2.72(m, 1H), 3.18(dd, J1 = 14.0 Hz,
acetone (3 mL) was added sodium iodide (128 mg, J2 = 11.4 Hz, 1H), 3.93–3.96(m, 2H), 4.11(d, J = 13.7 Hz, 1H),
0.85 mmol). The reaction mixture was stirred for 20 min and 4.26–4.30(m, 2H), 4.45(dd, J1 = 8.0 Hz, J2 = 6.9 Hz, 1H),
then filtered. The filtrate was evaporated to give the corres- 5.89–5.95(m, 4H), 6.35(s, 1H), 6.56(s, 1H), 6.63(s, 1H), 6.79(s,
ponding benzyl iodide. To 25 (0.66 mmol) in 1,2-dichloro- 2H), 7.48–7.52(m, 1H), 7.62–7.67(m, 2H), 8.01(d, J1 = 8.1 Hz,
ethane (15 mL) were added substituted benzyl iodide J2 = 0.9 Hz, 1H); 13C NMR (125 MHz, CDCl3): δ 42.08, 45.11,
(0.79 mmol) and anhydrous barium carbonate (0.79 mmol) 45.95, 51.38, 56.28, 67.90, 101.01, 101.32, 108.18, 108.50,
under nitrogen. After stirring for 40 h at 75–80 °C, the mixture 109.13, 109.99, 123.03, 125.05, 128.66, 131.25, 131.55, 133.20,
was filtered. The filtrate was evaporated to give a crude product 133.49, 134.82, 136.89, 146.54, 146.63, 147.80, 147.83, 149.19,
which was purified by silica column chromatography (ethyl 176.10; HRMS (ESI): calc. for C27H22O8N2Na (M + Na)+
acetate/petroleum ether, 3 : 1). The characterization and spec- 525.1268, found: 525.1291.
tral data of the final compounds 27g–m are given below.
5-(Benzo[d][1,3]dioxol-5-yl)-9-(benzylamino)-5,5a,8a,9-tetra-
4-(((9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexa-
hydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)amino)methyl)-
hydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-6(8H)-one (27g). benzonitrile (27k). White solid, yield: 47%, m.p. 213–215 °C;
1
White solid, yield: 48%, m.p. 196–199 °C; H NMR (500 MHz, 1H NMR (500 MHz, CDCl3) δ 2.63–2.70(m, 1H), 3.180(dd, J1 =
CDCl3) δ 2.58–2.65(m, 1H), 3.23(dd, J1 = 13.9 Hz, J2 = 11.4 Hz, 13.9 Hz, J2 = 11.4 Hz, 1H), 3.88(d, J = 3.1 Hz, 1H), 3.91–3.95(m,
1H), 3.89(d, J = 3.1 Hz, 1H), 3.90–3.92(m, 3H), 4.31(dd, J1
=
2H), 4.10(d, J = 13.9 Hz, 1H), 4.26(dd, J1 = 11.0 Hz, J2 = 8.2 Hz,
10.9 Hz, J2 = 8.2 Hz, 1H), 4.35–4.38(m, 1H), 5.87(dd, J1 = 4.5 1H), 4.37–4.40(m, 1H), 5.89–5.93(m, 4H), 6.35(s, 1H), 6.54(s,
Hz, J2 = 1.1 Hz, 2H), 5.93(d, J = 0.7 Hz, 2H), 6.32(s, 1H), 6.45(s, 1H), 6.58(s, 1H), 6.77(s, 2H), 7.50(d, J = 8.1 Hz, 2H), 7.66(d, J =
1H), 6.60(s, 1H), 6.77(s, 2H), 7.30–7.33(m, 1H), 7.36–7.40(m, 8.2 Hz, 2H); 13C NMR (125 MHz, CDCl3): δ 42.11, 45.02, 45.88,
4H); 13C NMR (125 MHz, CDCl3): δ 42.08, 45.20, 45.92, 54.52, 53.72, 55.99, 67.74, 101.06, 101.38, 108.22, 108.38, 108.98,
55.44, 67.99, 101.01, 101.25, 108.18, 108.45, 109.12, 109.91, 110.16, 111.43, 118.71, 123.03, 128.77, 131.16, 132.50, 133.24,
123.01, 127.58, 128.36, 128.73, 131.77, 133.20, 136.99, 139.83, 136.78, 145.16, 146.61, 146.64, 147.89, 175.91; HRMS (ESI):
146.53, 146.55, 147.64, 147.83, 176.28; HRMS (ESI): calc. for calc. for C28H22O6N2Na (M + Na)+ 505.1370, found: 505.1404.
C27H23O6NNa (M + Na)+ 480.1418, found: 480.1463.
3-(((9-(Benzo[d][1,3]dioxol-5-yl)-8-oxo-5,5a,6,8,8a,9-hexa-
5-(Benzo[d][1,3]dioxol-5-yl)-9-((4-fluorobenzyl)amino)-5,5a,8a,- hydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)amino)-
9-tetrahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-6(8H)-one methyl)benzonitrile (27l). White solid, yield: 40%, m.p.
(27h). White solid, yield: 45%, m.p. 187–189 °C; 1H NMR 201–203 °C; 1H NMR (500 MHz, CDCl3) δ 2.62–2.69(m, 1H),
(500 MHz, CDCl3) δ 2.59–2.66(m, 1H), 3.21(dd, J1 = 13.9 Hz, 3.17(dd, J1 = 14.0 Hz, J2 = 11.4 Hz, 1H), 3.88–3.90(m, 2H), 3.94
J2 = 11.4 Hz, 1H), 3.84–3.87(m, 2H), 3.90–3.95(m, 2H), 4.28(dd, (d, J = 11.3 Hz, 1H), 4.06(d, J = 13.5 Hz, 1H), 4.27(dd, J1 = 11.0
J1 = 11.0, J2 = 8.2 Hz, 1H), 4.35–4.38(m, 1H), 5.87–5.92(m, 4H), Hz, J2 = 8.2 Hz, 1H), 4.37–4.40(m, 1H), 5.88–5.92(m, 4H), 6.34
6.33(s, 1H), 6.50(s, 1H), 6.59(s, 1H), 6.77(s, 2H), 7.06(t, J = 8.6 (s, 1H), 6.54(s, 1H), 6.59(s, 1H), 6.77(s, 2H), 7.48(t, J = 7.7 Hz,
Hz, 2H), 7.34(dd, J1 = 8.4 Hz, J2 = 5.5 Hz, 2H); 13C NMR 1H), 7.60(d, J = 7.7 Hz, 1H), 7.64(d, J = 7.9 Hz, 1H), 7.66(s, 1H);
(125 MHz, CDCl3): δ 42.09, 45.13, 45.90, 53.67, 55.57, 67.91, 13C NMR (125 MHz, CDCl3): δ 42.12, 45.00, 45.89, 53.37, 55.94,
101.02, 101.30, 108.19, 108.41, 109.07, 109.98, 115.44(d, 2JC–F
21.3 Hz), 123.01, 129.82(d, JC–F = 7.5 Hz), 131.64, 133.19, 118.68, 122.99, 129.53, 131.18, 131.27, 131.66, 132.64, 133.22,
=
67.76, 101.05, 101.38, 108.22, 108.41, 109.06, 110.14, 112.72,
3
135.55, 135.57, 136.94, 146.55, 146.58, 147.71, 147.85, 161.21 136.81, 141.28, 146.59, 146.63, 147.87, 175.95; HRMS (ESI):
1
(d, JC–F
=
243.0 Hz), 176.17; HRMS (ESI): calc. for calc. for C28H22O6N2Na (M + Na)+ 505.1370, found: 505.1411.
C27H22O6NFNa (M + Na)+ 498.1323, found: 498.1373.
5-(Benzo[d][1,3]dioxol-5-yl)-9-((4-methylbenzyl)amino)-
5-(Benzo[d][1,3]dioxol-5-yl)-9-((4-nitrobenzyl)amino)-5,5a,8a,- 5,5a,8a,9-tetrahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-6-
1
9-tetrahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-6(8H)-one (8H)-one (27m). White solid, yield: 43%, m.p. 184–186 °C; H
(27i). Light yellow solid, yield: 43%, m.p. 210–212 °C; 1H NMR NMR (500 MHz, CDCl3) δ 2.40(s, 3H), 2.59–2.66(m, 1H), 3.26
(500 MHz, CDCl3) δ 2.64–2.71(m, 1H), 3.19(dd, J1 = 13.8 Hz, (dd, J1 = 13.8 Hz, J2 = 11.5 Hz, 1H), 3.87–3.94(m, 4H), 4.33(dd,
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J1 = 10.9 Hz, J2 = 8.2 Hz, 1H), 4.37–4.40(m, 1H), 5.90(d, J = Dickinson flow cytometer (BD Biosciences, Franklin Lakes, NJ,
3.8 Hz, 2H)), 5.95(s, 2H), 6.35(s, 1H), 6.50(s, 1H), 6.63(s, 1H), USA).41
6.79(s, 2H), 7.22(d, J = 7.8 Hz, 2H), 7.28(d, J = 7.8 Hz, 2H); 13C
Cell apoptosis detection. Annexin V-FITC Apoptosis Detec-
NMR (125 MHz, CDCl3): δ 21.17, 42.09, 45.21, 45.92, 54.24, tion Kit (eBioscience/Bender MedSystems GmbH, Vienna,
55.38, 68.04, 101.01, 101.25, 108.17, 108.51, 109.15, 109.88, Austria) was used to detect apoptosis induced by compound
123.00, 128.33, 129.38, 131.87, 133.19, 136.82, 137.04, 137.24, 27g. A549 cells were seeded into 6-well plates (105 cells per
146.51, 146.53, 147.61, 147.83, 176.35; HRMS (ESI): calc. for well). After attachment, cells were treated with 1 μM, 3 μM,
C28H25O6NNa (M + Na)+ 494.1574, found: 494.1629.
10 μM of 27g for 24 h. All the cells were harvested and washed
with ice-cold PBS. Annexin V-FITC and propidium iodide (PI)
were added. The cells were incubated at room temperature for
Biology
Cell culture. The human cancer cell lines were incubated at 30 min. Annexin V-FITC and PI fluorescence were monitored
37 °C in a humidified incubator with 5% CO2, including by using a Becton Dickinson FAC scan flow cytometer and the
HL-60 (human promyelocytic leukemia cell line), SGC-7901 data were analyzed using Cell Quest software. The experiments
(gastric cancer), MCF-7 (breast cancer), HCT-116 (colon were performed in three replicates.42
cancer), A549 (non-small cell lung cancer) and HaCat (human
Colony formation assay. A549 cells were seeded in 6-well
skin cell line/keratinocyte). All the cells were brought from plates at a density of 103 cells per well. After 24 h, the culture
American Type Culture Collection (ATCC; Manassas, VA, USA). medium was removed and fresh medium with various concen-
The cells were cultured in RPMI-1640 (HL-60, SGC-7901, trations of 27g was added. The medium was changed every
HaCat) or Dulbecco’s modified Eagle’s medium (DMEM; three days. Colonies were fixed with 4% paraformaldehyde and
MCF-7, HCT-116, A549) supplemented with 10% fetal bovine stained with 0.5% crystal violet solution. The images of the
serum (FBS; HyClone Laboratories, Logan, UT) and 1% penicil- colonies were captured and the number of crystal violet
lin–streptomycin antibiotics (HyClone Laboratories, Logan, stained colonies was counted randomly. GraphPad Prism 5
UT).
was used to do all statistical calculations. We used One-way
Cell viability assay. Cell viability was measured by the sulfor- ANOVA (and nonparametric) with post hoc Tukey’s tests
hodamine B (SRB) assay as previously described.39 Briefly, cells (compare all pairs of columns) for more than two groups. The
in their log growth phase were harvested, counted and seeded data represented mean values SD. Statistical differences were
in 96-well microtitre plates. After 24 h of incubation to allow compared between the groups and noted as ***P < 0.001.43
cell attachment, the cells were incubated in fresh medium con-
Western blotting assay. After being treated with the indi-
taining various concentrations (0.01–300 μM) of the tested cated concentrations of compound 27g, the cells were har-
compounds for an additional 72 h. Five replicate wells were set vested and lysed in ice-cold radioimmunoprecipitation assay
up for each experimental condition. The attached cells were buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.1% SDS;
then fixed with chilled trichloroacetic acid for 1 h at 4 °C, 0.5% sodium deoxycholate; 0.5% Triton X-100) supplemented
washed with phosphate-buffered saline (PBS) and dried, fol- with 50 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA,
lowed by staining with 0.04% sulforhodamine B dye for 0.5 h. 1 mM phenylmethylsulfonyl fluoride, protease inhibitor and
The non-adsorbed dye was washed away by 1% acetic acid solu- phosphorylation protease inhibitor. Colchicine (about 4 times
tion and the adsorbed dye was solubilized in 10 mM Tris- IC50) and paclitaxel (about 8 times IC50) were taken as the posi-
buffer. Before the optical density was measured at 515 nm on tive control. The lysates were centrifuged at 12 000 rpm for
a SpectraMax 190 microplate reader (Molecular Devices, Sun- 20 min and the protein concentrations were measured using
nyvale, CA, USA) with a GraphPad Prism 5 (GraphPad Software, the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham,
San Diego, CA, USA),40 the plates were gently shaken for MA, USA). The lysates were mixed with loading buffer (10%
20 min on a mechanical shaker. The results are reported in SDS, 25% 2-mercaptoethanol, 50% glycerol, 0.05% bromo-
terms of the half maximal inhibitory concentration (IC50) phenol blue and 0.35 M Tris-HCl, pH 6.8), and then boiled for
values. Etoposide served as the positive control. DMSO (0.1%) 10 min immediately. Fifty mg of total protein were loaded onto
was used as a negative control unless special instructions are 12% SDS-polyacrylamide gels, transferred to nitrocellulose
given. Three or more independent experiments were carried membrane, incubated with BSA, and probed with primary anti-
out.
bodies [phospho-histone H3 (Ser10) (Abcam, Burlingame, CA,
Cell cycle analysis. A549 cells were seeded in 6-well plates USA), MPM-2 (Millipore, Billerica, MA, USA) and β-actin
and incubated with compound 27g at concentrations of 1 μM, (Sigma, St Louis, MO, USA)] overnight at 4 °C. Either a goat
3 μM, 10 μM for 12 h and 24 h. Then, both floating and adher- anti-mouse or goat anti-rabbit horseradish peroxidase-conju-
ent cells were collected, washed with ice-cold PBS and fixed in gated secondary antibody (Pierce) was incubated for 1 h at
70% ethanol at 4 °C overnight. The cells were then washed room temperature. Protein bands were visualized by enhanced
with PBS, incubated with 1 mg mL−1 DNase-free RNase A chemiluminescence detection using the Odyssey Infrared
(OMEGA, Norcross, GA, USA) at 37 °C for 30 min, and stained Imager (LI-COR® Biosciences, Lincoln, NE, USA).42
with 50 mg mL−1 propidium iodide (Sigma, St Louis, MO,
Xenograft mouse model. BALB/cA nude male mice used in
USA) in 0.1% Triton X-100 in the dark at room temperature for the study were purchased from National Rodent Laboratory
30 min. The stained cells were analyzed with a Becton Animal Resources (Shanghai, China). All treatments were
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administered according to the NIH standards established in
the Guidelines for the Care and Use of Experimental Animals,
and all the protocols were approved by East China Normal
University.
A xenograft model was set up to study the inhibitory effects
of 27g in vivo. Human lung cancer cells A549 (5 × 106 cells per
9 J. Sun, J. Yu, P. C. Zhang, F. Tang, Y. D. Yue, Y. N. Yang,
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and were treated intraperitoneally with different compounds
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dissolved in the solvent vehicle (sterile PBS containing 1% 13 J. Li, A. P. Meng, X. L. Guan, J. Li, Q. Wu, S. P. Deng,
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ences of tumors were measured every 3 days with a vernier
caliper until animals were sacrificed. The tumor volume was
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