Synthesis of novel lipophilic N-Substituted norcantharimide derivatives and evaluation of their anticancer activities

Article abstract of DOI:10.3390/molecules19066911

This research attempted to study the effect of lipophilicity on the anticancer activity of N-substituted norcantharimide derivatives. Twenty-three compounds were synthesized and their cytotoxicities against five human cancer cell lines studied. The lipophilicity of each derivative was altered by its substituent, an alkyl, alkyloxy, terpenyl or terpenyloxy group at the N-position of norcantharimide. Further, among all synthesized derivatives studied, the compounds N-farnesyloxy-7-oxabicyclo[2.2.1]heptane-2,3- dicarboximide (9), and N-farnesyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (18), have shown the highest cytotoxicity, anti-proliferative and apoptotic effect against human liver carcinoma HepG2 cell lines, yet displayed no significant cytotoxic effect on normal murine embryonic liver BNL CL.2 cells. Their overall performance led us to believe that these two compounds might be potential candidates for anticancer drugs development.

Full text of DOI:10.3390/molecules19066911

Molecules 2014, 19, 6911-6928; doi:10.3390/molecules19066911
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Novel Lipophilic N-Substituted Norcantharimide
Derivatives and Evaluation of Their Anticancer Activities
Jin-Yi Wu 1,*, Cheng-Deng Kuo 2, Chien-Yu Chu 1, Min-Shin Chen 1, Jia-Hua Lin 1,
Yu-Jen Chen 3,4 and Hui-Fen Liao 5
1
Department of Microbiology, Immunology and Biopharmaceutics, College of Life Sciences,
National Chiayi University, Chiayi 60004, Taiwan
2
Laboratory of Biophysics, Department of Medical Research, Taipei Veterans General Hospital,
Taipei 11217, Taiwan
3
Department of Radiation Oncology, Mackay Memorial Hospital, New Taipei City 25160,
Taiwan
4
Institute of Transitional Medicine, National Yang Ming University, Taipei 11221, Taiwan
5
Department of Biochemical Science and Technology, College of Life Sciences, National Chiayi
University, Chiayi 60004, Taiwan
* Author to whom correspondence should be addressed; E-Mail: jywu@mail.ncyu.edu.tw;
Tel: +886-5-2717925; Fax: +886-5-2717778.
Received: 27 March 2014; in revised form: 21 May 2014 / Accepted: 22 May 2014 /
Published: 26 May 2014
Abstract: This research attempted to study the effect of lipophilicity on the anticancer
activity of N-substituted norcantharimide derivatives. Twenty-three compounds were
synthesized and their cytotoxicities against five human cancer cell lines studied. The
lipophilicity of each derivative was altered by its substituent, an alkyl, alkyloxy, terpenyl or
terpenyloxy group at the N-position of norcantharimide. Further, among all synthesized
derivatives studied, the compounds N-farnesyloxy-7-oxabicyclo[2.2.1]heptane-2,3-
dicarboximide (9), and N-farnesyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (18),
have shown the highest cytotoxicity, anti-proliferative and apoptotic effect against human
liver carcinoma HepG2 cell lines, yet displayed no significant cytotoxic effect on normal
murine embryonic liver BNL CL.2 cells. Their overall performance led us to believe that
these two compounds might be potential candidates for anticancer drugs development.

Molecules 2014, 19
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Keywords: norcantharimide derivatives; lipophilic substitution; terpenyl group; anticancer
activity; HepG2; apoptosis
1. Introduction
Mylabris (Mylabris phalerata and M. cichorii), the dried body of the blister beetle, has been used in
Chinese medicine for thousands of years for the treatment of malignant tumors such as hepatoma,
breast cancer, colorectal cancer and abdominal malignancy (Figure 1) [1–3].
Figure 1. Chemical structures of cantharidin (1), norcantharidin (2) and norcantharimide (15).
O
O
O
O
O
O
O
O
NH
O
O
O
Cantharidin (1)
Norcantharidin (2)
Norcantharimide (15)
Cantharidin (exo-2,3-dimethyl-7-oxabicyclo[2.2.1] heptane-2,3-dicarboxylic acid anhydride), one
of the active compounds obtained from Mylabris, has been shown to have anticancer properties both
in vitro and in vivo [4–10]. It is a potent serine/threonine protein phosphatase 1 (PP1) and protein
phosphates 2A (PP2A) inhibitor [11–14]. Nevertheless, cantharidin (1) was also found to be poisonous
to the kidney and liver [15]. Though norcantharidin (2), the demethylated form of cantharidin, appears
to have less nephrotoxicity and liver toxicity, the demethylation also lowers its bioactivity. Meanwhile,
studies had also shown that norcantharidin has anticancer activity against various cancer cell lines
through the retardation of cell cycle progression and the inhibition of cell proliferation in vitro.
Further, even though it induces apoptosis in various human cancer cell lines, including melanoma,
cervical cancer, bladder cancer, leukemia, colon cancer, breast cancer, and hepatoma [16–28], the main
use of norcantharidin has been limited to the treatment of hepatoma [29–32]. Lastly, to enhance its
anticancer activity, various structural modifications of norcantharidin had been studied and mixed
results had been reported. For instance, an analog of norcantharidin was shown to inhibit the function
of protein phosphatase and had anti-proliferative activity while others bioactivities were erased [33–39].
In the previous study, it has been reported that norcantharimide analogues bearing a long alkyl chain at
N-position may have enhanced bioavailability and transportability through cell membrane. The highly
hydrophobic nature of the alkyl tails of norcantharimide analogues may improve their uptake and
biological activity [40]. In this study, we examined closely the effect of the lipophilicity of
N-substituted norcantharimide on their anticancer activities. Specifically, we prepared and evaluated
the cytotoxicity of a series of N-substituted norcantharimide derivatives that bear alkyl, alkyloxy,
terpenyl or terpenyloxy groups at the N-position of norcantharimide.

Molecules 2014, 19
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2. Results and Discussion
2.1. Chemistry
The key starting material in this study was 5,6-dehydronorcantharidin (5), which was prepared on a
large scale through exo-selective cycloaddition, or Diels-Alder reaction, of furan (3) and maleic
anhydride (4). Subsequent hydrogenation of 5 (H2, 10% Pd/C) using a modified procedure of
McChuskey et al. [33] provided the starting norcantharidin (2) in excellent yield, which then was
employed as the substrate for the synthesis our designed derivatives. First, norcantharidin (2) was
reacted with hydroxylamine hydrochloride in the presence of sodium methoxide in dry methanol
at room temperature to produce N-hydroxynorcantharimide (6), according to a previous report.
N-Hydroxynorcantharimide (6) was then reacted with terpenyl or alkyl bromide in dry acetone in the
presence of K2CO3 to afford NO-substituted derivatives 7–12 in moderate to good yields, as shown in
Scheme 1.
Scheme 1. Synthesis of norcantharidin (2) and compounds 5–12.
Reagents and conditions: (a) Ether, room temperature, 48 h; (b) 3 atm H2, 10% Pd/C, THF, room
temperature, 8 h; (c) NaOCH3, NH2OH·HCl, MeOH, room temperature, 20 h; (d) K2CO3, alkyl or terpenyl
bromide, acetone, reflux, 8–10 h.
Second, furan (3) and maleimide (13) were heated in toluene to afford dihydroxynorcantharimide
(14) by the Diels-Alder reaction. Hydrogenation was then performed as a reduction in dry THF in the
presence of catalytic 10% Pd/C to give norcantharimide (15). The lipophilic substitution of these
derivatives all took place at N-position of norcantharimide. The compounds 6, 14 or 15 reacted with
alkyl or terpenyl bromide in dry acetone in the presence of K2CO3 to produce N-substituted derivatives
16–24 in moderate to excellent overall yields, as shown in Scheme 2. Using compounds 22–24 as
precursors we obtained compounds 25–27 in moderate to excellent yields through hydrogenation
reactions. The synthetic reactions were also outlined in Scheme 2. The chemical structures of these
compounds were elucidated by 1H-NMR, 13C-NMR, 2D NMR and LC-MS spectroscopic methods.

Molecules 2014, 19
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Scheme 2. Synthesis of compounds 14–27.
Reagents and conditions: (a) Toluene, 80 °C, 6 h; (b) 3 atm H2, 10% Pd/C, THF, room temperature, 8–48 h;
(c) K2CO3, alkyl or terpenyl bromide, acetone, reflux, 8–10 h.
2.2. In Vitro Anti-Proliferative Activity by MTT Assay
The cytotoxic effects of the twenty-three norcantharimide derivatives, with 5-fluorouracil (5-Fu),
cisplatin and doxorubicin as positive control, against five human cancer cell lines was evaluated by the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay [41]. The cancer cells
under study were HepG2 (liver carcinoma), BFTC905 (bladder carcinoma), HT29 (colon carcinoma),
SW480 (colon carcinoma), and HL60 (leukemia). The data were summarized in Table 1. Of the
twenty-three compounds tested, we found eleven compounds; 7–9, 17, 18, 20, 21, 23, 24, 26, and 27,
that showed significant cell growth inhibition on all five human cancer cells. As a trend, the anticancer
activity of these compounds increased with the chain length of the substitutes which implied that the
lipophilicity of the norcantharimide derivatives at N-substitute affected the bioactivity of the
compounds. This result indicated that the lipophilic characteristics at N-position substitution of the
terpenyl moieties of norcantharimide derivatives can remarkably enhance its anticancer activity on a
panel of human cancer cell lines. Interestingly, a higher calculated lipophilicity values (clogP) of
norcantharimide derivatives is associated with a higher anticancer activity, which the longer terpenyl
moieties may enhance their bioavailability and improve their cell membrane permeability to raise their
cytotoxic activities. In particular, among the eleven bioactive ones, compounds 9, N-farnesyloxy-7-
oxabicyclo[2.2.1]heptane-2,3-dicarboximide, and 18, N-farnesyl-7-oxabicyclo[2.2.1]heptane-2,3-
dicarboximide, exhibited the highest activities against human liver carcinoma HepG2 cell lines, with
the IC50 values of 8.3 ± 1.3 and 16.4 ± 1.2 μM, respectively. Compared to norcantharidin (2)’s IC50
value of 42.0 ± 1.8 μM, these two derivatives evinced a two to five fold stronger cytotoxicity potency
against human liver carcinoma HepG2 cells. Meanwhile, when treated with norcantharidin (2),
compounds 9 or 18, no significant cell death was detected in normal murine embryonic liver BNL
CL.2 cell lines. Furthermore, only a marked effect on cell death (under 20%) was observed at the
maximum concentration (60 μM) of these compounds after 24 h or 48 h treatment (Figure S1), except
for norcantharidin (2) which exhibited slight toxic effect after 48 h. It thus appeared that both
compounds 9 and 18 are cytotoxic to human liver cancer cells with no significant adverse effects on
normal murine embryonic liver cells. In other words, through a simple structure-activity relationship

Molecules 2014, 19
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(SAR) analysis we demonstrated that the cell growth inhibitory potency closely related to the length of
the alkyl or terpenyl chain.
Table 1. Cytotoxic effects (IC50 values in μM) of norcantharidin (2) and its derivatives
(5–12, 14–27), 5-Fu, cisplatin and doxorubicin against five human cancer cell lines for 48 h.
IC50 (μM) (Mean ± SD)
Compound
clogP a
HepG2 b
42.0 ± 1.8
ND
BFTC905 c
18.9 ± 0.3
75.3 ± 2.4
ND
HT-29 d
19.5 ± 0.2
ND
SW480 d
49.1 ± 8.4
ND
HL-60 e
ND f
NCTD (2)
−0.86
−1.14
−1.64
1.23
5
ND
6
ND
ND
ND
ND
7
83.2 ± 2.2
32.5 ± 1.4
8.3 ± 1.3
ND
31.7 ± 2.6
24.7 ± 1.8
11.3 ± 1.0
ND
38.5 ± 5.9
22.0 ± 0.1
9.7 ± 1.7
ND
66.3 ± 2.4
44.7 ± 2.3
18.5 ± 2.3
ND
ND
8
3.26
90.2 ± 2.2
39.0 ± 1.1
ND
9
5.29
10
11
12
14
15
16
17
18
1.11
3.23
ND
ND
ND
ND
ND
5.35
ND
ND
ND
ND
ND
−1.35
−1.67
1.05
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.09
34.3 ± 4.9
16.4 ± 1.2
15.5 ± 3.9
9.3 ± 0.6
26.9 ± 2.2
14.8 ± 1.9
86.6 ± 2.6
33.1 ± 1.0
ND
5.12
79.8 ± 1.1
19
20
21
0.94
3.06
5.17
ND
ND
ND
ND
ND
ND
67.2 ± 8.5
34.0 ± 2.7
25.8 ± 1.2
21.9 ± 0.7
57.4 ± 1.6
21.9 ± 0.9
84.6 ± 1.3
46.8 ± 3.0
81.0 ± 2.1
0.77
2.80
4.83
22
23
ND
ND
ND
ND
ND
ND
ND
39.1 ± 2.5
53.0 ± 3.4
54.9 ± 1.5
24
25
26
16.5 ± 2.5
ND
10.2 ± 2.8
ND
20.2 ± 3.3
ND
42.8 ± 0.4
ND
ND
ND
ND
1.34
3.85
6.37
36.4 ± 1.5
ND
28.6 ± 2.1
ND
27
28.1 ± 8.3
40.2 ± 7.6
36.1 ± 3.1
0.3 ± 0.0
13.5 ± 1.9
ND
27.8 ± 4.5
ND
33.8 ± 1.9
32.7 ± 8.3
40.7 ± 1.2
ND
ND
5-Fu
−1.72
−2.50
0.87
g
Cisplatin
-
24.1 ± 0.1
ND
g
Doxorubicin
-
1.7 ± 0.2
0.5 ± 0.1
14.3 ± 0.9
a
b
c
d
Calculated with ChemDraw 11.0 software; Liver carcinoma; Bladder carcinoma; Colon carcinoma;
e
f
Leukemia; ND indicated that no appreciable inhibition value (IC50) was observed upon treatment of
maximal concentration at 100 μM. Data are expressed as the mean ± SD from the dose response curve of at
least three independent experiments; g -: No test.
In light of the cytotoxicity findings described above, we extended our study on the anti-proliferation
efficacy of compounds 9 and 18 against HepG2 cells to 24 h, 48 h and 72 h periods. The data on
norcantharidin (2) was used here as the base case. As shown in Table 2, first of all, the cytotoxicity of
compounds 9 and 18 demonstrated, once again, an impressed fivefold and twofold, respectively,
stronger bioactivity than that of norcantharidin (2) in vitro. In addition, the growth inhibition of HepG2
cells induced by these three samples was exhibited in a time-dependent manner.

Molecules 2014, 19
Table 2. Cytotoxic effects of norcantharidin (2), compounds 9 and 18 against human
6916
HepG2 cancer cell lines for 24, 48, and 72 h.
IC50 (μM) a (Mean ± SD)
48 h
Compound
24 h
72 h
NCTD (2)
48.0 ± 0.7
10.7 ± 0.2 ***
19.4 ± 2.1 **
42.0 ± 1.8
22.8 ± 0.6
9
8.3 ± 1.3 ***
16.4 ± 1.2 ***
8.2 ± 0.6 ***
12.8 ± 2.5 ***
18
a
Data are expressed as the mean ± SD from the dose response curve of at least three independent
experiments. ** p < 0.01, *** p < 0.001 versus NCTD (2).
2.3. Nuclear Morphological Changes of HepG2 Cells Treated with Norcantharidin (2),
Compounds 9 and 18
To further investigate the role of apoptosis in the cytotoxicity of N-farnesyloxy- (9) and N-farnesyl-
norcantharimide (18), we incubated HepG2 cells with norcantharidin (2), compounds 9 or 18,
separately, for 48 h. The cells were then stained with Hoechst 33,258, and examined by fluorescence
microscopy for topical morphological changes [41]. As shown in Figure 2, while the nuclei of the cells
were round in shape and stained homogenously in the control without testing compound, those treated
with norcantharidin (2), N-farnesyloxy- (9) and N-farnesylnorcantharimide (18) showed typical
morphological features of apoptosis such as cell shrinkage, chromatin condensation and DNA
fragmentation [16]. Evidently, the proliferation of the cancer cell was inhibited by the testing
compounds in vitro.
Figure 2. (Top) Cell morphological observations of nuclear change of human hepatoma
HepG2 cell lines after 48 h exposure (untreated), with 40 μM norcantharidin (2),
compounds 9 and 18. (Bottom) Cells were stained with Hoechst 33258 and examined using
a Nikon (Tokyo, Japan) fluorescence microscope. (Magnification, 400×).

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2.4. Cell Cycle Distribution Analysis Using Flow Cytometry
To probe the apoptotic effects of norcantharidin (2), compounds 9 and 18 on cell cycle progression,
HepG2 cells were treated once again with these compounds, separately, at different concentrations for
48 h. The cell cycle distribution of the cancer cells was analyzed by flow cytometry, and the subG1
phase was analyzed by flow cytometry with propidium iodide (PI) staining [42]. The control of the
experiment was the untreated cells. As illustrated in Figure 3, only a small fraction of apoptotic cells
(0.8%) was detected in the control as well as in norcantharidin (2) at a low concentration of 10 μM.
However, as norcantharidin (2) dosage was increased from 20 to 60 μM, the fraction of apoptotic cell
went up substantially from 1.16% to 16.7% in a dose-dependent manner. As for compound 18, the
apoptotic effect meagerly laid between 0.7% to 1.1% across the treatment range of 10–60 μM. On the
other hand, compound 9 gave rise to an impressive 3.7%–19.7% apoptosis from 10 to 60 μM and
induced cell accumulation in the G2/M phase.
Figure 3. Induction of cells arrest by norcantharidin (2), compounds 9 and 18. Effects of
different concentrations of norcantharidin (2), compounds 9 and 18 on cell-cycle
progression of HepG2 cells. HepG2 cells were untreated or treated with 10–60 μM
norcantharidin (2), compounds 9 and 18 for 48 h. After treatment, cells were fixed and
stained with PI, and the cell cycle distribution was examined by flow cytometer.
18
2.5. Apoptotic Analyses-Annexin V-FITC/PI Double Staining and Flow Cytometry Analyses
To study in depth the bioactivities of norcantharidin (2), compounds 9 and 18 against HepG2 cells,
the cancer cells were treated with vehicle alone as control or with one of the three testing compounds

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at different concentrations (10–60 μM). After 48 h, the samples were double-stained with annexin V-
FITC and PI [42]. The percentages of cell populations at various stages of apoptosis were exhibited in
Figure 4. Evidently the data pointed out that the distributions of apoptotic cell death resulting from the
treatment of compounds 9 and 18 were dose-dependent, but this was not the case for norcantharidin
(2). Starting from a dosage of 40 μM, both compounds 9 and 18 induced higher frequency of HepG2
cells apoptosis, as well as cytotoxic effects at both early and late stages by annexin V-FITC/PI staining
analysis. For norcantharidin (2), still the only discernible effect was seen at a higher threshold
(60 μM). We attributed this finding to and confirmed that the superior efficiency of both compounds 9
and 18 lies in its cytotoxicity and inhibitive function on human hepatoma cell proliferation.
Figure 4. Flow cytometry analysis of HepG2 cells treated with different concentrations of
norcantharidin (2), compounds 9 and 18 for 48 h. Treated cells were examined for
apoptotic cells using Annexin V-FITC apoptosis detection kit. Annexin V-positive/PI-
negative cells were in early stages of apoptosis and double positive cells were in late
apoptosis, whereas annexin V- negative/PI -positive cells were necrotic.
18
3. Experimental
3.1. General
In details, all chemical reagents in commercial quality were used as received (Sigma-Aldrich or
Acros Organics) and were used without further purification. Solvents were dried and the synthesized
compounds were purified using standard techniques. In general, the reactions were carried out under
anhydrous conditions in dry solvent and nitrogen atmosphere. Reactions-progression was monitored by
thin layer chromatography (TLC) on aluminum plates coated with silica gel with a fluorescent
indicator (Merck 60 F254). Unless otherwise stated, column chromatography was performed with silica

Molecules 2014, 19
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gel SiliaFlash® G60 (60–200 μm) purchased from SiliCycle Inc. (Quebec City, QC, Canada). Melting
points were determined in open capillaries using the Fargo MP-2D apparatus and were uncorrected.
1
13
NMR spectra were recorded in CDCl3 at 500.13 MHz for H and at 125.77 MHz for C (Bruker
AVANCE III plus 500 MHz), respectively. Chemical shift (δ) were reported in parts per million (ppm)
measured relative to the internal standards (TMS), and the coupling constant (J) were expressed in
Hertz (Hz). The purity of these compounds was more than 98% based on the analysis of HPLC with
RP-C18 column. The mass spectra were acquired using a Thermo Finnigan model LXQ (Thermo
Electron Co., Waltham, MA, USA) ion trap mass spectrometer equipped with ESI source interference
and controlled by Xcalibur 2.06. The mass spectra were acquired in a positive ion mode or a negative
ion mode.
3.2. General Procedure for the Preparation of Compounds 2, 5 and 6
7-Oxabicyclo[2.2.1]-5-heptene-2,3-dicarboxylic anhydride (5). A solution of furan (40 mL, 550 mmol)
and maleic anhydride (10 g, 102 mmol) were stirred together in ether (100 mL) at room temperature
for 48 h, after which the white precipitate was collected to give 5 as a colorless solid, yield 93.3%, mp
1
121–122 °C. H-NMR (CDCl3): δ 6.58 (s, 2H, H-5,6), 5.47 (s, 2H, H-1,4), 3.19 (s, 2H, H-2,3);
13C-NMR (CDCl3): δ 170.1, 137.2, 82.4, 48.9; LC-MS (ESI‒, m/z) calculated for C8H6O4: 166.03,
found for 164.90 [M–H]‒.
7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylic anhydride (2). To a solution of 5 (4.7 g, 28.3 mmol) in
THF (200 mL), 10% Pd/C (470 mg) was added, and the mixture was stirred at room temperature under
a hydrogen atmosphere (3 atm) for 8–12 h. The reaction mixture was filtered through Celite 545® and
concentrated in vacuo to give norcantharidin (2) as colorless crystals, yield 91.4%, mp 116–118 °C.
1H-NMR (CDCl3): δ 5.05 (t, J = 2.3 Hz, 2H, H-1,4), 3.18 (s, 2H, H-2,3), 1.91–1.89 (m, 2H, H-5,6),
1.65–1.63 (m, 2H, H-5,6); 13C-NMR (CDCl3): δ 171.1, 80.2, 50.6, 28.1; LC-MS (ESI‒, m/z) calculated
for C8H8O4: 168.04, found for 166.86 [M–H]‒.
N-Hydroxy-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (6). A solution of 2 (5.04 g, 30 mmol) in
dry methanol (200 mL), sodium methoxide (1.62 g, 30 mmol) and hydroxylamine hydrochloride (2.08 g,
30 mmol) was added, and the mixture was stirred at room temperature for 20 h. The reaction mixture
was filtered and concentrated in vacuo and recrystallized using CHCl3 to give 6 as a colorless crystals,
1
yield 63%, mp 168‒169 °C. H-NMR (D2O): δ 4.93 (s, 2H, H-1,4), 3.21 (s, 2H, H-2,3),
13
1.89–1.86 (m, 2H, H-5,6), 1.78–1.74 (m, 2H, H-5,6); C-NMR (D2O): δ 177.0, 78.8, 46.9, 28.0; LC-
MS (ESI+, m/z) calculated for C8H9NO4: 183.05, found for 206.00 [M+Na]+.
3.3. General Procedure for Synthesis of Target Compounds 7‒12
To a stirred solution of 5 or 6 (1 mmol) in dry acetone (25 mL) was added the appropriate alkyl or
terpenyl bromide (1 mmol) and K2CO3 (3 mmol), and the reaction mixture was refluxed for 8‒10 h.
Then, the reaction mixture was filtered and concentrated in vacuo, and the residue was purified by
column chromatography using silica gel with ethyl acetate/n-hexane as eluent to afford the
desired compounds.

Molecules 2014, 19
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N-Isoprenyloxy-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (7). Colorless crystals, yield 64%, mp
93–94 °C. 1H-NMR (CDCl3): δ 5.38 (t, J = 7.7 Hz, 1H, H-2'), 4.83 (t, J = 2.7 Hz, 2H, H-1,4), 4.57 (d,
J = 7.7 Hz, 2H, H-1'), 2.80 (s, 2H, H-2,3), 1.86–1.83 (m, 2H, H-5,6), 1.74 (s, 3H, CH3), 1.69 (s, 3H,
CH3), 1.59–1.56 (m, 2H, H-5,6); 13C-NMR (CDCl3): δ 171.9, 144.3, 116.7, 78.8, 73.3 47.6, 28.9, 26.1,
18.2; LC-MS (ESI+, m/z) calculated for C13H17NO4: 251.12, found for 274.05 [M+Na]+.
N-Geranyloxy-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (8). Colorless liquid, yield 56%.
1H-NMR (CDCl3): δ 5.40 (t, J = 7.6 Hz, 1H, H-2'), 5.05 (t, J = 6.0 Hz, 1H, H-6'), 4.86 (t, J = 2.6 Hz,
2H, H-1,4), 4.63 (d, J = 7.6 Hz, 2H, H-1'), 2.83 (s, 2H, H-2,3), 2.11–2.02 (m, 4H, H-5,6, H-4'),
1.90–1.86 (m, 2H, H-5'), 1.72 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.59 (s, 3H, CH3), 1.6–1.58 (m, 2H,
H-5,6); 13C-NMR (CDCl3): δ 171.9, 147.5, 132.1, 123.9, 116.4, 78.8, 73.3, 47.6, 39.9, 29.0, 26.4, 25.9,
17.9, 16.8; LC-MS (ESI+, m/z) calculated for C18H25NO4: 319.18, found for 342.13 [M+Na]+.
1
N-Farnesyloxy-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (9). Colorless liquid, yield 28%. H-
NMR (CDCl3): δ 5.42 (td, J = 0.9, 7.7 Hz, 1H, H-2'), 5.11–5.07 (m, 2H, H-6',10'), 4.85 (q, J = 2.4 Hz,
2H, H-1,4), 4.62 (d, J = 7.7 Hz, 2H, H-1'), 2.83 (s, 2H, H-2,3), 2.09–2.04 (m, 6H, H-5,6, H-4',8'),
1.99–1.96 (m, 2H, H-5'), 1.89–1.86 (m, 2H, H-6'), 1.72 (d, J = 0.7 Hz, 3H, CH3), 1.68 (s, 3H, CH3),
13
1.63–1.62 (m, 2H, H-5,6), 1.60 (s, 3H, CH3), 1.59 (s, 3H, CH3); C-NMR (CDCl3): δ 171.6, 147.3,
135.5, 131.3, 124.3, 123.5, 116.1, 78.5, 73.0, 47.4, 39.65, 39.63, 28.7, 26.7, 26.1, 25.7, 17.7, 16.5,
16.0; LC-MS (ESI+, m/z) calculated for C23H33NO4: 387.24, found for 410.21 [M+Na]+.
N-Butyloxy-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (10). Colorless solid, yield 24%, mp
113–114 °C. 1H-NMR (CDCl3): δ 4.86 (dd, J = 2.3, 2.4 Hz, 2H, H-1,4), 4.04 (t, J = 6.8 Hz, 2H, H-1'),
2.82 (s, 2H, H-2,3), 1.87–1.85 (m, 2H, H-5,6), 1.67 (p, J = 6.8 Hz, 2H, H-2'), 1.64–1.57 (m, 2H, H-
13
5,6), 1.43 (hexa, J = 7.4 Hz, 2H, H-3'), 0.92 (t, J = 7.4 Hz, 3H, H-4'); C-NMR (CDCl3): δ 171.4,
78.7, 77.2, 47.3, 29.9, 28.7, 18.7, 13.7; LC-MS (ESI+, m/z) calculated for C12H17NO4: 239.12, found
for 240.24 [M+H]+.
N-Octyloxy-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (11). Colorless solid, yield 22%, mp 96–97 °C.
1H-NMR (CDCl3): δ 4.89 (dd, J = 2.3, 2.4 Hz, 2H, H-1,4), 4.05 (t, J = 6.9 Hz, 2H, H-1'), 2.85 (s, 2H,
H-2,3), 1.91–1.87 (m, 2H, H-5,6), 1.69 (p, J = 6.9 Hz, 2H, H-2'), 1.64–1.60 (m, 2H, H-5,6), 1.43–1.38
(m, 2H, H-3'), 1.32–1.26 (m, 8H, H-4'-7'), 0.88 (t, J = 6.8 Hz, 3H, H-12'); 13C-NMR (CDCl3): δ 171.4,
78.7, 77.6, 47.3, 31.7, 29.2, 29.1, 28.7, 27.9, 25.4, 22.6, 14.1; LC-MS (ESI+, m/z) calculated for
C16H25NO4: 295.18, found for 296.24 [M+H]+.
N-Dodecyloxy-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (12). Colorless solid, yield 40%, mp
1
90–91 °C. H-NMR (CDCl3): δ 4.89 (dd, J = 2.3, 2.4 Hz, 2H, H-1,4), 4.05 (t, J = 6.9 Hz, 2H, H-1'),
2.85 (s, 2H, H-2,3), 1.91–1.87 (m, 2H, H-5,6), 1.73–1.67 (m, 2H, H-2'), 1.64–1.60 (m, 2H, H-5,6),
13
1.44–1.38 (m, 2H, H-3'), 1.30–1.26 (m, 16H, H-4'-11'), 0.88 (t, J = 6.8 Hz, 3H, H-12'); C-NMR
(CDCl3): δ 171.4, 78.7, 77.6, 47.3, 31.9, 29.6, 29.5, 29.4, 29.32, 29.26, 28.7, 27.9, 25.4, 22.7, 14.1;
LC-MS (ESI+, m/z) calculated for C20H33NO4: 351.24, found for 374.26 [M+Na]+.

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7-Oxabicyclo[2.2.1]-5-heptene-2,3-dicarboximide (14). A solution of maleimide (13) (1.5 g, 15 mmol)
in dry toluene (30 mL) was heated at 80 °C, then furan (5.4 mL, 75 mmol) was added and stirred at
80 °C for 6 h, cooled at room temperature and white precipitated was collected and purified by column
chromatography using silica gel (1:3 ethyl acetate/n-hexane as eluent) to give 14 (2.1 g, yield: 87%) as
1
a colorless crystals, mp 162–163 °C. H-NMR (CDCl3): δ 8.13 (s, 1H, NH), 6.50 (s, 2H, H-5,6), 5.29
(s, 2H, H-1,4), 2.87 (s, 2H, H-2,3); 13C-NMR (CDCl3): δ 176.2, 136.8, 81.2, 48.9; LC-MS (ESI‒, m/z)
calculated for C8H7NO3: 165.04, found for 164.02 [M–H]‒.
7-Oxabicyclo[2.2.1]heptane-2,3-dicarboximide (15). To a solution of 14 (0.5 g, 2.8 mmol) in THF
(20 mL) was added 10% Pd/C (50 mg), and the mixture was stirred at room temperature under a
hydrogen atmosphere for 4 h. The reaction mixture was filtered through Celite 545® and concentrated
in vacuo to give 15 (424 mg, yield: 84%) as a colorless crystals, mp 188–190 °C. 1H-NMR (CDCl3): δ
8.60 (s, 1H, NH), 4.91 (dd, J = 2.4, 3.3 Hz, 2H, H-1,4), 2.92 (s, 2H, H-2,3), 1.89–1.85 (m, 2H, H-5,6),
1.61–1.57 (m, 2H, H-5,6); 13C-NMR (CDCl3): δ 177.2, 79.1, 51.3, 28.5; LC-MS (ESI‒, m/z) calculated
for C8H9NO3: 167.06, found for 166.03 [M–H]‒.
3.4. General Procedure for Synthesis of Target Compounds 16–24
A mixture of 14 or 15 (1.0 mmol) in dry acetone (25 mL) was added the appropriate alkyl or
terpenyl bromide (1.0 mmol) and K2CO3 (3 mmol), and the reaction mixture was refluxed for
8–10 h. Then, the reaction mixture was filtered and concentrated in vacuo, and the residue was purified
by column chromatography using silica gel with ethyl acetate/n-hexane as eluent to afford the
desired compounds.
N-Isoprenyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (16). White solid, yield 36%, mp 87–88 °C.
1H-NMR (CDCl3): δ 5.09 (t, J = 7.1 Hz, 1H, H-2'), 4.85 (dd, J = 2.3, 3.0 Hz, 2H, H-1,4), 4.02 (d,
J = 7.1 Hz, 2H, H-1'), 2.83 (s, 2H, H-2,3), 1.85–1.81 (m, 2H, H-5,6), 1.72 (s, 3H, CH3), 1.67 (s, 3H,
CH3), 1.59–1.55 (m, 2H, H-5,6); 13C-NMR (CDCl3): δ 176.9, 137.4, 117.4, 79.0, 50.0, 37.0, 28.6, 25.6,
17.9; LC-MS (ESI+, m/z) calculated for C13H17NO3: 235.12, found for 235.12 [M+Na]+.
N-Geranyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (17). White solid, yield 24%, mp 58–59 °C.
1H-NMR (CDCl3): δ 5.09 (t, J = 7.0 Hz, 1H, H-2'), 5.03 (t, J = 6.8 Hz, 1H, H-6'), 4.84 (t, J = 2.3 Hz,
2H, H-1,4), 4.03 (d, J = 7.0 Hz, 2H, H-1'), 2.83 (s, 2H, H-2,3), 2.05–2.00 (m, 2H, H-5'), 1.97–1.94 (m,
2H, H-4'), 1.84–1.82 (m, 2H, H-5,6), 1.72 (s, 3H, CH3), 1.64 (s, 3H, CH3), 1.59–1.55 (m, 2H, H-5,6),
1.56 (s, 3H, CH3); 13C-NMR (CDCl3): δ 177.0, 141.1, 131.9, 124.0, 117.5, 79.2, 50.2, 39.7, 37.2, 28.8,
26.5, 25.9, 17.9, 16.6; LC-MS (ESI+, m/z) calculated for C18H25NO3: 303.18, found for 326.15 [M+Na]+.
N-Farnesyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (18). Colorless liquid, yield 20%. 1H-NMR
(CDCl3): δ 5.10–5.02 (m, 3H, H-2',6',10'), 4.84 (dd, J = 2.2, 2.3 Hz, 2H, H-1,4), 4.03 (d, J = 7.0 Hz,
2H, H-1'), 2.82 (s, 2H, H-2,3), 2.06–2.01 (m, 4H, H-4',8'), 1.97–1.92 (m, 4H, H-5',9'), 1.84–1.81 (m,
2H, H-5,6), 1.75 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.60 (s, 3H, CH3), 1.59 (s, 3H, CH3), 1.58–1.55 (m,
13
2H, H-5,6); C-NMR (CDCl3): δ 176.8, 140.9, 135.3, 131.2, 124.3, 123.7, 117.2, 78.9, 50.0, 39.7,

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39.5, 37.0, 28.6, 26.8, 26.3, 25.7, 17.7, 16.4, 16.0; LC-MS (ESI+, m/z) calculated for C23H33NO3:
371.25, found for 394.20 [M+Na]+.
1
N-Butyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (19). Colorless liquid, yield 67%. H-NMR
(CDCl3): δ 4.82 (t, J = 2.4 Hz, 2H, H-1,4), 3.42 (t, J = 7.4 Hz, 2H, H-1'), 2.81 (s, 2H, H-2,3), 1.83–1.80
(m, 2H, H-5,6), 1.58–1.54 (m, 2H, H-5,6), 1.51–1.45 (m, 2H, H-2'), 1.25 (sep, J = 7.4 Hz, 2H, H-3'),
13
0.87 (t, J = 7.4 Hz, 3H, H-4'); C-NMR (CDCl3): δ 177.3, 79.0, 49.9, 38.6, 29.6, 28.6, 19.9, 13.6;
LC-MS (ESI+, m/z) calculated for C12H17NO3: 223.12, found for 246.15 [M+Na]+.
1
N-Octyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (20). Colorless liquid, yield 52%. H-NMR
(CDCl3): δ 4.87 (dd, J = 2.3, 2.4 Hz, 2H, H-1,4), 3.45 (t, J = 7.5 Hz, 2H, H-1'), 2.85 (s, 2H, H-2,3),
1.87–1.84 (m, 2H, H-5,6), 1.62–1.60 (m, 2H, H-5,6), 1.55–1.52 (m, 2H, H-2'), 1.27–1.25 (m, 10H,
H-3'–7'), 0.87 (t, J = 7.0 Hz, 3H, H-8'); 13C-NMR (CDCl3): δ 177.2, 79.0, 49.9, 39.1, 31.7, 29.1, 29.0,
28.6, 27.6, 26.7, 22.6, 14.1; LC-MS (ESI+, m/z) calculated for C18H25NO3: 303.18, found for
302.20 [M‒H]+.
1
N-Dodecyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (21). Colorless liquid, yield 77%. H-NMR
(CDCl3): δ 4.86 (dd, J = 2.3, 3.0 Hz, 2H, H-1,4), 3.45 (t, J = 7.5 Hz, 2H, H-1'), 2.85 (s, 2H, H-2,3),
1.87–1.83 (m, 2H, H-5,6), 1.62–1.58 (m, 2H, H-5,6), 1.53 (p, J = 6.8 Hz, 2H, H-2'), 1.24 (m, 18H,
13
H-3'–11'), 0.88 (t, J = 6.9 Hz, 3H, H-12'); C-NMR (CDCl3): δ 177.2, 79.0, 49.8, 39.1, 31.7, 29.6,
29.5, 29.4, 29.3, 29.1, 28.6, 27.5, 26.6, 22.6, 14.1; LC-MS (ESI+, m/z) calculated for C20H33NO3:
335.25, found for 358.36 [M+Na]+.
N-Isoprenyl-7-oxabicyclo[2.2.1]-5-heptene-2,3-dicarboximide (22) White solid, yield 93%, mp
1
111–112 °C. H-NMR (CDCl3): δ 6.48 (s, 2H, H-5,6), 5.24 (s, 2H, H-1,4), 5.10 (td, J = 1.1, 7.0 Hz,
1H, H-2'), 4.05 (d, J = 7.0 Hz, 2H, H-1'), 2.80 (s, 2H, H-2,3), 1.73 (s, 3H, CH3), 1.67 (s, 3H, CH3);
13C-NMR (CDCl3): δ 176.1, 137.6, 136.8, 117.6, 81.1, 47.7, 37.2, 25.8, 18.2; LC-MS (ESI+, m/z)
calculated for C13H15NO3: 233.11, found for 256.06 [M+Na]+.
N-Geranyl-7-oxabicyclo[2.2.1]-5-heptene-2,3-dicarboximide (23) White solid, yield 53%, mp 80–81 °C.
1H-NMR (CDCl3): δ 6.48 (s, 2H, H-5,6), 5.24 (s, 2H, H-1,4), 5.10 (t, J = 6.9 Hz, 1H, H-2'), 5.02 (t,
J = 6.8 Hz, 1H, H-6'), 4.04 (d, J = 6.9 Hz, 2H, H-1'), 2.80 (s, 2H, H-2,3), 2.04–2.00 (m, 2H, H-4'),
13
1.97–1.94 (m, 2H, H-5'), 1.75 (s, 3H, CH3), 1.66 (s, 3H, CH3), 1.58 (s, 3H, CH3); C-NMR (CDCl3):
δ 176.1, 141.1, 136.8, 131.9, 124.0, 117.4, 81.1, 47.7, 39.7, 37.1, 26.5, 25.9, 17.9, 16.6; LC-MS (ESI+,
m/z) calculated for C18H23NO3: 301.17, found for 324.05 [M+Na]+.
N-Farnesyl-7-oxabicyclo[2.2.1]-5-heptene-2,3-dicarboximide (24) White solid, yield 30%, mp 76–77 °C.
1H-NMR (CDCl3): δ 6.48 (s, 2H, H-5,6), 5.24 (s, 2H, H-1,4), 5.13 (dt, J = 0.8, 7.0 Hz, 1H, H-2'),
5.07–5.02 (m, 2H, H-6',10'), 4.04 (d, J = 7.0 Hz, 2H, H-1'), 2.80 (s, 2H, H-2,3), 2.04–2.01 (m, 4H,
H-4',8'), 2.00–1.91 (m, 4H, H-5',9'), 1.73 (s, 3H, CH3), 1.65 (s, 3H, CH3), 1.57 (s, 3H, CH3), 1.55 (s,
13
3H, CH3); C-NMR (CDCl3): δ 176.1, 141.1, 136.8, 135.5, 131.5, 124.6, 123.9, 117.4, 81.1, 47.7,
39.9, 39.7, 37.1, 26.9, 26.5, 25.9, 17.9, 16.6, 16.2; LC-MS (ESI+, m/z) calculated for C23H31NO3:
369.23, found for 392.08 [M+Na]+.

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3.5. General Procedure for Synthesis of Target Compounds 25–27
A solution of 22–24 (0.86 mmol) in THF (15 mL) was added 10% Pd/C (10 mg), and the mixture
was stirred at room temperature under a hydrogen atmosphere for 48 h. The reaction mixture was
filtered through Celite 545® and concentrated in vacuo and purified by column chromatography using
silica gel with ethyl acetate/n-hexane as eluent to afford the desired compounds.
N-3′-Methylbutyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (25). White solid, yield 86%, mp
1
52–53 °C. H-NMR (CDCl3): δ 4.87 (dd, J = 2.3, 3.1 Hz, 2H, H-1,4), 3.47 (t, J = 7.7 Hz, 2H, H-1'),
2.85 (s, 2H, H-2,3), 1.84–1.81 (m, 2H, H-5,6), 1.61–1.56 (m, 2H, H-5,6), 1.54 (nonet, J = 6.6 Hz, 1H,
13
H-3'), 1.42 (dd, J = 6.6, 7.7 Hz, 2H, H-2'), 0.92 (d, J = 6.6 Hz, 6H, 2 × CH3); C-NMR (CDCl3): δ
177.4, 79.2, 50.1, 37.8, 36.5, 28.8, 26.1, 22.5; LC-MS (ESI+, m/z) calculated for C13H19NO3: 237.14,
found for 260.17 [M+Na]+.
N-3′,7′-Dimethyloctyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (26). Colorless liquid, yield
1
78%. H-NMR (CDCl3): δ 4.87 (dd, J = 2.2, 3.2 Hz, 2H, H-1,4), 3.47 (td, J = 2.1, 6.2 Hz, 2H, H-1'),
2.85 (s, 2H, H-2,3), 1.86–18.4 (m, 2H, H-5,6), 1.61–1.56 (m, 2H, H-5,6), 1.56–1.54 (m, 1H, H-3'),
1.53–1.46 (m, 1H, H-7'), 1.42–1.07 (m, 8H, H-2',4',5',6'), 0.90 (d, J = 6.5 Hz, 3H, CH3), 0.86 (dd, J =
6.6 Hz, 6H, 2 × CH3); 13C-NMR (CDCl3): δ 177.4, 79.2, 50.1, 39.4, 37.6, 37.1, 34.7, 30.9, 28.8, 28.1,
24.7, 22.9, 22.8, 19.5; LC-MS (ESI+, m/z) calculated for C18H29NO3: 307.21, found for 330.19 [M+Na]+.
N-3′,7′,11′-Trimethyldodecyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide (27). Colorless liquid,
yield 25%. 1H-NMR (CDCl3): δ 4.87 (dd, J = 2.2, 3.0 Hz, 2H, H-1,4), 3.49–3.45 (m, 2H, H-1'), 2.85 (s,
2H, H-2,3), 1.86–1.84 (m, 2H, H-5,6), 1.61–1.56 (m, 2H, H-5,6), 1.56–1.47 (m, 3H, H-3',7',11'),
1.40–1.00 (m, 14H, H-2',4',5',6',8',9',10'), 0.91 (d, J = 6.5 Hz, 3H, CH3), 0.86 (d, J = 6.6 Hz, 6H, 2 ×
13
CH3), 0.83 (dd, J = 0.9, 6.6 Hz, 3H, CH3); C-NMR (CDCl3): δ 177.2, 79.0, 49.9, 39.5, 37.4, 37.4,
37.3, 37.0, 34.5, 32.7, 30.7, 28.6, 28.0, 24.8, 24.2, 22.7, 22.6, 19.7, 19.3; LC-MS (ESI+, m/z) calculated
for C23H39NO3: 377.29, found for 400.28 [M+Na]+.
3.6. In Vitro Pharmacology
3.6.1. Cell Culture and Stock Solutions
Stock solutions were prepared as follows and stored at −20 °C: norcantharidin (2) as a 30 mM
solution in dimethylsulfoxide (DMSO) and norcantharimide derivatives as 20 mM solution in DMSO.
All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan,
UT, USA) and RPMI 1640 (HyClone, USA), supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and 1% penicillin/streptomycin in CO2 incubator with a humidified atmosphere of 95%
air and 5% CO2 at 37 °C.
3.6.2. Cell Cytotoxicity Assay Using MTT Assay
The cytotoxic activities of compounds were evaluated using HepG2 (liver carcinoma), BFTC905
(bladder carcinoma), HT-29 (colon carcinoma), SW480 (colon carcinoma), and HL-60 (leukemia). The

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cytotoxic activities were assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) assay [41]. HepG2, BFTC905, HT-29, SW480, and HL-60 cell lines were cultured at 37 °C
under 5% CO2 in air and were maintained in DMEM medium or RPMI 1640 medium, supplemented
with 10% FBS, 10 mM sodium bicarbonate, penicillin (100 IU/mL), streptomycin (100 mg/mL) and
glutamine (4 mM). The compounds were dissolved in DMSO as 20 mM stock solutions and diluted
with culture medium used before. The final concentration of DMSO in the medium was less than 0.2%
and it showed no interference with the biological activities tested. After the cells were seeded for 24 h,
the compounds were added and incubated for 48 h. Then 10 μL MTT (5 mg/mL, dissolved in culture
medium) was added to each cell and incubated for 1 h (37 °C). The inhibition rate was calculated. The
errors were quoted as standard deviations and three replicates were used in the calculation of these
errors. Briefly, 2 × 104 cells/well were seeded in 96-well plate; samples were then added to the wells at
different concentrations keeping untreated and vehicle treated wells as controls. After 48 h incubation,
10 μL of MTT reagent (5 mg/mL) was added to the wells to facilitate reaction. Formazan crystals
produced during the reaction were solubilized and the absorbance at 540 nm was measured with a
microplate reader (Spectramax 340PC384, Molecular Devices, Orleans, CA, USA). The IC50 values
were defined as the drug concentration that inhibits 50% cell growth by setting the viability of untreated
cell as 100%. Values were represented as the mean ± SD from at least three independent experiments.
3.6.3. Morphological Observations of Nuclear Change with Hoechst 33,258 Staining
Staining with Hoechst 33258 was performed according to the method described previously [27,41].
HepG2 cells were treated with 40 μM norcantharidin (2), compounds 9 or 18 for 48 h. The cells were
washed with phosphate-buffered saline (PBS) and stained with Hoechst 33258 (Sigma, St. Louis,
MO, USA) at a final concentration of 10 μg/mL. The slides were examined under a fluorescence
microscope. Cells with a small nucleus, a high fluorescence intensity (due to chromatin condensation),
or nuclear fragmentation were considered apoptotic.
3.6.4. Flow Cytometry Analysis
Flow cytometry was used to obtain the cell cycle distribution and the apoptotic rate.To determine
the effect of norcantharidin (2), compounds 9 or 18 on the cell cycle, parasites (1 × 106 cells) were
treated with norcantharidin (2), compounds 9 and 18 (10–60 μM) for 48 h. The cells were fixed in
chilled 70% ethanol and kept at −20 °C until analysis. After the cells were washed in PBS, the
resultant pellet was resuspended in 500 μL DNase (200 μg/mL) and incubated for 1.5 h at 37 °C. The
cells were then stained with PI (40 μg/mL) and incubated in the dark for 20 min at 20–25 °C. Data
acquisition was carried out using FACS scan and analyzed using the CellQuest pro software.
The percentage of apoptotic cells was determined according to the manufacture’s protocol by using
an annexin-V/FITC kit/propidium iodide (PI) flow cytometer [27,42]. To facilitate the detection of
apoptosis, the treated cells were centrifuged for 5 min, under 1000 g, at room temperature (18–24 °C),
and then resuspended and washed once with 5 mL phosphate-buffered saline before being stained with
annexin-V/PI (apoptosis detection kit; R&D Systems, Taipei, Taiwan).

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4. Conclusions
In summary, the lipophilicity of N-substituted norcantharimide derivatives plays a crucial role in
their bioactivity. Thus, through optimization of the type and chain length of the N-substituent group we
might be able to maximize the cytotoxicity of norcantharimide. We also found that compounds 9,
N-farnesyloxy-7-oxabicyclo[2.2.1]heptane-2,3-dicarboximide, and 18, N-farnesyl-7-oxabicyclo[2.2.1]-
heptane-2,3-dicarboximide, in our study had the highest cytotoxicity, anti-proliferative and apoptotic
effects, among the twenty three samples studied, against HepG2 human hepatoma cell lines, without
cytotoxic effect on murine embryonic liver BNL CL.2 cells. Should the same performance trends
established here reproduced in clinic testing, compounds 9 and/or 18 could be promising candidates for
anticancer drug development.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/6/6911/s1.
Acknowledgments
Gratitude is intended to Christopher Liu for his valuable editorial revision. This study was supported
by grants V101E2-001 and V101E2-005 from Taipei Veterans General Hospital, Taipei, Taiwan.
Author Contributions
Yu-Jen Chen and Hui-Fen Liao suggested the research work and discussed the experimental data;
Chien-Yu Chu, Min-Shin Chen and Jia-Hua Lin finalized the experimental work, interpreted the
results and prepared figures; Jin-Yi Wu and Cheng-Deng Kuo wrote the paper, edited and revised
manuscript. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2, 5–27 are available from the authors.
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