Therapeutic effects of cantharidin analogues without bridging ether oxygen on human hepatocellular carcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2010.05.053

Previous research indicates that cantharidin, norcantharidin and their analogues exhibit anticancer activity due to their inhibition of cancer cell lines such as HL60, HT29 and L1210. The anticancer activities of cantharidin, norcantharidin and their analogues involve the suppression of serine/threonine protein phosphatases (PPs) activity. However, cantharidin is not suitable for cancer therapy because of its high cytotoxicity in vitro (IC₅₀ = 21 μM in primary cultured rat hepatocytes). In this study, synthetic cantharidin analogues with a structure of aminothiazole compounds 3-9 and a structure of anhydride compounds 10-12 were screened for anticancer activities and cytotoxic effects on human hepatocellular carcinoma cell (HCC) lines HepG2, Sk-Hep1, and primary cultured rat hepatocytes. Experimental results indicated that compounds 3-9 did not perform as expected with regard to anticancer activity and exhibited lower cytotoxicity. Compound 10 promoted apoptosis in HepG2 (IC₅₀ = 62 μM) and SK-Hep1(IC₅₀ = 151 μM) cell lines. Compounds 11 and 12 had anticancer potential similar to that of compound 10. After treatment with compounds 3-12, primary cultured rat hepatocytes exhibited no cytotoxicity (IC₅₀ > 200 μM). By investigating the structure-activity relationship (SAR) of these analogues as a whole, this study suggests that the anhydride ether oxygen such as in cantharidin, norcantharidin and compounds 10-12 may be correlated with HCC survival suppression. The results further suggest that the elimination of bridging ether oxygen on the ring, such as in compounds 10-12, can decrease cytotoxicity. Elimination of bridging ether oxygen on the ring, such as in compound 10, can decrease their cytotoxicity. Anhydride ether oxygen is crucial for inducing HCC cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2010.05.053

European Journal of Medicinal Chemistry 45 (2010) 3981e3985
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Therapeutic effects of cantharidin analogues without bridging ether oxygen
on human hepatocellular carcinoma cells
,
b
,
,b,e,
Chao-Bin Yeh a d, Chi-Jung Su c, Jin-Ming Hwang c, Ming-Chih Chou a
*
a Institute of Medicine, Chung Shan Medical University, 110 Section 1, Chien-Kuo N. Road, South District, Taichung 402, Taiwan, ROC
b School of Medicine, Chung Shan Medical University, 110 Section 1, Chien-Kuo N. Road, South District, Taichung 402, Taiwan, ROC
c School of Apply Chemical, Chung Shan Medical University, 110 Section 1, Chien-Kuo N. Road, South District, Taichung 402, Taiwan, ROC
d Department of Emergency Medicine, Chung Shan Medical University Hospital, 110 Section 1, Chien-Kuo N. Road, South District, Taichung 402, Taiwan, ROC
e Department of Surgery, Chung Shan Medical University Hospital, 110 Section 1, Chien-Kuo N. Road, South District, Taichung 402, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous research indicates that cantharidin, norcantharidin and their analogues exhibit anticancer
activity due to their inhibition of cancer cell lines such as HL60, HT29 and L1210. The anticancer activities
of cantharidin, norcantharidin and their analogues involve the suppression of serine/threonine protein
phosphatases (PPs) activity. However, cantharidin is not suitable for cancer therapy because of its high
Received 24 December 2009
Received in revised form
21 May 2010
Accepted 22 May 2010
Available online 1 June 2010
cytotoxicity in vitro (IC50 ¼ 21
mM in primary cultured rat hepatocytes). In this study, synthetic
cantharidin analogues with a structure of aminothiazole compounds 3e9 and a structure of anhydride
compounds 10e12 were screened for anticancer activities and cytotoxic effects on human hepatocellular
carcinoma cell (HCC) lines HepG2, Sk-Hep1, and primary cultured rat hepatocytes. Experimental results
indicated that compounds 3e9 did not perform as expected with regard to anticancer activity and
Keywords:
Cantharidin anhydride analogues
Aminothiazole analogues
Hepatocellular carcinoma cell
exhibited lower cytotoxicity. Compound 10 promoted apoptosis in HepG2 (IC50 ¼ 62
(IC50 ¼ 151 M) cell lines. Compounds 11 and 12 had anticancer potential similar to that of compound 10.
After treatment with compounds 3e12, primary cultured rat hepatocytes exhibited no cytotoxicity
(IC50 > 200 M). By investigating the structureeactivity relationship (SAR) of these analogues as a whole,
mM) and SK-Hep1
m
m
this study suggests that the anhydride ether oxygen such as in cantharidin, norcantharidin and
compounds 10e12 may be correlated with HCC survival suppression. The results further suggest that the
elimination of bridging ether oxygen on the ring, such as in compounds 10e12, can decrease cytotoxicity.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
purposes. The detailed mechanism underlying tumor suppression
of PP2A remains unknown due to its involvement in diverse
Cantharidin (compound 1) is a terpenoid isolated from Chinese
blister beetles that exhibits significant toxicity against cancer cells
[1]. Its pharmacological action includes the inhibition of xanthine
oxides [2]. However, cantharidin has limited anticancer therapeutic
potential because of its high mammalian toxicity [3]. Previous
research shows that cantharidin (compound 1) and norcantharidin
(compound 2) are potent serine/threonine protein phosphatases 1
and 2A (PP1 and PP2A) inhibitors [4e6]. Both of these terpenoids
have a slight inhibitory effect on the activity of PP2B, which is
involved in intracellular signal transductions [7] and plays a major
role in cell cycle progression. PP2A suppresses tumor growth [8]
and is often used as a target molecule for tumor suppression
signaling pathways [9]. Regarding the binding mode, the structure
activity relationship (SAR) of cantharidin analogues exhibits
selective inhibitory activity on serine/threonine protein phospha-
tases [7]. The SAR of cantharidin analogues with inhibitory activity
identifies bridging oxygen on the ring and anhydride and dicar-
boxylate structures as critical function groups [10]. In addition, the
substitution of sulfur for oxygen in anhydride modified cantharidin
analogues enhances the inhibition of PP2A, as the bridging oxygen
on the ring structure creates a hydrogen-bonding site for PP2A [11].
Other studies consider PP2A to be a tumor suppressor protein [7].
A recent report shows that the antibiotic thiazole not only
targets FoxM1 and efficiently inhibits its growth, but also induces
apoptosis in human cancer cell lines A549 and SW480 [12]. FoxM1
inhibitors are potential anticancer drugs [13]. It is theoretically
possible to synthesize thiazole as a functional group for anticancer
activity with anhydrides and aminothiazole analogues 3e9. These
chemically synthesized compounds are comparable to cantharidin
* Corresponding author. Chung Shan Medical University, 110 Section 1, Chien-Kuo
N. Road, South District, Taichung 402, Taiwan, ROC. Tel.: þ886 4 24739595x11602;
fax: þ886 4 24731521.
E-mail address: csmuchou@gmail.com (M.-C. Chou).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.053

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C.-B. Yeh et al. / European Journal of Medicinal Chemistry 45 (2010) 3981e3985
Table 1
derivatives. In an attempt to decrease the toxicity of cantharidin,
we synthesized three derivatives without bridging oxygen on the
ring in this study. These derivatives were synthesized according to
previously described methods and investigated for their influence
on the protein phosphatase family and apoptosis in human
hepatocellular carcinoma cells. We also examined cantharidin, cis-
5-norbornene-endo-2,3-dicarboxylic anhydride (compound 10),
4,40-oxydiphthalic anhydride (compound 11) and 5-(5-methyl-
thiazol-2-ylimino)-4-oxa-tricyclo [5.2.2.02,6] undec-8-en-3-one
(compound 12) for cellular cytotoxicity in HepG2, SK-Hep1 and
primary cultured rat hepatocytes [14]. The study aimed to identify
compounds that decrease cellular cytotoxicity in normal rat
hepatocytes and increase apoptotic activity in human hepatocel-
lular carcinoma cells.
The cytotoxicity of all tested compounds 1e12 in terms of their IC50 (mM) for
different HCC cell lines or rat hepatocytes.
Compound
HepG2
SK-Hep1
Rat hepatocyte
1
2
3
4
5
6
7
8
11
74
34
126
21
75
>200
>200
>200
>200
>200
>200
>200
62
>200
>200
>200
>200
>200
>200
>200
151
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
9
10
11
12
121
92
199
>200
O
O
O
O
O
S
O
O
O
N
N
2
1
3
O
O
O
O
S
N
S
N
N
N
O
4
5
O
O
O
S
N
S
N
N
N
6
O
7
O
O
O
S
N
S
N
N
O
N
O
8
O
9
O
O
10
O
O
O
O
O
O
O
S
N
12
N
11
O
O
Fig. 1. Structures of cantharidin (compound 1), norcantharidin (compound 2), and compounds 3e12.

C.-B. Yeh et al. / European Journal of Medicinal Chemistry 45 (2010) 3981e3985
3983
2. Results and discussion
IC50 values were calculated (Table 1) for all 12 synthetic
compounds (Fig.1) in SK-Hep1, HepG2, and primary rat hepatocytes.
Compounds 1, 2, 10, 11, and 12 had the lowest IC50 values in the HCC
cell lines. Compounds 10, 11, and 12 were chemically modified after
48 h of exposure to anhydride and exerted cytotoxicity [15] in
a dose-dependent manner in the HCC, SK-Hep1, and HepG2 cell
lines, but not in the primary cultured rat hepatocytes (Fig. 2). The
primary rat hepatocytes and HCC cell lines treated with compounds
1 and 2, induced cytotoxicity at low concentrations (10e25
However, the incubation of primary rat hepatocytes with
compounds 10, 11, or 12 at a concentration of 200 M for 48 h
mM).
m
resulted in a nearly 80% survival rate. Fig. 2 shows the viability curve
of these chemicals in the HepG2, SK-Hep1, and primary hepatocytes.
The influence of compounds 1, 2, 10, 11, and 12, at a concentra-
tion of 50 mM for 48 h, on rat hepatocytes, HepG2 and SK-Hep1 cells
was determined by examining morphological changes by light
microscopy. DAPI staining [16] showed that treated HCC cell lines
had chromatin condensation and nucleolus condensation, whereas
the chromatin in the nuclei of untreated cells was homogeneously
distributed. These results suggest that cancer cells create an
apoptotic pathway in the presence of cantharidin analogues.
Nucleolar condensation appeared in both cancer cell lines and
primary cell cultures after treatment with cantharidin (50
norcantharidin (50 M). Interestingly, chromatin was homoge-
neously distributed within the nuclei of rat hepatocytes treated
with compounds 10, 11, or 12 at a concentration of 50 M for 48 h
mM) or
m
m
implying that these analogues are toxic to cancer cells, but not to
primary rat hepatocytes.
Effective anti-tumor agents such as 5-fluorouracil (5-FU) often
induce neutropenia, the most common side effect, followed by
gastrointestinal toxicity [17], especiallyat the high dosage necessary
to achieve anti-tumor efficacy. A series of cantharidin analogues
were synthesized and screened for their anticancer potential in an
attempt to achieve relatively low toxicity to normal cells [18,19].
This study hypothesizes that the chemical structure of thiazole
can be modified into cantharidin. This approach effectively
enhances the apoptosis of cancer cells using a thiazole functional
group. The IC50 values of compounds 3e9 initially seemed to show
a decrease in anticancer activity and exhibited low cytotoxicity in
primary rat hepatocytes. Motivated by these results, we redesigned
compounds 10e12 using an anhydride structure. These compounds
were easily synthesized by a solvothermal method.
The effects of the cantharidin analogues 10e12 were evaluated
usingHepG2andSK-Hep1andthencomparedwithprimarycultured
rat hepatocytes as the normal cells. After 48 h, cell survival curves
showed that compounds 10 (IC50 ¼ 253
12 (IC50 ¼ 262 M) generated low cytotoxicity in rat hepatocytes
whereas rat hepatocytes treated with cantharidin (IC50 ¼ 21 M) and
norcantharidin (IC50 ¼ 75 M) manifested the highest cytotoxicity.
mM),11 (IC50 ¼ 407
mM), and
m
m
m
Previous research indicates that removing the methyl group
from cantharidin to form norcantharidin decreases irritation and
toxicity [3]. This study shows that norcantharidin exhibited less
cellular toxicity than cantharidin (Fig. 2A), which is consistent with
the previous study. Nevertheless, norcantharidin remained toxic to
primary cultured rat hepatocytes. Compounds 1, 2, 10, 11, and 12
had different degrees of cytotoxicity on the HepG2 cell line (Fig. 2B),
suggesting that these analogues modulate apoptosis. A possible
explanation for this is that the selective suppression of protein
phosphatases causes treated liver cancer cells to undergo apoptosis
via different pathways. Treating SK-Hep1 with cantharidin
analogues including compounds 1, 2, 10, 11, and 12 at a concentra-
Fig. 2. Cell cytotoxicity assay of cantharidin (1), norcantharidin (2), and compounds 10,
11, and 12 on (A) primary cultured rat hepatocytes, (B) HepG2, and (C) SK-Hep1 cell
lines. (*P < 0.01, compared with the control).
in human hepatocellular carcinoma cells by the anhydride modified
cantharidin analogues [20]. These results shown that the bridging
ether oxygen on the ring and the anhydride structure of the
cantharidin analogues are both crucial for the inhibitory activity on
protein phosphatases [9]. Even though cantharidin analogues are
PP1, PP2A, and PP2B inhibitors, an anhydride or dicarboxylate
structure is crucial for inhibitory activity [10]. Cantharidin is
a potent PP1 and PP2A inhibitor, but not an efficient PP2B inhibitor.
tion of 25
mM for 48 h resulted in cytotoxicity (Fig. 2C). DAPI
staining and fluorescence microscopy detected induced apoptosis

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C.-B. Yeh et al. / European Journal of Medicinal Chemistry 45 (2010) 3981e3985
anhydride, and 4,40-oxydiphthalic anhydride (compounds 2,10, and 11
respectively) were purchased from SigmaeAldrich (USA) and were
used without further purification. Endo-bicyclo[2,2,2]oct-5-ene-2,
3-dicarboxylic anhydride and 2-amino-5-methylthiazole were
purchased from Acros (USA). Toluene and triethylamine were freshly
distilled from sodium under nitrogen. 5-(5-methyl-thiazol-2-ylimino)-
4-oxa-tricyclo [5.2.2.02,6] undec-8-en-3-one (compound 12) and
compounds 3e9 were synthesized by heating various anhydrides
and aminothiazole derivatives to 200 ꢀC with 3 mL of dry toluene and
1e2 mL of triethylamine in a high-pressure sealed tube (Büchi
glasuster 0032) to obtain a good yield of phenylphthalimide derivatives
(Scheme 1).
Scheme 1. Compounds 3e9 were synthesized in a high-pressure sealed tube.
Although PP2A might be a tumor suppressor, it is difficult to
manipulate with pharmaceutical agents because it acts via various
signaling pathways [21].
4.2. General procedure for preparing imides
Cantharidin and norcantharidin are promising PP1, PP2A, and
PP2B selective inhibitors, and induce serious cytotoxicity in
primary cultured rat hepatocytes. We modified compounds 3e9 for
examination. To confirm that the anhydride structure is a critical
structure for selective anticancer activity, IC50 values were used to
compare synthetic compounds 3e9, which have a thiazole struc-
ture, with compounds 10e12, which have an anhydride structure.
Table 1 shows that compounds 10e12 were cytotoxic to HCC, while
compounds 3e9 were not. Compounds 3, 8, and 12 were screened
with the same thiazole functional group, but only compound 12
inhibited the growth of HepG2 (Fig. 2B) and SK-Hep1 (Fig. 2C) and
induced apoptosis. These results suggest that the anhydride
structure is crucial for anticancer activity.
Scheme 1 shows the synthesis of the desired imide derivatives
of anhydrides and related compounds with primary aminothiazole.
Compounds 3e9 (at 0.2 mM) were dissolved in 3 mL of dry toluene
and 1e2 mL of triethylamine (TEA), after primary benzylamine and
various aromatic amines (1.2 eq. and 1.0 eq. respectively) were
added. The mixtures were heated to ca. 200 ꢀC for 2 h and then
allowed to cool to room temperature. The mixtures were heated
a second time to remove the solvent, and the residues were purified
by silica gel column chromatography.
4.2.1. 2-(5-Methyl-thiazol-2-yl)-isoindole-1,3-dione (3)
Yield 91%; m.p. 173e175 ꢀC. 1H NMR (CDCl3):
d 2.26 (3H, s, CH3),
When observing the cytotoxicity of cantharidin and norcan-
tharidin in primary cultured rat hepatocytes, the bridging ether
oxygen differed between compounds 3e12, but none of these
compounds were cytotoxic in primary hepatocytes using IC50
values for comparison (Table 1). This indicates that bridging ether
oxygen on the ring of cantharidin analogues is a critical factor in
inducing or increasing cytotoxicity in primary cultured rat hepa-
tocytes. Based on the SAR of these cantharidin analogues and
aminothiazole derivates, deleting the bridging ether oxygen on the
ring decreased the hepatocyte cytotoxicity of these derivates, and
the anhydride modified structure promoted apoptosis in human
hepatocellular carcinoma cells.
7.82 (2H, d, J ¼ 6.8 Hz, phenyl H-5, 6), 7.97 (2H, d, J ¼ 6.8 Hz, phenyl
H-4, 7), 8.02 (1H,s, thiazolyl H-3) 13C NMR: 11.53 (CH3), 124.36 (C-
500), 128.06 (C-4, C-7), 130.42 (C-3, C-8), 132.02 (C-5, C-6), 135.35 (C-
400),137.55 (C-200), 166.26 (C-2), 166.84 (C-9).
4.2.2. 5-Methyl-2-thiazol-2-yl-isoindole-1,3-dione (4)
Yield 90%; m.p. 137e142 ꢀC. 1H NMR (CDCl3):
d 2.42 (3H, s, CH3),
6.23 (1H, d, J ¼ 3.6 Hz, thiazolyl H-4), 6.78 (1H, d, J ¼ 3.5 Hz, thiazolyl
H-3), 7.49 (1H, d, J ¼ 6.8 Hz, phenyl H-5), 7.81 (1H, d, J ¼ 6.8 Hz,
phenyl H-7), 7.95 (1H, d, J ¼ 6.9 Hz, phenyl H-4) 13C NMR: 21.43
(CH3), 124.36 (C-500), 128.08 (C-4,C-7), 130.82 (C-3, C-8), 132.65 (C-5,
C-6), 135.93 (C-400), 139.90 (C-200), 160.64 (C-2), 167.0 (C-9).
3. Conclusion
4.2.3. 2-(3-Phenyl-[1,2,4]thiadiazol-5-yl)-isoindole-1,3-dione (5)
Yield 93%; m.p. > 300 ꢀC. 1H NMR (CDCl3):
d 2.42 (3H, s, CH3),
The results of this study show that cantharidin (compound 1)
and norcantharidin (compound 2) are potent serine/threonine
protein phosphatases inhibitors due to their anhydride structure.
The anhydride modified cantharidin analogues (compounds 10 and
11) and aminothiazole derivative (compound 12) reduced toxicity
in rat hepatocytes compared to their parent compounds. These
results suggest that removing the bridging ether oxygen on the ring
can decrease their cytotoxicity and preserving the anhydride
structure may provide the necessary structure for HCC apoptosis.
6.23 (1H. d, J ¼ 3.6 Hz, thiazolyl H-4), 6.78 (1H, d, J ¼ 3.5 Hz,
thiazolyl H-3), 7.45 (1H, d, J ¼ 6.9 Hz, phenyl H-5), 7.82 (1H, d,
J ¼ 6.9 Hz, phenyl H-7), 7.95 (1H, d, J ¼ 6.9 Hz phenyl H-4) 13C NMR:
124.36 (C-500), 128.08 (C-4, C-7), 130.82 (C-3, C-8), 132.65 (C-5, C-6),
135.93 (C-400), 139.90 (C-200), 160.64 (C-2), 167.0 (C-9).
4.2.4. 2-Benzothiazol-2-yl-5-methyl-isoindole-1,3-dione (6)
Yield 62%; m.p. 160e163 ꢀC.1H NMR (CDCl3):
d 1.27 (3H, s, CH3),
7.55 (1H, d, J ¼ 6.8 Hz, phenyl H-5), 7.61 (1H,d, phenyl H-20), 7.74 (1H,
d, J ¼ 6.9 Hz, phenyl H-7), 7.8 (1H, d, J ¼ 6.9 Hz, phenyl H-30), 7.81 (1H,
d, J ¼ 6.9 Hz, phenyl H-10), 7.89 (1H, d, phenyl H-40), 8.11 (1H, d,
phenyl H-4) 13C NMR: 21.28 (CH3), 121.1(C-500), 121.57 (C-800), 125.32
(C-600), 126.37 (C-700), 128.10 (C-4, C-7), 130.57 (C-900), 130.95 (C-3, C-
8), 132.78 (C-400), 136.05 (C-5,C-6), 166.42 (C-2), 166.88 (C-9).
4. Experimental
1H NMR spectra were recorded on a Bruker DRX-500 spec-
trometer in a CDCl3 solution using the corresponding solvent as the
internal reference. Compound purity was >95% determined by 1H
NMR spectral analysis.
4.2.5. 4-Benzothiazol-2-yl-4-aza-tricyclo [5.2.2.02,6]undec-8-ene-
3,5-dione (7)
4.1. Chemistry
Yield 60%; m.p. 193e195 ꢀC. 1H NMR (CDCl3):
d 1.45 (2H, d,
J ¼ 7.6 Hz, ethenyl H-8), 1.68 (2H, d, J ¼ 7.6 Hz, ethenyl H-9), 3.10
(2H, s, H-4, 7), 3.45 (2H, s, H-7a, 3a), 6.28 (2H, d, H-5, 6), 7.40 (1H, d,
phenyl H-20), 7.46 (1H, d, J ¼ 6.8 Hz, phenyl H-30), 7.84 (1H, d,
J ¼ 6.8 Hz, phenyl H-10), 8.08 (1H, d, phenyl H-40) 13C NMR: 23.54
Cantharidin was extracted from Chinese blister beetles using
previously described method [1] and was designated as
compound 1. Norcantharidin, cis-5-norbornene-endo-2,3-dicarboxylic
a

C.-B. Yeh et al. / European Journal of Medicinal Chemistry 45 (2010) 3981e3985
3985
(C-3, C-10), 32.11 (C-4, C-7), 44.16 (C-8, C-9), 121.13(C-500), 123.63(C-
800), 125.63(C-600), 126.37(C-700).
each treatment). Only primary rat hepatocytes freshly isolated with
a viability > 90% were used. Statistical analysis was performed
using one-way analysis of variance, while the Student’s t test was
applied to paired samples.
4.2.6. 4-(5-Methyl-thiazol-2-yl)-4-aza-tricyclo[5.2.2.02,6]undec-8-
ene-3,5-dione (8)
Yield 90%; m.p. 165e168 ꢀC. 1H NMR (CDCl3):
J ¼ 7.4 Hz, ethenyl H-8), 1.56 (3H, s, CH3), 1.66 (2H, d, J ¼ 7.6 Hz,
ethenyl H-9), 3.03 (2H, s, H-4, 7), 3.25 (2H, s, H-7a, 3a), 6.31 (1H, d,
H-5, 6), 7.54 (1H, s, thiazolyl H-3).
d 1.44 (2H, d,
Acknowledgments
We would like to thank Professor Jer-Yuh Liu from the Graduate
Institute of Cancer Biology, College of Medicine, China Medical
University and Professor Fang-Rong Chang from the Department of
Pharmacognosy, Graduate Institute of Natural Products, Kaohsiung
Medical University College of Pharmacy, for their consultation and
invaluable advice on this paper. This study was supported by Chung
Shan Medical University and Hospital.
4.2.7. 4-Thiazol-2-yl-4-aza-tricyclo [5.2.2.02,6] undec-8-ene-3,5-
dione (9)
Yield 88%, m.p. 244e245 ꢀC. 1H NMR (CDCl3):
d 1.35 (2H, d,
J ¼ 7.5 Hz, ethenyl H-8), 1.64 (2H, d, J ¼ 7.5 Hz, ethenyl H-9), 3.05
(2H, s, H-4, 7), 3.26 (2H, s, H-7a, 3a), 6.24 (2H, m, H-5, 6), 7.27 (1H, d,
13
thiazolyl H-4), 7.73 (1H, d, thiazolyl H-3) C NMR: 121.46(C-500),
126.73(C-6, C-8), 131.20(C-5, C-9), 131.38(C-7), 134.80 (C-4, C-10),
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(NEAA) and a 1% antibiotic mixture (50
mL of streptomycin, and 10
mg/mL of penicillin, 50 mg/
m
g/mL of neomycin/mL) at 37 ꢀC in
a humidified atmosphere of 5% CO2. The primary rat hepatocytes
were isolated from Wistar rats as previously described [14]. The
isolated hepatocytes (viability > 90%) were prepared by liver
perfusion with collagenase IV (Sigma) and separated by density
centrifugation in Percoll solution (GE Healthcare Bio-Sciences AB).
Cells were cultured in William’s E Medium (Sigma) supplemented
with 10% fetal bovine serum (Gibco), 2 mM Glutamine, 1% non
essential amino acids (NEAA) and a 1% antibiotic mixture (50
mg/mL
of penicillin, 50 g/mL of streptomycin, and 10 g/mL of neomycin/
m
m
mL) at 37 ꢀC in a humidified atmosphere of 5% CO2 and 95% air.
HepG2, SK-Hep1 cells and primary cultured rat hepatocytes were
seeded in 24-well plates or in 100-mm culture dishes. After the
cells adhered overnight, they were treated with reagents or vehicle
only (control samples) in triplicate.
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The 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay was used for measurement of cytotoxicity [16].
Primary rat hepatocytes, SK-Hep1, and HepG2 were incubated in
24-well plates at a density of 1 ꢁ 105 cells/well for 12 h and
treated with test chemicals or vehicle for 48 h. The culture
supernatants were removed and MTT (Sigma, USA) in phosphate
buffered saline (PBS; pH 7.4) was added to each well. After 4 h
of incubation at 37 ꢀC, the MTT solution was removed and
DMSO was added to dissolve the formazan crystals. The
absorbance was determined at 540 nm in a Flexstation 3 device
(MDS Analytical Technologies, Canada). The percentage of
viability was calculated using the following equation: (test-
background)/(control-background) ꢁ 100.
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a novel synthetic
4.5. Statistical analysis
The results are shown as the means ꢂ SD from at least three
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independent experiments (involving three to six observations for