Synthesis and biological evaluation of norcantharidin derivatives as protein phosphatase-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.11.032

Cantharidin and norcantharidin display anticancer activity against a broad range of tumor cell lines. In this study, we have synthesized a series of norcantharidin derivatives and evaluated their cytotoxic effects on four human tumor cell lines together with the genetically normal human diploid fibroblast line WI-38. One of our compounds (1S,4R)-3-((4-(4-(4-fluorophenyl)piperazin-1-ylsulfonyl) phenyl)carbamoyl)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (12) exhibited potent cytotoxic effects on the tumor cell lines A-549, HepG2, HeLa, and HCT-8, whereas it was less toxic to WI-38 cells than its parent compound, norcantharidin. In addition, this compound inhibited protein phosphatase-1 activity and microtubule formation in HeLa cells, and it also interacts with calf thymus DNA.

Full text of DOI:10.1016/j.bmcl.2014.11.032

Bioorganic & Medicinal Chemistry Letters 25 (2015) 363–366
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of norcantharidin derivatives as
protein phosphatase-1 inhibitors
b,c,
Jie Zhao a, Xiao-Wen Guan a, Shi-Wu Chen a, , Ling Hui
⇑
⇑
a School of Pharmacy, Lanzhou University, Lanzhou 730000, China
b Experimental Center of Medicine, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
c Key Laboratory of Stem Cells and Gene Drug of Gansu Province, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2014
Revised 27 October 2014
Accepted 10 November 2014
Available online 18 November 2014
Cantharidin and norcantharidin display anticancer activity against a broad range of tumor cell lines. In
this study, we have synthesized a series of norcantharidin derivatives and evaluated their cytotoxic
effects on four human tumor cell lines together with the genetically normal human diploid fibroblast line
WI-38. One of our compounds (1S,4R)-3-((4-(4-(4-fluorophenyl)piperazin-1-ylsulfonyl) phenyl)carbam-
oyl)-7-oxa-bicyclo[2.2.1]heptane-2-carboxylic acid (12) exhibited potent cytotoxic effects on the tumor
cell lines A-549, HepG2, HeLa, and HCT-8, whereas it was less toxic to WI-38 cells than its parent
compound, norcantharidin. In addition, this compound inhibited protein phosphatase-1 activity and
microtubule formation in HeLa cells, and it also interacts with calf thymus DNA.
Keywords:
Norcantharidin
Cytotoxicity
Protein phosphatase-1
Microtubules
Ó 2014 Elsevier Ltd. All rights reserved.
Cantharidin (exo,exo-bicyclo[2.2.1]heptane 2,3-dicarboxylic
acid anhydride) (CTD, 1) is the principle active ingredient of Epi-
canta gorhami or Mylabris (blister beetles), a traditional Chinese
medicine that has been used to treat liver, lung and digestive tract
tumors. However its applications are limited by its severe nephro-
toxic and inflammatory side effects, and it has mainly been used to
treat the gastrointestinal tract, the ureter, and the kidney.1,2 A ser-
ies of bioactive analogues of cantharidin have been synthesized in
an attempt to reduce its toxicity and thereby increase its use.3,4
Norcantharidin (NCTD, 2, Fig. 1), the demethylated analogue of
cantharidin, appears to cause the least nephrotoxic and inflamma-
tory side effects. NCTD is active in vitro against several tumor cell
lines, including cervical, hepatoma, ovarian, laryngocarcinoma,
colon, osteocarcinoma, and leukemia cell lines.1,5,6 NCTD has also
been used in vivo in the treatment of primary hepatoma, oesopha-
geal, gastric and cardiac carcinomas.1 The reported anticancer
mechanisms of NCTD include interruption of DNA replication,
retardation of cell cycle progression, and induction of apoptosis
via the regulation of p53 and Bcl-2 gene expression.7,8
activity of CTD and NCTD are thought to come from the inhibition
of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A),
two phosphatases that are known to be involved in many different
cellular processes, including DNA damage, cell cycle arrest, and
apoptosis.10–12
Recently, a series of NCTD analogues have been generated and
shown to exhibit a greater antiproliferative potency, with only a
modest PP1 inhibition, compared with that of NCTD.13–15 In this
study, which is part of our continuing effort to find new natural
product-based compounds with potent activities and minimal side
effects,16–19 we have synthesized a series of norcantharidin deriv-
atives and evaluated their cytotoxicities in vitro against four
human tumor cell lines, together with a human lung fibroblast cell
line. The most potent compound, 12, was further evaluated for its
suppression of protein phosphatase-1, its interaction with calf thy-
mus DNA (CT DNA), and its inhibition of microtubule formation.
The synthesis of compounds 6a–c and 9a–c proved to be
straightforward by following standard procedures for sulfonamide
analogues, as illustrated in Scheme 1. For example, a combination
of 4-nitro-benzenesulfonyl chloride with compounds 4a–c affor-
ded the 4-nitro-benzenesulfonamides 5a–c. These were reduced
by hydrogenation over 10% Pd/C to provide the 4-amino-benzene-
sulfonamides 6a–c, with good yields.20
Serine/threonine protein phosphatases (S/T-PPs) are thought to
be cancer suppressive, since the inhibition of the S/T-PPs can lead
to an increased phosphorylation and hence the activation of sub-
strate kinases. The activation of some of these kinases has been
associated with the acceleration of cell growth.9 The anticancer
The synthesis of compounds 11–20 was carried out according to
the procedure outlined in Scheme 2. First, the key starting material
in this work was the readily synthesizable 5,6-dehydronorcantha-
ridin 10, which was prepared on a large scale through the
⇑
Corresponding authors. Tel./fax: +86 931 8915686.
E-mail address: chenshw@lzu.edu.cn (S.-W. Chen).
http://dx.doi.org/10.1016/j.bmcl.2014.11.032
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

364
J. Zhao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 363–366
O
sation reaction to provide a series of cyclic imides 18–20 with
O
moderate yields.23 The structures of compounds 11–20 were iden-
tified by IR, 1H NMR spectra, 13C NMR spectra and high resolution
mass spectrometry (HRMS).
O
O
O
O
O
O
The in vitro cytotoxicity of the norcantharidin derivatives
11–20 were evaluated against a panel of four human cancer cell
lines (A-549, HepG2, HeLa, and HCT-8) together with the human
lung fibroblast cell line WI-38, using NCTD as a reference com-
pound. The screening procedure was based on the standard MTT
method,24 and the IC50 values are summarized in Table 1.
Generally, most of the target compounds exhibited moderated
cytotoxicities in vitro against the tumor cell lines A-549, HepG2,
HeLa, and HCT-8. As previously published results,8 the ring-opened
acid amides (11, 12 and 15) were more cytotoxic than the ring-
closed norcantharimides (18, 19 and 20). The IC50 values for com-
pound 12, which was the most promising of the ring-opened acid
2
1
Figure 1. The structures of cantharidin (1) and norcantharidin (2).
exo-selective Diels–Alder addition of furan to maleic anhy-
dride.21,22 Subsequently, NCTD was obtained from 5,6-dehydronor-
cantharidin via simple hydrogenation, as described in our previous
publication.22 Additionally, treatment of NCTD, in THF at room
temperature, with a series of substituted benzenesulfonic amides
afforded the corresponding amide–acid analogues 11–17 with
good yields.14c Simultaneously, the treatment of NCTD with an
appropriate primary amine and triethylamine facilitated a conden-
amides, were 18.4 1.5, 14.4 0.9, 6.3 1.7, and 16.6 1.2 lM
O
N
O
O
O
O
O
S
S
S
b
a
N
Cl
HN
X
X
X
NO2
NH2
NO2
6a-c
3
4a-c
5a-c
X = p-F-Ph-N
b
c
a
X = Et-N
b
X= O
O
O
O
O
O
O
S
S
S
a
HN
Cl
HN
H2N
n
NO2
NO2
NH2
n
n
3
7a-c
a
8a-c
9a-c
c
b
: n = 4
: n = 1
: n = 3
Scheme 1. Synthesis of compounds 6a–c and 9a–c. Reagents and conditions: (a) Py/CH2Cl2; (b) Pd/C (10%), H2 (50 psi), 1 h.
O
N
H
R
O
O
O
O
COOH
c
a
b
11-17
O
O
O
O
O
O
O
d
O
O
O
O
N R
2
10
O
18-20
Compds
R
Compds
R
O
S
O
O
S
O
H
N
11
12
16
17
N
O
O
S
O
O
N
N
N
F
O
S
O
S
O
O
S
O
13
18
N
N
Et
N
O
O
S
O
O
S
O
H
14
15
19
20
N
N
N
F
O
S
O
O
S
H
H
N
N
O
Scheme 2. Synthesis of compounds 11–20. Reagents and conditions: (a) Et2O, rt, 48 h; (b) H2, 10% Pd–C, rt, EtOH; (c) RNH2, THF, rt, overnight; (d) RNH2, toluene, reflux, 36 h.

J. Zhao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 363–366
365
Table 1
The cytotoxicity (IC50, l
M)a and logP of compounds 2 and 11–20
Compds
Tumor cell
A549b
Normal cell
WI-38b
LogP
HepG2b
HeLab
HCT-8b
2
11
12
13
14
15
16
17
18
19
20
12.1 0.8
24.2 2.1
18.4 1.5
28.4 1.6
34.3 1.0
20.1 3.2
36.2 1.7
>100
24.7 1.2
19.3 0.5
14.4 0.9
20.2 1.5
26.2 2.6
22.3 2.1
22.5 1.3
>100
12.4 0.6
17.2 1.5
6.3 1.7
15.7 2.8
20.9 1.5
17.6 0.9
18.7 2.2
>100
28.4 0.9
31.3 1.6
16.6 1.2
27.4 2.5
28.3 2.0
30.2 3.5
31.0 2.6
>100
26.7 2.1
48.8 2.6
50.2 3.2
73.1 2.2
52.1 1.4
59.0 3.3
62.5 4.0
>100
0.7
0.4
0.6
0.4
0.2
0.2
0.2
0.8
0.9
1.4
1.1
39.2 2.2
25.8 1.6
32.5 1.3
28.7 1.3
25.6 1.8
23.5 1.6
16.3 2.1
18.6 1.8
20.67 3.2
43.5 3.5
32.1 1.0
32.8 2.2
52.1 3.3
51.8 3.3
40.3 2.3
a
IC50 values are presented as the means SD of triplicate experiments.
MTT method.
b
for the A-549, HepG2, HeLa, and HCT-8 tumor cells, respectively.
Whereas compound 12 was less toxic than its parent compound
NCTD to the normal human lung fibroblast cell WI-38, the CC50 val-
600
500
400
300
200
100
0
ues for compound 12 and NCTD were 50.2 3.2 and 26.7 2.1 lM
0 µM
6 µM
for WI-38, respectively. The selectivity index (SI = CC50 for WI-38
cell line/IC50 for HeLa cell line) for compound 12 was 8.0. Interest-
ingly, we found that compound 17, in which the 4-amino-benzene-
sulfonamides was substituted with ethyl 2-aminothiazole-5-
carboxylate, showed much less active than its parent compound
NCTD for these four tumor cell lines. Furthermore, the octanol–
water partition coefficients of 11–20 were also determined to
evaluate the water-solubility of target compounds. The partition
coefficient logP (Table 1) indicated that most compounds exhibited
better water-solubility than that of NCTD, and that the ring-closed
compounds 18–20 showed less water-solubility than those of ring-
opened acid amides 11–17. These results indicated that compound
12 might serve as a potential anticancer drug, and we choose the
HeLa cell line to further investigate the mechanism of its cytotoxic
effect.
240
260
280
300
wavelength (nm)
Figure 3. Effects of compound 12 on CT DNA. Fluorescence spectra of 12 (1
increasing concentrations of CT DNA (0–6 M).
lM) by
l
CTD and NCTD are potent inhibitors of PP1 and PP2A.12 To
determine whether compound 12 inhibits the PP1, we evaluated
the suppressive effects of compounds 12, 19 and NCTD on PP1.16
As shown in Figure 2A, compound 12 displayed more potent inhib-
itory activity against PP1 than compound 19 or NCTD, the IC50 val-
ues of compounds 12, 19 and NCTD for PP1 were 0.2 0.6, 2.3 1.0
DNA is the important pharmacological target of antitumor
drugs. It is therefore essential to the development of effective che-
motherapeutic agents to explore their interaction with DNA. A
binding assay of compound 12 with CT DNA was performed by
monitoring changes in the fluorescence spectroscopy emission pat-
tern of compound 12 (1
concentrations of CT DNA (0–6
Figure 3, the fluorescence intensity of the complex decreases pro-
gressively with increasing concentration of DNA, suggesting that
compound 12 is bound to DNA.
and 2.6 0.7 lM, respectively. We also observed that the inhibi-
l
M, excited at 264 nm) with increasing
tion of PP1 by compound 12 was in dose-dependent effect
(Fig. 2B). These data indicate that compound 12 may represent a
potential PP1 inhibitor. The cyclic imide compound 19 may bound
to the catalytic domain of the protein phosphatase 5 (PP5c) other
than PP1 inhibitory activity.25
l
M) in pH 7.2 PBS.26 As shown in
3.0
62
A
B
60
58
56
54
52
50
48
2.5
2.0
1.5
1.0
0.5
0.0
NCTD
Compd 12
Compd 19
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Concentration (µM)
Figure 2. (A) Inhibition of protein phosphatase-1 (PP1) by NCTD, 12 and 19, data are presented as the means SD of triplicate experiments; (B) PP1 was treated by compound
12 with different concentration (0.1, 0.2, 0.4, 0.6, 0.8 lM), a dose–response curve of percentage enzyme inhibition activity versus drug concentration. P < 0.01 versus DMSO
group.

366
J. Zhao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 363–366
Figure 4. Effects of compound 12 on the formation of microtubule. HeLa cells were treated with 12 (0
lM and 5 lM) for 24 h. Microtubules (red) were stained with an anti-b-
tubulin antibody and a Rhodamine (TRITC)-conjugated secondary antibody. Chromosomal DNA (blue) was stained with DAPI.
3. Stanton, H. L. K.; Chung, H. C.; Wing, S. L.; Jien, C.; Fung, Y. L.; Raymond, S. M.
Cytoskeleton proteins, such as actin filaments and microtubules,
are potential targets for cancer chemotherapy.27 The morphology of
non-treated and compound 12-treated HeLa cells was examined by
means of indirect immunofluorescence using an anti-b-tubulin
antibody to stain microtubules and a 40,6-diamidino-2-phenylin-
dole (DAPI) staining of the nuclei.28 As shown in Figure 4, the
non-treated cells were elongated, and staining of thin bundles of
microtubules was observed throughout the cytoplasm. In contrast,
cells treated with compound 12 for 24 h became round and
contained short, dense microtubule networks; we also observed
cell shrinkage and nuclear fragmentation and condensation
dispersed throughout the cytoplasm, hallmarks of apoptosis.
In summary, we introduced benzene sulfonamides groups into
the norcantharidin chemotype for the design and discovery
of novel anticancer drugs. The biological results revealed that a
4-(4-(4-fluorophenyl)piperazin-1-ylsulfonyl)benzenamine group
was favored for increased cytotoxicity and decrease toxicity of
the ring-opened acid of norcantharidin. Taken together, the
cytotoxicity, protein phosphatase-1 inhibition, DNA interaction
and the inhibition of microtubule formation in HeLa cells suggest
that compound 12 might be suitable as a potential drug candidate,
a PP1 inhibitor for cancer chemotherapy.
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Financial support from National Natural Science Foundation of
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