Synthesis and anticancer activity of dichloroplatinum(II) complexes of podophyllotoxin

Article abstract of DOI:10.1016/j.bmcl.2013.04.089

A series of dichloroplatinum(II) complexes of podophyllotoxin (PPT) were prepared, and their cytotoxicity against sensitive (A-549, HeLa, HCT-8, Hep-G2, K562) and resistant (ADM/K562) cell lines were evaluated. Complex cis-[4α-O-(2″,3″-diaminopropanoyl)-podophyllotoxin] dichloride platinum(II) (12) displayed most potent cytotoxicity with IC₅₀ value in the range 0.071-2.98 μM. Complex 12 induces cell cycle arrest in the G₂/M phase, and inhibits the formation of microtubules in HeLa cells. Furthermore, this complex exhibits potent DNA cleavage capabilities.

Full text of DOI:10.1016/j.bmcl.2013.04.089

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3780–3784
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anticancer activity of dichloroplatinum(II) complexes
of podophyllotoxin
e,
a,d,
Xuan Liu a, Lin-Lin Zhang a, Xiao-Hui Xu a, Lin Hui b,c, , Jin-Bang Zhang , Shi-Wu Chen
⇑
⇑
⇑
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
d State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
e College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dichloroplatinum(II) complexes of podophyllotoxin (PPT) were prepared, and their cytotoxic-
ity against sensitive (A-549, HeLa, HCT-8, Hep-G2, K562) and resistant (ADM/K562) cell lines were eval-
Received 26 January 2013
Revised 23 April 2013
Accepted 30 April 2013
Available online 9 May 2013
uated. Complex cis-[4
displayed most potent cytotoxicity with IC50 value in the range 0.071–2.98
a
-O-(200,300-diaminopropanoyl)-podophyllotoxin] dichloride platinum(II) (12)
lM. Complex 12 induces cell
cycle arrest in the G2/M phase, and inhibits the formation of microtubules in HeLa cells. Furthermore, this
complex exhibits potent DNA cleavage capabilities.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Cisplatin
Podophyllotoxin
Microtubules inhibition
DNA cleavage
Anticancer drugs are rarely used singly to treat cancer, because
only a few tumours are sensitive enough to be cured by single
drugs. For a specific type of tumour, effective chemotherapy usu-
ally depends on suitable drugs combinations.1 Cisplatin is a widely
used chemotherapeutic agent for the treatment of testicular can-
cer, and it is used in combination regimens for a variety of other
tumors, including ovarian, cervical, bladder, lung, and those of
the head and neck.2 However, cisplatin has several shortcomings
that limit its application in clinic, such as toxicity (ototoxicity,
neurotoxicity, gastrointestinal toxicity, renal and hepatic toxicity),
low water-solubility, narrow spectrum of activity and intrinsic or
acquired resistance.3 Over the last 30 years, 23 other platinum-
based drugs have entered clinical trials with only two (carboplatin
and oxaliplatin) of these gaining international marketing approval,
and another three (nedaplatin, heptaplatin and lobaplatin) (Fig. 1)
gaining approval in individual nations.4 Although 40 years have
passed since the discovery of the anticancer activity of cisplatin,
its mechanism of action is unclear.5 Early investigations of the
mechanism of action of cisplatin, suggested that the distortions
in the DNA structure, caused by the covalent binding of cisplatin
to two adjacent guanines on the same strand of the nuclear DNA,
interfere with replication and trigger cellular events that lead to
the death of the cancer cell.6,7 Further study on non-DNA cellular
interactions of platinum drugs suggested that the mitochondria
might be the critical pharmacological target of cisplatin.8 Several
strategies have been used to increase tumor cell selectivity of plat-
inum drugs or to overcome one or more resistance mechanisms,
including their conjugation to potentially site-directing molecules,
such as folate, porphyrins, adenine, terpenoids, peptides and many
others.9
Podophyllotoxin (PPT, 1) has cathartic, antirheumatic, and anti-
viral properties.10 However, its antimitotic activity has proved to
be of the greatest interest to researchers.11 Because PPT is highly
toxic, it is not used as an anticancer drug itself, but several semi-
synthetic derivatives, including etoposide (VP-16, 2), teniposide
O
H2
N
O
O
O
O
O
O
H3N
H3N
O
O
H3N
H3N
Cl
Cl
H3N
H3N
Pt
Pt
Pt
Pt
N
O
H2
O
oxaliplatin
carboplatin
O
cisplatin
nedaplatin
O
O
O
O
NH2
O
NH2
Pt
Pt
O
N
N
H2
H2
O
O
heptaplatin
lobaplatin
⇑
Corresponding authors. Tel./fax: +86 931 8915686.
E-mail addresses: chenshw@lzu.edu.cn, chenshwu@gmail.com (S.-W. Chen).
Figure 1. Structures of platinum-based anticancer drugs.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.089

X. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3780–3784
3781
R'
O
OH
O
5
4
11
3
2
O
HO
O
A
O
R'
R''
H
O
HO
O
D
B
C
Me
Etoposide
Teniposide
Etopophos
2
3
O
O
1
8
O
O
2'
H
6'
E
S
O
O
OH
OH
MeO
OMe
4'
Me
P
4
OMe
MeO
OMe
Podophyllotoxin
1
OR''
Figure 2. Podophyllotoxin (1) and its based clinical drugs.
(3), and etopophos (4) (Fig. 2), are in clinical use against various
cancers, including small cell lung cancer, testicular carcinoma,
lymphoma, and Kaposi’s sarcoma.12 The mechanism of action of
etoposide involves the inhibition of topoisomerase-II (TOP-II), un-
like the parent compound which inhibits mitosis.13 With the goal
of developing more-potent analogues and overcoming drug resis-
tance, several potential drug candidates based on PPT, such as
NK-611, GL-331, tafluposide and F14512, are being evaluated in
clinical trials.14
presence of N,N-dicyclohexylcarbodiimide (DCC) and 4-dimethyl-
aminopyridine (DMAP) at room temperature.21 Compounds 10
and 11 were easily obtained though intermediates 8 and 9 reacted
with 2,3-bis(t-butoxycarbonylamino)propanoic acid under 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
in the presence of N-hydroxybenzotriazole (HOBT) in dichloro-
methane.19 Then, the protection group Cbz in compound 9 were re-
moved by catalytic hydrogenation over Pd/C; and the protection
group Boc in compounds 10 and 11 were easily removed using tri-
fluroacetic acid. Subsequently, these products were without fur-
ther purified, and used directly for complexation reaction with
potassium tetrachloroplatinate in a solution of THF and water
(1:1) affording target complexes 12–14 in moderate yields.22
The structures of complexes 12–14 were identified by 1H NMR
spectra, 13C NMR spectra, IR spectra and HRMS (SI). In infrared
spectrum of complexes 12–14, the fact that the amino groups are
coordinated to the platinum atom is confirmed by the presence
Combination chemotherapy with cisplatin and VP-16 is always
applied for non-small-cell lung carcinoma, malignant lymphoma
and other cancer.15 In this Letter, which is part of our continuing
effort to find new natural product-based compounds with potent
activities,16–21 we prepared a series of dichloroplatinum(II) conju-
gated podophyllotoxin complexes and evaluated their cytotoxici-
ties against a panel of cancer cell lines. The most active complex,
cis-[4a
-O-(200,300-diaminopropanoyl)-podophyllotoxin] dichloride
platinum(II) 12, was evaluated for its effect on cell cycle progres-
sion, inhibition of microtubules formation, and DNA cleavage.
The synthesis of complexes 12–14 was carried out according to
the procedure outlined in Scheme 1. Firstly, the intermediates 4b-
amino-4-deoxylpodophyllotoxin (7) and 4b-amino-40-demethyl-4-
deoxylpodophyllotoxin (8) were obtained from PPT as described in
previous publication.17 Compound 9 was prepared by PPT reacted
with 2,3-bis(benzoxycarbonylamino)propanoic acid in the
of m
(Pt–N) in the range 3250–3260 cmꢀ1. The strongest peaks of
complexes 12–14 solution in dimethylsulfoxide are the [M–
Cl+DMSO]+ in the mass spectrum (ESI) as reference reported.23
The in vitro cytotoxicity of complexes 12–14 and intermediates
9–11 along with the clinically used drug cisplatin and VP-16 as ref-
erences were evaluated on the six human cancer cell lines (A-549,
lung carcinoma; HeLa, cervical carcinoma; HCT-8, colon
carcinoma; Hep-G2, hepatic carcinoma; K562, human chronic
NH2
OH
N3
O
O
O
O
O
O
O
O
Ref. 16
Ref. 16
O
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
OR
OMe
OR
7 R = Me
1
5 R = Me 6 R = H
8 R = H
ii
i
O
NH2
Pt Cl
Cl
X
O
O
O
H2N
NHBoc
NHBoc
O
NHCbz
NHCbz
O
HN
O
O
O
O
O
O
O
v
v
iii,
O
iv,
O
O
MeO
OMe
OR
MeO
OMe
MeO
OMe
12 X = O, R = Me
13 X = NH, R = Me
14 X = NH, R = H
OR
OMe
9
10 R = Me
11 R = H
Scheme 1. Synthesis of complexes 12–14. Reagents and conditions: (i) 2,3-bis (benzoxycarbonyl amino)propanoic acid, DMAP, DCC, CH2Cl2, (ii) 2,3-bis(t-butoxycarbon-
ylamino)propanoic acid, EDCI, HOBt, CH2Cl2, (iii) H2, 10% Pd/C, MeOH, (iv) TFA, CH2Cl2, (v) K2PtCl4, THF/H2O, rt.

3782
X. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3780–3784
Table 1
cytotoxicity of 12 is 50 times more potent than those of VP-16
In vitro cytotoxicity (IC50, l
M)a of compounds 9–14
and Cisplatin. In Table 1, we also found that the 40-OH series of
podophyllotoxin derivatives were more cytotoxic than the corre-
sponding 40-OMe analogues (14 vs 13, 11 vs 10), the result con-
firmed the assumption that free hydroxyl group at the C-40
position in derivatives of PPT was favorable for their antitumor
activity.13
Compounds A-549b HeLab HCT-8b Hep-G2b K562c
ADM/K562c
9
10
11
12
13
14
Cisplatin
VP-16
2.78
27.0
29.6
0.59
85.4
26.0
16.3
23.9
2.35
55.2
37.4
0.27
48.4
8.30
4.64
38.8
28.1
2.98
30.7
25.2
10.0
11.4
10.2
39.9
27.2
0.15
29.5
12.3
0.071
33.8
12.4
0.20
55.6
21.2
0.091
29.2
9.56
4.55
0.38
71.6
26.2
12.5
PPT and its analogues are reported to induce apoptosis and cell
cycle arrest in the G2/M phase.24 To determine whether complexes
of PPT and cisplatin act in a similar way, the effects of 12 on cell
cycle progression were determined by FACS analysis in propidium
iodide-stained HeLa cells.21 As shown in Figure 3, treatment with
17.8
12.2
3.11
6.00
0.354 NA
a
b
c
Data are the mean of three independent experiments.
MTT method with 72 h drug exposure.
CCK-8 method with 72 h drug exposure.
0.25 lM 12 lead to a time-dependent accumulation of cells in the
G2/M phase with a concomitant decrease in the population of G1
phase cells. 48.8% and 94.0% of the cells were in G2/M phase after
12 exposed for 12 h (Fig. 3B) and 24 h (Fig. 3C), respectively, com-
pared with 15.0% in untreated cultures (Fig. 3A). These results
demonstrate that 12 have similar effects on the cell cycle arrest
with that of parent compound PPT.
As above mentioned, PPT induce apoptosis of tumor cells in a
time-dependent manner,24 we did not find obvious apoptosis in
FACS analysis of 12-treated HeLa cells, to confirm the effect of
myelogenous leukemia; and ADM/K562, K562 resistant to ADM)
used the standard MTT or CCK-8 method.21 The results expressed
as IC50 values are summarized in Table 1.
As illustrated in Table 1, after a 72 h exposure, complex 12
showed excellent in vitro cytotoxicities against various experimen-
tal cell lines compared to clinical drug VP-16 and Cisplatin. And
intermediate 9 also exhibited moderate in vitro cytotoxicities to
these six cell lines. Especially to ADM/K562 resistant cell line, the
Figure 3. Effects of 12 on cell cycle progression. (A) Control HeLa cells; (B) HeLa cells treated with 0.25 lM 12 for 12 h; (C) HeLa cells treated with 0.25 lM 12 for 24 h.
Figure 4. Effects of 12 on cell morphologic. (A) Control HeLa cells; (B) HeLa cells treated with 0.25
cells treated with 0.25 M 12 for 24 h.
lM 12 for 6 h; (C) HeLa cells treated with 0.25 lM 12 for 12 h; (D) HeLa
l

X. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3780–3784
3783
Figure 5. HeLa cells grown on coverslips were treated with 12 (0.01
l
M and 0.1
l
M) for 24 h. Microtubules (red) were stained by incubation with anti-
a-tubulin antibody for
90 min and then with Rhodamine (TRITC)-conjugated secondary antibody for 30 min. Chromosomal DNA (blue) was stained with DAPI.
complex 12 on induction of apoptosis, the morphology of HeLa
cells, which were treated with 0.25 lM 12 for 6 h, 12 h and 24 h,
respectively, were examined using Hoechst staining (Fig. 4). As
shown in Figure 4, untreated HeLa cells exhibited excellent growth
characteristic (Fig. 4A). Interestingly, we found mitotic catastrophe
in the HeLa cells treated with 0.25 lM 12 for 6 h, the typical fea-
tures is large cytoplasmic vacuoles, abnormal mitotic figures, mul-
tinucleation, and formation of large cells (Fig. 4B).25 However, HeLa
cells treated with 0.25 lM 12 for 12 h or 24 h evoked typical apop-
totic features, such as membrane blebbing, cell shrinkage and
detachment, and nuclear condensation even fragmentation
(Fig. 4C and D).
Figure 6. Cleavage of pBR322 plasmid DNA by complex 12. Lane 1: control DNA;
lane 2: DNA treated with 50 M 12 for 24 h; lane 3: DNA treated with 100 M 12
for 24 h; lane 4: DNA treated with 100 M cisplatin for 24 h.
l
l
l
To determine whether 12 was able to depolymerize cellular
microtubules as PPT does,26 we examined the morphology of un-
treated and 12-treated HeLa cells by means of indirect immunoflu-
orescence with antitubulin antibody staining of microtubules and
fluorescent staining of nuclei (Fig. 5). The untreated cells were
elongated, and staining of thin bundles of microtubules was ob-
served throughout the cytoplasm. In contrast, 24 h after treatment
with 12, the cells were round and contained short, dense microtu-
bule networks; in addition we observed cell shrinkage and nuclear
fragmentation and condensation dispersed throughout the cyto-
plasm, which are hallmarks of apoptosis. These effects increased
dose dependently.
caused cleavage of DNA. These results suggest that 12 induced
apoptosis by means of multiple pathways.
Acknowledgments
This work was financially supported by the Natural Science
Foundation of Gansu Province (No. 1208RJZA247) and The Science
and Technology Development Project of Lanzhou City (No. 08-1-
57).
The mechanism of action of many platinum-based chemothera-
peutic agents relies upon DNA damage.27 In order to study whether
complex 12 enables DNA cleavage, different concentrations (50
Supplementary data
Supplementary data associated with this article can be found, in
and 100
lM) of 12 were incubated with 0.5
lg pBR322 DNA for
the
online
version,
at
http://dx.doi.org/10.1016/
24 h in 20
l
L Tris–Cl buffer (pH 7.5). The samples were analyzed
j.bmcl.2013.04.089.
on 1% native agarose gel electrophoresis followed by ethidium bro-
mide staining of the DNA bands.28 As shown in Figure 6, the inten-
sity of DNA bands decrease with concomitant increase in
concentration of 12, the result suggesting 12 induces degradation
of pBR322 DNA similar to cisplatin.29
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-O-(200,300-diaminopropanoyl)-podophyllo-
toxin] dichloride platinum(II) exhibited promising in vitro cytotox-
icities against not only VP-16 sensitive A-549, HeLa, HCT-8, Hep-
G2 and K562 but also ADM resistant K562 cell lines. Fluores-
cence-activated cell sorting analysis indicated that 12 induced cell
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;
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J = 8.6 Hz, 1H, H-4b), 5.56 (br s, 1H, NH), 4.57 (m, 1H, a-H of propanoic acid),
4.40 (d, J = 4.8 Hz, 1H, H-1), 4.21–4.12 (m, 1H, H-11a), 3.88–3.76 (m, 1H, H-
11b), 3.65 (s, 6 H, 30,50-OMe), 3.61 (s, 3H, 40-OMe), 3.45–3.38 (m, 2H, b-H of
propanoic acid), 2.94–2.89 (m, 1H, H-2), 2.86–2.80 (m, 1H, H-3); 13C NMR
(100 MHz, DMSO-d6) d 173.9, 168.2, 152.0 (2C), 147.6, 146.9, 136.4, 135.6,
132.6, 127.8, 109.4, 107.9 (2C), 107.6, 101.5, 74.7, 70.8, 61.5, 60.6, 55.8 (2C),
48.7, 48.3, 44.0, 42.8; LR-MS (ESI): m/z 808.7 [MꢀCl+DMSO] (100%); HR-MS
(ESI): 814.1648 (calculated for 814.1644 of
C
27H28D6ClN2O10PtS+
[MꢀCl+DMSO-d6+]).
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a
-O-(200,300-diaminopropanoyl)-
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H2O (5 mL). Following K2PtCl4 (239 mg, 0.57 mmol) was added to the above
solution, and the pH of the mixture keep at 5–6. Then the reaction mixture was
stirred until the disappearance of the starting material as indicated by TLC. The
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