Synthesis and biological evaluation of 4β-(thiazol-2-yl)amino-4′-O-demethyl-4-deoxypodophyllotoxins as topoisomerase-II inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.12.012

A series of 4β-(thiazol-2-yl)amino-4′-O-demethyl-4-deoxypodophyllotoxins were synthesized, and their cytotoxicities were evaluated against four human cancer cell lines (A549, HepG2, HeLa, and LOVO cells) and normal human diploid fibroblast line WI-38. Som

Full text of DOI:10.1016/j.bmcl.2017.12.012

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 4b-(thiazol-2-yl)amino-40-O-
demethyl-4-deoxypodophyllotoxins as topoisomerase-II inhibitors
b,c,
Chun-Yan Sang a, Heng-Zhi Tian a, Yue Chen d, Jian-Fei Liu 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
d College of Life Science, Northwest Normal University, Lanzhou 730070, China
a r t i c l e i n f o
a b s t r a c t
A series of 4b-(thiazol-2-yl)amino-40-O-demethyl-4-deoxypodophyllotoxins were synthesized, and their
cytotoxicities were evaluated against four human cancer cell lines (A549, HepG2, HeLa, and LOVO cells)
and normal human diploid fibroblast line WI-38. Some of the compounds exhibited promising antitumor
activity and less toxicity than the anticancer drug etoposide. Among them, compounds 15 and 17 were
found to be the most potent synthetic derivatives as topo-II inhibitors, and induced DNA double-strand
breaks via the p73/ATM pathway as well as the H2AX phosphorylation in A549 cells. These compounds
also arrested A549 cells cycle in G2/M phase by regulating cyclinB1/cdc2(p34). Taken together, these
results show that a series of compounds are potential anticancer agents.
Article history:
Received 3 November 2017
Revised 5 December 2017
Accepted 6 December 2017
Available online xxxx
Keywords:
Podophyllotoxin
Topo-II inhibitors
DSBs
Ó 2017 Elsevier Ltd. All rights reserved.
Cell cycle arrest
Cancer has become one of the leading causes of death world-
wide, according to information from the World Health Organiza-
tion (WHO), it is estimated that there will be 12 million deaths
from cancer in 2030. Among them, it is estimated that more than
1 million people die of lung cancer annually and approximately
1.4 million individuals are diagnosed per year, 12% of whom are
new cases.1 Thus, there is an unmet need for novel therapies to
improve the prognosis of patients with lung cancer. Plant-derived
compounds are known to have curative potential.
DNA is the most vulnerable material in the cell, and DNA dam-
age induces a prominent route of cell death known as apoptosis.2
In clinical treatment, other than surgery, the mainstay of cancer
treatment to date has involved the use of DNA-damaging agents
in the form of radiation and systemic chemotherapy. Radiation is
responsible for approximately 40% of all cures achieved in cancer
patients.3 Commonly used DNA-damage-inducing chemotherapies
include platinum salts (carboplatin, cisplatin, and oxaliplatin) that
generate covalent cross-links between DNA bases,4 and topoiso-
merase-II (topo-II) inhibitors (etoposide and doxorubicin) that
generate topo–DNA adducts and DNA strand breaks.5 Topo-I inhi-
bitors induce DNA single-strand breaks, while topo-II inhibitors
induce DNA double-strand breaks (DSBs). H2AX, an evolutionarily
conserved variant of histone H2A, is a key histone that undergoes
various posttranslational modifications in response to DSBs.6 By
virtue of phosphorylation, H2AX marks the damaged DNA double
helix to facilitate local recruitment and retention of DNA repair
and chromatin remodeling factors and thus restore genomic
integrity.
Podophyllotoxin (PPT, 1), derived from the roots and rhizomes
of Podophyllum species, has cathartic, antirheumatic, and antiviral
properties, and pesticidal and antimitotic activity.7 Etoposide
(VP-16, 2) and teniposide (VM-26, 3, Fig. 1) are semisynthetic glu-
cosidic cyclic acetals of PPT currently used in chemotherapy for
various types of cancer, including small-cell lung cancer, testicular
carcinoma, lymphoma, and Kaposi’s sarcoma.8 Both of these com-
pounds block the catalytic activity of DNA topo-II by stabilizing a
cleavable enzyme–DNA-complex in which the DNA is cleaved
and covalently linked to enzyme.9 Although they are widely used
in the clinic, several problems hinder their clinical efficacy such
as drug resistance and poor water solubility. Therefore, there
remains a need for new PPT derivatives with anticancer activity
and improved water solubility. Extensive efforts have been made
researchers to address these limitations.10 Structure–activity rela-
tionship (SAR) experiments have unambiguously demonstrated
that C4 is the major molecular site tolerant to significant structural
diversification.11 Furthermore, the comparative molecular field
analysis (‘CoMFA’) models generated by Lee and coworkers
⇑
Corresponding authors at: School of Pharmacy, Lanzhou University, Lanzhou
730000, China (S.-W. Chen).
E-mail addresses: chenshw@lzu.edu.cn (S.-W. Chen), zyhuil@hotmail.com
(L. Hui).
https://doi.org/10.1016/j.bmcl.2017.12.012
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
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C.-Y. Sang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. The structures of podophyllotoxin (1), etoposide (2), and teniposide (3).
demonstrated that bulky substituent at C4 of PPT might favor DNA
topo-II inhibition.12
The intermediates 2-(2-aminothiazol-4-yl)acetic carbamate
8a–f were synthesized from ethyl 2-(2-aminothiazol-4-yl)acetate
(compound 6) using previously published methods24 as outlined
in Scheme 1. Briefly, the amino group of compound 6 was pro-
tected with trityl chloride (TrtCl) and then saponified to produce
compound 7, followed by reaction with different amines under
1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride
(EDCI) in the presence of N-hydroxybenzotriazole (HOBT) in
dichloromethane. Last, the protecting group was removed in the
presence of acetic acid to provide the intermediates 8a–f.
Recently, kinds of 4b-N-substituted 40-O-demethyl-4-
deoxypodophyllotoxins were generated and have been shown to
exhibit more potent anticancer activity and better binding ability
to DNA topo-II compared with etoposide.13–16 As part of our
ongoing efforts to develop new podophyllotoxin derivatives with
potent biological activities,17–23 herein we report the synthesis
and cytotoxicities of
a
series of 4b-(thiazol-2-yl)amino-40-O-
demethyl-4-deoxypodophyllotoxins. Compound 15 and 17
were further evaluated for its effect on topo-II enzymes, H2AX
phosphorylation as well as cell cycle progression.
The target compounds 10–18 were synthesized from PPT
according to previously published methods (Scheme 2).22 Briefly,
Scheme 1. Synthesis of compounds 8a–f. Reagents and conditions: (i) TrtCl, Et3N, r.t (ii) NaOH, MeOH, reflux; (iii) amines, EDCI, HOBT, CH2Cl2, r.t; (iv) CH3COOH, 60 °C.
Scheme 2. Synthesis of target compounds 10–18.
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C.-Y. Sang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
methanesulfonic acid/sodium iodide was used for both 40-O-
demethylation and C-4 epimerization of the podophyllotoxin ring
system to provide the required key iodo intermediate (compound
9). Compound 9 then underwent further one-pot reactions with
the appropriate amine 8a–f in the presence of triethylamine in
refluxing tetrahydrofuran (THF) to produce the corresponding
compounds 10–15. Furthermore, to study the effects of carbamate
substituted with different esters of 2-aminothiazol, we also pre-
pared 40-demethy-4-epipodophyllotoxin (DMEP) derivatives of
ethyl 2-aminothiazol-4-yl esters 16 and 17, as well as of 2-
aminothiazol-4-yl acid 18.
H-4. While H-3 was irradiated in 1H NMR spectrum for compound
10–18, the H-4 was enhanced 3–5% (eg: 3.9% for compound 10),
however, H-2 was not enhanced. Thus we ensured that H-4 is cis
to H-3 and H-2 is trans to H-3.
The in vitro anti-proliferative activities of compounds 10–18
were evaluated against a panel of four human cancer cell lines
(lung carcinoma A549, hepatocellular carcinoma HepG2, cervical
carcinoma HeLa, colon adenocarcinoma LOVO) together with the
human lung fibroblast cell line WI-38, etoposide was used as a pos-
itive reference compound. The screening procedure was based on
The structure of all target compounds was confirmed by 1H
NMR, 13C NMR, and HRMS (Supporting Information). The assign-
ment of the configuration at C-4 position for compounds 10–18
was based on coupling constants J3,4. The C-4b substituted com-
pounds have J3,4 ꢀ 4.0 Hz due to a cis relationship between H-3
and H-4 (eg: J3,4 = 4.2 Hz for compound 10), the C-4
a substituted
compounds, however, have J3,4 ꢁ 10.0 Hz because of H-3 is trans
to H-4.25,26 The relative stereochemistry at C-4 position was also
demonstrated by the observation of 1D NOE between H-3 and
Table 1
The in vitro anti-proliferative activities (IC50, l
M)a,b of compounds 10–18.
Compds
Tumor cell
A549
Normal cell
WI-38
HepG2
HeLa
LOVO
10
11
12
13
14
15
16
17
11.1 0.7
9.1 0.9
9.4 0.5
18.7 0.8
22.3 1.3
1.3 0. 9
9.3 0.7
0.16 0.06
8.3 1.1
9.1 0.7
10.2 0.6
19.1 1.2
16.3 0.9
15.1 0.8
18.7 1.2
8.5 0.9
0.12 0.03
0.13 0.05
9.3 0.9
17.2 1.0
14.1 0.7
10.7 0.9
14.8 1.1
16.5 1.0
4.4 0.9
12.5 0.7
5.6 0.7
8.7 0.7
19.1 1.2
21.0 0.8
19.1 0.8
37.2 1.3
27.4 1.3
9.2 0.5
7.0 0.9
5.0 0.5
10.3 1.0
18.7 0.9
56.0 2.4
29.0 1.2
77.8 4.6
33.5 3.9
28.1 2.2
66.7 1.2
44.4 1.4
55.3 2.5
42.5 4.0
25.7 1.9
18
VP-16
9.2 0.9
11.2 1.0
a
Data are the mean of three independent experiments.
MTT method.
b
Fig. 3. Effects of 15 and 17 on H2AX phosphorylation. (A) Western blotting analysis
of H2AX phosphorylation in A549 cells treated with compound 15 and 17 (vehicle,
1
c
l
M, 5
H2AX antibodies and counterstained with DAPI after treatment with compound
M and 5 M) for 12 h.
lM and 10 lM) for 12 h; (B) A549 cells were immunostained with anti-
15 and 17 (vehical, 1
l
l
Fig. 2. The effect of compounds 15 and 17 on topo-II. (A) The effect of compounds
15 and 17 on topo-II and topo-IIb protein expression in A549 cells. (B) Effect of
compounds 15 and 17 on topo-II mediated DNA relaxation. Lane a, supercoiled
pBR322 DNA. Lane b, pBR322 DNA + topo-II ; lane c, pBR322 DNA + topo-II + 100
M etoposide; lane d, pBR322 DNA + topo-II + 100 M compound 17; lane e,
pBR322 DNA + topo-II + 100 M compound 15.
a
a
a
a
Fig. 4. Western blotting analysis p73 and pATM levels in A549 cells treated with
l
a
l
vehical, VP16 (5
for 12 h.
lM), 15 (1 lM, 5 lM and 10 lM) and 17 (1 lM, 5 lM and 10 lM)
a
l
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C.-Y. Sang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
the standard MTT method,23 and the results are summarized in
Table 1.
more potent compared with those of amides (compounds 16, 17
vs compounds 10–15). Second, the 2-(2-aminothiazol-4-yl)acetate
substituted derivative of DMEP appear to be less potent than the 2-
aminothiazole-4-carboxylate derivative (compound 16 vs com-
pound 17). Last, the 2-(2-aminothiazol-4-yl)acetic acid substituted
compound 18 also showed moderate potent antiproliferation.
These data suggested that compounds 15 and 17 might serve as
potential anticancer agents. We decided to investigate the mecha-
nism responsible for the cytotoxic effects of these compounds in
A549 cells.
The compounds 10–18 generally showed greater or equal anti-
proliferation toward these four cancer cell lines and less toxicity to
the normal human lung fibroblast cell WI-38 compared with VP-16
(Table 1). Based on these results, it was possible to deduce some
preliminary SARs. First, the large range of activities observed for
compounds 10–18 indicated that the substituent at C-4 of the
2-aminothiazol markedly affects the activity profile of this
compound class, with 2-aminothiazol-4-yl esters appearing to be
Fig. 5. Effects of 15 and 17 on cell cycle progression. A) flow cytometric analysis. a) A549 cells treated with vehical; b) A549 cells treated with 1
lM compound 15 for 12 h; c)
A549 cells treated with 5 M compound 15 for 12 h; d) A549 cells treated with 1 M compound 17 for 12 h; e) A549 cells treated with 5 M compound 17 for 12 h; f) Bar
l
l
l
graphs showing compound 15 and 17 on cell cycle progression. B) Western blotting analysis G2/M cell-cycle regulators expression of cyclin B1, cdc2(p34) treated with
compounds 15 and 17 various concentration in A549 cells for 12 h.
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C.-Y. Sang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Etoposide is well-known drug that causes double-strand DNA
Acknowledgements
breaks and is a topo-II inhibitor.7,8 As these 4b-(thiazol-2-yl)
amino-40-O-demethyl- 4-deoxypodophyllotoxins are closely
related molecules to etoposide, it was considered of interest to
examine the effects of topo-II and DSBs. Thus A549 cells were trea-
We gratefully acknowledge the financial support from the
National Natural Science Foundation of China (Grant No.
21372110, 21672093 and 81372177).
ted with etoposide (5
Interestingly both of compounds 15 and 17 decreased the expres-
sion of either topo-II or topo-IIb in A549 cells in a dose-dependent
lM), 15 and 17 at 1, 5 and 10 l
M for 12 h.23
A. Supplementary data
a
manner (Fig. 2A). Furthermore, compounds 15 and 17 induced
supercoiled pBR322 DNA relaxation to form linear DNA (Fig. 2B).
This was strongly supported by the fact that these derivatives of
podophyllotoxin can act as topo-II inhibitor, and the percentage
of cells with DSBs were greater than those of etoposide (at the
same molarity).
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.12.012.
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