Design and synthesis of novel 4'-demethyl-4-deoxypodophyllotoxin derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2015.06.089

A group of podophyllotoxin (PPT) derivatives (7a-j) were synthesized by conjugating aryloxyacetanilide moieties to the 4'-hydroxyl of 4'-demethyl-4-deoxypodophyllotoxin (DDPT), and their anticancer activity was evaluated. It was found that the most potent compound 7d inhibited the proliferation of three cancer cell lines with sub to low micromolar IC₅₀ values. Furthermore, it was demonstrated that 7d induced cell cycle arrest in G2/M phase in MGC-803 cells, and regulated the expression of cell cycle check point proteins, such as cyclin A, cyclin B, CDK1, cdc25c, and p21. Finally, 4 mg/kg of 7d reduced the weights and volumes of HepG2 xenografts in mice. Our findings suggest that 7d might be a potential anticancer agent.

Full text of DOI:10.1016/j.bmcl.2015.06.089

Accepted Manuscript
Design and synthesis of novel 4’-demethyl-4-deoxypodophyllotoxin derivatives
as potential anticancer agents
Xiong Zhu, Junjie Fu, Yan Tang, Yuan Gao, Shijin Zhang, Qinglong Guo
PII:
S0960-894X(15)00694-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.089
BMCL 22879
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
24 January 2015
30 May 2015
24 June 2015
Please cite this article as: Zhu, X., Fu, J., Tang, Y., Gao, Y., Zhang, S., Guo, Q., Design and synthesis of novel 4’-
demethyl-4-deoxypodophyllotoxin derivatives as potential anticancer agents, Bioorganic & Medicinal Chemistry
Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.089
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Design and synthesis of novel 4’-demethyl-4-deoxypodophyllotoxin derivatives as
potential anticancer agents
Xiong Zhua,#, Junjie Fub,#, Yan Tanga, Yuan Gaoc, Shijin Zhanga, and Qinglong Guoc,∗
aMedicinal and Chemical Institute, China Pharmaceutical University, Nanjing 210009, PR China
bDepartment of Medicinal Chemistry, University of Florida, Gainesville, FL 32610, United States
cState Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009,
PR China
ARTICLE INFO
ABSTRACT
Article history:
Received
A group of podophyllotoxin (PPT) derivatives (7a–j) were synthesized by conjugating
aryloxyacetanilide moieties to the 4′-hydroxyl of 4′-demethyl-4-deoxypodophyllotoxin (DDPT),
and their anticancer activity was evaluated. It was found that the most potent compound 7d
inhibited the proliferation of three cancer cell lines with sub to low micromolar IC50 values.
Furthermore, it was demonstrated that 7d induced cell cycle arrest in G2/M phase in MGC-803
cells, and regulated the expression of cell cycle check point proteins, such as cyclin A, cyclin B,
CDK1, cdc25c, and p21. Finally, 4 mg/kg of 7d reduced the weights and volumes of HepG2
xenografts in mice. Our findings suggest that 7d might be a potential anticancer agent.
Revised
Accepted
Available online
Keywords:
Podophyllotoxin
Anticancer
2015 Elsevier Ltd. All rights reserved.
Aryloxyacetanilide
Cell cycle
Cyclin
Podophyllotoxin (PPT, 1, Figure 1) is a non-alkaloid toxin
lignin extracted from the roots and rhizomes of Podophyllum
species. Since the first elucidation of its structure in the 1930s,
PPT has been extensively studied as a cytotoxic agent, showing
strong anticancer activity toward numerous cancer cell lines [1].
Mechanism studies unravel that PPT exerts its cytotoxicity
through the inhibition of both tubulin polymerization and DNA
topoisomerase-II [2, 3]. However, the systemic toxicity of PPT
significantly limits its clinical application as a medicinal drug [4].
This highlights the importance of further structure modifications
on PPT. Over the last 30 years, a large number of PPT
derivatives have been reported, leading to the discovery of
several clinically valuable anticancer drugs, such as etoposide
(VP-16), etoposide phosphate and teniposide (VM-26).
arrest, activation of caspase-3 and caspase-7, etc [6, 7]
Figure 1. Chemical structures of podophyllotoxin (PPT), 4-
deoxypodophyllotoxin (DPT), and 4′-demethyl-4-deoxypodophyllotoxin
(DDPT).
Moreover,
it
was
described that
4′-demethyl-4-
Structure activity relationship (SAR) revealed that ring C and
E are the two most preferable portions of PPT for structure
modification while the integrity of ring A, B and D is essential
for retaining anticancer activity. Therefore, most of the current
derivatization on PPT focuses on C-4 and C-4′ positions. 4-
Deoxypodophyllotoxin (DPT, 2, Figure 1), first isolated from the
root of Anthriscus sylvestris, has received substantial interest
from researchers due to its diverse biological functions including
potent anticancer activity [5]. The anticancer mechanisms include
inhibition of tubulin polymerization, induction of cell cycle
deoxypodophyllotoxin (DDPT, 3, Figure 1) exerted a comparable
in vitro potency with DPT, and derivation on 4′-hydroxyl further
increased in vivo activity. Based on this, a number of DDPT
derivatives have been synthesized by coupling additional
chemical moieties or drugs, such as amino acids [8], substituted
piperazines [9], and 5-FU [10], to the 4′-hydroxyl of DDPT.
Importantly, some of the conjugates showed superior bioactivity
and reduced toxicity compared with DPT.
Inspired by the above background, herein, we present the
design, synthesis, and biological evaluation of a novel series of
———
# These authors contributed equally to this work
∗ Corresponding author. Tel.: +086-025-000-83271055; e-mail: anticancer_drug@163.com

DDPT 4′-derivatives, which bear an aryloxyacetanilide moiety in
their structures (Figure 2). Aryloxyacetanilide represents an
important skeleton in medicinal chemistry. There has been
substantial evidence supporting that numerous compounds
containing this moiety showed moderate to high anticancer
activities in vitro toward various cancer cell lines [11-15], and
were able to inhibit tumor growth in vivo [16-18]. More
importantly, some of the compounds were proved to effectively
inhibit microtubule assembly at both molecular and cellular
levels [14]. Therefore, it is highly expected that conjugating
aryloxyacetanilide moieties to the 4′-hydroxyl of DDPT would
result in novel molecules with improved anticancer activity due
to the synergetic effects of the two components.
then converted to iodines by refluxing in acetone with potassium
iodide, giving iodoacetanilide derivatives 6a–j. Finally, idodines
atoms in 6a–j were conjugated with the 4-hydroxy group in
DDPT by refluxing in acetone using potassium carbonate as the
base, resulting in final products 7a–j. It is noteworthy that a
separate step for the conversion of 5a–j to 6a–j by Finkelstein
reaction is necessary, since direct conjugation of 5a–j with 3
either with or without potassium iodide resulted in incomplete
reaction.
The antiproliferative effects of target compounds 7a–j were
tested on three human cancer cell lines (colon carcinoma HCT-
116, hepatocellular carcinoma HepG2, and gastric cancer MGC-
803). Etoposide, a PPT-derived and clinically used anticancer
drug was selected as positive control. The IC50 values for all the
tested compounds are shown in Table 1. It was found that the
introduction of a fluorine atom as R1 on the aniline ring resulted
in the most potent compound 7d, which owned comparable IC50
value (2.20 µM) with etoposide (1.70 µM) on HCT-116 cells, and
exhibited much lower IC50 values than etoposide on HepG2 and
MGC-803 cells. Replacement of the fluorine atom in 7d with
methyl (7b), chloro (7e), or nitro (7f) groups led to diminished
cytotoxicity, indicating that a fluoro group might be the optimal
R1. However, as for R2, the best activity was observed when a
chloro group was selected (7h), instead of the fluoro counterpart
7g. The dose-response curves of 7d, the most cytotoxic
compound among this panel of molecules, on the three tested
cancer cell lines are shown in Figure 4.
O
O
O
O
O
O
O
MeO
OMe
O
4'
O
H
MeO
OMe
DDPT
O
O
R
N
H
O
R
O
Ar
N
H
Aryloxyacetanilide
Figure 2. Design of 4′-demethyl-4-deoxypodophyllotoxin 4′-hydroxy
derivatives containing an aryloxyacetanilide moiety.
The synthetic route to the target compounds is shown in
Figure 3. DDPT was generated from the starting material PPT
using the reported method. First, the 4-hydroxyl group of PPT
was removed by reduction in the presence of sodium borohydride
and trifluoroacetic acid in anhydrous THF [19]. Next, the
demethylation on the C-4′ position was achieved using
methylsulfonic acid and sodium iodide in anhydrous
dichlormethane [20]. In order to construct the aryloxyacetanilide
moiety, a series of ten phenyl amines (4a–j) with various
substitutes on the benzene ring were employed as building
blocks. The amino groups of 4a–j were reacted with 2-
chloroacetyl chloride in the presence of sodium acetate and acetic
acid to introduce the chloroacetamide linker, yielding
intermediates 5a–j, respectively. Chlorine atoms in 5a–j were
Figure 4. Dose-response curves showing the anti-proliferative effects of 7d
on (A) HCT-116, (B) HepG2, and (C) MGC-803 cell lines after an incubation
of 48 h. Data are means SD of the inhibition (%) from three independent
MTT assays.
Figure 3. Synthetic route for target compounds 7a–j. Reagents and conditions: (a) NaBH4, CF3COOH, anhydrous THF, 40 °C, 2 h. (b)
NaI, CH3SO3H, anhydrous CH2Cl2, 25 °C, 4 h. (c) ClCOCH2Cl, CH3COOH, CH3COONa, 25 °C 2 h. (d) KI, acetone, 60 °C, 2 h. (e)
K2CO3, acetone, 60 °C, 2 h.

Table 1. Structures of 7a–j and IC50 values on human cancer cell lines
IC50 (µM)a
Compounds
R1
R2
R3
R4
HCT-116
> 10
HepG2
> 10
MGC-803
2.54 0.32
4.05 0.33
6.16 0.51
0.22 0.02
> 10
7a
H
H
H
H
H
Cl
H
H
H
H
H
H
H
7b
CH3
CH3
F
H
H
> 10
7.80 0.52
> 10
7c
H
H
> 10
7d
H
H
2.20 0.17
> 10
1.08 0.08
> 10
7e
Cl
NO2
H
H
H
7f
H
H
> 10
> 10
> 10
7g
F
H
> 10
> 10
> 10
7h
H
Cl
NO2
H
H
2.68 0.21
3.20 0.43
> 10
2.97 0.29
> 10
1.24 0.20
2.82 0.11
> 10
7i
H
H
7j
H
OCH3
> 10
Etoposide
1.70 0.15
5.62 0.61
> 10
aDetermined using MTT assay after incubation with indicated compound for 48 h. Data are shown as mean SD from three separate assays.
Inhibition of tubulin polymerization has been well recognized
as one of the most important mechanisms of action underlying
the cytotoxicity of PPT and its derivatives. Meanwhile, all
inhibition of tubulin polymerization has been implicated in G2/M
cell cycle arrest in various cancer cell lines [21]. In this regard,
we next investigated the effects of 7d on cell cycle distribution in
MGC-803 cells. To this end, MGC-803 cells were treated with
10, 20, and 40 µM of 7d for 24 h, and the percentages of each
cell cycle were evaluated by flow cytometry analysis. As shown
in Figure 5, treatment of cancer cells with 7d led to a
concentration-dependent accumulation of cells in the G2/M
phase with a concomitant decrease in the population of G1 phase
cells. Cell cycle arrest in G2/M phase was initially detectable at a
concentration of 10 µM (7.29%), and showed significantly
difference at 20 µM (72.30%) compared with untreated control
group (1.89%). These data demonstrate that 7d induced G2/M
arrest in MGC-803 cell.
To further elucidate the roles of 7d in cell cycle related
signaling pathways, MGC-803 cells were treated with different
concentrations (0, 10, 20, and 40 µM) of 7d for 24 h, and the
expression levels of cell cycle related proteins were detected
using Western blot assay. The cyclin-CDK (cyclin dependent
kinase) complexes play essential roles in cell cycle progression.
Cyclin A associates with CDK1 from late S phase, and is
replaced by cyclin B when progressing to late G2 phase [22].
Cyclin B-CDK1 is the primary complex responsible for the
transition of cell cycle phase from G2 to M [23]. It was found
that 7d inhibited the expression of cyclin A, cyclin B and CDK1
in a dose-dependent manner, while the level of CDK2 was not
affected (Figure 6). Furthermore, 7d suppressed the expression of
CDK activator cdc25c [24], but increased the expression of CDK
inhibitor p21 [25]. Taken together, it was demonstrated that 7d
regulated the expressions of cell cycle related proteins.
Figure 6. Effects of 7d on the expression of cell cycle related proteins in
cancer cells. MGC-803 cells were incubated with indicated concentrations of
7d for 24 h, and the levels of the indicated protein expression were detected
by a Western blot assay using specific antibodies. *P < 0.05 vs 0 µM, **P <
0.01 vs 0 µM.
Figure 5. Effect of 7d on cell cycle progression in MGC-803 cells. MGC-803
cells were treated with indicated concentrations (0, 10, 20, and 40 µM) of 7d
for 24 h, and the percentages of each cell cycle were analyzed by flow
cytometry. Data are expressed as means
SD from three independent
experiments. *P < 0.05 vs control, **P < 0.01 vs control.

Table 2. Effects of 7d on HepG2 tumor weights and mouse body weights in vivoa
Weight (g)
Entry
Dose
Day 1
Day 14
Tumor weight (g)
1.54 0.22
Inhibitory ratio (%)
Control
0.4 mL/mouse
1 mg/kg
2 mg/kg
4 mg/kg
21.00 0.89
20.82 1.34
20.91 1.31
21.30 0.64
30.10 2.39
29.09 1.56
28.36 2.80
27.20 2.23
1.45 0.24
6.09
7d
1.13 0.23b
0.84 0.24b
26.59
45.56
aData shown are means SD of tumor weights and mouse body weights for each group of mice (n = 10). bP < 0.01 vs control.
Finally, the anticancer activity of 7d was evaluated in mice.
HepG2 xenograft models were established on ICR mice, which
were then randomized into four groups. Three groups were
injected with different doses of 7d (1, 2, 4 mg/kg, respectively)
intravenously every two days, and the fourth group was treated as
vehicle control. It was found that 7d exhibited tumor-retarding
effects in a concentration-dependent way as measured by tumor
weight after two weeks (Table 2). Meanwhile, 4 mg/kg of 7d
significantly reduced tumor volume as compared with control
(Figure 7). In addition, treatment of 7d showed no significant
effects on mouse body weights after two weeks (Table 2),
indicating the safety of 7d in vivo.
This study was supported by Social Development Foundation
of Jiangsu, China (BE20137080).
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PPT represents an attracting lead compound for anticancer
drug development. In current study, diverse aryloxyacetanilide
moieties were conjugated to the 4′-hydroxy of 4′-demethyl-4-
deoxypodophyllotoxin (3), generating a panel of novel PPT
derivatives (7a–j). MTT assays led to the discovery of the most
potent compound 7d, which inhibited the proliferation of cancer
cells with sub to low micromolar IC50 values. Further mechanism
studies revealed that 7d induced cancer cell cycle arrest in G2/M
phase. Western blot assays indicated that 7d suppressed the
expression of cyclin A, cyclin B, CDK1, and cdc25c, while
induced the expression of p21. Moreover, in vivo study
demonstrated that 4 mg/kg of 7d reduced the volumes and
weights of HepG2 mice xenografts without dramatically
affecting mouse body weights. In summary, our study suggested
7d as a potential anticancer compound, and further evaluation of
7d is on-going.
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Supplementary Material
1
Supplementary data (synthesis of 2, 3, 5a–j, 6a–j, 7a–j, H
NMR and IR spectra of 7a–j, and biological assay methods) can
online
be
http://dx.doi.org/10.1016/j.bmcl.
found,
in
the
version,
at

Graphical Abstract
Design and synthesis of novel 4′-demethyl-4-
deoxypodophyllotoxin derivatives as
potential anticancer agents
Xiong Zhua,#, Junjie Fub,#, Yan Tanga, Yuan Gaoc, Shijin Zhanga, and Qinglong Guoc,*