A rational design strategy of the novel topoisomerase II inhibitors for the synthesis of the 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin with antitumor activity by diminishing the relaxation reaction of topoisomerase II-DNA decatenation

Article abstract of DOI:10.1016/j.bmc.2014.03.048

A rational design strategy of the novel podophyllum topoisomerase II (Topo II) inhibitors for the synthesis of the esterification and amidation substituted 4′-demethylepipodophyllotoxin (DMEP) derivates was developed in order to discover the potential ant

Full text of DOI:10.1016/j.bmc.2014.03.048

Bioorganic & Medicinal Chemistry 22 (2014) 2998–3007
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A rational design strategy of the novel topoisomerase II inhibitors
for the synthesis of the 4-O-(2-pyrazinecarboxylic)-
40-demethylepipodophyllotoxin with antitumor activity
by diminishing the relaxation reaction of topoisomerase
II-DNA decatenation
Wei Zhao a, , Lu Chen a,b, , Hong-Mei Li a, Duan-Ji Wang a, Dong-Sheng Li a, Tao Chen c,
Zhan-Peng Yuan b, , Ya-Jie Tang a,
⇑
⇑
a Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, China
b School of Public Health, Wuhan University, Wuhan 430071, China
c Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A rational design strategy of the novel podophyllum topoisomerase II (Topo II) inhibitors for the synthesis
of the esterification and amidation substituted 40-demethylepipodophyllotoxin (DMEP) derivates was
developed in order to discover the potential antitumor prodrug. Firstly, according to the structure–
activity relationship, drug combination principle and bioisosterism, the –COO– and the –NH– bond
substituents at the 4 position of cycloparaffin would be a great modification direction to improve
antitumor activity of 40-demethylepipodophyllotoxin (DMEP). Secondly, from the prodrug principle view,
the esterification and amidation at the C-4 position of DMEP would be two useful structure modifications
for improve solubility. Thirdly, from the activity pocket in Topo II-DNA cleavage complex point of view, a
series of heterocyclic with pharmacological activity were chosen as module for improving antitumor
activity by binding with Topo II. Finally, nine novel esterification and amidation DMEP derivates were
designed and synthesized for the potential Topo II inhibitors with the superior biological activity. All
the novel compounds exhibited promising in vitro antitumor activity, especially 4-O-(2-pyrazinecarboxylic)-
40-demethylepipodophyllotoxin (compound 1). The antitumor activity of compound 1 against tumor cell
Received 30 January 2014
Revised 22 March 2014
Accepted 31 March 2014
Available online 13 April 2014
Keywords:
Rational drug design strategy
40-Demethylepipodophyllotoxin derivates
Antitumor activity
Topoisomerase II
Cell cycle arrest
Apoptosis
line HeLa (i.e., the IC50 value of 0.60 0.20
the IC50 value of 1.21 0.05 M), and BGC-823 (i.e., the IC50 value of 4.15 1.13
improved by 66, 16, 12, and 6 times than that of the clinically important podophyllum anticancer
drug etoposide (i.e., the IC50 values of 15.32 0.10, 59.38 0.77, 67.25 7.05, and 30.74 5.13 M),
l
M), A549 (i.e., the IC50 value of 3.83 0.08
lM), HepG2 (i.e.,
l
lM) was significantly
l
respectively. Compound 1 could arrest HeLa cell cycle G2/M and induce apoptosis by strongly diminishing
the relaxation reaction of Topo II-DNA decatenation. The correctness of rational drug design was strictly
demonstrated by the bioactivity test.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
II (Topo II) and then diminishing the relaxation reaction of Topo
II-DNA decatenation. The clinical success of etoposide and tenipo-
40-Demethylepipophyllotoxin (DMEP) was a well known natu-
rally occurring cyclolignans with antitumor activities,1 which can
bind at the pocket between the link site of DNA and topoisomerase
side has triggered the search for compounds with a similar
mechanism of action but without their inconveniences.2 This has
resulted in the discovery of many compounds with very different
chemical structures. Despite most of currently numerous studies
focus on the structure modification of DMEP in order to generate
derivatives with superior pharmacological profiles and broader
therapeutic scope,3–5 those have only begun to appreciate the full
potential of the prodrug approach in modern drug development,
and many novel prodrug innovations await discovery.
⇑
Corresponding authors. Tel./fax: +86 27 6875 9982 (Z.-P.Y.), +86 27 5975 0491
(Y.-J.T.).
E-mail addresses: zpyuan@whu.edu.cn (Z.-P. Yuan), yajietang@hotmail.com
(Y.-J. Tang).
Equally contributed to this work.
http://dx.doi.org/10.1016/j.bmc.2014.03.048
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
2999
To discover a series of the novel 40-demethylepipophyllum Topo
2. Materials and methods
2.1. Chemistry
II inhibitors, a rational prodrug design strategy should be devel-
oped to the structure modification of DMEP with the superior inhi-
bition on Topo II, the higher antitumor activity, the lower
cytotoxicity on the human normal cells and the better solubility.
Firstly, the steric clash and electrostatic contour plots of the com-
parative molecular field analysis models6 shows that the carbon
atom at the 4 position of cycloparaffin (C ring) of DMEP is more
reactive by comparing other carbon atoms in the tetranap skeleton
of DMEP, and is easier nucleophillic attacked by the molecule with
the electro negativity. Thus, from the structure–activity relation-
ship point of view, the structure modifications at the 4 position
of C ring of DMEP maybe a very effective pathway for improving
the antitumor activity. Secondly, according to drug combination
principle,7,8 the combination of two kinds of drugs through the
ester bond can gain some new compounds with better bioactivity.
Many famous drugs in clinic use such as sultamicillin and benori-
late were discovered through this way. So, DMEP could be com-
bined with other compounds through ester bond for the better
bioactivity and attenuated toxicity. Thirdly, according to bioiso-
sterism,9,10 bioisosteres are substituents with similar physical or
chemical properties which produce broadly similar biological
properties to a chemical compound for the activity improvement
and the toxicity reduction of the lead compound. Both of the ester
bond (–COO–) and the –NH– bond are the bioisosteres with the
same numbers of the outermost electrons. So, the –NH– bond at
the C-4 position of DMEP may be also a potential modification
direction for improving the antitumor activity of DMEP. Fourthly,
the prodrug approach is a very versatile strategy to increase the
utility of pharmacologically active compounds.11 Thus, according
to the prodrug principle,12 both of esters (–COOR) or amides
(–NHCOR) are commonly used ionizable groups, which can be
introduced into the hydroxyl, thiol, amine, or carboxylic acid
functionalities of the parent drug molecule to compensate for poor
aqueous solubility. A variety of esterases, amidases, and/or pepti-
dases in plasma or in other tissues can bioconvert these prodrugs
to their active counterparts. Quite often, esters and amides can also
be used to enhance absorption and consequently oral drug delivery
of parent drugs, because the brush-border membrane of intestinal
epithelium possesses a considerable number of transporters for
amino acids and peptides.13,14 Take these into consideration, many
famous drugs in clinic were discovered by using esterification
and amidation such as sultamicillin and benorilate. So, esterifica-
tion and amidation of DMEP at the C-4 position were permitted
for two useful DMEP structure modifications. Finally, from an
antitumour mechanistic point of view, substituents with the high
electron density at the C-4 position of DMEP derivatives (i.e.,
etoposide) has been demonstrated to be bound via the hydro-
gen bonding interactions with the tyrosine residue of Topo II and
the bases of DNA breakage.15,16 In order to design the novel
topoisomerase II inhibitors with better affinity for the active site
of DNA Topo II as anti-tumor agents, a series of heterocyclic with
pharmacological activity and the high electron density were
chosen as module at the C-4 position of DMEP for improving the
stabilization and inhibition of the complex of Topo II-DNA cleavage
complex.
Standard 40-demethylepipodophyllotoxin (DMEP) (98%) was
purchased from Shanxi Huisheng Medicament Technology Com-
pany, Ltd (Shanxi, China). N,N-dicyclohexylcarbodii mide (DCC)
(98%), 4-dimethylami-nopyridine (DMAP) (98%), 1-ethyl-3-(3-
dimethyllaminopropyl) carbodiimide hydrochloride (EDCꢀHCl)
(98%), 1-hydroxybenzotriazole hydrate (HOBT) (98%), 2-Pyrazine-
carboxylic acid (98%), 3,4,5-Trihydroxybenzoic acid (98%), 2-Quin-
oline carboxylic acid (98%), Fluoroquinolonic acid (98%), 9-
Anthracenecarboxylic acid (98%) and 4-Methyl-1-naphthoic acid
(98%) were purchased from Shanghai Jingchun Medicament Tech-
nology Company, Ltd (Shanghai, China). Dichloromethane (DCW)
was distilled from CaH2. Precoated silica gel G plates for TLC were
purchased from Merck Inc. (Darmstadt, Germany). Column
chromatography was performed with Sephadex LH-20 gel (20–
150 lm, Pharmacia & Upjohn Co., Switzerland). Flash column
chromatography (FC) silica gel (SiO2; 200–300 meshes) was pur-
chased from Qing Dao Haiyang Chemical Group Co. (Shandong,
China). Methanol and acetonitrile were of high-performance
liquid chromatography (HPLC) grade, and all other chemicals
used for extraction and isolation were of analysis grade and com-
mercially available. Deionized water was used throughout the
study.
2.2. Analytical method
The chemical reaction solution was alternately washed in
deionized water and saturated NaHCO3. After removing the deion-
ized water layer, drying the AcOEt layer by NaSO4, and removing
AcOEt by rotary evaporation, the residue was ground by aether
to obtain a white powder product. The powder product (2 mg) dis-
solved in 1 mL methanol/water (50:50 v/v) as pre-separated sam-
ple. Pre-separated sample was filtered (0.45-
and transferred into a sampling vial for HPLC analysis. The samples
were filtered with a 0.45 m micropore filter and transferred to a
lm-micropore filter)
l
sampling vial for HPLC analysis. HPLC analysis was carried out on
a Waters 600 Series HPLC system, equipped with 2487 UV detector.
An Akasil C18 column (5
l
m, 4.6 mm ꢁ 150 mm) was used. Mobile
phase was methanol/water (50:50 v/v) for the detection of alkane
amination DMEP derivatives. Mobile phase was methanol/water
(40:60 v/v) for the detection of esterification DMEP derivatives.
The pH of mobile phase was adjusted to 3.00 with formic acid.
The HPLC oven temperature was maintained at 45 °C, and the
detection wavelength was 230 nm or 219 nm. The flow rate was
0.8 mL/min. All 1H and 13C NMR spectra were recorded using a
mercury-300BB spectrometer (Varian, USA). Chemical shifts (d)
are reported in ppm relative to the TMS internal standard. Abbre-
viations are as follows: s (singlet), d (doublet), dd (doublet of dou-
blets), t (triplet), q (quartet) and m (multiple). The synthesized
compounds are at least 95% pure according to HPLC analysis. ESI-
MS spectra were obtained with an agility MSD trap mass spectrom-
eter. The separation was carried on a reversed-phase column with
dimensions 150 ꢁ 4.6 mm and a particle size of 5
lm. Thin layer
Based on the above analysis, this work provided an effective
method in the development of the novel podophyllum Topo II
inhibitors for discovery potential antitumor prodrug. Most of
compounds exhibit higher antitumor activities than etoposide,
which is a clinically important Topo II inhibitory anticancer
drug. These results provided the determinants of DNA Top II
binding affinity for this important class of anti-tumor
agents and pave the way for further rational structural
modification.
chromatography (TLC) analysis was carried out with a precoated
silica gel G plate, and a small spot of solution containing the sam-
ples was applied onto the plate, approximately 1.5 cm from the
bottom edge. The plate was then placed in a chamber with a sol-
vent system consisting of chloroform/acetone (10:1 to 1:1, v/v).
TLC spots were observed under an ultraviolet light (UV254) and
then observed after spraying with H2SO4/MeOH (10:1 v/v) and
heating to 110 °C.

3000
W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
2.3. General procedure for the synthesis of compounds
2.3.5. Compound (4) 4-O-(fluoroquinolonic)-40-
demethylepipodophyllotoxin
2.3.1. Synthesis of esterification 40-demethylepipodophyllotoxin
derivatives
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR: d = 8.73 (s, 1H; CH), 8.09 (d, 1H; Ar-
H), 7.88 (d, 1H; Ar-H), 6.82 (s, 1H; Ar-H), 6.36 (s, 1H; Ar-H), 6.28
(s, 2H; Ar-H), 5.85 (s, 2H, OCH2O), 5.22 (s, 1H; OH), 4.77 (s, 1H;
CH), 4.25 (s, 1H; CH), 4.00 (s, 1H; CH), 3.59 (d, 3H; OCH3), 3.45
(s, 6H; CH), 3.29 (d, 1H; CH), 3.24 (s, 1H; CH), 1.41 (s, 2H; CH2),
1.08 (s, 2H; CH2). 13C NMR: d = 175.92 (COOCH2), 161.86 (COO),
154.42 (CO), 151.81 (CCH), 149.95 (CCH), 148.26 (COCH3), 147.46
(CH), 138.84 (CN), 137.38 (CH), 132.86 (CN), 131.48 (C), 128.62
(C), 127.44 (C), 119.46 (C), 114.01 (C),110.26 (CH), 109.95 (CH),
109.29 (CH), 107.80 (OCH2O), 101.67 (OCH2O), 68.37 (CH), 66.23
(CH), 56.40 (OCH3), 49.45 (CH2), 44.24 (CH), 40.77 (CH), 38.88
(CH), 35.39 (CH3), 28.17 (CH3), ppm (CH). MS (ESI): m/z: 664
[M+H]+; C34H27ClFO10N.
DMEP (400 mg, 1 mmol), carboxylic acid (3 mmol), N,N0-dicy-
clohexylcarbodiimide (6 mmol) and 4-dimethylaminopyridine
(0.05 mmol) were mixed in 5 mL dichloromethane. All reactions
were stirred under nitrogen at room temperature for 24 h and
monitored by TLC (20 cm ꢁ 20 cm glass plates coated with a
0.5 mm layer of Merck silica gel GF 254). The reaction mixture
was diluted with dichloromethane (100 mL), washed with satu-
rated aqueous sodium bicarbonate (40 mL ꢁ 2), and water
(40 mL ꢁ 2). The organic layer was dried over anhydrous Na2SO4,
and then the liquid was removed by rotary evaporation in crude
solid. The product was purified by silica gel column chromatogra-
phy to give as a white solid.
2.3.2. Compound (1) 4-O-(2-pyrazinecarboxylic)-40-
demethylepipodophyllotoxin
2.3.6. Compound (5) 4-O-(2-quinoline carboxylic)-40-
demethylepipodophyllotoxin
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR d = 9.37 (s, 1H; CH), 9.07 (s, 1H; CH),
8.86 (s, 1H; CH), 7.05 (s, 1H; Ar-H), 6.65 (s, 1H; Ar-H), 6.38 (s,
2H; Ar-H), 6.07 (s, 1H; OCH2O), 6.02 (s, 1H; OCH2O), 5.73 (s, 1H;
OH), 4.79 (d, 2J (4, 4) = 4.8 Hz, 1H; CH), 4.47 (t, 1H; CH), 4.05 (t,
1H; CH), 3.85 (s, 6H; OCH3), 3.49 (d, 2J (1, 1) = 4.8 Hz, 1H; CH),
3.47 (d, 2J (2, 2) = 4.8 Hz, 1H; CH), 3.26–3.18 (m, 1H; CH). 13C
NMR: d = 174.42 (COOCH2), 164.05 (COO), 149.54 (CCH), 148.48
(CCH), 147.80 (COCH3), 146.87 (COCH3), 146.68 (CCH3), 145.04
(CHN), 143.06 (CHN), 134.45 (CCH), 133.87 (CCH), 130.17 (CCH),
127.15 (COH), 110.69 (CH), 110.05 (CH), 107.98 (CH), 102.07
(OCH2O), 70.65 (CH), 67.66 (CH2), 56.74 (OCH3), 43.96 (CH),
41.99 (CH), 37.12 ppm (CH). MS (ESI): m/z: 507 [M+H]+, 524
[M+NH4] +; C26H22O9N2.
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR d = 8.87 (s, 1H; CH), 8.52 (s, 1H; CH),
8.26 (s, 2H; Ar-H), 7.75 (s, 1H; Ar-H), 7.34 (s, 1H; Ar-H), 7.05 (s,
1H; Ar-H), 6.65 (s, 1H; Ar-H), 6.38 (s, 2H; Ar-H), 6.02 (d, 2H; OCH2-
O), 5.73 (s, 1H; OH), 4.79 (d, 1H; CH), 4.47 (t, 1H; CH), 4.05 (t, 1H;
CH), 3.85 (s, 6H; OCH3), 3.49 (d, 1H; CH), 3.47 (d, 1H; CH), 3.22 (m,
1H; CH). 13C NMR: d = 174.42 (COOCH2), 164.05 (COO), 149.54
(CCH), 148.48 (CCH), 147.80 (COCH3), 146.87 (COCH3), 146.68
(CCH3), 145.04 (CHN), 143.06 (CHN), 134.45 (CCH), 133.87 (CCH),
130.17 (CCH), 127.15 (COH), 110.69 (CH), 110.05 (CH), 107.98
(CH), 102.07 (OCH2O), 70.65 (CH), 67.66 (CH2), 56.74 (OCH3),
43.96 (CH), 41.99 (CH), 37.12 ppm (CH). MS (ESI): m/z: 556
[M+H]+; C31H25O9N.
2.3.7. Synthesis of amination 40-demethylepipodophyllotoxin
derivatives
2.3.3. Compound (2) 4-O-(5-methylpyrazine-2-carboxylic)-40-
demethylepipodophyllotoxin
DMEP (400 mg, 5 mmol), triethylamine (1.68 mL), 4-toluene-
sulfonylchloride (0.96 mL) were mixed in 40 mL dichloromethane.
All reactions were stirred under nitrogen at room temperature for
3 h and monitored by TLC (20 cm ꢁ 20 cm glass plates coated with
a 0.5 mm layer of Merck silica gel GF 254). The reaction mixture
was washed with saturated aqueous sodium bicarbonate
(40 mL ꢁ 2), and deionized water (40 mL ꢁ 2). The organic layer
was dried over anhydrous Na2SO4, and then the liquid was
removed by rotary evaporation in crude solid. Then, the crude solid
(1 mmol), module (3 mmol) were mixed in 10 mL acetonitrile. All
reactions were stirred under nitrogen at 80 °C for 48 h and moni-
tored by TLC and then the liquid was removed by rotary evapora-
tion. The product was purified by silica gel column
chromatography to give as a white solid.
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR: d = 9.16 (s, 1H; CH), 8.60 (s, 1H;
CH), 6.95 (s, 1H; Ar-H), 6.67 (s, 1H; Ar-H), 6.47 (s, 1H; Ar-H),
6.30 (s, 1H; Ar-H), 5.99 (d, 2H; OCH2O), 5.94 (s, 1H; OH), 4.71 (d,
1H; CH), 4.38 (t, 1H; CH2), 4.45 (t, 1H; CH2), 3.77 (s, 6H; OCH3),
3.42 (d, 2J (1, 1) = 5.1 Hz, 1H; CH), 3.39 (d, 2J (2, 2) = 4.8 Hz, 1H;
CH), 3.13–3.09 (m, 1H; CH). 2.67 (s, 3H; CH3). 13C NMR:
d = 174.44 (COOCH2), 163.46 (COO), 158.83 (CCH3), 149.43 (CCH),
147.72 (CCH3), 146.75 (COCH3), 145.69 (CHN), 144.83 (CHN),
134.34 (CH), 133.74 (CH), 130.14 (CH), 127.19 (CCH), 110.58
(CH), 109.96 (CH), 107.90 (CH), 101.96 (OCH2O), 70.30 (CH),
67.62 (CH2), 56.66 (OCH3), 43.90 (CH), 41.92 (CH), 37.05 ppm
(CH). MS (ESI): m/z: 521 [M+H]+; C27H24O9N2.
2.3.4. Compound (3) 4-O-(theophylline-7-acetic)-40-
demethylepipodophyllotoxin
2.3.8. Compound (6) 4-N-(2-pyrazinecarboxylic) amino-40-
demethylepipodophyllotoxin
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR: d = 8.49 (s, 1H; CH), 6.63 (s, 1H; Ar-
H), 6.25 (s, 1H; Ar-H), 6.07 (s, 2H; Ar-H), 5.72 (d, 2J (13,
13) = 3.6 Hz, 2H; OCH2O), 5.19(s, 1H; OH), 4.92 (s, H; CH), 4.60 (s,
1H; CH), 4.10 (t, 1H; CH), 3.45 (d, 3H; CH3); 3.35 (s, 6H; OCH3),
3.12 (m, 1H; CH), 2.64 (s, 1H; CH), 2.54 (s, 1H; CH), 1.34 (s, 3H;
CH3). 13C NMR: d = 175.32 (COOCH2), 165.32 (COO), 155.38 (CO),
151.92 (CCH), 151.35 (CCH), 148.68 (COCH3), 147.72 (CH), 142.32
(CH), 138.93 (CN), 132.24 (CH), 131.54 (CN), 127.21 (C), 110.57
(CH), 109.41 (CH), 107.73 (CH), 101.86 (OCH2O), 67.94 (CH),
66.72 (CH), 56.43 (OCH3), 47.10 (CH2), 44.17 (CH), 40.62 (CH),
38.55 (CH), 30.09 (CH3), 28.17 (CH3), ppm (CH). MS (ESI): m/z:
621 [M+H]+; C30H28O11N4.
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR d = 9.37 (s, 1H; CH), 9.07 (s, 1H; CH),
8.86 (s, 1H; CH), 7.05 (s, 1H; Ar-H), 6.65 (s, 1H; Ar-H), 6.38 (s,
2H; Ar-H), 6.02 (d, 2H; OCH2O), 5.73 (s, 1H; OH), 4.79 (d, 1H;
CH), 4.47 (t, 1H; CH), 4.05 (t, 1H; CH), 3.85 (s, 6H; OCH3), 3.49
(d, 1H; CH), 3.47 (d, 1H; CH), 3.22 (m, 1H; CH). 13C NMR:
d = 174.42 (COOCH2), 164.05 (COO), 149.54 (CCH), 148.48 (CCH),
147.80 (COCH3), 146.87 (COCH3), 146.68 (CCH3), 145.04 (CHN),
143.06 (CHN), 134.45 (CCH), 133.87 (CCH), 130.17 (CCH),
127.15 (COH), 110.69 (CH), 110.05 (CH), 107.98 (CH), 102.07
(OCH2O), 70.65 (CH), 67.66 (CH2), 56.74 (OCH3), 43.96 (CH),
41.99 (CH), 37.12 ppm (CH). ESI MS: m/z: 507 [M+H]+;
C26H22O9N2.

W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
3001
2.3.9. Compound (7) 4-N-(5-methylpyrazine-2-carboxylic)
amino-40-demethylepipodophyllotoxin
cytotoxic effects. Drug stock solutions (the each drug concentra-
tion was 0.5, 1, 10, 20, 50, 100, 200, and 500 M, respectively) were
l
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR: d = 8.97 (s, 1H; CH), 8.55 (s, 1H;
CH), 8.33 (s, 1H; NH), 6.79 (s, 1H; Ar-H), 6.53 (s, 1H; Ar-H), 6.24
(s, 1H; Ar-H), 5.94 (d, 2J (13, 13) = 9.3 Hz, 2H; OCH2O), 5.41 (t,
1H; OH), 4.47 (d, 2J (4, 4) = 4.8 Hz, 1H; CH), 4.33 (t, 1H; CH), 3.80
(t, 6H; OCH3), 3.47 (d, 2J (1,1) = 5.4 Hz, 1H; CH), 3.39 (d, 2J (2,
2) = 6.9 Hz, 1H; CH), 3.02 (s, 1H; CH), 3.25 (s, 1H; CH3), 2.60 (s,
3H; CH3). 13C NMR: d = 175.43 (COOCH2), 164.22 (NHCO), 157.75
(CCH3), 147.96 (CCH), 147.80 (CCH), 147.16 (COCH3), 143.46
(CHN), 142.57 (CHN), 135.21 (CH), 133.52 (CH), 130.96 (CH),
130.29 (CCH), 110.17 (CH), 109.57 (CH), 108.99 (CH), 101.90
(OCH2O), 68.97 (CH2), 56.65 (OCH3), 47.95 (CH), 43.63 (CH),
41.27 (CH), 37.29 ppm (CH). MS (ESI): m/z: 521 [M+H]+;
prepared in DMSO and the final solvent concentration was 62%
DMSO (v/v), a concentration without effect on cell replication. Ini-
tial seeding densities varied among the cell lines to ensure a final
absorbance reading in control (untreated) cultures in the range
0.6–0.8 A492 units. Drug exposure was for 2 days, and the IC50
value, the drug concentration that reduced the absorbance by
50%, was interpolated from dose–response data. Each test was per-
formed in triplicate, and absorbance readings varied no more than
5%. Values were averaged from six treated wells and values for
medium alone (lane 1) were subtracted. These values were then
normalized to control (unstimulated cells, lane 2) and used to cal-
culate the effective half-maximal concentrations for each drug as
well as the percentage of cell death (treated values, normalized
17
C27H24O9N2.
to control ꢁ 100) induced at ICmax
.
2.3.10. Compound (8) 4-N-(theophylline-7-acetic) amino-40-
demethylepipodophyllotoxin
2.5. Statistical evaluation
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR: d = 8.71 (d, 2J = 8.1 Hz, 1H; CH),
6.81 (s, 1H; Ar-H), 6.55 (s, 1H; Ar-H), 6.28 (d, 2J (2’, 6’) = 11.7 Hz,
2H; Ar-H), 6.02 (t, 2H; OCH2O), 5.18(m, 1H; OH), 4.99 (t, 2H;
CH2), 4.53 (d, 2J (4, 4) = 5.1 Hz, 1H; CH), 4.23 (t, 1H; CH), 3.94 (t,
1H; NH); 3.63 (s, 6H; OCH3), 3.44 (s, 3H; CH3), 3.32 (s, 1H; CH),
3.21 (s, 3H; CH3), 3.15 (d, 2J (2 ,2) = 5.1 Hz, 1H; CH), 2.96(d, 2J (3,
3) = 9.6 Hz, 1H; CH). 13C NMR: d = 175.18 (COOCH2), 166.84
(NHCO), 151.72 (CO), 147.83 (CCH), 147.26 (CCH), 144.31 (COCH3),
135.40 (CH), 133.03 (CH), 130.77 (CH), 110.12 (CH), 109.15 (CH),
107.23 (CH), 101.96 (OCH2O), 68.95 (CH2), 56.69 (OCH3), 48.96
(CH), 47.87 (CH2), 43.58 (CH), 41.60 (CH), 37.28 ppm (CH). MS
(ESI): m/z: 621 [M+H]+; C30H28O11N4.
Data presented as means SD. Statistical analyses were per-
formed by the analysis of variance (ANOVA). All statistical analyses
were performed using Origin version 8.0 (GraphPad Software, Orig-
inLab Corp., Northampton, MA, USA). Sigmoidal dose responses
and non-linear regression analyses were undertaken to identify
half-maximal concentrations for each of the drugs. To evaluate dif-
ferences in IC50 concentrations, analysis of variance combined with
Tukey’s multiple range test was used.
2.6. Octanol–water partition coefficients
LogP is one criterion used in medicinal chemistry to assess the
drug likeness of a given molecule, and used to calculate lipophilic
efficien⁄⁄cy. It was calculated with following equation: logP = log
2.3.11. Compound (9) 4-N-(3,4,5-trihydroxybenzoic) amino-40-
demethylepipodophyllotoxin
[Cin 1-octanol/(Cin water⁄Y)]. Cin
is the compound concentra-
1-octanol
tion in 1-octanol, Cin water is the compound concentration in water,
Y is a correction for index error = 2.555. The partition coefficient P
was determined according to the standard method18 and the
reported P values are an average of three measurements. For a
water-soluble compound, the reported logP values could be low.
1H NMR (300 MHz, DMSO, 25 °C, TMS) and 13C NMR (75 MHz,
DMSO, 25 °C, TMS). 1H NMR: d = 6.84 (s, 2H; Ar-H), 6.77 (s, 2H;
Ar-H), 6.53 (s, 1H; Ar-H), 6.24 (s, 1H Ar-H), 5.97 (d, 2J (13,
13) = 8.1 Hz 2H; OCH2O), 5.77 (s, 3H; OH), 5.36 (s, 1H; OH), 4.47
(d, 2J (4, 4) = 4.8 Hz, 1H; CH), 4.32 (s, 2H; CH2), 4.32 (s, 1H; CH),
3.64 (d, 2J (3’, 5’) = 16.2 Hz, 6H; OCH3), 3.45 (d, 2J (2, 2) = 5.1 Hz
2H, CH), 3.41 (s, 1H; CH), 3.32 (s, 1H; CH). 13C NMR: d = 175.44
(COOCH2), 167.35 (CONH), 147.88 (CCH), 146.09 (CCH), 137.21
(COCH3), 135.41 (CCH), 133.11 (CCH), 131.39 (COH), 130.99 (C),
124.85 (CH), 110.03 (CH), 109.58 (CH), 109.19 (CH), 107.84 (CH),
101.84 (OCH2O), 69.14 (CH2), 56.71 (OCH3), 47.80 (CH), 43.68
(CH), 41.44 (CH), 37.38 ppm (CH). MS (ESI): m/z: 567 [M+H]+;
2.7. kDNA decatenation assay
Topoisomerase II activity was determined using kit (Topogen
Inc., USA, Cat No. 2000H). The substrate kDNA (134 ng) and
10
l
M drug were combined in assay buffer (5 ꢁ Topo II assay buf-
fer (A+B)) and incubated for 10 min on ice. Next, 4 U of topoisomer-
ase II was added and the reaction was allowed to proceed for
60 min at 37 °C. The reaction was stopped by the addition of 1/
10 volume of 1 mg/mL proteinase K and incubated for 15 min at
37 °C. The reaction was quenched via the addition of loading buffer
(1% sarkosyl, 0.025% bromophenol blue, and 5% glycerol) and was
then analyzed by electrophoresis on a 1% agarose gel in TBE buffer
(89 mM Tris, 89 mM borate, and 2 mM Na-EDTA, pH 8.3) for 0.5 h
at 140 V. The gel was visualized under UV illumination and photo-
graphed on an Alpha Imager.
C29H26O12.
2.4. Cytotoxicity assay
Cytotoxicity assay was performed on human lung adenocarci-
noma epithelial cell line (A549), human henrietta lacks strain of
cancer cells (HeLa), human gastric carcinoma cell line (BGC-823),
human hepatocellular liver carcinoma cell line (HepG2), and
human normal cell Hepatic immortal cell line (HL-7702). Cells
(3500–13,000) in a 100
lL culture medium per well were seeded
2.8. Cell cycle arrest assay
into 96-well microtest plates (Falcon, CA). A549 cell lines, HeLa cell
lines and HepG2 cell lines were cultured in DMEM supplemented
with 10% CS, 100 mg/L penicillin G, 100 mg/L streptomycin. BGC-
823 cell lines were cultured in RPMI Medium 1640 supplemented
with 10% CS, 100 mg/L penicillin G, and 100 mg/L streptomycin.
The cells were incubated at 37 °C, in a humidified atmosphere with
5% CO2 for 48 h. For all cell lines, the microculture tetrazolium
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
MTT; Sigma, St. Louis, MO] assay was performed to measure the
HeLa were seeded in six multiwell plates at a density of
2 ꢁ 106 cells/plate. After 48 h of incubation with compound 1 with
various concentration(0, 0.1, 0.5, 1, 5 lM) in DMEM without serum
at 37 °C, cells were washed in PBS, pelleted, centrifuged, and
directly stained in a propidium iodide solution (50 mg of PI in
0.1% sodium citrate, 0.1% NP40, pH 7.4) for 30 min at 4 °C in the
dark. Flow cytometric analysis was performed using a FAC Scan
flow cytometer (Becton Dickinson, San Jose, CA). To evaluate the

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W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
cell cycle, PI fluorescence was collected as FL2 (linear scale) by the
Mod FIT software (Becton Dickinson). For the evaluation of intra-
cellular DNA content, at least 20,000 events for each point were
analyzed in at least three different experiments giving a SD of less
than 5%.
were computed by using AutoGrid 4, which was included in the
Autodock4 software. The graphical user interface ADT was
employed to set up the enzymes; in that, all hydrogen atoms were
added, the Gasteiger charges were calculated, and nonpolar hydro-
gen atoms were merged with the carbon atoms. For macromole-
cules, the generated pdbqt files were saved. Then, based on the
Auto-Dock software, a docking procedure was applied to position
the conformation of these compounds correctly with regard to
their active sites. Grid maps representing the topoisomerase II pro-
tein in the actual docking process were constructed with Auto Grid.
The points of the grids were thus set at 60 ꢁ 60 ꢁ 60 with a grid
spacing of 0.375 Å to ensure that it was sufficiently large to include
not only the active site but also significant portions of the sur-
rounding surface.
2.9. Annexin V/PI apoptosis assay
An amount of 2 ꢁ 106 cells/mL was plated in 6 well plates, trea-
ted with compound 1 at a range of concentrations 0, 0.5, 1, 5, and
10
1000 rpm for 10 min. The supernatant was discarded, and the cells
were resuspended in 100 L PBS. Apoptosis using a MultiSciences
Biotech kit (Lot: 1200945) kit, and 10 L annexin V and 5 L PI
lM and incubated for 48 h. The cells were centrifuged at
l
l
l
was added. The samples in the labeling solution were transferred
into Falcon tubes and incubated in a water bath at 37 °C for
20 min. The samples were then analyzed using a Becton Dickinson
FAC scan flow cytometer with Cell Quest software. The results were
tabulated as percentage of annexin V-FITC positive apoptotic cells.
The experiments were performed in three replicates and repeated
at least twice for each compound.
3. Results and discussion
3.1. Drug molecular design
The process of drug molecular design was performed in three
steps. Firstly, numerous studies on 40-demethylepipodophyllotoxin
(DMEP) currently focus on its structure modification of the cyclo-
paraffin (C-ring) in the tetranap skeleton, and the steric clash and
electrostatic contour plots of the comparative molecular field anal-
ysis models6 shows that the C-4 position would be a great modifi-
cation position to improve antitumor activity of DMEP. Meanwhile,
in our previous work, the DMEP derivate 4b-S-(1,2,4-triazole-3)-4-
demethylepipodophyllotoxin that structurally modified at C-4
position of DMEP against tumor cell line BGC-823 (IC50 values of
2.10. Computational docking simulations
Molecular docking of compounds into the X-ray structure of the
human topoisomerase II, DNA and VP-16 complex (PDB: 3QX3)
from the RCSB Protein Data Bank was carried out by using the
Auto-Dock software (version 4.2) via the graphic user interface
Auto-Dock Tool Kit (ADT 1.5.4).19 The grid maps of docking studies
Scheme 1. Synthesis of esterification and amidation 40-demethylepipodophyllotoxin derivatives 1–9.

W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
3003
0.28
0.42
l
l
M), A549 (IC50 values of 0.76
M) were significantly improved by 91, 221 and 2.73 times
l
M) and HepG2 (IC50 values of
chemistry, the higher electron density of nitrogen atom making
them easily attack the action of target protein and improve the
antitumor activity of leading compounds.24,25 Benzene ring con-
than those of etoposide (IC50 values of 25.72, 167.97 and
1.15
l
M), respectively.20 Furthermore, the structure of the Topo
tains a large p bond, which can easily form p–p accumulation with
II-DNA cleavage complex finding that electrophilic group in the
C-4 position of DMEP rests in a spacious binding pocket with rela-
tively few interactions, so it can be either modified or replaced to
produce derivatives with enhanced Topo-poisoning activities, it
appears that those non-C4 substitutions would cause steric con-
flicts and impair the drug-binding site. Thus, the C-4 position of
DMEP was ensured for an effective modification site for generating
new bioactive DMEP derivatives. Secondly, according to drug com-
bination principle,7,8 the combination of two kinds of drugs
through the ester bond can gain some new compounds with better
bioactivity. Many famous drugs in clinic use such as sultamicillin
and benorilate were discovered through this way. So, DMEP could
be combined with other compounds through ester bond for the
better bioactivity and attenuated toxicity. Thirdly, according to
bioisosterism9,10, both of the ester bond (–COO–) and the –NH–
bond are the bioisosteres with the same numbers of the outermost
electrons but with different electronegativity. Electronegativity
(electronegativity = constant*(|EA| + |IE|)/2, EA: energy liberated
when an extra electron attaches to a single neutral atom, IE: energy
required to remove an existing valence electrons from the neutral
atom) generally increases from left to right on the periodic table
and decreases from top to bottom. The most commonly used scale
of electronegativity is that developed by Linus Pauling21,22 in
which the value 3.44 is assigned to oxygen, 3.04 is assigned to oxy-
gen, and 2.10 is assigned to hydrogen. Although nitrogen has low
EA, the chemical bond –NH– with the polarity has enough IE for
higher Electronegativity than oxygen. Because hydrogen has an
electrognegativity of 2.10 and nitrogen has an electronegativity
of 3.04, they could form a polar molecule with the nitrogen being
the negative side of the dipole. The –NH– with the greater electro-
negativity may be denser than that of the oxygen atom. So, the
amidation is another effective pathway to improve the antitumor
activity of DMEP. Fourthly, it is extensively reported that the cyto-
toxicity of the Topo II poisons etoposide stems from its ability to
stabilize the DNA-Topo II complex and prevent the relegation of
the double-stranded breaks, which also results in inhibition of
DNA replication and apoptotic cell death.23 Comparatively, the fur-
ther studies focused on the C-4 position modification of the sub-
stituents with high electron density were much more promising.
The substituents with high electron density were favorable for
stabilization the complex of Topo II-DNA cleavage complex. So, a
series of heterocyclic with pharmacological activity and electro-
negative atom (i.e., oxygen, nitrogen) were chosen as module.
N-Benzyl heterocycles, which were frequently found in medicinal
biological molecules, thus can enhance the interaction between
drugs and target proteins. The quinolones26,27 derivates were a
family of anti-tumor drugs target to DNA topoisomerase II, the
same as DMEP derivates. Combing quinolones with DMEP enable
to enhance the targeting of the new DMEP derivates to DNA topo-
isomerase II. Base on the above analysis, we ensured amination and
esterification for two modification directions. In this effort, nine
novel esterification and alkane amination DMEP derivates were
rationally designed as target compounds, the structure of target
compounds were shown in Scheme 1.
3.2. Synthesis of esterification and amidation
40-demethylepipodophyllotoxin derivatives
The synthetic route to the target compounds is depicted in
Scheme 1. DMEP was condensed with the appropriate carboxylic
acids in the presence of N,N-dicyclohexylcarbodiimide (DCC) and
4-(dimethylamino) pyridine (DMAP) to provide the ester linkage
compounds 1–5 under an atmosphere of nitrogen at room temper-
ature.28 Compounds 6–9 were synthesized by three steps as
described in the reported method.29,30 Firstly, using sodium azide
as catalyst, 40-demethylepipodophyllotoxin was reducted to 4-
azido-40-demethylepipodophyllotoxin. Then, the key intermediate
4-amino-40-demethylepipodophyllotoxin was obtained by
Figure 1. Inhibition of human topoisomerase II (Topo II) catalytic activity by
compound 1. All reaction samples were electrophoresed in 1% agarose gels as
described in the Topo II mediated kDNA relaxation assay.
Table 1
The cytotoxic activity and the solubility of the esterification and amidation podophyllum compounds
Compound
Cytotoxic activitya (IC50
A549b
,
lM)
LogPa
HepG2b
HeLab
BGC-823b
HL-7702b
1
2
3
4
5
6
7
8
1.21 0.05
14.22 0.68
7.91 1.67
16.03 2.54
9.18 1.21
6.64 1.42
6.78 1.08
2.35 0.99
27.41 3.98
18.74 2.71
15.32 0.10
0.88 0.20
9.56 2.44
0.65 0.11
0.60 0.08
20.53 3.50
10.84 1.82
16.05 2.70
3.18 0.19
22.27 0.77
15.96 1.22
59.38 0.77
3.83 0.08
10.51 2.91
6.91 1.15
10.05 2.43
19.20 4.04
16.46 1.82
18.71 2.24
7.32 0.56
33.54 4.71
52.08 0.85
67.25 7.05
4.15 1.13
1.50 0.09
2.00 0.97
17.41 5.07
28.81 3.34
14.38 2.91
3.49 1.12
5.88 1.07
24.17 3.42
21.26 2.42
30.74 5.13
52.43 5.32
18.63 5.23
38.01 3.42
41.77 3.91
20.09 3.11
53.63 6.92
20.68 1.05
50.82 4.53
14.82 3.40
13.04 1.55
24.61 3.82
0.33 0.11
0.38 0.23
0.43 0.15
0.69 0.21
0.80 0.05
0.43 0.12
0.59 0.08
0.56 0.19
0.29 0.02
0.73 0.08
0.52 0.13
9
DMEP
Etoposide
a
The value was the average of triplicates.
MTT methods, drug exposure was for 48 h.
b

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W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
reducting 4-azido-40-demethylepipodophyllotoxin, which was treated
zinc (Zn) in ammonium formate. At last, combination 4b-amino-
40-demethylepipodophyllotoxin with various carboxylic acids in
presence of 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide
hydrochloride (EDCꢀHCl) and 1-hydroxybenzotriazole hydrate
(HOBT), the target compounds were successfully synthesized. The
details were shown in Scheme 1.
HepG2 (i.e., the IC50 value of 1.21 0.05
lM), and BGC-823 (i.e.,
the IC50 value of 4.15 1.13 M) was significantly improved by
l
66, 16, 12, and 6 times than that of the clinically important podo-
phyllum anticancer drug etoposide (i.e., the IC50 values of
15.32 0.10, 59.38 0.77, 67.25 7.05, and 30.74 5.13
respectively.
As shown in Table 1, although the growth of all cell lines was
inhibited by any of the compounds used independently in a
dose-dependent manner, the data demonstrated that different
compounds display different sensitivity to the same cell. Firstly,
esterification derivates (e.g., compounds 1–3) displayed better
antitumor activities than amidiation derivates (e.g., compounds
6–8) against four cancer cell lines. Secondly, N-benzyl heterocy-
cle-substitution derivates (e.g., compounds 1–3 whose 50% effec-
lM),
3.3. Activity evaluation
3.3.1. Effect of compounds on the proliferation of human tumor
cell lines
In the present study, the antitumor activities in vitro of nine
novel esterification and alkane amination DMEP derivates on the
several human tumor cell lines were investigated. The antineoplas-
tic drug etoposide (VP-16) was used as reference standard. Cyto-
tive concentration between 1–10
activities than benzene ring-substitution derivates (e.g., compound
9 whose 50% effective concentration between 22–33 M). At last,
lM) showed better antitumor
toxicity values for DMEP were included in Table
1
for
l
comparative purposes. When these cells treated with different
concentrations of DMEP derivatives cultured up to 48 h, there
was an increase in the percentage of cells being shrunk observed
with inverted microscope.
As shown in Table 1, with few exception, the results of inhibi-
tory indicated that the most of nine novel esterification and alkane
amination DMEP derivates potential antitumor activity on the
HepG2, HeLa, A549 and BGC-823 tumor cell lines. N-Benzyl hetero-
cycle-substitution DMEP derivate 4-O-(2-pyrazinecarboxylic)-40-
demethylepipodophyllotoxin (compound 1). The antitumor activ-
ity of compound 1 against tumor cell line HeLa (i.e., the IC50 value
another important critical trend observed across the entire data
set was compounds possessing an electrondonating substituent
showed enhanced antitumor activity such as the presence of F, Cl
atom (i.e., compound 4) increased the antitumor activity against
four cell lines. On the contrary, starting from compounds 1 and
2, removing the methyl group resulted in a moderate drop of
potency (i.e., compounds 6 and 7) antitumor activity. Simulta-
neously, most of the compounds exhibited the low toxic-side
effects. As shown in Table 1, the IC50 value of compound 1 (i.e.,
52.43 5.32
4 (i.e., 41.77 3.91
compound 8 (i.e., 50.82 4.53 l
l
M), compound 3 (i.e., 38.01 3.42
M), compound 6 (i.e., 53.63 6.92
M) against on the human normal
l
M), compound
l
lM), and
of 0.60 0.20
lM), A549 (i.e., the IC50 value of 3.83 0.08
lM),
Figure 2. Effect of compound 1 on the HeLa cell cycle arrest. Cell cycle arrest detection in HeLa cells using propidium iodide (PI) double staining treatment with 0, 0.5, 1, and
M compound 1 for 24 h.
5
l

W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
3005
Figure 3. Compound 1 induced HeLa cell apoptosis. Apoptosis detection in HeLa cells using annexin V and propidium iodide (PI) double staining treatment with 0, 1, 5, and
10 M compound 1 for 48 h. The cell scatter could be composed of four subgroups as follows: Q1 quadrant was the necrotic cells, Q2 quadrant was late apoptotic cells, Q3
l
quadrant was the living cells late, and Q4 quadrant was the early apoptotic cells.
cell HL-7702 was improved 114%, 54%, 70%, 117%, and 106% than
that of etoposide (i.e., 24.61 3.82 lM), respectively. The IC50 val-
to test drug potential to inhibit DNA Top II activity in a cell-free
system. To understand the action mechanisms of antitumor activ-
ity of these compounds, the effect on the catalytic activity of
human topoisomerase II was evaluated. As shown in Figure 1, com-
pound 1 diminished the relaxation reaction at a concentration 10,
ues indicate that most of compounds have promising anti-tumor
activity and lower cytotoxic efficacy on normal human cell line
HL-7702 than the standard compounds etoposide. On the other
hand, As expected, the partition coefficient logP indicated that
most of compounds exhibited the higher the water-solubility than
that of etoposide, and compound 9 had the lowest the partition
coefficient (i.e., logP = 0.29 0.02) among the other compounds.
Compared with etoposide (i.e., logP = 0.52 0.13), the partition
coefficient of compound 9 (i.e., logP = 0.32 0.11) was significantly
decreased 79%. This maybe the trihydroxy benzene with three
hydroxyl groups at C-4 position of compound 9 resulted in the
improvement of the water-solubility. Furthermore, interestingly,
the water-solubility of compound 1(i.e., logP = 0.33 0.11) was
better than that of compound 6 (i.e., logP = 0.43 0.12), the
water-solubility of compound 2 (i.e., logP = 0.38 0.23) was better
than that of compound 7 (i.e., logP = 0.59 0.08), the water-solu-
bility of compound 3 (i.e., logP = 0.43 0.15) was better than that
of compound 8 (i.e., logP = 0.56 0.19). These demonstrate that
the same substitution with the ester bond at the C-4 position of
DMEP was greater effect than the amido bond for improving the
water-solubility.
5, 2.5, and 1
lM, respectively, and completely inhibited the cata-
lytic activity of topoisomerase II at 10
lM of concentration, which
was much better than the effect of etoposide at same concentra-
tion. These results provide strong evidence that compound 1 with
pyrazine exhibit antiproliferative properties against human cancer
cells by inhibiting the activity of topoisomerase II. These may be
also indicated a different mode of action for that pyridine is com-
petitive nonselective phosphodiesterase inhibitor which raises
intracellular cAMP and activates apoptosis pathway correlated
with antitumor activity.
3.3.3. Effect of compound 1 on the HeLa cells cycle arrest and
apoptosis
On the basis of the above results, further biological evaluations
have been focused on compound 1. To gain further insight into the
mode of action of compound 1, we examined the effects of com-
pound 1 on cell cycle by flow cytometry in HeLa cells. Interestingly,
a concentration dependent change was observed in the cell cycle
pattern (Fig. 2). Our results demonstrated that treatment of HeLa
3.3.2. Effect of compounds on the Topo II DNA decatenation
assay
DMEP derivates have been reported to induce cell death by
inhibiting DNA Top II activity. So we investigated the efficacy of
the newly synthesized compounds for human DNA Top II enzyme
inhibiting ability. The kDNA decatenation assay has been utilized
cells for 24 h with 0.5, 1 and 5 lM of compound 1 could cause cell
cycle arrest at G2/M phase from 15.44% to 66.85%. To probe
whether compound 1 induced HeLa apoptosis, Flow cytometry
was used to generate an apoptotic cell scatterplot of the control
groups treated with compound 1. As shown in Figure 3, different
concentrations (i.e., 1 , 5, and 10 lM) of compound 1 were treated

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W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
Figure 4. (A) Structure of the topoisomerase II cleavage complex stabilized by compound 1. (B) Active pocket of topoisomerase II dimer bound with compound 1; (C) 3D view
of binding mode (the green dotted lines are H-bond); (D) 2D view of binding mode.
in HeLa for 48 h, the cells count in Q2 quadrants were considered
to be late apoptotic cells and the total percentages were 19.1%,
19.6%, 39.1% and 63.8%, respectively. The results showed that
the effect was observed in a dose-dependent manner and the apop-
totic rates of HeLa cells increased with the increase of concentra-
tion. It demonstrated that compound 1 induced apoptosis of cells
and thus as expected, caused the anticancer effects. In summary,
we have prepared a series of 40-demethylepipodophyllotoxin
derivatives. Antitumor evaluation indicates most of them per-
formed better antitumor activity than etoposide. For the
most promising compound 1, antitumor mechanism study showed
that compound 1 completely arrested HeLa cell at G2/M and
eventually induce apoptosis through inhibiting the activity of
Topo II.
It is quite similar with the binding mode of etoposide as reported
earlier. Hence, the amino acid residues with dioxolane ring and
trans-lactone ring can match that of compound 1 in space to firm
the conformation of the complex.
A careful inspection of the binding pocket showed (Fig. 4D) that
the trimethoxyphenyl ring (E ring) of 1 was inserted in a bigger
hydrophobic cavity formed by Arg 503, Lys 456, Asp 479, Leu
502, Gly 478, Gly 504. This result indicated that the hydrophobic
property was one of the main forces governing the interaction
between 1 and Topo II. On the other hand, the interaction was
not exclusively hydrophobic. It should be noted that there were
several polar residues (Arg 503, Lys 456, Asp 479, Gly 478, Gly
504, Gln 778)in the proximity to 1, which played important roles
in stabilizing the Topo II and compound 1 complex via electrostatic
interaction and Van der Waals’ force (Fig. 4D). The hydrogen-bond-
ing or electrostatic interaction acted as an anchor, intensely deter-
mining the 3D space position of 1 in the binding pocket and
facilitating the hydrophobic interaction of the ligand 1 with the
side chain of protein. In addition, it can be seen from the Figure 4D,
the nitrogen heterocyclic-substituted ring of compound 1 made a
3.3.4. Docking studies
As for the most promising compound 1, antitumor mechanism
study showed that it completely arrested HeLa cell in G2/M with
5 lM and eventually induce apoptosis through inhibiting the activ-
ity of Topo II. So, to study the molecular basis of interaction and
affinity of binding of the 40-demethylepipodophyllotoxin ana-
logues, compound 1, the most active compound, was used as the
representative one to investigate the binding modes of synthesized
40-demethylepipophyllotoxin derivatives.
Compound 1 was docked into the active site of Topoisomerase-
II for etoposide as reported earlier16,31, docking results are given in
Figure 4. Compound 1 was found to have a score of ꢂ12.85 kg/mol
with 1 H-bonds with Arg 503 and 3 H bonds with Asp 479 (Fig. 4C).
p–p interaction with DC: 8, which may contribute to the binding
and stabilization of the compound 1 with DNA, thereby, resulting
in strengthening the stability of DNA, Topo II and compound 1 ter-
nary complex. From the results, it was evident that the in silico
findings were well related to the IC50 values obtained through
in vitro cytotoxicity assay, furthermore, it displayed that com-
pound 1 induced apoptosis potentially by the strong interaction
of compound 1 with Topo II.

W. Zhao et al. / Bioorg. Med. Chem. 22 (2014) 2998–3007
3007
(2013070204020049) are gratefully acknowledged. Ya-Jie Tang
also thanks the Chutian Scholar Program (Hubei Provincial Depart-
ment of Education, China) (2006), Training Program for the Youth
Leading Talents by Ministry of Science & Technology, Program for
New Century Excellent Talents in University (NCET-11-0961),
and Training Program for Top Talents in Hubei Province.
4. Conclusion
This work presented the rational design method of the novel
topoisomerase II (Topo II) inhibitors for the synthesis of the ester-
ification and amidation substituted 40-demethylepipodophyllotox-
in (DMEP) derivates exhibiting antitumor activity. The correctness
of rational design was strictly demonstrated by the biological
activity tests. The bioassay tests showed that the designed novel
4-O-(2-pyrazinecarboxylic)-40-demethylepipodophyllotoxin (com-
pound 1) had superior in vitro biological activities including the
stronger anti-tumor activity, the lower cytotoxic activity on nor-
mal human cells, and the higher solubility, cell cycle G2/M arrest,
and induce apoptosis. The antitumor activity of 4-O-(2-pyrazine-
carboxylic)-40-demethylepipodophyllotoxin (compound 1) against
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l
l
l
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Acknowledgements
Financial supports from the National Natural Science Founda-
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