Synthesis and evaluation of the apoptosis inducing and CT DNA interaction properties of a series of 4β-carbamoyl 4′-O- demethylepipodophyllotoxins

Article abstract of DOI:10.1016/j.ejmech.2013.09.053

A series of carbamate derivatives of 4′-demethylepipodophyllotoxin have been synthesized, and their cytotoxicities against several human cancer cell lines, including HeLa, A549, HCT-8, and HL-60 cells, evaluated. Some of these compounds exhibited higher levels of cytotoxicity than the anticancer drug etoposide. 4β-4′-Demethylepipodophyllotoxin 1-(4-nitrophenyl) piperazinyl carbamate (19) was found to be the most potent compound of those synthesized in the current study, and induced cell cycle arrest in the G2/M phase in HeLa cells, which was accompanied by apoptosis. Furthermore, this compound activated the expression of Bax, p53 and caspase-3 in HeLa cells, leading to changes in the conformation of calf thymus DNA from the B-form to a more compact C-form.

Full text of DOI:10.1016/j.ejmech.2013.09.053

European Journal of Medicinal Chemistry 70 (2013) 59e67
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of the apoptosis inducing and CT DNA
interaction properties of a series of 4
demethylepipodophyllotoxins
b
-carbamoyl 40-O-
,
d,
**
Chun-Yan Sang a, Jian-Fei Liu a, Wen-Wen Qin a, Jie Zhao a, Lin Hui b , Yong-Xin Jin
,
**
,c,
Shi-Wu Chen a
*
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 Lab of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou 730000, China
d Department of Medicine, Second People’s Hospital of Gansu Province, Lanzhou 730000, China
a r t i c l e i n f o
a b s t r a c t
A series of carbamate derivatives of 40-demethylepipodophyllotoxin have been synthesized, and their
cytotoxicities against several human cancer cell lines, including HeLa, A549, HCT-8, and HL-60 cells,
evaluated. Some of these compounds exhibited higher levels of cytotoxicity than the anticancer drug
Article history:
Received 26 July 2013
Received in revised form
25 September 2013
Accepted 30 September 2013
Available online 6 October 2013
etoposide. 4b
-40-Demethylepipodophyllotoxin 1-(4-nitrophenyl) piperazinyl carbamate (19) was found
to be the most potent compound of those synthesized in the current study, and induced cell cycle arrest
in the G2/M phase in HeLa cells, which was accompanied by apoptosis. Furthermore, this compound
activated the expression of Bax, p53 and caspase-3 in HeLa cells, leading to changes in the conformation
of calf thymus DNA from the B-form to a more compact C-form.
Keywords:
Podophyllotoxin
Carbamates
Cytotoxicity
Apoptosis
Ó 2013 Elsevier Masson SAS. All rights reserved.
CT DNA
1. Introduction
Podophyllotoxin (PPT, 1), extracted from the roots and rhizomes
of Podophyllum species such as Podophyllum hexandrum and Podo-
Current advances in cancer research consider the apoptosis,
other than occurring in various physiological events, as crucially
involved in the regulation of tumor growth as well as in the
treatment response. Most of the anticancer strategies used in
phyllum peltatum [4], has cathartic, antirheumatic and antiviral
properties, and antimitotic activity [5]. By using PPT as a lead
compound, structural modification of it leads to the emergence of
less toxic, useful anti-cancer drugs namely, etoposide (VP-16, 2)
and teniposide (VM-26, 3), which are thus successfully utilized in
treatment of a variety of cancers, including small-cell lung cancer,
testicular carcinoma, lymphoma, and Kaposi’s sarcoma [6]. How-
ever, their therapeutic uses are often hindered by problems such as
poor water-solubility and acquired drug-resistance [7]. To over-
come these problems, extensive synthetic efforts have been carried
out by a number of researchers, which leads to the development of
several potential drug candidates based on PPT applied in clinical
trial, such as, etopophos (4), NPF [8], GL-331 [9], tafluposide
(F11782, 5) [10] and F14512 (6) [11] (Fig. 1). Among them, etopo-
phos, a water-soluble prodrug of 2, was developed and approved
for intravenous use in 1996. As a phosphate, 4 is readily converted
in vivo by endogenous phosphatase to the active drug 2 and ex-
hibits pharmacological and pharmacokinetic profiles similar to
those of 2 [12].
clinical oncology such as chemotherapy,
g-irradiation, including
suicide gene therapy or immunotherapy, have been related to the
activation of apoptosis signal transduction pathways in cancer cells
[1]. Deeper investigations on molecular basis of apoptosis modu-
lation open promising ways for a more rational approach to develop
new therapeutic strategies [2]. Therefore, the design of new com-
pounds able to modulate the apoptotic process, both as activators
or inhibitors, represents an important strategic therapeutic
approach in cancer disease [3].
* Corresponding author. School of Pharmacy, Lanzhou University, Lanzhou
730000, China. Tel./fax: þ86 0931 8915686.
** Corresponding authors.
E-mail addresses: chenshw@lzu.edu.cn, chenshwu@gmail.com (S.-W. Chen).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.053

60
C.-Y. Sang et al. / European Journal of Medicinal Chemistry 70 (2013) 59e67
Fig. 1. Structures of podophyllotoxin (1), etoposide (2), teniposide (3), etopophos (4), NK-611 (5) and tafluposide (6).
Actually, since the discovery of PPT as an antimitotic antitumor
agent, investigation of the structureeactivity relationships of PPT
indicates that the trans-lactone, the 4 -substituted moiety, and the
formation of the carbamates of 7 was confirmed by the presence of
a peak for the C-4 proton of DMEP at a chemical shift of w6.05 ppm.
b
40-demethyl moieties are essential for TOP-II inhibitory activity
[13]. According to SAR of PPT, the replacement of the C-4 sugar
moiety of etoposide with a nonsugar substitution has proven to be
significant in overcoming the drug resistance of etoposide. The C-4
non-sugar substitutions can be linked through O-, S- or N-linkage
[14], compare with the N-linked and S-linked compounds, the O-
linked (ethers, esters) congeners show promising antitumor ac-
tivity [15].
3. Biological results and discussion
3.1. Cytotoxicities of derivatives of 11e27
The in vitro cytotoxicities of carbamates 11e27 were evaluated
against a panel of four human tumor cell lines (cervical carcinoma
HeLa, lung carcinoma A-549, human colorectal adenocarcinoma
HCT-8 and premyelocytic leukemia HL-60), with etoposide as a
reference compound. The screening procedure was based on the
standard MTT or CCK-8 method [21], and the results are summa-
rized in Table 1.
All the carbamates showed substantial cytotoxicity and dis-
played IC50 values in the nanomolar range against the HL-60 and
HCT-8 cell lines; that is, they were even more cytotoxic than eto-
poside. The IC50 value of compound 19, which is the most prom-
Recently, a series of 4b-carbamoyl epipodophyllotoxins have
been generated, and shown to exhibit more potent anticancer ac-
tivity and better binding ability to TOP-II than etoposide [16]. In this
study, which is part of our continuing effort to find new
podophyllotoxin-based compounds with potent activities [17e23],
we synthesized a series of 4b
-carbamoyl 40-O-demethylepipodo-
phyllotoxin and evaluated their cytotoxicities against a panel of
four human cancer cell lines. Especially, the most active carbamate
(19) was evaluated for its effect on apoptosis and interaction with
Calf thymus DNA (CT DNA).
ising in this group compounds, is 0.074, 1.67, <0.01 and 0.17 mM for
HeLa, A549, HCT-8 and HL-60, respectively. The other compounds
generally showed moderate cytotoxicities against HeLa and A-549
cells lines.
Based on these results, we deduced some preliminary struc-
tureeactivity relationships. First, the large activity range of com-
pounds 11e19 indicated that the substituent of the amines
markedly affected the activity profiles of this compound class, and
the compounds with piperazines incorporated appear to be more
potent than those with alkyl amines (11e14 vs 15e19). Second, the
compounds with amino acids incorporated appear to be less potent
than those with p-nitrobenzylpiperzine directly (20e25 vs 19), and
2. Results and discussion
2.1. Chemistry
Organic carbamates have frequently been employed as phar-
maceuticals in the forms of drugs and prodrugs [24]. In recent
years, several reports have indicated that the carbamate linkage
present in the active pharmacophores of various structurally
diverse molecules increases the biological activities of semi-
synthetic/synthetic natural/synthetic molecules [25,26]. In this
the compounds with
L-amino acids incorporated appear to be more
potent than those with
D
-amino acid (22 vs 23, 24 vs 25). Lastly,
compounds which contain only one substituent at the C-4 position
showed superior activities in comparison with compounds simul-
taneously substituted at the C-4 and C-40 position of DMEP (15 vs
26, 19 vs 27). This observation is in accord with the previously re-
ported SARs, the hydroxyl at C-40 position of PPT is necessary to
remain their anticancer activity [28]. The data above revealed that
19 might serve as a potential anti-cancer drug, and we chose HeLa
cells to further investigate the mechanism of its cytotoxic effect.
work, we synthesized 17 4b
-carbamates of 40-demethylepipodo-
phyllotoxin (DMEP, 7) (11e27) by the route depicted in Scheme 1.
PPT (1) was regioselectively demethylated with trimethylsilyl io-
dide (TMSI) and barium carbonate to afford 7 by means of a pre-
viously described procedure [27]. Then the 40-hydroxyl of 7 was
subsequently protected with chlorocarbonylbenzoate (Cbz-Cl) to
give 8. Then the 4-hydroxy of 8 was activated by treatment with p-
nitrophenylchloroformate in pyridine to provide 9. Lastly, reaction
of 9 with various amines in the presence of triethylamine and 4-
dimethylaminopyridine (DMAP) afforded carbamates 11e25 in
good to moderate yields. In addition, compounds 26 and 27 were
also prepared by two steps to investigate the effects of free 40-OH of
PPT derivatives. Specially, the 4,40-hydroxyl of 7 were activated
simultaneously to afford 10, and reacting 10 with morphine or p-
nitrobenzylpiperazine generated target compounds 26 and 27 as
similar above procedure. All the synthesized compounds were
characterized by IR, 1H- and 13C NMR spectroscopy, and high-
resolution mass spectrometry. In the 1H NMR spectra, the
3.2. Cell cycle arrest and apoptosis induction
In our previous publication, we found some carbamate de-
rivatives of 4b-(1,2,3-triazol-1-yl)podophyllotoxin induced cell cy-
cle arrest in the G2/M phase accompanied by apoptosis in A-549
cells [22]. To determine whether 19 have similar effects on tumor
cells, we investigated its effects on cell cycle progression by means
of fluorescence-activated cell sorting analysis of HeLa cells stained
with propidium iodide (Fig. 2) [22]. Treatment of the HeLa cells

C.-Y. Sang et al. / European Journal of Medicinal Chemistry 70 (2013) 59e67
61
OH
OH
OH
O
O
O
O
O
O
ii
i
O
O
O
O
O
O
OMe
OCbz
8
OMe
OMe
MeO
iii
MeO
MeO
OMe
1
OH
7
iii
O
O
O
R1
R2
O
O
O
NO2
N
O
O
O
O
iv
O
O
O
O
OMe
MeO
OMe
MeO
O
OR
R
O
R = Bzl
9
11-25
R = H
26-27
10
R = CONR1R2
R = p-NO2-Ph
Compd
NR1R2
Compd
NR1R2
Compd
NR1R2
HN
11
17
18
19
20
21
23
24
25
26
27
12
13
14
15
16
22
Scheme 1. Synthesis of compounds 11e27. Reagents and conditions: i) TMSI then BaCO3; ii) PhCH2OCOCl, Et3N, CH2Cl2; iii) p-NO2PhOCOCl, pyridine; iv) HNR2R3, DMAP/Et3N,
CH2Cl2.
with 19 resulted in time- and dose-dependent accumulation of cells
in the G2/M phase with a concomitant decrease in the population
of G1 phase cells. G2/M phase arrest was initially detectable after
12 h of treatment; 29.9% and 38.1% of the cells were in the G2/M
phase after exposure to 19 for 12 h and 24 h, respectively. In
contrast, 22.7% of the cells were in this phase in untreated cultures
(Fig. 2A). Furthermore, a population of sub-G1 phase cells, which
are characteristic apoptotic cells, was observed after 12 h of treat-
ment with 19. In control HeLa cells (Fig. 2A), only w1.8% of the cells
were in the sub-G1 phase, whereas about 4.1% and 11.1% of cells
this compound induced G2/M arrest accompanied by apoptosis in
HeLa cells.
3.3. Effects of 19 on Bax, p53 and caspase-3 activation
Apoptosis or programmed cell death is a physiological process
that provides an effective, non-inflammatory way to remove
redundant or damaged cells from tissues thereby securing tissue
homeostasis [29]. Inhibition of apoptosis is considered as an
essential step in tumorigenesis and is one of the hallmarks of
cancer, allowing the survival of cells that accumulate oncogenic
events that otherwise would have been removed by apoptosis [30].
A multitude of signals activated by variable triggers, such as pro-
apoptotic gene, tumor-suppressor gene and growth factors, celle
were in the sub-G1 phase after treatment with 5 mM 19 for 12 h and
24 h, respectively. Similar effects can be found in the dose-
dependent manner (Fig. 2B). These results demonstrate that 19
interfered with cell proliferation by arresting the cell cycle and that

62
C.-Y. Sang et al. / European Journal of Medicinal Chemistry 70 (2013) 59e67
Table 1
concentration of DNA (Fig. 4B), suggesting that compound 19-based
emission is enhanced when it is bound to DNA [39].
Cytotoxicities of compounds 11e27 (mM) at drugs exposure for 48 h.
Compounds
Cytotoxicity (IC50
HL-60b
, m
M)a
4. Conclusions
A-549c
HeLac
HCT-8c
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
VP-16
0.09
0.16
0.21
0.27
0.01
0.19
<0.01
0.33
0.17
0.13
0.26
0.01
0.02
0.03
0.04
<0.01
0.35
1.15
8.83
18.27
4.54
26.12
8.55
4.18
3.89
5.24
1.67
19.03
8.32
>100
>100
9.55
>100
>100
56.9
13.15
10.78
3.75
5.40
8.59
20.04
4.40
1.58
0.074
>100
>100
49.43
>100
4.13
28.60
>100
>100
2.91
0.185
0.08
0.33
1.24
0.15
0.03
1.62
0.01
<0.01
0.71
0.01
0.57
0.85
0.01
0.07
0.13
0.01
7.06
In summary, carbamate derivatives of 4b
-40-demethylepipodo-
phyllotoxin (11e27) showed promising in vitro cytotoxicities
against a panel of human cancer cell lines. Fluorescence-activated
cell sorting analysis indicated that 19 induced cell cycle arrest in
the G2/M phase and apoptosis by activated Bax, p53 and caspase-3
in HeLa cells. In addition, 19 interacted with CT DNA in PBS, and
caused the conformation of CT DNA changed from the B-form to a
more compact C-form. These results suggest that these compounds
have potential for further development as anticancer agents.
5. Experimental
5.1. Chemistry
3.20
Melting points were determined in Kofler apparatus and were
uncorrected. IR spectra were measured on a Nicolet 5DX-FT-IR
spectrometer on neat samples placed between KBr plates. 1H NMR
and 13C NMR spectra were recorded with a Varian Mecury-400BB
and Mecury-600BB spectrometer with TMS as an internal stan-
a
Data are the mean of three independent experiments.
b
c
CCK-8 method.
MTT method.
dard, all chemical shift values are reported as
d ppm. Optical rota-
cell interactions, changing nutrient conditions, hypoxic conditions,
and cytotoxic damage affect the status of the apoptotic machinery
[31]. To further explore the apoptotic pathway of 19 in cells, effects
of 19 on Bax, p53 and caspase-3 activation in HeLa cells were
performed by western blotting [32]. As shown in Figure 3, 19
significantly affected the expression of these three proteins, except
for a marked increase of the pro-apoptotic protein Bax, tumor-
suppressor gene p53, and caspase-3. We also observed a clear
activation of Bax, p53 and caspase-3 in HeLa cells in a time-
dependent manner, these results suggest that 19 induced
apoptosis by means of multiple pathways.
tions were measured on a Perkin Elmer 341 polarimeter in a 1 dm
cell at 23 ꢀC. Mass spectra were recorded on a Bruker Daltonics
APEXⅡ49e and VGZAB-HS (70 ev) spectrometer with ESI source as
ionization, respectively. All reactions were monitored by thin layer
chromatograph (TLC) on silica gel GF254 (0.25 mm thick). Column
chromatography (CC) was performed on Silica Gel 60 (230e400
mesh, Qingdao Ocean Chemical Ltd., China). Podophyllotoxin was
isolated from a Chinesis medicinal herb Podophyllum emodi Wall
var Chinesis Sprague, other starting materials and regents were
purchased commercially and used without further purified, unless
otherwise stated.
3.4. Interaction of 19 with CT DNA
5.1.1. General procedure of synthesis of 8
To 7 (384 mg, 0.96 mmol) in 6 ml acetone was added pydrine
(0.36 ml, 4.4 mmol) and CbzeCl (0.4 ml, 2.8 mmol) at 0 ꢀC. The
reaction mixture was stirred at r.t. for 3 h, and then ice was added.
The organic layer was washed with water, and dried over MgSO4.
The solvent was removed in vacuo and the crude product was
purified by silica gel chromatography to give 8.
Ordinary, the analogs of 2 were considered as inhibitors of TOP-
Ⅱ, however, other compounds, which showed potent inhibitions
against tumor cell lines, but not inhibitions to TOP-Ⅱ were also
found [33]. In order to study the interaction of 19 with CT DNA in
pH 7.2 PBS, the CD spectrum of CT DNA and its complexes with 19 at
various concentrations: 0 (1), 5 (2), 10 (3), 20 (4) and 40 (5) mg/mL
were recorded from 200 to 300 nm at 5 ꢀC on a Jasco-810 spec-
trometer, and the CD spectra of CT DNA in the presence and absence
of 19 in pH 7.2 PBS were showed in Figure 4A. The CD spectrum of
free CT DNA exhibited a negative peak at 245 nm due to the helicity
and a positive peak at 275 nm due to the base stacking (Fig. 4A,
curve 1), which is the characteristic of DNA in the right hand B form
5.1.2. General preparation of compounds 9/10
To a solution of p-nitrophenylchloroformate (36 mmol) in
anhydrous CH2Cl2 (35 ml) was added dry pyridine (4.4 ml).
Instantaneously a white precipitate was formed. A solution of 7 (or
8) (12 mmol) in anhydrous CH2Cl2 (10 ml) was added dropwise
under an argon atmosphere and allowed the mixture was stirred
for further 45 min at room temperature. The mixture was purified
by silica gel chromatography using a mixture of dichloromethane:
ethyl acetate ¼ 8:1 as the eluent to afford compounds 9 and 10.
[34,35]. Increasing the concentration of 19 (c ¼ 5, 10, 20, 40
mg/mL,
resp.) in above CT DNA solution, the intensity of positive band
decreases and negative band increases, as well as a gradual blue
shift of the negative band from 245 to 243 nm (Fig. 4, curve 1, 2, 3, 4,
resp.), suggesting that DNA bound with 19 induces certain confor-
mational changes, DNA double helix in solution is in a more helical
state belonging to C form DNA [36e38].
5.1.3. General preparation of compounds 11e27
To a stirred solution of 9 (or 10) (0.1 mmol) in dry dichloro-
methane (5 ml) was added the appropriate amine (0.15 mmol,
1.5 equiv), triethylamine (0.11 mmol) and DMAP (0.11 mmol) at
room temperature. After stirring for overnight, the reaction
mixture was washed with cold saturated NaHCO3 and then water
until pH 6e7. The extract was dried over MgSO4 and concentrated
in vacuo at 30 ꢀC. The residue was purified by silica gel column
chromatography using a mixture of petro ether/ethyl acetate 1:1 as
the eluent to afford compounds 11e27 in the reported yields.
A binding assay of with CT DNA was also performed by moni-
toring the changes in the emission spectral pattern of compound 19
(0.5 mM, excited at 383 nm) in the presence of increasing concen-
tration of CT DNA (0, 1, 2, 3, 4, 5, 6 mM) in pH 7.2 PBS. Even though no
appreciable change in the position of the charge transfer band of the
complex was observed upon addition of DNA, the fluorescence in-
tensity of the complex increases progressively with increasing

C.-Y. Sang et al. / European Journal of Medicinal Chemistry 70 (2013) 59e67
63
Fig. 2. Effect of 19 on cell cycle progression.
5.1.3.1. 40-Demethylepipodophyllotoxin 4
b
-[N-(n-propyl)] carbamate
5.1.3.2. 40-Demethylepipodophyllotoxin
4
b
-(N-cyclopropyl) carba-
(11). Yield: 56%; white powder solid; mp: 184e186 ꢀC; ½a D23
ꢁ
ꢂ29ꢀ
mate (12). Yield: 71%; white powder solid; mp: 182e184 ꢀC;
(c 0.1, CHCl3); IR (KBr cmꢂ1) 3417, 2963, 1776, 1714, 1613, 1515, 1482,
½
a 2D3
ꢁ
ꢂ32ꢀ (c 0.1, CHCl3); IR (KBr cmꢂ1) 3434, 2955, 2905, 1768, 1712,
1460,1332, 1233, 1115, 1038, 999; 1H NMR (400 MHz, CDCl3)
d
6.93
1612, 1519, 1482, 1460, 1309, 1222, 1185, 1112, 1030, 992; 1H NMR
(s, 1H), 6.54 (s, 1H), 6.29 (s, 2H), 6.01 (t, J ¼ 4.4 Hz, 2H), 5.97 (s, 1H),
4.78 (t, J ¼ 5.6 Hz,1H), 4.64 (d, J ¼ 4.8 Hz,1H), 4.37 (t, J ¼ 4.4 Hz,1H),
3.99 (t, J ¼ 5.2 Hz, 1H), 3.77 (s, 6H), 3.20e3.15 (m, 2H), 3.00e2.91
(m, 1H), 1.59e1.50 (m, 2H), 0.94 (t, J ¼ 14.4 Hz, 3H); 13C NMR
(600 MHz, CDCl3) d 6.94 (s, 1H), 6.54 (s, 1H), 6.28 (s, 2H), 6.00 (t,
J ¼ 4.8 Hz, 2H), 5.97 (s,1H), 5.44 (s,1H), 4.99 (s,1H), 4.63 (d, J ¼ 4.2 Hz,
1H), 4.38 (t, J ¼ 8.4 Hz,1H), 3.98 (t, J ¼ 3.0 Hz,1H), 3.78 (s, 6H), 3.14 (d,
J ¼ 3.6 Hz,1H), 2.96 (d, J ¼ 4.8 Hz,1H), 1.62 (s, 1H), 0.78 (d, J ¼ 5.4 Hz,
(100 MHz, CDCl3)
d
174.7, 156.0, 148.9, 147.5, 146.6, 134.2, 133.0,
2H), 0.55 (d, J ¼ 6.0 Hz, 2H); 13C NMR (150 MHz, CDCl3)
d 174.5,156.5,
130.2, 128.3, 110.2, 109.7, 107.8 (2C), 101.7, 68.6, 67.8, 58.6, 56.5 (2C),
43.8, 43.0, 41.7, 37.2, 23.2, 18.5, 11.3; HRMS (ESI) 508.1571 for
[M þ Na]þ (calcd 508.1578 for C25H27NNaO9).
148.9, 147.4, 146.5, 134.1, 132.9, 130.1, 128.1, 110.1, 109.7, 107.7 (2C),
101.6, 68.6, 67.6, 56.4 (2C), 53.8, 43.6, 41.6, 37.0, 31.7, 6.9 (2C); HRMS
(ESI) 506.1413 for [M þ Na]þ (calcd 506.1422 for C25H25NNaO9).

64
C.-Y. Sang et al. / European Journal of Medicinal Chemistry 70 (2013) 59e67
½
a 2D3
ꢁ
ꢂ52ꢀ (c 0.1, CHCl3); IR (KBr cmꢂ1) 3389, 2900,1770, 1692, 1612,
1520, 1505, 1480, 1462, 1245, 1222, 1113, 1027, 993; 1H NMR
(400 MHz, CDCl3)
d 6.93 (s, 1H), 6.55 (s, 1H), 6.29 (s, 2H), 6.07 (d,
J ¼ 3.2 Hz, 1H), 6.00 (dd, J ¼ 7.6, 4.8 Hz, 2H), 4.65 (t, J ¼ 4.8 Hz, 1H),
4.38 (t, J ¼ 5.6 Hz, 1H), 4.00e3.92 (m, 1H), 3.78 (s, 6H), 3.70 (s, 2H),
3.68 (s, 2H), 3.50 (s, 2H), 3.42 (s, 2H), 3.14 (dd, J ¼ 7.2, 4.8 Hz, 1H),
3.02e2.93 (m, 1H); 13C NMR (100 MHz, CDCl3)
d 174.6, 154.9, 149.0,
147.5, 146.7, 134.4, 133.0, 130.0, 128.2, 110.2, 109.7, 107.9 (2C), 101.7,
69.6, 67.8, 66.7, 58.5 (2C), 56.6 (2C), 43.7, 41.9, 37.2, 18.5 (2C); HRMS
(ESI) 531.1985 for [M þ NH4]þ (calcd 531.1973 for C26H31N2O10).
5.1.3.6. 40-Demethylepipodophyllotoxin
4b-[N-(N-methyl piper-
azinyl)] carbamate (16). Yield: 57%; white powder solid; mp: 210e
213 ꢀC; ½a 2D3
ꢁ
ꢂ56ꢀ (c 0.1, CHCl3); IR (KBr cmꢂ1) 3415, 2928, 1771,
Fig. 3. Effects of compound 19 on cellular levels of Bax, p53 and caspase-3. HeLa cells
were incubated with 5 mM 19 for 0 h, 12 h, 24 h, and cell lysates were prepared and
1694, 1612, 1519, 1481, 1460, 1426, 1233, 1111, 1048, 996; 1H NMR
(400 MHz, CDCl3)
d 6.93 (s, 1H), 6.54 (s, 1H), 6.29 (s, 2H), 6.05 (d,
analyzed by Western blotting.
J ¼ 3.6 Hz, 2H), 6.00 (d, J ¼ 3.6 Hz, 1H), 4.65 (d, J ¼ 4.8 Hz, 1H), 4.38
(t, J ¼ 4.4 Hz, 1H), 3.97e3.92 (m, 1H), 3.78 (s, 6H), 3.60e3.42 (m,
4H), 3.15 (dd, J ¼ 7.2, 4.8 Hz, 1H), 2.99e2.92 (m, 1H), 2.43e2.30 (m,
5.1.3.3. 40-Demethylepipodophyllotoxin
4b-(N-cylcopentyl) carba-
mate (13). Yield: 60%; white powder solid; mp: 241e243 ꢀC;
7H); 13C NMR (100 MHz, CDCl3)
d 174.6, 154.8, 148.9, 147.5, 146.7
½
a 2D3
ꢁ
ꢂ28ꢀ (c 0.1, CHCl3); IR (KBr cmꢂ1) 3421, 2957, 1776, 1710, 1613,
(2C), 134.3, 133.0, 130.1, 128.4, 110.2, 109.8, 107.8 (2C), 101.7, 69.3,
67.9, 58.6, 56.6 (2C), 54.8, 46.3, 44.0, 43.0, 41.9, 37.2, 18.6; HRMS
(ESI) 527.2021 for [M þ H]þ (calcd 527.2024 for C27H31N2O9).
1514, 1483, 1459, 1331, 1232, 1115, 1037, 998; 1H NMR (400 MHz,
CDCl3)
d
6.93 (s, 1H), 6.54 (s, 1H), 6.29 (s, 2H), 6.00 (t, J ¼ 4.8 Hz, 2H),
5.97 (s, 1H), 4.64 (d, J ¼ 4.8 Hz, 1H), 4.38 (t, J ¼ 5.2 Hz, 1H), 4.02e
3.96 (m,1H), 3.77 (s, 6H), 3.17 (dd, J ¼ 9.2, 4.4 Hz,1H), 3.00e2.88 (m,
1H), 2.00e1.95 (m, 2H), 1.69e1.60 (m, 5H), 1.42e1.36 (m, 2H); 13C
5.1.3.7. 40-Demethylepipodophyllotoxin 4
b-{N-[1-(4-cyclopentyl
piperazinyl)]} carbamate (17). Yield: 59%; white powder solid;
mp: 140e142 ꢀC; ½a D23
ꢁ
ꢂ33ꢀ (c 0.1, CHCl3); IR (KBr cmꢂ1) 3436,
NMR (100 MHz, CDCl3) d 174.7, 155.4, 149.0, 146.7 (2C), 134.3, 133.0,
130.2, 118.4, 110.3, 109.7, 107.9 (2C), 101.7, 68.5, 67.8, 56.5 (2C), 53.1,
43.8, 41.8, 37.3, 33.4, 33.3, 23.7 (2C); HRMS (ESI) 529.2189 for
[M þ NH4]þ (calcd 529.2181 for C27H33N2O9).
2956,1776, 1695,1611, 1509, 1483, 1460, 1427, 1288, 1241, 1122, 1039,
1000; 1H NMR (400 MHz, CDCl3)
d 6.94 (s, 1H), 6.54 (s, 1H), 6.29 (s,
2H), 6.05 (d, J ¼ 3.6 Hz, 1H), 5.99 (t, J ¼ 7.2 Hz, 2H), 4.66 (d,
J ¼ 4.8 Hz, 1H), 4.39 (t, J ¼ 6.4 Hz, 1H), 3.99e3.94 (m, 1H), 3.78 (s,
6H), 3.69 (s, 1H), 3.65e3.47 (m, 4H), 3.17 (dd, J ¼ 7.2, 4.8 Hz, 1H),
2.98e2.95 (m, 1H), 2.55e2.44 (m, 4H), 2.38 (s, 1H), 1.84 (s, 2H), 1.71
(s, 2H), 1.69e1.66 (m, 2H), 1.57e1.36 (m, 2H); 13C NMR (100 MHz,
5.1.3.4. 40-Demethylepipodophyllotoxin 4
b-(N-cyclohexyl) carbamate
(14). Yield: 71%; white powder solid; mp: 262e264 ꢀC; ½a D23
ꢁ
ꢂ29ꢀ
(c 0.1, CHCl3); IR (KBr cmꢂ1) 3432, 2931, 1776, 1711, 1617, 1513, 1484,
1457, 1230, 1117, 1036, 1001; 1H NMR (400 MHz, CDCl3)
d 6.92 (s,
CDCl3)
d 174.6, 156.2, 154.8, 148.9, 147.5, 146.7(2C), 134.3, 133.0,
1H), 6.54 (s, 1H), 6.29 (s, 2H), 6.00 (t, J ¼ 5.6 Hz, 2H), 5.97 (s, 1H),
4.64 (t, J ¼ 7.2 Hz, 1H), 4.37 (t, J ¼ 5.6 Hz, 1H), 3.98 (t, J ¼ 5.2 Hz, 1H),
3.78 (s, 6H), 3.17 (dd, J ¼ 8.0, 4.8 Hz, 1H), 3.14e2.92 (m, 1H), 1.96 (d,
J ¼ 11.2 Hz, 2H), 1.72 (t, J ¼ 13.6 Hz, 2H), 1.63e1.60 (m, 2H), 1.31 (s,
130.2, 128.5, 112.0, 111.8, 107.9 (2C), 101.7, 69.2, 68.7, 67.4, 58.5 (2C),
56.6 (2C), 44.1, 41.9, 40.0, 30.4 24.2 (2C), 18.5 (2C); HRMS (ESI)
581.2485 for [M þ H]þ (calcd 581.2494 for C31H37N2O9).
1H), 1.28e1.08 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 174.7, 155.1,
5.1.3.8. 40-Demethylepipodophyllotoxin 4
b-{N-[1-(4-fluorophenyl
148.9, 147.5, 146.7 (2C), 134.3, 133.0, 130.2, 128.4, 110.3, 109.7, 107.9
(2C),101.7, 68.5, 67.9, 56.6 (2C), 50.3, 43.8, 41.8, 37.3, 33.4, 25.5, 24.8,
18.5 (2C); HRMS (ESI) 543.2334 for [M þ NH4]þ (calcd 543.2337 for
piperazinyl)]} carbamate(18). Yield: 65%; white powder solid; mp:
152e153 ꢀC; ½a 2D3
ꢁ
ꢂ35ꢀ (c 0.1, CHCl3); IR (KBr cmꢂ1) 3436, 2916,
1778, 1696, 1613, 1510, 1483, 1460, 1428, 1231, 1116, 1039, 999; 1H
C
28H35N2O9).
NMR (400 MHz, CDCl3)
d
6.96 (t, J ¼ 4.4 Hz, 2H), 6.95 (s, 1H), 6.89e
6.86 (m, 2H), 6.56 (s, 1H), 6.30 (s, 2H), 6.08 (d, J ¼ 3.6 Hz, 1H), 6.00e
5.98 (m, 2H), 4.66 (d, J ¼ 4.8 Hz, 1H), 4.39 (t, J ¼ 6.4 Hz, 1H), 3.99e
3.94 (m, 1H), 3.78 (s, 6H), 3.74 (d, J ¼ 8.0 Hz, 2H), 3.59 (d, J ¼ 6.8 Hz,
5.1.3.5. 40-Demethylepipodophyllotoxin
mate (15). Yield: 67%; white powder solid; mp: 208e210 ꢀC;
4b-(N-morpholino) carba-
Fig. 4. Effects of 19 with CT-DNA. (A) Circular dichroism spectra of CT-DNA in the presence of increasing concentration of compound 19 in 10 mM PBS. 1. 25
1 þ 5 g/mL 19, 3. 1 þ 10 g/mL 19, 4. 1 þ 20 g/mL 19, 5. 1 þ 40 g/mL 19. (B) Fluorescence spectra of compound 19 in the presence of increasing concentration of CT DNA.
[compound 19] ¼ 1 M, [DNA] ¼ 0, 1, 2, 3, 4, 5 and 6 M.
mg/mL CT-DNA, 2.
m
m
m
m
m
m

C.-Y. Sang et al. / European Journal of Medicinal Chemistry 70 (2013) 59e67
65
2H), 3.17 (dd, J ¼ 7.2, 4.8 Hz,1H), 3.09 (s, 2H), 3.03e2.97 (m, 3H); 13
C
J ¼ 6.0 Hz, 2H), 3.17 (d, J ¼ 12.0 Hz,1H), 2.94 (t, J ¼ 9.0 Hz,1H), 2.60e
NMR (100 MHz, CDCl3)
d
174.5, 156.2, 154.8, 149.0, 147.8, 147.6, 146.7
2.52 (m, 2H), 2.14 (s, 3H), 1.98 (s, 1H), 1.87 (s, 1H), 1.64 (s, 2H); 13C
(2C), 134.3, 133.1, 130.1, 128.3, 119.0, 118.9, 116.0, 115.8, 110.2, 109.8,
108.0 (2C), 101.8, 69.5, 67.9, 56.6 (2C), 50.7 (2C), 44.2, 43.8(2C), 40.0,
37.2; HRMS (ESI) 607.2089 for [M þ H]þ (calcd 607.2086 for
NMR (150 MHz, CDCl3) d 174.2, 170.0, 155.7, 154.2, 148.9, 147.4, 146.4
(2C), 139.4, 134.1, 133.1, 130.0, 127.8, 125.9 (2C), 113.2 (2C), 110.2,
109.4, 107.8 (2C), 101.6, 69.2, 67.4, 56.4 (2C), 49.8, 47.2, 46.8, 44.8,
43.6, 41.6, 41.5, 37.0, 32.9, 30.2, 15.8; HRMS (ESI) 787.2256 for
[M þ Na]þ (calcd 787.2256 for C37H40N4NaO12S).
C
32H32FN2O9).
5.1.3.9. 40-Demethylepipodophyllotoxin 4
b-{N-[1-(4-nitrophenyl
piperazinyl)]} carbamate(19). Yield: 69%; yellow powder solid;
5.1.3.13. N-[1-Oxyl-(40-demethylepipodophyllotoxin)]-D-methine-4-
mp: 154e156 ꢀC; ½a D23
ꢁ
ꢂ53ꢀ (c 0.1, CHCl3); IR (KBr cmꢂ1) 3464,
(4-p-nitrophenyl -piperazine) carbomate (23). Yield: 65%; yellow
2908,1777,1697, 1596, 1506,1484, 1428, 1324,1232,1114,1039, 999;
powder solid; mp: 172e175 ꢀC; ½a D23
ꢁ
ꢂ50ꢀ (c 0.1, CHCl3); IR (KBr
1H NMR (600 MHz, CDCl3)
d
8.16e8.13 (m, 2H), 6.94 (s, 1H), 6.84e
cmꢂ1) 3411, 2918, 1776, 1713, 1645, 1596, 1507, 1484, 1454, 1325,
6.81 (m, 2H), 6.57 (s, 1H), 6.29 (s, 2H), 6.09 (d, J ¼ 3.6 Hz,1H), 5.99 (t,
J ¼ 3.0 Hz, 2H), 5.46 (s, 1H), 4.67 (d, J ¼ 5.4 Hz, 1H), 4.39 (q,
J ¼ 6.0 Hz, 1H), 3.96 (q, J ¼ 6.6 Hz, 1H), 3.76 (s, 6H), 3.68 (s, 2H), 3.61
(s, 2H), 3.48 (s, 2H), 3.39 (s, 2H), 3.17 (dd, J ¼ 7.2, 4.8 Hz, 1H), 3.15e
1232, 1114, 1034, 998; 1H NMR (600 MHz, CDCl3)
d 8.17 (d,
J ¼ 9.0 Hz, 2H), 6.89 (s, 1H), 6.86 (d, J ¼ 9.6 Hz, 2H), 6.54 (s, 1H), 6.28
(s, 2H), 5.98 (d, J ¼ 12.6 Hz, 2H), 5.70 (d, J ¼ 8.4 Hz, 1H), 5.43 (s, 1H),
4.95e4.91 (m, 1H), 4.40 (d, J ¼ 6.0 Hz, 1H), 4.38 (t, J ¼ 9.0 Hz, 1H),
4.02 (q, J ¼ 8.4 Hz, 1H), 3.89 (d, J ¼ 9.0 Hz, 2H), 3.77 (s, 8H), 3.53e
3.44 (m, 4H), 3.21 (dd, J ¼ 7.2, 4.8 Hz, 1H), 3.00e2.94 (m, 1H), 2.62e
2.54 (m, 2H), 2.14 (s, 3H), 2.02e1.97 (m, 1H), 1.93e1.97 (m, 1H), 1.58
2.99 (m, 1H); 13C NMR (150 MHz, CDCl3)
d 174.3, 154.6, 154.3, 148.9,
147.4, 146.5, 139.1, 134.2, 132.9, 129.9, 127.9, 125.9 (2C), 113.1 (2C),
110.1, 109.5, 107.7, 101.6, 69.7, 69.5, 67.6, 56.4 (2C), 53.7, 46.7, 43.6,
43.1, 41.7, 37.0, 31.7, 29.2; HRMS (ESI) 656.1852 for [M þ Na]þ (calcd
656.1851 for C32H31N3NaO11).
(s, 3H), 1.26 (s, 1H); 13C NMR (150 MHz, CDCl3)
d 174.3, 169.9, 155.7,
154.3, 148.9, 147.3, 146.5 (2C), 139.5, 134.2, 133.0, 127.8, 125.9 (2C),
113.3 (2C), 110.2, 109.7, 107.8 (2C), 101.6, 69.2, 67.4, 56.5 (2C), 49.6,
47.3, 46.9, 44.8, 43.6, 41.6, 41.5, 37.0, 33.1, 30.3, 29.7, 15.9; HRMS
(ESI) 787.2260 for [M þ Na]þ (calcd 787.2256 for C37H40N4NaO12S).
5.1.3.10. N-[1-oxyl-(40-demethylepipodophyllotoxin)]-L-alanine-4-
(4-p-nitrophenyl -piperazine) carbomate (20). Yield: 63%; yellow
powder solid; mp: 177e179 ꢀC; ½a D23
ꢁ
ꢂ42ꢀ (c 0.1, CHCl3); IR (KBr
cmꢂ1) 3429, 2914, 1777, 1716, 1646, 1596, 1507, 1484, 1456, 1326,
5.1.3.14. N-[1-Oxyl-(40-demethylepipodophyllotoxin)]-L-phenylala-
1230,1113,1018, 996; 1H NMR (600 MHz, CDCl3)
d
8.16 (d, J ¼ 9.0 Hz,
nine-4-(4-p- nitrophenyl-piperazine) carbomate (24). Yield: 72%;
2H), 6.92 (s, 1H), 6.85 (d, J ¼ 9.0 Hz, 2H), 6.55 (s, 1H), 6.28 (s, 2H),
6.00 (t, J ¼ 7.2 Hz, 2H), 5.98 (t, J ¼ 1.2 Hz,1H), 5.81 (d, J ¼ 6.0 Hz,1H),
5.45 (s, 1H), 4.68 (t, J ¼ 7.2 Hz, 1H), 4.65 (d, J ¼ 4.8 Hz, 1H), 4.32 (t,
J ¼ 8.4 Hz, 1H), 4.00 (q, J ¼ 4.8 Hz, 1H), 3.90e3.87 (m, 1H), 3.77 (s,
8H), 3.67 (t, J ¼ 4.8 Hz, 1H), 3.52e3.44 (m, 4H), 3.18 (dd, J ¼ 7.2,
4.8 Hz, 1H), 2.98e2.92 (m, 1H), 1.63 (s, 1H), 1.40 (d, J ¼ 7.2 Hz, 3H);
yellow powder solid; mp: 169e171 ꢀC; ½a D23
ꢁ
ꢂ31ꢀ (c 0.1, CHCl3); IR
(KBr cmꢂ1) 3428, 2908, 2360, 1776, 1713, 1643, 1596, 1506, 1484,
1454, 1325, 1231, 1114, 1037, 997; 1H NMR (600 MHz, CDCl3)
d 8.13
(d, J ¼ 6.0 Hz, 2H), 7.31 (t, J ¼ 6.0 Hz, 2H), 7.23 (q, J ¼ 9.0 Hz, 3H),
6.85 (s, 1H), 6.73 (d, J ¼ 6.0 Hz, 2H), 6.55 (s, 1H), 6.29 (s, 2H), 6.01 (d,
J ¼ 6.0 Hz,1H), 5.96 (d, J ¼ 6.0 Hz,1H), 5.71(d, J ¼ 6.0 Hz,1H), 5.44 (s,
1H), 4.88 (t, J ¼ 9.0 Hz,1H), 4.65 (d, J ¼ 6.0 Hz,1H), 4.30 (t, J ¼ 9.0 Hz,
1H), 3.97 (t, J ¼ 9.0 Hz, 1H), 3.77 (s, 8H), 3.70 (d, J ¼ 6.0 Hz, 1H), 3.50
(s, 1H), 3.22 (s, 2H), 3.09 (s, 2H), 2.99 (s, 1H), 2.93 (s, 1H), 2.73 (t,
13C NMR (150 MHz, CDCl3)
d 174.3, 170.7, 155.1, 154.2, 148.9, 147.4,
146.5 (2C), 139.4, 134.1, 133.0, 127.9, 125.9 (2C), 113.3 (2C), 110.2,
109.5, 107.8 (2C), 101.6, 69.0, 67.4, 56.4 (2C), 47.1, 47.0, 46.9, 44.7,
43.6, 41.5 (2C), 37.0, 19.1; HRMS (ESI) 727.2225 for [M þ Na]þ (calcd
727.2222 for C35H36N4NaO12).
J ¼ 6.0 Hz,1H); 13C NMR (150 MHz, CDCl3)
d 174.2,169.8,155.1,154.1,
148.9, 147.4 (2C), 146.5 (2C), 139.3, 135.7, 134.2, 133.0, 129.6 (2C),
128.8 (2C), 127.8, 127.5, 125.9 (2C), 113.0 (2C), 109.5 (2C), 107.8 (2C),
101.6, 69.1, 67.4, 56.5 (2C), 51.8, 46.5, 46.4, 44.6, 43.6, 41.5, 41.4, 40.3,
37.0; HRMS (ESI) 803.2539 for [M þ Na]þ (calcd 803.2534 for
5.1.3.11. N-[1-Oxyl-(40-demethylepipodophyllotoxin)]-L-lysine-4-(4-
p-nitrophenyl -piperazine) carbomate(21). Yield: 68%; yellow poꢂw1-
der solid; mp: 170e171 ꢀC; ½a D23
ꢁ
ꢂ45ꢀ (c 0.1, CHCl3); IR (KBr cm
)
C41H40N4NaO12).
3429, 2911, 1776, 1713, 1642, 1596, 1507, 1484, 1459, 1326, 1229,
1114, 1093, 998; 1H NMR (600 MHz, CDCl3)
d
8.16 (d, J ¼ 7.2 Hz, 2H),
5.1.3.15. N-[1-Oxyl-(40-demethylepipodophyllotoxin)]-D-phenylala-
6.90 (s, 1H), 6.85 (d, J ¼ 9.0 Hz, 2H), 6.56 (s, 1H), 6.29 (s, 2H), 6.01 (s,
1H), 5.98 (d, J ¼ 6.0 Hz, 2H), 5.60 (d, J ¼ 9.6 Hz, 1H), 5.45 (s, 1H), 4.66
(d, J ¼ 6.4 Hz, 1H), 4.55 (q, J ¼ 8.4 Hz, 1H), 4.29 (t, J ¼ 8.4 Hz, 1H),
3.97 (q, J ¼ 4.8 Hz, 1H), 3.85 (t, J ¼ 8.4 Hz, 2H), 3.77 (s, 8H), 3.49 (d,
J ¼ 8.4 Hz, 2H) 3.18 (dd, J ¼ 8.4, 4.8 Hz, 1H), 2.97e2.92 (m, 1H), 2.00
nine- 4-(4-p-nitrophenyl piperazine) carbomate (25). Yield: 69%;
yellow powder solid; mp: 166e167 ꢀC; ½a D23
ꢁ
ꢂ53ꢀ (c 0.1, CHCl3); IR
(KBr cmꢂ1) 3432, 2916, 1776, 1713, 1643, 1596, 1506, 1484, 1454,
1325, 1230, 1114, 1033, 996; 1H NMR (600 MHz, CDCl3)
d 8.13 (d,
J ¼ 9.0 Hz, 2H), 7.31 (t, J ¼ 7.2 Hz, 2H),7.23 (q, J ¼ 9.0 Hz, 3H), 6.89 (s,
1H), 6.75 (d, J ¼ 9.6 Hz, 2H), 6.55 (s, 1H), 6.31 (s, 2H), 6.04 (d,
J ¼ 6.0 Hz,1H), 5.92 (d, J ¼ 7.2 Hz,1H), 5.76 (d, J ¼ 5.4 Hz,1H), 5.40 (s,
1H), 4.83 (t, J ¼ 9.6 Hz, 1H), 4.67 (d, J ¼ 6.0 Hz,1H), 4.33 (t, J ¼ 7.2 Hz,
1H), 3.90 (t, J ¼ 7.8 Hz, 1H), 3.76 (s, 8H), 3.70 (d, J ¼ 6.0 Hz, 1H), 3.54
(s, 1H), 3.19 (s, 2H), 3.03 (s, 2H), 2.92 (s, 1H), 2.90 (s, 1H), 2.73 (t,
(q, J ¼ 8.4 Hz, 1H), 1.04 (d, J ¼ 6.6 Hz, 3H), 0.91 (d, J ¼ 7.2 Hz, 3H); 13
C
NMR (150 MHz, CDCl3) d 174.2, 170.1, 156.0, 154.3, 148.9, 147.4, 146.5
(2C), 139.4, 134.1, 133.0, 127.9, 125.9 (2C), 113.2 (2C), 110.2, 109.3,
107.8 (2C), 101.6, 69.1, 67.4, 56.4 (2C), 55.7, 47.2, 46.9, 45.0, 41.5, 37.0,
29.7, 19.7, 17.1; HRMS (ESI) 755.2534 for [M þ Na]þ (calcd 755.2535
for C37H40N4NaO12).
J ¼ 6.0 Hz, 1H); 13C NMR (150 MHz, CDCl3)
d 174.3, 169.7, 155.0,
154.1, 148.5, 147.3, 146.5, 146.4, 139.2, 135.7, 130.5, 130.0, 129.5 (2C),
128.7 (2C), 127.7, 127.5, 125.9 (2C), 112.9, 110.4, 110.1, 109.6, 109.0,
107.9, 107.8, 101.6, 101.5, 69.1, 67.6, 56.4 (2C), 51.6, 46.5, 44.6, 43.7,
41.4, 40.3, 38.2, 37.0; HRMS (ESI) 803.2539 for [M þ Na]þ (calcd
803.2535 for C41H40N4NaO12).
5.1.3.12. N-[1-Oxyl-(40-demethylepipodophyllotoxin)]-L-methine-4-
(4-p-nitrophenyl -piperazine) carbomate (22). Yield: 56%; yellow
powder solid; mp: 199e201 ꢀC; ½a D23
ꢁ
ꢂ41ꢀ (c 0.1, CHCl3); IR (KBr
cmꢂ1) 3436, 2919, 1776, 1713, 1638, 1598, 1507, 1484, 1325, 1233,
1115, 1035, 999; 1H NMR (600 MHz, CDCl3)
d
8.15 (d, J ¼ 12.0 Hz,
2H), 6.91 (s, 1H), 6.85 (d, J ¼ 12.0 Hz, 2H), 6.56 (s, 1H), 6.28 (s, 2H),
5.99 (q, J ¼ 6.0 Hz, 3H), 5.70 (d, J ¼ 6.0 Hz, 1H), 5.46 (s, 1H), 4.90 (q,
J ¼ 6.0 Hz,1H), 4.65 (d, J ¼ 6.0 Hz,1H), 4.30 (t, J ¼ 9.0 Hz,1H), 3.96 (s,
1H), 3.85 (d, J ¼ 12.0 Hz, 2H), 3.74 (s, 8H), 3.62 (s, 2H), 3.46 (t,
5.1.3.16. 40-Demethylepipodophyllotoxin 40-bis-(N-morpholino)
4b,
carbamate (26). Yield: 69%; white powder solid; m.p.:281e283 ꢀC;
½
a 2D3
ꢁ
ꢂ36ꢀ (c 0.3, CHCl3); IR (KBr cmꢂ1) 3438, 2920,1778,1727,1696,
1601, 1507, 1484, 1458, 1424, 1239, 1129, 1042, 997; 1H NMR

66
C.-Y. Sang et al. / European Journal of Medicinal Chemistry 70 (2013) 59e67
(400 MHz, CDCl3)
d
6.94 (s, 1H), 6.55 (s, 1H), 6.31 (s, 2H), 6.06 (d,
filters. The blots were incubated with the corresponding antibodies
and developed [32].
J ¼ 3.6 Hz,1H), 6.00 (t, J ¼ 5.2 Hz, 2H), 4.68 (d, J ¼ 5.4 Hz,1H), 4.39 (t,
J ¼ 14.8 Hz, 1H), 3.95 (t, J ¼ 8.8 Hz, 1H), 3.92e3.72 (m, 5H), 3.71 (s,
9H), 3.62 (s, 2H), 3.53 (d, J ¼ 8.4 Hz, 4H), 3.42 (s, 2H), 3.16 (dd,
J ¼ 14.0, 4.8 Hz, 1H), 3.00e2.92 (m, 1H); 13C NMR (100 MHz, CDCl3)
5.2.4. Circular dichroism studies
CT DNA was dissolved and annealed in 10 mM PBS (pH 7.2). To
d
174.3,154.9,153.5,152.2 (2C),149.0,147.6,137.0,132.7,128.7,128.3,
CT DNA in PBS (5 ml 50
0.5, 1.0, 2.0, 4.0 ml, 100
m
m
g/mL) was added compound 19 in PBS (0,
g/mL, respectively), and diluted to 10 ml.
110.4, 109.8, 107.8 (2C), 101.8, 69.5, 67.8, 66.8 (4C), 56.4 (2C), 44.5
(2C), 44.1 (2C), 41.9 (2C), 37.2; HRMS (ESI) 627.2189 for [M þ H]þ
(calcd 627.2185 for C31H35N2O12).
Same concentration sample of 19 in PBS was also prepared. The
above mixture was incubated for 24 h in the dark at 37 ꢀC. CD
spectra were recorded on a JASCO J-810 spectrometer at 20 ꢀC in
quartz cells of 1 cm path length at increasing compound 19/DNA
ratio. Each sample solution was scanned in the range of 200e
300 nm. The appropriate concentration samples of compound 19 in
PBS were also determined as blank.
5.1.3.17. 40-Demethylepipodophyllotoxin 4 ,40-bis-{N-[1-(4-
b
nitrophenyl piperazinyl)]} carbamate (27). Yield: 73%; yellow poꢂw1-
der solid; m.p.: 210e213 ꢀC; ½a D23
ꢁ
ꢂ42ꢀ (c 0.3, CHCl3); IR (KBr cm
)
3436, 2908, 1778, 1723, 1700, 1597, 1505, 1486, 1423, 1326, 1234,
1115, 1038, 997; 1H NMR (400 MHz, CDCl3)
d 8.17e8.13 (m, 4H), 6.94
(s, 1H), 6.87e6.81 (m, 4H), 6.55 (s, 1H), 6.32 (s, 2H), 6.05 (d,
J ¼ 3.6 Hz, 1H), 6.00 (d, J ¼ 6.4 Hz, 2H), 4.66 (d, J ¼ 4.8 Hz, 1H), 4.39
(t, J ¼ 16.4 Hz, 1H), 3.99 (t, J ¼ 8.8 Hz, 1H), 3.88 (s, 2H), 3.71 (s, 10H),
3.62 (s, 2H), 3.52 (s, 6H), 3.40 (s, 2H), 3.17 (dd, J ¼ 14.4, 4.8 Hz, 1H),
5.2.5. Fluorescence titrations
The binding of the compound 19 to CT DNA is performed by
monitoring the changes in the emission spectral pattern of com-
pound 19 in pH 7.2 BPS in the presence of increasing concentrations
of CT DNA. After addition of indicated amount of DNA (0, 1, 2, 3, 4, 5,
3.04e2.94 (m, 1H); 13C NMR (100 MHz, CDCl3)
d 174.3, 154.7, 154.6,
153.4, 152.1 (2C), 149.1, 147.7, 139.0, 137.1, 132.6, 128.4, 128.1, 126.1
(4C), 115.6, 113.1 (2C), 110.4, 109.7, 107.7 (2C), 101.9, 69.7, 67.8, 56.4
(2C), 47.0 (4C), 44.0 (2C), 43.5 (2C), 41.8 (2C), 37.2; HRMS (ESI)
889.2646 for [M þ Na]þ (calcd 889.2651 for C43H42N6NaO14).
6 mM) to the compound 19 (0.5 mM), the resulting solution was
allowed to equilibrate for 5 min at 25 ꢀC, excited at 383 nm fol-
lowed by recording the emission spectral changes in the range of
765e790 nm. The appropriate concentration samples of CT DNA in
PBS were also determined as blank.
5.2. Biological evaluation
Acknowledgments
5.2.1. Cytotoxicity assays
Cells were incubated at 37 ꢀC in a 5% CO2 atmosphere. The MTT
and Cell Counting Kit-8 (CCK-8) colorimetric assay were used to
determined growth inhibition. The synthetic compounds 11e27
and reference compound VP-16 were dissolved in saline for five
Financial support from National Natural Science Foundation of
China (No. 21372110, 81372177), and Natural Science Foundation of
Gansu Province (No. 1208RJZA247) are gratefully acknowledged.
concentrations (0.001e100 mM). For the HL-60, cells were plated in
Appendix A. Supplementary data
96-well plates and exposed in quadplex well for 48 h. Then the
CCK-8 was added to each well. After 4 h of incubation, the absor-
bance at l450 was determined with a plate reader. For the A-549,
HCT-8 and HeLa, cells were plated in 96-well plates and allowed to
attach for 4e6 h, then exposed in quadplex well for 48 h. The media
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.053.
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