Synthesis and biological evaluation of derivatives of 4- deoxypodophyllotoxin as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2011.06.004

In an attempt to generate compounds with superior bioactivity and reduced toxicity, a series of derivatives of deoxypodophyllotoxin were synthesized by reacting 4′-demethyl-4-deoxypodophyllotoxin with substituted piperazines or their amino acid amides. The cytotoxic activity of these compounds against three human cancer cell lines was evaluated. We found that p- nitrophenylpiperazine substitution (Compound 8b) led to an increase in the potency of the compound. Compound 8b exhibited the most potent cytotoxicity against A-549, HeLa and SiHa cells (IC₅₀ values were 0.102, 0.180 and 0.0195 μM, respectively). In addition, flow cytometric analysis showed that 8b induced cell cycle arrest in the G1 phase accompanied by apoptosis in A-549 cells.

Full text of DOI:10.1016/j.ejmech.2011.06.004

European Journal of Medicinal Chemistry 46 (2011) 4056e4061
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of derivatives of 4-deoxypodophyllotoxin
as antitumor agents
,
c,
Yan Jin a, Jie Liu a, Wen-Ting Huang b, Shi-Wu Chen b , Ling Hui
*
**
a College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
b School of Pharmacy & Key Lab of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou 730000, China
c Experimental Center of Medicine, General Hospital of Lanzhou Military Command, Key Laboratory of Stem Cells and Gene Drug of Gansu Province, Lanzhou 730050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to generate compounds with superior bioactivity and reduced toxicity, a series of deriv-
atives of deoxypodophyllotoxin were synthesized by reacting 40-demethyl-4-deoxypodophyllotoxin with
substituted piperazines or their amino acid amides. The cytotoxic activity of these compounds against
three human cancer cell lines was evaluated. We found that p-nitrophenylpiperazine substitution
(Compound 8b) led to an increase in the potency of the compound. Compound 8b exhibited the most
Received 16 March 2011
Received in revised form
31 May 2011
Accepted 1 June 2011
Available online 14 June 2011
potent cytotoxicity against A-549, HeLa and SiHa cells (IC50 values were 0.102, 0.180 and 0.0195 mM,
respectively). In addition, flow cytometric analysis showed that 8b induced cell cycle arrest in the G1
phase accompanied by apoptosis in A-549 cells.
Keywords:
Podophyllotoxin
4-Deoxypodophyllotoxin
Piperazine
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antitumor activity
1. Introduction
Deoxypodophyllotoxin (DPT, 2), an analogue of PT, has anti-
proliferative and antitumor activity in diverse cell types [6e10], as
Podophyllotoxin (PT, 1) and its many related derivatives have
been used for a variety of therapeutic purposes including cathartic,
antirheumatic and anti-viral treatments. Because of its inhibitory
activity on cell growth, it is often used as a lead compound for drug
design in the search for improved anti-proliferative agents [1].
Hundreds of derivatives have been prepared, together with some
semi-synthetic analogues such as etoposide (VP-16), teniposide,
and more recently etopophos, a more soluble prodrug of etoposide,
that is currently used in chemotherapy for various types of cancer,
including small cell lung cancer, testicular carcinoma, and Kaposi’s
sarcoma [2e5]. Surprisingly, these semi-synthetic derivatives
exert their actions through different mechanisms than the parent
compound, podophyllotoxin [2]. Podophyllotoxin inhibits the
assembly of tubulin into microtubules through interaction with the
protein at the colchicine binding site, preventing the formation of
the spindle. However, etoposide and congeners induce a premitotic
blockade in the late S stage of the cell cycle through the inhibition
of DNA topoisomerase II (Topo II), achieved by binding to and
stabilizing the DNA-protein complex, thus preventing re-ligation of
the double-stranded breaks [4].
well as anti-inflammatory [11] and anti-viral [12] activity. Recently,
it has been reported that DPT inhibits tubulin polymerization and
induces cell cycle arrest at G2/M in HeLa cells, followed by
apoptosis through multiple cellular processes, involving the acti-
vation of ATM, upregulation of p53 and Bax, activation of caspase-3
and -7, and accumulation of PTEN resulting in the inhibition of the
Akt pathway [13e15]. Furthermore, Ahn and co-workers have
described a number of DPT derivatives, such as alkyl and carboxyl
alkyl esters of 40-demethyl-4-deoxypodophyllotoxin (DDPT, 3)
(Fig. 1) and prodrugs of DPT including carbamate- and carbonate-
associated forms. The antitumor activities of these compounds
showed that most derivatives enhanced cytotoxic or antitumor
activity compared with that of the parent compound, DPT [16,17].
In our continuing efforts to find new compounds with potent
activities and low toxicity from natural products [18e24], we here
present the synthesis of nine new derivatives (8aei) of DPT and our
measurements of theircytotoxic activityagainst A-549, HeLa, SiHa cell
lines. Furthermore, the effect of 8b on A-549 cells was also investi-
gated by flow cytometry studies to reveal its actions on the cell cycle.
2. Chemistry
* Corresponding author. Tel./fax: þ86 931 8915686.
** Corresponding author.
The synthetic route to the target compounds first involved
E-mail address: chenshwu@gmail.com (S.-W. Chen).
generation of the intermediate DDPT (Scheme 1), which was
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.004

Y. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4056e4061
4057
A
B
R''
R
O
HO
O
O
HO
R
R'
R''
Me
R'
H
O
O
Etoposide
Teniposide
O
1
2
3
OH Me
O
O
H
H
Me
H
O
H
S
O
P
MeO
OMe
OH
OH
Me
Etopophos
MeO
OMe
OR'
OR'
Fig. 1. Structures of podophyllotoxin (1) and related compounds.
prepared from 1 isolated from the Chinese medicinal herb Podo-
phyllum emodi Wall. var. Chinesis Sprague. Usually, the intermediate
3 is prepared from 1 through successive 4-deoxylation by hydro-
genolysis [25] and 40-demethylation with methanesulfonic acid/
sodium iodide [26]. However, this procedure gives onlylow yield and
purity, so we modified the procedure to prepare 3 from 1 through 40-
demethylation with HBr gas in CH2Cl2 with subsequent 4-
deoxylation by hydrogenolysis with 10% Pd/C in HAc. A previous
report has also demonstrated that treatment of 3 with 4-nitrophenyl
chloroformate (]4-nitrophenyl carbonochloridate) in the presence
of pyridine provides the intermediate 7 in high yield [27].
Having synthesized intermediate 7, compounds 8aee were
prepared by reacting 7 with the appropriate substituted pipera-
zines (1-methylpiperazine, 1-p-nitrophenylpiperazine, 1-p-fluoro
phenylpiperazine, 1-p-hydroxylphenylpiperazine, 1-cyclopentyl-
piperazine, respectively) in the presence of triethylamine (Et3N)
with high or moderate yields.
Amino acids are often used as carrier vehicles for some drugs
because of their good water solubility when actively transplanted
into mammalian tissue. As a continuation of our previous work
[21,23], we also designed and synthesized compounds 8fei. 1-p-
Nitrophenylpiperazine was condensed with the appropriate
Boc-protected amino acids 4aed in the presence of benzo
triazole-1-yl-oxy-tris- (dimethylamino)-phosphonium hexafluoro-
phosphate (BOP) and N,N-diisopropylethylamine (DIEA), and
subsequent deprotected with trifluoroacetic acid (Scheme 2) to
provide 5aed. Compounds 8fei were generated by reacting 7 with
the appropriate amino acid amides 5aed in the presence of Et3N.
The structures of compounds 8aei were established using IR, 1H
and 13C NMR spectroscopy, and high-resolution mass spectrometry.
3. Biological results and discussion
The biological activities of the DPT derivative compounds 8aei
were evaluated by an in vitro cytotoxicity test carried out with
a panel of three human tumor cell lines (A-549 (lung carcinoma),
HeLa (cervical carcinoma), and SiHa (cervical squamous cell carci-
noma)). The results of these experiments are summarized in
Table 1.
As illustrated in Table 1, the DPT derivatives, 8aei, were gener-
ally more potent than DPT, DDPT and VP-16 in their cytotoxicity to
these three cell lines. As a group, these compounds were most
effective in SiHa cells, and had lowest potency in HeLa cells,
although the order of potency varied in each cell line. In Table 1, we
also show that different piperazine substitutions in compounds
8aee have markedly different effects on the activity of the resultant
compound. Notably, compound 8b (a 1-p-nitrophenylpiperazinyl
O
O
O
O
OH
ii
O
O
O
i
O
MeO
OMe
O
O
OMe
2
O
O
OH
MeO
OMe
MeO
OMe
O
O
iii
i
OH
3
O
O
OMe
1
iv
MeO
OMe
OH
6
O
O
O
O
O
O
O
v
O
MeO
OMe
MeO
OMe
O
O
H
O
O
O
N
N
N R'
O
R
NO2
8a-i
7
Scheme 1. Syntheses of compounds 8a-i.

4058
Y. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4056e4061
Reagents and conditions: i) BOP, DIEA, amine, CH2Cl2; ii) Trifluoroacetic acid,
CH2Cl2
O
O
H
N
H2N
i
ii
N
N
NO2
Boc
OH
R
R
4a-d
5a-d
Scheme 2. Synthesis of compounds 5aed
substituted derivative of DPT) was approximately 10 times more
potent than 8a, 8c, 8d and 8e.
after treatment with 8b (Fig. 3B). In addition, other changes were
observed including blebbing, nuclear fragmentation and the
formation of apoptotic bodies (Fig. 3C).
Compounds 8fei generally showed comparable or weaker
activity in comparisonwith 8b. Differentsubstituentsat thea-carbon
of theaminoacidinthis class ofcompounds(8fvs8h, 8g vs8i) didnot
appeartohave significanteffectsontheiranti-proliferativeactivityin
the in vitro assay. However, the compounds with l-amino acids
incorporated appear to be more potent than those with d-amino acid
substituents (8f vs. 8g, 8h vs. 8i).
DPT is reported to induce apoptosis and cell cycle arrest in the
G2/M phase in HeLa cells [14,15]. To determine whether derivatives
of DPT have similar effects on other tumor cells, the effects of 8b
and DPT on cell cycle progression were also determined by FACS
analysis in propidium iodide-stained A-549 cells. As shown in
4. Conclusion
In summary, the podophyllotoxin class of compounds is
a promising group of putative antitumor chemotherapeutics. In this
present work, nine novel derivatives of DPT were successfully
synthesized, and biological evaluation showed that most of these
compounds exhibited improved anticancer activity as compared
with etoposide and DPT. Compound 8b possessed the most potent
cytotoxicity against A-549, HeLa and SiHa cells. In addition, the
synthesized derivative 8b could induce cell cycle arrest in the G1
phase and caused apoptosis in A-549 cells. Further biological
evaluation is in progress to clarify the mechanism of 8b action and
to define its effects on tubulin polymerization.
Fig. 2, treatment with 0.1 mM 8b and 0.5 mM DPT led to accumu-
lation of cells in the G1 phase; 76.8% and 85.5% of the cells were in
G1 phase after 48 h treatment with 8b and DPT, respectively,
compared with 59.9% in untreated cultures. Furthermore, a pop-
ulation of sub-G1 phase cells, a characteristic of apoptotic cells, was
observed after 48 h treatment with 8b, DPT and VP-16 treatment. In
control A-549 cells (Fig. 2A), only w0.2% of the cells were in sub-G1
phase, whereas 21.7%, 13.1% and 4.1% of cells were in the sub-G1
5. Experimental
Melting points were determined in Kofler apparatus and were
uncorrected. Optical rotations were measured on a Perkin Elmer 341
polarimeter in a 1 dm cell at 23 ꢀC. IR spectra were measured on
a Nicolet 5DX-FT-IR spectrometer on neat samples placed between
KBr plates. NMR spectra were recorded on a Bruker AM 400 at
400 MHz for 1H and 100 MHz for 13C in deuteriochloroform with
TMS as an internal standard. Chemical shift values are expressed in
ppm followed by multiplicity and coupling constants (J) in Hz. High
resolution mass spectra were run in a Bruker Daltonics APEXⅡ49e
spectrometer with ESI source as ionization. LRMS were run in
a VG-TS-250 spectrometer working at 70 eV. All reactions were
monitored by thin layer chromatograph (TLC) on silica gel GF254
(0.25 mm thick). CC was performed on Silica Gel 60 (230e400 mesh,
Qingdao Ocean Chemical Ltd., China). Podophyllotoxin was isolated
from a Chinese medicinal herb P. emodi Wall var Chinesis Sprague,
other starting materials and regents were purchased commercially
and used without further purified, unless otherwise stated.
phase after 48 h treatment with 0.1
mM 8b (Fig. 2B), 0.5 mM DPT
(Fig. 2) and 0.1 M VP-16 (Fig. 2D), respectively. These data
m
demonstrate that DPT and 8b induce G1 arrest and apoptosis in A-
549 cells. In addition, 8b has more potent pro-apoptotic activity
than DPT and VP-16. These results demonstrate that DPT and its
derivatives interfere with cell proliferation by arresting the cell
cycle, and that the cycle is arrested in different phases than those
identified in previous studies [14,15].
Compound 8b induces apoptosis, as verified by the morpholog-
ical changes seen in A-549 cells treated with 0.1 mM 8b as compared
with control cells. As shown in Fig. 3, negative control (untreated)
cells exhibited typical non-adherent, round morphology with
vacuolization after 48 h incubation (Fig. 3A). Loss of cell membrane
asymmetry and attachment, cell shrinkage and chromatin conden-
sation (all of which are characteristics of apoptosis) were observed
Table 1
5.1. General procedure of synthesis of 5aed
Cytotoxicity of compounds 8aei, assessed by MTT method with 48 h drug exposure.
Compounds
Cytotoxicity (IC50
,
m
M)a
HeLa
0.703
To a stirred solution of the appropriate Boc-protected amino
acid (2 mmol) in CH2Cl2 was added the 1-p-nitrophenylpiperazine
(2.2 mmol, 1.1 equiv), BOP reagent (1.1 mmol) and DIEA (1.1 mmol)
at room temperature. After stirred overnight, the reaction mixture
was concentrated in vacuo, the residue was added 15 mL ethyl
acetate, washed with 5% citric acid solution (6 mL ꢁ 3), 5%
NaHCO3 solution (6 mL ꢁ 3), then washed with saturated brine,
dried over anhydrous Na2SO4 and the solvent was evaporated
under reduced pressure. The crude product was purified by flash
column chromatograph to afford the appropriate Boc-amino acid
amides. Boc-amino acid amides was treated with trifluoroacetic
acid in CH2Cl2 and used directly for next reaction without further
purified.
A-549
SiHa
8a
8b
8c
8d
8e
8f
8g
8h
3.96
0.102
1.62
2.39
1.72
0.0802
0.300
0.183
0.208
14.8
4.97
0.0195
1.23
0.180
5.28
7.01
4.76
3.07
0.135
0.160
0.211
0.160
0.0839
30.7
0.433
0.802
0.191
0.203
50.6
36.0
7.43
8i
VP-16
DPT
DDPT
0.470
3.75
10.2
2.07
a
Data are the mean of three independent experiments.

Y. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4056e4061
4059
Fig. 2. Effects of 8b, DPT and VP-16 on cell cycle progression.
5.2. Synthesis of 40-demethyl-4-deoxylpodophyllotoxin (3)
stirred for another 5 h at 95 ꢀC under 2 atm of hydrogen. The mixture
was filtered and the filtrate was evaporated. The residue was purified
bycolumnchromatographyonsilicagelyieldingDPT(5.6g, yield73%).
A mixture of 10% Pd/C (6.0 g) in acetic acid (150 mL) was stirred
underhydrogenuntilnofurtherhydrogenwasabsorbed. Following40-
demethyl- epipodophyllotoxin (8.0 g), which was obtained from
podophyllotoxin under HBr, was added and the reaction mixture was
1H NMR (400 MHz, CDCl3)
d 6.66 (s,1 H), 6.52 (s,1 H), 6.35 (s, 2 H), 5.94
(d, J ¼ 9.2 Hz, 2 H), 5.41 (s, 1 H), 4.60 (s, 1 H), 4.45e4.42 (m, 1 H),
3.93e3.90(m,1H), 3.78(s,6H),3.08e3.05(m,1H),2.77e2.72 (m, 3 H).
Fig. 3. Comparison of A-549 cell morphology after treatment with or without 8b.

4060
Y. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4056e4061
5.3. Synthesis of 40-demethyl-4-deoxylpodophyllotoxin 40-(p-
nitrophenyl) carbonate (7)
(m, 4 H), 3.06 (dd, J ¼ 14.4, 4.4 Hz,1 H), 2.40e2.73 (m, 3 H); 13C NMR
(100 MHz, CDCl3) d 174.4, 153.2, 151.4 (2 C), 147.0, 146.6, 134.5, 130.3,
124.3, 124.2, 114.5, 114.4, 115.7, 115.4, 110.5, 104.4, 107.4 (2 C), 101.1,
77.3, 72.0, 56.2 (2 C), 47.4, 43.7, 33.0, 32.6; HRMS (ESI) 591.2132 for
[M þ H]þ (calcd 591.2137 for C32H32FN2O4).
To a solution of p-nitrophenyl chloroformate (1.3 g, 6.35 mmol)
in anhydrous CH2Cl2 (5 ml) was added dry pyridine (0.6 ml).
Instantaneously a white precipitate was formed. A solution of DDPT
(7.2 g, 1.87 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 8 (8.6 g,
5.4.4. 40-Demethyl-4-deoxylpodophyllotoxin 40-(4-p-hydroxylphenyl-
piperazine) carbomate (4d)
Yield: 43%; white amorphous solid; m.p.:140e142 ꢀC; ½a D23
ꢂ
-65
(c 0.3, CHCl3); IR (cmꢃ1) 3361, 2920, 1771, 1711, 1599, 1512, 1424,
1223, 1124, 1034, 995; 1H NMR (400 MHz, CDCl3)
d 6.46
yield: 88%). 1H NMR (400 MHz, CDCl3)
d
8.30 (m, J ¼ 8.8 Hz, 2 H,
(d, J ¼ 4.0 Hz, 2 H), 6.77 (d, J ¼ 4.0 Hz, 2 H), 6.66 (s, 1 H), 6.52 (s, 1 H),
6.34 (s, 2 H), 5.93 (d, J ¼ 7.6 Hz, 2 H), 4.63 (d, J ¼ 3.6 Hz, 1 H),
4.46e4.40 (m, 1 H), 3.91e3.79 (m, 2 H), 3.79e3.72 (m, 3 H), 3.70
(s, 6 H), 3.10e3.00 (m, 5 H), 2.40e2.70 (m, 3 H); 13C NMR (100 MHz,
p-Ph), 7.50 (m, J ¼ 9.2 Hz, 2 H, p-Ph), 6.68 (s, 1 H, H-5), 6.52 (s, 1 H,
H-8), 6.43 (s, 1 H, H-20,60), 5.96 (m, 2 H, OCH2O), 4.66 (d, 1 H, 1eH),
4.49 (m, 1 H, H-11a), 3.96 (t, 1 H, H-11b), 3.77 (s, 6 H, 2OCH3), 3.11
(m, 1 H, H-2), 2.82 (m, 2 H, H-2,3,); 13C NMR (100 MHz, CDCl3)
CDCl3)
d 175.4, 153.4, 152.3 (2 C), 147.6, 147.2, 139.3, 130.4, 124.4
d
174.8, 155.6, 151.1, 150.1, 147.1, 145.3, 140.0, 130.0, 128.3, 127.5,
(2C), 117.0, 111.0, 104.9, 104.3 (2 C), 101.7, 77.3, 72.6, 56.7, 53.9, 47.9,
44.2, 33.6, 33.2; HRMS (ESI) 549.2173 for [M þ H]þ (calcd 549.2141
for C32H33N2O9).
125.2, 121.6, 110.4, 108.5, 107.62, 101.2, 77.32, 72.03, 56.21, 47.32,
43.27, 33.00.
5.4. General procedure of synthesis of 8aei
5.4.5. 40-Demethyl-4-deoxylpodophyllotoxin 40-(4-
cyclopentylpiperazine) carbomate (4e)
To a stirred solution of 7 (0.1 mmol, 55 mg) in dry dichloro-
methane (3 ml) was added the appropriate amine (0.12 mmol,
1.2 equiv) and triethylamine (0.12 mmol) at room temperature.
After stirring for overnight, the reaction mixture was purified
directly by silica gel column chromatography.
Yield: 47%; white amorphous solid; m.p.:130e132 ꢀC; ½a D23
ꢂ
s-41
(c 0.3, CHCl3); IR (cmꢃ1) 2955, 2913, 2469, 1774, 1721, 1600, 1442,
1423, 1335, 1222, 1129, 1034, 997; 1H NMR (400 MHz, CDCl3)
d 6.66
(s, 1 H), 6.52 (s, 1 H), 6.37 (s, 2 H), 5.93 (d, J ¼ 4.0 Hz, 2 H), 4.63 (d,
J ¼ 3.6 Hz, 1 H), 4.46 (t, J ¼ 4.4 Hz, 1 H), 3.90 (t, J ¼ 9.2 Hz, 1 H), 3.70
(brs, 4 H), 3.54 (brs, 2 H), 3.06 (dd, J ¼ 12.4, 4.4 Hz, 1 H), 2.40e2.70
(m, 3 H), 2.54 (brs, 5 H), 1.47 (s, 2 H), 1.95e1.45 (m, 2 H), 1.73e1.69
(m, 2 H), 1.60e1.55 (m, 2 H), 1.47e1.42 (m, 2 H); 13C NMR (100 MHz,
5.4.1. 40-Demethyl-4-deoxylpodophyllotoxin40-(4-methyl-piperazine)
carbomate (4a)
The yield: 68%; white amorphous solid; m.p.:232e234 ꢀC; ½a D23
ꢂ
CDCl3) d 174.9, 153.4, 151.9 (2 C), 147.1, 146.7, 134.5, 130.5, 124.5,
-37 (c 0.3, CHCl3); IR (cmꢃ1) 3397, 2923, 1771, 1722, 1599, 1443,
124.4, 110.7, 104.5, 107.9 (2 C), 101.2, 72.1, 67.5, 56.4 (2 C), 52.0 (2 C),
47.6, 44.9, 44.1, 43.4, 33.2, 32.7, 30.4 (2 C), 24.1 (2 C); HRMS (ESI)
565.2553 for [M þ H]þ (calcd 565.2544 for C31H37N2O4).
1454, 1421, 1224, 1034, 999; 1H NMR (400 MHz, CDCl3)
d 7.26 (s,
1 H), 6.66 (s, 1 H), 6.52 (s, 1 H), 6.37 (s, 2 H), 5.94 (d, J ¼ 6.4 Hz, 2 H),
4.71 (brs, 1 H), 4.63 (t, J ¼ 6.4 Hz,1 H), 4.47e4.42 (m, 1 H), 3.93e3.44
(m, 1 H), 3.71 (brs, 4 H), 3.57 (br, 2 H), 3.04e3.04 (m, 1 H), 2.40e273
(m, 3 H), 2.44 (t, J ¼ 4.4 Hz, 4 H), 2.33 (s, 3 H); 13C NMR (100 MHz,
5.4.6. N-(1-oxyl-40-demethyl-4-deoxypodophyllic)-
L-phenylalanine
40-(4-p-nitrophenyl -piperazine) carbomate (4f)
CDCl3)
d
174.4, 153.3, 151.4 (2 C), 147.0, 146.7, 134.4, 134.7, 130.4,
Yield: 32%; yellow amorphous solid; m.p.:132e134 ꢀC; ½a D23
ꢂ
-31
124.4, 124.3, 110.5, 104.4, 107.4 (2C), 101.1, 72.0, 56.2 (2 C), 54.6 (2 C),
47.4, 46.1, 44.7, 44.1, 43.7, 33.1, 32.6; HRMS (ESI) 511.2066 for
[M þ H]þ (calcd 511.2075 for C27H31N2O4).
(c 0.3, CHCl3); IR (cmꢃ1) 3307, 2920, 1771, 1734, 1710, 1645, 1597,
1502, 1446, 1324, 1224, 1124, 1035, 996; 1H NMR (400 MHz, CDCl3)
d
4.10 (d, J ¼ 9.2 Hz, 2 H), 7.30e7.27 (m, 5 H), 6.70 (d, J ¼ 9.2 Hz, 2 H),
6.67 (s,1 H), 6.52 (s,1 H), 6.34 (s, 2 H), 6.14 (d, J ¼ 4.4 Hz, NH), 5.95 (d,
J ¼ 4.4 Hz, 2 H), 4.94e4.47 (m, 1 H), 4.63 (d, J ¼ 3.6 Hz, 1 H), 4.46 (t,
J ¼ 6.4 Hz, 1 H), 4.12 (q, J ¼ 7.2 Hz, 2 H), 3.92 (t, J ¼ 4.4 Hz, 1 H), 3.70
(s, 6 H), 3.50e3.45 (m, 1 H), 3.39e3.34 (m, 1 H), 3.21e3.15 (m, 3 H),
3.10e3.04 (m, 3 H), 2.41e2.74 (m, 3 H), 2.69e2.61 (m, 1 H); 13C NMR
5.4.2. 40-Demethyl-4-deoxylpodophyllotoxin 40-(4-p-nitrophenyl-
piperazine) carbomate (4b)
The yield: 72%; yellow amorphous solid; m.p.:149e150 ꢀC; ½a D23
ꢂ
-45 (c 0.3, CHCl3); IR (cmꢃ1) 2916,1773,1719,1597,1505,1445,1423,
1324, 1224, 1127, 1034, 996; 1H NMR (400 MHz, CDCl3)
d
4.13 (d,
(100 MHz, CDCl3) d 174.4, 169.4, 154.0, 153.5, 151.4 (2 C), 147.0, 146.6,
J ¼ 9.2 Hz, 2 H), 6.44 (d, J ¼ 9.6 Hz, 2 H), 6.66 (s, 1 H), 6.51 (s, 1 H),
6.34 (s, 2 H), 5.93 (d, J ¼ 4.0 Hz, 2 H), 4.63 (d, J ¼ 4.0 Hz, 1 H), 4.45 (t,
J ¼ 6.4 Hz, 1 H), 3.90 (t, J ¼ 9.2 Hz, 1 H), 3.46 (br, 2 H), 3.77e3.3.73
(m, 2 H), 3.70 (s, 6 H), 3.51 (br, 4 H), 3.06 (dd, J ¼ 14.4, 5.6 Hz, 1 H),
134.4, 136.0, 130.2, 129.5 (2 C), 129.4, 124.6 (2 C), 124.3, 127.6, 127.1,
125.4 (2 C), 112.7 (2 C), 110.4, 104.4, 107.7 (2 C), 101.1, 72.0, 60.3, 56.1
(2 C), 51.9, 47.3, 46.3, 44.5, 43.7, 41.2, 39.4, 33.0, 32.6; HRMS (ESI)
765.2744 for [M þ H]þ (calcd 765.2766 for C41H41N4O11).
2.40e270 (m, 3 H); 13C NMR (100 MHz, CDCl3)
d 174.4, 154.5, 153.2,
151.4 (2 C), 147.1, 146.7, 134.9, 134.7, 130.3, 124.3, 124.1, 125.9 (2 C),
112.9 (2 C), 110.5, 104.4, 107.4 (2C), 101.2, 72.0, 56.2 (2 C), 47.4, 46.4
(2 C), 44.1, 43.4, 43.3, 33.1, 32.7; HRMS (ESI) 640.1915 for [M þ Na]þ
(calcd 640.1902 for C32H31N3NaO10).
5.4.7. N-(1-oxyl-40-demethyl-4-deoxypodophyllic)-
D
-phenylalanine
40-(4-p-nitrophenyl -piperazine) carbomate (4g)
ꢀ
Yield: 32%; yellow amorphous solid; m.p.:133e135 C; ½
aꢂ2D3 -53
(c 0.3, CHCl3); IR (cmꢃ1) 3274, 2914, 1737, 1594, 1500, 1449, 1331,
1230, 1115, 1035, 996; 1H NMR (400 MHz, CDCl3)
d 4.10
5.4.3. 40-Demethyl-4-deoxylpodophyllotoxin 40-(4-p-flurophenyl-
piperazine) carbomate (4c)
(d, J ¼ 9.2 Hz, 2 H), 7.30e7.27 (m, 5 H), 6.69 (d, J ¼ 9.6 Hz, 2 H), 6.66
(s, 1 H), 6.49 (s, 1 H), 6.34 (s, 2 H), 6.07 (d, J ¼ 4.4 Hz, NH), 5.93
(d, J ¼ 6.4 Hz, 2 H), 4.91e4.49 (m, 1 H), 4.62 (d, J ¼ 4.0 Hz, 1 H), 4.45
(t, J ¼ 6.4 Hz, 1 H), 4.12 (q, J ¼ 7.2 Hz, 2 H), 3.92 (t, J ¼ 4.4 Hz, 1 H),
3.70 (s, 6 H), 3.52e3.47 (m, 1 H), 3.39e3.34 (m, 1 H), 3.20e3.15 (m,
3 H), 3.10e3.02 (m, 3 H), 2.74e2.71 (m, 3 H), 2.66e2.63 (m, 1 H); 13C
Yield: 47%; white amorphous solid; m.p.:142e143 ꢀC; ½a D23
ꢂ
-72
(c 0.3, CHCl3); IR (cmꢃ1) 2911, 1774, 1723, 1600, 1509, 1423, 1336,
1223, 1157, 1129, 1039, 995; 1H NMR (400 MHz, CDCl3)
d 7.00e6.96
(m, 2 H), 6.92e6.40 (m, 2 H), 6.66 (s, 1 H), 6.52 (s, 1 H), 6.34 (s, 2 H),
5.93 (d, J ¼ 4.0 Hz, 2 H), 4.63 (d, J ¼ 3.6 Hz, 1 H), 4.46 (t, J ¼ 4.4 Hz,
1 H), 3.93 (t, J ¼ 9.2 Hz,1 H), 3.44 (brs, 2 H), 3.70 (brs, 4 H), 3.15e3.10
NMR (100 MHz, CDCl3)
765.2766 for C41H41N4O11).
d
HRMS (ESI) 765.2762 for [M þ H]þ (calcd

Y. Jin et al. / European Journal of Medicinal Chemistry 46 (2011) 4056e4061
4061
5.4.8. N-(1-oxyl-40-demethyl-4-deoxypodophyllic)-
L
-methine 40-(4-p-
5.7. Analysis of morphological changes
A-549 cells treated with 0.1 M 8b or vehicle were harvested
after 48 h, fixed with glutaraldehyde and osmium tetroxide,
dehydrated with acetone, then embedded, sectioned, stained and
examined for morphological changes using a transmission electron
microscope (JEOL JEM-1230, Japan) [30].
nitrophenyl- piperazine) carbomate (4h)
Yield: 32%; yellow amorphous solid; 32%. m.p.:134e139 ꢀC; ½a D23
ꢂ
m
-41 (c 0.3, CHCl3); IR (cmꢃ1) 3406, 2973, 2925, 1690, 1596, 1501,
1452, 1395, 1326, 1235, 1167, 1129, 1027, 994; 1H NMR (400 MHz,
CDCl3)
d
4.14 (d, J ¼ 4.4 Hz, 2 H), 6.45 (d, J ¼ 4.4 Hz, 2 H), 6.66 (s,1 H),
6.52 (s, 1 H), 6.34 (s, 2 H), 6.30 (d, J ¼ 4.4 Hz, NH), 5.94 (d, J ¼ 4.4 Hz,
2 H), 4.63 (d, J ¼ 3.6 Hz, 1 H), 4.45 (t, J ¼ 7.2 Hz, 1 H), 3.92
(t, J ¼ 9.6 Hz, 1 H), 3.90e3.46 (m, 2 H), 3.74e3.72 (m, 2 H), 3.70
(s, 6 H), 3.50 (brs, 4 H), 3.09e3.04 (m, 1 H), 2.40e2.66 (m, 3 H),
2.63e2.59 (m, 2 H), 2.09 (s, 3 H), 2.04e2.02 (m, 2 H); HRMS (ESI)
749.2464 for [M þ H]þ (calcd 749.2447 for C37H41N4O11S).
Acknowledgements
This work was financially supported by the Doctoral Found of
Qingdao University of Science and Technology (QUST 0022380),
Open Funds of Key Lab of Preclinical Study for New Drugs of Gansu
Province (GSKFKT-00801) and the Fundamental Research Funds for
the Central Universities, Lanzhou University (lzujbky-2010-136).
5.4.9. N-(1-oxyl-40-demethyl-4-deoxypodophyllic)- -methine 40-(4-p-
D
nitrophenyl- piperazine) carbomate (4i)
Yellow amorphous solid, Yield:; m.p.:134e139 ꢀC; ½a D23
ꢂ
-50
(c 0.3, CHCl3); IR (cmꢃ1) 3343, 2962, 2916, 1771, 1734, 1710, 1645,
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1597, 1503, 1445, 1460, 1326, 1229, 1127, 1035, 997; 1H NMR
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d
4.14 (d, J ¼ 9.2 Hz, 2 H), 6.43 (d, J ¼ 9.2 Hz, 2 H),
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d 174.4,
169.4, 154.2, 153.4, 151.4, 147.0, 146.7, 139.1, 134.9, 130.2, 124.3,
125.9, 113.0, 110.5, 104.5, 107.7, 101.2, 72.0, 56.1, 53.7, 49.6, 47.4, 47.2,
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a log plot of percent of control versus concentration.
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