Synthesis and biological evaluation of conjugates of deoxypodophyllotoxin and 5-FU as inducer of caspase-3 and -7

Article abstract of DOI:10.1016/j.ejmech.2011.12.005

In order to generate compounds with superior antitumor activity and reduced toxicity, a series of conjugates of deoxypodophyllotoxin and 5-FU were synthesized by coupling 4′-demethyl-4-dexoypodophyllotoxin with N-(5-fluorouracil-N¹-ly acetic)-

Full text of DOI:10.1016/j.ejmech.2011.12.005

European Journal of Medicinal Chemistry 49 (2012) 48e54
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of conjugates of deoxypodophyllotoxin
and 5-FU as inducer of caspase-3 and -7
,
d
,
a,b,
Wen-Ting Huang a, Jie Liu a, Jian-Fei Liu a, Ling Hui c , Yi-Lan Ding
*
**
, Shi-Wu Chen
***
a School of Pharmacy & Key Lab of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou 730000, China
b State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
c Experimental Center of Medicine & Key Laboratory of Stem Cells and Gene Drug of Gansu Province, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
d Department of Medicine, Lanzhou Maternal and Child Health Care Hospital, Lanzhou 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to generate compounds with superior antitumor activity and reduced toxicity, a series of
conjugates of deoxypodophyllotoxin and 5-FU were synthesized by coupling 40-demethyl-4-
dexoypodophyllotoxin with N-(5-fluorouracil-N1-ly acetic)- amino acids (or 5-fluorouracil-N1-ly acetic
acid). The cytotoxic activity of these compounds against four human cancer cell lines (HL-60, A-549, HeLa
and SiHa) were evaluated, and results indicated that these compounds were more potent in terms of
cytotoxicity than either parent compound DPT or anticancer drug VP-16 and 5-FU. In addition, we found
that 14d induced cell cycle arrest in the G2/M phase accompanied by apoptosis in A-549 cells, and 14d
activated caspase-3 and -7. These results suggested that caspase-mediated pathways are involved in 14d
induced apoptosis.
Received 4 November 2011
Received in revised form
30 November 2011
Accepted 3 December 2011
Available online 9 December 2011
Keywords:
Podophyllotoxin
5-FU
Antineoplastic agents
Apoptosis
Ó 2011 Elsevier Masson SAS. All rights reserved.
Caspase
1. Introduction
prodrug, etoposide phosphate (4). These drugs are presently in
clinical use for the treatment of small cell lung cancer, testicular
Cancer is a major global health problem, representing the
second-leading cause of death worldwide [1]. According to infor-
mation from the World Health Organization (WHO), it is estimated
that there will be 12 million deaths from cancer in 2030. Over the
last few years, improvements in treatment and prevention have led
to a decrease in cancer deaths, and a number of chemotherapeutic
drugs have been developed to treat cancer.
Podophyllotoxin (PPT, 1), a naturally occurring cyclolignan iso-
lated from Podophyllum species, is a well-known cytotoxic deriva-
tive that acts as a potent anti-microtubule agent [2]. In spite of
potential uses as a medicinal drug, human trials with PPT were
discontinued due to its systemic toxicity [3]. Over the last 30 years,
extensive structural modifications of podophyllotoxin have led to
the development of the clinically valuable anticancer drugs, eto-
poside (VP-16, 2), teniposide (VM-26, 3) and the water-soluble
carcinoma, acute leukemia, Kaposi’s sarcoma and lymphoma. The
cytotoxic mechanism of VP-16 is the inhibition of topoisomerase II
(TOP II), unlike the parent compound which inhibits mitosis [4]. VP-
16 induces cell death by enhancing the TOP II-mediated DNA
cleavage through the stabilization of the transient DNA/TOP II
cleavage complex. In such a complex, DNA is cleaved on both
strands and covalently linked to the enzyme; the TOP II poison
prevents it from dissociating [5]. Recent researches reveal that the
antitumor activity of VP-16 is attributed primarily to its inhibition
of TOP II
the isoform [6]. Besides, after the approval of VP-16, there are
a, whereas the carcinogenic effect has been attributed to
b
several potential drug candidates based on PPT applied in clinical
trial, such as, NK-611 (5), GL-331 (6), tafluposide (F11782, 7) and
F14512 (8) (Fig. 1). Among them, F11782 is a dual inhibitor of top-
oisomerases I and II which impairs the binding of the enzyme to
DNA, but does not stabilize the cleavage complex [7,8]. The
spermine-conjugated epipodophyllotoxin derivative F14512 is
a topoisomerase II poison that exploits the polyamines transport
system (PTS) to target preferentially tumor cells [9,10].
* Corresponding author.
** Corresponding author.
*** Corresponding author. School of Pharmacy & Key Lab of Preclinical Study for
New Drugs of Gansu Province, Lanzhou University, Lanzhou 730000, China.
Tel./fax: þ86 931 8915686.
Deoxypodophyllotoxin (DPT, 9), an analog of PPT, possess
potential anti-proliferative and antitumor activity in diverse cell
types [11e13], as well as anti-inflammatory [14] and anti-viral [15]
E-mail address: chenshwu@gmail.com (S.-W. Chen).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.005

W.-T. Huang et al. / European Journal of Medicinal Chemistry 49 (2012) 48e54
49
R
R3
O
NO2
O
5
8
11
HN
O
O
O
HO
4
1
3
2
O
O
O
O
2
R1 = H, R2 = OH, R3 = Me
R2
O
O
S
O
O
3
4
5
R1 = H, R2 = OH, R3 =
2'
6'
O
O
O
R1 = PO3H2, R2 = OH, R3 = Me
R1 = H, R2 = NMe2, R3 = Me
MeO
OMe
4'
MeO
OMe
OMe
MeO
OMe
OH
1
R = OH
O
9
R = H
OR1
6
R2
O
H
R2
O
H
N
N
HN
N
NH2
H
O
O
O
O
O
O
O
F
F
O
R1 = PO3H
O
F
F
F
O
O
R2
=
O
MeO
OMe
MeO
OMe
O
OH
OR1
7
8
Fig. 1. Structures of podophyllotoxin (1), etoposide (2), teniposide (3), etopophos (4), NK-611 (5), GL-331 (6), tafluposide (7), F14512 (8) and deoxypodophyllotoxin (9).
activity. It has been revealed that DPT inhibits tubulin polymeri-
zation and induces G2/M cell cycle arrest followed by apoptosis
through multiple cellular processes, involving the activation 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 [16,17]. Besides, it is found that DPT also inhibits migration
2. Results and discussion
2.1. Chemistry
The synthetic route to the target compounds first involved the
generation of 5-Fluorouracil-N1-ly acetic acid (10) and its amino
acids derivatives 12aef, they were synthesized from 5-FU as
reference reported (Scheme 1) [30,33]. Briefly, 5-FU was treated
initially with 2-chloride acetic acid in the presence of aqueous
potassium hydroxide to afford compound 10. The activated ester 11
was obtained through esterification of 10 with p-nitrophenol using
N,N’-dicyclohexylcarbodiimide (DCC) in dry N,N-dimethylforma-
mide (DMF). The intermediate 11 was further reacted with appro-
priate amino acids in alkaline DMF-H2O solvent to yield N-(5-
fluorouracil-N1-ly acetyl)-amino acids 12aef.
Synthesis of 4-dexoydopodophyllotoxin conjugates was carried
out from the key intermediate 40-demethy-4-deoxypodophyl
lotoxin (DDPT, 13), which was prepared as described in our previ-
ously reported procedure [31]. The intermediate DDPT was coupled
to various N-(5-fluorouracil-N1-ly acetic)-amino acids (or 5-
Fluorouracil-N1-ly acetic acid) in the presence of DCC and 4-
dimethylamino pyridine (DMAP) to provide the corresponding
conjugates 14aef (or 15) with high or moderate yields as depicted
in Scheme 2. All the synthesized compounds were characterized by
IR, 1H and 13C NMR spectroscopy, and high-resolution mass
spectrometry.
and MMP-9 via MAPK pathways in TNF-a-induced HASMC [18].
In clinical, anticancer drugs are usually used as suitable
combinations of different drugs to treat cancer, because only a few
tumors are sensitive enough to be cured by single drugs [19]. Many
combinations, which were named mutual drug, in clinical use
consist of an antimetabolite with one or more other anticancer
agents. A mutual drug usually consists of two different synergistic
drugs joined together directly or by means of a linker [20]. In order
to generate more efficient anticancer drugs based on PPT, many
pharmacologist and chemists had synthesized a number of conju-
gates of PPT with other antitumor agents, such as conjugates of
podophyllotoxin-camptothecin [21], taxoid- epipodophyllotoxin
[22], polymer-linked podophyllotoxin [23], vinorelbine- podo-
phyllotoxin [24], thiocolchicine-podophyllotoxin [25], the biolog-
ical evaluation showed that most of these conjugates exhibited
superior cytotoxicity in vitro than parent compound PPT.
Recently, we have reported some conjugates of podophyllotoxin
with antimetabolite 5-FU [26e30] and derivatives of DPT [31,32]. In
our continuing efforts to find new compounds with potent activities
and low toxicity from natural products, considering amino acids are
often used as carrier vehicles for some drugs because of their good
water solubility when actively transplanted into mammalian tissue,
in this work we described the synthesis of a series of conjugates of
DPT and 5-FU joined by suitable amino acid spacers and tested their
cytotoxicity against a panel of four human cancer cell lines.
Furthermore, compound 14d was evaluated for its effect on cell cycle
progression, apoptosis, and induction to caspase-3 and -7.
2.2. Biological activities
The biological activities of the DPT conjugates 14aef and 15
were evaluated by an in vitro cytotoxicity test carried out with
a panel of four human tumor cell lines (premyelocytic leukemia HL-
60, lung carcinoma A-549, cervical carcinoma HeLa, and cervical
O
N
O
N
O
F
F
O
F
HN
HN
HN
F
HN
i
iii
ii
O
O
O
O
N
H
N
O
O
N
H
OH
OH
O
O
R
O
NO2
5-FU
12a-f
10
11
Scheme 1. Synthesis of 12aef. Reagents and conditions: i) 2-chloride acetic acid, 10% KOH, reflux; ii) p-nitro-phenol, DCC/DMF, rt; iii) amino acids, DMF, rt.

50
W.-T. Huang et al. / European Journal of Medicinal Chemistry 49 (2012) 48e54
O
OH
O
O
O
O
O
O
O
O
O
10 or 12a-f
Ref 31
O
O
DCC,DMAP
MeO
OMe
R
O
N
O
MeO
OMe
NH
F
MeO
OMe
O
N
H
O
OH
OMe
O
13
1
14a-f, 15
Compds Configuration
R
Compds Configuration
R
L-
L-
L-
L-
Me
CHMe2
L-
D-
--
CH2CH2SMe
CH2CH2SMe
--
14a
14b
14c
14d
14e
14f
15
CH2CHMe2
CH2C6H5
Scheme 2. Synthesis of 14aef and 15.
squamous cell carcinoma SiHa), using VP-16 and 5-FU as reference
compounds. The screening procedure was based on the standard
MTT or CCK-8 method [34], and the results of these experiments are
summarized in Table 1.
As illustrated in Table 1, the DPT conjugates, 14aef and 15, were
generally more potent than 5-FU, DPT, DDPT and VP-16 in their
cytotoxicity to these four cell lines. As a group, these compounds
were most effective in HL-60 cells, and had lowest potency in HeLa
cells, although the order of potency varied in each cell line. The IC50
value of compound 14d, which is the most potent conjugate in this
apoptosis in FACS analysis of 14d-treated A-549 cells. To confirm
the effect of conjugates of DPT and 5-FU on induction of apoptosis,
the morphology of A-549 cells were examined using Hoechst
staining. As shown in Fig. 3, negative control (untreated) cells
exhibited excellent growth characteristic after 24 h incubation
(Fig. 3A). However, A-549 cells treated with 0.5 mM 14d evoked
typical apoptotic features, such as membrane blebbing, cell
shrinkage and detachment, and nuclear condensation even frag-
mentation (Fig. 3B). A similar effect was observed when cells
were exposed to 0.5 mM PPT [16].
group compounds, is 0.023, 0.56, 0.83 and 0.76
549, HeLa and SiHa, respectively. In Table 1, we also found that
different substituent at the -carbon of the amino acid in this class
of compounds (14aee) appeared to have significant effects on their
anti-proliferative activity in the in vitro assay. Compounds 14cee
exhibited more potent activity against four human cancer cell lines
m
M for HL-60, A-
Caspases are cysteinyl aspartate proteinases that cleave
substrate proteins at aspartate residues. Upon receiving an
apoptotic signal, the precursor caspases undergo proteolytic
processing to generate an active subunit. Among the 11 caspases
characterized in humans, caspase-3 and -7 are the main down-
stream effector caspases that play essential roles in degrading
the majority of key cellular components in apoptotic cells [38].
To determine whether caspase is involved in 14d-induced
apoptosis, western blot analysis was performed using antibodies
to recognize the cleaved forms of caspase-3 and caspase-7 [39].
a
than those of 14a and 14b. In addition, the conjugates with
L-amino
acids incorporated appear to be more potent than those with
D
-
amino acid substituent (14e vs. 14f). Notably, conjugates 15 incor-
porated of DDPT with 5-Fluorouracil- N1-ly acetic acid also
exhibited high cytotoxities against these four tumor cell lines.
Results presented herein and elsewhere [35,36] suggested that
esters of DDPT will improve the antitumor activity compared with
that of the starting compound DDPT. These results also confirmed
the assumption that free hydroxyl group at the 40 position in DDPT
was not favorable for antitumor activity [35].
We found that treatment of A-549 cells with 0.5 mM 14d grad-
ually increased the cleavage of both caspase-3 (Fig. 4A) and
caspase-7 (Fig. 4B) in a time-dependent manner. These results
demonstrate the involvement of caspases-3 or -7 in 14d-induced
apoptosis.
DPT is reported to induce apoptosis and cell cycle arrest in the
G2/M phase in HeLa cells [16,17]. To determine whether conjugates
of DDPT and 5-FU have similar effects on tumor cells, the effects of
14d on cell cycle progression were determined by FACS analysis in
propidium iodide-stained A-549 cells [37]. As shown in Fig. 2,
Table 1
Cytotoxicity of compounds 14aed and 15 with 48 h drug exposure.
Compounds
Cytotoxicity (IC50
HL-60b
, m
M)a
Aꢀ549c
HeLab
SiHac
0.6
treatment with 0.5 mM 14d lead to a time-dependent accumulation
14a
14b
14c
14d
14e
14f
15
DPT
DDPT
VP-16
5-Fu
0.19
0.14
0.063
0.023
0.23
0.94
0.035
0.47
1.4
0.36
1.07
0.56
0.83
2.6
0.66
1.38
1.8
0.2
1.23
0.43
0.83
0.78
1.97
0.18
6.01
53.3
40.4
82.2
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; 52.6% and 64.8% of the cells were
in G2/M phase after 14d exposure for 12 h (Fig. 2B) and 24 h
(Fig. 2C) respectively, compared with 15.6% in untreated cultures
(Fig. 2A). These results demonstrate that 14d interfere with cell
proliferation by arresting the cell cycle, and induce G2/M arrest in
A549 cells, and that these conjugates of DDPT and 5-FU have similar
effects on the cell cycle arrest with that of parent compound DPT
[16,17].
1.65
0.35
0.76
0.36
1.51
0.11
1.98
43
2.76
218
2.96
2.85
68.3
24.9
54.8
a
Date are the mean of three independent experiment.
CCK-8 method.
MTT method.
b
c
As above mentioned, DPT induce apoptosis of tumor cells in
a time-dependent manner, however, we did not find obvious

W.-T. Huang et al. / European Journal of Medicinal Chemistry 49 (2012) 48e54
51
3. Conclusions
4.1.1. Procedure of synthesis of 10
Into a solution of 5-fluorouracil (6.5 g, 50 mmol) and potassium
hydroxide (5.6 g, 100 mmol) in water (40 mL) was added the
solution of chloroacetic acid (4.75 g, 50 mmol) in water (20 mL) and
stirred for 30 min at room temperature. The pH value of the reac-
tion mixture was adjusted to, and kept at 10 by the drop-by-drop
addition of a 10% potassium hydroxide aqueous solution. The
mixture was then refluxed for 2 h, cooled, and acidified to pH 2 by
the addition of concentrated hydrochloric acid and the crystals of
10 were isolated by suction. Recrystallization was completed by
dissolving the product in the saturated aqueous sodium bicar-
bonate and reprecipitated with concentrated hydrochloric acid to
result in white needles.
In summary, the cytoxicity assay for most of these conjugates
demonstrated many fold increase of activity in comparison to
either parent compound DPT or clinical drug etoposide and 5-FU;
Furthermore, the promising conjugate 14d could induce cell cycle
arrest in the G2/M phase and apoptosis by activated caspase-3 and
-7 in A-549 cells. Despite these potential therapeutical benefits of
combining such agents through suitable amino acid linkers are
still unclear, the results presented herein promoted us to believe
that the approach should be applicable for other antitumor agents,
and it is worthwhile to explore the antitumor potential of these
and similar types of compounds. Our current results shed more
light on the potential of these conjugated compounds to overcome
drug resistance frequently encountered with chemotherapeutic
agents.
4.1.2. Procedure of synthesis of 11
A mixture of 2-N1-5-FU acetic acid (9.8 g, 0.05 mol), p-nitro-
phenol (13.9 g, 0.1 mol) and DCC (10.3 g, 0.05 mol) was stirred in
anhydrous DMF (100 mL) for 4 h at 0 ꢁC, then stirred for another
12 h at room temperature. The reaction mixture was filtered and
filtrate was evaporated in vacuo, the crude product was further
recrystallized in acetonitrile-ethanol to yield compound 11.
4. Experimental section
4.1. Chemistry
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
spectrometer with TMS as an internal standard, all chemical shift
4.1.3. General procedure of synthesis of 12aef
p-Nitrophenyl 2-N1-5-fluorouracil-ylacetate 11 (1.4 g, 5 mmol)
was dissolved in DMF (10 mL), then the solution of aqueous 5%
NaOH (5 mL) of appropriate a-amino acid (6 mmol) was added and
the mixture stirred for 2 h at 0 ꢁC, then continuously stirred for 10 h
at room temperature while the pH value of the reaction mixture
was kept at 8e10. The solvent was removed under vacuum, the
residue was crystallized from water, and compounds 12aef were
provided in needle crystal.
values are reported as
d ppm. Optical rotations were measured on
a Perkin Elmer 341 polarimeter in a 1 dm cell at 23 ꢁC. Mass
spectra were recorded on a Bruker Daltonics APEXk49e and
VGZAB-HS (70 ev) spectrometer with ESI source as ionization,
respectively. All reactions were monitored by thin layer chro-
matograph (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.
4.1.4. General preparation of compounds 14aef and 15
A mixture of 13 (0.5 mmol),10 (or the appropriate N-5-FU-acetic
amino acid 12aef) (0.5 mmol) and N,N-dimethylaminopyridine
(DMAP, 20 mg) was stirred in dry dichloromethane (10 mL) for
5
min at room temperature under argon. N,N0-dicyclohexyl
carbodiimide (DCC, 104 mg) was added and the reaction mixture
Fig. 2. Effect of 14d on cell cycle progression. A) control A-549 cells; B) A-549 cells treated with 0.5 mM 14d for 12 h; C) A-549 cells treated with 0.5 mM 14d for 24 h.

52
W.-T. Huang et al. / European Journal of Medicinal Chemistry 49 (2012) 48e54
Fig. 3. Hoechst staining in A-549 cell line. A) control A-549 cells; B) A-549 cells treated with 0.5 mM 14d for 24 h.
was stirred for 3e40 h and monitored by TLC. The reaction mixture
was filtered and the filtrate was evaporated. The residue was
separated to afford compound 14aef and 15 by column chroma-
tography on silica gel with dichloromethane-acetone as eluent.
33.0, 32.7, 24.7, 22.7, 22.0, 21.9; HRMS (ESI) 685.2526 for
[M þ NH4]þ (calcd 685.2516 for C33H38FN4O11).
4.1.4.4. 40-O-(5-FU-acetic)- -phenylalanine 4-deoxyl-40-O-demethyl
L
podophyllic ester (14d). Yield: 48%; m.p.:186e187 ꢁC; ½a D23
ꢂ
-71 (c
4.1.4.1. 40-O-(5-FU- acetic)-
L
-alanine 4-deoxyl-40-O-demethylpodo-
0.3, CHCl3); IR (cmꢀ1) 3304, 3201, 3067, 3032, 3006, 2934, 2846,
1768, 1702, 1668, 1602, 1539, 1505, 1483, 1462, 1421, 1378, 1338,
phyllic ester (14a). Yield: 70%; m.p.:168e170 ꢁC; ½a D23
ꢂ
-66 (c 0.3,
CHCl3); IR (cmꢀ1) 3518, 3316, 3206, 3071, 2920, 2846, 1768, 1696,
1670, 1601, 1505, 1483, 1462, 1422, 1379, 1338, 1227, 1130, 1037, 996;
1227, 1155, 1131, 1094, 1037, 996; 1H NMR (400 MHz, CDCl3)
d 9.52
(brd, 1H, NH), 7.24e7.13 (m, 6H), 7.07 (t, J ¼ 8.8 Hz, 1H), 6.66 (s, 1H),
6.48 (s, 1H), 6.38 (s, 2H), 5.93 (d, J ¼ 7.6 Hz, 2H), 5.17 (q, J ¼ 6.0 Hz,
1H), 4.60 (d, J ¼ 3.2 Hz, 1H), 4.45 (t, J ¼ 8.0 Hz, 1H), 4.36 (ddq, J ¼ 16,
3.6 Hz, 2H), 3.89 (t, J ¼ 8.8 Hz, 1H), 3.67 (s, 6H, 2 OMe), 3.39e3.32
(m, 1H), 3.21e3.15 (m, 1H), 3.08 (dd, J ¼ 12.8, 4.4 Hz, 1H), 2.79e2.69
1H NMR (400 MHz, CDCl3)
d 9.71 (brs, 1H, NH), 7.36e7.33 (m, 1H,
NH), 7.17 (d, J ¼ 7.2 Hz, 1H), 6.66 (s, 1H), 6.49 (s, 1H), 6.37 (s, 2H),
5.93 (d, J ¼ 7.6 Hz, 2H), 4.88e4.83 (m, 1H), 4.60 (d, J ¼ 4.0 Hz, 1H),
4.45 (t, J ¼ 6.4 Hz, 1H), 4.36e4.33 (m, 2H), 3.90 (t, J ¼ 8.8 Hz, 1H),
3.66 (s, 6H, 2 OMe), 3.06 (dd, J ¼ 12.0, 4.0 Hz, 1H), 2.80e2.72 (m,
(m, 3H); 13C NMR (100 MHz, CDCl3)
d 174.9, 174.8, 169.2, 165.7, 157.0
3H), 1.55 (d, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d
175.0 (2C),
(d, J ¼ 26 Hz, 1C), 151.1, 149.6, 147.1, 146.7, 140.2 (d, J ¼ 234 Hz, 1C),
139.7, 135.6, 130.1, 129.7, 129.0 (d, J ¼ 37 Hz, 1C), 128.4 (2C), 127.0,
110.4, 108.5, 107.7, 101.2, 72.1, 69.5, 56.0 (2C, 2OMe), 53.8, 53.1, 49.9,
47.3, 43.8, 37.5, 33.0, 32.7, 31.6, 29.6, 29.2; HRMS (ESI) 719.2345 for
[M þ NH4]þ (calcd 719.2359 for C36H36FN4O11).
170.7, 165.9, 157.4 (d, J ¼ 26 Hz, 1C), 151.1, 151.0, 149.9, 147.0, 146.7,
140.2 (d, J ¼ 236 Hz, 1C), 139.5, 130.0, 129.6 (d, J ¼ 33 Hz, 1C), 128.4,
127.2, 110.3, 108.5, 107.8, 101.2, 72.1, 56.2 (2 OMe), 49.9, 48.4, 47.2,
43.7, 32.9, 32.7, 18.1; HRMS (ESI) 643.2039 for [M þ NH4]þ (calcd
643.2046 for C30H32FN4O11).
4.1.4.5. 40-O-(5-FU- acetic)- -methine 4-deoxyl-40-O-demethylpodo-
L
4.1.4.2. 40-O-(5-FU- acetic)-
L
-valine 4-deoxyl-40-O-demethylpodo-
phyllic ester (14e). Yield: 56%; m.p.:168e169 ꢁC;½a D23
ꢂ
-83 (c 0.3,
phyllic ester (14b). Yield: 65%; m.p.:183e185 ꢁC;½a D23
ꢂ
-69 (c 0.3,
CHCl3); IR (cmꢀ1) 3304, 3201, 3068, 3003, 2918, 2844, 1767, 1701,
CHCl3); IR (cmꢀ1) 3316, 3204, 3070, 3002, 2966, 2938, 2844, 1765,
1703,1601,1505,1483,1465,1422,1378,1338,1227,1154,1131,1038,
1601, 1505, 1483, 1463, 1379, 1338, 1227, 1154, 1130, 1037, 996; 1H
NMR (400 MHz, CDCl3) d 7.37e7.31 (m, 2H), 6.67 (s,1H), 6.49 (s,1H),
996; 1H NMR (400 MHz, CDCl3)
d
7.35 (d, J ¼ 5.6 Hz, 1H), 7.05 (br,
6.37 (s, 2H), 5.94 (d, J ¼ 4.6 Hz, 2H), 5.01 (q, J ¼ 6.8 Hz, 1H), 4.59 (d,
J ¼ 3.2 Hz, 1H), 4.45 (t, J ¼ 6.8 Hz, 1H), 4.38e4.31 (m, 2H), 3.90 (t,
J ¼ 8.8 Hz, 1H), 3.67 (s, 6H, 2 OMe), 3.07 (dd, J ¼ 15.6, 4.0 Hz, 1H),
2.80e2.72 (m, 3H), 2.64 (t, J ¼ 7.6 Hz, 2H), 2.35e2.27 (m, 1H),
1H), 6.67 (s, 1H), 6.50 (s, 1H), 6.37 (s, 2H), 5.94 (d, J ¼ 3.2 Hz, 2H),
4.88e4.84 (m, 1H), 4.61 (d, J ¼ 3.2 Hz, 1H), 4.46 (t, J ¼ 6.8 Hz, 1H),
4.36 (q, J ¼ 3.6 Hz, 2H), 3.90 (t, J ¼ 8.8 Hz, 1H), 3.66 (s, 6H, 2 OMe),
3.07 (dd, J ¼ 13.2, 4.4 Hz,1H), 2.80e2.67 (m, 3H), 2.46e2.35 (m, 1H),
2.21e2.12 (m, 1H), 2.09 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 175.0,
1.01 (d, J ¼ 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3)
d
175.0, 174.9,
169.5, 166.0, 157.3 (d, J ¼ 26 Hz, 1C), 151.0 (2C), 149.9, 147.1, 146.7,
140.3 (d, J ¼ 236 Hz, 1C), 139.7, 130.0, 129.5 (d, J ¼ 32 Hz, 1C), 128.3,
126.9, 110.3, 108.5, 107.1, 101.2, 72.1, 56.1 (2C), 51.9, 50.1, 47.3, 43.8,
32.9, 32.7, 31.7, 29.5, 15.2; HRMS (ESI) 703.2072 for [M þ NH4]þ
(calcd 703.2080 for C32H36FN4O11S).
169.4, 166.2, 157.2 (d, J ¼ 27 Hz, 1C), 151.1, 151.0, 149.9, 147.1, 146.7,
140.3 (d, J ¼ 236 Hz, 1C), 139.6, 130.1, 129.5 (d, J ¼ 28 Hz, 1C), 128.4,
126.9, 110.4, 108.5, 107.7, 101.2, 72.1, 57.4, 56.0 (2 OMe), 50.1, 47.3,
43.8, 32.9, 32.7, 31.4, 18.8, 17.1; HRMS (ESI) 671.2348 for [M þ NH4]þ
(calcd 671.2359 for C32H36FN4O11).
4.1.4.6. 40-O-(5-FU- acetic)- -methine 4-deoxyl-40-O-demethylpodo-
D
4.1.4.3. 40-O-(5-FU- acetic)-
L
-leucine 4-deoxyl-40-O-demethylpodo-
phyllic ester (14f). Yield: 48%; m.p.:169e171 ꢁC;½a D23
ꢂ
-77 (c 0.3,
phyllic ester (14c). Yield: 74%; m.p.:173e175 ꢁC;½a D
ꢂ
23-65 (c 0.3,
CHCl3); IR (cmꢀ1) 3303, 3225, 3069, 3303, 2918, 2844, 1766, 1698,
CHCl3); IR (cmꢀ1) 3313, 3207, 3072, 2958, 2926, 2847, 1766, 1699,
1601, 1505, 1482, 1421, 1379, 1337, 1226, 1153, 1130, 1037, 996; 1H
1601, 1505, 1483, 1379, 1338, 1227, 1154, 1130, 1037, 996; 1H NMR
(400 MHz, CDCl3)
6.67 (s, 1H), 6.50 (s, 1H), 6.37 (s, 2H), 5.94 (d, J ¼ 8.8 Hz, 2H), 5.02 (q,
d
7.35 (d, J ¼ 5.6 Hz, 1H), 7.22 (d, J ¼ 8.0 Hz, 1H),
NMR (400 MHz, CDCl3)
d
7.35 (d, J ¼ 5.6 Hz, 1H), 7.05 (br, 1H), 6.66
(s, 1H), 6.49 (s, 1H), 6.36 (s, 2H), 5.93 (d, J ¼ 8.0 Hz, 2H), 4.89e4.86
(m, 1H), 4.59 (d, J ¼ 4.2 Hz, 1H), 4.45 (t, J ¼ 7.2 Hz, 1H), 4.36 (d,
J ¼ 3.6 Hz, 2H), 3.90 (t, J ¼ 8.8 Hz, 1H), 3.66 (s, 6H, 2 OMe), 3.06 (dd,
J ¼ 16.0, 4.0 Hz, 1H), 2.80e2.72 (m, 3H), 1.90e1.75 (m, 2H),
1.68e1.65 (m,1H), 0.96 (d, J ¼ 5.2 Hz, 6H, 2 Me); 13C NMR (100 MHz,
CDCl3) d 175.0, 170.3, 165.8, 157.2, 151.1 (2C), 149.7, 147.1, 146.7, 140.4
(d, J ¼ 236 Hz, 1C), 139.5, 130.1, 129.3 (d, J ¼ 33 Hz, 1C), 128.3, 127.1,
110.4, 108.5, 107.8, 101.2, 72.1, 56.2 (2C), 51.2, 50.0, 47.3, 43.8, 41.6,
Fig. 4. Effects of 14d on the activation of caspase-3 and caspase-7.

W.-T. Huang et al. / European Journal of Medicinal Chemistry 49 (2012) 48e54
53
J ¼ 6.8 Hz,1H), 4.61 (d, J ¼ 2.4 Hz,1H), 4.46 (t, J ¼ 6.4 Hz,1H), 4.37 (d,
J ¼ 4.4 Hz, 2H), 3.92 (t, J ¼ 8.8 Hz, 1H), 3.67 (s, 6H, 2 OMe), 3.08 (dd,
J ¼ 16.0, 4.4 Hz, 1H), 2.80e2.72 (m, 3H), 1.90e1.75 (m, 2H), 0.96 (d,
Chromosomal condensation and morphological changes were
observed under a fluorescence microscope image system (Olympus,
Tokyo Japan).
J ¼ Hz, 2H); 13C NMR (100 MHz, CDCl3)
d 175.0,169.4,166.0,157.3 (d,
J ¼ 26 Hz, 1C), 151.0 (2C), 149.8, 147.1, 146.7, 140.3 (d, J ¼ 236 Hz, 1C),
139.7, 130.0, 129.5 (d, J ¼ 32 Hz, 1C), 128.4, 126.8, 110.3, 108.5, 107.1
(2C), 101.2, 72.1, 56.1 (2C, 2OMe), 51.9, 50.1, 47.3, 43.8, 32.9, 32.7,
31.7, 29.5, 15.2; HRMS (ESI) 703.2070 for [M þ NH4]þ (calcd
703.2080 for C32H36FN4O11S).
4.2.4. Western blot analysis
A-549 Cells (1 ꢃ 106 cells) exposed to compound 14d were
collected into tubes and then washed with PBS. Cell pellets were
lysesed with lyses buffer (50 mM TriseHCl, pH 7.5, 10 mM EDTA,
0.2M NaCl, 1.5 mM PMSF and 1% SDS). Cell lysates were boiled for
10 min, centrifuged and stored at ꢀ20 ꢁC. Cell lysates containing
4.1.4.7. 40-O-5-FU- acetic acid 4-deoxyl-40-O-demethylpodophyllic
10e20 mg protein were separated and transferred to nitrocellulose
ester (15). Yield: 62%; m.p.:180e182 ꢁC;½a D23
ꢂ
-50 (c 0.3, CHCl3); IR
filters. The blots were incubated with the corresponding antibodies
and developed [39].
(cmꢀ1) 3194, 3072, 3004, 2943, 2915, 2844, 1773, 1706, 1670, 1601,
1505, 1483, 1463, 1420, 1378, 1337, 1227, 1159, 1130, 1093, 1037, 996;
1H NMR (400 MHz, CDCl3)
d
9.40 (br, 1H, NH), 7.31 (d, J ¼ 5.6 Hz,
Acknowledgments
1H), 6.67 (s, 1H), 6.51 (s, 1H), 6.39 (s, 2H), 5.95 (d, J ¼ 7.6 Hz, 2H),
4.77 (s, 2H), 4.63 (d, J ¼ 4.4 Hz, 1H), 4.46 (t, J ¼ 8.8 Hz, 1H), 3.92 (t,
J ¼ 8.8 Hz, 1H), 3.70 (s, 6H, 2 OMe), 3.07 (dd, J ¼ 15.6, 4.8 Hz, 1H),
This work was financially supported by the Fundamental
Research Funds for the Central Universities, Lanzhou University
(lzujbky-2010-136), and the Open Funds of Key Lab of Preclinical
Study for New Drugs of Gansu Province (GSKFKT-00801).
2.80e2.67 (m, 3H); 13C NMR (100 MHz, CDCl3)
d 174.8, 165.2, 157.1
(d, J ¼ 26 Hz, 1C), 150.9 (2C), 149.4, 147.1, 146.7, 140.4 (d, J ¼ 237 Hz,
1C), 139.8, 130.1, 129.5 (d, J ¼ 32 Hz, 1C), 128.3, 126.7, 110.4, 108.5,
107.6, 101.2, 72.0, 56.1 (2 OMe), 48.0, 47.3, 43.7, 33.0, 32.7, 30.7;
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C27H277FN3O11).
þ
H]þ (calcd 572.1675 for
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