Synthesis and cytotoxic activity on human cancer cells of carbamate derivatives of 4β-(1,2,3-triazol-1-yl)podophyllotoxin

Article abstract of DOI:10.1016/j.ejmech.2013.03.068

Carbamate derivatives of 4β-(1,2,3-triazol-1-yl)podophyllotoxin were synthesized by means of click chemistry, and their cytotoxicities against human cancer cell lines HL-60, A-549, HeLa, and HCT-8 were evaluated. Some compounds were more potent than the anticancer drug etoposide. 4′-O-Demethyl- 4β-[(4-hydroxymethyl)-1,2,3-triazol-1-yl]-4-deoxypodophyllotoxin cyclopentyl carbamate, the most potent compound, induced cell cycle arrest in the G2/M phase accompanied by apoptosis in A-549 cells. Furthermore, this compound inhibited the formation of microtubules in A-549 cells and caused the inhibition of DNA topoisomerase-II.

Full text of DOI:10.1016/j.ejmech.2013.03.068

Accepted Manuscript
Synthesis and cytotoxic activity on human cancer cells of carbamate derivatives of
4β-(1,2,3-triazol-1-yl)podophyllotoxin
Jian-Fei Liu, Chun-Yan Sang, Xiao-Hui Xu, Lin-Lin Zhang, Xuan Yang, Lin Hui, Jin-
Bang Zhang, Shi-Wu Chen
PII:
S0223-5234(13)00228-6
10.1016/j.ejmech.2013.03.068
EJMECH 6106
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 January 2013
Revised Date: 29 March 2013
Accepted Date: 31 March 2013
Please cite this article as: J.-F. Liu, C.-Y. Sang, X.-H. Xu, L.-L. Zhang, X. Yang, L. Hui, J.-B. Zhang,
S.-W. Chen, Synthesis and cytotoxic activity on human cancer cells of carbamate derivatives of 4β-
(1,2,3-triazol-1-yl)podophyllotoxin, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.03.068.
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*Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphical Abstract.doc
ACCEPTED MANUSCRIPT
Synthesis and cytotoxic activity on human cancer cells of carbamate
derivatives of 4β-(1,2,3-triazol-1-yl)podophyllotoxin
Jian-Fei Liu, Chun-Yan Sang, Xiao-Hui Xu, Lin-Lin Zhang, Xuan Yang, Lin Hui*, Jin-Bang
Zhang*, Shi-Wu Chen**
O
R3
O
N
N
R2
N
N
O
O
O
O
OMe
MeO
OR1
Carbamate derivatives of 4β-(1,2,3-triazol-1-yl)podophyllotoxin exhibited promising in vitro
cytotoxicities. Compound 22 induced cell cycle arrest in the G2/M phase accompanied by apoptosis in
A-549 cells, and inhibited the formation of microtubules and DNA topoisomerase-II.

*Highlights (for review)
ACCEPTED MANUSCRIPT
Research highlights:
1. Eleven carbamates of 4β-(1,2,3-triazol-1-yl)podophyllotoxin were
synthesized by click chemistry.
2. Some carbamates exhibited potent cytotoxic activity compared with VP-16.
3. Compound 22 arrests cell cycle in the G2/M phase accompanied by apoptosis
in A-549 cells.
4. Compound 22 inhibits the formation of microtubules in A-549 cells.
5. Compound 22 inhibits DNA topoisomerase-II.

*Revised Manuscript
Click here to view linked References
ACCEPTED MANUSCRIPT
Synthesis and cytotoxic activity on human cancer cells of carbamate
derivatives of 4β-(1,2,3-triazol-1-yl)podophyllotoxin
a
a
a
a
a
Jian-Fei Liu , Chun-Yan Sang , Xiao-Hui Xu , Lin-Lin Zhang , Xuan Yang , Lin
Hui b,*, Jin-Bang Zhang c,*, Shi-Wu Chen a,c,**
a
School of Pharmacy & Key Lab of Preclinical Study for New Drugs of Gansu Province, Lanzhou
University, Lanzhou 730000, China
b
Experimental Center of Medicine & Key Laboratory of Stem Cells and Gene Drug of Gansu
Province，General Hospital of Lanzhou Military Command, Lanzhou 730050, China
c
State Key Laboratory of Applied Organic Chemistry & College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou 730000, China
Abstract: Carbamate derivatives of 4β-(1,2,3-triazol-1-yl)podophyllotoxin were
synthesized by means of click chemistry, and their cytotoxicities against human
cancer cell lines HL-60, A-549, HeLa, and HCT-8 were evaluated. Some compounds
were more potent than the anticancer drug etoposide. 4'-O-Demethyl-
4β-[(4-hydroxymethyl)-1,2,3-triazol-1-yl]-4-deoxypodophyllotoxin
cyclopentyl
carbamate, the most potent compound, induced cell cycle arrest in the G2/M phase
accompanied by apoptosis in A-549 cells. Furthermore, this compound inhibited the
formation of microtubules in A-549 cells and caused the inhibition of DNA
topoisomerase-II.
Keywords: Podophyllotoxin; Triazole; Microtubules; Topoisomerase-II
________________________
* Corresponding author.
** Corresponding author. School of Pharmacy, Lanzhou University, Lanzhou 730000,
China. Tel./fax: +86 931 8915686.
E-mail address: chenshwu@gmail.com; chenshw@lzu.edu.cn (S.-W. Chen).
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1. Introduction
Natural products show a broad spectrum of biological activities against a variety
of diseases, including infections, immune system disorders, neurological disorders,
and cancer [1]. Podophyllotoxin (PPT, 1), the most abundant naturally occurring
cyclolignan, is isolated mainly from Podophyllum peltatum and P. hexandrum, and
has cathartic, antirheumatic, and antiviral properties [2]. However, its antimitotic
activity has proved to be of the greatest interest to researchers [3]. PPT is an
antimicrotubule agent that acts at the colchicine-binding site on tubulin [4]. Because
PPT is highly toxic, it is not used as an anticancer drug itself, but several
semisynthetic derivatives, including etoposide (VP-16, 2), teniposide (3), and
etopophos (4) (Fig. 1), are in clinical use against various cancers, including small cell
lung cancer, testicular carcinoma, lymphoma, and Kaposi’s sarcoma [5–7]. The
mechanism of action of etoposide involves the inhibition of topoisomerase-II
(TOP-II), unlike the parent compound which inhibits mitosis. Etoposide induces cell
death by enhancing TOP-II-mediated DNA cleavage through the stabilization of the
transient DNA/TOP-II cleavage complex [8]. In this complex, DNA is cleaved on
both strands and covalently linked to the enzyme, which prevents dissociation of the
complex [9]. With the goal of developing more-potent analogues and overcoming
drug resistance, several potential drug candidates based on PPT, such as NK-611 (5),
GL-331, tafluposide (F11782, 6), and F14512 (Fig. 1), are being evaluated in clinical
trials [10]. F11782 inhibits both TOP-I and -II; it impairs the binding of the enzyme to
DNA but does not stabilize the cleavage complex [11,12]. F14512, a
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spermine-conjugated epipodophyllotoxin derivative is a TOP-II poison that exploits
the polyamine transport system to preferentially target tumor cells [13,14].
Investigation of the structure-activity relationships of PPT indicates that the
trans-lactone, the 4β-substituted moiety, and the 4'-demethyl moieties are essential for
TOP-II inhibitory activity [3,5]. 4β-N-Substituted derivatives of PPT have attracted
much interest owing to their improved cytotoxicity relative to PPT [15]. Recently,
4β-1,2,3-triazole derivatives of PPT have been shown to exhibit more potent
anticancer activity and better binding ability to TOP-II than etoposide [16–18].
We have discovered derivatives of PPT that show both higher antitumor activity
and greater solubility than PPT [19–22]. In this study, which is part of our continuing
effort to find new natural product–based compounds with potent activities and
minimal side effects, we synthesized a series of carbamate derivatives of
4β-(1,2,3-triazol-1-yl)podophyllotoxin by click chemistry and evaluated their
cytotoxicities against a panel of four human cancer cell lines. The most active
carbamate (22) was evaluated for its effect on cell cycle progression, apoptosis,
inhibition of microtubules formation, and inhibition of TOP-II.
2. Results and discussion
2.1. Chemistry
Click chemistry is an effective approach to generate novel pharmacophores [23].
Of particular interest is the Cu2SO4·5H2O-sodium ascorbate-catalyzed alkyne–azide
cycloaddition provides 1,4-disubstituted 1,2,3-triazoles [24]. Using this reaction, we
synthesized 11 carbamate derivatives of 4β-(1,2,3-triazol-1-yl)podophyllotoxin (15-
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–25) by the route depicted in Scheme 1. PPT (1) was regioselectively demethylated
with trimethylsilyl iodide and barium carbonate to afford 7 by means of a previously
described procedure [25]. The C-4' hydroxy group was then protected with
chlorocarbonylbenzoate to afford benzyloxycarbonyl derivative 8 [25]. Both PPT and
8 were converted into the corresponding 4β-azides 9 and 10 by reaction with HN3 in
the presence of Et2O·BF3 [26]. The 4β-triazole of PPT (11) and that of
4'-O-demethyl-4'-benzyloxycarbonyl-4-epipodophyllotoxin (12) were then prepared
in yield 88%‒ 90% by the reaction of 9 and 10, respectively, with propargyl alcohol in
the presence of CuSO4·5H2O and sodium ascorbate in water and t-butyl alcohol (1:1)
at room temperature [16,17]. The 4-hydroxymethyltriazole moieties of 11 and 12 were
activated by treatment with p-nitrophenylchloroformate in pyridine to afford 13 and
14. Reaction of 13 (or 14) with various amines in the presence of triethylamine and
4-dimethylaminopyridine (DMAP) afforded carbamates 15–25 in good to moderate
yields. Note that 21–25 were obtained directly by deprotection of the
benzoxycarbonyl groups in the presence of excess amine; that is, catalytic
hydrogenation over Pd/C was unnecessary.
1
All the carbamates were characterized by IR spectroscopy, H- and 13C-NMR
1
spectroscopy, and high-resolution mass spectrometry. In the H-NMR spectra, the
formation of the triazoles of PPT was confirmed by the presence of a peak for the C-5
proton of the triazole ring in the aromatic region at a chemical shift of ~7.33 ppm. In
addition, the 4α-protons of the triazoles were shifted downfield (to 6.08–6.10 ppm)
relative to the corresponding proton of PPT, and the coupling content was ~4.4 Hz.
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2.2. Biological Activity
The in vitro cytotoxicities of carbamates 15–25 and intermediate 11 were
evaluated against a panel of four human tumor cell lines (promyelocytic leukemia
HL-60, lung carcinoma A-549, cervical carcinoma HeLa, and human colorectal
adenocarcinoma HCT-8), with etoposide as a reference compound. The screening
procedure was based on the standard MTT or CCK-8 method [22], and the results are
summarized in Table 1.
All the carbamates except 25 showed substantial cytotoxicity against HL-60 cells.
Carbamates 18, 22, and 23 displayed IC50 values in the nanomolar range against the
A-549, HeLa, and HCT-8 cell lines; that is, they were even more cytotoxic than
etoposide and intermediate 11. The other compounds (15–17, 19–21, 24 and 25)
generally showed moderate cytotoxicities against the A-549, HeLa, and HCT-8 cell
lines.
Based on these results, we deduced some preliminary structure–activity
relationships. First, the 4′-OH compounds were more cytotoxic than the
corresponding 4′-OMe analogues (compare 15 and 21, 16 and 22, 17 and 23, and 19
and 24). This observation is in accord with the previously reported structure–activity
relationships [15]; although Kumar et al. recently reported that the
4β-(1,2,3-triazol-1-yl)podophyllotoxin
and
4′-demethyl-4β-(1,2,3-triazol-1-yl)
podophyllotoxin derivatives show no obvious differences in cytotoxicity [17]. Second,
the introduction of a large amine group, such as 4-cyclopentylpiperazine,
1-(4-nitrophenyl)piperazine, or 1-(4-fluorophenyl)piperazine, resulted in
a
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considerable reduction in cytotoxicity. Our previous work indicated that the
introduction of amino acid moieties into podophyllotoxin derivatives increases their
solubility and cytotoxicity [20], so we also synthesized methine carbamate derivative
25. Regrettably, although 25 was more soluble in water than PPT and 24 (data not
shown), it was less cytotoxic, perhaps because the substituent was too large allow the
molecule to cross cell membranes.
Our in vitro cytotoxicity studies indicated that carbamate 22 markedly inhibited
the growth of human lung cancer cell line A-549, so we studied this compound further
to determine the mechanism by which it inhibited cell growth. PPT is known to
inhibit mitosis by depolymerizing cellular microtubules, and etoposide inhibits
TOP-II. Microtubules are involved in cell shape determination, cell motility, vesicle
motility, and cell division [27]. During mitosis, microtubules rearrange to form a
mitotic spindle that is essential for cell division [28], and disruption of microtubules
can arrest the cell cycle at the M phase, and eventually induce apoptosis [29]. To
determine whether 22 was able to depolymerize cellular microtubules as PPT does,
we examined the morphology of untreated and 22-treated A-549 cells by means of
indirect immunofluorescence with antitubulin antibody staining of microtubules and
fluorescent staining of nuclei (Fig. 2). The untreated cells were elongated, and
staining of thin bundles of microtubules was observed throughout the cytoplasm. In
contrast, 48 h after treatment with 22, the cells were round and contained short, dense
microtubule networks; in addition we observed cell shrinkage and nuclear
fragmentation and condensation dispersed throughout the cytoplasm, which are
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hallmarks of apoptosis. These effects increased dose dependently.
4β-[(4-Alkyl)-1,2,3-triazol-1-yl]podophyllotoxin derivatives induce apoptosis and
cell cycle arrest in the G2/M phase in MCF-7 cells [17]. To determine whether 22 had
similar effects on tumor cells, we investigated its effects on cell cycle progression by
means of fluorescence-activated cell sorting analysis of A-549 cells stained with
propidium iodide (Fig. 3) [22]. The FACS analysis showed that A549 cells treated
with 5 μM 22 accumulated in the S phase after treatment, then were blocked in the
G2/M phase with a concomitant decrease in the population of G1 phase cells (Fig. 3).
G2/M phase arrests were initially detectable after 24 h of treatment; 20.6% and 22.2%
of the cells were in the G2/M phase after exposure to 22 for 24 h (Fig. 3B) and 48 h
(Fig. 3C), respectively. In contrast, 16.4% 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 24 h of treatment with 22. In control
A-549 cells (Fig. 3A), only ~1.5% of the cells were in the sub-G1 phase, whereas
about 24.3% and 26.8% of cells were in the sub-G1 phase after treatment with 5 μM
22 for 24 h (Fig. 3B) and 48 h (Fig. 3C), respectively. These results demonstrate that
22 interfered with cell proliferation by arresting the cell cycle and that this compound
induced S and G2/M arrests accompanied by apoptosis in A-549 cells.
DNA TOP-II is a pharmacological target of clinical relevance for the PPT lignans,
so we evaluated the ability of 22 to inhibit TOP-II-mediated DNA relaxation by
means of a procedure described in the literature [30]. We found that 22 exhibited in
vitro inhibition of TOP-II activity similar to that of etoposide (Fig. 4).
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3. Conclusions
Carbamate derivatives of 4β-(1,2,3-triazol-1-yl)podophyllotoxin (15–25)
showed promising in vitro cytotoxicities against a panel of human cancer cell lines.
Fluorescence-activated cell sorting analysis indicated that 22 induced cell cycle arrest
in the G2/M phase accompanied by apoptosis in A-549 cells. In addition, 22 inhibited
the formation of microtubules in A-549 cells and TOP-II-mediated DNA relaxation.
These results suggest that 22 induced apoptosis by means of multiple pathways. Our
results suggest that these compounds have potential for further development as
anticancer agents.
4. Experimental
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
1
between KBr plates. H NMR and 13C NMR spectra were recorded with a Varian
Mecury-400BB spectrometer with TMS as an internal standard, all chemical shift
values are reported as δ ppm. Optical rotations were measured on a Perkin Elmer 341
polarimeter in a 1dm cell at 23℃. 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 (230-400 mesh, Qingdao Ocean Chemical Ltd., China). Podophyllotoxin
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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.
4.1.1. General procedure of click chemistry
To a solution of propargyl alcohol (20 mmol) in t-butyl alcohol and water (1:1,
250 mL) was added CuSO4·5H2O (20 mmol), sodium ascorbate (50 mmol) followed
by 4β-azido-podophyllotoxin 9 (or 4β-azido-4'-demethyl-4'-benzyloxycarbonyl-
podophyllotoxin 10) (10 mmol). The reaction mixture was stirred at room temperature
for 10 h. After completion, the reaction mixture was diluted with 50 ml of water and
extracted with chloroform (3×50 mL). The combined extracts were washed with brine,
dried over anhydrous Na2SO4 and evaporated in vacuo. The crude product obtained
was washed with petro ether to remove excess propargyl alcohol, and recrystallized
with ether to yield the pure product 11 (or 12).
4.1.2.1. 4β-[(4-Hydroxy methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin (11).
1
Yield: 88%; mp: 148-150 ℃; H NMR (400 MHz, CDCl3) δ 7.24 (s, 1 H), 6.65 (s, 1
H), 6.62 (s, 1 H), 6.32 (s, 2 H), 6.07 (d, J = 4.8 Hz, 1 H), 6.03 (s, 1 H), 6.01 (s, 1 H),
4.75 (d, J = 4.8 Hz,1 H), 4.40 (t, J = 6.8 Hz, 1 H), 3.82 (s, 3 H), 3.75 (s, 6 H), 3.74 (s,
2 H), 3.28-3.16 (m, 2 H), 3.06 (dd, J = 9.0, 5.0 Hz, 1 H); ESI-MS: 496 (M+1, 100%).
4.1.2.2. 4'-O-Demethyl-4'-benzyloxycarbonyl-4β-[(4-hydroxy
methyl)-1,2,3-
triazol-1-yl]-4-desoxypodophyllotoxin (12). Yield: 90%; white powder solid; mp:
256-258 ℃; 1H NMR (400 MHz, DMSO) δ 7.87 (s, 1 H), 7.42-7.37 (m, 5 H), 6.77 (s,
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1 H), 6.71 (s, 1 H), 6.40 (s, 2 H), 6.25 (d, J = 5.2 Hz, 1 H), 6.04 (s, 1 H), 6.02 (s, 1 H),
5.25 (s, 2 H), 5.17 (t, J = 5.2 Hz, 1H), 4.80 (d, J = 5.2 Hz, 1 H), 4.51 (d, J = 5.6 Hz, 2
H), 4.39 (t, J = 8.0 Hz, 1 H), 3.67 (s, 6 H), 3.53 (dd, J = 15.6, 5.2 Hz, 1 H), 3.31-3.25
(m, 1 H), 2.98 (t, J = 10.4 Hz, 1 H); MS-ESI: 616 ([M+H], 100 %).
4.1.2. General procedure of synthesis of 13 and 14
To a solution of p-nitrophenyl chloroformate (6.35 mmol) in anhydrous CH2Cl2
(5 mL) was added dry pyridine (0.6 mL). Instantaneously a white precipitate was
formed. A solution of 11 (or 12) (1.9 mmol) in anhydrous CH2Cl2 (10 mL) was added
dropwise under an argon atmosphere and the mixture was stirred for further 45 min at
room temperature. The mixture was purified by silica gel chromatography using a
mixture of PE: ethyl acetate = 3:1 as the eluant to afford compounds 13 and 14.
4.1.2.1. 4β-[(4-Hydroxy
methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
1
p-nitrophenyl carbonate (13). Yield: 82%; white powder solid; mp: 139-141 ℃; H
NMR (400 MHz, CDCl3) δ 8.33 (d, J = 6.8 Hz, 2 H), 8.04 (s,1 H), 7.50 (d, J = 6.8 Hz,
2 H), 6.81 (s, 1 H), 6.70 (s, 1 H), 6.46 (s, 2 H), 6.39 (d, J = 4.4 Hz, 1 H), 6.03 (s, 2 H),
5.40 (s, 2 H), 4.83 (d, J = 4.4 Hz, 1 H), 4.39 (t, J = 6.8 Hz, 1 H), 3.96-3.90 (m, 1 H),
3.72 (s, 6 H), 3.69 (s, 3 H), 3.55-3.45 (m, 2 H); MS (ESI): 660 ([M], 80%), 683
([M+Na], 100%).
4.1.2.2. 4'-Demethyl-4'-benzyloxycarbonyl-4β-[(4-hydroxy
methyl)-1,2,3-triazol-
1-yl]-4-desoxypodophyllotoxin p-nitrophenyl carbonate (14). Yield: 80%; white
1
powder solid; mp: 162-164 ℃; H NMR (400 MHz, CDCl3) δ 8.30-8.27 (m, 2 H),
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7.44-7.33 (m, 7 H), 6.66 (s, 1 H), 6.61 (s, 1 H), 6.34 (s, 2 H), 6.13 (d, J = 4.8 Hz, 1 H),
6.04 (s, 1 H), 6.02 (s, 1 H), 5.40 (s, 2 H), 5.27 (s, 2 H), 4.79 (d, J = 5.4 Hz, 1 H), 4.43
(q, J = 4.2 Hz, 1 H), 3.71 (s, 6 H), 3.30 (q, J = 4.2 Hz, 1 H), 3.26-3.22 (m, 1 H), 3.06
(dd, J = 13.8, 5.4 Hz, 1 H); MS (ESI): 803 (M+Na, 100%).
4.1.3. General preparation of com pounds 15-25
To a stirred solution of 13 (or 14) (0.1 mmol) in dry dichloromethane (5 mL) was
added the appropriate amine (0.11 mmol, 1.1 equiv), triethylamine (0.11 mmol) and
catalytical amount of DMAP at room temperature. After stirring for overnight, the
reaction mixture was washed with cold saturated NaHCO3 and then water until pH
6-7. 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 15–20 in the reported yields.
In the cases of compounds 21–25, while the reaction was finished, the appropriate
amine (0.11 mmol, 0.5 equiv) was added to the reaction mixture and stirred for
another 2 h, then similar procedure was done and afforded pure title compounds.
4.1.3.1. 4β-[(4-Hydroxy methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin propyl
carbamate (15). Yield: 65%; white powder solid; mp: 141-142 ℃; [α]2D3 -29° (c 0.1,
CHCl3); IR (KBr cm-1) 3375, 2961, 1780, 1718, 1589, 1507, 1485, 1420, 1331, 1236,
1
1126, 1036, 1001, 767; H NMR (400 MHz, CDCl3) δ 7.31 (s, 1 H), 6.65 (s, 1 H),
6.60 (s, 1 H), 6.32 (s, 2 H), 6.09 (d, J = 4.4 Hz, 1 H), 6.03 (s, 1 H), 6.00 (s, 1 H),
5.20-5.15 (m, 2 H), 4.85-4.78 (m, 1 H), 4.75 (d, J = 4.8 Hz, 1 H), 4.39 (t, J = 6.8 Hz, 1
H), 3.82 (s, 3 H), 3.77 (s, 6 H), 3.22-3.29 (m, 2 H), 3.13 (dd, J = 7.2 Hz, 2 H),
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3.02-3.06 (m, 1 H), 1.49 (d, J = 7.2 Hz, 2 H), 0.89 (t, J =7.2 Hz, 3 H); 13C NMR (100
MHz, CDCl3) δ 173.0, 156.0, 152.8, 149.4, 148.1, 143.7, 137.6, 134.1, 133.2, 124.5,
124.0, 110.5, 108.8, 108.2, 101.9, 67.3, 60.7, 58.7, 57.5, 56.3, 43.6, 42.7, 41.5, 37.1,
23.1, 11.1; HRMS (ESI) 581.2245 for [M+H]+ (calcd 581.2242 for C29H32N4O9).
4.1.3.2. 4β-[(4-Hydroxy
methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
cyclopentyl carbamate (16). Yield: 68%; white powder solid; mp: 139 ℃; [α]2D3 -29° (c
0.1, CHCl3); IR (KBr cm-1) 3363, 2955, 2924, 1780, 1714, 1589, 1507, 1331, 1236,
1126, 1036, 1003, 932, 772, 583; 1H NMR (400 MHz, CDCl3) δ 7.31 (s, 1 H), 6.65 (s,
1 H), 6.60 (s, 1 H), 6.32 (s, 2 H), 6.09 (d, J = 4.4 Hz, 1 H), 6.03 (s, 1 H), 6.00 (s, 1 H),
5.15 (s, 2 H), 4.75 (d, J = 5.2 Hz, 1 H), 4.40 (t, J = 6.8 Hz, 1 H), 3.92-3.97 (m, 1 H),
3.82 (s, 3 H), 3.77 (s, 6 H), 3.22-3.28 (m, 2 H), 3.02-3.07 (m, 1 H), 1.36-1.93 (m, 9
13
H) ; C NMR (100 MHz, CDCl3) δ 173.0, 152.8, 149.4, 148.1, 143.7, 137.6, 134.1,
133.2, 124.5, 124.0, 110.5, 108.8, 108.3, 101.9, 67.38, 60.7, 58.7, 57.5, 52.8, 43.6,
41.5, 37.1, 33.1, 29.6, 23.4; HRMS (ESI) 607.2409 for [M+H]+ (calcd 607.2399 for
C31H34N4O9).
4.1.3.3. 4β-[(4-Hydroxy
methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
N-morpholino carbamate (17). Yield: 70%; white powder solid; mp: 165-166 ℃; [α]2D3
-49° (c 0.1, CHCl3); IR (KBr cm-1) 3441, 2922, 1780, 1701, 1589, 1506, 1485, 1424,
1239, 1160, 1125, 1035, 1001, 758; 1H NMR (400 MHz, CDCl3) δ 7.33 (s, 1 H), 6.65
(s, 1 H), 6.60 (s, 1 H), 6.32 (s, 2 H), 6.08 (d, J = 4.4 Hz, 1 H), 6.03 (s, 1 H), 6.01 (s, 1
H), 5.20 (q, J = 16.0 Hz, 2 H), 4.76 (d, J = 5.2 Hz, 1 H), 4.40 (t, J = 6.8 Hz, 1 H), 3.82
(s, 3 H), 3.77 (s, 6 H), 3.64 (s, 4 H), 3.46 (t, J = 9.6 Hz, 4 H), 3.22-3.27 (m, 2 H),
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3.05-3.09 (m, 1 H); 13C NMR (100 MHz, CDCl3) δ 173.1, 155.0, 152.3, 149.7, 148.1,
143.4, 137.6, 134.1, 133.2, 124.4, 124.3, 110.5, 108.8, 108.2, 102.0, 67.3, 66.4, 60.7,
58.7, 58.1, 56.3, 56.3, 43.6, 41.5, 37.1; HRMS (ESI) 609.2108 for [M+H]+ (calcd
609.2191 for C30H32N4O10).
4.1.3.4. 4β-[(4-Hydroxy
methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
1-(4-cyclopentyl piperazinyl) carbamate (18). Yield: 63%; white powder solid; mp:
144-146 ℃; [α]2D3 -33° (c 0.1, CHCl3); IR (KBr cm-1) 3436, 2956, 1780, 1698, 1589,
1484, 1240, 1125, 1035, 1003, 758; 1 H NMR (400 MHz, CDCl3) δ 7.34 (s, 1 H), 6.65
(s, 1 H), 6.61 (s, 1 H), 6.32 (s, 2 H), 6.08 (d, J = 4.4 Hz, 1 H), 6.02 (s, 1 H), 6.00 (s, 1
H), 5.19 (q, J = 10 Hz, 2 H), 4.76 (d, J = 5.2 Hz, 1 H), 4.40 (t, J = 6.8 Hz, 1 H), 3.82
(s, 3 H), 3.77 (s, 6 H), 3.50 (s, 4 H), 3.22-3.27 (m, 2 H), 3.06-3.10 (m, 1 H), 2.47-2.53
(m, 5 H), 1.26-1.72 (m, 8 H); 13C NMR (100 MHz, CDCl3) δ 173.1, 154.9, 152.8,
149.4, 148.1, 143.6, 137.6, 134.2, 133.2, 124.5, 124.2, 110.5, 108.8, 108.2, 101.9,
67.3, 60.7, 58.7, 56.3, 51.8, 43.6, 41.5, 37.1, 30.2, 24.0; HRMS (ESI) 676.2963 for
[M+H]+ (calcd 676.2977 for C35H41N5O9).
4.1.3.5. 4β-[(4-Hydroxy
methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
1-(4-nitrophenyl piperazinyl) carbamate (19). Yield: 65%; yellow powder solid; mp:
140-141 ℃; [α]2D3 -36° (c 0.1, CHCl3); IR (KBr, cm-1) 3324, 2916, 1779, 1702, 1594,
1
1505, 1326, 1269, 1233, 1120, 1037, 1001, 752; H NMR (400 MHz, CDCl3) δ 8.13
(d, J = 9.2 Hz, 2 H), 7.36 (s, 1 H), 6.81 (d, J = 9.6 Hz, 2 H), 6.65 (s, 1 H), 6,61 (s, 1
H), 6,32 (s, 2 H), 6.10 (d, J = 4 Hz, 1 H), 6.02 (s, 1 H), δ 6.00 (s, 1 H), 5.20 (q, J = 10
Hz, 2 H), 4.75 (d, J = 5.2 Hz, 1 H), 4.41 (t, J = 6.8 Hz, 1 H), 3.82 (s, 3 H), 3.77 (s, 6
13

ACCEPTED MANUSCRIPT
H), 3.66 (t, J = 5.2 Hz, 4 H), 3.43 (s, 4 H), 3.23-3.28 (m, 2 H), 3.04-3.09 (m, 1 H); 13C
NMR (100 MHz, CDCl3) δ 173.2, 154.9, 154.5, 152.8, 149.5, 148.1, 143.2, 139.0,
137.7, 134.1, 133.3, 126.1, 125.9, 124.4, 115.6, 113.0, 110.6, 108.8, 108.3, 102.0,
67.4, 60.7, 58.8, 58.3, 56.4, 46.8, 43.6, 41.6, 37.1; HRMS (ESI) 729.2436 for [M+H]+
(calcd 729.2451 for C36H37N6O11).
4.1.3.6. 4β-[(4-Hydroxy
methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllotoxin
1-(4-fluorophenyl piperazinyl) carbamate (20). Yield: 66%; white powder solid; mp:
141-143 ℃; [α]2D3 -35° (c 0.1, CHCl3); IR (KBr cm-1) 3436, 2920, 1780, 1589, 1609,
1459, 1235, 1037, 1002, 937, 828, 757, 531; 1H NMR (400 MHz, CDCl3) δ 7.36 (s, 1
H), 6.97 (t, J = 17.6 Hz, 2 H), 6.86-6.88 (m, 2 H), 6.65 (s, 1 H), 6.61 (s, 1 H), 6.32 (s,
2 H), 6.10 (d, J = 4 Hz, 1 H), 6.02 (s, 1 H), 5.99 (s, 1 H), 5.22 (q, J = 10.0 Hz, 2 H),
4.76 (d, J = 5.2 Hz, 1 H), 4.41 (t, J = 6.8 Hz, 1 H), 3.82 (s, 3 H), 3.77 (s, 6 H), 3.62 (t,
J = 5.2 Hz, 4 H), 3.01-3.22 (m, 2 H), 3.05-3.10 (m, 5 H); 13C NMR (100 MHz, CDCl3)
δ 173.1, 154.9, 152.8, 149.4, 147.7, 143.5, 137.6, 134.1, 133.2, 124.4, 124.3, 118.7,
118.6, 115.7, 115.5, 110.5, 108.8, 108.2, 101.9, 67.3, 60.7, 58.7, 58.1, 56.3, 50.4, 43.6,
41.5, 37.1; HRMS (ESI) 702.2561 for [M+H]+ (calcd 702.2570 for C36H36FN5O9).
4.1.3.7. 4'-O-Demethyl-4β-[(4-hydroxy
methyl)-1,2,3-triazol-1-yl]-
4-desoxypodophyllotoxin propyl carbamate (21). Yield: 60%; white powder solid; mp:
142-143 ℃; [α]2D3 -29° (c 0.1, CHCl3); IR (KBr cm-1) 3398, 2963, 1779, 1714, 1610,
1
1514, 1459, 1329, 1232, 1160, 1114, 1036, 1001, 789, 761; H NMR (400 MHz,
CDCl3) δ 7.32 (s, 1 H), 6.64 (s, 1 H), 6.60 (s, 1 H), 6.33 (s, 2 H), 6.09 (d, J = 4.4 Hz, 1
H), 6.03 (s, 1 H), 6.00 (s, 1 H), 5.30 (s, 1 H), 5.15 (s, 2 H), 4.75 (d, J = 5.2 Hz, 1 H),
14

ACCEPTED MANUSCRIPT
4.37 (t, J = 6.8 Hz, 1 H), 3.80 (s, 6 H), 3.21-3.25 (m, 2 H), 3.14 (dd, J = 7.2 Hz, 2 H),
13
3.00-3.09 (m, 1 H), 1.49 (dd, J = 7.2 Hz, 2 H), 0.89 (t, J =15.0 Hz, 4 H); C NMR
(100 MHz, CDCl3) δ 173.14, 156.0, 149.4, 148.0, 146.6, 143.7, 134.5, 133.4, 129.7,
124.5, 124.0, 110.5, 108.7, 107.9, 101.9, 67.3, 58.7, 57.5, 56.5, 53.3, 43.5, 42.7, 41.6,
37.0, 23.1, 11.0; HRMS (ESI) 567.2080 for [M+H]+ (calcd 567.2086 for
C28H30N4O9).
4.1.3.8. 4'-O-Demethyl-4β-[(4-hydroxy
methyl)-1,2,3-triazol-1-yl]-
4-desoxypodophyllotoxin cylcopentyl carbamate (22). Yield: 56%; white powder solid;
mp: 146-147 ℃; [α]2D3 -28° (c 0.1, CHCl3); IR (KBr cm-1) 3400, 2957, 1778, 1711,
1607, 1512, 1388, 1330, 1233, 1161, 1115, 1036, 760; 1H NMR (400 MHz, CDCl3) δ
7.32 (s, 1 H), 6.64 (s, 1 H), 6.60 (s, 1 H), 6.33 (s, 2 H), 6.09 (d, J = 4.4 Hz, 1 H), 6.03
(s, 1 H), 6.00 (s, 1 H), 5.53 (s, 1 H), 5.14 (s, 2 H), 4.74 (d, J = 5.2 Hz, 1 H), 4.37 (t, J
= 6.8 Hz, 1 H), 3.92-3.97 (m, 1 H), 3.80 (s, 6 H), 3.17-3.28 (m, 2 H), 3.00-3.05 (m, 1
13
H), 1.36-1.93 (m, 9 H) ; C NMR (100 MHz, CDCl3) δ 173.1, 149.4, 148.0, 146.6,
143.7, 134.5, 133.4, 129.7, 128.4, 128.1, 124.5, 124.0, 110.5, 108.7, 107.9, 101.9,
67.3, 58.7, 57.5, 56.5, 43.5, 41.6, 37.0, 33.1, 23.4; HRMS (ESI) 593.2250 for [M+H]+
(calcd 593.2242 for C30H32N4O9).
4.1.3.9. 4'-O-Demethyl-4β-[(4-hydroxy
methyl)-1,2,3-triazol-1-yl]-
4-desoxypodophyllotoxin morpholino carbamate (23). Yield: 58%; white powder
solid; mp: 167-169 ℃; [α]2D3 -52° (c 0.1, CHCl3); IR (KBr cm-1) 3415, 2918, 1779,
1
1700, 1645, 1606, 1512, 1484, 1329, 1239, 1162, 1115, 1035, 767; H NMR (400
MHz, CDCl3) δ 7.32 (s, 1 H), 6.65 (s, 1 H), 6.60 (s, 1 H), 6.33 (s, 2 H), 6.08 (d, J =
15

ACCEPTED MANUSCRIPT
4.4 Hz, 1 H), 6.03 (s, 1 H), 6.00 (s, 1 H), 5.46 (s, 1 H), 5.20 (q, J = 16.0 Hz, 2 H),
4.75 (d, J = 5.2 Hz, 1 H), 4.39 (t, J = 6.8 Hz, 1 H), 3.80 (s, 6 H), 3.64 (s, 4 H), 3.46 (t,
J = 9.6 Hz, 4 H), 3.20-3.27 (m, 2 H), 3.02-3.06(m, 1 H); 13C NMR (100 MHz, CDCl3)
δ 173.1, 155.0, 149.8, 148.0, 146.6, 143.5, 134.5, 133.4, 129.6, 124.5, 124.2, 110.6,
108.7, 107.9, 101.9, 67.3, 66.4, 60.3, 58.8, 58.1, 56.5, 43.5, 41.6, 37.0, 29.6; HRMS
(ESI) 595.2043 for [M+H]+ (calcd 595.2035 for C29H30N4O10).
4.1.3.10.
4'-O-Demethyl-4β-[(4-hydroxy
methyl)-1,2,3-triazol-1-yl]-
4-desoxypodophyllotoxin 1-(4-nitrophenyl piperazinyl) carbamate (24). Yield: 54%;
yellow powder solid; mp: 140-141 ℃; [α]2D3 -53° (c 0.1, CHCl3); IR (KBr, cm-1) 3324,
1
2916, 1779, 1702, 1594, 1505, 1326, 1269, 1233, 1120, 1037, 1001, 752; H NMR
(400 MHz, CDCl3) δ 8.12 (d, J = 9.2 Hz, 2 H), 7.35 (s, 1 H), 6.82 (d, J = 9.2 Hz, 2 H),
6.66 (s, 1 H), 6.62 (s, 1 H), 6.32 (s, 2 H), 6.10 (d, J = 4.0 Hz, 1 H), 6.02 (s, 1 H), 6.00
(s, 1 H), 5.20 (q, J = 9.8 Hz, 2 H), 4.75 (d, J = 5.2 Hz, 1 H), 4.41 (t, J = 6.8 Hz, 1 H),
3.77 (s, 6 H), 3.68-3.55 (m, 1 H), 3.43 (s, 4 H), 3.23-3.28 (m, 2 H), 3.04-3.09 (m, 1 H);
13C NMR (100 MHz, CDCl3) δ 173.2, 154.9, 154.5, 152.8, 149.5, 148.1, 143.2, 139.0,
137.7, 134.1, 133.3, 126.1, 125.9, 124.4, 115.6, 113.0, 110.6, 108.8, 108.3, 102.0,
67.4, 60.7, 58.3, 56.4, 46.8, 43.6, 41.6, 37.1; HRMS (ESI) 715.2372 for [M+H]+
(calcd 715.2364 for C35H35N6O11).
4.1.3.11.
4-Nitrophenyl piperazinyl N-[1-oxyl-(4'-O-demethyl-4β-[(4-hydroxy
methyl)-1,2,3-triazol-1-yl]-4-desoxypodophyllic)]-L-methine amide (25). Yield: 63%;
yellow powder solid; mp: 155-157 ℃; [α]2D3 -45° (c 0.1, CHCl3); IR (KBr cm-1) 3417,
1
2910, 1777, 1701, 1647, 1606, 1513, 1474, 1327, 1235, 1161, 1116, 1039, 764; H
16

ACCEPTED MANUSCRIPT
NMR (400 MHz, CDCl3) δ 8.14 (d, J = 9.2 Hz, 2 H), 7.34 (s, 1 H), 6.82 (d, J = 9.2 Hz,
2 H), 6.65 (s, 1 H), 6.60 (s, 1 H), 6.32 (s, 2 H), 6.10 (d, J = 4.8 Hz, 1 H), 6.03 (d, J =
0.8 Hz, 1 H), 6.00 (d, J = 1.2 Hz, 1 H), 5.23 (q, J = 6.8 Hz, 2 H), 4.72 (d, J = 5.2 Hz, 1
H), 4.40 (q, J = 6.4 Hz, 1 H), 3.80 (s, 6 H), 3.67-3.64 (m, 4 H), 3.42-3.39 (m, 4 H),
13
3.28-3.23 (m, 2 H), 3.06 (dd, J = 12.8, 4.8 Hz, 1 H); C NMR (100 MHz, CDCl3) δ
173.1, 170.3, 155.8, 154.3, 152.9, 149.6, 148.2, 146.6, 143.4, 139.4, 137.7, 134.3,
133.4, 126.0, 124.5, 123.3, 113.3, 110.7, 108.9, 108.4, 102.3, 67.5, 66.8, 58.8, 56.4,
49.6, 47.3, 46.9, 44.8, 43.8, 41.7, 37.2, 32.9, 30.2, 15.9; HRMS (ESI) 860.2928 for
[M+H]+ (calcd 860.2925 for C41H46N7O12S).
4.2. Biological evaluation
4.2.1. Cytotoxicity assays
Cells were incubated at 37 ℃ in a 5% CO2 atmosphere. The MTT and CCK-8
colorimetric assay were used to determined growth inhibition. The synthetic
compounds 15–25 and reference compound VP-16 were dissolved in saline for five
concentrations (0.01–100 μM). For the HL-60, cells were plated in 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 absorbance at λ450 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
4-6 h, then exposed in quadplex well for 48 h. The media was aspirated, and 10 μL of
5 mg/mL MTT solution (dilute in sterile PBS) diluted in serum-free media was added
to each well. After 4 h of incubation, the solution was centrifuged for 10 min under
2000 rpm. The supernatant was mixed with 150 μL DMSO, and shaken on an
17

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oscillator. The absorbance at λ570 was determined on a plate reader. IC50 values were
determined from a log plot of percent of control versus concentration.
4.2.2. Immunofluorescence microscopy
A-549 cells plated on coverslips were either untreated or treated with 0.5 μM
compound 22. After various lengths of time, the cells were fixed with 4%
paraformaldehyde and permeabilized in 0.1% Triton X-100 and 2% bovine serum
albumin as described previously [31]. For microtubule staining, the samples were
incubated with primary anti-α-tubulin antibody for 90 min and then with
Rhodamine(TRITC)-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA)
for 30 min. The nuclear DNA was then stained with 4′,6-diamidino-2-phenylindole
(DAPI) (Molecular Probes Inc., Eugene, OR, USA). Cells were analyzed under a
confocal microscope (FV-1000; Olympus, Tokyo, Japan).
4.2.3. Analysis of cell cycle by flow cytometry
For cell cycle analysis, we used the human lung carcinoma A-549 cell line grown
in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM
L-glutamine, 100 units/mL penicillin, and 24 μg/mL gentamicin and incubated at
37 °C in a humidified atmosphere of 5% CO2 and 95% air. Untreated and drug-treated
cells ((3–5)×105) were harvested and fixed overnight in 70% ethanol at 4 °C. Cells
were then washed three times with PBS, incubated for 1 h with 1 mg/mL RNase A
and 20 μg/mL propidium iodide at room temperature, and analyzed with a flow
cytometer (COULTER EPICS XL, USA) as described previously [22, 32].
18

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4.2.4. DNA Topoisomerase-II inhibition assay
Topoisomerase-II activity was determined using kit (Topogen Inc., USA, Cat No.
2000H). Reaction was assembled in micro centrifuge tube that contains super coiled
DNA 250 ng/μL & Topoisomerase-II (4 units) in assay buffer (A 0.1 volume and B 1
volume). In each reaction 2 μL sample was added then volume was made up to 20 μL
with water and then incubated at 37 ℃. Reaction was terminated by addition of 2 μL
of 10% SDS. Each sample tube was treated with proteinase K and extracted once with
chloroform/isoamyl alcohol (24:1). Products were resolved by 1% agarose gel
electrophoresis in TAE buffer (40 mM tris-acetate, pH 8.0, and 1 mM EDTA) and
stained with 0.5 μg/mL ethidium bromide [30].
Acknowledgments
Financial support from The Natural Science Foundation of Gansu Province (No.
1208RJZA247) and The Science and Technology Development Project of Lanzhou
City (No. 08-1-57) are gratefully acknowledged.
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Figure Captions
Fig. 1. Structures of podophyllotoxin (1), etoposide (2), teniposide (3), etopophos (4),
NK-611 (5), tafluposide (6)
Fig. 2. Immunofluorescence images of untreated A-549 cells and cells treated with 22
(5 µM and 10 µM) for 48 h. Microtubules (red) were stained by incubation with
anti-α-tubulin antibody for 90 min and then with Rhodamine (TRITC)-conjugated
secondary antibody for 30 min. Chromosomal DNA (blue) was stained with DAPI.
Fig. 3. Effects of 22 on cell cycle progression: (A) control A-549 cells, (B) A-549
cells treated with 5 μM 22 for 24 h, and (C) A-549 cells treated with 5 μM 22 for 48
h.
Fig. 4. Inhibition of TOP-II by compound 22. Lane 1: supercoiled pBR322 DNA.
Lane 2: pBR322 DNA relaxed by TOP-II; Lane 3: inhibitory effect of 100 µM
etoposide; Lane 4: inhibitory effect of 100 M 22.
Scheme 1. Synthesis of carbamates 15–25. Reagents and conditions: (i) trimethylsilyl
iodide followed by BaCO3; (ii) PhCH2OCOCl, Et3N, CH2Cl2; (iii) Et2O·BF3/HN3; (iv)
propargyl alcohol, CuSO4·5H2O, sodium ascorbate, 1:1 t-BuOH/H2O; (v)
p-NO2PhOCOCl, pyridine; (vi) HNR2R3, 4-dimethylaminopyridine/Et3N, CH2Cl2.
23

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Table 1 Cytotoxicities of carbamates 15–25 at drugs exposure for 48 h
Cytotoxicity (IC50, μM) a
Compounds
HL-60 b
A-549 c
46.8
14.8
23.3
8.70
56.6
22.1
23.0
0.14
1.46
5.58
45.6
22.1
3.20
HeLa c
7.42
1.16
35.7
<0.01
6.48
3.84
6.91
0.51
0.03
3.50
10.3
0.92
2.91
HCT-8 c
6.29
14.9
2.05
0.35
31.3
1.47
16.2
0.05
0.37
6.15
40.8
2.76
7.06
2.77
15
0.375
0.629
<0.01
0.625
<0.01
0.183
<0.01
0.045
0.32
16
17
18
19
20
21
22
23
24
15.7
25
0.298
11
1.15
VP-16
a Data are the mean of three independent experiments.
b CCK-8 method.
c MTT method.
24

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Fig. 1.
R4
O
OH
O
2 R1 = H, R2 = OH, R3 = OH, R4 = Me
5
8
11
O
R3
O
O
O
4
1
3
2
S
O
R2
3 R1 = H, R2 = OH, R3 =OH, R4 =
O
O
O
4 R1 = PO3H2, R2 = OH, R3 = OH, R4 = Me
5 R1 = H, R2 = NMe2, R3 = OH, R4 = Me
O
2'
6'
O
MeO
OMe
4'
OMe
6 R1 = PO3H2, R2 = R3 = OCOCH2OC6F5, R4 = Me
MeO
OMe
1
OR1
Fig. 1. Structures of podophyllotoxin (1), etoposide (2), teniposide (3), etopophos (4),
NK-611 (5), tafluposide (6)
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Fig. 2.
Fig. 2. Immunofluorescence images of untreated A-549 cells and cells treated with 22
(5 µM and 10 µM) for 48 h. Microtubules (red) were stained by incubation with
anti-α-tubulin antibody for 90 min and then with Rhodamine (TRITC)-conjugated
secondary antibody for 30 min. Chromosomal DNA (blue) was stained with DAPI.
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Fig. 3.
Compounds Sub-G1
G1
S
G2/M
Control
24 h
1.5%
58.1% 25.5% 16.4%
24.3% 43.1% 36.3% 20.6%
26.8% 22.7% 55.1% 22.2%
48 h
Fig. 3. Effect of 22 on cell cycle progression. A) Control A-549 cells; B) A-549 cells
treated with 5 μM 22 for 24 h; C) A-549 cells treated with 5 μM 22 for 48 h.
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