Synthesis of hybrid 4-deoxypodophyllotoxin-5-fluorouracil compounds that inhibit cellular migration and induce cell cycle arrest

Article abstract of DOI:10.1016/j.bmcl.2016.02.013

A series of deoxypodophyllotoxin-5-fluorouracil hybrid compounds were synthesized, and their cytotoxic activity was evaluated using four human cancer cell lines (HeLa, A549, HCT-8, and HepG2) and the human normal cell line WI-38. The synthesized compounds exhibited greater cytotoxic activity in tumor cells and reduced toxicity in the normal cell line compared with the anticancer drug VP-16 and 5-FU. Additionally, the most potent of these compounds - 4′-O-demethyl-4-deoxypodophyllotoxin-4′-yl 4-((6-(2-(5-fluorouracil-yl) acetamido) hexyl) amino)-4-oxobutanoate (compound 22) - induced cell-cycle arrest in the G2/M phase by regulating levels of cdc2, cyclinB1, and p-cdc2 in A549 cells. Furthermore, compound 22 may inhibited the migration of A549 cells via down-regulation of MMP-9 and up-regulation of TIMP-1.

Full text of DOI:10.1016/j.bmcl.2016.02.013

Accepted Manuscript
Synthesis of hybrid 4-deoxypodophyllotoxin–5-fluorouracil compounds that
inhibit cellular migration and induce cell cycle arrest
Xiao-Wen Guan, Xiao-Hui Xu, Shi-Liang Feng, Zhen-Bo Tang, Shi-Wu Chen,
Ling Hui
PII:
S0960-894X(16)30116-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.013
BMCL 23564
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 November 2015
26 January 2016
5 February 2016
Please cite this article as: Guan, X-W., Xu, X-H., Feng, S-L., Tang, Z-B., Chen, S-W., Hui, L., Synthesis of hybrid
4-deoxypodophyllotoxin–5-fluorouracil compounds that inhibit cellular migration and induce cell cycle arrest,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.013
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Synthesis of hybrid 4-deoxypodophyllotoxin–5-fluorouracil
compounds that inhibit cellular migration and induce cell cycle
arrest
Xiao-Wen Guan a, Xiao-Hui Xu a, Shi-Liang Feng a, Zhen-Bo Tang a, Shi-Wu Chen a,*,
Ling Hui b,c,
*
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 Laboratory of Stem Cells and Gene Drug of Gansu Province, General Hospital of Lanzhou
Military Command, Lanzhou 730050, China
Abstract: A series of deoxypodophyllotoxin–5-fluorouracil hybrid compounds were
synthesized, and their cytotoxic activity was evaluated using four human cancer cell
lines (HeLa, A549, HCT-8, and HepG2) and the human normal cell line WI-38. The
synthesized compounds exhibited greater cytotoxic activity in tumor cells and reduced
toxicity in the normal cell line compared with the anticancer drug VP-16 and 5-FU.
Additionally, the most
potent of these compounds—4′-O-demethyl-
4-deoxypodophyllotoxin-4'-yl 4-((6-(2-(5-fluorouracil-yl) acetamido) hexyl) amino)-
4-oxobutanoate (compound 22)—induced cell-cycle arrest in the G2/M phase by
regulating levels of cdc2, cyclinB1, and p-cdc2 in A549 cells. Furthermore,
compound 22 may inhibited the migration of A549 cells via down-regulation of
MMP-9 and up-regulation of TIMP-1.
Key word: Podophyllotoxin; 5-fluorouracil; cell cycle arrest; cell migration
* Corresponding author. Tel. & fax: +86 931 8915686.
E-mail address: chenshw@lzu.edu.cn (S.-W Chen)
1

Molecular hybridization involves the rational design of new chemical entities
based upon recognition of pharmacophoric subunits within the molecular structure of
two or more known bioactive derivatives. Successful fusion of these subunits
produces a new hybrid architecture that maintains desired characteristics of the
original subunits.1 Selection of two chemical entities for fusion is usually based on
their observed synergistic or additive pharmacological activities. Fusion allows
creation of a novel chemical entity that can limit or counter-balance known side
effects associated with one or both hybrid parts.2 The development of hybrid
compounds can improve drug affinity and efficacy when compared with the parent
drugs.
Podophyllotoxin (PPT, 1, Figure 1) is a naturally-occurring cyclolignan present
in the roots and rhizomes of Podophyllum species. PPT exhibits strong cytotoxic
activity against various cancer cell lines.3 Structural modifications to PPT have
yielded the anticancer drugs etoposide (VP-16, 2), teniposide (VM-26, 3), and
etopophos (4). These drugs are widely used in clinical settings, though drug resistance
and poor water solubility are significant concerns.4 The PPT analog
4-deoxypodophyllotoxin (DPT, 5) possesses potential antiproliferative and antitumor
activity in diverse cell types,5,6 in addition to its anti-inflammatory and antiviral
activity.7-9 Moreover, 4′-demethyl-4-deoxypodophyllotoxin (DDPT, 6)—which is
derived from DPT—exhibits a similar potency to DPT in vivo. Based on the idea of
combination therapy in clinical, many pharmacologist and chemists had synthesized a
number of conjugates of DDPT with other antitumor agents or 4'-O-linked derivates
2

at C-4' of DDPT, such as conjugates of DDPT-FU,10 DDPT-Nitroxides,11 and
4′-O-linked derivates of DDPT.12,13 The biological evaluation showed that most of the
modified compounds exhibited superior cytotoxicity in vitro than parent compound
DDPT.
Figure 1. Structures of podophyllotoxin (1), etoposide (VP-16, 2), teniposide (VM-26,
3), etopophos (4), DPT (5) and DDPT (6).
Diamine compounds play important cellular roles and exhibit variety of
biological activities, such as anticancer and antitubercular.14 Use of diamines as
linkers between conjugated molecules facilitates a balance between water-soluble and
fat-soluble states in vivo, which can improve anticancer activity.15 As in our
continuing efforts to find new compounds with potent activities and low toxicity from
natural products, we selected diamines as linker to design and synthesize a series of
novel some hybrids of DDPT and 5-FU. We found these DDPT–5-FU hybrid
compounds showed superior cytotoxicities against cancer cell lines and less toxicity
to normal cell line. Here, we describe the synthesis of these hybrid compounds and
evaluate their cytotoxic activity against a panel of four human cancer cell lines
(HepG2, A549, HeLa and HCT-8) and one human normal cell line WI-38.
3

Furthermore, to further investigate the mechanisms of toxicity of these DDPT–5-FU
hybrid compounds, the most potent compound 22 was selected to evaluate its effect
on cell-cycle arrest, cell nuclei morphology and cell migration.
The synthetic route to the target compounds first involved the generation of
N1-(4-(piperazin-1-yl)butyl)-5-fluorouracil
(9),
2-(5-fluorouracil-yl)acetamides
(12a–c) and N1-(2-oxo-2-(piperazin-1-yl)ethyl)-5-fluorouracil (13), they were
synthesized from 5-FU as reference reported (Scheme 1).10,16 Briefly,
1-(4-bromobutyl)-uracil 7, which was easily obtained from 5-FU under Cs2CO3 in
dimethylformamide (DMF) at 40℃, reacted with N1-Boc-piperazidine to provide 8.
Compound 10 were prepared through 5-FU was treated initially with chloroacetic acid
in the presence of aqueous potassium hydroxide. Compound 10 was then reacted with
different N1-Boc-diamines under 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (EDCI), N-hydroxybenzotriazole (HOBt) in DMF to yield compounds
11a–c and N1-BOC-(2-oxo-2-(piperazin-1-yl)ethyl)-5-FU. The protective groups of 8,
11a–c, and N1-BOC-(2-oxo-2-(piperazin-1-yl)ethyl)-5-FU were removed using
trifluoroacetic acid in dichloromethane to yield compounds 9, 12a–c, and 13,
respectively. These products were used in subsequent reactions without further
purification.
4

Scheme 1. Synthesis of compounds 9, 12a–c, and 13. Reagents and conditions: i)
Br(CH2)4Br, Cs2CO3, DMF, 40℃; ii) 1-N-BOC-piperazidine, K2CO3, acetone, 45℃;
iii) TFA, DCM, rt; iv) ClCH2COOH, 10% KOH; v) amines, HOBt, EDC, DMF.
The target compounds 16–22 were synthesized from the intermediate DDPT,
which was prepared from PPT as described in a previous publication (Scheme 2).13
Treatment of DDPT with 4-nitrophenyl chloroformate in the presence of pyridine
produced a high yield of intermediate 14. Subsequent introduction of carbamate
chains was achieved by reacting 14 with compounds 9, 12a–c, or 13 in the presence
of 4-N,N-dimethylaminopyridine (DMAP) and triethylamine (Et3N). This process
resulted in moderate or high yields of the 1-oxyl-4′-demethyl-4-
deoxypodophyllotoxin-4'-yl carbamate compounds 16–20. Additionally, intermediate
6 was reacted with succinic anhydride under DMAP and TEA in dichloromethane to
generate intermediate 15.17 Finally, 15 was reacted with 13 and 12c to produce 21 and
1
22, respectively. All synthesized target compounds were characterized by H NMR
5

and 13C NMR spectroscopy, and high-resolution mass spectrometry.
Scheme 2. Synthesis of target compounds 17–22. Reagents and conditions: i)
4-nitrophenyl chloroformate, pyridine, CH2Cl2; ii) succinic anhydride, DMAP, TEA,
DCM; iii) DMAP, TEA, DCM; iv) HOBt, EDC, DMF.
The in vitro cytotoxic activities of hybrids 16–22 were evaluated against a panel
of four human cancer cell lines (hepatocellular carcinoma HepG2, lung carcinoma
A549, cervical carcinoma HeLa, and human colorectal adenocarcinoma HCT-8 cells)
6

and one human normal cell line (human lung fibroblast cell line WI-38), using VP-16,
DDPT and 5-FU as reference compounds. The screening procedure was based on the
standard MTT method,10 and the results are summarized in Table 1.
Table 1 Cytotoxic activity of compounds 16–22
Cytotoxicity (IC50, μM)a
Compds
HeLab
A549b
HCT-8b
HepG2b
WI-38b
13.21±0.83
2.14±0.21
9.18±0.75
20.18±1.54
104.2±2.9
16
11.19±0.82
15.78±1.25
9.58±1.10
13.25±1.13
14.98±0.97
4.03±0.35
53.3±1.12
82.27±2.71
12.12±2.13
2.41±0.18
11.70±1.71
23.86±1.24
0.14±0.05
1.35±0.52
0.27±0.11
3.9±0.32
17.62±3.21
5.26±3.13
8.53±2.72
2.04±0.53
9.01±1.47
1.01±0.13
9.81±0.75
32.31±1.76
10.89±1.14
14.33±1.36
>100
121.0±2.8
109.1±1.8
85.47±1.15
87.03±3.12
112.1±2.8
113.8±2.6
78.52±2.11
38.91±1.26
35.80±1.29
17
18
22.28±1.32
11.74±2.31
11.52±1.13
0.45±0.13
16.53±1.02
41.62±1.34
27.07±2.51
19
20
21
22
DDPT
5-FU
VP16
54.82±1.57
23.94±2.13
a Data are the mean of three independent experiments.
b MTT method with 48 h drug exposure.
Hybrids 16–22 were generally more potent than patent compounds 5-FU and
DDPT in their cytotoxicity to these four tumor cell lines. The cytotoxic activity of
these compounds was generally highest in A549 cells, while the lowest potency was
7

observed in HepG2 cells. However, potency varied in each cell line. Compound 22
appeared most promising, with IC50 values of 4.03, 0.27, 1.01, and 0.45 μM in HeLa,
A549, HCT-8, and HepG2 cells, respectively. Additionally, 22 was less toxic to
normal human lung fibroblast WI-38 cells than VP-16 and 5-FU, with IC50 values for
compound 22 and VP-16 or 5-FU of 113.8 μM and 35.8 μM or 78.52 μM, respectively.
In Table 1, we also found that different linker in this class of compounds (18–20)
appeared to have moderate effects on their anti-proliferative activity in the in vitro
assay. Compounds 20 exhibited more potent activity against four human cancer cell
lines than those of 18 and 19. In addition, the hybrids with succinic anhydride
incorporated appear to be more potent than those with carbamate condensation (21 vs.
17, 22 vs. 20). Other target compounds exhibited moderate levels of cytotoxicity in
HepG2, HeLa, and HCT-8 cells, although the IC50 value of compound 20 in A549
cells was 0.14 μM—less than that of compound 22. These results confirmed the
assumption that free hydroxyl group at the 4' position in DDPT was not favorable for
antitumor activity.18 The results also suggest that compound 22 could serve as a
potential anti-cancer drug candidate.
To further investigate the mechanisms of toxicity of 22 in A549 cells, the
morphology of A549 cells treated with 22 was examined by indirect
immunofluorescence. A549 cells were treated with 22 (0, 0.3 μM, 1 μM) for 24 h, and
then the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI). As shown in
Figure 2, control cells exhibited intact nuclear structures characterized by round,
homogeneous nuclei, while the nuclei of the cells treated with 22 lost the normal
8

morphology, exhibited nuclear fragmentation.
Figure 2. Effects of 22 on the morphology of cell nuclei. (A) Control A549 cells; (B)
A549 cells treated with 0.3 μM 22 for 24 h; (C) A549 cells treated with 1 μM 22 for
24 h. Magnification: 200×; scale bar, 100 μm. Fragmented nuclei were indicated as
arrows.
We next explored the effects of 22 on cell-cycle progression using
fluorescence-activated cell sorting (FACS) analysis of propidium iodide-stained A549
cells. Treatment with 22 led to a dose-dependent accumulation of cells in the G2/M
phase with a concomitant decrease in the population of cells in G1 (Figure 3).
Approximately 47.2% or 64.5% of cells were in G2/M following exposure to 0.1 μM
(Figure 3B) or 0.5 μM (Figure 3C) 22 for 24 h, respectively, compared with 22.4% in
untreated cultures (Figure 3A). These results demonstrate that 22 induced cell-cycle
arrest in the G2/M phase in A549 cells. This is consistent with previous findings that
DPT and its analogs could induce cell-cycle arrest in the G2/M phase.19
9

Control 0.1μM 0.5μM
G1
S
58.3% 7.7% 0.1%
19.3% 45.1% 35.4%
G2/M 22.4% 47.2% 64.5%
Figure 3. Compound 22 impairs cell-cycle progression in A549 cells. Values indicate
the percentage of the cell population at the phase of the cell cycle. (A) Control A549
cells; (B) A549 cells treated with 0.1 μM 22 for 24 h; (C) A549 cells treated with 0.5
μM 22 for 24 h.
During the normal cell cycle, progression of cells in the G2 phase to M phase is
triggered by activation of the cyclinB1-dependent cdc2 kinase.20 Cdc2 is regulated by
a
series of phosphorylation–dephosphorylation events and protein–protein
interactions.21 Therefore, we used western blotting to measure expression levels of
cdc2, p-cdc2, and cyclinB1 in A549 cells exposed to increasing concentrations of 22.
Marked increases in cyclinB1 and cdc2 were observed, while a decrease in p-cdc2
protein levels was apparent (Figure 4). These results suggest that 22 likely promote
10

cell-cycle arrest in the G2/M phase by influencing the cell-cycle regulators cyclinB1,
cdc2, and p-cdc2.
Figure 4. Compound 22 influences levels of the G2/M cell-cycle regulator cyclin B1,
cdc2, and p-cdc2 in A549 cells treated with vehicle, 0.5 μM 22 or 2 μM 22 for 24 h.
Globally, metastasis accounts for more than 90% of cancer-related death.22
Progression from localized tumor to metastatic cancer requires enhanced migration,
invasion, and angiogenic properties.23 Cell migration occurs via chemotaxis, and is
required for angiogenesis.24 As DPT exerts an anti-angiogenic effect,25 we used a
transwell migration assay to investigate whether compound 22 possesses a similar
inhibitory effect on cell migration. Treatment with 1 μM of compound 22 resulted in
an approximate 47% inhibition of migration compared with untreated control cells
(Figure 5).
11

Figure 5. Effect of 22 on the migration of A549 cells by a transwell assay.
Representative images of A549 cells were treated with (A) vehicle, (B) 0.3 μM 22, (C)
1 μM 22 for 12 h, Magnification: 200×; scale bar, 100 μm. (D) Summary data (bar
graph) of the stained A549 cell numbers were counted as an average of three
independent experiments. The results are expressed as the mean ± SD; *P<0.05, **P<
0.001 vs control.
Matrix metalloproteinases (MMPs)—MMP-9 in particular—play critical roles in
remodeling the extracellular matrix (ECM) by digesting various ECM proteins in the
basement membrane.26,27 Through their MMP-inhibitory activities, tissue inhibitors of
metalloproteinases (TIMPs) such as TIMP-1 are capable of inhibiting tumor invasion
and metastasis in experimental cancer models.28,29 We used western blotting and
enzyme-linked immunosorbent assay (ELISA) to measure expression levels of
12

MMP-9 and TIMP-1 in A549 cells exposed to 22. A549 cells were treated with 0, 0.5,
or 2 μM 22 for 24 h. Western blotting revealed decreased levels of MMP-9, and
increased levels of TIMP-1 in A549 cells following exposure to 22 (Figure 6A).
Similar results were obtained using ELISA (Figure 6B). These findings suggest that
22 presumably inhibit migration of A549 cell by down-regulation the activity of
MMP-9 and up-regulation TIMP-1. The result is consistent with previous findings
that DPT has an inhibitory effect on the MMP-9 production.30
Figure 6. (A) Analysis of expression of MMP-9 and TIMP-1 in A549 cells exposed
to 0, 0.5, or 2 μM 22 for 24 h by western blotting. (B) ELISA analysis of MMP-9
from A549 cells exposed to indicated concentrations (0 μM, 0.1 μM, 0.5 μM, 1.5 μM,
5.0 μM) of 22 for 24 h. The results are expressed as the mean ± SD; n = 3; **P<
0.001 vs control.
13

In summary the majority of hybrid compounds examined in this study
demonstrated increased cytotoxic activity in cancer cells and decreased toxicity in
non-cancerous cells compared with the clinical drug etoposide. Furthermore, hybrid
22 can make the cell nuclei lost the normal morphology and induce cell-cycle arrest in
the G2/M phase by influencing levels of the cell-cycle regulators cyclin B1, cdc2, and
p-cdc2 in A549 cells. Additionally, 22 inhibits cell migration by down-regulation
MMP-9 and up-regulation TIMP-1. While the potential therapeutic benefits of
combining such agents through suitable linkers remain unclear, our results suggest
that this approach could be applied to other antitumor agents. Such studies may
produce enhanced therapeutic anticancer agents.
Acknowledgments
We gratefully acknowledge the financial support from the National Natural
Science Foundation of China (Grant No. 21372110, 81372177 and 21302079).
Supplementary data
Supplementary data associated with this article can be found, in the online
version, at doi:
14

References and notes
[1] Viegas-Junior, C.; Danuello, A.; da Silva Bolzani, V.; Barreiro, E. J.; Fraga, C. A. Curr.
Med. Chem. 2007, 14, 1829.
[2] Prinka, S.; Vijay, L.; Kamaldeep, P. Bioorg. Med. Chem. 2015, 23, 1691.
[3] Canel, C.; Moraes, R. M.; Dayan, F. E.; Ferreira, D. Phytochemistry 2000, 54, 115.
[4] Gordaliza, M.; García, P. A.; del Corral, J. M. M.; Castro, M. A.; Gómez-Zurita, M. A.
Toxicon 2004, 44, 441.
[5] Kim, Y.; Kim, S.-B.; You, Y.-J.; Ahn, B.-Z. Planta Med. 2002, 68, 271.
[6] Masuda, T.; Oyama, Y.; Yonemori, S.; Takeda, Y.; Yamazaki, Y.; Mizuguchi, S.;
Nakata, M.; Tanaka, T.; Chikahisa, L.; Inabak, Y. ; Okada, Y. Phytother. Res. 2002, 16,
353.
[7] Jin, M.; Lee, E.; Yang, J.-H.; Lu, Y.; Kang, S.-G.; Chang, Y.-C.; Lee, S.-H.; Suh, S.-J.;
Kim, C.-H.; Chang, H.-W. Biol. Pharm. Bull. 2010, 33, 1.
[8] Lee, S.-H.; Son, M.-J.; Ju, H.-K.; Lin, C.-X.; Moon, T.-C.; Choi, H.-G.; Son, J.-K.;
Chang, H.-W. Biol. Pharm. Bull. 2004, 27, 786.
[9] Charlton, J. L. J. Nat. Prod. 1998, 61, 1447.
[10]Huang, W.-T.; Liu, J.; Liu, J.-F.; Hui, L.; Ding, Y. -L.; Chen, S.-W. Eur. J. Med. Chem.
2012, 49, 48.
[11]Zhang, Z.-W.; Zhang, J.-Q.; Hui, L.; Chen, S.-W.; Tian, X. Eur. J. Med. Chem. 2010,
45, 1673.
[12]You, Y.-J.; Kim, Y.; Nam, N.-H.; Ahn, B.-Z. Bioorg. Med. Chem. Lett. 2003, 13, 2629.
[13]Chen, S.-W.; Gao, Y.-Y.; Zhou, N.-N.; Liu, J.; Huang, W-T.; Hui, L.; Jin, Y.; Jin, Y.-X.
Bioorg. Med. Chem. Lett. 2011, 21, 7355.
[14]Bogatcheva, E.; Hanrahan, C.; Nikonenko, B.; Samala, R.; Chen, P.; Gearhart J.,
15

Barbosa, F.; Einck, L.; Nacy, C. A.; Protopopova, M. J. Med. Chem., 2006, 49, 3045.
[15]Guddneppanavar, R.; Choudhury, J. R.; Kheradi, A. R.; Steen, B. D.; Saluta, G.;
Kucera, G. L.; Day, C. S.; Bierbach, U. J. Med. Chem. 2007, 50, 2259.
[16]Chen, S.-W.; Xiang, R.; Liu, J.; Tian, X. Bioorg. Med. Chem. 2009, 17, 3111.
[17]Passarella, D.; Giardini, A.; Peretto, B.; Fontana, G.; Sacchetti, A.; ilvani, A.; Ronchi,
S. C.; Cappelletti, G.; Cartelli, D.; Borlak, J.; Danieli, B. Bioorg. Med. Chem. 2008,
16, 6269.
[18]You, Y.-J.; Kim, Y.; Nam, N.-H.; Bang, S.-J.; Ahn, B.-Z. Eur. J. Med. Chem. 2004, 39,
189.
[19]Shin, S.-Y.; Yong, Y.; Kim, C.-G.; Lee, Y.-H.; Lim, Y. Cancer Lett. 2010, 287, 231.
[20]Abad, A.; López-Pérez, J. L.; del Olmo, E.; García-Fernández, L. F.; Francesch, A.;
Trigili, C.; Barasoain, I.; Andreu, J. M.; Díaz, J. F.; Feliciano, A. S. J. Med. Chem.
2012, 55, 6724.
[21]Lew, D. J.; Kornbluth, S. Curr. Opin. Cell. Biol. 1996, 8, 795.
[22]Sethi, N.; Kang, Y. Nat. Rev. Cancer. 2011, 11, 736.
[23]Ho, C. S.; Yap, S. H.; Phuah, N. H.; In, Lionel L. A.; Hasima, Noor. Lung Cancer
2014, 83, 154.
[24]Nabeshima, K.; Inoue, T.; Shimao, Y.; Sameshima, T. Pathol. Int. 2002, 52, 255.
[25]Jiang, Z.-Z.; Wua, M.-J.; Miao, J.-S.; Duan, H.-Q.; Zhang, S.; Chen, M.; Sun, L.-X.;
Wang, Y.-R.; Zhang, X.-J. Int. J. Biochem. Cell. B. 2013, 45, 1710.
[26]Lobo, G.; Monasterios, M.; Rodrigues, J.; Gamboa, N.; Capparelli, M. V.;
Martínez-Cuevas, J.; Lein, M.; Jung, K.; Abramjuk, C.; Charris, J. Eur. J. Med. Chem.
2015, 96, 281.
[27]Lenger, J.; Kaschani, F.; Lenz, T.; Dalhoff, C.; Villamor, J. G.; Köster, H.; Sewald, N.;
16

van der Hoorn, R. A. Bioorg. Med. Chem. 2012, 20, 592.
[28]Vincenti, M. P. Methods Mol. Biol. 2001, 151, 121.
[29]Gomez, D. E.; Alonso, D. F.; Yoshiji, H.; Thorgeirsson, U. P. Eur. J. Cell. Biol. 1997,
74, 111.
[30]Suh, S.-J.; Kim, J.-R.; Jin, U.-H.; Choi, H.-S.; Chang, Y.-C.; Lee, Y.-C.; Kim, S.-H.;
Lee, I.-S.; Moon, T. C.; Chang, H.-W.; Kim, C.-H. Vasc. Pharmacol. 2009, 51, 13.
17

Figure Capture:
Fig. 1. Structures of podophyllotoxin (1), etoposide (VP-16, 2), teniposide (VM-26,
3), etopophos (4), DPT (5) and DDPT (6)
Fig. 2. Effects of 22 on the morphology of cell nuclei. (A) Control A549 cells; (B)
A549 cells treated with 0.3 μM 22 for 24 h; (C) A549 cells treated with 1 μM 22 for
24 h. Magnification: 200×; scale bar, 100 μm. Fragmented nuclei were indicated as
arrows.
Fig. 3. Compound 22 impairs cell-cycle progression in A549 cells. Values indicate the
percentage of the cell population at the phase of the cell cycle. (A) Control A549 cells;
(B) A549 cells treated with 0.1 μM 22 for 24 h; (C) A549 cells treated with 0.5 μM 22
for 24 h.
Fig. 4. Compound 22 influences levels of the G2/M cell-cycle regulator cyclin B1,
cdc2, and p-cdc2 in A549 cells treated with vehicle, 0.5 μM 22 or 2 μM 22 for 24 h.
Fig. 5. Effect of 22 on the migration of A549 cells by a transwell assay.
Representative images of A549 cells were treated with (A) vehicle, (B) 0.3 μM 22, (C)
1 μM 22 for 12 h, Magnification: 200×; scale bar, 100 μm. (D) Summary data (bar
graph) of the stained A549 cell numbers were counted as an average of three
independent experiments. The results are expressed as the mean ± SD; *P<0.05, **P<
0.001 vs control.
Fig. 6. (A) Analysis of expression of MMP-9 and TIMP-1 in A549 cells exposed to 0,
0.5, or 2 μM 22 for 24 h by western blotting. (B) ELISA analysis of MMP-9 from
A549 cells exposed to indicated concentrations (0 μM, 0.1 μM, 0.5 μM, 1.5 μM, 5.0
18

μM) of 22 for 24 h. The results are expressed as the mean ± SD; n = 3; **P< 0.001 vs
control.
19

Graphical abstrct
Synthesis of hybrid 4-deoxypodophyllotoxin–5-fluorouracil compounds that
inhibit cellular migration and induce cell cycle arrest
Xiao-Wen Guan, Xiao-Hui Xu , Shi-Liang Feng, Zhen-Bo Tang, Shi-Wu Chen*, Ling Hui*
Compound 22 inhibits the migration of A549 cells regulating MMP-9 and TIMP-1, and induces cell cycle arrest in
the G2/M phase involving cdc2, cyclinB1 and p-cdc2.
20