Synthesis and evaluation of novel podophyllotoxin derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2014.08.006

Cancer multidrug resistance (MDR) is a common cause of treatment failure in cancer patients. Increased expression of permeability glycoprotein (P-gp), which is also known as MDR-1, is the main cause of multidrug resistance. Podophyllotoxin derivatives hold great promise in the battle to overcome multidrug resistance, as they can induce cytotoxicity through multiple mechanisms. Here, we synthesized sixteen novel podophyllotoxin derivatives and evaluated their cytotoxicities in human cancer cell lines, HeLa, K562 and K562/A02. Some of these compounds were more potent than etoposide, a clinically relevant inhibitor of DNA repair enzymes. In particular, compound 5p exhibited the most potent activity toward drug-resistant K562/A02 cells, as it robustly inhibited tumor cell proliferation and induced apoptosis. Furthermore, preliminary investigation suggested that 5p inhibited the expression of MDR-1 in K562/A02 cells more effectively than etoposide.

Full text of DOI:10.1016/j.ejmech.2014.08.006

European Journal of Medicinal Chemistry 85 (2014) 498e507
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of novel podophyllotoxin derivatives as
potential antitumor agents
Wei-Hua Cheng a, Bo Cao b, Hai Shang a, Cong Niu b, Li-Ming Zhang c,
,
c
,
, *
Zhong-Heng Zhang c, Dan-Li Tian d, Shi Zhang b, Hong Chen b **, Zhong-Mei Zou a
a Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China
b Pharmacognosy Division, Medical College of Chinese People's Armed Police Force, Tianjin 300162, PR China
c Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University,
Tianjin 300070, PR China
d Tianjin First Central Hospital, Tianjin 300192, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cancer multidrug resistance (MDR) is a common cause of treatment failure in cancer patients. Increased
expression of permeability glycoprotein (P-gp), which is also known as MDR-1, is the main cause of
multidrug resistance. Podophyllotoxin derivatives hold great promise in the battle to overcome multi-
drug resistance, as they can induce cytotoxicity through multiple mechanisms. Here, we synthesized
sixteen novel podophyllotoxin derivatives and evaluated their cytotoxicities in human cancer cell lines,
HeLa, K562 and K562/A02. Some of these compounds were more potent than etoposide, a clinically
relevant inhibitor of DNA repair enzymes. In particular, compound 5p exhibited the most potent activity
toward drug-resistant K562/A02 cells, as it robustly inhibited tumor cell proliferation and induced
apoptosis. Furthermore, preliminary investigation suggested that 5p inhibited the expression of MDR-1
in K562/A02 cells more effectively than etoposide.
Received 23 April 2014
Received in revised form
24 July 2014
Accepted 4 August 2014
Available online 4 August 2014
Keywords:
Podophyllotoxin
Cytotoxicity
Apoptosis
© 2014 Elsevier Masson SAS. All rights reserved.
Multidrug resistance
MDR-1
1. Introduction
antitumor activity, such as etoposide (VP-16, 2) and teniposide
(VM-26, 3) have been widely used as anticancer drugs for clinical
Prolonged cancer chemotherapy treatment can result in an ac-
quired resistance toward multiple drugs, which is known as
multidrug resistance (MDR) [1]. The development of MDR is a
major factor of mortality following failed chemotherapy; as such,
intense efforts are underway to develop novel synthetic antitumor
agents with cytotoxicity against MDR cancer cell lines.
Podophyllotoxin (PPT, 1), an abundant naturally occurring
cyclolignan isolated mainly from Podophyllum peltatum and Podo-
phyllum hexandrum, has important antineoplastic and antiviral
properties [2]. However, its antimitotic activity proved to be of the
greatest interest to researchers [3]. Because of its toxic side effects,
extensive modifications to the original structure have been carried
out over several decades. Podophyllotoxin derivatives with
chemotherapy [4]. However, their low water solubility, induction of
acquired drug-resistance and severe gastrointestinal disturbances
prompted a search for new podophyllotoxin derivatives [5]. In or-
der to obtain better therapeutic agents, a great number of podo-
phyllotoxin analogs have been synthesized. This has led to the
development of NK-611 (4) andGL-331 (5) (Fig. 1); the latter com-
pound entered phase II clinical trials for the treatment of various
cancers [6]. Recently, several novel podophyllotoxin derivatives
showed a potent anti-tumor activity [7e9].
Two alternative molecular mechanisms are generally involved
in the antineoplastic activity of podophyllotoxin analogs: preven-
tion of the assembly of tubulin into microtubules and inhibition of
the catalytic activity of DNA topoisomerase II (Topo II) [10,11].
The investigation of the structureeactivity relationships of PPT
indicates that the trans-lactone, the 4
40-demethyl moieties are essential for TOP-II inhibitory activity
[2,11,12]. 4 -N-substituted derivatives of PPT have attracted much
interest owing to their improved cytotoxicity relative to PPT [13]. In
this regard, we have discovered 4 -N-substituted derivatives of PPT
that show both higher antitumor activity and greater solubility
b-substituted moiety, and the
* Corresponding author. Institute of Medicinal Plant Development, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR
China.
** Corresponding author. Pharmacognosy Division, Medical College of Chinese
People's Armed Police Force, Tianjin 300162, PR China.
E-mail addresses: chenhongtian06@163.com (H. Chen), zmzou@implad.ac.cn
(Z.-M. Zou).
b
b
http://dx.doi.org/10.1016/j.ejmech.2014.08.006
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

W.-H. Cheng et al. / European Journal of Medicinal Chemistry 85 (2014) 498e507
499
Fig. 1. Structures of podophyllotoxin (1), etoposide (2), teniposide (3), NK611(4) and GL331(5).
than VP-16 [14e16]. However, these novel podophyllotoxin analogs
are subject to rapid metabolic turnover in vivo [16]. In order to
address this limitation, we synthesized additional 4b-N-substituted
derivatives of PPT by means of a Schiff base reaction, and evaluated
their cytotoxicities against a panel of three human cancer cell lines.
The toxicity of compound 5p was 10 times higher than that of VP-16
in K562/A02 tumor cells. This was reflected by the resistance index
of 0.905 for 5p, but 5.667 for podophyllotoxin and 69.29 for VP-16.
Our preliminary results indicate that compound 5p may overcome
MDR by reducing the expression of MDR-1.
formaldehyde aqueous solution. Then, compound 7 was reacted
with active dioxide manganese to give the intermediate 8.
The synthetic route (Scheme 2) to the target compounds 5aep
involved the intermediate 3, which was prepared from 1. In the
presence of sodium azide, compound 2 was derived from com-
pound 1 [17]. Then, compound 3 was derived from 2 through a
reduction azide reaction. Next, 3 was combined with one of com-
pounds 8aee in the presence of absolute methanol and a catalytic
amount of glacial acetic acid to provide the intermediate 4. Then,
compounds 5aep was derived from 4 through a reduction reaction.
The structures of the target compounds were identified by HRMS,
1H NMR, and 13C NMR spectral analysis.
2. Results and discussion
2.1. Chemistry
2.2. Biological activity
The synthesis of compounds 8aee is outlined in Scheme 1.
Compound 7 was prepared by means of a Mannich reaction with
secondary amine in the presence of glacial acetic acid and
2.2.1. In vitro cytotoxicity assay
The evaluation of the cytotoxicities of derivatives 5aep was
carried out with a panel of three tumor cell lines (HeLa, K562, and
Scheme 1. Reagents and conditions: (i) CH3COOH/HCHO/amines; (ii) CH2Cl2/MnO2.

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W.-H. Cheng et al. / European Journal of Medicinal Chemistry 85 (2014) 498e507
Scheme 2. Reagents and conditions: (i) NaN3, CF3COOH, CH2Cl2; (ii) Pd/C, HCOONH4, CH2Cl2; (iii) CH3COOH, MeOH; (iv) NaBH4, MeOH.
K562/A02) using the MTT assay. As a comparison, we used the
cytotoxic podophyllotoxin and its derivative, VP-16. The results are
summarized in Table 1, where it can be seen that compounds 5a
and 5i were more cytotoxic towards HeLa cells than the positive
We were also able to deduce some preliminary structur-
eeactivity relationships. First, the 4'-OH compounds were more
cytotoxic than the corresponding 4'-OMe analogues (compare 5a
and 5b, 5c and 5d, 5e and 5f, and 5g and 5h). This observation is in
accord with previously reported activities of closely-related struc-
tures [13]. Second, the introduction of an amine group, such as
dimethylamine, diethylamine, pyrrolidine, 1-methylpiperazine, or
1-ethylpiperazine, resulted in a considerable increase in cytotox-
icity in HeLa and K562. However, 5l, 5n and 5p, which lack the
amide group, exhibited stronger activity against K562/A02 tumor
cells than did the positive control, VP-16. We thus infer that
control, VP-16. The IC50 value of compound 5a was 0.51
mM,
whereas compounds 5e, 5f, 5l and 5n displayed more moderate
cytotoxicities (i.e., higher IC50) in both HeLa and K562 cell lines. The
derivatives 5e, 5i, 5n and 5p appear to be more cytotoxic to the
MDR cell line, K562/A02, when compared to VP-16. In particular,
compound 5p significantly inhibited proliferation of K562/A02,
which likely explains its ability to abrogate drug resistance.

W.-H. Cheng et al. / European Journal of Medicinal Chemistry 85 (2014) 498e507
501
Table 1
Cytotoxicities of podophyllotoxin derivatives in three cancer cell lines.
Comp.
IC50
(m
mol/L)a,b
K562
RF
HeLa
K562/A02
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
0.51
14.2
6.38
>100
1.21
1.40
2.42
15.3
0.67
>100
70.46
1.53
60.45
1.94
6.64
7.55
0.10
6.27
7.89
6.91
8.25
>100
5.28
6.25
8.13
>100
7.45
>100
>100
5.78
65.91
6.79
9.24
5.99
0.03
3.39
70.12
35.33
>1000
>1000
24.66
40.23
200.12
>1000
123.21
>1000
>1000
10.94
>1000
10.08
59.24
5.42
8.89
5.11
>100
>100
4.67
6.43
24.61
>100
16.54
>100
>100
1.89
34.23
1.48
6.41
0.905
5.667
69.29
Podophyllotoxin
VP-16
0.17
234.7
RF: resistance factor was calculated from the ratio of the growth inhibition constant
(IC50) of the resistant cell subline to that of the parental cell line.
a
The value is the average of three replicates.
IC50: Concentration that causes a 50% reduction of cell growth.
b
removal of the amine group might increase the cytotoxicity of
podophyllotoxins in K562/A02 tumor cells. Conversely, an excessive
number of amino groups may engender multidrug resistance,
although this hypothesis requires further testing.
Previous studies indicate that VP-16 and VM-26, which have a
free hydroxyl group at the E-ring, are inhibitors of topoisomerase II
[18e21]. The derivatives we prepared also have free hydroxyl
groups at the E-ring (5a, 5c, 5e, 5g, 5i, 5k, 5m and 5o). It was
suggested that these derivatives were inhibitors of topoisomerase II
[22], whereas podophyllotoxin targets microtubules [23]. In
contrast, and analogous to podophyllotoxin, the derivatives (5b, 5d,
5f, 5h, 5j, 5l, 5n and 5p) do not have free hydroxyl groups at the E-
ring, which is consistent with our previous finding that they also
target microtubules [22].
Fig. 2. Time-dependent effects of 5p on cell growth and viability. (A) Treatment of
K562 cells; (B) Treatment of K562/A02 cells.
2.2.2.3. Apoptosis-specific marker induction in response to com-
pounds. The nuclear morphology of treated cells suggested that 5p
triggered apoptosis. To confirm that 5p induced apoptosis in K562/
A02 cells, we performed double staining with Annexin V-FITC and
propidium iodide (PI). This revealed an increase in the percentage
of apoptotic cells when cells were treated with either 5p or eto-
poside for 48 h. We found that a 48 h treatment with 12.0 mM 5p
greatly enhanced the apoptosis of K562/A02 cells (65.35 1.35%
compared with 7.9 1.34% in the control group; P < 0.01) (Fig. 4).
2.2.2. Study of the pharmacological mechanism-of-action
Our in vitro cytotoxicity studies indicated that compound 5p
markedly inhibited the growth of K562/A02 tumor cells. We
therefore studied this compound further in order to elucidate the
mechanism underlying its anti-MDR activity.
2.2.2.4. Effect of 5p on MDR-1 mRNA expression in K562/A02 and
K562 cells. The permeability glycoprotein (P-gp), encoded by MDR-
1 is a170 kDa, 1280-residue polypeptide chain organized into two
homologous halves. The putative ATP-dependent export pump
function of P-gp, which results in the translocation of small mole-
cules from the cell interior, was deduced based on sequence ho-
mology to bacterial ABC transporters [24]. The highest MDR-1
mRNA level was found in K562/A02, while K562 expressed only low
levels of MDR-1(P < 0.001). Strikingly, MDR-1 mRNA was down
regulated in a dose dependent manner upon treatment of K562/
2.2.2.1. Cell growth. Cell growth assays indicated that 5p inhibited
the growth of cells in a concentration dependent manner. Indeed,
5p was able to block the growth of both parental K562 cells and
their multidrug-resistant derivatives (K562/A02) in a dose depen-
dent fashion (Fig. 2).
2.2.2.2. Morphological evaluation of apoptosis. As shown in Fig. 3,
untreated K562/A02 cells exhibited normal growth characteristics
(Fig. 3A). Interestingly, we found alterations in the structure, size,
A02 with 5p. Specifically, high-dose (12 mM) 5p treatment signifi-
cantly reduced MDR-1 mRNA to 31.17% of the levels found in control
cells (P < 0.01). As shown in Fig. 5, compound 5p was much more
effective in down regulating MDR-1 when compared with etopo-
side at an equimolar concentration.
and shape of the nuclei in K562/A02 cells treated with 6.0
mM
etoposide or 5p for 48 h (Fig. 3B), with large cytoplasmic vacuoles,
abnormal mitotic figures, multinucleation and formation of large
cells being typical (Fig.3C and D). However, HeLa cells treated with
12.0
m
M of 5p for 48 h developed typical apoptotic features, such as
2.2.2.5. Effect of compounds 5c, 5e and 5p on MDR-1 mRNA
expression in K562/A02 and K562 cells. We also evaluated the effect
of other synthetic compounds on the expression of MDR-1. K562/
membrane blebbing, cell shrinkage and detachment, and nuclear
condensation and fragmentation (Fig. 3E). These hallmarks of cell
death, which increased concomitantly with increasing drug con-
centration, are indicated by red arrows.
A02 cells were treated with 6
mM 5c, 5e, 5p, or VP-16. All com-
pounds elicited a reduction in MDR-1 levels compared to the

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W.-H. Cheng et al. / European Journal of Medicinal Chemistry 85 (2014) 498e507
Fig. 3. Chromatin condensation and nuclear fragmentation typical for apoptosis induction were visualized by fluorescence microscopy following Hoechst 33342 staining. (A)
Control K562/A02 cells; (B) K562/A02 cells treated with 6 M etoposide; (C) K562/A02 cells treated with 3 M 5p; (D) K562/A02 cells treated with 6 M 5p; (E) K562/A02 cells
treated with 12
M 5p. Magnification 400ꢀ.
m
m
m
m
control group (P < 0.05). Of note, 5p lead to a greater reduction
(~70%) than did 5c (~35%), which indicates that the structure of 5p
may be more suitable for further anti-MDR drug development
(Fig. 6).
our results suggest that these novel compounds have the potential
to be developed into anticancer agents.
4. Experimental
4.1. Chemistry
3. Conclusion
All materials and reagents were obtained from commercial
sources and used without further purification unless stated.
Podophyllotoxin (98% purity) was purchased from Qingze Corpo-
ration of Nanjing in China. CH2Cl2 was redistilled over P2O5. Melting
points were determined on an electric X-4 digital visual melting
point apparatus. The 1H NMR and 13C NMR spectra were obtained
using a Bruker ARX-300 instrument (300 MHz, 400 MHz). Chemical
shifts (d) are reported in ppm downfield from internal TMS stan-
dard, and the multiplicities were marked as s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, m ¼ multiplet. Mass spectra data were
obtained on Agilent 6210 TOP-MS and are reported as m/z.
Here, we synthesized sixteen novel podophyllotoxin derivatives
5aep, most of which showed promising cytotoxicity against a panel
of cancer cell lines in vitro. Our studies indicate that 5p markedly
inhibited the growth of the MDR human cancer cell line K562/A02
in vitro. This was associated with both reduced proliferation and
induction of apoptosis, as revealed by colorimetric tests, morpho-
logical analysis, and flow cytometry. Our data suggest that the
mechanism-of-action of compounds 5p, 5c and 5e may involve the
inhibition of MDR-1 expression, although further mechanistic
research must be performed to confirm this hypothesis. Together,

W.-H. Cheng et al. / European Journal of Medicinal Chemistry 85 (2014) 498e507
503
Fig. 4. 5p induces apoptosis in K562/A02 cells. (A) Control K562/A02 cells; (B) K562/A02 cells treated with 6
m
M etoposide; (C) K562/A02 cells treated with 3
mM 5p; (D) K562/A02
cells treated with 6
m
M 5p; (E) K562/A02 cells treated with 12
m
M 5p. *P < 0.05, **P < 0.01 compared with the control.
4.1.1. General procedure for the synthesis of compounds 8aee
Compounds (8aee) were synthesized by means of a Mannich
reaction. Formaldehyde (1.2 mmol) was added to a mixture of
furfuryl alcohol (1 mmol) and corresponding secondary amine
(1.2 mmol) in glacial acetic acid (15 mL) and stirred for 4 h at
40 ꢁC. After completion of the reaction was confirmed by thin
layer chromatography (TLC), the solvent was evaporated and
glacial acetic acid was recovered before neutralization with
saturated aqueous NaOH and extraction with ethyl acetate
(3 ꢀ 30 mL). The combined organic fractions were washed with
water followed by brine, dried over Na2SO4. Then, to a stirred
solution of compound 7 (1 mmol) and active dioxide manga-
nese (10 mmol) in dry CH2Cl2 (20 mL) at room temperature for
2h, purified by column chromatography on silica gel using pe-
troleum ether and ethyl acetate to afford the yellow solids
8aee.

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W.-H. Cheng et al. / European Journal of Medicinal Chemistry 85 (2014) 498e507
mixture was stirred at 60 ꢁC for 8 h. After completion of the reaction
was confirmed by TLC, the solvent was washed with saturated
brine, dried over anhydrous Na2SO4, and then removed under
reduced pressure. The residue was dissolved in CH2Cl2 and purified
on silica gel to afford the product 3.
A mixture of one of the compounds 8aee (1.5 mmol), compound
3 (1.0 mmol), and glacial acetic acid (60 mL) was stirred in dry MeOH
(15 mL) for 12 h at room temperature. Then, NaBH4 (4 mmol) was
added and the mixture was stirred for 4 h at 0 ꢁC.The reaction
mixture was neutralized with 1 M HCl, and extracted with CH2Cl2
(3 ꢀ 30 mL). The combined organic fraction was evaporated. The
residue was separated by column chromatography on silica gel with
CH2Cl2eMeOH to afford compounds 5aej.
4.1.2.1. 4b-N-[5-{(Dimethylamino)methyl)furan-2-yl}methyl]-
amido-40-demethyl-4-desoxy-podophyllotoxin (5a). Yield: 65%;
white powder solid; mp: 223e224 ꢁC; [
NMR (300 MHz, CDCl3)
a
]
25 ꢂ 52ꢁ (c 0.1 CH3CN); 1H
D
d
6.43 (s, 1H), 6.33 (s, 1H), 6.25 (s, 2H), 6.19
(d, J ¼ 3.1 Hz, 2H), 5.91 (dd, J ¼ 6.1, 1.3 Hz, 2H), 4.50 (d, J ¼ 5.1 Hz,
1H), 4.35e4.17 (m, 2H), 3.95e3.83 (m, 2H), 3.75 (s, 6H), 3.60 (d,
J ¼ 14.0 Hz, 1H), 3.53e3.44 (m, 2H), 3.31 (dd, J ¼ 13.0, 5.1 Hz, 1H),
2.84e2.67 (m, 1H), 2.30 (s, 6H). 13C NMR (75 MHz, CDCl3)
d 175.1,
153.0, 151.7, 147.5, 147.1, 146.2, 134.0, 132.4, 131.8, 131.2, 110.2, 109.7,
108.6, 108.5, 108.3, 101.5, 69.0, 57.1, 56.6, 55.4, 47.4, 45.7, 44.3, 42.2,
39.5, 31.8. HRMS (ESI) 537.2249 for [MþH]þ (calcd. 537.2237 for
Fig. 5. Effect of 5p on MDR-1 mRNA expression in K562A and K562 cells. (A) Control
K562/A02 cells; (B) Control K562 cells; (C) K562/A02 cells treated with 6
side; (D) K562/A02 cells treated with 3 M 5p; (E) K562/A02 cells treated with 6
M 5p. *P < 0.05, **P < 0.01, ***P < 0.01
mM etopo-
m
mM
C29H32N2O8).
5p; (F) K562/A02 cells treated with 12
m
compared with the control.
4.1.2.2. 4
b
-N-[5-{(Dimethylamino)methyl)furan-2-yl}methyl]-
amido-4-desoxy-podophyllotoxin (5b). Yield: 62%; white powder
25
solid; mp: 226e227 ꢁC; [
(300 MHz, CDCl3)
a
]
D
ꢂ 48ꢁ (c 0.1 CH3CN); 1H NMR
d
6.43 (s, 1H), 6.34 (s, 1H), 6.24 (s, 1H), 6.19 (d,
J ¼ 3.2 Hz,1H), 5.98e5.81 (m, 2H), 4.51 (d, J ¼ 5.3 Hz, 1H), 4.36e4.18
(m, 2H), 3.95e3.82 (m, 2H), 3.78 (s, 3H), 3.72 (s, 6H), 3.61 (d,
J ¼ 14.9 Hz, 1H), 3.53e3.45 (m, 2H), 3.32 (m, 1H), 2.85e2.67 (m, 1H),
2.30 (s, 6H). 13C NMR (75 MHz, CDCl3)
d 174.9, 153.5, 152.3, 147.5,
147.2, 145.4, 135.6, 134.3, 132.3, 131.7, 110.7, 110.3, 108.7, 101.6, 69.0,
61.4, 57.0, 56.2, 55.6, 47.5, 45.3, 44.5, 42.1, 39.6, 31.8. HRMS (ESI)
(551.2413 for [MþH]þ (calcd. 551.2393for C30H34N2O8).
4.1.2.3. 4b-N-[5-{(Diethylamino)methyl)furan-2-yl}methyl]-amido-
40-demethyl-4-desoxy-podophyllotoxin (5c). Yield: 72%; white
powder solid; mp: 230e231 ꢁC; [
(300 MHz, CDCl3)
a
]
25 ꢂ 69ꢁ (c 0.1 CH3CN); 1H NMR
D
d
6.43 (s, 1H), 6.34 (s, 1H), 6.25 (s, 2H), 6.18 (s, 2H),
5.91 (dd, J ¼ 6.5, 1.3 Hz, 2H), 4.50 (d, J ¼ 5.2 Hz, 1H), 4.26 (dd, J ¼ 9.1,
4.0 Hz, 2H), 3.93e3.84 (m, 2H), 3.74 (s, 6H), 3.69 (d, J ¼ 5.2 Hz, 2H),
3.60 (d, J ¼ 14.0 Hz,1H), 3.30 (dd, J ¼ 14.0, 5.2 Hz,1H), 2.80e2.69 (m,
1H), 2.64e2.50 (m, 4H), 1.09 (t, J ¼ 7.1 Hz, 6H). 13C NMR (75 MHz,
CDCl3)
d 174.5, 152.0, 146.6, 146.3, 145.3, 133.0, 131.4, 130.7, 130.1,
109.0, 107.4, 107.0, 100.2, 67.4, 55.4, 53.7, 48.0, 45.8, 45.6, 42.5, 40.3,
Fig. 6. Effect of compounds 5c, 5e and 5p on MDR-1 mRNA expression in K562A and
K562 cells. (A) Control K562/A02 cells; (B) K562/A02 cells treated with 6 M etoposide;
(C) K562/A02 cells treated with 6 M 5c; (D) K562/A02 cells treated with 6 M 5e; (E)
K562/A02 cells treated with 6 M 5p; (F) Control K562 cells. *P < 0.05, **P < 0.01,
37.6, 10.5. HRMS (ESI) 565.2637 for [MþH]þ (calcd. 565.2550 for
m
m
m
C31H36N2O8).
m
***P < 0.01 compared with the control.
4.1.2.4. 4
b
-N-[5-{(Diethylamino)methyl)furan-2-yl}methyl]-amido-
4-desoxy-podophyllotoxin (5d). Yield: 70%; white powder solid;
ꢂ 58ꢁ (c 0.1 CH3CN); 1H NMR (300 MHz,
25
mp: 233e235 ꢁC; [
CDCl3)
a]
4.1.2. General procedure for the synthesis of compounds 5aej
A mixture of podophyllotoxin 1 (10 mmol), NaN3 (40 mmol), and
CF3COOH (10 mL) was stirred in dry CH2Cl2 (10 mL) for in ice bath
for 15 min under nitrogen. Then, the mixture was stirred for 4 h at
room temperature. The reaction mixture was neutralized with
saturated aqueous NaHCO3. The organic extracts were dried over
anhydrous Na2SO4, concentrated, and crystallized from CH2Cl2/
acetic ether (1:1) to give a crystalline material 2.
D
d
6.43 (s, 1H), 6.34 (s, 1H), 6.24 (s, 2H), 6.19 (s, 2H), 5.91 (dd,
J ¼ 6.0, 1.4 Hz, 2H), 4.52 (d, J ¼ 5.2 Hz, 1H), 4.36e4.19 (m, 2H),
3.94e3.88 (m, 1H), 3.86 (s, 1H), 3.78 (s, 3H), 3.72 (s, 6H), 3.70 (d,
J ¼ 5.2 Hz, 2H), 3.64e3.55 (m, 1H), 3.41e3.26 (m, 1H), 2.81e2.69 (m,
1H), 2.65e2.53 (m, 4H), 1.10 (t, J ¼ 7.1 Hz, 6H). 13C NMR (75 MHz,
CDCl3)
d 174.4, 152.0, 151.4, 146.6, 146.3, 136.1, 134.7, 131.4, 130.5,
109.0, 108.6, 107.4, 107.4, 107.3, 100.3, 67.4, 59.7, 55.2, 53.7, 48.0,
45.8, 45.6, 42.7, 40.2, 37.7, 10.5. HRMS (ESI) 579.2696 for [MþH]þ
(calcd. 579.2706 for C32H38N2O8).
To a solution of compound 2 (10 mmol) in ethyl acetate (20 mL),
10% Pd/C (1.00 g) and HCOONH4 (40 mmol) were added, and the

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505
4.1.2.5. 4
b
-N-[5-{(Pyrrolidin-1-yl)methyl)furan-2-yl}methyl]-amido-
4.1.2.10. 4b-N-[5-{(4-Ethylpiperazin-1-yl)methyl)furan-2-yl}
40-demethyl-4-desoxy-podophyllotoxin (5e). Yield: 60%; white
methyl]-amido-4-desoxy-podophyllotoxin (5j). Yield: 70%; white
powder solid; mp: 222e223 ꢁC; [
(300 MHz, CDCl3)
a
]
25 ꢂ 68ꢁ (c 0.1 CH3CN); 1H NMR
powder solid; mp: 226e227 ꢁC; [
(400 MHz, CDCl3)
a
]
25 ꢂ 59ꢁ (c 0.1 CH3CN); 1H NMR
D
D
d
6.43 (s, 1H), 6.30 (s, 1H), 6.25 (s, 2H), 6.19 (d,
d
6.45 (s, 1H), 6.35 (s, 1H), 6.26 (s, 2H), 6.23e6.16
J ¼ 5.4 Hz, 2H), 5.91 (dd, J ¼ 6.4, 1.3 Hz, 2H), 5.41 (s, 1H), 4.50 (d,
J ¼ 5.2 Hz, 1H), 4.32e4.18 (m, 2H), 3.94e3.84 (m, 2H), 3.75 (s, 6H),
3.69 (s, 2H), 3.59 (d, J ¼ 14.9 Hz, 1H), 3.31 (dd, J ¼ 13.7, 5.2 Hz, 1H),
2.82e2.67 (m, 1H), 2.61 (s, 4H), 1.82 (dd, J ¼ 6.6, 3.4 Hz, 4H). 13C
(m, 2H), 5.93 (dd, J ¼ 5.0, 1.4 Hz, 2H), 4.53 (d, J ¼ 5.2 Hz, 1H),
4.34e4.20 (m, 2H), 3.96e3.85 (m, 2H), 3.79 (s, 3H), 3.73 (s, 5H), 3.64
(d, J ¼ 5.2 Hz, 1H), 3.62e3.55 (m, 2H), 3.32 (dd, J ¼ 13.7, 5.2 Hz, 1H),
2.82e2.71 (m, 1H), 2.68e2.49 (m, 8H), 2.46 (t, J ¼ 7.0 Hz, 2H), 1.10 (t,
NMR (75 MHz, CDCl3)
d
175.5, 153.3, 147.6, 147.2, 146.3, 133.9, 132.3,
J ¼ 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 175.46, 153.17, 152.46,
131.8, 131.2, 110.1, 108.6, 108.4, 107.9, 101.3, 68.4, 56.4, 54.7, 53.7,
46.6, 43.5, 41.3, 38.6, 30.9, 23.4. HRMS (ESI) 563.2863 for [MþH]þ
(calcd. 563.2393 for C31H34N2O8).
147.65, 147.30, 137.08, 135.70, 132.43, 131.62, 110.11, 109.89, 108.53,
108.45, 108.31, 101.30, 68.48, 60.75, 56.25, 54.99, 54.71, 52.42,
52.24, 46.69, 43.71, 41.28, 38.70, 14.39. HRMS (ESI) 620.2986 for
[MþH]þ (calcd.620.2972 for C34H41N3O8).
4.1.2.6. 4b-N-[5-{(Pyrrolidin-1-yl)methyl)furan-2-yl}methyl]-amido-
4-desoxy-podophyllotoxin (5f). Yield: 70%; white powder solid; mp:
4.1.3. General procedure for the synthesis of compounds 5kep
A mixture of the appropriate intermediate (1.5 mmol), 3
(1.0 mmol), and glacial acetic acid (60 mL) was stirred in dry MeOH
(15 mL) for 12 h at room temperature. Then NaBH4 (4 mmol) was
added and the mixture was stirred for 4 h at 0 ꢁC.The reaction
mixture was neutralized with 1 M HCl, and extracted with CH2Cl2
(3 ꢀ 30 mL). The combined organic fractions were evaporated. The
residue was separated by column chromatography on silica gel with
petroleum ether-acetic ether to afford compounds 5kep.
220e221 ꢁC; [
a]
25 ꢂ 66ꢁ (c 0.1 CH3CN); 1H NMR (300 MHz, CDCl3)
D
d
6.43 (s, 1H), 6.30 (s, 1H), 6.24 (s, 2H), 6.18 (d, J ¼ 3.0 Hz, 2H), 5.91
(dd, J ¼ 5.5, 1.3 Hz, 2H), 4.51 (d, J ¼ 5.2 Hz, 1H), 3.93e3.85 (m, 2H),
3.79 (s, 3H), 3.72 (s, 6H), 3.60 (d, J ¼ 14.9 Hz, 1H), 3.41e3.23 (m, 2H),
2.82e2.69 (m, 1H), 2.63 (s, 4H), 1.82 (dd, J ¼ 6.6, 3.4 Hz, 4H). 13C
NMR (75 MHz, CDCl3)
d 175.4, 152.5, 147.7, 147.3, 137.1, 135.7, 132.3,
131.6,110.1,108.7,108.4,108.2,101.3, 68.4, 60.7, 56.2, 54.8, 46.6, 43.7,
41.2, 38.6, 30.9, 23.5. HRMS (ESI) 577.3030 for [MþH]þ (calcd.
577.2550 for C32H36N2O8).
4.1.3.1. 4b
-N-[{Furan-2-yl}methyl]-amido-40-demethyl-4-desoxy-
podophyllotoxin (5k). Yield: 75%, white powder solid; mp
4.1.2.7. 4
methyl]-amido-40-demethyl-4-desoxy-podophyllotoxin
Yield: 73%; white powder solid; mp: 211e212 ꢁC; [
CH3CN); 1H NMR (400 MHz, CDCl3)
(s, 2H), 6.20 (d, J ¼ 7.4 Hz, 2H), 5.93 (d, J ¼ 6.1 Hz, 2H), 4.51 (d,
J ¼ 5.2 Hz, 1H), 4.29e4.20 (m, 2H), 3.93e3.85 (m, 2H), 3.76 (s, 6H),
3.66e3.60 (m, 1H), 3.58 (d, J ¼ 5.2 Hz, 2H), 3.31 (dd, J ¼ 13.8, 5.2 Hz,
1H), 2.83e2.71 (m,1H), 2.59 (s, 8H), 2.32 (s, 3H). 13C NMR (100 MHz,
b
-N-[5-{(4-Methylpiperazin-1-yl)methyl)furan-2-yl}
201e203 ꢁC; [
a
]
25 ꢂ 73ꢁ (c 0.1, CH3CN); 1H NMR (400 MHz, CDCl3)
D
(5g).
d 6.45e6.47 (m, 1H), 6.38e6.40 (m, 1H), 6.25e6.27 (m, 1H), 6.43 (s,
a
]
25 ꢂ 61ꢁ (c 0.1
1H), 6.30 (s, 1H), 6.25 (s, 2H), 5.93 (dd, J ¼ 5.2, 1.3 Hz, 2H), 4.51 (d,
J ¼ 5.2 Hz, 1H), 4.24e4.27 (m, 2H), 3.75e3.78 (m, 1H) 3.88e3.94 (m,
2H), 3.75 (s, 6H), 3.27e3.34 (m, 1H), 2.69e2.81 (m, 1H). 13C NMR
D
d
6.45 (s, 1H), 6.36 (s, 1H), 6.27
(100 MHz, CDCl3) d 175.1,153.1,147.1,147.5,146.2,142.2,133.9,132.3,
131.2,131.8,110.7,110.2,108.5,108.1,101.6, 69.0, 57.1, 55.5, 47.4, 44.3,
42.2, 39.5. HRMS (ESI) 480.1648 for [MþH]þ(calcd. 480.1658 for
CDCl3)
d
175.5, 153.2, 147.6, 147.2, 146.3, 133.9, 132.4, 131.8, 131.2,
C26H25NO8).
110.1, 108.5, 108.4, 107.9, 101.2, 68.4, 56.5, 56.4, 54.9, 54.7, 54.7, 46.7,
43.5, 41.3, 38.6. HRMS (ESI) 592.2984 for [MþH]þ (calcd.592.2659
for C32H37N3O8).
4.1.3.2. 4
(5l). Yield: 78%, white powder solid; mp 205e206 ꢁC; [
0.1, CH3CN); 1H NMR (400 MHz, CDCl3)
b-N-[{Furan-2-yl}methyl]-amido-4-desoxy-podophyllotoxin
a]
25 ꢂ 67ꢁ (c
D
d
6.45e6.47 (m, 1H),
6.38e6.40 (m, 1H), 6.25e6.27 (m, 1H), 6.43 (s, 1H), 6.30 (s, 1H), 6.25
(s, 2H), 5.93 (dd, J ¼ 5.2, 1.3 Hz, 2H), 4.51 (d, J ¼ 5.2 Hz, 1H),
4.24e4.27 (m, 2H), 3.75e3.78 (m, 1H), 3.88e3.94 (m, 2H), 3.78 (s,
3H), 3.72 (s, 6H), 3.24e3.38 (m, 1H), 2.74e2.88 (m, 1H). 13C NMR
4.1.2.8. 4b-N-[5-{(4-Methylpiperazin-1-yl)methyl)furan-2-yl}
methyl]-amido-4-desoxy-podophyllotoxin (5h). Yield: 73%; white
powder solid; mp: 214e215 ꢁC; [
(400 MHz, CDCl3)
a
]
25 ꢂ 55ꢁ (c 0.1 CH3CN); 1H NMR
D
d
6.46 (s, 1H), 6.36 (s, 1H), 6.26 (s, 2H), 6.23e6.17
(100 MHz, CDCl3)
d 175.4, 153.4, 152.4, 147.6, 147.3, 142.2, 137.2,
(m, 2H), 5.93 (dd, J ¼ 5.2, 1.3 Hz, 2H), 4.53 (d, J ¼ 5.3 Hz, 1H),
4.32e4.21 (m, 2H), 3.93e3.86 (m, 2H), 3.79 (s, 3H), 3.73 (s, 6H),
3.69e3.61 (m, 1H), 3.59 (d, J ¼ 5.3 Hz, 2H), 3.41e3.29 (m, 2H),
2.82e2.69 (m, 1H), 2.62 (s, 8H), 2.37 (s, 3H). 13C NMR (100 MHz,
135.7, 132.4, 131.6, 110.5, 110.1, 108.4, 108.3, 107.9, 101.3, 68.4, 60.7,
56.2, 54.8, 43.7, 41.3, 38.7. HRMS (ESI) 494.1808 for [MþH]þ (calcd.
494.1815 for C27H27NO8).
CDCl3)
d 175.4, 153.2, 152.5, 147.6, 147.3, 137.1, 135.6, 132.4, 131.6,
4.1.3.3. 4b
-N-[5-{Methylfuran-2-yl}methyl]-amido-40-demethyl-4-
desoxy-podophyllotoxin (5m). Yield: 72%, white powder solid; mp
110.1, 110.0, 108.5, 108.4, 108.3, 101.3, 68.4, 60.7, 56.2, 54.7, 54.5,
54.2, 46.7, 43.7, 41.2, 38.6. HRMS (ESI) 606.3324 for [MþH]þ
(calcd.606.2815 for C33H39N3O8).
211e212 ꢁC; [
a
]
D
25 ꢂ 58ꢁ (c 0.1, CH3CN); 1H NMR (400 MHz, CDCl3)
d
6.44 (s, 1H), 6.32 (s, 1H), 6.24 (s, 2H), 6.13 (d, J ¼ 3.0 Hz, 2H), 5.94
(dd, J ¼ 5.2, 1.3 Hz, 2H), 4.50 (d, J ¼ 5.2 Hz, 1H), 4.25e4.27 (m, 2H),
3.93 (d, J ¼ 3.1 Hz, 1H), 3.80e3.88 (m, 2H), 3.75 (s, 6H), 3.32 (dd,
J ¼ 13.8, 5.2 Hz, 1H), 2.34 (s, 3H), 2.34e2.84 (m, 1H). 13C NMR
4.1.2.9. 4
b-N-[5-{(4-Ethylpiperazin-1-yl)methyl)furan-2-yl}methyl]-
amido-40-demethyl-4-desoxy-podophyllotoxin
(5i). Yield:
71%;
white powder solid; mp: 222e224 ꢁC; [
NMR (300 MHz, CDCl3)
a
]
25 ꢂ 56ꢁ (c 0.1 CH3CN);1H
(100 MHz, CDCl3) d 176.4, 153.5, 152.5, 147.7, 147.4, 142.3, 137.2,
D
d
6.43 (s, 1H), 6.32 (s, 1H), 6.25 (s, 2H),
135.6, 133.9, 131.6, 110.2, 110.1, 108.3, 108.0, 107.9, 101.4, 68.4, 60.7,
56.3, 54.7, 43.5, 41.3, 38.7, 13.6. HRMS (ESI) 494.1841for [MþH]þ
(calcd. 494.1815 for C27H27NO8).
6.22e6.15 (m, 2H), 5.91 (d, J ¼ 4.6 Hz, 2H), 4.50 (d, J ¼ 5.2 Hz, 1H),
4.33e4.18 (m, 2H), 3.93e3.82 (m, 2H), 3.75 (s, 4H), 3.66e3.52 (m,
2H), 3.30 (dd, J ¼ 13.8, 5.2 Hz,1H), 2.81e2.71 (m, 1H), 2.65e2.49 (m,
8H), 2.44 (q, J ¼ 7.1 Hz, 2H),1.09 (t, J ¼ 7.1 Hz, 3H). 13C NMR (75 MHz,
4.1.3.4. 4b-N-[5-{Methylfuran-2-yl}methyl]-amido-4-desoxy-podo-
CDCl3)
d
175.5, 153.2, 147.6, 147.2, 146.3, 133.9, 132.4, 131.7, 131.1,
phyllotoxin (5n). Yield: 65%, white powder solid; mp 213e214 ꢁC;
110.1, 109.9, 108.5, 108.4, 107.9, 101.2, 68.4, 56.4, 54.9, 54.7, 52.3,
52.2, 46.6, 43.5, 41.3, 38.6, 11.6. HRMS (ESI) 606.2809 for [MþH]þ
(calcd.606.2815 for C33H39N3O8).
[
a
]
25 ꢂ 63ꢁ (c 0.1, CH3CN); 1H NMR (400 MHz, CDCl3)
d 6.45 (s, 1H),
D
6.34 (s, 1H), 6.25 (s, 2H), 6.03 (d, J ¼ 3.2 Hz, 2H), 5.95 (dd, J ¼ 5.2,
1.3 Hz, 2H), 4.52 (d, J ¼ 5.2 Hz, 1H), 4.27e4.30 (m, 2H), 3.93 (d,

506
W.-H. Cheng et al. / European Journal of Medicinal Chemistry 85 (2014) 498e507
J ¼ 2.7 Hz 1H), 3.80e3.88 (m, 2H), 3.80 (s, 3H), 3.75 (s, 6H), 3.32 (dd,
J ¼ 13.8, 5.2 Hz, 1H), 2.34 (s, 3H), 2.34e2.84 (m, 1H). 13C NMR
were collected by centrifugation at 1000 ꢀ g for 5 min, washed with
ice-cold PBS and then incubated with Hoechst-33342 (10 g/mL)
m
(100 MHz, CDCl3)
d
175.5,151.9,150.5,147.7,146.2,142.3,137.1,135.7,
for 15 min in the dark, then placed on slides, and observed under a
fluorescence microscope (excitation 346 nm, emission 460 nm;
NIKON TE2000-E). Apoptotic cells were identified by condensation
of chromatin and fragmentation of nuclei. Pictures were obtained
using a video camera Q-imaging (Burnaby, BC, Canada).
133.9, 131.6, 110.2, 110.1, 108.4, 108.0, 107.9, 101.4, 68.5, 60.7, 56.3,
54.7, 43.5, 41.3, 38.1, 13.6. HRMS (ESI) 508.2005 for [MþH]þ (calcd.
508.1971 for C28H29NO8).
4.1.3.5. 4b
-N-[5-{Nitrofuran-2-yl}methyl]-amido-40-demethyl-4-
desoxy-podophyllotoxin (5o). Yield: 68%, yellow powder solid; mp
4.2.4. Apoptosis detection using Annexin V/PI staining
222e223 ꢁC; [
a]
25 ꢂ 78ꢁ (c 0.1, CH3CN); 1H NMR (300 MHZ, CDCl3)
D
Apoptosis can be detected by translocation of phosphatidylser-
ine to the cell surface using an Annexin V-FITC antibody. K562/A02
cells were seeded into 6-well plates (1 ꢀ 105 cells/well), incubated
d
7.35 (d, J ¼ 3.8 Hz, 1H), 7.04 (d, J ¼ 3.8 Hz, 1H), 6.56 (s, 1H), 6.53 (s,
1H), 6.33 (s, 2H), 5.94 (dd, J ¼ 5.2,1.3 Hz, 2H), 4.71 (d, J ¼ 5.2 Hz,1H),
4.67e4.28 (m, 2H), 3.97e4.05 (m, 2H), 3.89 (d, J ¼ 3.2 Hz 1H), 3.79
(s, 6H), 3.56 (dd, J ¼ 13.8, 5.2 Hz, 1H), 3.04e3.18 (m, 1H). 13C NMR
overnight, treated with 3.0, 6.0, or 12.0 mM of 5p for 48 h; 6.0 mM
etoposide was used as the positive control. To assess apoptosis, cells
were washed with ice-cold PBS (pH 7.4). Annexin V-FITC and PI
staining were performed using an Annexin V-FITC kit (Beyotime
Co., Ltd). Annexin V-FITC and PI fluorescence were monitored using
a Beckman Coulter flow cytometer. Ten thousand events were
collected per sample. Data were analyzed using FlowJo analysis
software.
(75 MHZ, CDCl3)
d 174.9,152.1,152.4,148.4,147.5, 134.2, 132.6, 132.6,
131.0, 128.2, 110.8, 108.3, 108.6, 101.6, 67.8, 67.7, 56.5, 56.5, 43.9,
HRMS (ESI) 525.1545 for [MþH]þ (calcd. 525.1509 for C26H24N2O10).
4.1.3.6. 4b-N-[5-{Nitrofuran-2-yl}methyl]-amido-4-desoxy-podo-
phyllotoxin (5p). Yield: 66%, yellow powder solid; mp 229e230 ꢁC;
25
[
a]
ꢂ 73ꢁ (c 0.1, CH3CN); 1H NMR (300 MHz, CDCl3)
d 7.23 (d,
D
J ¼ 3.9 Hz, 1H), 6.43 (d, J ¼ 3.9 Hz, 1H), 6.51 (s, 1H), 6.26 (s, 1H), 6.16
(s, 2H), 5.84 (dd, J ¼ 5.2, 1.3 Hz, 2H), 4.46 (d, J ¼ 5.2 Hz, 1H),
4.27e4.18 (m, 2H), 3.85e3.93 (m, 2H), 3.80 (d, J ¼ 2.9 Hz, 1H), 3.71
(s, 3H), 3.65 (s, 6H), 3.21 (dd, J ¼ 13.8, 5.2 Hz, 1H), 2.69e2.82 (m,
4.2.5. Reverse transcriptase-polymerase chain reaction (RT-PCR)
Since we determined that compound 5p was the most effective
among the podophyllotoxin derivatives with respect to abrogation
of MDR, we decided to evaluate MDR-1 expression by RT-PCR. K562
and K562/A02 cells were seeded on 6-well plates at a density of
1 ꢀ105 cells/well in 10% FCS/RPMI-1640, and varying amounts of 5p
were added 24 h later; cells were then cultured for another 48 h.
Total RNA was extracted by Trizol on Day 3 and absorbance was
measured in an RNA/DNA calculator at 260 and 280 nm cDNA was
1H). 13C NMR (75 MHZ, CDCl3)
d 174.9, 152.1, 152.4, 148.4, 147.5,
134.2, 132.6, 132.6, 131.0, 128.2, 110.8, 108.6, 108.3, 101.6, 67.8, 67.7,
56.5, 56.5, 43.9. HRMS (ESI) 539.1710 for [MþH]þ (calcd. 539.1666
for C27H26N2O10).
4.2. Biological evaluation
synthesized from 2 mg of total RNA using TIANScript RT Kit reverse
transcription reagents (TIANEN BIOTECH, RT120420, Beijing).
Amplification of MDR-1 was performed under the following cycling
conditions: 94 ꢁC for 3 min and then 30 cycles of 94 ꢁC for 40 s,
55 ꢁC for 40 s, 72 ꢁC for 1 min, finally 72 ꢁC for 10 min, followed by
cooling to 4 ꢁC by Program Temp Control System (ASTSC. PC-818s.
Japan). Primers for MDR-1 (157bp product) amplification were 50-
CCC ATA ATT GCA ATA GCA GC-30 and 50-GTT CAA ACT TCT GCT CCT
CA-30, while 50-GTG GGG CGC CCC AGC CAC CA -30 and 50-CTT CCT
4.2.1. Cell viability assay
HeLa, K562, and K562/A02 cells were cultured in RPMI-1640
containing 10% FBS, 2 mmol/L glutamine, 100 U/mL penicillin, and
100 m
g/mL streptomycin at 37 ꢁC in a humidified atmosphere with
5% CO2. The cells were seeded at a density of 5 ꢀ 103 cells/well in
96-well plates and allowed to attach for 24 h. The thiazolyl blue
tetrazolium blue (MTT) assay was performed to quantify cell
viability following treatment with the synthetic compounds or
TAA TGT CAC GCA CGA TTT C-30 were for
b-actin (540bp) PCR target
reference compound etoposide (VP-16) [25]. After 48 h, 20 mL MTT
products were then separated electrophoretically on a 5% agarose
DNA gel and stained with Gold View. The mRNA levels were
normalized to the levels of b-actin.
(5 mg/mL) solution was added for 4 h at 37 ꢁC. Then, the super-
natant was discarded and dimethylsulfoxide (DMSO) was added to
dissolve the formazan product. The intensity was measured at a
wavelength of 490 nm.
Acknowledgments
4.2.2. Cell growth curves
We used the K562 and K562/A02 cell lines for cell growth
curves. The cells were cultured on 96-well plates with 5p or eto-
This work has been financially supported by National S & T
Major Special Project on Major New Drug Innovation
(2011ZX09307-002-01 and 2013ZX09508104), PUMC Youth Fund
(33320140074), the Fundamental Research Funds for the Central
Universities and Program for Innovative Research Team in IMPLAD
(IT1305).
poside at varying concentrations of 3, 6, and 12 mM on Day 1. This
was followed by examination of their inhibitory effects on cell
survival recorded for each plate every 24 h. The assessment was
performed using a modification of the MTT assay. Following the
addition of 5 mg/mL MTT, samples were cultured for a further 4 h.
After the removal of culture medium, followed by the addition of
DMSO, absorption at 490 nm was measured.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.006.
4.2.3. Morphological analysis with fluorescence microscopy
Apoptosis is one of the major pathways leading to cell death,
and is associated with classical morphologies, including chromatin
condensation and nuclear fragmentation. To evaluate the apoptotic
activity of 5p, we performed nuclear staining with the DNA-binding
dye, Hoechst-33342. Briefly, K562/A02 cells were plated into 6-well
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