First synthesis and biological evaluation of novel spin-labeled derivatives of deoxypodophyllotoxin

Article abstract of DOI:10.1016/j.ejmech.2009.12.032

Deoxypodophyllotoxin inhibits tubulin polymerization and induces cell cycle arrest at G2/M, followed by apoptosis. In order to find compounds with superior bioactivity and less toxicity, a series of spin-labeled derivatives of deoxypodophyllotoxin were synthesized by reacting 4′-demethyl-4-deoxypodophyllotoxin (DPPT) with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl) amino acids. The cytotoxic activities against three tumor cell lines (HL-60, RPMI-8226, A-549) in vitro and the antioxidative activities in tissues of Sprague Dawley (SD) rats of target compounds were evaluated, and the results indicated that compounds 11a-h were more potent in terms of cytotoxicities and antioxidative activities than either parent compound DPPT or anticancer drug VP-16.

Full text of DOI:10.1016/j.ejmech.2009.12.032

European Journal of Medicinal Chemistry 45 (2010) 1673–1677
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
First synthesis and biological evaluation of novel spin-labeled derivatives
of deoxypodophyllotoxin
,
b
,
a,
*
Zhi-Wei Zhang a, Jia-Qiang Zhang a, Ling Hui c, Shi-Wu Chen a , Xuan Tian
*
a State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
b School of Pharmacy, Lanzhou University, Lanzhou 730000, China
c Experimental Center of Medicine, Lanzhou General Hospital, Lanzhou Command, Lanzhou 730050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Deoxypodophyllotoxin inhibits tubulin polymerization and induces cell cycle arrest at G2/M, followed by
apoptosis. In order to find compounds with superior bioactivity and less toxicity, a series of spin-labeled
derivatives of deoxypodophyllotoxin were synthesized by reacting 40-demethyl-4-deoxypodophyllotoxin
(DPPT) with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl) amino acids. The cytotoxic activi-
ties against three tumor cell lines (HL-60, RPMI-8226, A-549) in vitro and the antioxidative activities in
tissues of Sprague Dawley (SD) rats of target compounds were evaluated, and the results indicated that
compounds 11a–h were more potent in terms of cytotoxicities and antioxidative activities than either
parent compound DPPT or anticancer drug VP-16.
Received 4 October 2009
Received in revised form
4 December 2009
Accepted 12 December 2009
Available online 23 December 2009
Keywords:
Deoxypodophyllotoxin
Spin-labeled
Ó 2009 Elsevier Masson SAS. All rights reserved.
Antitumor activity
Antioxidative activity
1. Introduction
40-demethyl-4-deoxypodophyllotoxin (DDPT, 3) and prodrugs of
DPT including carbamates and carbonate. The antitumor activities
Podophyllotoxin (PT, 1) and its many related derivatives are well
known to have pronounced antineoplastic and anti-viral properties
[1]. PT preferentially binds to tubulin and inhibits tubulin poly-
merization, while some semi-synthetic derivatives of PT, such as
etoposide (VP-16), teniposide (VM-26) and etopophos, have potent
inhibitory activity of DNA topoisomerase II. And these derivatives of
PT currently used in chemotherapy for various types of cancer,
including small cell lung cancer, testicular carcinoma, and Kaposi’s
sarcoma [2–9]. Deoxypodophyllotoxin (DPT, 2), as an analogue of
PT, has antiproliferative and antitumor activities in diverse cell
types [10–14], as well as anti-inflammatory [15] and anti-viral [16]
activities. The mechanism studies showed that DPT inhibits tubulin
polymerization and induces cell cycle arrest at G2/M, followed by
apoptosis through multiple cellular processes, involving the acti-
vation of ATM, upregulation of p53 and Bax, activation of caspase-3
and -7, and accumulation of PTEN resulting in the inhibition of the
Akt pathway [17,18]. Recently, Ahn and co-workers synthesized
many derivatives of DPT, such as alkyl, carboxylalkyl esters of
of all these compounds showed that most derivatives enhanced
cytotoxic or antitumor activity compared with that of parent
compound DPT [19,20].
The nitroxyl free radicals have a wide range of activities in
biology. Previous studies have shown that the introduction of
nitroxyl moiety can lead to a series of positive effects, such as
higher alkylating, lower carbamoylating activity, better anti-
melanomic activity and lower general toxicity. These types of
compounds can move through cell membranes as a transport
[21–23]. In addition, the nitroxyl free radicals possess low toxicity
and are not mutagenic or carcinogenic by themselves [24]. Our
group had been aiming at the discovery of new drugs of PT deriv-
atives with improved bioactivities and less toxicity in recent years
[25–34]. A series of spin-labeled podophyllotoxin analogues, via
modification of different positions, had been prepared and these
compounds showed significant antitumor activity with marked
decrease in toxicity when compared with the parent compound
[25–28]. Especially, GP-11 (4) (Fig. 1) was reported as low immu-
nosuppressive antitumor agent, which increased the mitotic index
and resulted in G2/M phase, and to a lesser extent, S arrest. This
compound has the possibility of becoming a promising new anti-
tumor drug. Inspired by previous work as well as the fact that
* Corresponding authors. State Key Laboratory of Applied Organic Chemistry,
Lanzhou University, Lanzhou 730000, China. Tel.: þ86 931 8912410; fax: þ86 931
8912582.
L-amino acids, possessing good water solubility if actively trans-
E-mail addresses: chenshwu@gmail.com (S.-W. Chen), xuant@lzu.edu.cn
(X. Tian).
planted into mammalian tissue, are often used as carrier vehicles
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.032

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Z.-W. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 1673–1677
OH
R
11
5
B
8
4
3
2
O
O
O
OH
R
R'
O
H
O
A
C
D
N
OH
1
2
3
Me
N
H
N
H
O
N
1
O
O
H
H
Me
H
O
2'
6'
E
MeO
OMe
MeO
OMe
4'
OR'
OMe
4
Fig. 1. Structures of podophyllotoxin (1) and related compounds.
for some drugs, a series of spin-labeled derivatives of DDPT were
synthesized and the cytotoxic activities against three tumor cells
(A-549, RPMI-8226 and HL-60) were evaluated. We also deter-
mined malondialdehyde (MDA) on liver and kidney homogenate of
SD rats by the TBA method.
methanesulfonic acid/sodium iodide [39]. Then 3 was condensed
with the appropriate 10a–h in the presence of N,N0-dicyclohex-
ylcarbodiimide (DCC) and catalytic amount of 4-dimethylamino
pyridine (DMAP) to provide the target compounds 11a–h. The
structures of the target compounds were identified by HRMS, ESR
and IR spectral analysis.
2. Chemistry
3. Biological results and discussion
The synthesis of compounds 10a–h is outlined in Scheme 1.
4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (6) was prepared
in excellent yield by means of oxidation of 4-hydroxyl-2,2,6,6-
tetramethylpiperidine (5) with sodium tungstate–hydrogen
peroxidate–EDTA [35]. Then the compound 6 reacted with N,N0-
carbonyldiimidazole to give the intermediate N-(1-oxyl-2,2,6,6-
tetramethyl- piperidinyloxycarbonyl)-imidazole (7) [36]. The
intermediate 7 without isolation, further reacted with p-toluene-
sulfonic acid monohydrate to give its higher reactive tosylate (8).
The salt 8 was so reactive that it was instantaneously converted into
compound 9 when dissolved in an aqueous solution of sodium
azide. Compounds 10a–h were obtained in good yield by reaction
of the alkoxycarbonyl azide (9) with free amino acids in the pres-
ence of MgO [37].
The biological activities of this series of spin-labeled derivatives
of DDPT 11a–h were evaluated by an in vitro cytotoxicity test,
which was carried out with a panel of three tumor cell lines that
comprise human premyelocytic leukemia (HL-60), human multiple
myeloma (RPMI-8226) and human lung carcinoma (A-549). The
antioxidative activity on liver and kidney homogenate of SD rats
was also tested and the results are summarized in Table 1.
As illustrated in Table 1, the spin-labeled derivatives of DDPT
11a–h exhibited basically stronger inhibition of these three cell
lines comparing to DDPT and VP-16. In these three tumor cell lines,
the inhibition of all these spin-labeled compounds showed
decreased activity in the following order: HL-60 > RPMI-8226 >
A-549, such as, the IC50 value of compound 11b is 0.012, 0.044 and
The synthetic route to the target compounds 11a–h involved the
intermediate 40-demethyl-4-deoxypodophyllotoxin 3 which was
prepared from 1. Compound 1 was isolated from a Chinese medic-
inal herb Podophyllum emodi Wall var Chinesis Sprague (Scheme 2).
The intermediate 3 was prepared from 1 through successive 4-
deoxylation by hydrogenolysis [38] and 40-demethylation with
0.67 mM for HL-60, RPMI-8226 and A-549, respectively. In Table 1,
we could also see that the target derivatives 11a–h showed superior
or comparable antioxidative activities in both tissues of kidney and
liver homogenate of SD rats in comparison with parent compound
DDPT and VP-16. The values of IC50 are ranged from 6.89 to
20.33
mM. However, different substituents at a-carbon of amino
O
O
O
OH
O
N
OH
N
NH
N
TosO-
i
ii
iii
N
O
N
O
N
H
N
O
8
7
5
6
O
R
O
O
N
COOH
a R=H
O
N3
H
v
iv
b R=Me
c R=CHMe2
d R=CH2CHMe2
f
N
O
N
O
9
e R=CHMeCH2Me
g R=CH2Ph
Proline
h R=CH2(C6H4)OH
10a-h
Scheme 1. Reagents and conditions: (i) Na2WO4/H2O2/EDTA; (ii) N,N0-carbonyldiimidazole/THF; (iii) p-toluenesulfonic acid monohydrate; (iv) NaN3/water, stir; (v) amino acids/
MgO, stir, 24 h.

Z.-W. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 1673–1677
1675
OH
O
O
O
O
O
O
O
O
O
i
ii
O
O
O
OMe
OMe
OMe
MeO
MeO
MeO
OMe
OH
OMe
2
3
1
O
O
O
a R=H
b R=Me
d R=CH2CHMe2
Proline
h R=CH2(C6H4)OH
c R=CHMe2
iii
O
e R=CHMe)CH2Me
g R=CH2Ph
f
OMe
R
MeO
O
O
N O
N
H
O
O
11a-h
Scheme 2. Reagents and conditions: (i) Pd/C 10%, H2; (ii) MeSO3H/NaI, CH2Cl2; (iii) 10a–h, DCC, DMAP, CH2Cl2, N2.
reported work, eight spin-labeled derivatives of deoxypodo-
phyllotoxin were first synthesized successfully, the results of bio-
logical evaluation showed that the vast majority of compounds
exhibited more potent cytotoxicities against HL-60, RPMI-8226 and
A-549. Compound 11a showed most potent cytotoxicity against HL-
Table 1
Biological activity of compounds 11a–h.
Compounds
Cytotoxicity (IC50
,
m
M)a
Antioxidate activity
(IC50
M)a
,
m
60 with IC50 at 0.0087 mM. In addition, the synthesized derivatives
A-549b
RPMI-8226c
HL-60c
Kidney
Liver
11a–h showed either similar or better antioxidative activities in both
tissues of kidney and liver homogenate of SD rats than that of the
parent compound DDPT, and anticancer drug VP-16. Further biolog-
ical evaluation is in progress to define their tubulin polymerization
activity and to clarify whether spin-labeled deoxypodophyllotoxin
analogues might counteract the toxicity of this class antitumor drug.
11a
11b
11c
11d
11e
11f
0.62
0.67
0.55
0.77
0.83
0.60
0.50
0.27
0.80
0.83
0.073
0.044
0.16
0.11
0.058
0.13
0.031
0.043
0.23
0.0087
0.012
0.024
0.085
0.036
0.14
0.018
0.032
0.11
13.70
15.78
20.33
12.08
6.89
14.56
15.05
16.27
22.99
64.63
14.37
15.32
19.30
18.01
13.16
9.70
10.38
15.2
33.75
46.38
11g
11h
DDPT
VP-16
5. Experimental
0.70
0.18
a
The value is the average of triplicate.
MTT method, drugs exposure was for 48 h.
CCK-8 method, drugs exposure was for 72 h.
Melting points were determined in Kofler apparatus and were
uncorrected. IR spectra were measured on a Nicolet 5DX-FT-IR spec-
trometer on neat samples placed between KBr plates.1H NMR spectra
were obtained by using a Bruker AM400 with TMS as an internal
b
c
acid in this class of compounds did not show obviously different
effects on the inhibition of the three tumor cell lines in vitro and on
antioxidative activity.
standard, all chemical shifts are reported as d ppm. Mass spectra were
recorded on a Bruker Daltonics APEXII49e spectrometer with ESI
source as ionization. Optical rotations were measured on a Perkin
Elmer 341 polarimeter in a 1 dm cell at 23 ꢀC. The electron spin
resonance (ESR) spectra were obtained with a Bruker A300 X-band
EPR spectrometer. The N-(1-oxyl-2,2,6,6-tetramethyl-4-piper-
idinyloxycarbonyl)-amino acids10a–hused for theexperiments were
prepared by following a modified previous procedure [27,35–37].
These results indicated that the structures of L-amino acids have
potential effects on the bioactivity of these compounds. Hence,
a systemic, predictable correlation could be made between the
nature of amino acids and anticancer and antioxidant activities. As
can be seen, as a whole, the introduction of a stable nitroxyl radical
into the molecule of 3 with L-amino acids led to potentiate their
antitumor and antioxidative activity, which also indicated that the
design and synthesis of these compounds should be beneficial for
therapeutic values of deoxypodophyllotoxin and they probably
have synergistic action to tumor cell lines.
5.1. Synthesis of DDPT (3)
A mixture of 10% Pd/C (6.0 g) in acetic acid (150 mL) was stirred
under hydrogen until no further hydrogen was absorbed. Then PT
(8.0 g) was added and the reaction mixture was stirred for another
5 h at 95 ꢀC under 2 atm of hydrogen. The mixture was filtered and
the filtrate was evaporated. The residue was purified by column
chromatography on silica gel (elution with CH2Cl2) yielding DPT
(5.6 g, yield 73%).
4. Conclusion
In summary, novel spin-labeling of podophyllotoxin class is
a promising direction in antitumor chemotherapy, not only because
these compounds exhibit superior activities, but also because they
can be monitored by ESR in pharmacological experiments. In this
To a solution of DPT (3.98 g, 10 mmol) in dry CH2Cl2 (100 mL),
NaI (4.47 g, 30 mmol) was added and stirred for 5 min. To this

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Z.-W. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 1673–1677
stirred suspension, MeSO3H (2.88 g, 30 mmol) was added dropwise
with syringe at 0 ꢀC and stirring was continued for another 5 h at
room temperature. Nitrogen was bubbled through the solution to
drive off the excess hydrogen iodide. The solution was then evap-
orated in vacuo and purified by chromatography to give yellow oil.
Further purification by recrystallization with methanol afforded
2.1 g of 3 as a white solid. Yield 55%; 1H NMR (400 MHz, CDCl3):
1342 (N–O.), 1226, 1129, 926; MS m/z 679 (Mþ); ESR: An ¼ 15.95 G,
g0 ¼ 2.006; HRMS (ESI) 702.2759 for [M þ Na]þ (calcd 702.2763 for
C36H43O11N2).
5.2.7. N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-
phenylalanine 40-demethyl-4-deoxypodophyllic ester (11g)
The yield: 54%; red powder; mp: 119–126 ꢀC; [
a
] ꢁ62 (c 0.1,
d
6.66 (s, 1H), 6.52 (s, 1H), 6.35 (s, 2H), 5.94 (d, J ¼ 9.2 Hz, 2H), 5.41
CHCl3); IR (cmꢁ1) 3330, 2933, 2848, 1773, 1721, 1506, 1484, 1460,
1338 (N–O.), 1226, 1129, 936; MS m/z 731 (M þ 2Hþ); ESR:
An ¼ 15.78 G, g0 ¼ 2.0061; HRMS (ESI) 752.2914 for [M þ Na]þ
(calcd 752.2916 for C40H45O11N2).
(s, 1H), 4.60 (s, 1H), 4.45–4.42 (m, 1H), 3.93–3.90 (m, 1H), 3.78 (s,
6H), 3.08–3.05 (m, 1H), 2.77–2.72 (m, 3H).
5.2. General procedure of synthesis of 11a–h
5.2.8. N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-
A mixture of the appropriate 10a–h (0.5 mmol), DDPT (0.192 g,
0.5 mmol) and DMAP (20 mg) was stirred in dry CH2Cl2 (10 mL) for
5 min at room temperature under nitrogen. N,N-Dicyclohex-
ylcarbodiimide (DCC,106 mg, 0.5 mmol) was added and the mixture
was stirred for 4 h. The reaction mixture was filtered and the filtrate
was evaporated. The residue was separated by column chromatog-
raphy on silica gel with CH2Cl2–acetone to afford compounds 11a–h.
tyrosine 40-demethyl-4-deoxypodophyllic ester (11h)
The yield: 44%; red powder; mp: 166–168 ꢀC; [
a] ꢁ80 (c 0.1,
CHCl3); IR (cmꢁ1) 3433, 2974, 2937, 1766, 1723, 1508, 1485, 1463,
1341 (N–O.), 1234, 1130, 937; MS m/z 747 (M þ 2Hþ); ESR:
An ¼ 15.88 G, g0 ¼ 2.006; HRMS (ESI) 745.2977 for [M þ Na]þ (calcd
745.2967 for C40H45O11N2).
5.3. Cytotoxicity assays
5.2.1. N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-
glycin 40-demethyl-4-deoxypodophyllic ester (11a)
Cells were incubated at 37 ꢀC in a 5% CO2 atmosphere. The MTT
and Cell Counting Kit-8 (CCK-8) colorimetric assay were used to
determined growth inhibition [40]. The spin-labeled derivatives
The yield: 65%; red powder; mp: 149–152 ꢀC; [
a
] ꢁ65 (c 0.1,
CHCl3); IR (cmꢁ1) 3329, 2933, 2849, 1778, 1723, 1507, 1484, 1462,
1337 (N–O.), 1227, 1130, 931; MS m/z 639 (M þ 2Hþ); ESR:
An ¼ 16.02 G, g0 ¼ 2.0061; HRMS (ESI) 662.2446 for [M þ Na]þ
(calcd 662.2436 for C33H39O11N2).
were dissolved in saline for five concentrations (0.005–50 mM). For
the HL-60 and RPMI-8226, cells were plated in 96-well plates and
exposed in quadplex well for 48 or 72 h. Then the CCK-8 was added
to each well. After 4 h of incubation, the absorbance at l450 was
determined with a plate reader. For the A-549, cells were plated in
96-well plates and allowed to attach for 24 h, then exposed in
5.2.2. N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-
alanine 40-demethyl-4-deoxypodophyllic ester (11b)
The yield: 69%; red powder; mp: 139–142 ꢀC; [
a
] ꢁ67 (c 0.1,
quadplex well for 48 h. The media were aspirated, and 10 mL of
CHCl3); IR (cmꢁ1) 3328, 2932, 2849, 1773, 1720, 1507, 1484, 1461,
1338 (N–O.), 1227, 1130, 930; MS m/z 653 (Mþ); ESR: An ¼ 16.16 G,
g0 ¼ 2.006; HRMS (ESI) 676.2603 for [M þ Na]þ (calcd 676.2614 for
C34H41O11N2).
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 mL DMSO, then was shaken on an oscillator. The
absorbance at l570 was determined on a plate reader. IC50 values
were determined from a log plot of percent of control versus
concentration.
5.2.3. N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-
valine 40-demethyl-4-deoxypodophyllic ester (11c)
The yield: 75%; red powder; mp: 138–140 ꢀC; [
a
] ꢁ67 (c 0.1,
CHCl3); IR (cmꢁ1) 3329, 2932, 2850, 1771, 1721, 1507, 1484, 1463,
1338 (N–O.), 1227, 1130, 936; MS m/z 682 (M þ Hþ); ESR:
An ¼ 15.98 G, g0 ¼ 2.006; HRMS (ESI) 704.2916 for [M þ Na]þ (calcd
704.2924 for C36H45O11N2).
5.4. In tissues antioxidative activity
The extent of lipid peroxidation was assessed using a modifica-
tion of the thiobarbituric acid (TBA) assay [41]. Determination of
malondialdehyde (MDA) content was carried out in 5% and 1% of
liver and kidney homogenate of SD rats in 0.9% NaCl solution,
respectively. The tissue homogenate 1 mL was incubated with test
compounds or vehicle control (DMSO 2.5 mL) at 37 ꢀC for 10 min
incubation, MDA was determined by the TBA colormetric analysis
method. The absorbance at l532 was determined on a UV spectro-
photometer. IC50 values were determined from 50% of control
versus concentration.
5.2.4. N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-
leucine 40-demethyl-4-deoxypodophyllic ester (11d)
The yield: 63%; red powder; mp: 139–143 ꢀC; [
a
] ꢁ68 (c 0.1,
CHCl3); IR (cmꢁ1) 3335, 2934, 2873, 1773, 1722, 1507, 1484, 1463,
1338 (N–O), 1226, 1130, 935; MS m/z 695 (M þ 2Hþ); ESR:
An ¼ 16.04 G, g0 ¼ 2.0061; HRMS (ESI) 718.3072 for [M þ Na]þ
(calcd 718.3066 for C37H47O11N2).
5.2.5. N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-
Acknowledgement
isoleucine 40-demethyl-4-deoxypodophyllic ester (11e)
The yield: 42%; red powder; mp: 138–140 ꢀC; [
a
] ꢁ59 (c 0.1,
This work is financially supported by the State Key Laboratory of
Applied Organic Chemistry, Lanzhou University.
CHCl3); IR (cmꢁ1) 3335, 2934, 1771, 1721, 1507, 1484, 1462, 1338 (N–
O.), 1227, 1130, 936; MS m/z 695 (Mþ); ESR: An ¼ 16.10 G,
g0 ¼ 2.006; HRMS (ESI) 718.3072 for [M þ Na]þ (calcd 718.3075 for
C37H47O11N2).
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