Novel semisynthetic spin-labeled derivatives of podophyllotoxin with cytotoxic and antioxidative activity

Article abstract of DOI:10.1016/j.bmcl.2009.12.048

A series of novel spin-labeled podophyllotoxin derivatives were synthesized by reacting the corresponding N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxy carbonyl)-amino acids with 4β-amino-4′-demethylepipodophyllotoxin. The synthesized derivatives 12a-g were evaluated for the partition coefficients, cytotoxicities in vitro against three tumor cell lines (A-549, HL-60, and RPMI-8226) and antioxidative activities in tissues of SD rats by the TBA method. The vast majority of target compounds have shown superior or comparable activities against A-549, HL-60, and RPMI-8226 compared to VP-16, and they have shown more significant antioxidative activities and superior water solubility than VP-16.

Full text of DOI:10.1016/j.bmcl.2009.12.048

Bioorganic & Medicinal Chemistry Letters 20 (2010) 983–986
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel semisynthetic spin-labeled derivatives of podophyllotoxin
with cytotoxic and antioxidative activity
a,
Jia-Qiang Zhang a, Zhi-Wei Zhang a, Ling Hui c, Shi-Wu Chen a,b, , 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:
A series of novel spin-labeled podophyllotoxin derivatives were synthesized by reacting the corresponding
N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxy carbonyl)-amino acids with 4b-amino-40-demethylepip-
odophyllotoxin. The synthesized derivatives 12a–g were evaluated for the partition coefficients, cytotoxic-
ities in vitro against three tumor cell lines (A-549, HL-60, and RPMI-8226) and antioxidative activities in
tissues of SD rats by the TBA method. The vast majority of target compounds have shown superior or com-
parable activities against A-549, HL-60, and RPMI-8226 compared to VP-16, and they have shown more
significant antioxidative activities and superior water solubility than VP-16.
Received 27 August 2009
Revised 26 November 2009
Accepted 14 December 2009
Available online 22 December 2009
Keywords:
Podophyllotoxin
Nitroxides
Ó 2009 Elsevier Ltd. All rights reserved.
Antitumor
Antioxidants
Podophyllotoxin (1), as well as its congeners and derivatives ex-
hibit pronounced biological activity mainly as antineoplastic drugs
and as strong antiviral agents.1 The podophyllotoxin derivatives,
such as etoposide (VP-16, 2), teniposide (VM-26, 3), and etopophos
(etoposide phosphate, 4) have been used as DNA topoisomerase II
inhibitors in chemotherapy for various types of cancer.2,3 Recently,
more complex and diverse analogues have been synthesized either
to get more potent compounds or to overcome drug resistance.4 As
the results of previous structure–activity relationship studies, it
was also found that the trans-lactone, 4b-N-substituted and 40-
demethyl moieties of podophyllotoxin were essential to maintain
the antineoplastic activity as topoisomerase II inhibitors.5 In addi-
tion, podophyllic aldehyde and its analogues were also found to be
a highly selectivity against the HT-29 colon carcinoma. Additional
biological studies indicate that these derivatives induce microtu-
bule depolymerization, arrest cells at the G2/M phase of cell cycle,
and are able to induce a delayed apoptosis after 48 h of treatment,
characterized by caspase-3 activation.6 In our previous studies, we
have synthesized lots of spin-labeled podophyllotoxin derivatives
and found that introduction of a stable nitroxyl radical into the
molecular of podophyllotoxin or its analogues could increase anti-
tumor activity and marked decrease in toxicity compared with par-
ent compounds.7–10 Among them, GP-11 (Fig. 1, 5) was reported as
a low immunosuppressive antitumor agent, which increases the
mitotic index and results in G2/M, and to a lesser extent, S arrest.11
Furthermore, -amino acids are actively transported into mamma-
lian tissue, have good water solubility, and are often used as carrier
vehicles for some drugs.
L
Basedupontheaboveresults, asanimportantpartofourprogram
aimed at the discovery and development of bioactive molecules de-
rived from natural product podophyllotoxin,7–16 in this Letter, a ser-
ies of novel spin-labeled podophyllotoxin derivatives (12a–g) were
designed, synthesized, andpreliminarilyevaluatedfor theircytotox-
icities in vitro against three tumor cells (A-549, HL-60, and RPMI-
8226) and antioxidative activity in tissues of SD rats. Also, the octa-
nol–water partition coefficients (log P) were measured.
The synthesis of nitroxide free radical 10a–g is outlined in
Scheme 1. Briefly, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl
(6) was prepared by catalytic oxidation of 4-hydroxy-2,2,6,6-tetra-
methylpiperidine with sodium tungstate–hydrogen peroxide–
EDTA in yield 85%.17 Following, the reaction of 6 with N,N0-carbon-
yldiimidazole proceeds to give N-(1-oxyl-2,2,6,6-tetramethyl pipe-
ridinyloxycarbonyl)-imidazole (7) by the modified method.18
Compound 7, without further purification, was further reacted
with p-toluenesulfonic acid monohydrate to give its higher reac-
tive tosylate (8). Compound 8 is instantaneously converted into
alkoxycarbonyl azide (9) when dissolved in an aqueous solution
of sodium azide. Compounds 10a–g were obtained in good yield
by reaction of 9 with free amino acids in presence of MgO.19
The synthetic route to the target compounds from 1 was
depicted in Scheme 2. The intermediate 4b-NH2-40-demethylepip-
* Corresponding authors. Tel./fax: +86 931 8915686.
E-mail addresses: chenshwu@gmail.com (S.-W. Chen), xuant@lzu.edu.cn (X.
odophyllotoxin (11) was prepared stereo-selectively from
1
Tian).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.048

984
J.-Q. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 983–986
R'
O
O
O
HO
R'
R''
H
O
HO
Me
Etoposide
Teniposide
Etopophos
2
3
O
O
O
H
S
O
O
OH
OH
Me
P
4
MeO
OMe
OR''
11
OH
4
OH
5
3
2
O
O
O
OH
O
O
D
A
O
B
8
C
H
N
N
N
N
H
1
H
O
O
O
2'
6'
E
MeO
OMe
MeO
OMe
4'
OMe
OMe
Podophyllotoxin
1
GP-11 5
Figure 1. Structures of podophyllotoxin (1) and related compounds.
O
O
O
R
O
TsO
NH
N
O
N
OH
O
N
O
N
COOH
R
H
OH
O
N3
H
i
ii
iii
v
iv
10a
10b Me
N
O
N
O
N
O
N
O
N
H
N
O
9
10c CHMe2
10d CH2CHMe2
10e CH(OH)Me
10f CH2CH2SMe2
10g CH2Ph
6
7
8
Scheme 1. Reagents: (i) Na2WO4/H2O2/EDTA; (ii) N,N0-carbonyl-dimidazole/THF; (iii) TsOHꢀH2O; (iv) NaN3/H2O; (v) amino acids/MgO.
O
H
N
O
N O
NH2
HN
O
O
R
O
Ref.14
O
O
O
10a-g
EDCI/HOBT
1
O
R
H
a
b
c
d
e
f
O
Me
O
CHMe2
CH2CHMe2
CH(OH)Me
CH2CH2SMe
CH2Ph
MeO
OMe
MeO
OMe
OH
OH
g
11
12a-g
Scheme 2. Synthesis of compounds 12a–g.
through 40-demethylation, 4-iodination, azidation, and catalytic
hydrogenation according to the previously reported method.14
Compound 11 was then condensed with the appropriate nitroxide
free radical 10a–g in the presence of 1-ethyl-3-(3-dimethyllamino-
propyl) carbodiimide hydrochloride (EDCꢀHCl) and 1-hydroxyben-
zotriazole hydrate (HOBT) to provide the target compounds 12a–g
in moderate yield.20
For the new compounds 12a–g, ESR spectra were recorded to
prove the presence of the nitroxide group in the target molecules.
The EPR spectra were recorded from freshly prepared 0.3 g/mL
chloroform solutions, using a Bruker ER200D-SRC spectrometer
at room temperature. The ESR spectra of 12a–g showed triplet with
hyperfine coupling constants (aN) in the range of 15.90–16.28 G
due to interaction between an unpaired electron and nitrogen

J.-Q. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 983–986
985
Table 1
Physical and spectroscopy characters of compounds 12a–g
½ ꢁ
a 2D3
a
Compds
Molecular formula
Mp (°C)
Yield (%)
HRMS (ESI)
IR
ESRb
aN (G)
16.04
Calcd
Found
g
DH (G)
12a
12b
12c
12d
12e
12f
C33H40O11N3
156–158
144–146
118–120
152–154
148–150
138–140
142–144
52
47
40
35
37
45
49
ꢂ80
ꢂ59
ꢂ78
ꢂ57
ꢂ78
ꢂ56
ꢂ39
677.2543
([M+Na]+)
669.2892
([M+H]+)
698.3283
(M+2H]+)
733.3181
([M+Na]+)
700.3076
([M+2H]+)
746.3191
([M+NH4]+)
745.3205
([M+H]+)
677.2555
([M+Na]+)
669.2896
([M+H]+)
698.3285
([M+2H]+)
733.3175
([M+Na]+)
700.3082
([M+2H]+)
746.3200
([M+NH4]+)
745.3199
([M+H]+)
1335(N–OÅ)
1718(NHCO)
1312(N–OÅ)
1714(NHCO)
1331(N–OÅ)
1664(NHCO)
1330(N–OÅ)
1717(NHCO)
1333(N–OÅ)
1713(NHCO)
1330(N–OÅ)
1717(NHCO)
1330(N–OÅ)
1717(NHCO)
2.0061
3.02
2.79
3.19
2.80
2.82
2.73
2.72
C
C
C
C
C
C
34H42O11N3
36H46O11N3
37H48O11N3
35H44O12N3
36H46O11N3S
40H46O11N3
2.0060
2.0060
2.0061
2.0060
2.0060
2.0061
15.90
16.12
16.12
16.01
16.02
16.28
12g
a
Concentration is 0.3 g/100 mL in CHCl3.
ESR determined in 10ꢂ3 M EtOH solution.
b
Table 2
Biological evaluation of compounds 12a–g (IC50, lM)
Compounds
A-549a
Cytotoxic activity
HL-60a
Antioxidative activity
Kidney
Log P
RPMI-8226a
Liver
Heart
12a
12b
12c
12d
12e
12f
12g
2
0.21
0.12
0.15
0.19
0.21
0.42
0.21
0.29
0.32
0.22
0.24
0.16
0.21
0.67
0.21
0.42
0.33
0.26
7.47
7.64
8.69
8.85
7.12
6.85
6.55
43.89
8.9
7.68
8.88
8.47
7.45
7.55
9.63
23.77
7.19
6.39
8.04
ND
8.79
5.89
7.39
40.67
0.11
0.12
0.15
0.21
0.10
0.161
0.183
0.68
0.061
0.089
0.078
0.48
0.090
0.14
ND = not determined.
a
Drugs exposure was for 48 h.
nucleus of the aminoxyl radical (g value = 2.006). Further charac-
terization of the final spin-labeled compounds was achieved by
infrared spectroscopy, and high revolution mass spectrometry
(ESI). Optical rotations and melting points were also determined
(Table 1).
Compounds 12a–g reported here were evaluated in vitro for
their cytotoxicities21 against the following tumor cell lines: A-
549 (human lung cancer), HL-60 (human premyelocytic leukemia),
and RPMI-8226 (human multiple myeloma). Their antioxidative
activities of malondialdehyde (MDA) (liver, heart, and kidney
homogenate of SD rats)22 were also tested. The results obtained
are summarized in Table 2. The vast majority of 12a–g showed
comparable or superior inhibition of A-549, HL-60, and RPMI-
8226 than VP-16. Remarkably, compounds 12c–e and 12g exhib-
ited significant inhibitory activities against RPMI-8226 with the
our initial design purpose, introduction of amino acid as linkers to
podophyllotoxin may increase the aqueous solubility of target
compounds and penetrate tumor cell membrane easily.7,13
In summary, novel spin-labeling of podophyllotoxin is a prom-
ising direction in antitumor chemotherapy, not only because they
exhibit superior activities, but also because they can be monitored
by ESR in pharmacological experiments. In this work, seven novel
spin-labeling amino acid-linked derivatives of podophyllotoxin
were designed and synthesized. Compared to VP-16, the vast
majority compounds exhibited comparable cytotoxic activity
against A-549, HL-60, and RPMI-8226, and more pronounced anti-
oxidative activities in tissues of SD rats and superior water
solubility.
Acknowledgment
IC50 value in the range of 0.06–0.09 lM. Furthermore, all desired
compounds showed 3–6-fold more potent antioxidative activities
in tissues of liver, heart, and kidney homogenate of SD rats than
We thank State Key Laboratory of Applied Organic Chemistry of
Lanzhou University for financial support.
that of VP-16, with the IC50 values in the range of 5.89–9.63
lM.
In previous Letter, we found that different -amino acid as linker
L
Supplementary data
markedly affected the activity of podophyllotoxin derivatives, the
hydroxyl groups in the amino acid side chains may decrease the
antineoplastic activity.13 However, as seen in Table 2, different al-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.12.048.
kyl or aryl substituted at
a-carbon of L-amino acid did not show
obviously different effects on tumor cell and antioxidative activity
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