Design, synthesis and cytotoxic activity of novel spin-labeled rotenone derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.12.024

Three series of novel spin-labeled rotenone derivatives were synthesized and evaluated for cytotoxicity against four tumor cell lines, A-549, DU-145, KB and KBvin. All of the derivatives showed promising in vitro cytotoxic activity against the tumor cell lines tested, with IC₅₀ values ranging from 0.075 to 0.738 μg/mL. Remarkably, all of the compounds were more potent than paclitaxel against KBvin in vitro, and compounds 3a and 3d displayed the highest cytotoxicity against this cell line (IC₅₀ 0.075 and 0.092 μg/mL, respectively). Based on the observed cytotoxicity, structure-activity relationships have been described.

Full text of DOI:10.1016/j.bmcl.2011.12.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 920–923
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and cytotoxic activity of novel spin-labeled
rotenone derivatives
Ying-Qian Liu a,c, , Emika Ohkoshi b, Lin-Hai Li a, Liu Yang d, Kuo-Hsiung Lee b,c,
⇑
⇑
a School of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
b Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
d Environmental and Municipal Engineering School, Lanzhou Jiaotong University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of novel spin-labeled rotenone derivatives were synthesized and evaluated for cytotoxicity
against four tumor cell lines, A-549, DU-145, KB and KBvin. All of the derivatives showed promising
in vitro cytotoxic activity against the tumor cell lines tested, with IC50 values ranging from 0.075 to
Received 19 September 2011
Revised 1 December 2011
Accepted 5 December 2011
Available online 9 December 2011
0.738
in vitro, and compounds 3a and 3d displayed the highest cytotoxicity against this cell line (IC50 0.075
and 0.092 g/mL, respectively). Based on the observed cytotoxicity, structure–activity relationships have
been described.
lg/mL. Remarkably, all of the compounds were more potent than paclitaxel against KBvin
l
Keywords:
Rotenone
Spin-labeled
Nitroxide
Ó 2011 Elsevier Ltd. All rights reserved.
Cytotoxicity
Cancer is currently the second most important disease leading
to death in both developing and developed countries. However,
the rising resistance to available chemotherapeutic agents, com-
bined with their adverse side effects and high cost, is driving the
search for new alternative anticancer compounds from natural
products. Investigation of natural sources not only enhances diver-
sity in the search for new prototype antitumor agents, but also may
lead to commercially available marketed products as underscored
by the prominent examples of etoposide and vinblastine.1–5 Natu-
ral products and their closely related analogs are thus an important
resource for new antitumor agents and are also regularly used as
the templates for further sequential chemical modifications and
structure optimization.
lymphomas,13 promyelocytic leukemias,14 and neuroblastomas.15
In addition, rotenone can inhibit microtubule assembly and arrest
cells in mitosis by binding directly to tubulin,16 and thus, it is a
possible scaffold for the design of potent microtubule assembly
inhibitors. These findings have made rotenone a very attractive
candidate for the clinical treatment of various forms of cancer.
However, so far, not much attention has been paid to rotenone as
a starting material for further transformations.
As part of an ongoing effort to identify potential antitumor mol-
ecules derived from natural products, we have successfully pre-
pared spin-labeled antitumor drugs.3,17–19 Herein, we report the
design, synthesis, and preliminary in vitro cytotoxicity testing of
three series of novel spin-labeled rotenones.
Rotenone is a naturally occurring flavonoid derived from the
roots of cubé (Lonchocarpus utilis and urucu) or derris (Derris
elliptica) or from other Leguminosa species. It has been used for at
least 150 years as a botanical insecticide to control crop pests.6,7
Its pesticidal activity is attributed to irreversible binding and
inactivation of complexes in the mitochondrial electron transport
chain. This action can block electron transfer from the complex
to ubiquinone, thus, blocking the oxidative phosphorylation
process, as well as increasing reactive oxygen species (ROS).8,9
Other studies have found that rotenone displays anticancer activity
by inducing apoptosis10–12 in cells derived from human B-cell
The synthetic chemical routes to compounds 3a–d and 5a–d
are depicted in Scheme 1. Commercially available rotenone (1)
was first reduced with NaBH4 to yield the intermediate rotenol
(2) in 85% yield.20 Moreover, the keto moiety of 1 was converted
to an oxime (4) by treatment with hydroxylamine hydrochloride
(NH2OHÁHCl) in pyridine in 70% yield.21 The intermediates 2 and
4 were then condensed with the appropriate piperidine (pyrroline)
nitroxyl acids in the presence of N,N-dicyclohexylcarbodiimide
(DCC) and 4-dimethylaminopyridine (DMAP) to provide target
compounds 3a–d and 5a–d, respectively.
Based on previous work,19,22 as well as the fact that
L-amino
acids are actively transplanted into mammalian tissues, have good
water solubility, and are often used as carrier vehicles for some
drugs, we also used an amino acid spacer as a linkage between
⇑
Corresponding authors. Tel.: +1 919 962 0066; fax: +1 919 966 3893.
E-mail addresses: khlee@email.unc.edu, khlee@unc.edu (K.-H. Lee).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.024

Y.-Q. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 920–923
921
O
O
O
O
O
R
O
O
O
O
O
OH
H
O
O
O
H
H
H
H
RCOOH
NaBH4/MeOH
O
DCC/DMAP
O
O
O
O
H
2
3a-d
1
NH2OH
pyridine
O
R
N
O
O
O
O
O
O
H
OH
O
N
O
RCOOH
DCC/DMAP
H
H
O
O
H
O
5a-d
4
R=
N
O
N
O
a
N
O
N
O
b
d
c
Scheme 1. Synthesis of compounds 3a–d and 5a–d.
rotenone core and the nitroxyl radical moiety. The starting materials
[N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl-oxycarbonyl) amino
acids 10a–g for the preparation of the target compounds 11a–g
were synthesized according to our previous procedure as shown
in Scheme 2.19 Briefly, 4-hydroxy-2,2,6,6-tetramethylpiperidine-
1-oxyl (6) was prepared by catalytic oxidation of 4-hydroxy-
2,2,6,6-tetramethylpiperidine with sodium tungstate–hydrogen
peroxide–EDTA in yield 85%. Subsequently, the reaction of 6 with
N,N-carbonyldiimidazole gave N-(1-oxyl-2,2,6,6-tetramethyl pipe-
ridinyloxycarbonyl)-imidazole (7). Without further purification,
compound 7 was reacted with p-toluenesulfonic acid monohydrate
to give its more reactive tosylate (8). Compound 8 was instanta-
neously converted into the corresponding alkoxycarbonyl azide
(9) when dissolved in an aqueous solution of sodium azide. Com-
pounds 10a–g were obtained in good yield by reaction of 9 with
various amino acids in presence of MgO (Scheme 2). The 12-hydro-
xyl of intermediate 2 was then condensed with 10a–g to afford
compounds 11a–g by a similar carbodiimide procedure as used in
the preparation of 3a–d (Scheme 3).
All synthesized target compounds 3a–d, 5a–d and 11a–g were
purified by column chromatography, and their structures were
confirmed unambiguously from mp, IR, ESR and HRMS analyses.
Target compounds 3a–d, 5a–d and 11a–g were evaluated for
in vitro cytotoxic activity against four different tumor cell lines, KB
(nasopharyngeal), A-549 (lung), DU-145 (prostate), and KBvin (an
MDR KB subline), using a sulforhodamine B colorimetric assay with
triplicate experiments.23 Compound 1 and paclitaxel were used as
reference compounds. The screening results are shown in Table 1.
Our preliminary investigation showed that 1-related derivatives
are potential lead compounds for new antitumor agents. As
O
O
O
O
N
O
N
OH
O
N3
NH
TosO
N
carbonyldiimidazole
NaN3/water
TsOH
N
O
N
O
7
N
O
6
N
O
9
8
O
R
R
H
O
N
H
COOH
CH2
10d CH2CH(CH3)2
10a
10g
amino acids/MgO
stir, 24 h
N
H
10e CH(CH3)CH2CH3
N
O
10b CH3
10c CH(CH3)2
CH2Ph
10f
10a-g
Scheme 2. Synthesis of compounds 10a–g.

922
Y.-Q. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 920–923
H
R
O
N
O
O
O
O
O
O
O
N
O
O
O
OH
O
H
H
10a-g
H
H
R
11a H
11d CH2CH(CH3)2
DCC/DMAP
O
O
O
11e
11b CH3
CH(CH3)CH2CH3
CH2Ph
11f
11c CH(CH3)2
CH2
11a-g
2
11g
N
H
Scheme 3. Synthesis of compounds 11a–g.
Table 1
In vitro cytotoxicity assay against four cancer cell lines
Entry
IC50 (lg/mL)
A-549
DU-145
KB
KBvin
3a
3b
3c
3d
5a
5b
5c
5d
11a
11b
11c
11d
11e
11f
11g
1
0.408 0.150
0.533 0.091
0.578 0.062
0.523 0.109
0.738 0.202
0.471 0.038
0.542 0.109
0.651 0.051
0.624 0.111
0.650 0.068
0.615 0.133
0.521 0.238
0.649 0.088
0.713 0.057
0.631 0.021
0.901 0.192
0.0052 0.0017
0.223 0.186
0.277 0.170
0.322 0.158
0.266 0.180
0.426 0.030
0.322 0.053
0.358 0.037
0.488 0.122
0.450 0.084
0.417 0.086
0.451 0.059
0.443 0.077
0.396 0.083
0.462 0.082
0.477 0.101
0.465 0.082
0.0037 0.0011
0.273 0.138
0.443 0.093
0.427 0.085
0.408 0.142
0.633 0.146
0.560 0.093
0.575 0.066
0.523 0.144
0.510 0.070
0.442 0.030
0.497 0.144
0.483 0.124
0.516 0.092
0.509 0.047
0.565 0.069
0.513 0.087
0.0048 0.0009
0.075 0.015
0.241 0.103
0.299 0.045
0.092 0.063
0.620 0.400
0.298 0.045
0.162 0.118
0.358 0.044
0.410 0.054
0.357 0.045
0.381 0.023
0.355 0.024
0.399 0.054
0.383 0.053
0.414 0.046
0.595 0.177
1.145 0.236
Paclitaxel
illustrated in Table 1, all new compounds exhibited significant
in vitro cytotoxic activity against all tested tumor cell lines, with
IC50 values ranging from 0.075 to 0.738 lg/mL. Most of the deriv-
atives displayed increased cytotoxic activity compared with the
parent compound 1. Remarkably, all of the compounds also were
more potent than paclitaxel against KBvin. With IC50 values of
vitro. As a whole, the introduction of nitroxides into the rotenone
molecule potentiated the antitumor activity. Thus, the design and
synthesis of these compounds provides valuable information to
potentially increase the therapeutic value of rotenone. Synergistic
action might also be found against tumor cell lines.
In summary, novel spin-labeling of the rotenone class is a prom-
ising direction in antitumor chemotherapy, not only because these
compounds exhibit superior cytotoxic activity, but also because
they can be monitored by ESR in pharmacological experiments. In
this paper, three series of novel spin-labeled derivatives of rotenone
were first synthesized successfully, and their antitumor activity
was evaluated against for four tumor cell lines using an SRB-assay.
The cytotoxic results showed that most of the new spin-labeled
compounds exhibited more potent cytotoxicity against A-549,
DU-145, KB and KBvin compared to rotenone. Compounds 3a and
3d were the most promising derivatives and were selected as lead
molecules for further development. More systematic structural
modifications will carried out to further clarify these initial inter-
esting findings.
0.075 and 0.092
showed the greatest cytotoxicity against KBvin, compared to 1
and paclitaxel (IC50 0.595 and 1.145 g/mL, respectively). Esterifi-
lg/mL, respectively, compounds 3a and 3d
l
cation of the C-12-hydroxyl of rotenol with the nitroxides of differ-
ent ring classes (piperidine, pyrroline) groups (3a–d) led to
significantly improved cytotoxic activity in comparison to 1.
Conversion of 1 into the oxime esters 5a–d also resulted in active
derivatives, but 5a–d were generally less potent compared to
esters 3a–d. This finding indicated that the cytotoxic profile of
1-derivatives may be sensitive to the size and electronic density
of the substituents at C-12. Furthermore, the cytotoxicity of these
compounds (3a–d, 5a–d) was distinctly correlated with the nitrox-
ide. Also, the ring size and degree of unsaturation did not affect the
bioactivity of the target compounds against three (KB, A-549 and
DU-145), of the four tested tumor cell lines, which was consistent
with the literature.24 Interestingly, compounds 11a–g, with an
amino acid linker moiety at the C-12 position, showed slightly de-
creased activity in all the tested cell lines compared to compounds
3a–d, suggesting a size limitation at position C-12. In our previous
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (30800720); the Post-Doctor Research
Foundation (20090450142); the Fundamental Research Funds for
the Central Universities (lzujbky-2009-98); Key Scientific Research
Programme of EPPI (2010hzsZDZX003) of Chinese Academy of
Tropical Agricultural Science from the Ministry of Agriculture. This
study was also supported in part by the Cancer Research Center of
Excellence, Taiwan (DOH-100-TD-C-111-005).
paper,19 we found that using different
markedly affected the biological activity of cytotoxic agents.
However, as seen in Table 1, the different substituents at -carbon
L-amino acid as linkers
a
of the amino acid in compounds 11a–g did not lead to obviously
different effects on the inhibition of the four tumor cell lines in

Y.-Q. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 920–923
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