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,⇑ aSchool of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
bNatural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA cChinese 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
Article history:
Received 19 September 2011 Revised 1 December 2011 Accepted 5 December 2011 Available online 9 December 2011 Keywords: Rotenone Spin-labeled Nitroxide Cytotoxicity
a b s t r a c t
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 IC50values ranging from 0.075 to
0.738lg/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 (IC500.075
and 0.092lg/mL, respectively). Based on the observed cytotoxicity, structure–activity relationships have been described.
Ó 2011 Elsevier Ltd. All rights reserved.
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.
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
lymphomas,13promyelocytic leukemias,14and 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.
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.20Moreover, the keto moiety of 1 was converted to an oxime (4) by treatment with hydroxylamine hydrochloride (NH2OHHCl) 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,19,22as well as the fact thatL-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 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.024
⇑Corresponding authors. Tel.: +1 919 962 0066; fax: +1 919 966 3893. E-mail addresses:[email protected],[email protected](K.-H. Lee).
Bioorganic & Medicinal Chemistry Letters 22 (2012) 920–923
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Bioorganic & Medicinal Chemistry Letters
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b m c lrotenone 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, 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.23Compound 1 and paclitaxel were used as reference compounds. The screening results are shown inTable 1.
Our preliminary investigation showed that 1-related derivatives are potential lead compounds for new antitumor agents. As
N OH O carbonyldiimidazole N O O N N O N O O N NH O N O O N3 O NaN3/water N O O N H COOH O R 7 6 8 9 10a-g TosO TsOH amino acids/MgO stir, 24 h 10a H 10e CH(CH3)CH2CH3 CH2Ph 10d CH2CH(CH3)2 10c CH(CH3)2 10g N H CH2 10b CH3 R 10f
Scheme 2. Synthesis of compounds 10a–g.
O O O O O O H H O O O OH O O H H NaBH4/MeOH DCC/DMAP O O O O O O H H R O R= N O N O N O N O a b c d 1 2 3a-d RCOOH O O O N O O H H OH
NH
2OH
pyridine
O O O N O O H H O O R DCC/DMAP 5a-d RCOOH 4Scheme 1. Synthesis of compounds 3a–d and 5a–d.
illustrated in Table 1, all new compounds exhibited significant in vitro cytotoxic activity against all tested tumor cell lines, with IC50values ranging from 0.075 to 0.738
l
g/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 0.075 and 0.092l
g/mL, respectively, compounds 3a and 3d showed the greatest cytotoxicity against KBvin, compared to 1 and paclitaxel (IC500.595 and 1.145l
g/mL, respectively). Esterifi-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 paper,19 we found that using different L-amino acid as linkers markedly affected the biological activity of cytotoxic agents. However, as seen inTable 1, the different substituents ata
-carbon of the amino acid in compounds 11a–g did not lead to obviously different effects on the inhibition of the four tumor cell lines invitro. 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.
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).
Table 1
In vitro cytotoxicity assay against four cancer cell lines
Entry IC50(lg/mL)
A-549 DU-145 KB KBvin
3a 0.408 ± 0.150 0.223 ± 0.186 0.273 ± 0.138 0.075 ± 0.015 3b 0.533 ± 0.091 0.277 ± 0.170 0.443 ± 0.093 0.241 ± 0.103 3c 0.578 ± 0.062 0.322 ± 0.158 0.427 ± 0.085 0.299 ± 0.045 3d 0.523 ± 0.109 0.266 ± 0.180 0.408 ± 0.142 0.092 ± 0.063 5a 0.738 ± 0.202 0.426 ± 0.030 0.633 ± 0.146 0.620 ± 0.400 5b 0.471 ± 0.038 0.322 ± 0.053 0.560 ± 0.093 0.298 ± 0.045 5c 0.542 ± 0.109 0.358 ± 0.037 0.575 ± 0.066 0.162 ± 0.118 5d 0.651 ± 0.051 0.488 ± 0.122 0.523 ± 0.144 0.358 ± 0.044 11a 0.624 ± 0.111 0.450 ± 0.084 0.510 ± 0.070 0.410 ± 0.054 11b 0.650 ± 0.068 0.417 ± 0.086 0.442 ± 0.030 0.357 ± 0.045 11c 0.615 ± 0.133 0.451 ± 0.059 0.497 ± 0.144 0.381 ± 0.023 11d 0.521 ± 0.238 0.443 ± 0.077 0.483 ± 0.124 0.355 ± 0.024 11e 0.649 ± 0.088 0.396 ± 0.083 0.516 ± 0.092 0.399 ± 0.054 11f 0.713 ± 0.057 0.462 ± 0.082 0.509 ± 0.047 0.383 ± 0.053 11g 0.631 ± 0.021 0.477 ± 0.101 0.565 ± 0.069 0.414 ± 0.046 1 0.901 ± 0.192 0.465 ± 0.082 0.513 ± 0.087 0.595 ± 0.177 Paclitaxel 0.0052 ± 0.0017 0.0037 ± 0.0011 0.0048 ± 0.0009 1.145 ± 0.236 11a H 11e CH(CH3)CH2CH3 CH2Ph 11d CH2CH(CH3)2 11c CH(CH3)2 11g N H CH2 11b CH3 R 11f O O O OH O O H H DCC/DMAP 2 11a-g O O O O O O H H O H N O O N O R 10a-g
Scheme 3. Synthesis of compounds 11a–g. 922 Y.-Q. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 920–923
Supplementary data
Supplementary data associated with this article can be found, in the online version, atdoi:10.1016/j.bmcl.2011.12.024.
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