Atovaquone derivatives as potent cytotoxic and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bmcl.2009.07.044

2-Piperazinyl naphthoquinones (2) and 2-piperidinyl naphthoquinones (3) were designed and synthesized as new cytotoxic and apoptosis inducing agents by utilizing the anti-parasite drug atovaquone as lead compound. Several compounds displayed significantly

Full text of DOI:10.1016/j.bmcl.2009.07.044

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5091–5094
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Atovaquone derivatives as potent cytotoxic and apoptosis inducing agents
Jing Zhou a,b, , Lei Duan a,b, , Huaming Chen c, Xiaomei Ren a,b, Zhang Zhang a,b, Fengtao Zhou a,b,d
,
Jinsong Liu b, Duanqing Pei a, Ke Ding a,b,
*
a Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, International Business Incubator A-3,
Guangzhou Science Park, Guangzhou 510663, China
b Institute of Chemical Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, International Business Incubator A-3,
Guangzhou Science Park, Guangzhou 510663, China
c China Pharmaceutical University, # 24 Tongjiaxiang, Nanjing 210009, PR China
d Graduate School of the Chinese Academy of Sciences, 19 Yuquan Rd, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Piperazinyl naphthoquinones (2) and 2-piperidinyl naphthoquinones (3) were designed and synthe-
sized as new cytotoxic and apoptosis inducing agents by utilizing the anti-parasite drug atovaquone as
lead compound. Several compounds displayed significantly improved cytotoxic activities against a panel
of cancer cell lines than that of atovaquone. These compounds also induced apoptosis through activating
pro-apoptotic caspases 9 and 3.
Received 28 April 2009
Revised 30 June 2009
Accepted 2 July 2009
Available online 10 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Atovaquone
Apoptosis
Cytotoxic
Given the extraordinarily high costs and poor successful rates of
drug development, repositioning (or repurposing) of existed drugs,
in which to find new indications of old drugs, has become an
attractive approach to accelerate drug development process.1 As
part of our chemical biology program, we aimed to identify small
molecules with potent anti-tumor activity. By using cell growth
inhibitory assay, a number of FDA approved anti-infectious drugs
and antibiotics were found to display promising anticancer activity
in vitro, among which atovaquone had moderate anti-proliferation
potential sites for structural optimization. Based on these consider-
ations, 2-piperazinyl naphthoquinones (2) and 2-piperidinyl naph-
thoquinones (3) were designed as new atovaquone derivatives. Of
note, the 2-hydroxyl group in atovaquone was erased for the syn-
thetic feasibility and chemical stability of the designed derivatives
(Fig. 1).
The designed compounds were readily prepared by a condensa-
tion of naphthoquinones 4 with different arylpiperazines or arylpi-
peridines (Scheme 1). For the coupling reaction of 5-hydroxyl
naphthoquinone 6 with arylpiperazine or arylpiperidine 5, both
5-hydroxyl and 8-hydroxyl substituted products were obtained.
The major products were identified as 8-hydroxyl analogues by
using an X-ray crystallographic analysis of 8-hydroxy-2-(4-phen-
ylpiperazin-1-yl)naphthalene-1,4-dione (2q)5 (Fig. 2).
activity against different cancer cells with IC50 values of 15–30 lM.
Herein, we report the structural optimization and in vitro biologi-
cal evaluation of atovaquone derivatives as new cytotoxic and
apoptosis inducing agents.
Atovaquone is an anti-parasite drug selectively targeting the
mitochondrial respiratory chain of malaria parasite.2 This drug
has poor water solubility and oral bioavailability because of its
structurally hydrophobic nature. The chemical structure of ato-
vaquone comprised a naphthoquinonyl head, a cyclohexanyl linker
and a hydrophobic 4-chlorophenyl tail (Fig. 1). Many compounds
consisted of naphthoquinonyl moiety have been reported to be
cytotoxic,3,4 which indicated that the naphthoquinonyl head might
be the crucial part for atovaquone to display anti-tumor activity.
The cyclohexanyl linker and/or the hydrophobic tail would provide
Cl
4'
R1
R2
8
O
1
2
X
O
O
hydrophobic tail
N
7
6
3
OH
5
4
O
R3
2. X = N
3. X = CH
1.
Atovaquone
* Corresponding author. Fax: +86 20 32290485.
E-mail address: ding_ke@gibh.ac.cn (K. Ding).
These authors contributed equally.
Figure 1. Chemical structures of atovaquone and designed derivatives.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.044

5092
J. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5091–5094
R1
R1
R2
O
O
R2
R3
O
O
X
N
EtOH, K2CO3, reflux
X
+
R3
N
H
4
5
2 or 3
OH
O
O
OH
O
O
N
O
O
N
N
N
EtOH, K2CO3, reflux
N
+
N
H
OH
6
7
2q
2r
Scheme 1. Synthesis of 2-piperazinyl naphthoquinones (2) and 2-piperidinyl naphthoquinones (3).
Figure 2. X-ray structure of compound 2q.
The anti-tumor activity of the designed compounds was prelim-
inarily screened by cell growth inhibitory assay (MTT assay)
against Du145 human prostate carcinoma cells. From Table 1, it
was clear that almost all the compounds designed display good
or moderate anti-proliferation activity against Du145 cells. 2-(4-
Table 1
Anti-proliferation activities of compounds 2 and 3 against prostate Du145 cells
Compds
R1
R2
R3
X
Cell growth
inhibitory
activitiesa
(40-chlorophenyl)piperazin-1-yl)naphthalene-1,4-dione (2a),
a
IC50 (lM)
structurally close derivative of atovaquone, display almost identi-
cal anti-tumor activity with that of atovaquone. When the 40-Cl
group was removed (2f) or replace with F, methyl groups (2b,
2e), the activity was improved 3–4 folds. However, the Br or meth-
oxyl group substituted analogues (2c, 2d) were much less active.
Further structure–activity relationship analysis indicated that
o-position of the phenyl tail in compound 2 might be the optimal
position for substitution. For examples, compound 2a had an IC50
Atovaquone
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
2u
2v
2w
2x
29.3 1.2
29.5 2.7
11.7 1.8
>50
p-Cl
p-F
p-Br
p-MeO
p-Me
H
m-Cl
o-Cl
m-Me
o-Me
m-OMe
o-OMe
3,5-di Methoxyl
3,4-di Methoxyl
3,4,5-tri Methoxyl
4-F-3-Cl
H
H
4-F
4-F
o-Cl
o-OMe
o-Me
H
H
H
p-Cl
p-Cl
o-Me
o-Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
OH
H
OH
OH
OH
OH
H
OH
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
OH
H
H
H
OH
H
H
H
H
H
H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
46.1 2.0
8.2 1.6
9.9 1.3
8.3 1.7
2.9 0.8
7.4 1.0
5.7 0.8
15.0 0.7
8.0 1.9
14.9 2.6
17.8 2.4
11.6 3.2
32.2 4.9
2.8 0.3
15.2 1.2
8.4 1.3
22.4 3.5
2.3 0.2
1.8 0.2
2.3 0.3
1.3 0.3
6.0 1.1
3.0 0.3
7.3 1.6
2.6 0.4
6.1 0.9
2.5 0.3
value of 29.5
lM, while the IC50 value for m-Cl (2g) and o-Cl (2h)
analogues were 8.3 and 2.9
lM respectively. Compounds with
multiple substitutions (2m, 2n, 2o, 2p) were less potent.
5-Hydoxyl and 8-dydroxyl derivatives were also designed on
the basis of 2f. The results revealed that 8-dydroxyl compound
2q is 3.5-fold more potent than 2f, while 5-hydoxyl analogue 2r
was less active. Other 8-dydroxyl analogues 2s, 2u, 2v and 2w also
displayed promising anti-tumor activity. The 5, 8-didydroxyl com-
pound 2x had an IC50 value of 1.3 lM.
The 2-piperidinyl naphthoquinones 3a–3f also displayed good
cell anti-tumor activity. Similar to that of 2-piperazinyl analogues
(2), the introduction of 8-hydroxyl group obviously improved the
anti-tumor activity (3b, 3d and 3f).
The anti-tumor activity of compounds 2q, 2u, 2v, 2w, 2x, 3b, 3d
and 3f was further evaluated on various human cancer cell lines by
using doxorubicin (adriamycin) as a positive control. As shown in
Table 2, almost all the compounds displayed broad-spectrum
3a
3b
3c
3d
3e
3f
anti-proliferation activity against solid tumor cells with low
lM
OH
IC50 values. Although doxorubicin is highly cytotoxic to T-cell leu-
kemia Molt-4 cells in our cell growth assay, most of the deigned
a
Values are means of three experiments.

J. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5091–5094
5093
Table 2
Anti-proliferation activities of compounds 2q, 2u, 2v, 2w, 2x, 3b, 3d and 3f against 13 human cancer cell lines
a
Compds
IC50
(
lM)
IMR-90 A549
Hela
1.0 (0.77)
6.9
11.3
7.2
6.5
7.2
2.1
8.6
HepG2
HT-1080
0.1 (0.29)
8.2
15.4
5.8
5.1
11.0
2.8
HT-29
KB
L-78 MCF-7
MOLT-4
OS-RC-2 SGC-7901 U251c
Doxorubicinb 1.0
1.9 (7.97)
4.2
0.3 (1.28)
5.4
11.1
7.7
3.9
10.3
5.4
9.7
5.4
16.6
1.3 (1.110
2.9
18.9
1.4
2.8
2.8
2.6
5.4
6.9
6.7
)
0.3 (0.211
7.0
3.7
4.4
2.8
6.6
1.4
2.2
14.0
4.4
)
2.4
3.6
0.7 (0.710
5.2
)
0.002 (0.0612
>50
>50
27.6
25.8
>50
>50
2.7
>50
>50
)
5.4
1.5
8.9
2.5
1.9
4.4
1.5
0.7
5.2
3.2
0.9 (3.713
3.7
41.8
8.2
6.8
5.3
)
0.6
6.1
30.8
8.0
4.4
10.2
6.1
10.8
6.1
30.4
2q
2h
2u
2v
2w
2x
3b
3d
3f
>50
>50
16.9
>50
>50
>50
>50
>50
>50
10.3
4.5
3.6
28.8 10.3
5.2 23.8
4.6
15.1
1.9
5.6
9.7
3.0
8.8
5.3
1.7
2.5
>50
17.2
5.2
11.1
22.4
>50
13.4
11.1
>50
3.9
18.2
9.7
40.6 20.5
4.4 8.9
a
b
c
Values are means of three independent experiments.
Reported values are given in parentheses.
A549: Lung adenocarcinoma cell line; hela: epithelial carcinoma cell line; HepG2: hepatocellular carcinoma cell line; HT1080: fibrosarcoma cell line; HT-29: colon
adenocarcinoma cell line; KB: mouth epidermoid carcinoma cell line; L-78: lung squamous carcinoma cell line; MCF-7: breast adenocarcinom cell line; MOLT-4: acute
lymphoblastic leukemia cell line; OS-RC-2: renal carcinoma cell line; SGC-7901: gastric carcinoma cell line; U251: human glioma cell line; IMR-90: a human lung fibroblast
cell line.
Figure 3. Induction of apoptosis by 2q in the human prostate cancer Du145 cell line. Cells were treated for 72 h, and apoptosis was determined using AnnexinV conjugated 7-
AAD staining by flow cytometry.
compounds did not showed obvious inhibition against Molt-4 cells.
It was noteworthy that compound 2x had comparable cell growth
inhibitory activity with doxorubicin against several cancer cell
lines such as A549 lung cancer cells, Hela epithelial carcinoma
cells, HT-29 colon adenocarcinoma cells, L-78 lung squamous
carcinoma cells,6 and OS-RC-2 renal carcinoma cells in a parallel
comparison. In order to monitor the potential toxicity of the com-
pounds to non-cancerous cells, their inhibitory activities against
IMR-90 human lung fibroblast cells were evaluated. Different from
that of doxorubicin, almost all the compounds did not showed
obvious cytotoxic activity to non-cancerous IMR-90 fibroblast cells
tive to Annexin V than to 7-AAD, was also increased. For example,
about 84% cells were in healthy condition before treatment, but the
number was dramatically decreased to 2.8% after treatment with
10 lM compound 2q. Over 58% cells were found to be dead, and
37.6% cells were under apoptosis.
Apoptosis can be characterized by morphological and biochem-
ical changes in the cell nucleus, such as chromatin condensation
and nuclear shrinking.14 Morphological analysis of treated cells
by using fluorescent micrograph with Hoechst 33342 staining
clearly revealed compound 2q significantly induced chromatin
condensation after a 24-h treatment (Fig. 4). While western blot
analysis indicated compound 2q dose-dependently caused the
activation of pro-apoptotic caspase 9 and 3 (Fig. 5). These data fur-
ther verified the apoptosis inducing effect of compound 2q.
under high concentrations (IC50 >50 lM), which indicated that the
compounds might selectively target tumor cells.
The apoptosis inducing activity of the compounds was investi-
gated by using flow cytometric analysis to detect the phosphatidyl-
serine content (PS) on the outer leaflet of the plasma cell
membrane (PS externalization) in Du145 cells. Annexin V is a PS-
binding protein that can be conjugated to fluorescent groups and
used in flow cytometric analysis to determine cell viability.
7-AAD (7-amino-actinomycin) is a fluorescent molecule that is
impermeable to cells with intact membranes but permeable to
dead cells. As shown in Figure 3, the majority of untreated cells
appeared negative for staining with Annexin V and 7-AAD, indicat-
ing that the cells were healthy. Almost all the compounds dose-
dependently induced the cells to be positive for both Annexin V
and 7-AAD, which indicated these cells were dead either by necro-
sis or apoptosis (Fig. 3 and Supplementary data). In addition, the
population of cells undergoing apoptosis, which were more sensi-
Figure 4. Fluorescent micrographs of Hoechst 33342 staining (100ꢀ). The arrows
indicate apoptotic cells.

5094
J. Zhou et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5091–5094
2. Srivastava, I. K.; Rottenberg, H.; Vaidya, A. B. J. Biol. Chem. 1997, 272,
Caspase 9
Activated caspase 9
Caspase 3
3961.
3. Papageorgiou, V. P.; Assimopoulou, A. N.; Couladouros, E. A.; Hepworth, D.;
Nicolaou, K. C. Angew. Chem., Int. Ed. 1999, 38, 270.
4. Recent examples see: (a) Salaski, E. J.; Krishnamurthy, G.; Ding, W.; Yu, K.;
Insaf, S. S.; Eid, C.; Shim, J.; Levin, J. I.; Tabei, K.; Toral-Barza, L.; Zhang, W.;
McDonald, L. A.; Honores, E.; Hanna, C.; Yamashita, A.; Johnson, B.; Li, Z.;
Laakso, L.; Powell, D.; Mansour, T. S. J. Med. Chem. 2009, 52, 2181; (b)
Bolognese, A.; Correale, G.; Manfra, M.; Esposito, A.; Novellino, E.; Lavecchia, A.
J. Med. Chem. 2008, 51, 8148; (c) Bolognesi, M. L.; Calonghi, N.; Mangano, C.;
Masotti, L.; Melchiorre, C. J. Med. Chem. 2008, 51, 5463; (d) He, J.; Roemer, E.;
Lange, C.; Huang, X.; Maier, A.; Kelter, G.; Jiang, Y.; Xu, L.; Menzel, K.; Grabley,
S.; Fiebig, H.; Jiang, C.; Sattler, I. J. Med. Chem. 2007, 50, 5168; (e) Wang, W.; Dai,
M.; Zhu, C.; Zhang, J.; Lin, L.; Ding, J.; Duan, W. Bioorg. Med. Chem. Lett. 2009, 19,
735; (f) Eyong, K. O.; Kumar, P. S.; Kuete, V.; Folefoc, G. N.; Nkengfack, E. A.;
Baskaran, S. Bioorg. Med. Chem. Lett. 2008, 18, 5387; (g) Yamashita, M.; Kaneko,
M.; Iida, A.; Tokudab, H.; Nishimura, K. Bioorg. Med. Chem. Lett. 2007, 17, 6417;
(h) Hadden, M. K.; Hill, S. A.; Davenport, J.; Matts, R. L.; Blagg, B. S. J. Bioorg. Med.
Chem. 2009, 17, 634; (i) Bhasin, D.; Cisek, K.; Pandharkar, T.; Regan, N.; Li, C.;
Pandit, B.; Lin, J.; Lia, P. Bioorg. Med. Chem. Lett. 2008, 18, 391; (j) Lee, E.; Lee, H.;
Park, H. J.; Min, H.; Suh, M.; Chung, H.; Lee, S. K. Bioorg. Med. Chem. Lett. 2004,
14, 5175.
GAPDH
0 M 1.0 M 2.0 M 5.0 M 10.0 M
Figure 5. Compound 2q induced the activation of caspase 9 and capase 3 after 24-h
treatment.
In summary, atovaquone derivatives were designed and synthe-
sized as new cytotoxic and apoptosis inducing agents. Both 2-pip-
erazinyl analogues (2) and 2-piperidinyl analogues (3) displayed
good cytotoxic activities against a panel of cancer cell lines. The
study also indicated that the cell growth inhibitory activity might
be related to apoptosis induced by the compounds. Further mech-
anism studies are under going and will be reported in due course.
5. CCDC # 727371 contains the Supplementary data for this paper. These data can
be obtained free of charge at www.ccdc.cam.ac.uk/conts/ retrieing.html [or
from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge
CB2 1EZ, UK; fax: +44(1223)336033; E-mail: deposit@ccdc.cam.ac.uk].
6. L-78 Cells: kindly provided by Beijing Institute for Cancer Research, Peking
University; OS-RC-2 cells and SGC-7901 cells: ordered from Shanghai Cellular
Institute of the Chinese Academy of Sciences; Other cells: ordered from
American Type Culture Collection (ATCC, USA).
Acknowledgments
This work was supported by 100-talent program of CAS, CAS grant
(# KSCX2-YW-R-27), and 863 program (grant # 2007AA02Z490).
Supplementary data
7. Li, F.; Awale, S.; Tezuka, Y.; Kadota, S. Bioorg. Med. Chem. 2008, 16, 5434.
8. Hasegawa, T.; Bai, J.; Dai, J.; Bai, L.; Sakai, J.; Nishizawa, S.; Bai, Y.; Kikuchi, M.;
Abe, M.; Yamori, T.; Tomida, A.; Tsuruo, T.; Hirose, K.; Ando, M. Bioorg. Med.
Chem. Lett. 2007, 17, 3722.
9. De Groot, F. M.; Broxterman, H. J.; Adams, H. P.; van, Vliet A.; Tesser, G. I.;
Elderkamp, Y. W.; Schraa, A. J.; Kok, R. J.; Molema, G.; Pinedo, H. M.; Scheeren,
H. W. Mol. Cancer Ther. 2002, 1, 901.
10. Serova, M.; Galmarini, C. M.; Ghoul, A.; Benhadji, K.; Green, S. R.; Chiao, J.;
Faivre, S.; Cvitkovic, E.; Le, T. C.; Calvo, F.; Raymond, E. Br. J. Cancer 2007, 97,
628.
Supplementary data (experimental procedures, characteriza-
tion of final compounds and flow cytometric analysis results for
compounds 2h, 2n, 2v, 2w, 2x, 3h, 3b and 3f) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2009.07.044.
11. Wu, J.; Lee, A.; Lu, Y.; Lee, R. J. Int. J. Pharm. 2007, 337, 329.
12. Svensson, S. P.; Lindgren, S.; Powell, W.; Green, H. Pigment Cell Res. 2003, 16,
351.
13. He, Q. C.; Zhang, G. Y.; Cao, W. J. Ai Zheng (Chin. J. Cancer) 2008, 27, 337.
14. Kerr, J. F.; Winterford, C. M.; Harmon, B. V. Cancer 1994, 73, 2013.
References and notes
1. (a) Chong, C. R.; Chen, X.; Shi, L.; Liu, J. O.; Sullivan, D. J. Nat. Chem. Biol. 2006, 2,
415; (b) Chong, C. R.; Sullivan, D. J. Nature 2007, 448, 645; (c) Ashburn, T. T.;
Thor, K. B. Nat. Rev. Drug Disc. 2004, 3, 673.