Combretastatin A4-β-Galactosyl Conjugates for Ovarian Cancer Prodrug Monotherapy

Article abstract of DOI:10.1021/acsmedchemlett.6b00427

Chemotherapy for ovarian cancer often causes severe side effects. As candidates for combretastatin A4 (CA4) prodrug for ovarian cancer prodrug monotherapy (PMT), we designed and synthesized two β-galactose-conjugated CA4s (CA4-βGals), CA4-βGal-1 and CA4-βGal-2. CA4 was liberated from CA4-βGals by β-galactosidase, an enzyme more strongly expressed in ovarian cancer cells than normal cells. CA4-βGal-2, which has a self-immolative benzyl linker between CA4 and the β-galactose moiety, was more cytotoxic to ovarian cancer cell lines than CA4-βGal-1 without a linker. Therefore, CA4-βGal-2 can serve as a platform for the design and manufacture of prodrugs for ovarian cancer PMT.

Full text of DOI:10.1021/acsmedchemlett.6b00427

Letter
pubs.acs.org/acsmedchemlett
Combretastatin A4-β-Galactosyl Conjugates for Ovarian Cancer
Prodrug Monotherapy
Tomohiro Doura,*,‡ Kazuaki Takahashi,‡ Yasumitsu Ogra, and Noriyuki Suzuki*
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
S
* Supporting Information
ABSTRACT: Chemotherapy for ovarian cancer often causes
severe side effects. As candidates for combretastatin A4 (CA4)
prodrug for ovarian cancer prodrug monotherapy (PMT), we
designed and synthesized two β-galactose-conjugated CA4s
(CA4-βGals), CA4-βGal-1 and CA4-βGal-2. CA4 was
liberated from CA4-βGals by β-galactosidase, an enzyme
more strongly expressed in ovarian cancer cells than normal
cells. CA4-βGal-2, which has a self-immolative benzyl linker
between CA4 and the β-galactose moiety, was more cytotoxic
to ovarian cancer cell lines than CA4-βGal-1 without a linker.
Therefore, CA4-βGal-2 can serve as a platform for the design and manufacture of prodrugs for ovarian cancer PMT.
KEYWORDS: Antitumor agent, prodrug monotherapy, ovarian cancer, tubulin polymerization inhibitor, β-galactosidase,
combretastatin A4
varian cancer is called a “silent killer”, and its late
the three methoxy substituents on ring A, the cis-stilbene
moiety, and hydrogen bond donors, such as the hydroxy group
on ring B, are necessary for CA4 to exert potent cytotoxicity
(Figure 1a).11 Based on this knowledge, we speculated that the
cytotoxicity of CA4 would be weakened by substituting a β-
1
Odiagnosis reduces the overall cure rate. Metastasis of
ovarian cancer cells to the peritoneal cavity occurs easily
because no anatomical barrier exists between them.2 Chemo-
therapy for stage 3 patients (in whom ovarian cancer cells have
metastasized to the abdomen) revealed that intraperitoneal
delivery of cisplatin and paclitaxel is more effective than
intravenous delivery of those drugs. However, the intra-
peritoneal delivery method causes more side effects than the
intravenous delivery method.3 This indicates that chemo-
therapy for advanced-stage ovarian cancer patients requires
antitumor agents that exhibit selective cytotoxicity to cancer
cells.
In prodrug monotherapy (PMT), a nontoxic prodrug is
administered, which releases a cytotoxic drug in response to
enzymatic activity enhanced in tumor tissues, realizing cancer
cell selective chemotherapy.4 It is known that β-galactosidase
activity is enhanced in some cancer cell lines, such as ovarian,5
breast,6 colon,6 and larynx7 cancers and gliomas.8 Utilizing this
characteristic, Asanuma et al. developed a fluorescence probe
that is transformable into a fluorescent dye by β-galactosidase
and applied it to the visualization of small peritoneal metastases
in mouse models of ovarian cancer.9 Based on this result, we
hypothesized that noncytotoxic molecules that are transformed
into cytotoxic molecules by β-galactosidase are suitable
prodrugs for ovarian cancer PMT.
We focused on combretastatin A4 (CA4), a potent tubulin
polymerization inhibitor, because microtubule disrupting
agents, such as paclitaxel, are used for ovarian cancer
chemotherapy. CA4 is a natural product isolated from the
bark of the African willow tree Combretum caffrum.10
Structure−activity relationship studies of CA4 revealed that
Figure 1. (a) Structures of CA4-βGal-1 and CA4-βGal-2 based on the
structure−activity relationships of CA4. (b) Release pathway of CA4
from CA4-βGal-2.
Received: October 27, 2016
Accepted: January 20, 2017
Published: January 20, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00427
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthetic Sequence to CA4-βGal-1 and CA4-βGal-2
a
Reagents and conditions: (a) CA4, NaH, THF/DMF (7:1), reflux, 23 h, yield 40%; (b) NaOMe, MeOH, 0 °C, 5 h, yield 41%; (c) CA4, NaH,
THF/DMF (36:5), RT, 42 h, yield 71%; (d) NaOMe, MeOH, 0 °C, 3 h, yield 89%.
galactose moiety for the hydroxy group on ring B. In this letter,
we report the design and syntheses of β-galactose-conjugated
CA4s (CA4-βGals) for ovarian cancer PMT and their potent
cytotoxicity to ovarian cancer cell lines.
We designed two CA4-βGals, CA4-βGal-1 and CA4-βGal-2,
which release their β-galactose moieties and are transformed
into CA4 in response to β-galactosidase activity (Figure 1b). In
the case of CA4-βGal-1, a β-galactose moiety is directly
introduced to the hydroxy group on ring B through a glycoside
bond. In contrast, in the case of CA4-βGal-2, a self-immolative
benzyl linker is inserted between CA4 and the β-galactose
moiety. When the β-galactose moiety is removed, 1,6-benzyl
elimination of the linker subsequently occurs, resulting in the
formation of CA4.12,13 Some reports about prodrugs activated
by a tumor-associated protease plasmin showed the importance
of self-immolative linkers to avoid steric hindrance near the
tripeptide substrate moiety for effective release of drugs.14−16
Therefore, we speculated that CA4-βGal-1 would be metabo-
lized slowly by β-galactosidase because the methoxy group on
ring B is located near the β-galactose moiety, whereas CA4-
βGal-2 would be decomposed rapidly by β-galactosidase due to
little steric hindrance near the β-galactose moiety.
CA4 was synthesized according to a reported scheme.17
Williamson ether syntheses of CA4 with 1 or 3 and removal of
the acetyl groups using sodium methoxide produced CA4-βGal-
Figure 2. (a) HPLC profiles of enzymatic reactions of 0.2 mM CA4-
βGal-1 (left column) and 0.2 mM CA4-βGal-2 (right column) with β-
galactosidase (0.1 U) in 0.2 M PBS(−) (pH 7.4) at 37 °C. Reactions
were monitored by measuring the absorption at 300 nm. (b) Yields of
CA4 (left) and consumption of CA4-βGal-1 and CA4-βGal-2 (right)
by β-galactosidase activity. Yields were determined on peak integrals.
1 and CA4-βGal-2, respectively (Scheme 1).
First, we evaluated the inhibitory activities of CA4-βGal-1
and CA4-βGal-2 against tubulin polymerization. As results,
neither CA4-βGal-1 nor CA4-βGal-2 showed the tubulin
polymerization inhibitory activity (Figure S1). These results
are coincident with the structure−activity relationship of CA4
derivatives.
Next, we investigated whether CA4-βGals would be
metabolized into CA4 by β-galactosidase. HPLC analyses
revealed that β-galactosidase recognized both CA4-βGals as
substrates and transformed them mainly into CA4 (Figure 2).
In addition, under the same enzymatic reaction conditions, the
yields of CA4 from CA4-βGal-1 were lower than those from
CA4-βGal-2, and CA4-βGal-2 was more rapidly consumed by
β-galactosidase than CA4-βGal-1 (Figure 2b). This difference
probably results from the steric hindrance existing near their β-
galactose moieties. This result demonstrates the suitability of
the molecular design using 1,6-benzyl linker as a self-
immolative benzyl linker to facilitate the access of β-
galactosidase to the β-galactose moiety of a substrate.
Next, we evaluated the cytotoxicity levels of CA4-βGal-1,
CA4-βGal-2, and CA4 in two ovarian cancer cell lines,
OVCAR3 and OVK18, using a WST-8-based colorimetric cell
cytotoxicity assay. The EC50 values of CA4-βGal-2 against
OVCAR3 and OVK18 were 2.47 and 2.67 nM, respectively,
and were equivalent to the EC50 values of CA4 against the same
cell lines (3.45 and 1.99 nM, respectively). However, the EC50
values of CA4-βGal-1 against the same cell lines were 69.1 and
252 nM, respectively (Figure 3a, Table 1). These results
revealed that CA4-βGal-2 exhibits higher cytotoxicity than
B
DOI: 10.1021/acsmedchemlett.6b00427
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GA, the cytotoxicity levels of CA4-βGals in OVCAR3 and
OVK18 were decreased substantially (Figure 3b). These results
revealed that the cytotoxicity levels of CA4-βGals in ovarian
cancer cell lines are dependent on intracellular β-galactosidase
activity, and their high cytotoxicity levels are caused by CA4,
the product of β-galactosidase enzymatic activity. The
inhibition condition, 1 mM β-GA, suppressed β-galactosidase
activity of OVK18 to that of normal cells.9 Therefore, these
results also demonstrated that CA4-βGals disrupt ovarian
cancer cells preferentially.
CA4 exhibits potent inhibition of tubulin polymerization and
disrupts intracellular microtubule architecture.10 To elucidate
whether the cytotoxicity of CA4-βGals to ovarian cancer cells is
caused by the disruption of intracellular microtubule
architecture, we evaluated intracellular α-tubulin and DNA,
which were stained with suitable fluorescent dyes, with a
confocal fluorescence microscope after incubating OVCAR3
with CA4-βGal-1 or CA4-βGal-2 for 24 h. When OVCAR3 was
incubated with CA4-βGal-2 at a concentration below its EC50
(1 nM), contraction of cytoskeleton was observed, but
intracellular microtubule architecture was maintained (Figure
4c, left). When CA4-βGal-2 above its EC50 was added (10 nM),
Figure 3. (a) Cell viability curves of ovarian cancer cell lines,
OVCAR3 (left) and OVK18 (right), treated with CA4 and CA4-βGals.
(b) Inhibition effects of intracellular β-galactosidase activity on cell
viabilities of OVCAR3 (top) and OVK18 (bottom) with β-GA (1
mM). ***p < 0.01 by Welch’s t-test (n = 6 for each group).
Table 1. EC50 Values of CA4-βGals and CA4 against Ovarian
a
Cancer Cell Lines
EC50 (nM)
cell line
CA4-βGal-1
CA4-βGal-2
CA4
Figure 4. Fluorescence microscopy images of the ovarian cancer cell
line OVCAR3 (a) treated with CA4 at 10 nM (b) and CA4-βGal-2 at
1 and 10 nM (c). Nuclei were directly stained with DAPI (blue), and
α-tubulin was detected with Alexa Fluor 647 (red) conjugated with
secondary antibody. Scale bars, 10 μm.
OVCAR3
OVK18
69.06 14.67
252.42 17.56
2.47 0.21
2.67 0.19
3.45 0.97
1.99 0.05
a
Values represent the average and SE of six experiments performed in
triplicate.
CA4-βGal-1 in ovarian cancer cell lines. The enzymatic reaction
rates of the two CA4-βGals in vitro might have led to these
differences in cytotoxicity. The cellular permeability of CA4-
βGals also might have affected their cytotoxicity. The estimated
log P values of CA4-βGal-1 and CA4-βGal-2 are 1.5 and 3.1
(calculated by ChemBioDraw13), respectively, which indicate
that CA4-βGal-2 is more hydrophobic than CA4-βGal-1. For
compounds whose molecular weights are higher than 400, a
certain level of hydrophobicity is required to enhance their
cellular permeability.18 Therefore, CA4-βGal-2 is probably
more cell-permeable than CA4-βGal-1.
we observed disruption of microtubule architecture in addition
to contraction of cytoskeleton. Some cells showed nuclear
contraction and fragmentation (Figure 4c, right). Similar
abnormal morphological changes of microtubule architecture,
cytoskeleton, and nucleus were observed in OVCAR3 treated
with 10 nM CA4 for 24 h (Figure 4b). We also observed similar
morphological changes in the case of CA4-βGal-1 (Figure S2).
These cytoskeletal and nuclear changes are characteristic of
apoptosis. These findings demonstrate that the cytotoxicity of
CA4-βGals to ovarian cancer cells is a result of the disruption of
microtubule architecture induced by CA4, which is produced
from CA4-βGals by intracellular β-galactosidase activity, and
the subsequent apoptosis.
We also investigated whether the cytotoxicity levels of CA4-
βGals are dependent on the intracellular β-galactosidase activity
by introducing β-GA, a β-galactosidase inhibitor.19 When
intracellular β-galactosidase activity was inhibited by 1 mM β-
In conclusion, we designed and synthesized two CA4-βGals
for ovarian cancer PMT, CA4-βGal-1 and CA4-βGal-2, and
C
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Letter
(4) Bosslet, K.; Czech, J.; Hoffmann, D. A novel one-step tumor-
selective prodrug activation system. Tumor Target. 1995, 1, 45−50.
(5) Chatterjee, S. K.; Bhattacharya, M.; Barlow, J. J. Glycosyltransfer-
ase and glycosidase activities in ovarian cancer patients. Cancer Res.
1979, 39, 1943−1951.
demonstrated that CA4-βGal-2 exhibited more potent cytotox-
icity to ovarian cancer cell lines than CA4-βGal-1. This result
indicates the suitability of introducing the self-immolative 1,6-
benzyl linker in the design of prodrugs for ovarian cancer PMT.
Because high β-galactosidase activity is reported in not only
ovarian cancer but also other malignant tumors,6−9 CA4-βGal-2
can be also used as a prodrug candidate for PMT for these
malignant tumors. Our design strategy for CA4-βGal-2 is
valuable for the development of prodrugs for PMT targeting
various cancers.
(6) Bosmann, H. B.; Hall, T. C. Enzyme activity in invasive tumors of
human breast and colon. Proc. Natl. Acad. Sci. U. S. A. 1974, 71, 1833−
1837.
(7) Olszewska, E.; Borzym-Kluczyk, M.; Rzewnicki, I.; Wojtowicz, J.;
Rogowski, M.; Pietruski, J. K.; Czajkowska, A.; Sieskiewicz, A. Possible
role of α-mannosidase and β-galactosidase in larynx cancer.
Wspolczesna Onkol. 2012, 16, 154−158.
(8) Wielgat, P. Activity of lysosomal exoglycosidases in human
gliomas. J. Neuro-Oncol. 2006, 80, 243−249.
(9) Asanuma, D.; Sakabe, M.; Kamiya, M.; Yamamoto, K.; Hiratake,
J.; Ogawa, M.; Kosaka, N.; Choyke, P. L.; Nagano, T.; Kobayashi, H.;
Urano, Y. Sensitive β-galactosidase-targeting fluorescence probe for
visualizing small peritoneal tumors in vivo. Nat. Commun. 2015, 6,
6463.
(10) Pettit, G. R.; Cragg, G. M.; Herald, D. L.; Schmidt, J. M.;
Lohavanijaya, P. Isolation and structure of combretastatin. Can. J.
Chem. 1982, 60, 1374−1376.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00427.
■
S
Chemical synthesis and characterization of target
compounds; protocols of biological assays (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: n-suzuki@chiba-u.jp (N.S.).
■
(11) Lin, C. M.; Singh, S. B.; Chu, P. S.; Dempcy, R. O.; Schmidt, J.
M.; Pettit, G. R.; Hamel, E. Interactions of tubulin with potent natural
and synthetic analogs of the antimitotic agent combretastatin: a
structure-activity study. Mol. Pharmacol. 1988, 34, 200−208.
(12) Meyer, Y.; Richard, J.-A.; Delest, B.; Noack, P.; Renard, P.-Y.;
Romieu, A. A comparative study of the self-immolation of para-
aminobenzylalcohol and hemithioaminal-based linkers in the context
of protease-sensitive fluorogenic probes. Org. Biomol. Chem. 2010, 8,
1777−1780.
*E-mail: doura_t@yamaguchi-u.ac.jp (T.D.).
ORCID
Noriyuki Suzuki: 0000-0002-9345-6335
Author Contributions
‡T.D. and K.T. contributed equally to this work. T.D. and K.T.
designed this research; T.D. and K.T. performed experiments
and analyzed data; N.S. and Y.O. supervised this research; and
T.D., K.T., N.S., and Y.O. cowrote the manuscript.
̀
(13) Alouane, A.; Labruere, R.; Le Saux, T.; Schmidt, F.; Jullien, L.
Self-immolative spacers: kinetic aspects, structure-property relation-
ships, and applications. Angew. Chem., Int. Ed. 2015, 54, 7492−7509.
(14) Chakravarty, P. K.; Carl, P. L.; Weber, M. J.; Katzenellenbogen,
J. A. Katzenellenbogen. Plasmin-activated prodrugs for cancer
chemotherapy. 2. Synthesis and biological activity of peptidyl
derivatives of doxorubicin. J. Med. Chem. 1983, 26, 638−644.
(15) de Groot, F. M. H.; de Bart, A. C. W.; Verheijen, J. H.;
Scheeren, H. W. Synthesis and biological evaluation of novel prodrugs
of anthracyclines for selective activation by the tumor-associated
protease plasmin. J. Med. Chem. 1999, 42, 5277−5283.
Funding
This research was supported in part by Chiba University
Research Promotion Program (a grant to T.D.), by The
Mochida Memorial Foundation for Medical and Pharmaceutical
Research (a grant to T.D.), by the Ministry of Education,
Culture, Sports, Science and Technology of Japan Grant-in-Aid
for Scientific Research (KAKENHI) (a grant 16K18909 to
T.D.), and JSPS KAKENHI Grants-in-Aid for Scientific
Research (a grant 26460032 to N.S.).
(16) de Groot, F. M. H.; Loos, W. J.; Koekkoek, R.; van Berkom, L.
W. A.; Busscher, G. F.; Seelen, A. E.; Albrecht, C.; de Bruijn, P.;
Scheeren, H. W. Elongated multiple electronic cascade and cyclization
spacer systems in activatible anticancer prodrugs for enhanced drug
release. J. Org. Chem. 2001, 66, 8815−8830.
Notes
The authors declare no competing financial interest.
́ ́
(17) Furst, R.; Zupko, I.; Berenyi, A.; Ecker, G. F.; Rinner, U.
̈
ACKNOWLEDGMENTS
We thank Prof. Yasuteru Urano for measurements of high-
resolution mass spectra.
Synthesis and antitumor-evaluation of cyclopropyl-containing com-
bretastatin analogs. Bioorg. Med. Chem. Lett. 2009, 19, 6948−6951.
(18) Waring, M. J. Lipophilicity in drug discovery. Expert Opin. Drug
Discovery 2010, 5, 235−248.
■
(19) Guo, W.; Hiratake, J.; Ogawa, K.; Yamamoto, M.; Ma, S.-J.;
Sakata, K. β-D-Glycosylamidines: potent, selective, and easily
accessible β-glycosidase inhibitors. Bioorg. Med. Chem. Lett. 2001, 11,
467−470.
ABBREVIATIONS
■
PMT, prodrug monotherapy; CA4, combretastatin A4; HPLC,
high-performance liquid chromatography; WST-8, water-
soluble tetrazolium salt-8; EC50, half maximal effective
concentration
REFERENCES
■
(1) Cannistra, S. A. Cancer of the ovary. N. Engl. J. Med. 2004, 351,
2519−2529.
(2) Bast, R. C.; Hennessy, B.; Mills, G. B. The biology of ovarian
cancer: new opportunities for translation. Nat. Rev. Cancer 2009, 9,
415−428.
(3) Armstrong, D. K.; Bundy, B.; Wenzel, L.; Huang, H. Q.; Baergen,
R.; Lele, S.; Copeland, L. J.; Walker, J. L.; Burger, R. A. Intraperitoneal
cisplatin and paclitaxel in ovarian cancer. N. Engl. J. Med. 2006, 354,
34−43.
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DOI: 10.1021/acsmedchemlett.6b00427
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX