Reengineered epipodophyllotoxin

Article abstract of DOI:10.1039/c2cc35044k

A variant structural skeleton of epipodophyllotoxin was synthesized and found to rival the natural cyclolignan in antiproliferative and microtubule destabilizing properties. This discovery leads to a new structural class of tubulin targeting agents.

Full text of DOI:10.1039/c2cc35044k

View Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 10416–10418
www.rsc.org/chemcomm
COMMUNICATION
Reengineered epipodophyllotoxinw
Igor V. Magedov,*a Nikolai M. Evdokimov,a Menuka Karki,b Amanda S. Peretti,c
Dustin T. Lima,a Liliya V. Frolova,a Mary R. Reisenauer,c Anntherese E. Romero,c
Paul Tongwa,d Alexandr Fonari,d Jeff Altig,a Snezna Rogelj,c Mikhail Yu. Antipin,d
Charles B. Shusterb and Alexander Kornienko*ae
Received 14th July 2012, Accepted 29th August 2012
DOI: 10.1039/c2cc35044k
A variant structural skeleton of epipodophyllotoxin was syn-
thesized and found to rival the natural cyclolignan in antiproli-
ferative and microtubule destabilizing properties. This discovery
leads to a new structural class of tubulin targeting agents.
However, its glycosylated derivatives etoposide (3) and teniposide
(4), commonly referred to as epipodopyllotoxins, are currently
used in clinics for the treatment of lung and testicular cancers,
lymphoma, non-lymphocytic leukemia and glioblastoma
multiforme.3 Due to the development of drug resistance by
cancer cells as well as side effects associated with the use of these
agents in clinics, the search for new effective anticancer drugs
with 1 and 2 as lead agents remains an intense area of research.4–6
Unfortunately, the complex chemical structure of podo-
phyllotoxin prevents the generation of libraries of its analogues
from simple commercially available materials. At the same
time, the derivatization of podophyllotoxin as a means to
obtain structure–activity relationship (SAR) information has
the drawback of producing non-systematic data because it is
limited by the type of chemistry that podophyllotoxin can undergo.
For example, substitutions of rings AB and E by other moieties
have been scarcely explored due to the synthetic challenge of
removing the profuse oxygenation in these parts of the molecule
(Fig. 2). Limited literature examples include ring E phenyl analogue
5 synthesized by Berkowitz and co-workers in 19 steps from
commercially available materials9 and phenazine 6 prepared
by way of oxidation of the AB subunit to an ortho-quinone
and its subsequent condensation with 1,2-phenylenediamine.10
These reports constitute rare synthetic accomplishments illus-
trating the challenge of a systematic exploration of the
chemical space occupied by these oxygenated moieties.11
Computer modeling simulations utilizing the tubulin–
podophyllotoxin crystal structure12 revealed that the
Cyclolignan podophyllotoxin (1, Fig. 1), isolated from American
mayapple (Podophyllum peltatum), was extensively investigated as
an antitumor agent, but the clinical results were disappointing due
to severe gastrointestinal side effects.1 Its C-4 epimer epipodophyl-
lotoxin (2), also found in the above-mentioned plant,2 is generally
5–10 times less potent as an antiproliferative agent against cancer
cells and as an inhibitor of in vitro mitotic spindle assembly.1,3–8
Fig. 1 Structures of podophyllotoxin (1), epipodophyllotoxin (2),
etoposide (3) and teniposide (4).
a Department of Chemistry, New Mexico Institute of Mining and
Technology, Socorro, New Mexico 87801, USA.
E-mail: imagedov@nmt.edu
b Department of Biology, New Mexico State University, Las Cruces,
New Mexico 88003, USA
c Department of Biology, New Mexico Institute of Mining and
Technology, Socorro, New Mexico 87801, USA
d Department of Biology and Chemistry, New Mexico Highlands
University, Las Vegas, New Mexico 87701, USA
e Department of Chemistry and Biochemistry,
Texas State University – San Marcos, San Marcos, TX 78666,
USA. E-mail: a_k76@txstate.edu
w Electronic supplementary information (ESI) available: Synthetic
procedures, molecular biology methods, X-ray data, details of tubulin
polymerization assay and computational work. CCDC 873143–873145.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c2cc35044k
Fig. 2 Is systematic interrogation of the chemical space occupied by
rings AB and E possible?
c
10416 Chem. Commun., 2012, 48, 10416–10418
This journal is The Royal Society of Chemistry 2012

View Online
of ring E was not critical as 3-bromopyridine and 3,5-dibromo-
phenyl rings were superior in many cases (e.g., 8 and 10). Although
our MCR-based mimetic scaffold approach led to promising novel
antitubulin agents (i.e., 8, 9 and 10) in its own right, we were
intrigued by the possibility of utilizing this method as a tool to
rapidly probe drastic skeleton modifications in the natural cyclo-
lignans. With this idea in mind, we conceived reengineered
epipodophyllotoxin 11, whose new naphthalene and 3,5-dibromo-
phenyl rings AB and E were inspired by the MCR product 10. In
this communication, we report synthesis and anticancer evaluation
of this unorthodox epipodophyllotoxin analogue.
The synthesis relied on an exo-selective intramolecular Diels–
Alder reaction of ester 17, prepared using standard chemistry
(Scheme 1). A simple reflux of 17 in toluene gave a 1.6 : 1
mixture of exo and endo products 18 and 19. These were easily
separable and their structures were confirmed by single-crystal
X-ray analyses. The desired C-4 hydroxyl was installed by the
dihydroxylation of 18, which proceeded exclusively from the
b-face furnishing diol 20 as a single diastereomer (Scheme 2).
The synthesis of the epipodophyllotoxin analogue 11 was then
completed by a TFAA-promoted dehydration to give dihydro-
naphthalene 21 and its subsequent oxidation with DDQ. The
structure of 11 was unambiguously established with X-ray
crystallography. The synthetic work was completed by the
preparation of additional analogues in the cis-lactone series.
To this end, endo Diels–Alder product 19 was subjected to the
same three-reaction sequence leading to cis-lactone 24.
Fig. 3 Strategy leading to reengineered epipodophyllotoxin 11.
podophyllotoxin’s pentacyclic framework can be mimicked with a
dihydropyridine-based scaffold having comparable binding modes
at the colchicine site.13 Because this mimetic dihydropyridine
skeleton is easily accessible by a multicomponent reaction
(MCR) of commercially available aromatic/heteroaromatic
amines with aldehydes and tetronic acid, we prepared a diverse
library of podophyllotoxin mimetics and generated systematic
anticancer SAR data.13 In this method, the utilization of selected
amine and aldehyde starting materials leads directly to the desired
modifications of rings AB and E, respectively (Fig. 3). We found
that compounds incorporating pyrazole (8), indole (9) and
naphthalene (10) AB ring substitutions possessed nanomolar
antiproliferative potencies. In addition, the trimethoxy substitution
Evaluation of the synthesized analogues for antiprolifera-
tive activity was performed using HeLa and MCF-7 cancer
cell lines as models for human cervical and breast adeno-
carcinomas, respectively (Table 1). In addition, we assessed the
ability of these compounds to induce apoptosis in Jurkat cells
Scheme 1 Synthesis of Diels–Alder products 18 (exo) and 19 (endo).
Scheme 2 Elaboration of the Diels–Alder products 18 and 19 to reengineered epipodophyllotoxin 11 and its C-3 epimer 24.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10416–10418 10417

View Online
Table 1 Antiproliferative and apoptosis-inducing properties of the
synthesized compounds, and their binding energies at the colchicine site
analogue 21 bound to the colchicine site in orientations similar to
that of 1. It should be noted that cis-lactone derivatives are
significantly less active as revealed by literature reports4–6 as well
as the results of the present investigation demonstrating decreased
activity associated with cis-lactone analogues 22–24 and the
absence of comparable binding modes in docking simulations.
Our mechanistic experiments confirm that compounds 21 and 11
retain the antitubulin mode of action of epipodophyllotoxin. Thus,
11 was found to arrest cancer cells in the G2/M phase of the cell
cycle, exhibit little toxicity toward normal primary blood lympho-
cytes and (together with 21) display microtubule-destabilizing
activity in vitro using a fluorimetry-based tubulin polymerization
assay13 (see ESIw for details). In addition, to determine the extent
of microtubule disruption in whole cells, HeLa cells were cul-
tured in the presence of a carrier control (DMSO) or compounds
2, 10, 11 and 21 at the half maximal growth inhibitory concen-
tration and examined for microtubule morphology (Fig. 5). In
contrast to DMSO-treated cells that displayed normal interphase
and mitotic microtubule organization (A and F), compounds 2,
10, 11 and 21 all affected spindle morphology (G–J). Reengi-
neered epipodophyllotoxin 11 exhibited the most potent desta-
bilizing activity, completely blocking spindle formation (G) and
disrupting all but the most stable microtubules in interphase
cells (B). Compounds 10, 2 and 21 had no effect on the
relatively stable interphase microtubules (C–E), but did result
in shorter spindle lengths, as evidenced by 13, 7.5 and 15.2%
decreases in pole-to-pole distances, respectively (H–J).
GI50a/mM
Apoptosis (%) Binding
Jurkat
#
HeLa
MCF-7
energiesc/kJ molÀ1
1
2
0.030 Æ 0.002 0.018 Æ 0.002 37.5 Æ 1.9
À9.3
À9.2
À8.1
nd
0.36 Æ 0.09 0.24 Æ 0.00 30.0 Æ 1.2
10 0.016 Æ 0.002 0.014 Æ 0.001 27.7 Æ 3.2
18 30.8 Æ 7.9
23.7 Æ 0.8
4.4 Æ 0.2
46.9 Æ 0.7
8.0 Æ 1.3
7.0 Æ 1.0
7.5 Æ 0.4
19
3.4 Æ 0.1
À7.0
20 42.2 Æ 1.0
21 0.22 Æ 0.00
11 0.56 Æ 0.01
22 56.9 Æ 3.4
23 41.4 Æ 3.0
24 12.9 Æ 2.4
nd
0.23 Æ 0.01 28.5 Æ 1.3
0.43 Æ 0.05 39.7 Æ 0.8
À8.6
À8.4
nd
nd
nd
79.7 Æ 0.1
24.1 Æ 1.4
5.9 Æ 0.1
7.3 Æ 0.6
7.7 Æ 1.4
7.4 Æ 0.7
a
Concentration required to reduce the viability of cells by 50% after
48 h of treatment with indicated compounds, relative to DMSO
b
control Æ SD, determined by MTT assay. % Apoptotic cells after
24 h of treatment with indicated compounds at the concentration of
300 nM relative to DMSO control Æ SD, determined by flow cyto-
c
metric Annexin-V/propidium iodide assay. Binding energies revealed
by Autodock Vina simulations, nd = no comparable binding pose.
(a model for human T-cell leukemia) and calculated their binding
energies using docking simulations (Table 1). The results indicated
that in accordance with the literature data 2 was about an order of
magnitude less potent in its antiproliferative effects than 1. Analo-
gue 11, together with its dihydronaphthalene variant 21, exhibited
submicromolar antiproliferative activity and potent apoptosis-
inducing properties rivaling those of 2, in spite of their racemic
nature. In addition, these four compounds displayed good binding
affinities as revealed by Autodock simulations. This can also be
seen in Fig. 4 visually demonstrating that the matching enantiomer
of reengineered epipodophyllotoxin 11 and its dihydronaphthalene
In conclusion, using our MCR-based mimetic scaffold
approach we conceived and synthesized an epipodophyllotoxin
analogue possessing a variant structural skeleton. Despite being
racemic, this compound rivals the natural cyclolignan in its
antiproliferative and apoptosis-inducing properties and induces
dramatic microtubule disorganization effects in whole cells.
Thus, compound 11 represents a new structural class of tubulin
targeting agents.
Notes and references
1 T. F. Imbert, Biochimie, 1998, 80, 207–222.
2 J. K. Bastos, C. L. Burnadt, N. P. D. Nanayakkara, L. Bryant and
J. D. McChesney, J. Nat. Prod., 1996, 59, 406–408.
3 L. Bohlin and B. Rosen, Drug Discovery Today, 1996, 1, 343–351.
4 Y. J. You, Curr. Pharm. Des., 2005, 11, 1695–1717.
5 M. Gordaliza, M. A. Castro, J. M. M. Corral and A. San
Feliciano, Curr. Pharm. Des., 2000, 6, 1811–1839.
6 M. Lv and H. Xu, Mini-Rev. Med. Chem., 2011, 11, 901–909.
7 M. Gordaliza, M. A. Castro, M. D. Garcia-Gravalos, P. Ruiz,
J. M. M. del Corral and A. San Feliciano, Arch. Pharm.
(Weinheim, Ger.), 1994, 175–179.
Fig. 4 Molecular docking poses (black) of 21 (A) and 11 (B) overlaid
with that of 1 (red).
8 C. F. Brewer, J. D. Loike, S. B. Horwitz, H. Sternlicht and
W. J. Gensler, J. Med. Chem., 1979, 22, 215–221.
9 D. B. Berkowitz, J.-H. Maeng, A. H. Dantzig, R. L. Shepard and
B. H. Norman, J. Am. Chem. Soc., 1996, 118, 9426–9427.
10 S. J. Cho, Y. Kashiwada, K. F. Bastow, Y. C. Cheng and
K.-H. Lee, J. Med. Chem., 1996, 39, 1396–1402.
11 For related pyridazine and indole A,B-ring analogues, see
(a) E. Bertounesque, T. Imbert and C. Monneret, Tetrahedron,
1996, 52, 14235–14246; (b) S. D. Cline, T. L. Macdonald and
N. Osheroff, Biochemistry, 1997, 36, 13095–13101.
12 R. B. J. Ravelli, B. Gigant, P. A. Curmi, I. Jourdain, S. Lachkar,
A. Sobel and M. Knossow, Nature, 2004, 428, 198–202.
13 I. V. Magedov, L. Frolova, M. Manpadi, U. D. Bhoga, H. Tang, N. M.
Evdokimov, O. George, K. H. Georgiou, S. Renner, M. Getlic, T. L.
Kinnibrugh, M. A. Fernandes, S. van Slambrouck, W. F. A. Steelant,
C. B. Shuster, S. Rogelj, V. L. A. van Otterlo and A. Kornienko, J. Med.
Chem., 2011, 54, 4234–4246, and references cited therein.
Fig. 5 Microtubule organization in HeLa cells during interphase
(A–E) and mitosis (F–J). Hela cells were treated for 3 hours with the
indicated compounds at their MTT-related GI50 concentrations (see
Table 1). Following drug treatment, cells were probed for microtubules
(green), centromeres (red) and DNA (blue). Bar, 10 m.
c
10418 Chem. Commun., 2012, 48, 10416–10418
This journal is The Royal Society of Chemistry 2012