Synthesis of A83586C analogs with potent anticancer and β-catenin/TCF4/osteopontin inhibitory effects and insights into how A83586C modulates E2Fs and pRb

Article abstract of DOI:10.1021/ol802818f

(Chemical Equation Presented) The synthesis of three potent new antitumor agents is described: the A83586C -citropeptin hybrid (1), the A83586C-GE3 hybrid (2), and L-Pro-A83586C (3). Significantly, compounds 1 and 2 function as highly potent inhibitors of β-catenin/TCF4 signaling within cancer cells, while simultaneously downregulating osteopontin (Opn) expression. A83586C antitumor cyclodepsipeptides also inhibit E2F-mediated transcription by downregulating E2F1 expression and inducing dephosphorylation of the oncogenic hyperphosphorylated retinoblastoma protein (pRb).

Full text of DOI:10.1021/ol802818f

ORGANIC
LETTERS
2009
Vol. 11, No. 3
737-740
Synthesis of A83586C Analogs with
Potent Anticancer and ꢀ-Catenin/
TCF4/Osteopontin Inhibitory Effects and
Insights Into How A83586C Modulates
E2Fs and pRb
Karl J. Hale,*,† Soraya Manaviazar,*,† Linos Lazarides,‡ Jonathan George,‡
Marcus A. Walters,‡ Jiaqiang Cai,‡ Vern M. Delisser,‡ Gurpreet S. Bhatia,‡
S. Andrew Peak,‡ Stephen M. Dalby,‡ Amandine Lefranc,‡ Ying-Nan P. Chen,§
Alexander W. Wood,§ Paul Crowe,† Pauline Erwin,† and Mohamed El-Tanani†
The School of Chemistry & Chemical Engineering and the Centre for Cancer Research
and Cell Biology (CCRCB), Queen’s UniVersity Belfast (QUB), Stranmillis Road,
Belfast BT9 5AG, U.K., The Department of Oncology, NoVartis Institutes for
BioMedical Research, Cambridge, Massachusetts 02139, and The Christopher Ingold
Laboratories, UCL, 20 Gordon Street, London WC1H 0AJ U.K.
k.j.hale@qub.ac.uk; s.manaViazar@qub.ac.uk
Received December 6, 2008
ABSTRACT
The synthesis of three potent new antitumor agents is described: the A83586C-citropeptin hybrid (1), the A83586C-GE3 hybrid (2), and
L-Pro-A83586C (3). Significantly, compounds 1 and 2 function as highly potent inhibitors of ꢀ-catenin/TCF4 signaling within cancer cells, while
simultaneously downregulating osteopontin (Opn) expression. A83586C antitumor cyclodepsipeptides also inhibit E2F-mediated transcription
by downregulating E2F1 expression and inducing dephosphorylation of the oncogenic hyperphosphorylated retinoblastoma protein (pRb).
In the preceding paper, we reported the first total syntheses
of (+)-azinothricin and (+)-kettapeptin via a new synthetic
approach that gives greatly improved access to molecules
of the A83586C class, as well as A83586C itself.1 In this
Letter, we now report on the application of this new strategy
to the synthesis of three novel A83586C analogues: the
A83586C-citropeptin hybrid (1), the A83586C-GE3 hybrid
(2), and L-Pro-A83586C (3); molecules which, collectively,
have provided powerful new insights into the functioning
of this family as antitumor agents.
In this regard, we show here, for the Very first time, that
the hybrids 1 and 2, and A83586C, are all highly potent
inhibitors of ꢀ-catenin/TCF4-mediated signaling.2 For 1 and
2 we also correlate these effects with a significant reduction
† Queen’s University Belfast.
(2) For a previous Novartis report on less potent small molecule
inhibitors of the oncogenic TCF4/ꢀ-catenin protein-protein interaction, see:
Lepourcelet, M.; Chen, Y-N. P.; France, D. S.; Wang, H.; Crews, P.;
Petersen, F.; Bruseo, C.; Wood, A. W.; Shivdasani, R. A. Cancer Cell 2004,
5, 91.
§ Novartis Institutes for Biomedical Research.
‡ UCL.
(1) Hale, K. J.; Manaviazar, S.; George, J. H.; Walters, M. A.; Dalby,
S. M. Org. Lett. 2009, 11, 733.
10.1021/ol802818f CCC: $40.75
Published on Web 01/06/2009
2009 American Chemical Society

in osteopontin (Opn) expression in metastatic rat Rama-37-
Opn mammary epithelial cells. Opn is a metastasis-inducing
protein whose transcription is instigated by the ꢀ-catenin/
TCF4 interaction.3
Foremost among the A83586C analogues that we wished
to synthesize were the A83586C-citropeptin hybrid 1, the
A83586C-GE3 hybrid 2, and L-Pro-A83586C (3). All were
chosen in the hope that they might have enhanced antitumor
effects and be more readily synthesizable than their natural
congeners. While the construction of 1 would inevitably
mean that a route to the previously unsynthesised citropeptin
cyclodepsipeptide 12 would have to be developed, in the
case of 2 and 3, we had already devised an effective route
to the GE37 and L-Pro-A83586C cyclodepsipeptides,8 and
so the main issue now centered around whether we could
successfully perform the final chemoselective couplings of
these peptides with 131 (see Schemes 1 and 2) in decent
yield and thereafter hydrate.
We also clarify here the mechanism by which A83586C
and its congeners potently repress E2F-mediated gene trans-
cription within human cancer cells. Specifically, we show
that E2F inhibition occurs directly as a result of the comp-
ounds downregulating E2F1 protein expression, while simul-
taneously promoting the dephosphorylation of oncogenic
hyperphosphorylated pRb.
A83586C was the first member of the azinothricin family
of depsipeptides to have its potent in vitro antitumor
properties recorded (IC50 ) 13.5 nM vs a CCRF-CEM human
T-cell leukemia cell line).4 Its sister molecule, (+)-citropep-
tin, was likewise reported to have pronounced antitumor
effects in 1990. It inhibited the growth of murine P388
lymphocytic leukemia and B16 melanoma cell lines (IC50
) 0.1 and 0.02 µg/mL, respectively), and it conferred a 123%
life extension on mice with the P388 leukemia tumor when
administered intraperitoneally at doses of 2 mg/kg/day.5 The
third member of this family to have its antitumor charac-
teristics profiled was GE3.6 Its IC50 values ranged from
3.6-16 nM against three different human tumor cell lines,
and remarkably, it produced a 47% reduction in tumor
volume in mice xenografted with the incurable PSN-1 human
pancreatic carcinoma when administered at 2 mg/kg/day.6
As such, molecules of this genre (Figure 1) have now
Scheme 1. Route Used to the A83586C-Citropeptin Hybrid (1)
Our route to 1 is presented in Scheme 1. It commenced
with a BOP-Cl/sym-collidine mediated coupling7 between
41 and 57 which proceeded in 54-58% yield. Following
deprotection with diethylamine in MeCN, the requisite
tetrapeptide 6 was isolated in 67-72% yield. The key union
with acid chloride 71 furnished hexapeptide 8 in 70-72%
yield. The Boc-groups were next detached with trifluoroacetic
Figure 1. A83586C family of antitumor antibiotics.
emerged as important new chemical genomics tools and
medicinal chemistry leads.
738
Org. Lett., Vol. 11, No. 3, 2009

carcinoma cell line with IC50’s of 47, 139, and 40 nM,
respectively (Figure 2). However, compound 3 was a much
Scheme 2. Chemoselective Couplings Used for Construction of
the A83586C-GE3 Hybrid (2) and L-Pro-A83586C (3)
Figure 2. IC50 values (nM) of 1, 2, 3, and A83586C on human
tumor cell line growth (3 day MTS assay).
less potent growth inhibitor (IC50 ) 880 nM). Significantly,
all four molecules also potently inhibited ꢀ-catenin mediated
transactivation from the TCF4 promoter in HCT-116 cells
after 24 h of drug exposure (IC50 for 1 ) 5 nM, IC50 for 2
) 3 nM, IC50 for A83586C ) 3 nM). Again, 3 was
approximately a 100-fold less active inhibitor (IC50 ) 430
( 150 nM). The latter results were gathered via TOP-
FLASH/FOP-FLASH assaying in HCT-116 colon cancer
cells (see Supporting Information).2 For 1 and 2, we further
validated our observations by examining the expression of
a relevant ꢀ-catenin/TCF4 downstream target gene, os-
teopontin (Opn),9 in Rama-37-Opn cells (Figure 3). Signifi-
acid in CH2Cl2, and the resulting acyl hydrazide was
converted into the carboxylic acid by treatment with N-
bromosuccinimide in aqueous THF.1 The crude seco-amino
acid 9 was then directly macrolactamized1 under high-
dilution conditions with HATU. The Troc-group was there-
after excised from 10, and the crude amine temporarily
blocked with a Z-group, to allow the zinc salts to be removed
and the highly pure hexapeptide 11 obtained. The latter was
then globally O-debenzylated by catalytic hydrogenolysis
over 10% Pd/C in methanol containing 1 equiv of anhydrous
HCl. The hydrochloride salt 12 was mixed with the activated
ester 13,1 and DMF was added. The resulting solution was
then cooled to -78 °C, and excess Et3N (10 equiv) was
introduced. After 5 min at -78 °C and gradual warming to
rt, the coupled glycal was obtained in 44% yield from the
Z-protected hexapeptide hydrogenation precursor. Prolonged
storage in CDCl3 cleanly hydrated this glycal to give the
A83586C-citropeptin hybrid 1 quantitatively.
The coupling of 131 with the GE3 cyclodepsipeptide 147
was also performed in DMF, but in this instance, only 2 equiv
ofEt3Nwasused(Scheme2).Again,thedesiredA83586C-GE3
hybrid 2 was readily formed; it was isolated in 41% overall
yield for the last three steps. To our great surprise, the
coupling of 13 with 158 only proceeded in low yield (17%).
However, as we shall see, the incorporation of an L-Pro unit
not only was detrimental to the chemistry but also greatly
attenuated antitumor potency.
Figure 3. Quantitative real-time PCR data for two rat breast cell
lines treated with the A83586C-citropeptin hybrid 1 and the
A83586C-GE3 hybrid 2 at a concentration of 10 nM in DMSO/
DMEM.
cantly, while Opn expression was markedly downregulated
by 1 and 2, ꢀ-catenin expression was left untouched
suggesting that 1 and 2 are somehow blocking the ꢀ-catenin/
TCF4 interaction.
Given Sakai’s report that GE3 can prevent E2F/DP
transcription factors from binding to target DNA,6 we next
examined whether synthetic A83586C could behave simi-
In the latter regard, the hybrids 1 and 2, and A83586C
itself, all potently inhibited the growth of a HCT-116 colon
(3) Mason, C. K.; McFarlane, S.; Johnston, P. G.; Crowe, P.; Erwin,
P. J.; Domostoj, M. M.; Campbell, F. C.; Manaviazar, S.; Hale, K. J.; El-
Tanani, M. Mol. Cancer Ther. 2008, 7, 548.
(4) Smitka, T. A.; Deeter, J. B.; Hunt, A. H.; Mertz, F. P.; Ellis, R. M.;
Boeck, L. D.; Yao, R. C. J. Antibiot. 1988, 41, 726.
(5) Hayaka, Y.; Nakagawa, M.; Toda, Y.; Seto, H. Agric. Biol. Chem.
1990, 54, 1007.
(6) Sakai, Y.; Yoshida, T.; Tsujita, T.; Ochiai, K.; Agatsuma, T.; Saitoh,
Y.; Tanaka, F.; Akiyama, T.; Akinaga, S.; Mizukami, T. J. Antibiot. 1997,
50, 659.
(9) (a) El-Tanani, M.; Barraclough, R.; Wilson, M. C.; Rudland, P. S.
Cancer Res. 2001, 61, 5619. (b) Jin, D.; El-Tanani, M.; Campbell, F. C.
Int. J. Oncol. 2006, 29, 1591. (c) Suzuki, M.; Mose, E.; Galloy, C.; Tarin,
Am. J. Pathol. 2007, 171, 682.
(7) Hale, K. J.; Lazarides, L. Org. Lett. 2002, 4, 1903.
(8) Hale, K. J.; Lazarides, L. Chem. Commun. 2002, 1832.
Org. Lett., Vol. 11, No. 3, 2009
739

larly. Active E2F transcription factors10 are generally formed
when one of four E2F proteins heterodimerize with one of
two DP proteins, and the pRb lies in its hyperphosphorylated
tumor-promoting state, due to upregulated cyclin/cdk growth
signaling. Under such circumstances, the E2F transcription
factor is free to switch on the transcription of a range of
genes involved in cancer cell growth and proliferation. To
probe whether A83586C could disrupt the E2F-DP interac-
tion, we performed pull down experiments using GST-E2F.
Figure 4. Results of GST-E2F1 (full length) pull down experiments
with 35S-labeled DP1 in the presence of A83586C dissolved in
DMSO that had been diluted to the desired concentration in the
pull down buffer.
The results (Figure 4) suggested that A83586C does not
perturb this interaction.
We next evaluated whether A83586C in DMSO/ RP-
MI1640 culture medium could inhibit E2F-mediated tran-
scription in HCT-116 colon carcinoma cells through an
induction of pRb hypophosphorylation (Figure 5). Following
Figure 6. Western blotting indicates that, after 24 h, synthetic
A83586C induces cyclin A and E2F-1 downregulation in HCT-
116 human colon carcinoma cells at 0.3 µM. p53 levels remain
unaffected.
in cancer patients.3,9,11 Upregulated ꢀ-catenin/TCF4 signal-
ing2,3 and deregulated E2F/DP transcription factor activity10
are also now strongly implicated in cancer onset and
progression. The fact that A83586C and its hybridized
analogues, 1 and 2, can potently and concurrently operate
on the ꢀ-catenin/TCF4/Opn and E2F/pRb oncological targets
makes these molecules of outstanding interest for probing
the functional interactions between these two signaling
pathways.
In summary, our combined biological data, gathered on a
range of A83586C analogues, and synthetic A83586C itself,
all very strongly point to the antitumor properties of this
class emanating, at least in part, from a beneficial modulation
of the human ꢀ-catenin/TCF4/Opn and E2F/pRb oncological
targets. With regards to the former, molecules of the A83586C
class are the most potent Wnt/ꢀ-catenin/TCF4 signaling
antagonists so far discoVered.2
Figure 5. After 24 h, western blotting indicates that A83586C in
DMSO/ RPMI1640 culture medium induces pRb-hypophosphory-
lation in HCT-116 human colon carcinoma cells at 0.3 µM.
exposure of such cells to a 0.3 µM solution of A83586C for
24 h, A83586C was indeed found to cause pRb hypophos-
phorylation.
Finally, the data in Figure 6 confirm that 0.3 µM solutions
of synthetic A83586C in DMSO/RPMI1640 culture medium
can strongly downregulate E2F1 protein expression in
HCT116 cells over a 6-24 h period. However, cyclin A is
only partially downregulated after 24 h of drug treatment,
and p53 levels are left essentially undisturbed.
Acknowledgment. We thank Novartis Pharma AG,
AVERT (Mrs. Annabel Kanabus), Cancer Research-UK, and
the EPSRC for their generous financial support.
Supporting Information Available: Full experimental
procedures and detailed spectral data of all key compounds
are reported. Copies of 500 MHz 1H and 125 MHz 13C NMR
spectra are provided along with IR spectra and HRMS data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Significantly elevated Opn expression is associated with
the most aggressive of metastatic human tumors, with high
Opn levels correlating closely with poor prognostic outcome
(10) Iaquinta, P. J.; Lees, J. A. Curr. Opin. Cell Biol. 2007, 19, 649.
(11) Rudland, P. S.; Platt-Higgins, A.; El-Tanani, M.; DeSilva Rudland,
S.; Barraclough, R.; Winstanley, J. H.; Howitt, R.; West, C. R. Cancer
Res. 2002, 62, 3417.
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