A synthetic strategy for the cyclodepsipeptide core of the antitumor antibiotic verucopeptin.

Article abstract of DOI:10.1021/ol016440s

[reaction: see text]. An efficient [2 + 2 + 2]-fragment condensation strategy is described for obtaining the cyclodepsipeptide core of verucopeptin. The 19-membered macrocycle was established through a Carpino HATU mediated macrolactamization, which proce

Full text of DOI:10.1021/ol016440s

ORGANIC
LETTERS
2001
Vol. 3, No. 18
2927-2930
A Synthetic Strategy for the
Cyclodepsipeptide Core of the
Antitumor Antibiotic Verucopeptin
Karl J. Hale,* Linos Lazarides, and Jiaqiang Cai
The Christopher Ingold Laboratories, Department of Chemistry,
UniVersity College London, 20 Gordon Street, London WC1H 0AJ, United Kingdom
k.j.hale@ucl.ac.uk
Received July 16, 2001
ABSTRACT
An efficient [2 + 2 + 2]-fragment condensation strategy is described for obtaining the cyclodepsipeptide core of verucopeptin. The 19-
membered macrocycle was established through a Carpino HATU mediated macrolactamization, which proceeded in good yield under high-
dilution conditions.
Verucopeptin is a powerful antitumor antibiotic isolated from
the fermentation broths of Philippino soil microorganism
Actinomadura Verrucospora.1 It significantly prolongs the
life expectancy of mice with B16 melanoma, conferring a
162% life extension when given at the low dosage of 2 mg/
kg/day. Structurally, verucopeptin belongs to the A83586C/
GE3 family2 of pyranylated cyclodepsipeptides, which also
count azinothricin3 and citropeptin4 among their number. At
present, the relative and absolute stereochemistry of veru-
copeptin remains unknown, notwithstanding extensive chemi-
cal and spectroscopic investigations by the Bristol-Myers1a,b
and Concordia University1c groups.
It is thought that molecules of the A83586C/GE3 class
function as anticancer agents by selectively preventing
deregulated E2F-DP transcription factors from binding to and
activating genes encoding for proteins involved in cell growth
and proliferation (e.g., DNA-polymerase-R, dihydrofolate
reductase, thymidine kinase, etc.).2b Understanding how they
do this is currently a topic of great interest, as it could
potentially lead to the detailed characterization of an
important new drug target. In this regard, several modes of
action are presently being considered. One is that members
of the A83586C family are binding to individual E2F and
DP proteins and preventing them from heterodimerizing. The
latter is considered essential for high-affinity binding to target
DNA, which in turn is a prerequisite for efficient transcrip-
tional activation.5 Alternatively, molecules of this class might
be complexing to individual E2F-DP heterodimers and
sterically blocking their interaction with various target genes.
E2F/DP-drug binding could also be halting the recruitment
(1) Verucopeptin. (a) Isolation and biological properties: Nishiyama, Y.;
Sugawara, K.; Tomita, K.; Yamamoto, H.; Kamei, H.; Oki, T. J. Antibiot.
1993, 46, 921. (b) Structure determination: Suguwara, K.; Toda, S.;
Moriyama, T.; Konishi, M.; Oki, T. J. Antibiot. 1993, 46, 928. (c)
Biosynthesis and partly revised spectroscopic assignments: Tsantrizos, Y.
S.; Shen, J.; Trimble, L. A. Tetrahedron Lett. 1997, 38, 7073.
(2) (a) A83586C: 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. (b)
GE3: 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.
(3) Azinothricin: Maehr, H.; Liu, C.; Palleroni, N. J.; Smallheer, J.;
Todaro, L.; Williams, T. H.; Blount, J. F. J. Antibiot. 1986, 39, 17.
(4) Citropeptin: Hayakawa, Y.; Nakagawa, M.; Toda, Y.; Seto, H. Agric.
Biol. Chem. 1990, 54, 1007.
10.1021/ol016440s CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/09/2001

of “essential” transcriptional coactivators such as the CREB
transcription factor.6
with A83586C suggested that a [2 + 2 + 2]-fragment
condensation between 7, 6, and 5 (in that order) could take
us toward the cyclization precursor 4, which could then be
macrolactamised between its Gly and Sar termini without
the risk of epimerization. Global deprotection would sub-
sequently yield 3, appropriately tailored for future chemose-
lective coupling to a range of activated esters or acid
chlorides, which could include 2, for a total synthesis of
verucopeptin itself. Such a “biogenetically modeled” cou-
pling strategy had previously been used to great effect during
our total syntheses of A83586C7 and 4-epi-A83586C,8 and
so it was anticipated that such an approach would proceed
equally satisfactorily here. With this as background, we will
now present details of our route to 3.
Some time ago, we initiated a total synthesis program,7,8
on the A83586C-GE3-verucopeptin family of antitumor
antibiotics, for the purpose of preparing novel analogues that
could probe these mechanistic issues further. It is hoped that
our analogue work will lead to the identification of several
new, structurally less elaborate, E2F inhibitors with enhanced
antineoplastic effects. In furtherance of these goals, we now
report a synthetic strategy for the cyclodepsipeptide core of
verucopeptin.
Although eight diastereomeric possibilities exist for this
core region, the fact that all other family members have a
(3R)-piperazic acid unit linked to a (2S,3S)-3-hydroxyleucine
suggests that an identical sequence prevails in verucopeptin.
As a consequence, we made diastereoisomer 3 the object of
our initial synthetic planning (Scheme 1). Past experience
Early attention was directed at the preparation of dipeptide
6 (Scheme 2). For this, the protected hydroxamic acid 9 was
Scheme 2. Preparation of Dipeptide 6a
Scheme 1. Retrosynthetic Planning for the Verucopeptin
Cyclodepsipeptide Core
a Reagents and conditions: (a) (COCl)2 (20 equiv), C6H6 (0.55
M), rt (20 min), then warm to 50 °C, stir 1.5 h. (b) Ph2CdN2 (2
equiv), Me2CO (0.4 M), rt, 24 h. (c) AgCN (1.5 equiv), C6H6 (0.43
M), 70 °C, 40 min. (d) CF3CO2H (36 equiv), PhOH (1.5 equiv),
CH2Cl2 (0.09 M), 0 °C, 2 h.
needed; it was synthesized according to the method of Kolasa
and Chimiak.9 Its acid grouping was temporarily blocked as
a diphenylmethyl ester,10 and the remaining NH coupled to
the known piperazic acid chloride 10 under AgCN-assisted
amidation conditions.7,8,11 Dipeptide 12 was isolated in
excellent yield (89-96%). The diphenylmethyl ester group-
ing was then cleaved from 12 with trifluoroacetic acid (TFA)
to obtain acid 6.
Dipeptide 7 was synthesized by coupling 13 with 14 under
standard DCC conditions12 and cleaving the Z-group from
15 by catalytic hydrogenolysis (Scheme 3). A tert-butyl
carbazate group was selected for protecting the Gly acid
residue, as this circumvented diketopiperazine formation
2928
Org. Lett., Vol. 3, No. 18, 2001

17 was isolated after the Fmoc group14 had been cleaved
from 16 with diethylamine in MeCN.
Scheme 3. Route to Tetrapeptide 17a
The next fragment assembled was acid chloride 5. It was
prepared from the known amino acids 18 and 19 according
to Scheme 4. The key steps were a DCC-DMAP mediated
Scheme 4. Route to Verucopeptin Cyclodepsipeptide 3a
a Reagents and conditions: (a) DCC (1.05 equiv), DMAP (0.2
equiv), CH2Cl2 (0.25 M), rt, 24 h. (b) H2, 20% Pd(OH)2/C (0.2
equiv), MeOH (0.26 M), rt, 8 h. (c) BOP-Cl (1.3 equiv), Et3N (2
equiv), CH2Cl2 (0.07 M), -20 °C (20 min), then warm to 0 °C, 3
h. (d) Et2NH (40 equiv), MeCN (0.12 M), rt, 20 min.
during hydrogenolysis of the Z-group; the latter was prob-
lematic when ester protecting groups were employed in the
sequence. With 6 and 7 in hand, their union was effected
with BOP-Cl13 and Et3N at low temperature. Tetrapeptide
(5) Reviews on E2F/DP complexes, cell cycle, and cancer: (a) Nevins,
J. R. Human Mol. Gen. 2001, 10, 699. (b) Yamasaki, L. Biochim. Biophys.
Acta 1999, 1423, M9-M15. (c) Bandara, L. R.; Girling, R.; La Thangue,
N. B. Nature Biotech. 1997, 15, 896. (d) Monograph: Transcriptional
Control of Cell Growth, The E2F Gene Family. Farnham, P. J., Ed.,; Curr.
Top. Microbiol. Immunol. 1996, 208. (e) Lam, E. W.; La Thangue, N. B.
Curr. Opin. Cell. Biol. 1994, 6, 859. (f) Johnson, D. G.; Schneider-
Broussard, R. Front. Biosci. 1998, 3, d447. (g) Helin, K. Curr. Opin. Gen.
DeV. 1998, 8, 28.
a Reagents and conditions: (a) DCC (1.1 equiv), DMAP (1 equiv),
CH2Cl2 (0.17 M), rt, 24 h. (b) (Ph3P)4Pd (0.1 equiv), morpholine
(8.5 equiv), THF (0.17 M), rt, 30 min. (c) (COCl)2 (35 equiv), C6H6
(0.3 M), rt, 2.5 h. (d) AgCN (1.5 equiv), C6H6 (0.15 M), 75-80
°C, 2-3 min. (e) CF3CO2H (200 equiv), CH2Cl2 (0.08 M), rt, 2 h.
(f) NBS (2 equiv), THF/H2O (1:1) (0.04 M), rt, 2 h. (g) Add 22
and NEM (13.5 equiv) in CH2Cl2 over 8 h to HATU (10 equiv) in
CH2Cl2 at 0 °C, then warm to rt, 48 h (total final CH2Cl2
concentration = 0.0004 M). (h) Zn (85 equiv), AcOH/H2O (10:1)
(0.02 M), rt, 1.5 h. (i) Z-Cl (3 equiv), 10% sat. aq. NaHCO3, CH2Cl2
(0.13 M), rt, 1 h. (j) H2, 10% Pd/C (Aldrich wet Degussa type)
(0.1 equiv), MeOH (0.01 M), HCl in MeOH (1 equiv), 24 h.
(6) Magnaghi-Jaulin, L.; Ait-Si-Ali, S.; Harel-Bellan, A. Cell DeVel. Biol.
1999, 10, 197.
(7) Total synthesis of A83586C: (a) Hale, K. J.; Cai, J. J. Chem. Soc.,
Chem. Comm 1997, 2319. (b) Hale, K. J.; Cai, J.; Delisser, V. M.
Tetrahedron Lett. 1996, 37, 9345-9348. (c) Hale, K. J.; Cai, J. Tetrahedron
Lett. 1996, 37, 4233-4236. (d) Hale, K. J.; Cai, J.; Manaviazar, S.; Peak,
S. A. Tetrahedron Lett. 1995, 36, 6965-6968. (e) Hale, K. J.; Delisser, V.
M.; Yeh, L.-K.; Peak, S. A.; Manaviazar, S.; Bhatia, G. S. Tetrahedron
Lett. 1994, 35, 7685-7688. (f) Hale, K. J.; Manaviazar, S.; Delisser, V.
M. Tetrahedron 1994, 50, 9181-9188. (g) Hale, K. J.; Bhatia, G. S.; Peak,
S. A.; Manaviazar, S. Tetrahedron Lett. 1993, 34, 5343-5346. (h) For a
detailed account of our A83586C synthesis and the synthesis of other
complex cyclodepsipeptides, see: Hale, K. J.; Bhatia, G. S.; Frigerio, M.
The Chemical Synthesis of Natural Products; Hale, K. J., Ed.; Sheffield
Academic Press: Sheffield, 2000; Chapter 12, p 349.
O-esterification7a,8 to obtain 20, a Kunz-Waldmann O-
deallylation reaction with Pd(0) and morpholine to unmask
the acid,15 and a chlorination with excess oxalyl chloride in
benzene to obtain acid chloride 5. The chemoselective
(8) Total synthesis of 4-epi-A83586C: Hale, K. J.; Cai, J.; Williams, G.
Synlett 1998, 149-152.
(9) Kolasa, T.; Chimiak, A. Tetrahedron 1974, 30, 3591.
(10) Stelakatos, G. C.; Paganou, A.; Zervas, L. J. Chem. Soc. C 1966,
1191.
Org. Lett., Vol. 3, No. 18, 2001
2929

N-acylation of 17 with 5 was effected by heating in benzene
at 80 °C for 2-3 min in the presence of AgCN. Prolonging
the reaction much beyond this time frame is not recom-
mended, as it generally leads to a diminution in the yield of
hexadepsipeptide 21.
chromatography. All the benzyl protecting groups were then
cleaved from 24 by hydrogenolysis under mildly acidic
conditions. The structure of 3 was confirmed by its high-
resolution FAB mass spectrum, which contained an [M]+
ion at m/e 514.2600 (calcd for C21H36N7O8 [M]+, 514.2625),
and by its IR spectrum, which showed an intense CdO
stretching absorption at 1746 cm-1 indicative of a cyclodep-
sipeptide ester linkage. Additional evidence to support the
assigned structure was provided by the room temperature
500 MHz 1H NMR spectrum of 3 in CD3OD, which clearly
showed that it was a single compound, and that it existed as
one conformer.
We now had to address the potentially troublesome issue
of having to convert compound 21 into the hexapeptide
amino acid 22. This was accomplished nonproblematically
by TFA treatment and by chemoselective oxidation of the
Gly hydrazide grouping (in the presence of the Sar amine)
with N-bromosuccinimide in aqueous THF.16 It is presumed
that the latter reaction selectively converts the acyl hydrazine
into a highly reactive acyl diazene, which immediately is
intercepted by the massive excess of water that is present.
The success of this transformation underpinned our eventual
synthesis of 3. The macrolactamisation of 22, to obtain 23,
was achieved in high yield using Carpino’s HATU reagent,17
under conditions of high dilution. The Troc group was now
detached from 23 with Zn dust in aqueous acetic acid. To
facilitate the final purification of 3, we temporarily capped
the product amine with a Z-group under classical Schotten-
Baumann conditions and carefully purified 24 by SiO2 flash
In conclusion, we have developed a highly efficient
chemical pathway to molecules with the verucopeptin core
structure. We anticipate that our efforts will expedite future
parallel-synthesis work aimed at identifying novel new E2F-
inhibitory anticancer drugs. Further work in this direction
and on the synthesis of a diastereoisomer of verucopeptin
will be presented in due course.
Acknowledgment. We thank AVERT for a postgraduate
studentship (L.L.), Pfizer for generous financial support, and
ULIRS for HRMS measurements.
(11) Total synthesis of L-156,602: Durette, P. L.; Baker, F.; Barker, P.
L.; Boger, J.; Bondy, S. S., Hammond, M. L.; Lanza, T. J.; Pessolano, A.
A.; Caldwell, C. G. Tetrahedron Lett. 1990, 31, 1237.
(12) Sheehan, J. C.; Hess, G. P. J. Am. Chem. Soc. 1955, 77, 1067.
(13) Tung, R. D.; Rich, D. H. J. Am. Chem. Soc. 1985, 107, 4342.
(14) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404.
(15) Kunz, H.; Waldmann, H. Angew. Chem., Int. Ed. Engl. 1984, 23,
71.
Supporting Information Available: Low-resolution mass
spectra and 500 MHz 1H and 125 MHz 13C NMR spectra of
all new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(16) Cheung, H. T.; Blout, E. R. J. Org. Chem. 1965, 30, 315.
(17) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4937.
OL016440S
2930
Org. Lett., Vol. 3, No. 18, 2001