Verticilide: Elucidation of absolute configuration and total synthesis

Article abstract of DOI:10.1021/ol0623365

Verticilide (1) is a 24-membered cyclic depsipeptide isolated from the culture broth of Verticillium sp. FKI-1033. It inhibits ryanodine binding to ryanodine receptor (RyR) and has insecticidal activity. The stereochemistry of 2-hydroxyheptanoic acid in v

Full text of DOI:10.1021/ol0623365

ORGANIC
LETTERS
2006
Vol. 8, No. 24
5601-5604
Verticilide: Elucidation of Absolute
Configuration and Total Synthesis
Souichi Monma, Toshiaki Sunazuka, Kenichiro Nagai, Takahiro Arai,
Kazuro Shiomi, Ryosuke Matsui, and Satoshi Ohmura*
Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences,
Kitasato UniVersity and The Kitasato Institute, 5-9-1 Shirokane, Minato-ku,
Tokyo 108-8641, Japan
omura-s@kitasato.or.jp
Received September 22, 2006
ABSTRACT
Verticilide (1) is a 24-membered cyclic depsipeptide isolated from the culture broth of Verticillium sp. FKI-1033. It inhibits ryanodine binding
to ryanodine receptor (RyR) and has insecticidal activity. The stereochemistry of 2-hydroxyheptanoic acid in verticilide was elucidated by
chiral HPLC analysis of the degradation product 6 and synthetic (+) and (−)-6. We also describe the practical total synthesis of verticilide.
A plant alkaloid, ryanodine, is an agonist of ryanodine
receptor (RyR) and exhibits insecticidal activity. RyR is a
Ca2+-release channel of the sarcoplasmic reticulum. There
are three isoforms, RyR1, RyR2, and RyR3, in mammalian
RyR. However, insects have a single but distinct RyR.
Therefore, insect RyR is a potential target for new insecti-
cides.1 In the course of screening for ryanodine binding
inhibitors, verticilide 1 was isolated from the culture broth
of Verticilium sp. FKI-10332 (Figure 1). Verticilide 1 is a
new 24-membered cyclic depsipeptide consisting of four
2-hydroxyheptanoic acids and four N-methyl-L-alanines.
Verticilide 1 selectively inhibited the binding of ryanodine
to insect RyR with an IC50 value of 4.2 µM (mammalian
RyR IC50 ) 54 µM). Verticilide 1 is expected to be a
potential lead compound for insecticides with a new mode
(1) (a) Fill, M.; Copello, J. A. Physiol. ReV. 2002, 82, 893-922. (b)
Schmitt, M.; Turberg, A.; Londershausen, M; Dorn, A. Pestic. Sci. 1996,
48, 375-378. (c) Xu, X.; Bhat, M. B.; Nishi, M.; Takeshima, H.; Ma, J.
Biophys. J. 2000, 78, 1270-1281.
(2) Ohmura, S.; Shiomi, K.; Masuma, R. Patent PCT WO2004044214,
2004.
Figure 1. Structure of verticilide (1).
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Published on Web 10/31/2006

of action. However, the stereochemistry of 2-hydroxy-
heptanoic acid in 1 could not be elucidated by spectroscopic
analysis because of the small value of [R]D.3
Scheme 2. Resolution of Verticilide into Two Building
Blocks
A practical method for synthesis of 1 is required to allow
further biological evaluation, and the synthesis of its analogs
is impotant for the study of the structure-activity relation-
ships. Herein we report the elucidation of stereochemistry
of 2-hydroxyheptanoic acid by chiral HPLC analysis and an
efficient total synthesis of verticilide 1, which can be applied
for scaled-up production of verticilide.
In investigating the stereochemistry of 2-hydroxyheptanoic
acid and to confirm the absolute configulation of 1, we
synthesized the enantiomers of 2-hydroxyheptanoic acid,
which can be prepared from L- and D-malic acids, respec-
tively, and compared them with a fragment obtained by
degradation of natural product directly. Synthesis of (S)-2-
hydroxyheptanoic acid benzyl ester 6 is summarized in
Scheme 1.
with Cs2CO3 and BnBr to provide benzyl ester 6 (Scheme
1
2). H and 13C NMR and MS spectral data of 6 from the
natural product were found to be identical to those of
synthetic 6. The result of chiral HPLC analysis is shown in
Figure 2.
Scheme 1. Preparation of (S)-2-Hydroxyheptanoic Acid (-)-6
l-Malic acid 2 was converted into aldehyde 3 using a
method reported by Dutton.4 Wittig olefination of 3 with
n-propyltriphenylphosphonium bromide5 and NaHMDS gave
4, which was used without further purification because of
its instability. Hydrogenation of 4 followed by transesteri-
fication of 5 with benzyl alcohol and p-TsOH condition
afforded (S)-2-hydroxyheptanoic acid benzyl ester (-)-6.
Furthermore, we synthesized enantiomer (+)-6 from D-malic
acid using the same procedure.
Degradation of 1 in 6 M HCl at 110 °C afforded
2-hydroxyheptanoic acid 7 and N-methyl-L-alanine 8, which
was determined by amino acid analysis. Then, 7 was treated
Figure 2. HPLC analysis of 2-hydroxyheptanoic acid. Column,
Daicel OD10C; column temperature, 10 °C; eluent, n-hexane/2-
propanol ) 400:15; detection, UV 210 nm; flow rate, 1 mL/min.
(a) Fragment 6 from natural product; (b) 1:1 mixture of fragment
6 and synthetic (-)-6; (c) 1:1 mixture of fragment 6 and synthetic
(+)-6.
(3) (a) Nugteren, D. H.; Van Dorp, D. A.; Bergstrom, S.; Hamburg, M.;
Samuelsson, B. Nature 1966, 212, 38. (b) Hauser, F. M.; Coleman,
M. L.; Huffman, R. C.; Carroll, F. I. J. Org. Chem. 1974, 39, 3426. (c)
Townsend, C. A.; Christensen, S. B. Tetrahedron Lett. 1986, 27, 887-
888.
(4) Dutton, F. E.; Lee, B. H.; Johnson, S. S.; Coscarelli, E. M.; Lee, P.
H. J. Med. Chem. 2003, 46, 2057-2073.
(5) (a) Ainai, T.; Matsuumi, M.; Kobayashi, Y. J. Org. Chem. 2003, 68,
7825-7832. (b) Stockman, R. A.; Sinclair, A.; Arini, L. G.; Szeto, P.;
Hughes, D. L. J. Org. Chem. 2004, 69, 1598-1602.
We could achieve separation of synthetic enantiomers
(+)-6 and (-)-6 by using chiral HPLC. After several
attempts, enantiomers (+)-6 and (-)-6 were separated by
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Org. Lett., Vol. 8, No. 24, 2006

imidazole, transesterification with BnOH7 afforded 14 in
excellent yield. Finally, deprotection of the TBDMS group
by 4 M HCl/dioxane gave (R)-2-hydroxyheptanoic acid
benzyl ester (+)-6 efficiently. As the synthetic route of (+)-6
was developed, we turned to total synthesis of verticilide 1.
Esterification of (+)-6 and alanine 158 with EDCI in the
presence of DMAP gave the key intermediate 16 in quantita-
tive yield. A benzyl group of one 16 was deprotected and a
Boc group of another 16 was deprotected by 4 M HCl/
dioxane. Condensation of 17 and 18 with PyBrop and
i-Pr2NEt produced tetradepsipeptide 19 in 98% yield for two
steps. Use of other coupling reagents instead of PyBrop
reduced the yields. Octadepsipeptide 22 was prepared in
similar manner using 19 in quantitative yield. Finally,
deprotection of the benzyl and Boc group, followed by the
cyclization of linear depsipeptide in highly diluted CH2Cl2
by PyBop and i-Pr2NEt, provided verticilide (1) in 94% yield
for 3 steps (Scheme 4). Its spectral properties were identical
in all respects (1H and 13C NMR, IR, HR-FABMS, optical
rotation, TLC, HPLC, and bioactivity) to those of the natural
product.
Scheme 3. Preparation of (R)-2-Hydroxyheptanoic Acid
Benzyl Ester (+)-6
Daicel OD10C column with an eluent of n-hexane/2-propanol
(400:15). A 1:1 mixture of fragment 6 and synthetic (+)-6
was subjected to the chiral HPLC analysis and produced
duplicate peaks (Figure 2c). This result indicated that
fragment 6 was identical with synthetic (+)-6. Therefore we
confirmed that the absolute configuration at C2 in 2-hy-
droxyheptanoic acid is R. However, this synthetic route of
(R)-2-hydroxyheptanoic acid appeared to be inadequate for
practical synthesis of verticilide, because the overall yield
of (+)-6 was very low and D-malic acid was expensive as a
starting material. Accordingly, we employed diastereoselec-
tive oxidation reported by Evans6 for an effective synthesis
of (+)-6 (Scheme 3).
In conclusion, the elucidation of the stereochemistry
for 2-hydroxyheptanoic acid and the total synthesis of
verticilide were completed. The longest linear synthetic
sequence for the synthesis of verticilide comprised 13 steps
and proceeded in 66% overall yield (corresponding to a 97%
average yield per step). Our synthesis used a diastereo-
selective Davis oxidation for the effective synthesis of
2-hydroxyheptanoic acid. We have synthesized 10 g of
verticilide by using this synthetic route for further bio-
logical evaluations. Studies on the mode of action and the
structure-activity relationships of verticilide are currently
under way.
Condensation of (R)-oxazolidinone 9 and heptanoyl chlo-
ride 10, followed by diastereoselective oxidation of the imide
with NaHMDS and Davis reagent 12 gave 13 in 94% yield.
After protection of the hydroxyl group by TBDMSCl and
Scheme 4. Total Synthesis of Verticilide
Org. Lett., Vol. 8, No. 24, 2006
5603

Acknowledgment. This work was supported by a Grant
from the 21st Century COE Program, Ministry of Education,
Culture, Sports, Science and Technology. We also thank Ms.
A. Nakagawa, Ms. C. Sakabe, and Ms. N. Sato (School of
Pharmaceutical Sciences, Kitasato University) for the various
instrumental analysis.
Supporting Information Available: Experimental pro-
cedures and spectral data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(6) Evans, D. A.; Morrissey, M. M.; Dorow, R. L. J. Am. Chem. Soc.
1985, 107, 4346-4348.
(7) Ghosh, A. K.; Liu, C. J. Am. Chem. Soc. 2003, 125, 2374-2375.
(8) Schutkowski, M.; Mrestani-Klaus, C.; Neubert, K. Int. J. Peptide
Protein Res. 1995, 45, 257-265.
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