Structure of FD-895 revealed through total synthesis

Article abstract of DOI:10.1021/ol3023006

The total synthesis of FD-895 was completed through a strategy that featured the use of a tandem esterification ring-closing metathesis (RCM) process to construct the 12-membered macrolide and a modified Stille coupling to append the side chain. These stu

Full text of DOI:10.1021/ol3023006

ORGANIC
LETTERS
2012
Vol. 14, No. 21
5396–5399
Structure of FD-895 Revealed through
Total Synthesis
Reymundo Villa, Alexander L. Mandel, Brian D. Jones, James J. La Clair, and
Michael D. Burkart*
Department of Chemistry and Biochemistry, University of Calfornia, San Diego,
9500 Gilman Drive, La Jolla, California 92093-0358, United States
mburkart@ucsd.edu
Received August 17, 2012
ABSTRACT
The total synthesis of FD-895 was completed through a strategy that featured the use of a tandem esterification ring-closing metathesis (RCM)
process to construct the 12-membered macrolide and a modified Stille coupling to append the side chain. These studies combined with detailed
analysis of all four possible C16ÀC17 stereoisomers were used to confirm the structure of FD-895 and identify an analog with an enhanced
subnanomolar bioactivity.
First described in 1994, FD-895 (1, Figure 1) introduced
a family of 12-membered macrolides identified with potent
cytostatic activity during hypoxia response.1 A decade
later, studies at Eisai Co. Ltd. led to the isolation of related
macrolides including pladienolides B (2a) and D (2b).2 In
2007, the complete structure of 2b (Figure 1) was reported3
and confirmed by total synthesis.4 Fueled by a series of
mode of action studies,5 the entry into clinical trials of
E7107 (2c)6 sparked wide interest in the spliceosome as a
new chemotherapeutic target (Figure 1).7 The early sus-
pension of this trial suggests the need for further studies
into this potent class of macrolides. The planar structure
and in vitro cytotoxicity of FD-895 (1) was reported in
1994.1 We now report the use of total synthesis to identify
and validate the structure of FD-895 (1) and apply our
synthetic methods to identify analogs with enhanced ac-
tivity in select tumor cell lines.
Our studies began with evaluating the structure of FD-
895 (1) by NMR methods to naturally isolated material.
After screening solvents, we collected a 2D data set on 1 in
C6D6, as it provided optimal stability and peak resolution.
We first assigned the protons and carbons in 1 using a
combination of gCOSY, TOCSY, HSQC, and HMBC
data. We then applied proton coupling constants and
NOE correlations to evaluate the stereochemistry. Cou-
pling constant analyses confirmed the cis- or trans- rela-
tionships at C8ÀC9, C10ÀC11, C14ÀC15, C18ÀC19,
C20ÀC21, and C21ÀC22. Through-ring NOEs obtained
from NOESY spectra indicated that the stereochemistry
(1) Seki-Asano, M.; Okazaki, T.; Yamagishi, M.; Sakai, N.; Takayama,
Y.; Hanada, K.; Morimoto, S.; Takatsuki, A.; Mizoue, K. J. Antibiot.
(Tokyo) 1994, 47, 1395–1401.
(2) Mizui, Y.; Sakai, T.; Iwata, M.; Uenaka, T.; Okamoto, K.;
Shimizu, H.; Yamori, T.; Yoshimatsu, K.; Asada, M. J. Antibiot.
(Tokyo) 2004, 57, 188–196.
(3) Asai, N.; Kotake, Y.; Niijima, J.; Fukuda, Y.; Uehara, T.; Sakai,
T. J. Antibiot. (Tokyo) 2007, 57, 188–196.
(4) (a) Kanada, R. M.; Itoh, D.; Nagai, M.; Niijima, J.; Asai, N.;
Mizui, Y.; Abe, S.; Kotake, Y. Angew. Chem., Int. Ed. 2007, 46, 4350–
4355. (b) Kanada, R. M.; Itoh, D.; Nagai, M.; Niijima, J.; Asai, N.;
Mizui, Y.; Abe, S.; Kotake, Y. Angew. Chem., Int. Ed. 2007, 46, 8734.
(5) (a) Kotake, Y.; Sagane, K.; Owa, T.; Mimori-Kiyosue, Y.;
Shimizu, H.; Uesugi, M.; Ishihama, Y.; Iwata, M.; Mizui, Y. Nat. Chem.
Biol. 2007, 3, 570–575. (b) Kaida, D.; Motoyoshi, H.; Tashiro, E.;
Nojima, T.; Hagiwara, M.; Ishigami, K.; Watanabe, H.; Kitahara, T.;
Yoshida, T.; Nakajima, H.; Tani, T.; Horinouchi, S.; Yoshida, M. Nat.
Chem. Biol. 2007, 3, 576–583. (c) Yokoi, A.; Kotake, Y.; Takahashi, K.;
Kadowaki, T.; Matsumoto, Y.; Minoshima, Y.; Sugi, N. H.; Sagane, K.;
Hamaguchi, M.; Iwata, M.; Mizui, Y. FEBS J. 2011, 278, 4870–4880.
(6) Folco, E. G.; Coil, K. E.; Reed, R. Genes Dev. 2011, 25, 440–444.
(7) (a) Ward, A. J.; Cooper, T. A. J. Pathol. 2010, 220, 152–163. (b)
Fan, L.; Lagisetti, C.; Edwards, C. C.; Webb, T. R.; Potter, P. M. ACS
Chem. Biol. 2011, 6, 582–589.
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10.1021/ol3023006
Published on Web 10/16/2012
2012 American Chemical Society

at C6, C7, C10, and C11 was comparable to that in
pladienolide B (2a).3 While the C16ÀC17 diad was as-
signed as transÀ due to a 12.6 Hz coupling constant for
H16ÀH17, C17ÀC18 could not be assigned due to the
mid-ranged 5.5 Hz coupling constant for H17ÀH18. After
further inspection through examination of 1D and 2D
NMR spectra in different solvents, we concluded that
centers C3, C16 and C17 could not be unambiguously
assigned by NMR methods.
chain vinyl stannane B.9 Component B was derived by
applying a Marshall allenylÀaddition10 to C followed by
subsequent hydrostannylation, as this method could be
tuned to deliver each of the four C16ÀC17 stereoisomers.
Scheme 1. Construction of Side Chain 8 in 13 Steps and 19%
Overall Yield from L-Phe
We began with epoxy-aldehyde component C by devel-
oping a route to prepare aldehyde 88 in 13 steps from L-Phe
(Scheme 1). Application of a Crimmins auxiliary set the
C20ÀC21 stereodiad by providing non-Evans syn-adduct
3 with ∼5:1 de, which after chromatographic purification
provided pure 3 in 68% yield.11
Conversion of 3 to Weinreb’s amide 4 followed by
methylation provided amide 5. Reduction of 5 with
DIBAL-H, followed by a HornerÀWadsworthÀEmmons
olefination,12 provided ester 6. Potential isomerization
of the R-methyl group in the intermediate aldehyde was
avoided by conducting the HWE reaction immediately after
isolation of the aldehyde.
Reduction to 7 with DIBAL-H set the stage for installa-
tion of the C18ÀC19 epoxideby a Sharpless epoxidation,13
which after IBX oxidation14 completed the synthesis of
component 8. Encouragingly, the coupling constants in 8
were comparable to that at C18ÀC21 in 1 (see Supporting
Information).
Figure 1. NMR analysis of FD-895 (1), in C6D6 revealed key
COSY interactions, 3J-coupling constants, and select NOE
interactions. FD-895 (1) was the first member of a family of
12-memebered ring polyketides that includes pladienolides B
(2a) and D (2b), as well as clinical entry E7107 (2c).
While considerable effort was made to determine the
structure of 1 via X-ray crystallography and degradative
methods, all attempts proved unsuccessful. Based on prior
studies on 2aÀ2b,3,4 we tentatively assigned C3 as R. The
quest then remained to validate the stereochemistry within
the core (C3, C6, C7, C10, and C11), confirm the assign-
ment in the side chain terminus (at C18ÀC21), and identify
the C16ÀC17 stereodiad.
Our attention next turned to the core component A.
Building on our initial route4b,8 and approaches developed
by Kotake, Maier, and Skaanderup,4,15 we identified a
(10) (a) Marshall, J. A. J. Org. Chem. 2007, 72, 8153–8166. (b)
Marshall, J. A.; Perkins, J. J. Org. Chem. 1994, 59, 3509–3511.
(11) Crimmins, M. T.; King, B. W.; Tabet, E. A. J. Am. Chem. Soc.
1997, 119, 7883–7884.
(12) Wadsworth, W. S.; Emmons, W. D. J. Am. Chem. Soc. 1961, 83,
1733–1738.
We tailored our retrosynthesis such that installation
of the unassigned C16ÀC17 stereocenters occurred at the
end of the synthesis (see Abstract).4b,8 A Stille coupling
dissected the molecule into a core vinyl iodide A and side
(13) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974–
5976.
(8) Synthesis of intermediate 8 and a first-generation demonstration
of this approach can be found in: Mandel, A. L.; Jones, B. D.; La Clair,
J. J.; Burkart, M. D. Bioorg. Med. Chem. Lett. 2007, 17, 5159–5164.
(9) Stille, J. K.; Groh, B. L. J. Am. Chem. Soc. 1987, 109, 813–817.
(14) More, J. D.; Finney, N. S. Org. Lett. 2002, 4, 3001–3003.
€
(15) (a) Muller, S.; Mayer, T.; Sasse, F.; Maier, M. E. Org. Lett. 2011,
13, 3940–3943. (b) Skaanderup, P. R.; Jensen, T. Org. Lett. 2008, 10,
2821–2124.
Org. Lett., Vol. 14, No. 21, 2012
5397

stereoselective route to macrolide 21 that began with
Brown addition16 of the MEM ether 10 to aldehyde 9 in
order to set the C7 stereocenter in 11 (Scheme 2).
workup. We next applied an esterification and RCM
protocol derived from our prior synthetic endeavors.19,8
Acid 17 was coupled to alcohol 18 to afford ester 19. The
conversion from 14 to 19 was ideally conducted in a single
direct sequence without long-term storage or purification.
Removal of the PMP acetal enabled the preparation of
lactone 20 by means of an RCM reaction using HoveydaÀ
Grubbs second generation catalyst.20 The desired core unit
21 was obtained by acylation of the secondary carbinol at
C7 and TBS deprotection at C3.
Scheme 2. Synthesis of Core 21 in 14 Steps and 2% Yield
The final objective in our synthesis involved the assem-
bly of components 8 and 21 to prepare the desired
C16ÀC17 stereoisomer in FD-895 (1). As the stereochem-
istry at C16ÀC17 was not defined, we explored the versa-
tile allenylstannane methods developed by Marshall to
prepare each of the possible terminal alkynes 24a, 24b,
24c, and 24d (Scheme 3).10 Allenylstannanes 23a and
23b were prepared from the respective mesylates 22R
(Scheme 3) and 22S (not shown). Addition of 23a or 23b
to 8 in the presenceof BF3•Et2O led tothe formation of 24b
or 24d, respectively, as a single diasteromeric product
(Scheme3). NMR analysesclearlydemonstratedthatthese
materials contained a cis-configuration at C16ÀC17, as
given by a 3JHH value of 4À5 Hz.
Alkynes 24b and 24d were then hydrostannylated21 to
their corresponding vinylstannanes sequentially coupled
to core 21 under modified Stille conditions8 to afford the
cis-isomers 1RS and 1SR from 24b and 24d, respectively
(Scheme 3). NMR studies indicated that neither matched 1
(Figure 1). 1H NMR, gCOSY, 13C NMR data confirmed
that while 1RS and 1SR were isomers of 1, protons H16
and H17 were most likely trans, as exemplified by a 3JHH
value of 12.6 Hz in 1 versus 4À5 Hz in 1RS and 1SR.
We then shifted our focus to the two trans-isomers 1SS
and 1RR. Using a mixture ofInI and Pd(OAc)2,10b we were
able to obtain alkyne 24a as a single product (>99% de)
and in sufficient yield from 22R. The structure of 24a
was confirmed by a combination of 1H NMR, 13C NMR,
gCOSY, and HRMS spectral data. Hydrostannylation
and Stille coupling to21 afforded trans-isomer 1SS. Again,
NMR data collected from 1SS (Figure 2) did not match 1.
Unfortunately, application of the InI and Pd(OAc)2
conditions to 22S failed to provide alkyne 24c. After
screening conditions, we found that treating mixtures of
23b and 8 with SnCl4 at À78 °C in hexane10b provided 24c
in 83% yield (Scheme 3). Hydrostannylation 24c followed
by Stille coupling to 21 afforded the final isomer 1RR.
Fortunately, the spectral properties of this isomer matched
those of the natural material (Figure 2), confirming the
synthesis of FD-895 (1) in 30 total steps, with the longest
linear sequence of 15 steps. All but one of the steps, pre-
paration of 3, occurred with highstereocontrol (>99% de).
Finally, cytotoxicity analyses via the MTT assay22 in-
dicated that all four isomers displayed sub-nM to nM
We then used this C7 center to guide the construction at
C6 by oxidation of 11 to ketone 12, followed by a chelateÀ
controlled addition17 of MeMgBr, to afford adduct 13.
Treatment of 13 with p-anisaldehyde dimethylacetal and
ZnBr2 both converted the MEM ether to PMP acetal and
deprotected the TBS ether in one operation, affording
carbinol 14.
At this stage, we were set to install the C3 center
(Scheme 2). Oxidation of 14 to aldehyde 15, followed by
an acetate aldol addition of the Sammakia auxiliary 16,18
provided acid 17 after TBS protection and hydrolytic
(16) Ramachandran, P. V.; Liu, H.; Ram Reddy, M. V.; Brown,
H. C. Org. Lett. 2003, 5, 3755–3757.
(17) Still, W. C.; McDonald, J. H. Tetrahedron Lett. 1980, 21, 1031–
1034.
(20) Stewart, I. C.; Ung, T.; Pletnev, A. A.; Berlin, J. M.; Grubbs,
R. H.; Schrodi, Y. Org. Lett. 2007, 9, 1589–1592.
(21) Ichinose, Y.; Oda, H.; Oshima, K.; Utimoto, K. Bull. Chem. Soc.
Jpn. 1987, 60, 3468–3470.
(18) Zhang, Y.; Phillips, A. J.; Sammakia, T. Org. Lett. 2003, 6, 23–
25.
(19) Ko, K. S.; Alexander, M. D.; Fontaine, S. D.; Biggs-Houck,
J. E.; La Clair, J. J.; Burkart, M. D. Org. Biomol. Chem. 2010, 8, 5159–
5165.
(22) Mosmann, T. J. Immunol. Meth. 1983, 65, 55–83.
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Org. Lett., Vol. 14, No. 21, 2012

Scheme 3. Syntheses of the Four C16ÀC17 Stereoisomers 1SS, 1RS, 1RR, and 1SR
activity when screened against the HCT-116 tumor cell line
(IC50 value of 23.0 ( 1.2 nM for 1SS, 0.80 ( 0.05 nM for
1RS, 24.2 ( 0.9 nM for 1RR, and 3.7 ( 0.2 nM for 1SR).
While the activity for 1RR matched that of natural FD-895
(IC50 value of 23.5 ( 0.2 nM), isomers with the natural
R-configuration at C16 were less active than those with S
at C16. Additional activity was also observed when C17
was left in its naturalR configuration. Remarkably, isomer
1RS was nearly 25 times more active than FD-895 (1),
providing a strong foundation for further medicinal chem-
ical optimization.
Acknowledgment. This work was financially supported
by the American Cancer Society (RSG-06-011-01-CDD)
and the NIH (3R01GM086225-01S1).
Supporting Information Available. Experimental pro-
cedures, characterization data for all new compounds,
and full acknowledgments. This material is available free
of charge via the Internet at http://pubs.acs.org.
Figure 2. 1H NMR spectra of synthetic isomers 1SS, 1RS, 1RR,
1SR in C6D6 indicated that 1RR matched natural FD-895 (1).
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 21, 2012
5399