Confirmation of the stereostructure of (+)-cytostatin by fluorous mixture synthesis of four candidate stereoisomers

Article abstract of DOI:10.1002/anie.200704893

Mix and match: Four closely related candidate isomers for cytostatin have been synthesized by using the technique of fluorous mixture synthesis. Comparison of NMR, thin-layer chromatography, and optical rotation data enabled the three isomers that were not cytostatin to be identified. Thus, by a process of elimination (disproof), the isomer corresponding to cytostatin was determined, and the 4S,5S,6S,9S,10S,11S configuration confirmed. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200704893

Communications
DOI: 10.1002/anie.200704893
Natural Products
Confirmation of the Stereostructure of (+)-Cytostatin by Fluorous
Mixture Synthesis of Four Candidate Stereoisomers**
Won-Hyuk Jung, Sabrina Guyenne, Concepción Riesco-Fagundo, John Mancuso,
Shuichi Nakamura, and Dennis P. Curran*
Dedicated to Professor Yoshito Kishi on the occasion of his 70th birthday
Cytostatin (1) is a potent and selective inhibitor of protein
phosphatase 2A that was isolated from the cultured broth of
Streptomyces sp. by Ishizuka and co-workers (Scheme 1).[1] It
anti at C5/C6 and C10/C11 by the research groups of
Waldmann and Boger by comparison of key features of the
1H NMR spectra of cytostatin with those of relevant
models.[2,3] This approach lowered the number of structure
candidates down to four, namely: 1ss, 1rs, 1sr, and 1rr.[4]
Based on an analogy to fostriecin 2[5] and related compounds
(which share three stereocenters with 1), the research groups
of Waldmann and Boger independently synthesized
(4S,5S,6S,9S,10S,11S)-1 (hereafter called 1ss), and concluded,
by comparison of spectroscopic, physical, and biological
properties, that this was the natural product.
We have recently commented on the logic of proof and
disproof of stereostructures by comparison of synthetic and
natural samples.[6] If two or more stereoisomers of a natural
product can reasonably be expected to have substantially
identical spectra, then a rigorous assignment of the structure
should include proof that the candidate isomer matches the
natural product and, more importantly, proof that the other
isomers do not. Acetogenins, such as the murisolins have very
remote groups of stereocenters (10 or more methylene groups
apart), so it can be expected that diastereomers might exhibit
substantially identical spectra.[6] In contrast, the two groups of
stereocenters in cytostatin (1) are only insulated by an
ethylene group. Will enantiomers 1ss and 1rr have different
spectra from their diastereomers 1rs and 1sr? To answer this
question, and thereby to prove which three isomers were not
cytostatin, we undertook the fluorous mixture synthesis[7,8] of
all four isomers of 1.
Scheme 1. Candidate structures of cytostatin (1) and structure of
fostriecin (2).
inhibits lung metastasis of melanoma cells in mice and
displays potent cytotoxic activity toward leukemia cell lines
(inhibitory concentration; IC50 = 42–65 nm). Ishizuka and co-
workers assigned the two-dimensional structure (constitu-
tion) of cytostatin (1) by analysis of 1D and 2D NMR spectra.
The relative configurations of C4–C6 and C9–C11 of
cytostatin were assigned as syn at C4/C5 and C9/C10 and as
The fluorous mixture synthesis (FMS) strategy to make
the four isomers of cytostatin is briefly outlined in Scheme 2.
Late introduction of the triene is dictated by its chemical
instability, so we followed the strategy of Bialy and Wald-
mann[2d] by planning to prepare four individual vinyl iodides 3
by coupling with alkenyl stannane 4.
[*] W.-H. Jung, S. Guyenne, C. Riesco-Fagundo, J. Mancuso,
S. Nakamura, Dr. D. P. Curran[+]
Department of Chemistry
University of Pittsburgh
These four isomers are made over several steps from a
single four-compound mixture of fluorous-tagged quasiisom-
ers M-5[9] (“quasi” because the compounds have different
fluorous tags and are not true isomers[10]). In turn, M-5 is
made by coupling between fluorous-tagged quasiracemic
aldehydes M-6 with quasiracemic keto phosphonates M-7
by a Horner–Wadsworth–Emmons (HWE) reaction.
Pittsburgh, PA 15260 (USA)
Fax: (+1)412-624-9861
E-mail: curran@pitt.edu
Homepage: http://www.radical.chem.pitt.edu
[+] Bayer Prof. and Distinguished Service Prof. of Chemistry.
[**] We thank the National Institute of General Medical Science of the
National Institutes of Health for funding of this work. C.R.-F. thanks
the Spanish Ministry of Education and Science (MECD) for a
postdoctoral fellowship. We thank Prof. Dale Boger and Dr. Brian
Lawhorn for providing a sample of natural cytostatin along with
spectra of synthetic and natural samples as well as for helpful
advice.
From the FMS standpoint, the configurations of the
stereocenters at C4–C6 (SSS or RRR) and C9–C11 (SSS or
RRR) are encoded by differing silyl groups. Demixing[11]
a
late-stage mixture by fluorous HPLC to provide its individual
components will be possible because each quasiisomer has a
different number of fluorine atoms. The fluorine atoms are
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Scheme 3. Synthesis of quasiracemate M-6. Bn=benzyl, DDQ=2,3-
dichloro-5,6-dicyano-1,4-benzoquinone, DMAP=4-dimethylaminopyri-
dine, DMP=Dess–Martin periodinane, PMB=para-methoxybenzyl,
Tf =triflate, TIPS=triisopropylsilyl, Tr=trityl=triphenylmethyl.
Scheme 2. Fluorous mixture synthesis plan.
distributed over two silyl groups, so their approximate
additivity upon HPLC demixing is important.[8f,12]
The syntheses of quasiracemates M-6 and M-7 are
summarized in Schemes 3 and 4, respectively. To make the
left-side quasiracemate M-6 (Scheme 3), readily available
Evans aldol[13] adduct (R,R,S,S)-8 and its enantiomer were
tagged with different fluorous silyl groups to encode the
configurations, and the tagged products 9s and 9r were then
mixed. Reductive removal of the Evans auxiliary with LiBH4
afforded the primary alcohol M-10 in 77% yield, which was
subsequently protected with TrCl to give M-11 in 86% yield.
Selective deprotection of the PMB group with DDQ provided
M-12 in 75% yield. Subsequent treatment with DMP gave the
fluorous quasiracemic mixture of aldehyde M-6 in 84% yield.
To make the right-side quasiracemate M-7 (Scheme 4)
isomerically pure enantiomers (R,R)-13 and (S,S)-13 (pre-
pared by Brown–Ramachandran allylboration)[14] were
tagged to encode configuration information, and the tagged
quasienantiomers 14s and 14r were then mixed. The mixture
of M-14 was treated with OsO4 and NMO in tBuOH/H2O to
provide a mixture of diols, which was directly treated with
NaIO4 to afford M-15 in 90% yield over two steps. Treatment
of aldehyde M-15 with the lithium anion of (MeO)2P(O)CH3,
followed by oxidation with DMP, afforded the fluorous
quasiracemic mixture of keto phosphonate M-7 in 82%
yield over two steps.
Scheme 4. Synthesis of quasiracemate M-7. NMO=4-methylmorpho-
line N-oxide, TMS=trimethylsilyl.
reagent[15] gave a saturated ketone. Reduction of this ketone
with LiAl(tBuO)3H gave M-16 with a syn relationship
between C9 and C10, wherein the configuration of the C9
stereocenters was controlled by the C10 stereocenters (sub-
strate control). Removal of the terminal TMS group from the
alkyne with KOH gave M-17, and installation of Fm-
protected phosphonate with a phosphoramidite (iPr)2NP-
(OFm)2 provided M-18. Selective cleavage of the trityl group
with CSA followed by oxidation using DMP afforded M-19.
The mixture of four aldehydes M-19 was subjected to Still–
Gennari olefination[16] to give a,b-unsaturated esters M-20 in
95% yield. Just prior to removal of the fluorous tags, the
mixture M-20 was demixed into four individual quasiisomers
The fragment coupling and subsequent seven-step syn-
thesis were carried out with mixtures of four quasiisomers
(Scheme 5). A HWE reaction between M-6 and M-7 provided
the enone M-5 in 80% yield, then 1,4-reduction using Stryker
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Scheme 5. Fluorous mixture synthesis and demixing. CSA=camphorsulfonic acid, Fm=fluorenylmethyl, KHMDS=potassium 1,1,1,3,3,3-
hexamethyldisilazane.
(20ss, 20sr, 20rs, 20rr) by preparative fluorous HPLC. As
expected, the quasiisomers were well separated and eluted in
order of increasing fluorine content.
The remaining steps were carried out on each isomer
individually; Scheme 6 illustrates these steps for the prepa-
ration of 1ss. Two silyl groups in 20ss were removed with
with diimide. The Stille coupling was performed with the
stannane 4 and [Pd2(dba)3] to provide the (12Z,14Z,16E)-
triene, which was carefully purified by preparative HPLC.
Cleavage of the fluorenylmethyl group under basic conditions
followed by ion-exchange using Dowex provided cytostatin
stereoisomer 1ss. Likewise, the other three candidate isomers
(1rs, 1sr, and 1rr; see Scheme 1) were made by the same
sequence of reactions starting from the appropriate demixed
product 20.
We then compared the four synthetic samples of 1 with
each other and with a natural sample to prove which three
samples were not the natural products. The measurement of
optical rotations provided what proved to be misleading
information. The optical rotation of 1ss ([a]2D0 = + 45; c = 0.08,
MeOH) agreed well with data from the research groups of
Waldmann and Boger,[2] while the rotation of its diastereomer
1sr was [a]2D5 = + 29 (c = 0.09, MeOH). The rotation value of
1sr is in better agreement with that reported for an aged[17]
natural product sample (+ 20; c = 0.27, MeOH).[2d] These
values could be taken as evidence that cytostatin is 1sr.
However, subsequent experiments disproved this assignment.
Comparisons of high-field 1H NMR spectra (500 MHz) of
the five samples gave unexpected results. The 1H NMR
spectra were very similar, but none of the five spectra exactly
matched another one (see the Supporting Information for
copies of the spectra). Since two pairs of synthetic samples are
enantiomers and one of the four synthetic samples has to be
the natural product, the tiny differences in the chemical shift
that were observed between identical compounds or enan-
tiomers must arise as a consequence of concentration or pH
differences between the samples. Despite this complication,
we still identified two reliable differences in the spectra of the
Scheme 6. Final steps on individual isomers illustrated in the “ss”
series. NIS=N-iodosuccinimide, pyr=pyridine.
HF·pyr to provide lactone 21ss in 59% yield. As prescribed
by Bialy and Waldmann,[2d] iodination of the triple bond with
NIS in the presence of a catalytic amount of AgNO3 afforded
an iodoalkyne, which was reduced to the Z-iodoalkene 3ss
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Bialy, H. Waldmann, Chem. Eur. J. 2004, 10, 2759 – 2780; e) B. G.
two diastereomers. Surprisingly, the two protons that have
clearly different chemical shifts are not attached to any of the
stereocenters, but are instead the diastereotopic protons at
C8. The H8 and H8’ resonances of 1ss matched those of the
natural sample, while the resonances of 1sr were different.
Finally, simple TLC experiments on standard silica gel
conclusively proved that cytostatin was not 1sr. Cospotting of
the natural sample with synthetic samples showed that it was
inseparable from 1ss. In contrast, 1sr was slightly faster
moving on the plate and separated from both the natural
sample of cytostatin and the sample of 1ss. These experiments
prove that 1sr is not cytostatin, and accordingly its enantio-
mer 1rs cannot be cytostatin either. The large differences in
the size and magnitude of the optical rotation prove that 1rr is
not cytostatin. No information was obtained to prove that 1ss
is not cytostatin and, accordingly, we concur with the research
groups of Waldmann and Boger that it is.
In summary, we have synthesized four closely related
candidate isomers for cytostatin (1) by using the technique of
fluorous mixture synthesis. With the isomers in hand, we
proved by a combination of NMR, TLC, and optical rotation
experiments that three of the isomers were not cytostatin, and
accordingly we confirmed structure 1ss for this important
natural product. Rigorous proof of the stereostructure of
natural products by disproof of all the candidate stereoiso-
mers but one is today not common because traditional “one at
a time” approaches to stereoisomer synthesis are too long and
tedious. The emerging techniques of fluorous mixture syn-
thesis offer a respite from this tedium by providing additional
stereoisomers without a proportional increase in effort.
Lawhorn, S. B. Boga, S. E. Wolkenberg, D. L. Boger, Hetero-
cycles 2006, 70, 65 – 68; f) B. G. Lawhorn, S. B. Boga, S. E.
Wolkenberg, D. A. Colby, C. M. Gauss, M. R. Swingle, L.
Amable, R. E. Honkanen, D. L. Boger, J. Am. Chem. Soc.
2006, 128, 16720 – 16732.
[3] Key enabling methods for cytostatin synthesis have been
described: a) J. A. Marshall, K. Ellis, Tetrahedron Lett. 2004,
45, 1351 – 1353; b) A.-F. Salit, C. Meyer, J. Cossy, Synlett 2007,
934 – 938.
[4] In the candidate isomers for 1, the two groups of stereocenters
either have all S or all R absolute configurations. The pair of
small letters “s/r” after the numbers abbreviate the configura-
tions of the three centers at C4–C6 (left letter) and C9–C11
(right letter). As
a result of Cahn–Ingold–Prelog priority
changes, synthetic intermediates may have different configura-
tions from the natural product.
[5] a) D. L. Boger, M. Hikota, B. M. Lewis, J. Org. Chem. 1997, 62,
1748 – 1753; b) D. S. Lewy, C. M. Gauss, D. R. Soenen, D. L.
Boger, Curr. Med. Chem. 2002, 9, 2005 – 2032; c) M. Shibasaki,
M. Kanai, Heterocycles 2005, 66, 727 – 741.
[6] D. P. Curran, Q. S. Zhang, H. J. Lu, V. Gudipati, J. Am. Chem.
Soc. 2006, 128, 9943 – 9956.
[7] Short reviews: a) W. Zhang, Chem. Rev. 2004, 104, 2531– 2556;
b) D. P. Curran in The Handbook of Fluorous Chemistry (Eds.:
J. A. Gladysz, D. P. Curran, I. T. Horvath), Wiley-VCH, Wein-
heim, 2004, pp. 128 – 156.
[8] a) Z. Y. Luo, Q. S. Zhang, Y. Oderaotoshi, D. P. Curran, Science
2001, 291, 1766 – 1769; b) W. Zhang, Z. Luo, C. H. T. Chen, D. P.
Curran, J. Am. Chem. Soc. 2002, 124, 10443 – 10450; c) S.
Dandapani, M. Jeske, D. P. Curran, Proc. Natl. Acad. Sci. USA
2004, 101, 12008 – 12012; d) S. Dandapani, M. Jeske, D. P.
Curran, J. Org. Chem. 2005, 70, 9447 – 9462; e) Y. Fukui, A. M.
Brueckner, Y. Shin, R. Balachandran, B. W. Day, D. P. Curran,
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[9] The prefix “M” stands for a mixture of two or four quasiisomers.
Numbers without prefixes are individual compounds.
[10] Q. S. Zhang, D. P. Curran, Chem. Eur. J. 2005, 11, 4866 – 4880.
[11] D. P. Curran, in The Handbook of Fluorous Chemistry (Eds.:
J. A. Gladysz, D. P. Curran, I. T. Horvath), Wiley-VCH, Wein-
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[12] S. Manku, D. P. Curran, J. Org. Chem. 2005, 70, 4470 – 4473.
[13] J. S. Johnson, D. A. Evans, Acc. Chem. Res. 2000, 33, 325 – 335.
[14] R. V. Ramachandran, Aldrichimica Acta 2002, 35, 23 – 35.
[15] W. S. Mahoney, D. M. Brestensky, J. M. Stryker, J. Am. Chem.
Soc. 1988, 110, 291– 293.
[16] W. C. Still, C. Gennari, Tetrahedron Lett. 1983, 24, 4405 – 4408.
[17] Waldman and Boger have both commented that the rotation of
the natural sample may not be accurate because of its modest
level of purity; see Ref. [2].
Received: October 22, 2007
Published online: January 3, 2008
Keywords: antitumor agents · cytostatin · fluorous tags ·
.
natural products · structure elucidation
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