Total Synthesis of the Potent HIF-1 Inhibitory Antitumor Natural Product, (8R)-Mycothiazole, via Baldwin-Lee CsF/CuI sp3-sp2-Stille Cross-Coupling. Confirmation of the Crews Reassignment

Article abstract of DOI:10.1021/acs.orglett.5b01966

A convenient asymmetric total synthesis of the potent HIF-1 inhibitory antitumor natural product, (-)- or (+)-(8R)-mycothiazole (1), is described. Not only does our synthesis confirm the 2006 structural reassignment made by Crews (Crews, P., et al. J. Nat

Full text of DOI:10.1021/acs.orglett.5b01966

Letter
pubs.acs.org/OrgLett
Total Synthesis of the Potent HIF‑1 Inhibitory Antitumor Natural
Product, (8R)‑Mycothiazole, via Baldwin−Lee CsF/CuI sp3−sp2‑Stille
Cross-Coupling. Confirmation of the Crews Reassignment
Liping Wang and Karl J. Hale*
The School of Chemistry & Chemical Engineering and the Centre for Cancer Research and Cell Biology (CCRCB), the Queen’s
University Belfast, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, United Kingdom
S
* Supporting Information
ABSTRACT: A convenient asymmetric total synthesis of the
potent HIF-1 inhibitory antitumor natural product, (−)- or
(+)-(8R)-mycothiazole (1), is described. Not only does our
synthesis confirm the 2006 structural reassignment made by
Crews (Crews, P., et al. J. Nat. Prod. 2006, 69, 145), it revises
the [α]D data previously reported for this molecule in MeOH
from −13.7° to +42.3°. The newly developed route to (8R)-1
sets the C(8)−OH stereocenter via Sharpless AE/2,3-epoxy
alcohol reductive ring opening and utilizes two Baldwin−Lee
CsF/cat. CuI Stille cross-coupling reactions with vinyl-
stannanes 8 and 3 to efficiently elaborate the C(1)−C(4)
and C(14)−C(18) sectors.
(−)-(8R)-Mycothiazole (1) is a novel naturally occurring
thiazolo-polyene first encountered by Crews and co-workers1 in
anthelmintic extracts of Spongia mycofijiensis, a sponge
indigenous to coastal waters off Vanuatu island, west of Fiji.
The precise chemical structure of (−)-mycothiazole has been
the subject of much debate over the years, its most recent
structural assignment2 being stereoisomer 1 (Figure 1).
be incorrectly assigned to the C(14)−C(15)-alkene. Crews also
never specified C(8) stereochemistry in his original 1988
structure 2, which inevitably led to several teams attempting to
do this by total synthesis of the individual enantiomers.3−5
The first asymmetric total synthesis of (−)-(8R)-2 was
accomplished by Shioiri and co-workers in 2003.3 It stood out
for the excellent stereocontrol it imparted to the setting of the
target’s stereodefined olefins and for its use of a Nagao
asymmetric aldol reaction to efficiently control C(8)-hydroxy
stereochemistry. Somewhat disconcertingly, however, the final
[α]D that was recorded for (−)-(8R)-2 by Shioiri et al.3 was
−26.0° (c 0.64, CHCl3), which differed substantially from the
value that had originally been measured for natural
(−)-mycothiazole by Crews et al.1 ([α]D −3.8° (c 2.9,
CHCl3)). Shioiri’s 13C NMR data3 for (−)-(8R)-2 in CDCl3
also deviated significantly from the data reported for natural
(−)-mycothiazole by Crews.1 In particular, the 13C chemical
shifts for C(13) and C(16) in 2 were some 5 ppm further
downfield3 than those listed for the same carbons in the natural
product (see Figure 1).1 Despite the significant chemical
differences that were observed, Shioiri and co-workers still
continued to maintain that a confirmatory total synthesis of the
natural product had been achieved,3 attributing these
discrepancies to impurities in Crews’ original (−)-mycothiazole
sample and to several resonance misassignments by Crews (see
p S-9 of the Supporting Information of ref 4a).4a As a result, the
Cossy group, who also reported a racemic total synthesis of 2 in
2005,4 again incorrectly concluded4 that they had achieved a
Figure 1. Crews’ corrected (−)-mycothiazole structure 1 and his
originally proposed structure 2 for the (8R)-enantiomer.
However, for more than two decades, (−)-mycothiazole had
its structure incorrectly formulated as 2, due to its complex
polyolefinic network giving rise to a series of highly overlapped
multiplets in the various 1H NMR spectra that were recorded.1
This led to an erroneous J value of 18 Hz being determined for
H14/H15, which inevitably caused an (E)-olefin geometry to
Received: July 9, 2015
© XXXX American Chemical Society
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total synthesis of natural mycothiazole, only to later find that
this collective claim would be challenged by Crews2 in 2006.
In this connection, and having noted the significant 13C
NMR differences that existed between natural mycothiazole
and incorrectly formulated synthetic (−)-(8R)-2, Crews and his
team eventually decided to revisit their original 1988 structural
assignment, while attempting a second large-scale isolation of
the natural product from Dactylospongia sp.2 The detailed NMR
analysis that followed used 600 MHz 1H NMR NOE difference
spectroscopy and spectral simulation to resolve the various
spectral inconsistencies, and eventually produced a new
structural formulation for (−)-mycothiazole, namely,
(−)-(8R)-1, where the C(14)−C(15) alkene was now of (Z)-
rather than (E)-geometry.2 Despite this important advance,
still, the issue of C(8) absolute stereochemistry remained
unresolved, with Crews’ new assignment resting solely on a
tentative [α]D comparison with Shioiri’s (−)-(8R)-2, rather
than on a definitive X-ray or Mosher ester NMR determination.
Crews’ revision of the (−)-mycothiazole structure2 and the
biological reports that followed6 have since helped fuel
synthetic interest in (−)-(8R)-1 as a target.7 Yet, despite this,
no group has so far achieved a full total synthesis of (−)-(8R)-
1, notwithstanding significant progress having been made on
the construction of various subregions.7 Biologically, (−)-(8R)-
mycothiazole (1) is a molecule of growing pharmacological
importance6 due to the powerful, yet selective, growth
inhibitory effects it exerts against human tumor cell lines
(IC50 values = 0.36−10 nM), and for its potent HIF-1
inhibitory effects within hypoxic T47D human breast carcinoma
cells (IC50 = 1 nM). The discovery of these properties for 1
could be of considerable pharmaceutical worth, since 1 does
appear to be a potentially tractable new drug lead from where
to commence much future discovery effort. However, before
drug development can begin in earnest, the issue of
(−)-mycothiazole structure and stereochemistry has to be
resolved beyond all doubt, and a reliable asymmetric total
synthesis of (−)-(8R)-1 must be developed. Herein, we now
report success in the latter of these two endeavors.
In our retrosynthetic planning for (−)-(8R)-1 (Scheme 1),
the C(8)-hydroxy stereocenter of 1 would be set through a
Sharpless asymmetric epoxidation on 13 allied with a C(2) site-
selective reductive ring opening of the 2,3-epoxy alcohol 12 and
protection. Compound 11 would thereafter be advanced
toward thionoamide 9 and thiazole ring construction
completed by a Hantzsch cyclization with the bromopyruvate
10. Subsequent elaboration of the vinyl iodide 7 and Stille
cross-coupling with Shioiri’s vinylstannane 83 would then
provide a product whose ester could be reduced and
brominated to yield bromide 4. A second Stille coupling with
the skipped dienylstannane 3 and an O-desilylation would then
finalize the synthesis of (−)-(8R)-mycothiazole 1. The known
(Z)-vinylstannane 38 would itself be accessed via Yamamoto’s
ZrCl4-cod alkyne allylstannation procedure.8
With this in mind, chiral 2,3-epoxy alcohol 12 (Scheme 2)
was prepared following Masamune’s published procedure for
the opposite enantiomer,9 and its absolute configuration was
unambiguously confirmed by [α]D comparison with Masa-
mune’s published data.9 Although the literature records a 92%
ee for formation of the opposite enantiomer, our Mosher ester
analysis of 12 revealed that it was virtually optically pure.
Epoxy-alcohol 12 was then taken forward and reduced with
REDAL in THF/PhMe at −40 °C. Diol 14 was formed almost
exclusively and was isolated in 93% yield as an oil after SiO2
Scheme 1. Our Retrosynthetic Analysis for (−)-(8R)-
Mycothiazole 1
flash chromatography. It was then doubly O-silylated with
TBSOTf (2.2 equiv) and 2,6-lutidine (4.4 equiv) in dry
CH2Cl2. Although the TBSOTf addition was done at −78 °C, a
prolonged reaction period at rt (2 h) was subsequently required
to drive the process to completion, whereafter the di-O-silyl
ether 11 was obtained in 93% yield. The benzyl ether was then
cleaved from 11 by Pd/C catalyzed hydrogenolysis in EtOAc
over 30 min. The product alcohol 15, isolated in 92% yield, was
thereafter submitted to a two-stage oxidation under cat. TPAP/
NMO and Pinnick conditions. The resulting carboxylic acid 17
was then converted to the pentafluorophenyl ester with DCC/
pentafluorophenol in CH2Cl2, and ammonolysis was performed
with ammonia in THF to give the amide 18. Amide 18 was
subsequently converted into the thionoamide 9 by treatment
with Lawesson’s reagent in THF at 60 °C, and although this
reaction did typically proceed in fair yield (58%), still, this did
not markedly hamper overall material throughput.
Thiazole formation was accomplished by implementing
either one of the following two Hantzsch protocols.10 In our
slightly lower yielding procedure (56%, 2 steps, one pot), 9 was
treated with ethyl bromopyruvate 10 in THF for 2 h. This
process also caused a partial O-desilylation of the primary
OTBS group, a reaction that could be driven all the way
through to completion by exposing the crude product to PPTS
in MeOH at rt for 4 h. The great advantage of this protocol lay
in its ability to be executed rapidly (6 h overall). However, the
alternative, slower, NaHCO3-mediated procedure also procured
19 in a slightly higher yield (61%, 3 steps), but it completed the
key thiazole ring-forming step by treatment of the intermediary
hydroxy-thiazoline with NaHCO3/(CF3CO)2O,10 prior to
performing the PPTS/MeOH induced selective O-desilylation.
With alcohol 19 in hand, the synthesis advanced with a Swern
oxidation to 20 and a (Z)-selective Stork−Zhao Wittig
B
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coupling proved to be fully compatible with the carboxyethyl
and TBS substituents that were present in 7 and 21.
Scheme 2. Our Asymmetric Pathway to Thiazolo-Ester 21
Our synthesis progressed further (Scheme 3) with a DIBAL-
H reduction of the ester in 21 (73−80% yield) and
Scheme 3. Completion of Our Total Synthesis (−)-(8R)-
Mycothiazole (1) and Our Baldwin−Lee Model SN2 Study
bromination of the product alcohol 22 with Ph3P and CBr4
in THF, which yielded the bromomethyl thiazole 4 in 63−68%
yield. The requisite Stille cross-coupling partner, dienyl-
stannane 3, was itself stereoselectively prepared by application
of Yamamoto’s ZrCl4/1,5-cyclooctadiene-promoted alkyne
allylstannation addition reaction to acetylene 6,8 which afforded
the desired cis-addition product 3 in 47−57% yield. Vinyl-
stannane 3 efficiently coupled to 4 in 94% yield within 50 min
at rt under Baldwin−Lee conditions,11 without F− displacement
of the bromide compromising the overall success of the
process. In the latter regard, prior model studies with the
closely related bromomethylthiazole 24 had already shown that
CsF (2 equiv) in DMF at rt for 1 h did not SN2 displace the
primary bromide to give 25 (Scheme 3). A subsequent
Baldwin−Lee coupling between 24 and 3 was also shown to
be viable, a 63% yield of 26 being furnished after 1.5 h at rt. In
our view, this novel extension of the CsF/cat. CuI-accelerated
Stille protocol to bromomethylthiazoles represents a most
useful, and potentially general, advance for coupling activated
sp2-vinylstannanes with bromides of this sort. However, it is
iodoolefination; the latter occurred with high stereocontrol,
furnishing 7 as a single stereoisomer without causing damage to
the ester in either 20 or 7.
With the requisite iodoolefin 7 in hand, the C(1)−C(13)
sector of (8R)-mycothiazole was elaborated by performing a
Baldwin−Lee variant of the Stille cross-coupling11 with known
vinylstannane 8 (1.8 equiv), prepared according to Shioiri’s
method.3 Importantly, this union worked very well indeed on a
quite decent scale (0.65 g with respect to 7), delivering the
desired diene 21 in 88% yield after 40 min of stirring at rt.
Significantly, as well, the aforementioned Baldwin−Lee cross-
C
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Letter
equally important for us to state that we were unsuccessful
when we attempted to perform a similar sp3−sp3 cross-coupling
between 24 and Me4Sn under prolonged rt conditions!
The final step in our (8R)-mycothiazole synthesis was O-
desilylation of 23 with n-Bu4NF (5 equiv) in THF, which
required 96 h to reach completion; it afforded 1 in 66% yield.
Importantly, the spectral data for synthetic 1 now closely
matched those originally reported by Crews’ and co-workers in
1988,1 so confirming his 2006 structural revision.2
Even so, the [α]D value that we recorded for synthetic
(−)-(8R)-mycothiazole (1) (−21.4°, c 0.47 CHCl3) still
differed substantially from the value reported by Crews et al.
for natural material ([α]D −3.5°, c 0.6 CHCl3) in 2006 (see
Scheme 3).2 Even more surprisingly, as well, our synthetic
sample of (−)-(8R)-mycothiazole (1) also gave rise to a large
positive [α]D of +42.3° in MeOH (c 1.13), which contrasted
sharply with Crews’ 2006 [α]D report of −13.7° in MeOH (c
0.6).2
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b01966.
■
S
1
Full experimental procedures, and copies of the H and
13C NMR spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: k.j.hale@qub.ac.uk.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank QUB for a studentship (to L.W.) and start-up
funding.
■
Our observations suggest that in a strongly hydrophobic
solvent such as CHCl3 strong internal H-bonding exists
between the C(8)−OH and the thiazole ring N atom, which
helps restrict conformational freedom within the molecule,
leading to a substantial negative [α]D. In MeOH, however, this
strong internal H-bonding is almost certainly disrupted. Such a
significant conformational perturbation could account for the
dramatic large positive [α]D that is now observed for (8R)-
mycothiazole in this solvent. Similar arguments probably apply
to the seemingly anomalous positive [α]D value that was
observed for (−)-(3R)-inthomycin C by Taylor et al.12 when
their [α]D was recorded in CHCl3 in the presence of a small
quantity (20%) of the H-bond-disrupter, tetramethylurea.12
In conclusion, an unambiguous total synthesis of (−)-(8R)-
mycothiazole (1) has been accomplished that spectrally
confirms Crews’ recently revised structure for this natural
product. Our [α]D measurements for the (8R)-enantiomer of
mycothiazole (1) have also revealed that it can exhibit both a
large negative and a large positive [α]D, depending upon for
which solvent the [α]D is recorded. Quite clearly, our [α]D data
deviate significantly from the [α]D values reported by Crews et
al. on lower purity natural mycothiazole.1,2 Given the lack of a
good [α]D correlation, our latest findings still do not allow a
confident assignment of absolute stereochemistry to be made
for natural mycothiazole which must, we believe, remain only
tentatively assigned as (8R). While future biological testing of
synthetic 1, and its enantiomer, may allow a definitive
assignment of absolute stereochemistry for the natural product,
a substantial difference in potency will have to be observed for
such a protocol to be credible.
REFERENCES
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seminal application of the Baldwin−Lee cat. CuI/CsF Stille
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important new synthetic advance. Our synthesis has also
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mediated cis-selective alkyne allylstannation reaction8 and
compliments Yamamoto’s own elegant efforts at applying this
reaction in natural product total synthesis.8b Finally, with the
new synthetic route we have developed to (8R)-mycothiazole,
the gross structure of 1 has been confirmed. Efficient medicinal
chemistry exploitation of 1 should now prove possible.
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