Preparation of a 1,2-isoxazolidine synthon for the synthesis of zetekitoxin AB

Article abstract of DOI:10.1016/j.tetlet.2015.09.070

A synthesis of the 1,2-isoxazolidine fragment of the potent voltage gated sodium channel blocker, zetekitoxin AB is described. The synthesis utilizes an intramolecular nitrone-olefin 1,3-dipolar cycloaddition to establish the stereochemistry of the cis-1,

Full text of DOI:10.1016/j.tetlet.2015.09.070

Accepted Manuscript
Preparation of a 1,2-isoxazolidine synthon for the synthesis of zetekitoxin AB
Srinivas R. Paladugu, Ryan E. Looper
PII:
S0040-4039(15)30112-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.09.070
TETL 46742
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 August 2015
10 September 2015
16 September 2015
Please cite this article as: Paladugu, S.R., Looper, R.E., Preparation of a 1,2-isoxazolidine synthon for the synthesis
of zetekitoxin AB, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.2015.09.070
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Graphical Abstract
Preparation of a 1,2-isoxazolidine synthon
for the synthesis of zetekitoxin AB.
Leave this area blank for abstract info.
Srinivas R. Paladugu and Ryan E. Looper

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Preparation of a 1,2-isoxazolidine synthon for the synthesis of zetekitoxin AB.
Srinivas R. Paladugu and Ryan E. Looper*
aDepartment of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112
ARTICLE INFO
ABSTRACT
Article history:
Received
A synthesis of the 1,2-isoxazolidine fragment of the potent voltage gated sodium channel
blocker, zetekitoxin AB is described. The synthesis utilizes an intramolecular nitrone –olefin
1,3-dipolar cycloaddition to establish the stereochemistry of the cis-1,2-isoxazolidine. The
oxidative cleavage of an all anti-triol with the excision of the central carbon is central to using
α-D-glucopyranoside as a traceless stereochemical template. This route furnishes a suitably
protected synthon for the synthesis of zetekitoxin AB.
Received in revised form
Accepted
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
Zetikitoxin AB
saxitoxin
dipolar cycloaddition
1,2-isoxazolidine
nitrone
1. Introduction
In 1969, H. S. Mosher and colleagues isolated the potent
Although neither report had the correct carbon framework of
the 1,2-isoxazolidine fragment, it is clear that factors that affect
the 13C environment are unique and suggests that ZTX will
provide a rich medium for structural and synthetic studies.
sodium ion channel blocker zetekitoxin AB (ZTX) from the skin
extracts of a Panamanian golden frog, Atelopus zeteki.1 ZTX was
isolated by bioassay guided fractionation for acute toxicity (LD50
= 11 µg/kg, i.p. mouse) and shown to be a very potent (pM)
antagonist of the Nav, with potencies ranging from ~60-500 fold
more than those for STX (2) based on tissue type and isoform
distribution. In 2004, 30+ years after isolation, Yamashita and
coworkers disclosed the magnificent structure of ZTX (1) using
just 0.3 mg of the isolated sample.2 Similar to the related natural
product saxitoxin (2), ZTX also contains a tricyclic bis-guanidine
core. However, ZTX also includes a 1,2-isoxazolidine group
O
OH
NH2
O
N
H
H2N
HN
H2N
HN
HO
O
O
N
1
NH
NH
OH
8
NH2
NH
NH2
4
N
N 13
O
HO
11
H
O
N
HO
OSO3
within
a 10 membered macrocyclic ring, a pendant N-
hydroxycarbamate and a sulfate ester (Figure 1).2 Due to the
classification of A. zeteki as an endangered species, this 0.3 mg
sample remains the only purified of ZTX. Given the utility of
ZTX as a probe for ion channel physiology, its unique structure
and its exquisite rarity, total synthesis provides the only current
avenue to secure more material for biological investigations and
several groups have initiated programs in pursuit of the total
synthesis of ZTX.3,4 These synthetic approaches have also
revealed unique spectroscopic anomalies in the structural features
in ZTX. For example, Nishikawa first noted the discrepancy of
the 13C chemical shift of the amide carbonyl (C13) in ZTX (δ =
156.5 ppm) compared to most N-alkoxyamides (δ = 170-175
ppm) and showed that inductive effects of a tethered guanidinium
ion in model 1,2-isoxazolidine systems could still not account for
the unsusual chemical shift (δ = 167-173 ppm).4a This was further
confirmed in a more elaborate bicyclic guanidine model.4b
14
H
1
2
OH
(+)-saxitoxin (STX)
zetekitoxin AB
key biosynthetic
disconnection ?
(ZTX)
Figure 1. Structures of zetekitoxin AB 1 and saxitoxin 2.
This and our immediate access to STX (2) and its analogues,
piqued our interest in the structure of ZTX (1).5 It is known that
the pyrrolidine ring in STX is derived from arginine and thus
likely the same in ZTX and further unlikely that nature elaborates
this ring until late in its production.6 Since C10 in STX is
oxidized one could imagine that enol/keto tautomerization
produces a high enol content at C11 and this reacts with an
electrophilic carbon at C14 in the biosynthesis of ZTX (Figure
1). Herein we describe the synthesis of a suitably protected 1,2-
isoxazolidine derivative, with the correct carbon framework, to
explore this strategy for the synthesis of ZTX.
* Corresponding author. Tel. +01 801 585 0408
E-mail address: r.looper@utah.edu

2
Tetrahedron
2. Results and discussion
Scheme 2. Synthesis of a differentially protected diol.
Our synthetic approach toward the 1,2-isooxazolidine
fragment began by regioselectively converting the primary
hydroxyl group of methyl α-D-glucopyranoside (3) to the iodide
and acetylation of the remaining alcohols to give α-D-2,3,4
as an oxazolidinone. Protection of two primary hydroxyl groups
with tert-butyldiphenylsilyl chloride gave the di-TBDPS
protected species 10 in 97% yield. Von Braun type cleavage of
triacetoxy-6-deoxy-6-iodoglucopyranoside
4
in 70% yield.6
the
PMB
group
via
the
trichloroethoxycarbonyl chloride (TrocCl)
reaction
with 2,2,2-
gave the Troc
According to methods developed by Bernet and Vasella,
reductive fragmentation of 4 with zinc in refluxing ethanol gave
the aldehyde 5.7 Without further purification, the aldehyde was
condensed with p-methoxybenzylhydroxylamine to afford the
nitrone. Heating in ethanol triggered an intramolecular nitrone-
olefin [3+2] cycloaddition, proceeding through a predictable
transition state in which all acetates adopt an equatorial
arrangement, to afford the cis-fused bicyclic isoxazolidine 6 in
52% yield as a single stereoisomer.7,8 Deacetylation of 6 was
carried out with sodium methoxide to afford the product triol 7 in
80% yield (Scheme 1).8
protected 1,2-isoxazolidine derivative 11 in 82% yield (Scheme
3). The Troc group allowed us to effectively distinguish the two
primary hydroxyl groups by selectively trapping one of the
primary hydroxyl groups as a cyclic carbamate. This was
accomplished by treatment of 11 with tetra-n-butylammonium
fluoride to give 12 in 92% yield.
Re-silylation of the C4 hydroxyl group as the TBDPS ether
gave the protected cyclic carbamate 13 in 85% yield (Scheme 3).
Base mediated hydrolysis (Cs2CO3 in EtOH) of cyclic carbamate
13 furnished the amino alcohol 14 in 72% yield. Using N-Boc-
glycine as a surrogate for the saxitoxin core, we attempted a
coupling with the amino alcohol 14 using EDCI, HOBt or
HATU. This resulted in a mixture of undesired isoxazolidine-
glycine ester and amide. Whether this mixture arises from direct
acylation of the alcohol or a facile N→O acyl migration from the
isoxazolidine is unclear but argued for activation of the C6
hydroxyl group prior to peptide coupling. Treatment of 14 with
carbon tetrabromide and triphenylphosphine provided the amino-
bromide 15 which was unstable when isolated. Coupling of this
intermediate, in situ, with N-Boc-Gly-OH using standard EDCI,
HOBt coupling conditions afforded the protected isoxazolidine-
glycine amide 16 in 40% overall yield.
Scheme 3. Synthesis of a peptide coupling substrate.
We had some concern with the stability of the sensitive bis-
Scheme 1. The key 1,3-dipolar cycloaddition.
Having set the requisite cis-stereochemistry of the isoxazolidine
we focused our attention on the cleavage of anti-triol 7 with the
excision of C5 to remove the initial stereochemical information
provided by the pyranoside. After exploring various conditions,
sodium periodate and NaHCO3 gave an unstable bis-aldehyde
hydrate 8 as a mixture of diastereomers. In-situ reduction with
LiAlH4 reduction at room temperature afforded the 3,4-
dihydroxymethyl isoxazolidine 9 in 70% overall yield.9,10
Attempts to differentially protect the C4 and C6 alcohols at this
point proved unsuccessful. Oxidative strategies to cyclize the C6
alcohol on the benzylic carbon of the PMB group were also
unsuccessful, leading to decomposition of the substrate. An
alternative strategy would engage the C6 alcohol and the amine
aldehyde hydrate
8 and the potential for epimerization.
Comparison of the coupling constants for H3 in our final product
16 (H3: J = 14, 8, 5 Hz) with Nishikawa’s N-acetylisoxazolidine
17 ((H3: J = 12, 8, 6 Hz) indicates that the cis-stereochemistry
has been maintained. The 13C chemical shift of the isoxazolidine
amide carbonyl in 16 is 171.0 ppm, consistent with the
previously published models4a,b and suggest that if the structure
of ZTX is correct, a significant decoupling of the isoxazolidine
nitrogen non-bonding electrons from the carbonyl, or complex
shielding phenomenon imposed by the rigid three dimensional
structure of ZTX, significantly effects this carbonyl.

3
In conclusion we have developed a synthesis of the 1,2-
isoxazolidine fragment of ZTX. Our synthesis features an
intramolecular nitrone-olefin cycloaddition to establish the cis-
fused isoxazolidine. This strategy uses α-D-glucopyranoside as a
sterochemical template to relay the absolute stereochemistry
required for this fragment. The oxidative cleavage of an all anti-
triol with excision of the central carbon serves to remove all of
the stereochemical information presented in the original template.
This approach allows us to prepare gram quantities of 14, a
suitably protected synthon to explore a C11 - C14 coupling
strategy for the synthesis of ZTX.
References and notes
1. a) Shindelman, J.; Mosher, H. S.; Fuhrman, F.A. Toxicon. 1969,
7, 315. b) Fuhrman, F. A.; Fuhrman, G. J.; Mosher, H. S. Science,
1969, 165, 1376.
2. Yotsu-Yamashita, M.; Kim, Y. H.; Dudley, Jr. S. C.; Choudhary,
G.; Pfahnl, A.; Oshima, Y.; Daly, J. W. Proc. Natl. Acad. Sci. USA
2004, 101, 4346.
3. Peasrson, A. D.; Williams, R. M. Tetrahedron, 2014, 70, 7942.
4. a) Nishikawa, T.; Wang, C.; Akimoto, T.; Koshino, H.; Nagasawa,
K. Asain J. Org. Chem. 2014, 3, 1308. b) Nishikawa, T.; Urabe,
D.; Isobe, M. Heterocycles, 2009, 79, 379.
5. Bhonde, V. R.; Looper, R. E. J. Am. Chem. Soc. 2011, 133, 20172.
6. Miller, J. N.; Pongdee, R. Tetrahedron Lett. 2013, 54, 3185.
7. Bernet, B.; Vasella, A. Helv. Chim. Acta. 1979, 62, 1990.
8. a) Ferrier, R. J.; Furneaux, R. H.; Prasit, P.; Tyler, P. C. J. Chem.
Soc., Perkin Trans. 1 1983, 1621. b) Dransfield, P. J.; Moutel, S.;
Shipman, M.; Sik, V. J. Chem. Soc., Perkin Trans. 1 1999, 3349.
9. Ogawa, S.; Orihara, M. Carbohydrate Research, 1989, 189, 323.
10. Baumgartner, H.; O’Sullivan, A. C.; Schneider, J. Heterocycles,
1997, 45, 1537.
Acknowledgments
We are grateful to the NIH, General Medical Sciences (R01
GM090082) for financial support. We are grateful to Dr. Paul R.
Sebahar for insightful discussions.
Supplementary Material
Supplementary data (experimental procedures, characterization
data for new compounds) associated with this article can be
found, in the online version.
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