Function-oriented studies targeting pectenotoxin 2: Synthesis of the GH-ring system and a structurally simplified macrolactone

Article abstract of DOI:10.1021/acs.orglett.7b02435

A chemical foundation for function-oriented studies of pectenotoxin 2 (PTX2) is described. A synthesis of the bicyclic GH-system, and the design and synthesis of a PTX2-analogue, is presented. While maintaining critical features for actin binding, and lacking the Achilles' heel for the natural product's anticancer activity (the AB-spiroketal), this first-generation analogue did not possess the anticancer properties of PTX2, an observation that indicates the molecular significance of features present in the natural product's CDEF-tetracycle.

Full text of DOI:10.1021/acs.orglett.7b02435

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Letter
pubs.acs.org/OrgLett
Function-Oriented Studies Targeting Pectenotoxin 2: Synthesis of
the GH-Ring System and a Structurally Simplified Macrolactone
Natasha F. O’Rourke,† Mu A,‡ Henry N. Higgs,‡ Alan Eastman,‡ and Glenn C. Micalizio*,†
†Department of Chemistry, Dartmouth College, Burke Laboratory, Hanover, New Hampshire 03755, United States
‡Geisel School of Medicine, Dartmouth College, Hanover, New Hampshire 03755, United States
S
* Supporting Information
ABSTRACT: A chemical foundation for function-oriented studies of pectenotox-
in 2 (PTX2) is described. A synthesis of the bicyclic GH-system, and the design
and synthesis of a PTX2-analogue, is presented. While maintaining critical features
for actin binding, and lacking the Achilles’ heel for the natural product’s anticancer
activity (the AB-spiroketal), this first-generation analogue did not possess the
anticancer properties of PTX2, an observation that indicates the molecular
significance of features present in the natural product’s CDEF-tetracycle.
ectenotoxin 2 (PTX2) is an exceedingly scarce marine-
derived natural product that has become a target of interest
for many chemical synthesis laboratories across the globe due to
its potentially valuable anticancer activity and its daunting
molecular structure (Figure 1).1 While isolated over 30 years ago
P
Figure 2. Structure of PTX2 bound to actin.3
system is known to undergo rapid isomerization to other PTX
congeners that have markedly dampened anticancer activity
(Figure 1). Further, it has been shown that oxidation at C43, or
saponification of the macrolactone, results in family members
that lack the toxicity profile of PTX2.6
We have been fascinated by the chemical challenge that
members of this natural product class present, and our early
efforts established synthetic chemistry capable of addressing the
C1−C26 hexacyclic subunit.7 While we have found this domain
to be appealing from an architectural standpoint, the published
PTX2−actin structure (Figure 2) reveals that this region of the
natural product appears to merely cap a shallow hydrophobic
surface and engage in a hydrogen bond with actin through the C-
ring ketone. In stark contrast, the C32−C40 domain that houses
the GH-ring system projects into a narrow hydrophilic pocket
and is secured by numerous hydrogen-bond interactions. Here,
we describe a concise synthesis of the GH-bicyclic heterocycle of
PTX2 and secure chemistry capable of fueling function-oriented
Figure 1. Introduction to the pectenotoxins.
in an attempt to identify causative agents of diarrhetic shellfish
poisoning,2 PTX2 has more recently been shown to possess
affinity to G-actin, binding to a unique site with respect to other
known marine-derived actin-targeting agents, and capping the
barbed end without filament severing properties.3 Distinct from
other actin-disrupting agents, PTX2 has been claimed to possess
selective toxicity toward p53 mutant and p53 null cancers
(including variants that are highly chemoresistant).4 While this
natural product has been embraced as an intriguing challenge for
numerous campaigns in total synthesis,5 parts of its structure
define significant liabilities for programs aimed at advancing it as a
therapeutically relevant agent. For example, the AB-spiroketal
Received: August 7, 2017
© XXXX American Chemical Society
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studies8 in the area, delivering macrocyclic PTX2-analogues in
∼20 linear steps from commercially available material.
tion [SmI2, (CH2OH)2, HMPA] provided the secondary alcohol
5 in 82% yield (dr ≥20:1).12
At the outset of these studies, it was recognized that the shape
of the hydrophobic capping region (C1−C26) is clearly
important for toxicity, as acid-mediated equilibration of the C7
acetal results in isomers with markedly dampened toxicity
profiles, and hydrolysis of the macrolactone abolishes activity. As
a result, PTX2-analogues of interest would retain the critically
important GH-ring system, yet offer different hydrophobic
macrocycles that could serve to mitigate the acid-triggered
deactivation of the natural product (i.e., be devoid of the AB-
spiroketal system altogether). Unfortunately, the C31−C40
region of the pectenotoxin structure that contains the GH-
bicyclic heterocycle has proven tobe quite challenging to prepare,
requiring longest linear sequences of ∼21 to >30 chemical steps
to establish only 10 backbone carbons.9
Installation of the tetrahydrofuran and alkyne motif of 1 was
accomplished by semireduction of the lactone with DIBALH and
Wittig reaction of the intermediate lactol with ylide 6.13 Enyne 7
was then stereoselectively epoxidized to furnish 9,14 which was
immediately used in a stereoretentive ring-closing event via BF3·
OEt2-mediated cyclization of the intermediate Co-complexed
alkyne.15 Finally, selective desilylation of the TMS-alkyne and
protection of the G-ring secondary alcohol as its corresponding
TES ether delivered the fully functionalized bicyclic target 1.
Notably, this reaction sequence proceeds in just 14 steps from
furfural and has been employed to generate multigram quantities
of this critically important PTX2-fragment.
With a route to the GH-system secured, we focused our
attention on establishing a general chemical synthesis pathway
capable of fueling function-oriented8 studies. These efforts began
by defining a model substrate to guide our chemical efforts. For
this, we employed a combination of molecular modeling and in
silico docking studies to arrive at a reasonable first-generation
synthetic target. First, PTX2 was docked to G-actin using
AutoDock.16 To our delight, this in silico experiment resulted in a
predicted PTX2−actin structure that resembled the known
PTX2−actin complex (Figure 5A vs Figure 2). Further in silico
studies predicted that a simple enyne-derivative of PTX2 would
associate with actin in a similar manner as the natural product
(Figure 5B), and that the drastically simplified macrolactone 10
could be a good starting point for function-oriented pursuits
(Figure 5C). In this latter case, however, notable differences in
the “docked” structure include a flipping of the H-ring in the
hydrophilic pocket to more closely resemble that seen in the
actual PTX2−actin structure (Figure 2), and a lack of
functionality in the C3−C26 PTX2-prosthetic capable of
achieving a hydrogen bond similar to that seen with the natural
product’s C-ring ketone. Despite these variations, and an
uncertainty regarding thesignificanceof docking scores produced
from AutoDock for the analysis of complex macrocyclic species
like pectenotoxin 2, we moved forward with 10 as a target to drive
the development of a general pathway to synthetic PTX2-
inspired agents.
Due to this lack of step ecomony, efforts were first directed at
defining a more concise approach to an intermediate containing
the GH-bicyclic system. As illustrated in Figure 3, the substituted
Figure 3. Retrosynthetic strategy for the C30−C40 subunit.
heterocycle 1 rose as an early target for synthesis. It was thought
that this system could be accessed from substrate-directed
functionalization of spiroketal 2. The stereodefined spiroketal 2
was then reasoned to be readily available from the crotylation
product of furfuraldehyde (3), through sequential silylation,
oxidation, and cyclization processes.
The successful execution of this design is illustrated in Figure 4.
Protection of the secondary alcohol of 3 (TBSCl, imidazole) was
followed by regioselective hydroboration and oxidation to deliver
4. Subsequent oxidation of the furan with m-CPBA, followed by
oxidation with PDC, provided the spiroketal intermediate 2 (dr
≥20:1) in 78% yield for the two-step sequence.10 With this
intermediate in hand, conditions for substrate controlled
stereoselective oxidation were explored. While initial attempts
at conjugate borylation [B2(pin)2, (PPh3)3RhCl or a variety of
copper catalysts] indicated a substantial lack of reactivity of the
system, and subsequent attempts at dihydroxylation with OsO4/
NMO were not promising, it was later found that treatment with
RuCl3/NaIO4 generated a valuable diol intermediate with
exquisite levels of stereocontrol.11 Finally, reductive deoxygena-
As depicted in Figure 6A, synthesis of 10 was reasoned to be
possible by macrolactonization of seco-acid 11, a compound
deemed to be readily available from the coupling of 1 to the
triaryl-ether fragment 12. Synthesis of 12 was accomplished as
depicted in Figure 6B and proceeded by stepwise homologation
of 1,4-bis(bromomethyl)benzene 13. Sequential nucleophilic
displacement, first with the anion of 4-hydroxybenzyl alcohol and
then with the phenoxide of 14, was effective for generating gram
Figure 4. Synthesis of the GH-bicyclic domain of the pectenotoxins from the crotylation product of furfuraldehyde.
B
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Figure 5. In silico docking experiments for design of a first-generation PTX2-inspired agent.
Figure 6. Establishment of a synthesis pathway capable of fueling function-oriented studies: convergent assembly of a PTX2-inspired agent.
quantities of the desymmetrized benzylic alcohol 15. Oxidation
to the corresponding aldehyde (MnO2, PhH), followed by
stereoselective propargylation17 and silylation of the resulting
homopropargylic alcohol, gave 17 (dr = 10:1). Regioselective
hydrostannylation18 of the internal alkyne (n-Bu3SnH, PCy3,
Pd(OAc)2) was followed by conversion to the vinyl iodide 18 (I2,
CH2Cl2).19 Selective desilylation of the primary silyl ether (CSA,
CH2Cl2−MeOH), oxidation to the aldehyde (MnO2, CH2Cl2),
and subsequent catalytic anti-Evans’ aldol reaction with 19
generated 20 in 65% yield over the three steps.20 Finally,
conversion of 20 to the planned coupling partner 12 was
accomplished by cleavage of the TMS ether with citric acid in
methanol, protection of the resulting secondary alcohol as a TBS
ether, and reduction to the primary alcohol with LiBH4 (72%
overall yield for this final three-step sequence).
illustrated in Figure 6C, Sonogashira coupling21 was highly
effective for uniting the two fragments (89% yield). Sequential
oxidation to the carboxylic acid and selective deprotection of the
TES ether then delivered seco-acid 11. To our delight, cyclization
under the conditions of Yamaguchi22 was effective for generating
the macrolactone, and a simple two-step deprotection sequence
converted the intermediate macrolactone to 10 in 82% yield.
With chemistry established to advance the synthetic GH-
system 1 to a macrocyclic analogue of PTX2 in just six additional
linear transformations, we elected to explore the anticancer and
actin-perturbing properties of this first-generation analogue.
While docking studies suggested that 10 was capable of
mimicking the binding of PTX2 to actin, dosing of this
compound in four different human cancer cell lines (MDA-
MB-231, MALME, HT29, and H522) at concentrations up to 10
μM led to no observable inhibition of growth. While this lack of
cellular activity could be related to distinct permeability
With gram quantities of 1 and 12 in hand, effort was directed
toward completing the synthesis of PTX2-analogue 10. As
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(5) For the total synthesis of PTX2, see: (a) Fujiwara, K.; Suzuki, Y.;
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b02435.
Procedures and spectroscopic data (PDF)
Muller, R.; Paquette, L. A. Org. Lett. 2005, 7, 1813. (r) Halim, R.;
Brimble, M. A. Org. Biomol. Chem. 2006, 4, 4048−4058 and references
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̈
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: glenn.c.micalizio@dartmouth.edu.
ORCID
Glenn C. Micalizio: 0000-0002-3408-5570
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
The authors acknowledge financial support from Dartmouth
College and the National Institutes of Health (GM080266).
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