Synthesis of (+)-7,20-Diisocyanoadociane and Liver-Stage Antiplasmodial Activity of the Isocyanoterpene Class

Article abstract of DOI:10.1021/jacs.6b03899

7,20-Diisocyanoadociane, a scarce marine metabolite with potent antimalarial activity, was synthesized as a single enantiomer in 13 steps from simple building blocks (17 linear steps). Chemical synthesis enabled identification of isocyanoterpene antiplasmodial activity against liver-stage parasites, which suggested that inhibition of heme detoxification does not exclusively underlie the mechanism of action of this class.

Full text of DOI:10.1021/jacs.6b03899

Communication
pubs.acs.org/JACS
Synthesis of (+)-7,20-Diisocyanoadociane and Liver-Stage
Antiplasmodial Activity of the Isocyanoterpene Class
Hai-Hua Lu,†,§ Sergey V. Pronin,†,§,∥ Yevgeniya Antonova-Koch,‡ Stephan Meister,‡
Elizabeth A. Winzeler,‡ and Ryan A. Shenvi*,†
†Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
‡Department of Pediatrics, University of California, San Diego, School of Medicine, 9500 Gilman Drive 0741, La Jolla, California
92093, United States
S
* Supporting Information
ABSTRACT: 7,20-Diisocyanoadociane, a scarce marine
metabolite with potent antimalarial activity, was synthe-
sized as a single enantiomer in 13 steps from simple
building blocks (17 linear steps). Chemical synthesis
enabled identification of isocyanoterpene antiplasmodial
activity against liver-stage parasites, which suggested that
inhibition of heme detoxification does not exclusively
underlie the mechanism of action of this class.
socyanoterpene (ICT) metabolites1 are secreted from soft-
bodied marine organisms and are thought to protect against
I
predation or colonization.2 ICTs also kill the malaria parasite
Plasmodium falciparum at low-nanomolar concentrations with
high selectivity over human (KB) cells.3 Neither the mechanism
of antiplasmodial activity nor the structural requirements for
potency are fully understood.1,4 One mechanism of action
ascribed to the amphilectene and adociane ICT classes involves
inhibition of heme detoxification (crystallization to hemazoin),
for which computational models of binding have been
reported.3c,4a−c Among the isonitrile metabolites screened for
inhibition of heme crystallization in vitro, isocyanoamphilec-
tene (1) and 7,20-diisocyanoadociane (2) were identified as the
most effective members.4c Increased heme binding was
correlated to higher antiplasmodial activity relative to their
congeners [1: IC50 = 47 nM (D6), 423 nM (W2); 2: IC50 = 5
nM (D6), 4 nM (W2)]. Further study of 1 and 2 has been
explicitly impeded by scarcity of material.4c Here we describe an
asymmetric chemical synthesis of (+)-2, whose previous
syntheses have been 40 steps with stereocontrol5 and 27
steps to produce a near-equimolar mixture of isonitrile
diastereomers.6 Access to 1 and 2 through synthesis enabled
the demonstration that heme detoxification cannot be the
exclusive mechanism of antiplasmodial activity for the
amphilectene and adociane class, as recently claimed.4c
Figure 1. (a) Our approach to amphilectene ( )-1 via Danishefsky
dendralene [3]-Dd; (b) problems and solutions in the approach to
(+)-2.
annulation to access the fourth ring; (4) chemoselective
solutions to these problems to minimize functional group
interconversions (FGIs);8 and (5) full control of the relative
and absolute stereochemistry from double dienophile 3 and a
multiply substituted dendralene (4), a problem that appears to
be simple but is deceptively complex.
Access to the all-trans-fused scaffolds of the amphilectene,
adociane, and kalihinol ICT classes has been challenged by the
thermodynamic favorability of alternative cis-fused configura-
tions in synthetic intermediates.9 Control of the absolute
stereochemistry is also complicated by the double dienophile
targeted for cycloaddition. Two electron-withdrawing groups
are necessary for an efficient Diels−Alder reaction, but both
direct endo, rendering C2-symmetric chiral catalysts ineffec-
tive10 and preventing high diastereoselectivity in related
systems.11 After a screen of chiral auxiliaries, we identified
In 2012, we reported an approach to the amphilectane class
of terpenes, which includes the pseudopterosins, pseudopter-
oxazoles, and isocyanoamphilectenes like 1, by way of a
polarized Danishefsky-type dendralene ([3]-Dd; Figure 1a).7
Application of this overall strategy to an efficient synthesis of 2
required solutions to several problems: (1) simultaneous
incorporation of two tert-alkyl isonitriles, one axial and one
equatorial; (2) installation of the correct C1 stereochemistry
opposite to that of amphilectene 1; (3) stereoselective
Received: April 15, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.6b03899
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Journal of the American Chemical Society
Communication
(+)-8-phenylmenthol or (+)-2-trans-cumylcyclohexanol (TCC,
5)12 as ideal controllers for three reasons. First, incorporation
into the substrate was simple and even stabilized the reactive
dienophile (Scheme 1a). Second, control of the cycloaddition
process could be applied to simple alkynones. In the absence of
Lewis acid, 4-bromo-2-butynone (8) and ethyl vinyl ether do
not react, but trimethylaluminum catalyzes their cycloaddition
to form 9,15 and over time retroelectrocyclization occurs to
deliver 10 in 56% yield as a single geometrical isomer. We
anticipate this reaction to be a general route to many
substituted Danishefsky-type dendralenes. After some explora-
tion, we found that 10 could efficiently couple to commercially
available (Rieke Metals) zinc bromide 11 (99% ee) using
catalytic palladium(II) acetate, but only in the absence of
phosphine ligands. Enol silane formation completed the
synthesis of dendralene 4.
Scheme 1. Synthesis of Building Blocks 3 and 4
The merger of dendralene 4 and double dienophile 3
occurred at −78 °C in the presence of copper(II) triflate using
crude solutions of both components (yield calculated from 7).
The cycloaddition generated cyclohexenone 12 with 95:5
diastereoselectivity (relative to the auxiliary) after elimination
of the ethyl silyl ether, which occurred in the presence of the
Lewis acid. The second Diels−Alder reaction proceeded at 180
°C, and auxiliary removal via heteroretroene/decarboxylation
took place when the temperature was increased to 200 °C.16
Themolysis offered an excellent solution for auxiliary removal
since standard nucleophilic substitution was outcompeted by
both deconjugation of the strained cyclohexenone in 13 and
retro-Dieckmann ring scission at the intermediate β-keto ester.
The gross simplification of this disconnection and the overall
yield of these two processes (40%, 15:1 dr; 63% per step)
allowed us to push forward toward the target.
Conversion of 13 to target (+)-2 was not straightforward.
First, addition of standard methyl nucleophiles such as
Grignard and organocerium reagents to the saturated ketone
failed because of competitive addition to the enone. Eventually,
tetramethylaluminum magnesium bromide in the presence of
anisole was found to deliver the axial alcohol in good yield.
Second, hydrogenation of the enone was problematic because
of competitive deconjugation of the strained alkene using Pd,
Pt, or Rh catalysis. This deconjugated alkene delivered the
incorrect configuration at C1 upon hydrogenation. Instead, we
turned to hydrogen atom transfer (HAT) hydrogenation,17
which we thought would deliver the thermodynamic diaster-
eomer independent of any competing isomerization. Under
stereoselectivity proved to be high (95:5 dr; Scheme 2). Third,
we could not excise auxiliaries by saponification because of
competing substrate reactivity, whereas 5 offered an alternative
solution (see below).
A single enantiomer of double dienophile 3 was synthesized
from dioxenone 6 via γ-selective crotylation, acyl ketene
generation and capture with 5, and unsaturation via the
selenoxide. This short sequence could generate large quantities
of 3, but its instability prevented its isolation and storage, so it
was used crude and immediately. Highly substituted dendralene
4 proved to be more difficult to access,13 so we turned to the
tandem [2 + 2] cycloaddition/retroelectrocyclization reaction
reported for electron-deficient alkynes14 in the hope that this
Scheme 2. Synthesis of (+)-7,20-Diisocyanoadociane [(+)-2]
B
DOI: 10.1021/jacs.6b03899
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Journal of the American Chemical Society
Communication
HAT conditions,18 ketone 14 was produced in good yield as
the major diastereomer.19
We intended to directly access ketone 17 by tandem acyloin
cyclization and alkoxy ketone deoxygenation. Unfortunately, all
methods for ketyl generation yielded complex mixtures, so we
devised a workaround. Keto ester 14 was reduced with DIBAL
and oxidized to keto aldehyde 15. Cyclization catalyzed by the
N-heterocyclic carbene generated in situ from triazolium salt 16
generated the expected α-hydroxy ketone, and samarium(II)
iodide-mediated deoxygenation delivered ketone 17.
In previous work, the diaxial diol corresponding to 18 has
been converted by non-stereoselective displacement using
TMSCN/TiCl4 to (+)-2 and its three other diastereomers,
which were separated by HPLC.6 We have previously shown
that tert-alkyl trifluoroacetates can undergo stereoinversion via
Sc(OTf)3-catalyzed solvolysis with TMSCN.20 For such a
reaction to generate 2, we required an axial alcohol at C7 but an
equatorial alcohol at C20. Yamamoto’s MAD reagent achieved
this stereochemistry in model ketones but delivered only the
axial alcohol from 17 using organolithium, -magnesium, or
-cerium nucleophiles. Finally, we found that methylenation
followed by oxymercuration cleanly generated the equatorial,
axial diol 18 as a single diastereomer. Isocyanation according to
our solvolysis protocol yielded a 5:1 mixture of two
diastereomers in 60% yield, and the two other diastereomers
were not detected.
Access to ( )-1 and (+)-2 allowed their antiplasmodial
activities to be investigated. Strictly chemical experiments have
shown that 1 and 2 can bind free heme in aqueous solution and
prevent crystallization of β-hematin.4a,c The ability of several
isonitriles to prevent crystallization were correlated to their
ability to kill P. falciparum and taken as proof that inhibition of
biocrystallization is the mechanism of ICT antiplasmodial
activity.4c
The liver schizont of Plasmodia species does not rely on
catabolism of host hemoglobin for nutrition and therefore does
not rely on biocrystallization as a protective mechanism.
Consequently, liver-stage assays have been used to identify the
existence of alternative mechanisms of action for putative
biocrystallization disruptors.21 We assayed22 the metabolites
amphilectene ( )-17a and (+)-2 for activity against asexual-
blood-stage (P. falciparum, Dd2 strain) and liver-stage
(Plasmodium berghei) parasites in addition to the simple
isonitriles 19, 20, and 21 synthesized in our lab (Figure 2).20
The blood-stage inhibition values for 1 and 2 are comparable to
those reported in the literature for related strains.4 Remarkably,
isonitriles 1, 2, 20, and 21 are all active against liver-stage
parasites with IC50 values near or below 1 μM. Therefore, a
mechanism or mechanisms other than or in addition to heme
detoxification inhibition may also underlie the activity of 1 and
2, in contrast to prior theories about their activity.4 The marked
differences between the blood-stage and liver-stage potencies of
the different isonitriles underscore the functional role of
additional molecular architecture, e.g., (+)-20 vs (+)-21.
Abstraction of the presumed pharmacophore yields decalin
19, which is completely inactive in both liver-stage and blood-
stage assays. Clearly, hydrophobic isonitriles are not all
equivalent, possibly because of differences in productive
binding, cellular uptake, or intracellular pharmacokinetics, for
example.
Figure 2. Asexual-blood-stage (ABS, P. falciparum) and liver-stage
(PbLuc, P. berghei) values for 1, 2, and 19−21.22
target folate metabolism are precedented in malaria therapeu-
tics,25 and intracellular copper chelation has been shown to
arrest parasite maturation.26 However, since multiple pathways
may be targeted by the ICTs, an agnostic approach to target
identification may be the best strategy for mechanistic study.27
To summarize, we have reported a concise and fully
stereocontrolled synthesis of (+)-2, a potent antimalarial
metabolite.28 Highlights of the synthesis include (1) a short
route to substituted polarized dendralenes via Lewis acid-
mediated [2 + 2] cyclization/retroelectrocyclization; (2)
application of these reagents (which we termed Danishefsky-
type dendralenes) to the synthesis of the carbon scaffold of the
adocianes; (3) use of HAT hydrogenation to establish the
correct C1 stereochemistry when all other methods failed; (4)
stereocontrolled installation of the axial, equatorial bis-
(isonitrile) of 2; and (5) design of the reaction sequence to
minimize FGIs,8 which includes the absence of protecting
groups. In contrast to other approaches to this family, our route
through polarized dendralenes and tert-alkyl alcohol inversion is
highly stereocontrolled. Access to isonitriles 1, 2, and 19−21
allowed us to demonstrate that heme detoxification cannot
exclusively underlie the antiplasmodial activity of the ICT class
and that other mechanisms must operate, even for potent heme
crystallization disruptors like 1 and 2.29 Nevertheless, our data
also indicate that the structure−activity relationships within this
class are nebulous. We hope that these data may stimulate more
interest in the community and perturb the impression that all
hydrophobic isonitriles are functionally equivalent.30 The
identification of easily synthesized isonitriles (+)-20 and
(+)-21 as potent liver- and blood-stage inhibitors, respectively,
should significantly aid further studies.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b03899.
Detailed experimental procedures, spectral data, and
chromatograms (PDF)
Crystallographic data for ( )-18 and ( )-11 (PDF)
Crystallographic data for ( )-11 (CIF)
The related kalihinol class of ICTs has been shown to inhibit
bacterial folate biosynthesis23 and also to induce a copper-
deficient phenotype in zebrafish embryos.22b,24 Strategies to
C
DOI: 10.1021/jacs.6b03899
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Journal of the American Chemical Society
Communication
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Russ. Chem. Bull. 2009, 58, 1438.
AUTHOR INFORMATION
Corresponding Author
*rshenvi@scripps.edu
■
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Present Address
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∥S.V.P.: Department of Chemistry, University of California,
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§H.-H.L. and S.V.P. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this work was provided by the NIH
(GM105766). Additional support was provided by Eli Lilly,
Novartis, Bristol-Myers Squibb, Amgen, Boehringer-Ingelheim,
the Sloan Foundation, and the Baxter Foundation. We thank
Dr. Milan Gembicky, Dr. Curtis Moore, and Professor Arnold
L. Rheingold for X-ray crystallographic analysis.
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