Total Synthesis of Belizentrin Methyl Ester: Report on a Likely Conquest

Article abstract of DOI:10.1002/anie.201805125

The assigned structure of the dinoflagellate-derived toxin belizentrin was prepared by total synthesis in form of the corresponding methyl ester for stability reasons. The successful route features an unusual solution for the preparation of a recalcitrant ylide on a C-glycosidic segment; moreover, it involves an asymmetric hetero-Diels–Alder reaction en route to the tertiary hemiacetal substructure, a Negishi cross-coupling of two elaborate building blocks, and a macrocyclization based on an intramolecular aminolysis of a spirolactone. A modified Kocienski olefination ultimately allowed the polyol side chain to be attached to the macrocycle although this transformation faced the exceptional base sensitivity of this polyunsaturated target compound.

Full text of DOI:10.1002/anie.201805125

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Accepted Article
Title: Total Synthesis of Belizentrin Methyl Ester: Report on a Likely
Conquest
Authors: Alois Fürstner, Felix Anderl, Sylvester Größl, and Conny Wirtz
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201805125
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Total Synthesis of Belizentrin Methyl Ester: Report on a Likely Conquest
Felix Anderl,[+] Sylvester Größl,[+] Conny Wirtz, and Alois Fürstner*
Dedicated to Prof. Tohru Fukuyama on the occasion of his 70th birthday
Abstract: The assigned structure of the dinoflagellate-derived toxin belizentrin was prepared by total
synthesis in form of the corresponding methyl ester for stability reasons. The successful route features
an unusual solution for the preparation of a recalcitrant ylid on a C-glycosidic segment; moreover, it
involves an asymmetric hetero-Diels/Alder reaction en route to the tertiary hemiacetal substructure, a
Negishi cross coupling of two elaborate building blocks, and a macrocyclization based on an
intramolecular aminolysis of a spirolactone. A modified Kocienski olefination ultimately allowed the
polyol side chain to be attached to the macrocycle, although this transformation faced the
exceptional base-sensitivity of this polyunsaturated target compound.
Many marine dinoflagellates possess disproportionately large genomes that encode for impressive
biosynthetic capabilities of these morphologically rather simple organisms.1 A host of structurally
diverse secondary metabolites of utmost complexity was isolated from various members of this
taxon, including some of the most potent non-peptidic poisons known to date. Emblematic examples
are the alkaloid saxitoxin,2 an intriguing class of ladder polyethers (maitotoxin, ciguatoxin, brevetoxin
etc.),3 as well as numerous macrolides of polyketide origin, for which the amphidinolide family is
deemed representative.4,5 Because of their intriguing molecular architectures, structural diversity,
biological significance and paucity, dinoflagellate-derived natural products served as inspiring yet
highly demanding targets for synthesis, and rightfully continue to do so to the present day.
Belizentrin (1) is a particularly notable recent addendum to the list.6 This compound was obtained in
low isolation yield (3.1 mg) from a large-scale culture (1000 L) of the benthic dinoflagellate
Prorocentrum belizeanum.7 Although the small available quantity and low stability made a detailed
*
F. Anderl,[+] S. Größl,[+] C. Wirtz, Prof. A. Fürstner
Max-Planck-Institut für Kohlenforschung
45470 Mülheim/Ruhr (Germany)
E-Mail: fuerstner@kofo.mpg.de
[+]
These authors contributed equally to the project
Supporting Information #######################################
1
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biological assessment impossible, 1 was shown to disrupt the neuronal network of cerebellar cell
cultures in vitro. Despite a slow onset of action, serious neurite damage was observed at 100 nM and
complete degeneration of neuronal somas was induced at 300 nM.6 Since the compound partly
degraded during the course of the bioassay, these data points can only be taken as rough estimates
and the inherent activity of 1 might actually be (significantly) higher.
Scheme 1. Structure of belizentrin (1) and retrosynthetic analysis of belizentrin methyl ester (2)
The inherent instability also rendered the structure elucidation of belizentrin (1) challenging as some
desirable NMR data simply could not be acquired. Based on a masterful interplay of spectroscopic
analysis, detailed computational studies and careful comparison with literature data, the isolation
team proposed the constitution and stereostructure shown in Scheme 1.6 It was prudently pointed
2
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out, however, that only the relative configuration within the macrocycle and the sidechain were
assigned, whereas the relationship between these domains was arbitrarily assigned.
In any case, belizentrin (1) is rather unique in structural terms: it features an odd-numbered 25-
membered ring – which in itself is a remarkable structural attribute – that contains an ester as well as
an amide linkage and as such seems to be the first example of a dinoflagellate-derived macrolactam.
The tetraene subunit in the southern sector is partly skipped, comprises an exocyclic methylene
group, and is certainly labile since isomerization of the two remote olefins into conjugation with the
ester would entail a considerable enthalpic gain; at the same time, the scattered array of double
bonds rigidifies the macrocyclic frame. No less than 16 chiral centers decorate the carbon backbone
of 1, 11 of which reside in the side chain that is highly hydrophilic by virtue of the carboxylic acid
terminus, six secondary hydroxy groups and two ether rings. Interestingly, four of the five remaining
chiral centers are clustered together to form the unusual tertiary hemiacetal entity embedded into
the macrocyclic perimeter. These attributes render belizentrin (1) “first in class” in that it stands for
an unprecedented type of dinoflagellate-derived secondary metabolites.6
Apprehensive of the fact that the limited stability of 1 further augments the synthetic challenges, we
deliberately targeted the methyl ester 2 in the hope to improve the lifetime of the compound while
not interfering with its biological activity;8 it is reasonable to believe that hydrolytic enzymes might
release the parent compound in a given biological matrix under assay conditions. For the uncertainty
concerning the stereochemical correlation between tail and macrocycle, the C17-C18 double bond
constituted the obvious assembly site, which mandates an E-selective olefination that is compatible
with the numerous functionalities in either building block. The Kocienski variant of the Julia reaction
was deemed appropriate,9,10 not least because previous work from this laboratory had shown that a
tetrazolyl sulfone carrying a C-H acidic ester group can be successfully engaged in olefination;11 since
the metalated sulfone had not entailed any epimerization of an -alkoxylated aldehyde either,11,12
this precedent boded well for the projected coupling of segment A with the -acylated partner B,
which are of similar complexity yet largely different chemical character.
Various options for macrocyclization were considered, of which macrolactamization was given
priority over macrolactonization. Critical for this judgement was our previous experience with a
3
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skipped polyunsaturated seco-acid, which had been irreparably damaged upon activation as a mixed
anhydride.13 Moreover, it appeared to us that the amide linkage invites an intramolecular aminolysis
of a lactone precursor, which is a rather rare tactic for macrocyclization.14 The required precursor
could be assembled via cross coupling from building blocks C, D and E by a convergent route that
should allow the number of steps to be minimized where the arguably sensitive polyene motif needs
to survive. Symmetry considerations let us trace the tetrahydropyran entity embedded into the
polyhydroxylated tail A back to D-glucose, whereas the 2,5-trans-disubstituted tetrahydrofuran ring H
seemed accessible from I by adaptation of a sequence that had previously served our work in the
amphidinolide series well.15 We were confident that the all-syn triol unit connecting the two ether
rings could be formed by stereoselective olefination of fragments F and H followed by a catalyst-
controlled dihydroxylation event.
Scheme 2. a) NaNO2, HCl, H2O, 0 °C  RT, 79%; b) BH3SMe2, THF, 0 °C  RT, 69%; c) TrCl, pyridine,
73%; d) LDA, MeI, THF, 78 °C  30 °C, 99%; e) LDA, 78 °C, then H2O, 96%; f) Dibal-H, CH2Cl2, 78
°C, 98%; g) Ph3P=CHCOOEt, toluene, 80 °C, 69%; h) TBAF3H2O, THF, 0 °C, 82%; i) LiAlH4, THF, 20 °C
 RT, quant.; j) 1-phenyl-1H-tetrazolyl-5-thiol, DIAD, PPh3, THF, 0 °C  RT, 87%; k) (NH4)6Mo7O244
H2O, aq. H2O2, EtOH, 71%; l) TFA, CH2Cl2, 0 °C, 98%; m) (COCl)2, DMSO, DIPEA, CH2Cl2, 78 °C  RT,
94%; DIAD = di-isopropyl azodicarboxylate; DIPEA = di-isopropylethylamine; LDA = lithium di-
isopropylamide; TBAF = tetra-n-butylammonium fluoride; TFA = trifluoroacetic acid; Tr = trityl
In the forward sense, compound 5 was prepared on scale from L-glutamic acid (3) by following the
route previously described for the enantiomer (Scheme 2).15 The key step was a fluoride-promoted
oxa-Michael reaction, which furnished the 2,5-trans-disubstituted tetrahydrofuran ring 5 as the only
4
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isomer. Elaboration of the ester terminus to the required tetrazolylsulfone was uneventful, as was
the conversion of the trityl ether into the labile aldehyde 9, which had to be used without delay.
Scheme 3. a) allyl(trimethyl)silane, BF3OEt2, MeCN, RT  80 °C, 79% (: = 7:1); b) NaOEt, MeOH,
98%; c) TBSOTf, 2,6-lutidine, CH2Cl2, 0 °C  RT, 96%; d) HFpyridine, THF/pyridine, 0 °C  RT, 97% (dr
= 8:1); e) (COCl2), DMSO, DIPEA, CH2Cl2, 78 °C  RT, 90%; f) [MeOCH2PPh3]Cl, KOtBu, THF, MS 5Å,
50 °C, then 78 °C  RT, 88%; g) PCC, CH2Cl2, 79% (dr = 3:1); h) KMnO4, HOAc, aq. acetone, 77%; i)
CBr4, PPh3, CH2Cl2, 81%; j) PPh3, benzene, 20 °C, quant.; k) DIPEA, benzene; l) 9, benzene, 79% (over
both steps, E:Z = 18:1); m) 22, BH3SMe2, CH2Cl2, 20 °C, 91% (dr > 30:1); n) K2OsO4 (20 mol%), 23 (25
mol%), MeSO2NH2, K3[Fe(CN)6], K2CO3, tBuOH/H2O, 59% (dr = 4:1); o) TESOTf, 2,6-lutidine, CH2Cl2, 0
°C  RT, 82%; MS = molecular sieves; PCC = pyridinium chlorochromate; TBS = tert-
butyldimethylsilyl; TES = triethylsilyl; Tf = trifluoromethylsulfonyl
The required partner was derived from glucopyranose 10, which was transformed in three known
operations into C-glycoside 11 (Scheme 3).16 Selective desilylation of the primary –OTBS ether with
HF/pyridine furnished alcohol 12, which held various options for C1-homologation to the required
methyl ester 14. In practical terms, a sequence comprised of Swern oxidation followed by Wittig
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olefination with [MeOCH=PPh3] and treatment of the resulting enol ether 13 with PCC17 proved most
robust on scale. The subsequent oxidation of the terminal alkene to the corresponding hydroxy
ketone 15 with KMnO4 in aqueous acetone/HOAc worked well without noticeable CC bond cleavage
or deprotection of the silyl ethers.18
In sharp contrast, the seemingly trivial conversion of 15 into the stabilized ylid 18 was unexpectedly
difficult. Because -bromo ketone 16 is prone to debromination, many attempts at converting this
compound into the corresponding phosphonium salt 17 resulted in (partial) degradation. Recourse to
a somewhat counterintuitive method allowed this serious problem to be overcome: reminiscent of
early studies, which had shown that the N-alkylation of Et3N is particularly effective in frozen
benzene,19 we were hoping that the P-alkylation of PPh3 with 16 would benefit from the same effect.
The observed rate acceleration had been ascribed to phase segregation close to the eutectic
temperature, which entails high concentration of any solute reagent in the few remaining liquid
regions within an otherwise solid solvent matrix.19 Indeed, 17 was reliably formed in essentially
quantitative yield in benzene glass, simply by storing the reaction mixture at 20 °C.
The fact that the carbonyl group of 17 is notably enolized (NMR) explains why Hünig base sufficed to
release ylid 18, which reacted with aldehyde 9 to give the corresponding enone (E/Z = 18:1). For the
elaboration of the major E-isomer, it proved advantageous to first subject the ketone to CBS-
reduction,20 followed by Sharpless dihydroxylation of the resulting allylic alcohol 19 using
(DHQD)2AQN (23) as the ligand.21 This sequence furnished triol 20 in appreciable yield and selectivity.
Its configuration was unambiguously assigned by comparison with the other conceivable
diastereomers (prepared by two independent routes), the formation of cyclic derivatives for NMR
inspection, as well as on Mosher ester analyses; details will be reported in a future full paper. Final
TES-protection gave building block 21 representing the tail region of belizentrin methyl ester (2).
6
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Scheme 4. a) (COCl)2, then TMBOH, DMAP, K2CO3, CH2Cl2, 91%; b) 3-buten-2-one, HGII (0.1 mol%),
CH2Cl2, reflux, 96%; c) TESOTf, Et3N, Et2O, 0 °C, 89%; d) O2, [Cu(MeCN)4]BF4 (4.5 mol%), TEMPO (4.5
mol%), 2,2‘-bipyridine (4.5 mol%), N-methylimidazole (9 mol%), MeCN, quant.; e) 35 (9 mol%), MS
4Å, 76% (95% ee); f) O2, Pd(OAc)2 (10 mol%), DMSO, 78%; g) MeMgCl, THF, 65°C, 77%; h) AD-mix-,
tBuOH/H2O, 81%; i) TESCl, AgNO3, DMAP, DMF, pyridine, 76%; j) Ph3PBr2, CH2Cl2, 0 °C, 89%; DMAP =
4-dimethylaminopyridine; TEMPO = 2,2,6,6-tetramethylpiperidinyloxy; TMB = 3,4,5-trimethoxybenzyl
4-Pentenoic acid (24) served as point of departure for the preparation of the macrocyclic sector
(Scheme 4), which was elaborated into diene 27 by esterification, cross metathesis and enolsilane
formation. Compound 27 was subjected to an asymmetric hetero-Diels/Alder reaction with aldehyde
29 catalyzed by the chromium complex 35,22,23 which furnished adduct 30 with 95% ee on a
multigram scale. A modified Saegusa oxidation then gave enone 31,24 which reacted with MeMgCl to
provide the tertiary alcohol 32 as a single diastereomer;25 low temperature was necessary to avoid
competing attack of the Grignard reagent onto the ester group. The subsequent asymmetric
dihydroxylation under standard conditions21 was accompanied by spontaneous closure of the
butyrolactone ring, which we were hoping would later prove sufficiently electrophilic as to allow for
the formation of the macrocyclic ring by intramolecular aminolysis. Protection of the two remaining
hydroxy groups mandated in situ activation of TESCl with AgNO3.26 Treatment of the resulting
product 33 with Ph3PBr2 engaged only the primary silyl ether into functional group interchange to
furnish bromide 34 as an adequate pre-nucleophile for fragment coupling.
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Scheme 5. a) PhMe2SiLi, CuCN, THF, 78 °C; b) 39, BF3OEt2, 51%; c) 2,4,6-trichlorobenzoyl chloride,
Et3N, toluene, then 40, DMAP cat., 0 °C  RT, 63%; PMB = p-methoxybenzyl
The second required building block was obtained by silylcupration of propyne,27 which formed an
organocopper reagent of the formal composition 36 that was quenched with 39 in the presence of
BF3OEt2 to give alcohol 37 (Scheme 5). Although the yield was only 51%, the reaction was nicely
scalable; therefore this one-step operation was preferred over alternative routes to this compound.
Esterification of 37 with the stannylated acrylic acid 4028 under Yamaguchi conditions29 gave product
38 endowed with two orthogonal metalloid sites.
The third segment was secured by regioselective silylmetalation of 2-butyn-1-ol (41) (Scheme 6).30
Conversion of the –OH group of 42 into the corresponding allylic bromide 43 followed by an
alkylation/Knoevenagel reaction cascade31 furnished multigram amounts of 44, which was
transformed into the allylic acetate 45 by standard means. This compound was amenable to an allyl-
Suzuki reaction with the boronate derivative 49,32 obtained by hydroboration of N-Boc propargyl
amine with pinacol borane (see the SI). Since the NBoc group later proved inadequate for the
projected end game, it was transformed at this point into the highly practical –NTsoc group33 on
treatment of 46 with TIPSOTf/2,6-lutidine in CH2Cl2.34 Final iodo-desilylation35 gave alkenyl iodide 47
as adequate surrogate of the skipped triene motif of belizentrin (1).
With all required fragments in hand, the stage was set for the assembly of the macrocycle and the
completion of the total synthesis (Scheme 7). To this end, 38 and 47 were engaged in a Stille coupling
which was pleasingly productive even though the alkenylstannane 38 is rendered electron-poor by
conjugation with the ester group.36 The polyunsaturated nature of the resulting product 50
8
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notwithstanding, the alkenyl silane could be selectively iodinated with [Py2I]BF4, even though partial
isomerization of the resulting alkenyl iodide was observed. Compound 51 was then coupled with the
highly functionalized organozinc derivative 52 prepared by insertion of Rieke-Zn into bromide 34.37
Best results were obtained with a catalyst generated from [(dppf)PdCl2] and Ph3As by reduction with
Mn metal in DMF prior to use; this procedure is subject to further study in our laboratory. In any
case, the advanced Negishi reaction between the two elaborate partners was clean and productive,38
furnishing product 53 in readiness for the projected macrocyclization.
Scheme 6. a) PhMe2SiLi, AlEt3, CuCN (4 mol%), THF/hexane, 90%; b) CBr4, PPh3, CH2Cl2, 96%; c) (i) 48,
DBU, toluene; (ii) (CH2O)n, K2CO3, THF, reflux, 77%; d) Dibal-H, CH2Cl2; e) Ac2O, DMAP, Et3N, 94% (over
both steps); f) 49, [(tris-2-furylphosphine)2PdCl2] (2.7 mol%), KF, MeOH, 90%; g) TIPSOTf, 2,6-lutidine,
CH2Cl2, 0 °C  RT, 89%; h) NIS, 2,6-lutidine, 1,1,1,3,3,3-hexafluoroisopropanol, 0 °C, 63%; DBU = 1,8-
diaza-bicyclo[5.4.0]undec-7-ene; NIS = N-iodosuccinimide; TIPS = tri-isopropylsilyl
It was at this stage that the –NTsoc group proved critically important because it could be selectively
removed with excess HFpyridine without affecting the residual TES-ethers. In contrast, TBAF rapidly
decomposed the compound, probably causing instant isomerization of the exo-methylene group into
conjugation. This observation bore strategic relevance as it implied that basic conditions must be
avoided from this point onwards. Moreover, it was noticed that attempted isolation of the released
free amine 54 resulted in substantial material loss, which we ascribed to partial oligomerization upon
concentration of the mixture. While this finding made a rigorous purification of the macrocyclization
9
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precursor impossible, it augured well for the projected aminolysis. Indeed, heating of the crude
amine 54 in dilute toluene solution sufficed to form the desired macrolactam 55; it proved beneficial
to supplement the mixture with excess 2-pyridone which is thought to act as a proton shuttle that
accelerates the likely rate-determining breakdown of the tetrahedral intermediate formed upon
attack of the amine onto the lactone carbonyl group.14,39 Somewhat surprisingly though, the reaction
invariably stalled at about 60% conversion: it was a control experiment, in which pure 55 was re-
subjected to the reaction conditions, which proved that this composition represents the equilibrium
mixture: amide formation is obviously reversible by virtue of the favorable disposition of the –OH
group in 55 which facilitates re-formation of the spiro-lactone ring.40
Scheme 7. a) [(Ph3As)4Pd] (6 mol%), DMF, 82%; b) [Py2I]BF4, MeCN, 20 °C, MeCN, 62% (Z:E = 3:1); c)
Rieke-Zn, DMF; d) [(dppf)PdCl2] (20 mol%), Ph3As, Mn, DMF, then 52, 70%; e) HFpyridine, THF, 0°C; f)
2-pyridone, toluene (0.001 M), 90 °C, 45-52% (at ca. 60% conversion, see Text); g) [Ph3C]BF4, CH2Cl2, 0
°C, 44-52%; h) SO3pyridine, DMSO, DIPEA, CH2Cl2, 20 °C, quant.; i) 21, lithium hexamethyldisilazide,
ZnCl2, then 56, DMF/DMPU, 40 °C  RT, 25-30%; j) aq. HF, MeCN, then Me3SiOH, 36%
With a reliable entry into the macrocyclic fragment 55 secured, we tackled the attachment of the
side chain which proved highly demanding. Cleavage of the terminal PMB-ether failed under a variety
10
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of conditions and could only be achieved with trityl tetrafluoroborate.41 Considerably more difficult,
however, was the actual fragment coupling itself: since all attempts at protecting the sterically
hindered tertiary anomeric –OH group were to no avail (at the stage of 55 or before), the Kocienski
olefination had to be performed with the -acylated aldehyde carrying a free -OH group, which, on
top, is prone to hemiacetal or hydrate formation.42 By far most troublesome, however, was the
exceptional sensitivity of this polyunsaturated compound towards even moderately basic conditions,
which had been foreshadowed during the cleavage of the –NTsoc group: whereas the presence of
the ester in 21 did not interfere with the selective deprotonation of the sulfone group, as expected
based on prior art,11 aldehyde 56 got instantly destroyed when exposed to the resulting metalated
species; variation of the base, the solvent and/or the temperature remained unsuccessful.10
Inspired by a report that the outcome of a Kocienski olefination with an enolizable ketone was much
improved upon transmetalation of the lithiated sulfone with CeCl3,43 we tried to temper the basicity
of the nucleophile derived from 21 in an analogous fashion; these conditions did indeed provide a
tiny first crop of 57 ( 8%). A similar result ( 10%) was obtained with TMSCl as the additive, which
had previously been recommended for the olefination of a base-sensitive aldehyde.44 After much
experimentation, it was found that ZnCl2 in combination with LiHMDS in DMF/DMPU as the solvent45
was somewhat more effective, furnishing E-57 as the only detectable isomer in well reproducible 25-
30% yield. Although certainly modest, this outcome must be assessed against the chemical challenge
that it does address. Global deprotection of all eight silyl ethers in the resulting product 57 mandated
the use of aqueous HF in MeCN,46,47 whereas other commonly used reagents destroyed the valuable
compound; to account for the sensitivity and polarity of the resulting product 2, the work-up
procedure was modified by quenching the reaction with Me3SiOH so as to produce only volatile
byproducts (Me3SiF, H2O) that could be largely removed in high vacuum.
The recorded 13C NMR spectra of belizentrin methyl ester (2) seem to show a systematic drift ( 0.4
ppm) relative to the literature data of parent belizentrin (1);6 whether this is due to the fact that the
data of the natural product could only be extracted from 2D spectra for the lack of material and are
hence likely less accurate, or whether the difference is caused e. g. by different calibration cannot be
decided. Upon correction for this apparently systematic difference, almost all positions show a
reasonable to excellent match (except for C7, C9, C23), especially if one considers that the natural
11
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1
product is a carboxylic acid whereas our synthetic samples are methyl esters. Likewise, the H NMR
data  including the entire pattern of NOE/ROESY correlations  are in good accord with the reported
spectra (for details, see the SI). Therefore we tend to believe that structure 1 describes the correct
constitution and relative stereochemistry of belizentrin;48 it is emphasized, however, that a definitive
answer must await the synthesis of the other conceivable diastereomer and release of the
carboxylate termini from the corresponding esters.49 Any such venture is likely to benefit from the
intelligence gathered in this report about the Achilles heel of belizentrin and its immediate
precursors.
Acknowledgements
Generous financial support by the MPG is gratefully acknowledged. We thank the analytical
departments of our Institute for expert assistance and Dr. Antonio Hernández Daranas for sharing
with us additional information concerning the spectra of belizentrin.
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For the Table of Contents
Potent but fragile: The first total synthesis of the provisional structure of belizentrin methyl ester is
described. This sizeable metabolite from a tiny marine microorganism potently impacts on the
survival of cerebellar cells and the integrity of their neuronal networks at nanomolar concentrations.
The successful route had to cope with the sensitivity of this target, which let us recognize the partly
skipped polyene region as a veritable Achilles heel.
Keywords: cross coupling  macrocyclization  marine natural products  polyenes  Kocienski
olefination
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1
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42
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48
Because attempted hydrolysis of 2 has not yet been successful, it is currently impossible to decide on the
absolute configuration of belizentrin: 1 had been reported in the literature to be levorotatory, whereas
synthetic 2 proved dextrorotatory.
49
Actually, we experienced in the past that even the synthesis of both conceivable target structures may not
provide a fully conclusive answer: specifically, in the leiodermatolide case, it required a bioassay to decide
beyond doubt which of two conceivable but spectroscopically almost indistinguishable diastereomers
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16
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