Scalable Total Synthesis, IP3R Inhibitory Activity of Desmethylxestospongin B, and Effect on Mitochondrial Function and Cancer Cell Survival

Article abstract of DOI:10.1002/anie.202102259

The scalable synthesis of the oxaquinolizidine marine natural product desmethylxestospongin B is based on the early application of Ireland–Claisen rearrangement, macrolactamization, and a late-stage installation of the oxaquinolizidine units by lactam reduction. The synthesis serves as the source of material to investigate calcium signaling and its effect on mitochondrial metabolism in various cell types, including cancer cells.

Full text of DOI:10.1002/anie.202102259

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 11278–11282
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doi.org/10.1002/anie.202102259
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Total Synthesis
Scalable Total Synthesis, IP3R Inhibitory Activity of
Desmethylxestospongin B, and Effect on Mitochondrial Function and
Cancer Cell Survival
ˇ
Masa Podunavac, Artur K. Mailyan, Jeffrey J. Jackson, Alenka Lovy, Paula Farias,
Hernan Huerta, Jordi Molgꢀ, Cesar Cardenas,* and Armen Zakarian*
Abstract: The scalable synthesis of the oxaquinolizidine
marine natural product desmethylxestospongin B is based on
the early application of Ireland–Claisen rearrangement, mac-
rolactamization, and a late-stage installation of the oxaquino-
lizidine units by lactam reduction. The synthesis serves as the
source of material to investigate calcium signaling and its effect
on mitochondrial metabolism in various cell types, including
cancer cells.
Figure 1. Structures of representative 1-oxaquinolizidine alkaloids.
X
estospongins, along with araguspongins, are a group of
natural products isolated from marine sponges of Xestospon-
gia sp.[1] Structurally, these compounds are comprised of two
oxaquinolizidine units tethered by saturated alkylidene
chains to form a macrocyclic core, and have variable
oxidation and stereochemistry at C9/C9’ (Figure 1).[1] While
a range of biological activity was reported for xestospongins,
we became intrigued by the mounting evidence for the unique
ability of xestospongin B to influence mitochondrial metab-
olism by modulating calcium signaling between the endo-
plasmic reticulum (ER) and mitochondria through the
inhibition of inositol-1,4,5-triphosphate receptors (IP3Rs).[2]
The constitutive activity of IP3Rs is essential for cellular
bioenergetics in a variety of cell types. Its inhibition disrupts
the constitutive calcium transfer from ER to mitochondria
causing a drop in mitochondrial respiration that generates
a bioenergetic crisis characterized by AMPK and autophagy
activation.[3] It has been shown that interruption of this
communication leads to a selective, substantial cancer cell
death leaving normal cells unaffected.[4] One of the goals of
this study was to evaluate the effect of desmethylxestospon-
gin B on mitochondrial respiration in breast cancer cell lines,
and the resultant selectivity in inducing cancer cell death
leaving normal cells nearly unaffected.
While xestospongin B (XeB) is no longer available from
the natural sources, total synthesis provides a feasible source
of material. We targeted desmethylxestospongin B
(dmXeB),[1b] a natural product within this family of metab-
olites, based on the premise that the 3’-methyl group has no
effect on the IP3R inhibitory activity, and obviating the need
to install it would simplify the development of a scalable
synthesis. While XeC and ArB are more accessible, they
show lower specificity with notable side-effects on calcium
homeostasis, and no data is available for ArC.[2b]
[*] M. Podunavac, Dr. A. K. Mailyan, Dr. J. J. Jackson,
Prof. Dr. C. Cardenas, Prof. Dr. A. Zakarian
Department of Chemistry and Biochemistry
University of California Santa Barbara
Santa Barbara, CA 93106 (USA)
Previous syntheses in this area by Baldwin[5] and Hoye[6]
are characterized by remarkable brevity and strategic ele-
gance, targeting C2-symmetrical, non-hydroxylated congeners
such as araguspongine B (ArB), and XeC and A, and
resolving controversy about the absolute configuration of
these compounds. However, the simplifying C2-symmetry is
broken in the biologically more intriguing XeB and dmXeB
by the presence of 9-OH and, in the former case, the 3’-CH3
groups.
E-mail: zakarian@chem.ucsb.edu
Dr. A. Lovy, P. Farias, H. Huerta, Prof. Dr. C. Cardenas
Center for Integrative Biology, Faculty of Sciences, Geroscience
Center for Brain Health and Metabolism, Universidad Mayor
Santiago (Chile)
E-mail: julio.cardenas@umayor.cl
J. Molgꢀ
The synthesis design is depicted in Scheme 1. The final
assembly of the 1-oxaquinolizidine units was envisioned to
take place by intramolecular N-alkylation of the amide
groups in macrocyclic bis(lactam) 1 followed by lactam
semireduction and hemiaminal formation. The bis(lactam)
was planned to arise from combining two w-amino acid
precursors 2 and 3 by sequential amide formation. These
precursors will be prepared from a common starting material,
azidoalcohol 4. After its esterification with either 2-(benzy-
loxy)-5-chloropentanoic acid (5) or 5-chloropentanoic acid,
Universitꢁ Paris-Saclay, CEA, Institut des Sciences du Vivant Frꢁdꢁric
Joliot, ERL CNRS n 9004, Dꢁpartement Mꢁdicaments et Technologies
pour la Santꢁ, Service d’Ingꢁniere Molꢁculaire pour Santꢁ (SIMoS)
Batiment 152, Point courrier 24, 91191 Gif-sur-Yvette (France)
Prof. Dr. C. Cardenas
The Buck Institute for Research on Aging
Novato, CA (USA)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under https://doi.org/10.
1002/anie.202102259.
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reaction conditions. Cuprate coupling with the remainder of
chloro epoxide (S)-6 with the reagent formed from 8 was
accomplished in 78% yield.[10] Subsequent substitution of
chloride with sodium azide, O-silylation, and removal of the
PMB group delivered 58.1 grams of 4 in 87% yield.
In order to access non-symmetrical congeners such as
dmXeB possessing a C9 hydroxy group, the synthesis of an a-
benzyloxy acyl chloride 14 was required. Due to the thermal
instability of ester enolates generated from a-alkoxy esters,
alkylation of t-butyl a-benzyloxyacetate with iodide 10 was
accomplished at À958C in moderate yield of 45% under
optimized reaction conditions. After desilylation, mesylation,
and substitution with LiCl, ester 13 was obtained in 86%
yield. Cleavage of the t-butyl ester and chlorodehydroxylation
with oxalyl chloride completed the synthesis of acyl chloride
14 (93% yield, 10.2 grams).
In preparation for the bis(macrolacram) assembly, the
individual amino acid synthons were obtained by Ireland-
Claisen rearrangement (ICR) of two esters prepared from
allylic alcohol 4 (Scheme 3a and b).[11] Intermediate 2 was
obtained from ester 15 (82% yield, dr 10:1) under reaction
conditions developed specifically for a-alkoxy esters, where
we found that the best chelation-controlled outcome is
achieved with KN(SiMe3)2/PhMe combination, while, sur-
prisingly, LDA/THF affords non-chelation controlled diaste-
reomer with good selectivity, despite the expected higher
propensity of lithium to coordinate with oxygen Lewis
bases.[12] ICR of ester 16 gave rise to carboxylic acid 17
(87% yield, dr 6:1), which, after forming the methyl ester with
Me3SiCHN2, was advanced to amino ester 3 by azide
reduction with SnCl2/PhSH (82% yield).[13]
Fragment union was achieved by amide formation from
acid 2 and amine 3, followed by another azide reduction
under the same conditions, ester hydrolysis, and macro-
lactamization.[14] At this stage, the minor diastereomers
formed during ICR were separable, and macrocyclic bis-
(lactam) 1 was isolated in 35% yield as a single diastereomer.
Completion of the synthesis follows a bidirectional strat-
egy, starting with the formation of two valerolactam moieties
by intramolecular N-alkylation of 1 (LiN(SiMe3)2, THF, 85%
yield). Removal of the silyl groups preceded the crucial
reductive 1-oxaquinolizidine formation with Li-NH3 reagent,
which simultaneously accomplished O-debenzylation.[15]
Notably, numerous alternative reagents attempted to achieve
semireduction of the lactams (iBu2AlH,[16] NaAlH2(OR)2,[17]
IrCl(CO)(PPh3)2/(HMe2Si)2O),[18] consistently resulted in
complete reduction to piperidine. Partial a-dehydroxylation
leading to a minor amount of 22 was also observed.
Scheme 1. Synthesis design for dmXeB.
intermediates 2 and 3 will be accessed via Ireland-Claisen
rearrangement to establish stereogenic centers at C9’ and C9,
respectively.
This synthesis design has the advantage of flexibility in
accessing 1-oxaquinolizidine alkaloids with variable substitu-
tion at C9/9’ (with or without OH groups), as well as variable
stereochemistry at the same positions.
1-Chloro-3-butene served as the starting point for the
synthesis of 4 (Scheme 2a). Jacobsen hydrolytic kinetic
resolution of 1-chlorobutene oxide, obtained by epoxidation
with MCPBA, delivered (S)-6 in 58% yield and 92% ee.[7] The
epoxide was distributed divergently, with part of it being
advanced to allylic alcohol 7 with dimethylmethylenesulfur
ylide.[8] After reductive para-methoxybenzylation (PMB)
under electrophilic conditions and displacement of the
chloro group, iodide 8 was obtained in a 71% overall
yield.[9] The development of electrophilic benzylation was
required due to the propensity of 7 to undergo intramolecular
etherification during alkoxide formation under a number of
The final hydrogenation of the double bonds required
optimization, as the typical conditions using Pd/C in various
solvents proved inconsistent and occasionally resulted in an
intractable mixture of products. However, hydrogenation of
21 and 22 with Rh/Al2O3 in ethyl acetate[19] reliably and
cleanly delivered dmXeB (94% yield) and araguspongineB,
(ArB, 97% yield), respectively.
To further highlight the generality of the strategy, we
completed the synthesis of bis-hydroxylated ArC, which has
not been accessed previously by synthesis (Scheme 4, See
Supporting Information for the synthesis of 23 and 24). In this
Scheme 2. Preparation of early stage intermediates 4 and 14.
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case, we opted for p-methoxybenzyl (PMB) ether at C9/9’ to
test an alternative deprotection/reductive oxaquinolizine
construction method. We also utilized allyl ester in 24 to
simplify access to the free acid, as the methyl ester proved
resistant to hydrolysis. Thus, after acid 23 and amine 24 were
combined by amide formation, azide reduction, acid deal-
lylation, and macrolactamization afforded bis-lactam 25. Six-
membered lactam closure was accomplished with LiN-
(SiMe3)2 as previously, and removal of TBS and PMB
groups delivered 26 in high yield. Finally, reduction of the
lactam with Li-NH3 and hydrogenation completed the syn-
thesis of ArC.
Having established access to synthetic dmXeB, we
proceeded to evaluate its effect on IP3R-mediated calcium
release, mitochondrial respiration and selective anticancer
activity in breast cancer cell lines, and how it compares to
XeB isolated previously from a marine sponge. As shown in
Figure 2a, 30 min incubation with either dmXeB or XeB
completely abolished IP3R calcium release induced by ATP
in MDA-MB-231 cells. Subsequently, the normal cell line
MCF10A and a cancer cell line MDA-MB-231 were treated
with increasing concentrations of dmXeB and XeB for 24 h,
and oxygen consumption rates (OCR) were determined using
Seahorse system. As shown in Figure 2b, both compounds
decrease OCR similarly in a dose-dependent fashion. A
similar effect was observed in a triple-negative cell line BT-
549 and the luminal A MCF7 cell line (See Supporting
Information).
We have also demonstrated that dmXeB induces selective
cell death in breast cancer cell lines. Several breast cancer cell
lines that represent heterogeneity of breast cancer and the
normal cell line MCF10A were treated with different
concentrations of either XeB or dmXeB for 24 h and cell
death was measured by propidium iodide incorporation
through flow cytometry. As shown in Figure 2c, 7.5 mM
XeB induced a significant increase in cell death in MDA-
MB-231 cells, without affecting the normal cell line MCF10A.
At 10 mM, XeB is still more effective in MDA-MB-231 cells,
but some effects in MCF10A cells become measurable,
indicating reduced selectivity. On the other hand, dmXe B
in fact shows somewhat increase selectivity, inducing cell
death in MDA-MB-231 at 5 mM, which is sustained at 10 mM
with no effect on normal MCF10 cells (Figure 2c). At 20 mM,
MDA-MB-231 cells are still more sensitive, with almost 100%
of the population dead, whereas MCF10A shows an initial
increase in cell death. In cell lines representative of luminal A
(MCF7) and luminal B (ZR75) breast cancer cell lines XeB
and dmXeB display selective cell death at concentrations
between 5 and 10 mM. Similarly to the observations with
MDA-MB-231 cell line, dmXeB at 20 mM concentration
caused an almost complete cell death in ZR75 and MCF7 cell
lines, while also affecting the normal cell line MCF10A to
a much lesser extent. The BT-549 cells are more resistant to
both compounds, showing only selectivity at 7.5 mM. Finally,
we determined whether dmXeB affected the ability of cancer
cells to proliferate indefinitely (one of the hallmarks of
cancer), as demonstrated previously for XeB[4] MDA-MB-
231 cells were treated with either compound for 24 h and then
one thousand cells were collected and reseeded. After one
Scheme 3. Fragment union and completion of the synthesis.
Scheme 4. The synthesis of araguspongineC (ArC).
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Figure 2. Average cytosolic calcium increases in response to ATP (100 mM) in MDA-MB-231 cells treated with ethanol (vehicle, control) or 5 mM
XeB (upper panel, red trace) or dmXeB (bottom panel, blue trace) for 1 h. Mean Æ SEM of 3 independent experiments. In each experiment, 100
cells were analyzed. b) Basal oxygen consumption rate (OCR) of MDA-MB-231 (upper panel) and MCF10A (bottom panel) cells incubated for 24 h
with increasing concentration of either XeB (red) or dmXeB (blue). The black bar represents cells basal OCR without treatment. Mean Æ SEM of
3 independent experiments with 10 replicates each. *p<0.05, **p<0.01, ***p<0.001 compared to respective control. c) MDA-MB-231 (upper
panel) and MCF10A (lower panel) were treated with increasing concentrations of either Xe B (upper panel, red) or dmXeB (lower panel, blue) for
24 h and cell death was determined by propidium iodide incorporation by flow cytometry. Mean Æ SEM of 3 independent experiments, each in
triplicate. *p<0.05, ***p<0.001 compared to respective control. d) Representative plates of three different experiments with MDA-MB-231 cells
treated with 7.5 mM of either XeB (upper panel) or dmXeB (bottom panel).
Fellowship, Mabe Memorial Fellowship, and UCSB Travel
week, we evaluated colony formation and found that at
grant. We thank Dr. Hongjun Zhou for the assistance with the
7.5 mM the synthetic dmXeB, similarly to natural XeB,
completely abolished this phenomenon (Figure 2d).
NMR spectrometry, Dr. Dmitri Uchenik for the assistance
with mass spectroscopy, and Dr. Guang Wu for the assistance
In summary, we developed a scalable synthesis of dmXeB,
with X-ray crystallography.
taking advantage of a convergent strategy allowing the
preparation of xestospongins with variable stereochemistry
and oxidation at C9/9’, as attested to by the synthesis of ArC,
which has not been accessed by previously described strat-
Conflict of interest
egies. The hallmarks of the strategy include a strategic
application of ICR to achieve the desired makeup of the
The authors declare no conflict of interest.
C9/9’ stereocenters, assembly of the macrocyclic core by
macrolactamization, and late-stage 1-oxaquinolizidine con-
struction by amide reduction. With dmXeB in hand, we
established that it is an effective inhibitor of the constitutive
ER-to-mitochondria calcium transfer, and subsequently con-
firmed its ability to induce selective cancer cell death in
Keywords: cancer · IP3R receptor · metabolism · total synthesis ·
xestospongins
a variety of cell lines, including metastatic cancer, while
leaving normal cells nearly unaffected. According to a recent
study, these effects are associated with a drop in the
mitochondrial activity of the calcium-sensitive a-ketogluta-
rate dehydrogenase, a key enzyme in oxidative phosphoryla-
tion and reductive carboxylation required for cancer cell
survival.[20]
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Acknowledgements
This work was supported by ANID/FONDECYT grants
1160332 and 1200255, ANID/FONDAP grant 15150012
(C.C.), UC CRCC grant CRR-19-577249, and the NIGMS
R01-077379 (A.Z.). MP is a recipient of the Tantiwiwat
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Manuscript received: February 13, 2021
Accepted manuscript online: March 10, 2021
Version of record online: April 8, 2021
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