Enantioselective syntheses and application of 4-: Epi -galiellalactone and the corresponding activity-based probe: From strained bicycles to strained tricycles

Article abstract of DOI:10.1039/c8ob01915k

The [6,5,5] tricyclic fungal metabolite galiellalactone is a Michael acceptor that has been demonstrated to be a covalent inhibitor for Signal Transducer and Activator of Transcription 3 (STAT3). Recognizing the ring strain associated with the skeleton of this natural product, we utilized 1R-5S-bicyclo[3.1.0]hexan-2-one as the starting material and developed two novel approaches to accomplish the enantioselective total synthesis of the C4 epimer of galiellalactone in 5 and 7 steps, respectively, which capitalized on an efficient radical cyclization/fragmentation cascade reaction. Furthermore, an activity-based probe of 4-epi-galiellalactone with a terminal alkyne tag was successfully prepared to enable the experiments of activity-based protein profiling (ABPP). Through western blot and proteomic analysis, we not only confirmed the known target STAT3, but also identified a new target protein ataxin-7, which formed a covalent bond with the probe in intact cells via the Cys-129 residue.

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Enantioselective Syntheses and Application of 4-epi-
Galiellalactone and the Corresponding Activity-Based Probe: From
Strained Bicycles to Strained Tricycles
Received 00th January 20xx,
Accepted 00th January 20xx
Yandong Lu,a Shan Zhao,b Shijie Zhou,b Si-Cong Chen,b and Tuoping Luo*ab
DOI: 10.1039/x0xx00000x
www.rsc.org/
The [6,5,5] tricyclic fungal metabolite galiellalactone is a Michael acceptor that has been demonstrated to be a covalent
inhibitor for Signal Transducer and Activator of Transcription 3 (STAT3). Recognizing the ring strain associated with the
skeleton of this natural product, we utilized 1R-5S-bicyclo[3.1.0]hexan-2-one as the starting material and developed two
novel approaches to accomplish the enantioselective total synthesis of the C4 epimer of galiellalactone in 5 and 7 steps
respectively, which capitalized on an efficient radical cyclization/fragmentation cascade reaction. Furthermore, an activity-
based probe of 4-epi-galiellalactone with a terminal alkyne tag was successfully prepared to enable the experiments of
activity-based protein profiling (ABPP). Through western blot and proteomic analysis, we not only confirmed the known
target STAT3, but also identified a new target protein ataxin-7, which formed a covalent bond with the probe in intact cells
via the Cys-129 residue.
recently reported the syntheses of (+)-wortmannin and
maoecrystal P, a highly oxidized ent-kauranoid.5 Herein, we
Introduction
disclose our work in the chemotype of galiellalactone (1,
Small molecule natural products with electrophiles reacting
with specific nucleophilic protein residues are extremely
valuable tools for biomedical researches, and have led to
important discoveries in life sciences.1 Based on the structural
features, the majority of protein-reactive natural products
could be divided into two groups. One contains strained ring
systems including epoxides, β-lactams and β-lactones, and the
release of ring-strain drives the ring-opening reaction with the
protein residue, while the other group are Michael acceptors.2
Both groups, which are well horned by coevolution of natural
products and their macromolecular targets, have provided
numerous inspirations not only for the development of
covalent inhibitors but also for activity-based protein profiling
(ABPP) technology.3 It is noteworthy that nature also takes
advantage of the ring strain to enhance the reactivity of
Michael acceptors. One of the most famous examples is
wortmannin, a potent and selective PI3K inhibitor, which
possesses a strained energy of ~12.1 kcal/mol.4 Interested in
the organic synthesis and chemical biology of natural product
Michael acceptors with strain-enhanced reactivity, we have
Figure 1), a fungal metabolite first isolated from Galiella rufa
that embeds an α,β-unsaturated ester in the [6,5,5] tricyclic
skeleton.6
Galiellalactone has been reported to possess a broad
spectrum of biological activities, including antitumor7, anti-
inflammatory8 and anti-HIV9 properties. In particular, the
natural product significantly inhibits the growth of androgen-
insensitive prostate cancer cells including the stem cell-like
ALDH-positive cells both in vitro and in vivo.7b Besides
combating castration-resistant prostate cancer, galiellalactone
has showed promise in both allergic asthma8a and
atherosclerosis8b mouse models. In terms of the mechanism-
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of-action, it has been suggested that galiellalactone targets 2A, see Supporting Information for details), which might play a
the Signal transducer and activator of transcription 3 (STAT3), vital role in the reactivity of this unique natural product.
a critical transcription factor that is persistently activated in a Furthermore, the tendency of a Michael acceptor to undergo
variety of human cancers.10 Using a biotinylated probe
developed by Sterner group, Hellsten and co-workers The LUMO energy of
demonstrated that galiellalactone reacted with several and methyl vinyl ketone, respectively; the LUMO energy gap
cysteine residues of the protein to block STAT3-DNA around 0.28 eV between the bicycle and the strained tricycle
interaction independent of STAT3 phosphorylation status.11
may endow the latter with higher activity to undergo
Probe molecule also facilitated the investigation by Muñoz nucleophilic addition with residues of targeting proteins
group, which revealed galiellalactone could directly target the (Figure 2B, see Supporting Information for details).
3
nucleophilic addition could be estimated by its LUMO energy.
and is close to that of methyl acrylate
7
9
7
9
3
p65 subunit of NF-ĸB and impair the nuclear translocation of
Intrigued by the inherent chemical reactivity of the strained
tricyclic Michael acceptor observed in galiellalactone, we
of aimed at preparing an appropriate activity-based probe and
this transcription factor.9
Structure-activity
relationship
(SAR)
studies
galiellalactone have been conducted by several groups using the powerful mass spectrometry-based proteomic
regarding to its antitumor or STAT3-inhibitory activity.12-17 In profiling to shed light on the corresponding proteinaceous
general, the Michael acceptor system together with the reaction partners under physiologically relevant conditions in
heteroatom substitution at C10 are essential for the activity.12 addition to STAT3 and NF-ĸB. Furthermore, the positive results
It has also been reported that the C4-epimer
compared to (−)-galiellalactone (
).13, 14 However, extending developing a new and concise synthesis to access diversified
2
is equally active from previous SAR studies15-17 argue for the urgency of
1
side chains at C4 significantly reduced its ability to inhibit the analogues and expand research territory of such an important
proliferation of tumor cells, whereas the biological activity was natural product.
restored when a phenyl group was added at C8 (probe
example).15 It is noteworthy that oral administration of a so far. Sterner and co-workers achieved the first total synthesis
prodrug conjugate prepared by the addition of a cysteine of (+)-galiellalactone starting from (+)-pulegone, in which the
3
as an
Three total syntheses of galiellalactone have been reported
5
derivative to galiellalactone demonstrated much better lactone ring and the tertiary hydroxyl group at C10 were
pharmacokinetic properties than that of the parent natural furnished simultaneously by the opening of an epoxide
product on a mouse model.16 Most recently, Suh and co- embedded in a bicyclic intermediate.18 Later on, Sterner group
workers found that the truncation of the cyclohexene moiety deployed a palladium-catalyzed tandem reaction followed by
of galiellalactone to [3.3] bicyclic lactone (
improved the cytotoxic effects against triple-negative breast intermediate, thus completing the syntheses of the target
cancers (TNBCs).17
molecule and its analogs, including the aforementioned probe
Based on a comparison of the relative differences in the 3 14
In comparison, the latest synthesis by Suh and co-workers
energy of hydrogenation of and , we estimated the strain relied on an intramolecular palladium-catalyzed allylic
6 as an example) Diels-Alder cyclization to efficiently access the same bicyclic
.
7
9
energy of galiellalactone skeleton is around 4 kcal/mol (Figure alkylation and olefin metathesis to construct the tricyclic ring
system.19 Besides the de novo chemical synthesis, the
biosynthetic pathway20 of
1 inspired Sterner group to combine
the synthesis of desoxygaliellalactone with the enzymatic
hydroxylation at C10 for the production of (−)-galiellalactone,21
whereas Lebel group completed
a formal synthesis by
developing a one-pot copper-catalyzed methylenation-Diels-
Alder reaction.22
Results and Discussion
Retrosynthetic analysis
Our recent syntheses of (−)-hibiscone C and lysergine based on
the radical cyclization/fragmentation strategy inspired a novel
disconnection of 4-epi-galiellalatone (2) (Figure 3),23 which has
been reported to exhibit
aforementioned natural product 1 13,
intermediate 11 could not only provide
a similar activity with the
14
.
The late-stage
2
via reduction, but
also be converted to an activity-based probe by introducing a
terminal alkyne tag for the click chemistry conjugation. While
11 could be obtained from bicycle 12 via bromolactonization,
we hypothesized that 12 would be prepared in one step from
bromide 14 via the radical cyclization/fragmentation cascade
2 | J. Name., 2012, 00, 1-3
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Scheme 1. First Generaꢀon Synthesis of (−)-4-epi-Galiellalactonea
involving vinyl radical 13 as the intermediate. Due to
stereoelectronic effects, the ring-opening of the cyclopropane
would take place predominately by the scission of the axial C3-
C11 bond.24 The keto-tautomer of 14, ketoester 15, would be
afforded by a regioselective ring expansion of 17. At last,
ketone 17 could be obtained by the allylic alkylation of chiral
bicycle 18 in a stereoselective manner given the steric
hindrance of the fused cyclopropane ring. In comparison with
previous syntheses that introduced the tertiary hydroxyl group
at C10 through the opening of epoxide,14, 18 allylic oxidation19
or enzymatic hydroxylation,21 our strategy traced this motif
could be used directly in the next step and avoid the
protection maneuver. To our delight, the desired
cyclization/fragmentation reaction took place smoothly in the
presence of nBu3SnH and AIBN at 80°C, affording 12 as the final
product in 42% yield. To formulate the tricyclic ring, we
employed the widely-used bromolactonization approach on
pentenoic acid-type substrates.27 A series of conditions were
screened using the in-situ generated carboxylate from 12 as
the substrate (Table S3), but the desired product 11 was
isolated in only 30% yield, for which we hypothesized the
strained nature of [6,5,5]-tricycle played a role. Nonetheless,
back to the carbonyl group of substrate 18
.
Synthesis of 4-epi-Galiellalactone
We commenced our studies by preparing chiral (+)-18
according to known procedures on a multi-gram scale (Scheme
1).25 The alkylation of 18 with 1,3-dibromo-2-propene afforded
ketone 17 as a mixture of Z- and E-isomers. After substantial
optimization, treatment of ketone 17 with ethyl diazoacetate
16 and boron trifluoride etherate at room temperature
accomplished the one-carbon ring expansion, leading to
compound 14 as the only isolable product. The well-known
trend of the preferential migration aptitude in the insertion
debromination of 11 under radical condition provided 2, the
structure of which was determined unambiguously by X-ray
crystallography. Without using any protecting groups, our first
generation approach delivered (−)-4-epi-galiellalactone (2) in 5
steps starting from 18 with an overall yield of 2.1%, which
prompted us to develop the second generation of synthesis
with higher efficiency.
reaction of
a ketone by ethyl diazoacetate is the less
substituted or less bulky side.26 In our scenario, the addition of
ethyl diazoacetate 16 to the ketone would take place from the
side opposite to the cyclopropane, and therefore the
cyclopropyl carbon could be considered as the less bulky side.
Transition state 19 with minimized gauche steric repulsions
was therefore preferred, in which the cyclopropyl carbon was
To circumvent the low-yielding bromolactonization step in
the initial route, we envisioned a ketyl radical cyclization
approach28 to directly introduce the hydroxyl group at C9
(Scheme 2). Following similar transformations with the first
route, allylation of bicycle 18 with allyl iodide and subsequent
ring expansion using ethyl diazoacetate 16 afforded 21 in 43%
yield (2 steps), again as an enol tautomer. In the meantime, a
cursory screen of Lewis acid and solvent was conducted to
increase the efficiency of the latter step (Table S4). Protection
of the hydroxyl group with ethoxymethyl group followed by
+
anti-planar to the leaving group -N2 , hence giving rise to 14 as
the major product. Fortuitously, the enol-tautomer (14) was
significantly favored over the keto-tautomer (15) at room
temperature as indicated by NMR spectrums (see SI), which
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Scheme 2. Second Generation Synthesis of (−)-4-epi-Galiellalactonea
Scheme 3. Syntheses of Small-molecule Probe 29 and Pseudo-probe 30 a
The reaction of bromide 11 with excess acrylic acid in the
n
presence of Bu3SnH and AIBN at 80°C led to a crude product
of acid 28, which was direct subjected to amidation reaction
with propargylamine to afford 29 as our probe molecule.
Though the yield was low (26% over 2 steps), the expedient
approach still provided us enough quantities of 29
.
Furthermore, we treated 29 with DBU to effect the olefin
isomerization to prepare a pseudo-probe (30), which could be
used as a negative control of the Michael acceptor probe 29 in
ABPP studies.
Activities of Synthesized Galiellalactone Analogues against Cell
Proliferation
The anti-proliferation effects of galiellalactone and its
analogues have been extensively studied in prostate cancer
cells and recently in breast cancer cells,7,
12-17
whereas we
hydroboration/oxidation provided aldehyde 23 as the ketyl
radical precursor. The desired cyclization/fragmentation
reaction took place in the presence of tris(trimethylsilyl)silane
and AIBN at 85°C, affording 25 in 86% yield as a single
diastereomer. The preference of forming 25 could be
rationalized by invoking the transition state 24 that minimized
the dipole-dipole interaction. We were delighted to find
irradiation of 25 in methanol effected the deprotection of both
found
1 also demonstrated significant cytotoxicity in two
human T-cell acute lymphocytic leukemia (T-ALL) cell lines
(MOLT-4 and Jurkat), which were then used for our
subsequent investigation (Table 1; see Figure S1 for dose-
response curves). The potency of 4-epi-galiellalactone (2) was
hydroxyl groups followed by lactonization to furnish (−)-
yield in one step. Consequently, the second generation route
provided in 7 steps with an overall yield of 11%.
2 in 63%
2
Attempts to epimerize the C4 methyl groups under various
conditions were not successful. But we accidentally obtained
26 with shifted double bond by treating
2
with DBU,29 which
could be used as a negative control in our follow-up studies
due to the lack of active Michael acceptor. In a one-pot
procedure, 2 was further converted to tetracyclic compound 4,
a molecule previously prepared from
comparable STAT3 inhibitory activity.12
1 and reported to have
Synthesis of the Activity-based Probe (Scheme 3)
4 | J. Name., 2012, 00, 1-3
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similar to that of galiellalactone (1) (IC50s between 2~4 fold),
while probe 29 showed four-fold decreased cytotoxicity in
MOLT-4 cells (IC50 = 8.6 μM). In contrast, 26 and 30 with
shifted double bond hardly had any anti-proliferation activity
(IC50 > 100 μM), suggesting the critical role of the Michael
acceptor system. Interestingly, bicyclic compound 12 did not
show any activity (IC50 > 100 μM) in spite of the intact α, β-
unsaturated ester in the molecule. We hypothesized that
absence of the strain energy conferred by the [6,5,5] tricyclic
skeleton decreased the reactivity of Michael acceptor in 12
,
In-situ proteome labeling was carried out using 25 μM 29 in
which significantly diminished its bioactivity. Moreover,
the absence and presence of 2, and a photocleavable biotin-
bromide 11 and the Diels-Alder adduct
4 also exhibited
azide (Figure S2) was used to form a conjugate with the
targeting proteins via click reaction, resulting in the removal of
the connected biotin by UV light at 365 nm following the pull-
down by streptavidin beads.30 After trypsin digestion, the
resulting samples were subjected to LC-MS/MS analysis (see
Supporting Information for details). Again, STAT3 was clearly
identified as a target of 29 (see Table S5 for the list of hit
proteins), even though the covalent modification site was not
identified. In contrast, the proteome labeling of pseudo-probe
30 with shifted double bond turned out to be minimal (see
Figure S3 for silver staining), indicating that the necessity of
the Michael acceptor motif in 4-epi-galiellalactone probe 29
regarding the reaction with proteins in intact cancer cells.
Importantly, the chemical proteomics approach also allowed
for the discovery of target proteins that may not be relevant
for the anti-cancer or anti-inflammatory activities intensively
studied. From the MS/MS data, we found that Ataxin-7, a
protein mainly expressed throughout the central nervous
system,31 was selectively labeled by probe 29. Mutated Ataxin-
7, with the polyglutamine-tract expansion, is associated with a
rare neurodegenerative disease named spinocerebellar ataxia
type 7 (SCA7) that has no current therapies in the clinic.32 The
proteolysis of this mutated protein produces toxic fragments
that underlies the pathogenesis of the disease.33 To our
knowledge, neither small molecules interacting with Ataxin-7
moderate anti-proliferation activities.
Moreover, treatment of MOLT-4 cells with 4-epi-
galiellalactone did not affect the expression of p-
(2)
STAT3(Tyr705) and total STAT3 except those at 25 μM
compound concentration (Figure 4), which was consistent with
observations of galiellalactone (1
) in DU145 cells.11 The
appearance of the apoptotic marker cleaved poly(ADP
ribos)polymerase (PARP) was significant accompanying the
increasing concentrations of
2, while cell cycle regulatory
protein c-Myc and CDK6 were downregulated; in comparison,
the expression levels of cyclin D1 and Mcl-1 were not altered.
MS-Based ABPP Profiling of 29-Interaction Proteins
Before carrying out the activity-based protein profiling (ABPP)
of probe 29, we first validated the covalent binding of this
probe to STAT3 (Figure 5A). MOLT-4 or Jurkat cells were
treated with probe 29 for 2 h and then lysed. The cell lysates
were subjected to the CuSO4-catalyzed click reaction with
biotin-azide and then the binding proteins were successively
pulled down by streptavidin beads, separated by SDS-PAGE
and immunoblotted with STAT3 antibody. Pretreatment of
cells with galiellalactone (1) or 4-epi-galiellalactone (2) prior to
the addition of 29 significantly reduced the binding of STAT3 to
the probe, thus demonstrating a competitive binding mode.
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nor the targetable cysteines within this protein have been and 21672011). We thank Dr. Nengdong Wang and Prof.
reported before. The LC-MS/MS data suggested probe 29 Wenxiong Zhang (Peking University) for their help in analyzing
could form a covalent bond with Cys-129 of Ataxin-7 (Figure 5B; the X-ray crystallography data, Mr. Yi Wang and Prof. Zhi-Xiang
see Figure S4 for MS2 data). Subsequently, the binding Yu (Peking University) for their help in the density functional
interaction between ataxin-7 and the probe was further theory calculations, Prof. Peng Zou (Peking University) for
demonstrated by western-blot following pull-down providing the primary mouse brain cells, Prof. Chu Wang
experiments using both SK-N-SH neuroblastoma cells (Figure (Peking University) for helpful discussions, and support from
5C) and fresh isolated mouse neuro cells (Figure S5). Herein, the High-performance Computing Platform of Peking
for the first time, we discovered that small-molecule probe 29 University.
could covalently bind ataxin-7, and the labeling event was
competed away by both galiellalactone
(1) and 4-epi-
Notes and references
galiellalactone (2). Whether the biological functions of ataxin-7
is affected invites further investigations.
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In summary, we have accomplished the enantioselective total
synthesis of 4-epi-galiellalactone via two approaches in 5 (2.1%
overall yield) and 7 steps (11% overall yield), respectively. Key
3
,
5
for both syntheses was the application of
a radical
cyclization/fragmentation strategy, and the three membered
ring of starting material (+)-18 not only provided excellent
stereochemical control but also enabled the strain-release
fragmentation. Moreover, regioselectivity for the ring
enlargement of the strained bicyclic ketones 17 and 20 was
also notable. In leukemia cells lines MOLT-4 and Jurkat, 4-epi-
6
7
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, while the
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reactivity of Michael acceptor.
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Subsequently, the development of the activity-based probe
29 enabled the ABPP experiments, with the pseudo-probe 30
serving as a negative control. Through western blot and
proteomic analysis, we confirmed the known target STAT3 and
shed light on other potential interacting proteins. One
prominent example is the identification of the covalent
attachment of 29 to Cys129 of ataxin-7, which has not been
targeted by small molecules before. The proteolysis of
mutated Ataxin-7 produces toxic fragments that underlies the
pathogenesis of the neurodegenerative disease SCA7.33
Therefore, additional studies are urgently needed to elucidate
whether the reaction of galiellalactone and its analogues
intervene the proteolysis of mutated Ataxin-7.
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Acknowledgements
This work was supported by generous start-up funds from the
National “Young 1000 Talent Plan” Program, College of
Chemistry and Molecular Engineering, Peking University,
Peking-Tsinghua Center for Life Sciences, and the National
Science Foundation of China (Grant No. 21472003, 31521004
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