Pre-mRNA splicing-modulatory pharmacophores: The total synthesis of herboxidiene, a pladienolide-herboxidiene hybrid analog and related derivatives

Article abstract of DOI:10.1021/cb400695j

Herboxidiene is a natural product that has previously been shown to exhibit antitumor activity by targeting the spliceosome. This activity makes herboxidiene a valuable starting point for the development of anticancer drugs. Here, we report an improved enantioselective synthesis of herboxidiene and the first report of its biologically active totally synthetic analog: 6-norherboxidiene. The synthesis of the tetrahydropyran moiety utilizes the novel application of inverse electron-demand Diels-Alder chemistry and the Ferrier-type rearrangement as key steps. We report, for the first time, cytotoxicity IC₅₀s for synthetic herboxidiene and analogs in human tumor cell lines. We have also demonstrated that synthetic herboxidiene and its analogs can potently modulate the alternate splicing of MDM-2 pre-mRNA.

Full text of DOI:10.1021/cb400695j

Articles
pubs.acs.org/acschemicalbiology
Pre-mRNA Splicing-Modulatory Pharmacophores: The Total
Synthesis of Herboxidiene, a Pladienolide−Herboxidiene Hybrid
Analog and Related Derivatives
Chandraiah Lagisetti,† Maria V. Yermolina,† Lalit Kumar Sharma,† Gustavo Palacios, Brett J. Prigaro,‡
and Thomas R. Webb*,§
Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas PI, MS 1000,
Memphis, Tennessee 38105, United States
S
* Supporting Information
ABSTRACT: Herboxidiene is a natural product that has previously been shown to
exhibit antitumor activity by targeting the spliceosome. This activity makes
herboxidiene a valuable starting point for the development of anticancer drugs. Here,
we report an improved enantioselective synthesis of herboxidiene and the first report
of its biologically active totally synthetic analog: 6-norherboxidiene. The synthesis of
the tetrahydropyran moiety utilizes the novel application of inverse electron-demand
Diels−Alder chemistry and the Ferrier-type rearrangement as key steps. We report,
for the first time, cytotoxicity IC50s for synthetic herboxidiene and analogs in human tumor cell lines. We have also demonstrated
that synthetic herboxidiene and its analogs can potently modulate the alternate splicing of MDM-2 pre-mRNA.
he prominence of pre-mRNA splicing in gene expression
in higher eukaryotes makes it an attractive target for
derivatives of 2 (E7107) has shown remarkable preclinical
tumor regression efficacy and advanced to phase I clinical
trials,12 which stimulated considerable interest in the potential
of splicing modulators as potential therapeutic agents for the
treatment of cancer.4,27,28 Very recently, interesting active-
analogs of another natural product (FD-895), which is
structurally related to 2, have also been reported.27
T
therapeutic intervention, and recently this process has emerged
as a potentially important therapeutic target in cancer.1−4 The
pre-mRNA splicing process involves the removal of introns
(noncoding sequences) from pre-mRNA, which is followed by
ligation of exons (coding sequences).1 Alternative splicing is the
mechanism by which different forms of mature mRNAs are
generated from the same gene. Commonly, alternative splicing
patterns determine the inclusion or exclusion of portions of the
coding sequence in the mRNA, giving rise to protein isoforms
that differ in their peptide sequence and hence in their chemical
and biological activity.1,5 Alternative splicing plays important
roles in the development of multicellular organisms and in
numerous pathologies, including cancer.3,6 This splicing
process is catalyzed by a macromolecular complex called the
spliceosome, which is composed of five small nuclear
ribonucleoproteins (snRNPs) (U1, U2, U4, U5, and U6) and
over 150 associated proteins.
Several bacterial natural products such as herboxidiene (1
also known as GEX1A),7−9 pladienolide B (2),10−13
FR90146414−16 and the thailanstatins17 have been shown to
effect splicing by targeting the splicing factor 3b (SF3b)
subunit, an essential component of the spliceosome. Several of
these natural products also induce cell cycle arrest at the G1
and G2/M phase and show potent antitumor activity in human
tumor cell lines.11,14,18,19 Importantly, several of these natural
products have also been reported to show potent in vivo activity
in tumor xenograft models.11,23 Ongoing synthetic work20−27
has also provided novel synthetic spliceosome modulators such
as 1-deoxy FR901464,23 and the meayamycins23,24 (all analogs
of FR901464), as well as new structure−activity relationships
for FR901464 analogs.23 Indeed, one of the semisynthetic
RESULTS AND DISCUSSION
■
We have also been engaged in a successful effort to design new
effective and highly active synthetic analogs of FR901464 and 2
by the application of a consensus pharmacophore hypoth-
esis.29−31 We believed that we could extend our approach by
the generation of a new structurally simplified scaffold based on
herboxidiene. Thus, using the guidance of our pharmacophore
hypothesis, we designed a hybrid molecule (3) from
herboxidiene (1) and pladienolide B (2), (Figure 1, Scheme
1). This hybrid 3 has the tetrahydropyran core of 1 (red) and
side chain of 2 (blue). Based on our pharmacophore model, we
anticipated that compound 3 could incorporate the correct
molecular geometry as well as all of the key SF3B1 interaction
features.29
For the synthesis of the hybrid analog 3 we coupled aldehyde
16b with the Julia−Kocienski reagent 4 that has been used in
the total synthesis of pladienolide, which was prepared as
reported (Supporting Information, Scheme S1).32 We were
initially surprised when we tested the cytotoxicity of our hybrid
molecule 3 in various human tumor cell lines and it was found
Received: September 9, 2013
Accepted: December 20, 2013
Published: December 30, 2013
© 2013 American Chemical Society
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Herboxidiene (1) is a bacterial secondary metabolite,
originally isolated from the Streptomyces chromofuscus strain
A7847 by Isaac et al. and initially evaluated as a herbicide.7
Subsequently, Edmunds et al. determined the absolute
configuration of 1,8 which was further confirmed by the first
total synthesis by Blakemore and Kocienski.34 Importantly, a
study by Horiguchi et al. also reported the in vivo antitumor
activity of 1 in a murine tumor model.23 Of particular interest
to our lab was the recent photoaffinity-labeling study, which
found that the SF3b1 (SAP155) protein is also the major target
of 1.9 Because 1 has a simpler structure, when compared to the
other known splicing modulators, it is an especially attractive
starting point for the development of structure−activity
relationship (SAR) studies. Therefore, 1 represents a unique
opportunity for the discovery of drug-like synthetic agents for
SF3b modulation.
Figure 1. Structures of splicing modulators: herboxidiene (1),
pladienolide (2), and our synthetic hybrid molecule (3).
In the realm of herboxidiene total synthesis, impressive
progress has been made recently.35−40 However, to the best of
our knowledge, no study on the evaluation of the biological
activity of synthetic 1 and its analogs in human cancer cell lines
has been reported to date. A recent publication by Koide and
co-workers provides the only report on the biological activity of
synthetic 1 and this is in a nontumor (HEK293) cell line.41
Therefore, as a first step in exploring totally synthetic analogs of
1, as potential antitumor therapeutics, we embarked on the
total synthesis of the natural product herboxidiene and its close
analogs.
Scheme 1. Synthesis of the Pladienolide−Herboxidiene
a
Hybrid Molecule 3
Our assembly of 1 and its analogs took advantage of a
convergent strategy in which tetrahydropyran cores 16a/16b
would be coupled with a herboxidiene side-chain precursor
through the versatile Julia−Kocienski olefination reaction. We
envisioned the synthesis of aldehydes 16a/16b from 14a/14b,
which in turn could be derived via Ferrier-type rearrangement
of compounds 9a/9b. We planned to construct dihydropyr-
anones 9a/9b using an inverse electron-demand Diels−Alder
reaction between (S)-2-(benzyloxy)propanal (7) and diene 8a
or 8b. The sulfone 19 can be derived from alcohol 18, which is
conveniently prepared by an efficient method reported by Urpi
and co-workers.35
Our tetrahydropyran synthesis commenced with the
preparation of diene 8a/8b (as a 10:1 mixture of Z and E
isomers) using a method reported by Li et al.43 The MgBr2
mediated inverse electron-demand Diels−Alder reaction of
diene 8a or 8b and (S)-2-(benzyloxy)propanal (7) then
provided dihydropyranones 9a and 9b as single diaster-
eomers.42 The reduction of the ketone moiety was achieved
under Luche conditions to selectively provide the alcohols 10a
and 10b. The latter, in turn, were acetylated with Ac2O and
Et3N to provide the corresponding acetates 11a and 11b. In an
effort to obtain the tetrahydropyran core, we initially
investigated application of the Ireland−Claisen rearrangement
of 11a, which has been used in the construction of analogous
tetrahydropyrans.44 We anticipated that Ireland−Claisen
conditions, followed by esterification with CH2N2 would lead
to 12a. Unfortunately, in our hands this reaction sequence
failed to provide the desired products in acceptable yields. We
therefore pursued the alternative route to these intermediates
that installed the desired stereochemistry and ester moiety in
one step via a Ferrier-type rearrangement using a silylketene
acetal.45−47 The stereochemical outcome of this reaction has
previously been shown to be highly dependent on the
conditions employed.49 We developed conditions for this
transformation that provided a 2:1 mixture of cis and trans fused
a
Reagents and conditions: (a) 16b, KHMDS, THF, −78 °C, 1.5 h,
46%; (b) TBAF, THF, 76%; (c) KOSiMe3, THF, rt, 79%.
inactive (IC50 > 20 μM). In these assays, we used natural 1
(prepared by bacterial fermentation, purchased from Cfm
Oskar Tropitzsch e.K) as a standard. For comparison, we also
referred to the reported activity of 2 (IC50s in the nanomolar
range).10,33 Based on these data we hypothesized that the
hydrogen-bond donor (OH) at C18 represents an additional
new pharmacophore feature in herboxidiene and that this key
functionality is required in order to effectively interact with
SF3B1 (see Figure 2). To investigate our hypothesis, we next
turned our attention toward the synthesis of herboxidiene and
its analogs that maintain this hydrogen-bond donor feature.
Figure 2. Hypothetical pharmacophore features for herboxidiene.4
The features are F1, Donor (Don) and acceptor (Acc); F2, Acc; F3,
Hydrophobic (Hyd); F4, Acc (epoxide); F5, Acc; and F6, Don and
Acc. The pharmacophore is calculated using the Molecular Operating
System 2011.10 from the Chemical Computing Group, Inc.
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a
substituted dihydropyrans 12a (in 72% yield) and 12b (in 70%
yield). This ratio is identical to that reported in a related C-
glycolsylation reaction of this type.47 We found that cis and
trans isomers of 12a and 12b were readily separated by flash
chromatography to give the stereochemically pure products.
The relative stereochemistry of both isomers was established by
NOESY experiments and by single crystal X-ray structures of 4-
nitrophenyl ester derivatives (see Figure 3 and Supporting
Scheme 2. Synthesis of Tetrahydropyran 16a/16b
Figure 3. X-ray structure of nitrobenzoyl derivative of 13a.
Information (SI)). At this point, the simultaneous reduction of
the alkene moiety and benzyl ether cleavage of 12a and 12b
was accomplished by catalytic hydrogenation. Next, the
oxidation of the resulting alcohols 13a and 13b yielded the
corresponding ketones 14a and 14b. Grignard reaction of 14a
and 14b with vinylmagnesium bromide then furnished the
tertiary alcohols 15a and 15b as a mixture of diastereomers.
Finally, PCC oxidation of 15a or 15b afforded the desired
aldehydes 16a and 16b as inseparable mixtures of E:Z isomers
(7:2). (We found, however, that the final products could be
separated to give pure E derivatives, see Scheme 3 and
discussion below). This concise route toward the tetrahy-
dropyran core of 1 represents a significant advancement in the
syntheses of the key intermediate 14a.35−39
We found that the method shown in Scheme 2 could provide
compound 14b in 6 steps in an overall yield of 25% (see SI).
Our improved route shown in Scheme 2 was also more efficient
for the synthesis of aldehyde 16b. In addition to the route
shown in Scheme 2, we also successfully implemented a known
approach to 14b via oxa-Michael cyclization followed by
functionalization from the resulting cis fused pyran using a
published procedure (see SI, Scheme S2).48 We were also able
to confirm the relative and absolute stereochemistry of 14a and
14b using single crystal X-ray crystallography of derivatives (see
SI: Schemes S3 and S4, Figure S1, and Figure S2).
We then turned our attention to the acyclic side chain of 1.
Recently, Urpi and co-workers reported an efficient method for
the synthesis of alcohol 10 starting from the commercially
available ester 17 in nine linear steps.23 Using this procedure,
with some minor modifications, we were able to synthesize
compound 18 on a multigram scale (see SI). The resulting
substrate 18 was subjected to a Mitsunobu reaction with 1-
phenyl-1H-tetrazole-5-thiol, followed by ammonium molyb-
date-catalyzed oxidation, to provide sulfone 19. With sulfone 19
and aldehyde 16a and 16b in hand, we next focused on the
convergent coupling to provide 20a and 20b. These products
were obtained in moderate yields in the presence of KHMDS
via the Julia−Kocienski olefination. Removal of the tert-
butyldimethylsilyl group in 20a and 20b with HCl, followed
by C18 hydroxyl directed selective epoxidation of alcohols 21a
and 21b with catalytic VO(acac)2 provided the pure
diastereomers 22a and 22b in good yields, as expected based
on the literature precedence.35−38 The purification step at this
point also readily removed the minor C9,C10-Z isomer as
a
Reagents and conditions: (a) MgBr2, THF, rt, 12 h; (b) CeCl3·7H2O,
NaBH4, MeOH, 30 min; (c) Ac2O, Et3N, CH2Cl2, 0 °C, 1 h; (d) Ti(i-
OPr)2Cl2, toluene, −78 °C to rt, 16 h, (cis:trans = 2:1); (e) H2, Pd/C,
EtOAc, 1 h; (f) Dess−Martin periodinane (DMP), CH2Cl2, 30 min;
(g) vinylmagnesium bromide, CH2Cl2, −78 °C, 24 (h) Pyridinium
chlorochromate (PCC), CH2Cl2, 40 °C, 18 h, E:Z = 7:2.
shown in Scheme 3 (see SI), which had been carried over from
16a/16b. Hydrolysis of the methyl esters 22a and 22b under
basic conditions afforded pure herboxidiene (1) and C6-
norherboxidiene (23). This series of reactions also provided
important analogs for SAR studies, such as 21a, 22a, and 22b.
Additionally we prepared the C18 ketone derivative 24 by
oxidation of 22b with DMP, in order to confirm the importance
of the C18 hydrogen-bond donor feature in the herboxidiene
pharmacophore.
Next, we evaluated the cytotoxicity of both natural and
synthetic 1 and its analogs on six human cancer cell lines
(Table 1). The IC50 values of synthetic 1 were found to be in
the low nanomolar range (4.5−22.4 nM), which was
comparable to the results with the natural 1 (4.3−46.3 nM).
Surprisingly, the methyl ester precursor of 1 (compound 22a)
also displayed highly potent cytotoxicity (IC50 = 6.2−15.8 nM).
However, a great loss in activity (>200-fold) was observed for
triene 21a as compared to its epoxide derivative 22a. This result
is consistent with the early reports of the herbicidal activity
dependence on an intact epoxide group in several semisynthetic
analogs of herboxidiene.7 C6-Norherboxidiene (23) showed a
loss in activity (∼10-fold) indicating that this methyl group is
contributing to the potency of 1. Most importantly, when the
ketone analog 24 was evaluated, it was found to be essentially
inactive, with IC50 values in micromolar range (>5−25 μM).
This low activity of 24 clearly supports our hypothesis that the
hydrogen-bond donor (OH) interaction feature at C18 is
required for the potency of this class of compounds in addition
to the other features previously described (Figure 2).4,29 This
result is also consistent with the lack of activity seen in the
hybrid molecule 3.
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a
Scheme 3. Synthesis of Herboxidiene (1) and Its Analogs
a
Reagents and conditions: (a) Diisopropyl azodicarboxylate (DIAD), 5-mercapto-1-phenyltetrazole, PPh3, THF, rt, 3 h, 89%; (b) (NH4)6Mo7O24,
30% H2O2, EtOH, rt, 24 h, 88%; (c) KHMDS, 16a or 16b, THF, −78 °C, 1.5 h, (E:Z = 7:2); (d) 0.16 M HCl, MeOH, (e) VO(acac)2, t-BuOOH,
CH2Cl2; (f) KOSiMe3, THF; (g) Dess−Martin periodinane (DMP), CH2Cl2, 55%. PT = phenyltetrazole.
Table 1. Cytotoxicity of Herboxidiene and Its Analogs in Various Human Cancer Cell Lines
b
IC50 (nM)
a
entry
cell Lines
natural 1
synthetic 1
21a
22a
22b
23
24
1
2
3
4
5
6
JeKo-1
HeLa
4.3 1.5
14.7 2.5
46.3 5.2
34.0 32.0
12.7 2.3
12.0 1.9
4.5 0.7
6.8 1.4
22.4 4.2
14.0 11.0
6.5 0.9
7.2 0.3
1306 660
903 137
2022 562
>1800
12.0 1.8
11.0 2.3
18.9 1.9
20.0 18
14.7 2.0
8.1 1.2
310 57
310 37
266 41
1243 187
646 286
246 42
55.2 7.2
74.7 3.8
256 35.0
301 293
90.5 8.9
122 20
>4700
>7600
>28000
>240000
>5000
>5400
PC-3
SK-MEL-2
SK-N-AS
WiDr
859 165
1271 594
a
Cancer Type: JeKo-1 (Mantle cell lymphoma); HeLa (Cervical adenocarcinoma); PC-3 (Prostate adenocarcinoma); SK-MEL-2 (Malignant
b
melanoma); SK-N-AS (Neuroblastoma); WiDr (Colorectal adenocarinoma). Cells were treated continuously with the compound for 72 h.
Cytotoxicity was determined as the IC50 values calculated from the percentage of viable cells remaining at 72 h, measured with CellTiter-Glo reagent.
The IC50 values represent the average of three independent determinations SE.
Previously, we have reported the use of an assay for the
evaluation of the efficacy of alternative splicing modulator
drugs.31,49 This assay depends on our observation that the
splicing modulator drugs that we have evaluated induce exon
skipping in MDM2 pre-mRNA, which results in the formation
of shorter isoforms that can readily be detected (see SI, Figure
S3). In addition to our investigation of the cytotoxicity of these
compounds we also performed this MDM2 alternative splicing
assay for compounds 1, 22a, and 23, in order to determine
whether these compounds modulate the splicing machinery.
This assay was performed as previously described.49 The results
shown in Figure 4 demonstrate for the first time that both
natural and synthetic 1 potently induce MDM2 alternate
splicing of MDM2 pre-mRNA. Synthetic 1 appears to be
slightly more potent in this induction (Figure 4), we believe
this is due to the higher purity of the synthetic material when
compared to the commercially available fermentation product
(∼92% pure). Although 22a and 23 are less potent than 1, it is
clear that these two compounds are effective in the induction of
alternative splicing of MDM2 pre-mRNA and that the potency
of 22a is superior to sudemycin C1. All of these results are
consistent with the pharmacophore features shown in Figure 2.
In conclusion, the flexible and concise syntheses of
herboxidiene and its previously unexplored analogs were
accomplished by the orchestration of synthetic schemes that
build on published chemistry, which include the novel
application of the inverse electron-demand Diels−Alder
reaction, a Ferrier-type rearrangement as well as the established
Figure 4. Herboxidiene and its analogs modulate alternate splicing of
MDM2 in Rh18 cells. The cells are exposed to drug for 8 h in these
experiments (see SI for assay details).49
Julia−Kocienski reaction, as key steps. Initial cytotoxicity assays
of the synthetic compounds provided a good initial insight into
the pharmacophore features for this class of compounds. We
have also demonstrated that synthetic herboxidiene and its
potent analogs efficiently modulate the alternative mRNA
splicing. This study provides strong evidence that synthetic
herboxidiene analogs may serve as good anticancer drug lead
candidates in the discovery of new synthetic agents targeting
the pre-mRNA splicing process.
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METHODS
■
The methods are reported in the Supporting Information.
ASSOCIATED CONTENT
* Supporting Information
Detailed experimental procedures and methods, ORTEP
images for X-ray structures, full characterization data and
copies of spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
*E-mail: thomasr.webb@gmail.com.
■
Present Addresses
§SRI International, 333 Ravenswood Avenue, Menlo Park, CA
94025-3493.
‡LC Vision, 4150 Darley Ave #10, Boulder, CO 80305.
Author Contributions
†C.L., M.V.Y., and L.K.S. contributed equally to the chemistry
reported in this publication.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank J. Bollinger for his determination of the single crystal
X-ray structures of compounds S16 and S17. This work was
supported in part by National Institutes of Health Grant
CA140474, by the American Lebanese Syrian Associated
Charities (ALSAC), and by St Jude Children’s Research
Hospital (SJCRH).
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