A Carbohydrate-Derived Splice Modulator

Article abstract of DOI:10.1021/jacs.5b13427

Small-molecule splice modulators have recently been recognized for their clinical potential for diverse cancers. This, combined with their use as tools to study the importance of splice-regulated events and their association with disease, continues to fue

Full text of DOI:10.1021/jacs.5b13427

Article
pubs.acs.org/JACS
A Carbohydrate-Derived Splice Modulator
Sachin Dhar,† James J. La Clair,*,† Brian Leon,† Justin C. Hammons,† Zhe Yu,‡ Manoj K. Kashyap,‡
́
Januario E. Castro,‡ and Michael D. Burkart*,†
†Department of Chemistry and Biochemistry, University of California−San Diego, 9500 Gilman Drive, La Jolla, California
92093-0358, United States
‡Moores Cancer Center, University of California−San Diego, La Jolla, California 92093-0358, United States
S
* Supporting Information
ABSTRACT: Small-molecule splice modulators have recently
been recognized for their clinical potential for diverse cancers.
This, combined with their use as tools to study the importance
of splice-regulated events and their association with disease,
continues to fuel the discovery of new splice modulators. One
of the key challenges found in the current class of materials
arises from their instability, where rapid metabolic degradation
can lead to off-target responses. We now describe the
preparation of bench-stable splice modulators by adapting
carbohydrate motifs as a central scaffold to provide rapid access to potent splice modulators.
carbohydrate-derived 2, an analog of FD-895 whose core was
derived from D-glucose.
INTRODUCTION
■
In 2007, two Japanese teams revealed the importance of
targeting the SF3b unit of the spliceosome for antitumor
therapy.1,2 These breakthroughs not only forwarded the
spliceosome as an important target for cancer2 but also
identified two key families of polyketide spliceosome modu-
lators (Figure 1), including FD-895 (1a),2 pladienolide B (1b),3
and herboxidiene (1c).4 Since these discoveries, the develop-
ment of splicing assays has been instrumental in the advance of
next generation splice modulators. The success of these
discoveries was further marked by the translation of an analog
of pladienolide D, E7101, into phase I clinical trials.5 However,
many of these natural products are subject to low stability and/
or poor pharmacological properties.6 To this end, our laboratory
has been part of a growing medicinal chemistry effort to identify
next generation splice modulators with improved pharmaco-
logical properties.
An attractive proposal developed by Webb suggested a
method of analog development through the use of a consensus
motif.7 Pursuing this approach, their team has advanced the
sudemycins8 and a pladienolide−herboxidiene hybrid9 as next-
generation leads. This, along with efforts at the Eisai Co., Ltd.,10
H3 Biomedicine,11 Pfizer,12 Ghosh-Jurica laboratories,13 Koide
laboratory,14 and our laboratories,15 have provided a strong
foundation for achieving analogs with improved pharmaco-
logical properties. We now report on an advanced new class of
splice modulators that are more potent, stable, and rapidly
accessible analogs.
RESULTS
■
Our design began with FD-895 (1a) because of the fact that
earlier medicinal chemistry efforts in our laboratory provided
derivatives with improved pharmacological properties.6a From
these studies, we determined that manipulation of the
stereochemistry at the C16−C17 diad (blue shaded, Figure 1)
provided an effective means to improve activity. From this data,
we targeted a side chain that contained the exact terminus
(orange shading, Figure 1), epoxide (green shading, Figure 1),
and diene groups (green shading, Figure 1) as one of our more
active analogs of FD-895.6a Here, we incorporated already
known SAR data to improve activity by selecting an improved
C16−C17 stereodiad (blue shading in 2, Figure 1).
Clues Obtained from the Solution Structure Analysis
of FD-895 (1a). Although structural information on the
eukaryotic spliceosome has recently been published,18 repre-
sentation of the binding of a splice modulator with SF3b has yet
to be established. We began by exploring the solution structure
of the macrolide core of FD-895 (1a). Using multiple solvents
(CDCl3 and C6D6) for data acquisition, we identified a series of
NOE interactions that along with coupling constant data
allowed assignment of the solution structure of the macrolide
core (Figure 2a).
Design of a Carbohydrate Core Motif. Using this
structure and analyzing its space-filling model, we began to
search for motifs that accurately represented the macrolide core.
Our goal was to find a structural unit that offered an improved
In addition to carbohydrates being ubiquitous biological
scaffolds, they are also derived from the natural chiral pool,
making them ideal scaffolds for medicinal chemical optimiza-
tion.16 To expand on this hypothesis, we designed and prepared
Received: December 23, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b13427
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Figure 2. Models of FD-895 (1a, blue), herboxidiene (1c, magenta),
and carbohydrate-derived analog 2 (green). (a) Derived solution
structure of the FD-895 core motif as determined by evaluation of 1H
coupling constants and NOE interactions. NOE interactions were
observed both on the top face (red) and bottom face (blue). Data
shown was collected from 1H,1H NOESY spectral data of 1a in CDCl3
and C6D6 at 23 °C. (b) Model depicting the structure of 1a (blue)
superimposed on the surface of 1c (magenta). (c) Model depicting the
structure of 1c (magenta) superimposed on the surface of 1a (blue).
(d) Models depicting the structure of 1a (blue) superimposed on the
surface of 2 (green). (e) Model depicting the structure of 2 (green)
superimposed on the surface of 1a (blue). The structure of the core
motif in 1a was determined via NMR spectroscopy. Structures of 1b
and 1c were modeled by energy minimization.17
Figure 1. Structures of FD-895 (1a), pladienolide B (1b), herboxidiene
(1c), and carbohydrate-derived splicing modulator 2. Compound 2
contains the terminus (orange) of 1a, the epoxide and diene of 1a
(green), and a core derived from 1c (gold).
synthesis entry and diverse means of tailoring while also
addressing previous pharmacological issues (stability and
solubility). We began by inspecting the overlap between the
small herboxidiene (1c) and large FD-895 (1a) cores. Although
a clear overlay existed (Figure 1b,c), it was evident that space
occupied by the C4−C7 unit within 1a may not be a
requirement for activity. We soon realized that simple
monosaccharides such as D-glucose could provide a strong
overlap with these cores. After evaluating several structures, we
turned our attention to explore α-methyl-2,3,4-trimethoxy-D-
glucopyranoside (3) as a core mimetic.19
Scheme 1. Synthesis of Core Component 8
Structurally, glycoside 2 offered a functional map that lay
between the core sizes of 1a and 1c (Figure 2). As illustrated in
Figure 2d,e, the C2−C4 methoxy groups in 2 provided a fit
approximating 1a relative to that of herboxidiene (Figure 2b,c).
Specifically, the C2 methoxy group of 2 filled the space taken by
the C24 methyl group in 1a. Likewise, the C3 methoxy of 2 was
able to partially fill the space of the C29−C30 acetate, a region
that was already shown to tolerate functional modifications.20
With this support, we turned our attention to synthesize and
biologically evaluate analog 2.
Synthetic Strategy. Our plan focused on using a late-stage
Julia−Kocienski olefination to couple a side chain derived from
FD-895 to the monosaccharide core (see Abstract graphic).
Here, we envisioned that we could leverage the extensive efforts
from our own laboratory for development of pladienolide/FD-
895 side chain6a,15b as a means to probe the structure−activity
relationships (SARs) of side-chain-incorporating analogs. We
therefore set out toward preparation of core component 8
(Scheme 1) and side-chain component 16 (Scheme 2).
Synthesis of a Carbohydrate Core Component 8.
Synthesis of 8 was accomplished in five steps from 3, a
monosaccharide derivative that in turn was prepared in three
B
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16 in 6 steps and 58% overall yield from 9 and 12 steps and 21%
overall yield from auxiliary 11.6a
Scheme 2. Synthesis of Side Chain Component 16
Component Assembly and Completion of the Syn-
thesis of 2. With components 8 and 16 in hand, the
component coupling began by oxidizing allylic alcohol 8 to
the corresponding aldehyde 17. Purification via dry column
vacuum chromatography using Geduran 60 silica gel was
sufficient to afford pure 17 for coupling. After initial screening,
we found the optimum yield (68%) of 18 was obtained when
applying 1.05 equiv NaHMDS to 1.0 equiv of sulfone 8 and
following this by addition of 1.1 equiv of aldehyde 17. The yield
of this reaction also included recovery of 8% of sulfone 16 and
11% of the chromatographically separable cis isomer of 18
(structure not shown). With samples of pure 18 on hand,
deprotection with TBAF afforded alcohol 19 (Scheme 3).
Scheme 3. Component Assembly
steps (54% yield) from α-methyl-D-glucopyranoside.19 We
began by oxidizing to aldehyde 4. Advantageously, we were
able to reduce chromatographic purification efforts by using a
combination of an IBX oxidation followed by solvent-dependent
filtration through a pad of SiO2. The addition of MeLi to crude 4
occurred with minimal byproduct formation, providing alcohol
5 in 82% yield from 3.
Repetition of the IBX oxidation procedure provided ketone 6,
which could be rapidly purified by passage through a SiO2 plug.
Although small samples of 6 were purified for characterization,
the crude product was readily subjected to a Horner−
Wadsworth−Emmons olefination to afford ester 7. We were
able to convert 5 to 7 in two steps and 80% yield with a single
purification. Finally, ester 7 was reduced with DIBAL-H to
afford core unit 8, which could be stored for months at −20 °C
without decomposition. Overall, this process provides access to
8 in 66% in five steps from 3 (36% in 8 steps from α-methyl-D-
glucopyranoside).
Synthesis of the Side Chain Component 16. The
synthesis of the side chain component began with allylic alcohol
9, an intermediate that was prepared at >10 g scale for the
synthesis of FD-895 (1a).6a Allylic oxidation with MnO2
provided aldehyde 10, which was purified through a SiO2 plug
and directly submitted to a Crimmins aldol reaction with
auxiliary 11, to afford adduct 12 in 87% yield over two steps.
NMR analyses on the crude product indicated that this reaction
occurred without detectable formation of other isomeric
products.
With 12 in hand, the carbinol was protected as TBS ether 13,
and the auxiliary was removed by NaBH4 reduction.
Optimization studies indicated that a 5:1 ratio of THF:CH3OH
provided a reproducibly high yield of 14. Completion of this
component was accomplished by a two-step installation of the 1-
phenyl-1H-tetrazol-5-yl (PT) sulfone. Although Mitsunobu
conditions were effective at producing sulfone 15 in 82% yield
at a gram scale, oxidation often provided significant quantities of
the corresponding sulfoxides (incomplete oxidation byprod-
ucts). Typically, samples of these sulfoxides were collected after
chromatographic purification and resubmitted to the oxidation
conditions to afford a combined yield of 89% of component 16.
To date, we have completed the synthesis of gram quantities of
Next, we turned to the stereoselective VO(acac)2-catalyzed
epoxidation of allylic alcohols21 to install the C12−C13 epoxide.
As shown in Figure 3a, treatment of 19 with 4.5 equiv of
tBuOOH and 3 equiv of VO(acac)2 at −20 °C for 6 h provided
the desired stereoisomer with ∼4:1 diastereoselectivity.
Although this was the major product, over-warming or not
starting the reaction at −78 °C resulted in epoxidation at C8−
C9. This product was not as stable because the C11 carbinol was
capable of attacking the C8 center of the epoxide, forming the
corresponding furan analog (structure not shown). Although
additional optimization efforts will be required, the current
conditions returned 2 in 52% yield with only traces of the
undesired C8−C9 epoxidation (5−10% yield).
Validation of the Structural Assignment of 2. Although
the mechanism of the VO(acac)2/tBuOOH epoxidation
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Figure 4. Coupling constant analyses. NMR analysis in C6D6 provided
a coupling pattern at C12−C13 in 2 (blue) that matched that from the
corresponding FD-895 analog 1e (green) but not analogs 1a, 1f, or 1g
(black).6a,15b
(1a). Using capillary NMR and solvent 13C satellites (QSCS) as
a means of quantitation,22 we found that 2 was soluble up to 95
5 mM D2O/DMSO-d6 (10:1) at 23 °C, whereas FD-895 (1a)
was limited to 15
5 mM under the same conditions.
Additionally, we used NMR monitoring to show that although
1a had a half-life of ∼70 h in D2O/DMSO-d6 (10:1) at 37 °C,6a
carbohydrate analog 2 showed no signs of decomposition even
after 744 h (1 month) under the same conditions. Although
detailed partitioning, permeability, and metabolic stability
analyses are ongoing, this initial evidence suggests that 2 offers
the potential to improve pharmacological properties of this class
of splice modulator.
Epoxide 2 Displays Potent Activity in Primary CLL-B
Cells in Contrast to Its Precursor Alkene 19. We then
screened the activity of 19 and 2 in primary CLL-B cells
obtained from two CLL patients that displayed potent in vitro
cytotoxic response (Figure 1), measured in the form of specific
induced apoptosis (% SI), to 1a (IC50 value of 54.1 7.5 nM)
Figure 3. Stereoselectivity of the C12−C13 epoxidation. (a) Expanded
LC-MS trace via UV monitoring at 288 nm depicting the reaction
product from treatment of 19 with VO(acac)2/tBuOOH (blue). (b)
Comparable LC-MS trace depicting the formation of two isomers by m-
1
CBPA epoxidation (red). (c) Expansion of the H NMR spectra of
epoxide 2 from VO(acac)2/tBuOOH in CDCl3 (blue). (d) Expansion
of the 1H NMR spectra of epoxide 20 in CDCl3 from m-CBPA (red).
Traces of 2 denoted by a blue asterisk could not be completely removed
from 20 because of close retention times.
suggests delivery of the correct stereochemical outcome, we
wanted to gain further confirmation that the product was indeed
2. We began by exploring other epoxidation conditions to
prepare samples of the alternate isomer. Treatment of 19 with
1.2 equiv of m-CPBA at −78 °C in NaHCO3-buffered CH2Cl2
followed by slow warming to room temperature afforded a ∼1:1
mixture of both epoxides (Figure 3b) in 62% yield. After
chromatographic purification, we were able to obtain samples of
both isomers.
Comparative NMR analyses on the epoxides from VO-
(acac)2/tBuOOH and m-CPBA provided a solution (Figure 3c).
Using 2D NMR data set (Supporting Information), we were
able to fully assign the proton assignments in this product. We
identified that the chemical shift and coupling patterns of the
epoxide protons at C12−C13 provided the closest match to the
configuration of corresponding C16−C17 isomer 1e (Figure 4),
with comparable chemical shifts and coupling constants.
Stability and Solubility of 2. Early studies indicated that 2
was more soluble and stable in aqueous buffers than FD-895
and 1b (IC50 value of 84.4
1.2 nM). Interestingly, these
studies indicated that alkene 19 did not display any activity (IC50
value >50 μM in both sets of cells). Epoxide 2, however, was
active (IC50 value of 153.0 11.8 nM) with ∼3-fold loss of
activity when compared to FD-895 (1a).
Compound 2 Modulates Splicing. We then turned to RT-
PCR analysis to determine if the activity of 2 also included a
splice response comparable to 1a and 1b in CLL-B cells. Using
unspliced RNU6A and unmodulated GAPDH (top, Figure 5) as
controls, we were able to obtain complementary splice
modulation in all six genes selected from our prior studies to
provide a diversity of intron retention (IR) response.15a This
included comparable modification of IR in DNAJB1, ARF4,
PRPF4, RIOK3, SF3A1, and U2AF2 (Figure 4 and S2) from
primary CLL-B cells. In contrast, alkene precursor 19 was
inactive for all six genes (Figure 5), indicating that the epoxide in
2 is essential for activity.
Additionally, we were able to confirm that compound 2 also
induced alternate splicing (AS) events. As shown in Figure 6,
D
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Figure 6. Compound 2 modulates alternate splicing (AS) in CLL-B
cells, which is comparable to FD-895 (1a) and pladienolide B (1b).
The alkene precursor 19, which was shown inactive in screening assays,
did not induce AS. H2A gene was used as unspliced loading control as
depicted. S and L denote short and long isoforms, respectively.
CONCLUSIONS
■
We now describe the preparation of a carbohydrate-derived
spliceosome inhibitor 2 in a total of 22 steps, with the 12 longest
linear steps from common building blocks (auxiliary 11, D-
glucose and propionaldehyde). Although complete in vitro and
in vivo pharmacological analyses are underway, this analog's
improved solubility, along with improved solubility in aqueous
buffers, suggests an important advance in optimization of splice
modulators.
Overall, this development offers several medicinal chemical
benefits. First, it demonstrates that the pyran or macrolide core
commonly associated with the known spliceosome inhibitors
can be replaced with materials directly available from
monosaccharides in the chiral pool. Second, it provides a
means to prepare highly stable materials that lack rapid
degradation and/or metabolic instability. Third, the use of a
carbohydrate motif offers ready access to explore the SAR
contained within the core unit (macrolide of 1a and 1b or pyran
in 1c, Figure 1). Here, one can envision application of the
described route to provide derivatives with expanded function-
ality at the anomeric C1 center and stereoisomers at C1−C4
positions and to explore the use of deoxygenated materials and
alternative ethers at C1−C4. Although to date there has been no
structural information on the complex of any established splice
modulator with the Sf3b component of the spliceosome, the
subsequent integration of a rational design could deliver a
carbohydrate-derived splice modulator with enhanced binding
and reduced pharmacological risk.
Figure 5. Compound 2 induces splice modulation via intron retention
(IR) comparable to FD-895 (1a) and pladienolide B (1b) in CLL-B
cells. The alkene precursor 19, which was shown inactive in screening
assays, did not induce IR. Genes GAPDH and RNU6A were used as
unspliced and loading controls, respectively. Complementary qRT-
PCR data has been provided in Figure S2.
treatment of CLL-B cells with 1a, 1b, and 2 resulted in a
decrease in the long (anti-apoptotic) form and increased the
expression of short (pro-apoptotic) form of MCL1. Similarly,
BCL-X demonstrates comparable AS events (Figure 6). This
indicates that cell death induced by 1a, 1b, and 2 is not
exclusively derived through AS of MCL1 but may also include
other players. This fact is further supported by RNAseq
analyses,15a which implicates the involvement of other pathways.
Efforts are now underway to explore the roles of IR and AS
events as well as to identify structural motifs that enrich specific
events.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b13427.
Experimental procedures, characterization data for all new
compounds, and full acknowledgments. (PDF)
E
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AUTHOR INFORMATION
■
Corresponding Authors
*jlaclair@ucsd.edu
*mburkart@ucsd.edu
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was support by financial support from the Lymphoma
Research Foundation (#285871), the NIH (PO1-CA081534),
the University of California−San Diego Foundation Blood
Cancer Research Fund and the Bennett Family Foundation. B.
L. was supported by the NIH IRACDA K12 (GM068524)
award. We thank Dr. Yongxuan Su for mass spectral analyses
and Drs. Anthony Mrse and Xuemei Huang for assistance with
acquiring NMR spectral data. We thank Prof. Thomas J. Kipps
for providing the CLL patient-derived cells.
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DOI: 10.1021/jacs.5b13427
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