ll

OPEN ACCESS

Report
Scalable Synthesis of 17S-FD-895 Expands the
Structural Understanding of Splice Modulatory
Activity
Warren C. Chan, James J. La
Clair, Brian León, ..., Joshua S.
Figueroa, Catriona Jamieson,
Michael D. Burkart

jlaclair@ucsd.edu (J.J.L.C.)
mburkart@ucsd.edu (M.D.B.)

HIGHLIGHTS
Process scaled synthesis of a
complex polyketide

Complete control of
regioselective bond assembly

Installation of 11 stereocenters
with high enantioselectivity

Synthetic access to single

stereoisomeric and single-atom
isotopically labeled analogs

There is a pressing need for developing potent small molecule splice modulators
for the treatment of malignancies characterized by splice deregulation. Chan et al.
report a scalable strategy to prepare an in vivo active splice modulator, 17S-FD-
895, providing material to enable clinical translation and expanding our
understanding of its structure-activity relationships.

Chan et al., Cell Reports Physical Science 1,

100277
December 23, 2020 ª 2020 The Authors.
https://doi.org/10.1016/j.xcrp.2020.100277
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Report
Scalable Synthesis of 17S-FD-895
Expands the Structural Understanding
of Splice Modulatory Activity
Warren C. Chan,1 James J. La Clair,1,* Brian León,1 Kelsey A. Trieger,1 Martijn Q. Slagt,2
Mark T. Verhaar,2 Dominika U. Bachera,2 Minze T. Rispens,2 Remco M. Hofman,2 Vincent L. de Boer,2
Rory van der Hulst,2 Rutger Bus,2 Pieter Hiemstra,2 Michael L. Neville,1 Kyle A. Mandla,1
Joshua S. Figueroa,1 Catriona Jamieson,3 and Michael D. Burkart1,4,*

SUMMARY
While splice modulators have entered clinical trials, limited clinical
efficacy in splicing factor mutation-driven malignancies, such as
acute myeloid leukemia, has remained a challenge. There is a press-
ing unmet medical need for developing potent small molecule splice
modulators for the treatment of a broad array of malignancies char-
acterized by splicing deregulation. However, the inability to practi-
cally access gram-scale lead molecules with viable pharmacological
properties continues to hinder their application. Here, we report a
scalable approach to prepare 17S-FD-895, a potent in vivo active
splice modulator. The strategy described herein not only provides
material to enable clinical translation but also furthers lead valida-
tion by expanding the structure-activity relationships that guide
splice modulation.

INTRODUCTION
Since their first discovery in the mid-1990s, families of polyketide natural products,
including FD-895, the pladienolides, the spliceostatins, herboxidiene, and the thai-
lanstatins, have garnered interest due to selective antitumor activities.1–5 In recent
years, two lead candidates, E71076 and H3B-8800,7 have advanced to phase I clin-
ical trials for solid tumors and leukemia. Mode-of-action studies indicate that they
share similar abilities to modulate splicing8–10 through interactions within the
SF3B component of the spliceosome.11 First suggested as a consensus motif12
and later validated by structural analyses,13 these small molecules uniquely position
themselves at an interface between SF3B1, PHF5A, and SF3B3,14 a hinge region
involved in regulating the branch site adenosine-binding pocket.15,16 These splice
modulators all possess a similar structural backbone containing a macrolactone 1Department of Chemistry and Biochemistry,
ring linked by a diene to a side chain.17,18 Here, the importance and positioning University of California, San Diego, 9500 Gilman
Drive, La Jolla, CA 92093-0358, USA
of the stereochemical centers within these molecules clearly indicate a unique
2Symeres,Kadijk 3, 9747 AT Groningen, the
geometrical requirement for activity. Netherlands
3Division
of Regenerative Medicine, Moores
While many of these splice modulators display the necessary functional spatiality to Cancer Center and Sanford Consortium for
enable facile binding to the SF3B pocket in vitro, the high density of their functional Regenerative Medicine, University of California,
San Diego, La Jolla, CA 92093, USA
groups led to low stability in biological media, resulting in short half-lives (t1/2 % 4Lead Contact
30 min).19 Recent studies now indicate that synthetic modifications along the side chain
*Correspondence: jlaclair@ucsd.edu (J.J.L.C.),
are not only tolerated but also allow for access to a three-dimensional arrangement that mburkart@ucsd.edu (M.D.B.)
reduces the rate of degradation.19 These studies also indicate that synthetic analogs https://doi.org/10.1016/j.xcrp.2020.100277

Cell Reports Physical Science 1, 100277, December 23, 2020 ª 2020 The Authors. 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Figure 1. Synthetic Design

(A) The synthesis of 17S-FD-895 (1) arises through the coupling of side chain 2 and core 3. The 11 sp3 stereocenters and stereochemistry of the three
olefins of 1 arose from 12 precursors (inset) that are available on the kilogram scale. The key steps used to prepare each component are noted.
(B) The retro-analysis of the related macrolide, pladienolide B, as developed by Ghosh and Anderson 24 from core 5a and Kotake 28 from core 5b. Colored
highlights denote the sourced components as shown in gray inset.

meet the requirements for active binding to the spliceosome pocket in vivo.14,15 This ul-
timately led to our identification of 17S-FD-895 (1) as a therapeutic lead.20,21

While efforts have been developed to access gram-scale quantities of pladienolides

via fermentation,22 these approaches have been limited to the production of natural
materials. To access the non-natural C17 stereocenter in 17S-FD-895, we focused on
a synthetic approach. To date, reported gram-scale synthesis has enabled access
only to the less complex herboxidiene.23 The synthetic challenges in facing gram-
scale preparation of 17S-FD-895 (1, Figure 1A) include the following: 11 total stereo-
centers (6 contiguous), a substituted diene, remote functionality, a quaternary car-
bon, and a 12-membered lactone. Our approach (Figure 1A) expanded on prior
milligram-scaled campaigns (Figure 1B)24–28 that identified the importance of
component assembly. As 1 possesses potent biological activity, with a human
maximum tolerated dose (MTD) estimated at 4 mg/m2 based on E7107,6 we opted
for a route that avoided production of active materials until the final step. In general,
we targeted a process that would be amenable for large-scale synthesis by reducing
operations and chromatographic requirements.

RESULTS
Preparation of 2
We began by developing methods to prepare 20 g (0.039 mol) of side chain 2
(Scheme 1) to secure more than 15 g (0.027 mol) of 1. This started with optimization
and preparation of Crimmins’ auxiliary 7 on a kilogram scale.29 Diastereoselective
aldol addition, followed by aminolysis and subsequent methylation, enabled the
successful transition to 155 g (0.82 mol) of Weinreb amide 10 per batch from
235 g (0.94 mol) of 7.20 Fortunately, we were able to recover 65% G 5% of 6. At
this point, we encountered our first challenge: the high volatility of aldehyde 11.
This was circumvented by a solvent change to 2-methyltetrahydrofuran (m-THF),
enabling reduction of 10 and homologation to 12 without isolation of 11. Next,

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Scheme 1. Side Chain 2 Was Synthesized in 11 Steps Beginning from Crimmins’ Auxiliary 6
The yields and stereoselectivities indicated reflect the improvements made that enabled gram-scale production. Compounds 6 and 7 were purified by
recrystallization. Colored highlighting denotes carbons from sourced precursors (Figure 1). Abbreviations are as follows: DMAP, 4-
dimethylaminopyridine; DIBAL-H, diisobutylaluminium hydride; DET, diethyltartrate; m-THF, 2-methyltetrahydrofuran; i-Pr, iso-propyl; t-Bu, tert-butyl;
TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl; n-Bu, butyl.

diisobutylaluminium hydride (DIBAL-H) reduction afforded alcohol 13, which could

be stored at 4
C for more than 2 years. Sharpless epoxidation of 13 provided 14
with a 6:1 dr (diastereomeric ratio), which was oxidized to 15 by use of 2,2,6,6-tetra-
methylpiperidine-N-oxyl (TEMPO). As shown in Scheme 1, condensation of alde-
hyde 15 with Marshall allenylstannane 1630 provided alkyne 17.

The next issue arose in the hydrostannylation of 17, where the use of a palladium catalyst
generated only a 1:5 a:b regioselectivity. This led to contamination by traces of the un-
desired a-vinylstannane, which was reduced by use of Figueroa’s molybdenum cata-
lyst31 (inset, Scheme 1) to a 1:10 dr favoring the desired b-stannane. Ultimately, effective
chromatographic conditions assisted access to 2 with 95+% purity that was established
by liquid chromatography-mass spectrometry (LC-MS). To date, we have stocked
more than 200 g (1.3 mol) of 13. Over multiple repetitions, we were able to synthesize
6.5 G 0.5 g (0.013 mol) of 2 from 25 g (0.16 mol) of 13 in a week.

Preparation of 3
Parallel efforts were also launched to produce 20 g (0.043 mol) of 3. We developed
scalable methods to prepare intermediate 2220 in 300 g batches from mono-pro-
tected 18 (Scheme 2). To achieve this, TEMPO oxidations enabled scalable conver-
sion of 18 to 19 and 20 to 21 without chromatography. Reducing the reaction tem-
perature (78 to 94
C) improved the dr (85% improved to 95%) of the allylboration
of aldehyde 19 to 20. Solvent change (THF to Et2O) and reaction temperature opti-
mization (78 to 94
C) improved the selectivity of the Grignard addition (85%
improved to 90% dr) to 21 affording 22. This process currently requires a single chro-
matographic step (20, Scheme 2). With a stability of more than 4 years at 20
C,
compound 22 provides an ideal storage point for batch preparation of core 3.

The conversion of 22 to 3 provided the most significant challenge. Previously estab-

lished methods20 to convert 22 to 23 relied on extremely pure ZnBr2, which caused

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Scheme 2. Synthesis of Core 3 from Mono-Protected 1,4-Butanediol 18

The yields and stereoselectivities indicated reflect the improvements made that enabled gram-scale production. Colored highlighting denotes carbons
from sourced precursors (Figure 1). The yield of the ring closing metathesis step could be improved by recovery and resubmission of unreacted starting
material (30% of the mass balance). Abbreviations are as follows: Ipc, isopinocampheyl; Ph, phenyl; TBSOTf; tert-butyldimethylsilyl
trifluoromethylsulfonyl; HGII, 2 nd -generation Hoveyda-Grubbs catalyst; CSA, (1S)-(+)-10-camphorsulfonic acid.

issues due to its hygroscopicity. After reaction screening, we observed that the in
situ decomposition of CBr4 in iso-propyl alcohol32 reproducibly returned 65% G
5% of 23, enabling three transformations in one step. The next challenge arose in
the installation of the C1-C3 fragment. Upon oxidation to 24, we installed the remote
C3 stereocenter in 9:1 dr using a chiral t-leucine-derived thiazolidinethione auxil-
iary.33 Subsequent protection and saponification afforded acid 27, which was ester-
ified with alcohol 3334 in neat pivalic anhydride35 to afford 34. These conditions
operated without solvent and were high yielding and reproducible. This six-step
sequence could be conducted in 3 days, accessing 10 g (0.015 mol) batches of 34
from 25 g (0.069 mol) of 22. At this point, we had installed the remaining five stereo-
centers required for 1 with 95+% purity in 34.

Ring Closing Metathesis

Next, we turned our attention to the challenging ring closing metathesis (Scheme 2). Pre-
viously, the reaction had been performed at a maximum of 1 g28 and suffered from allylic
isomerization, despite the use of additives.36 After screening catalysts and reaction con-
ditions, we discovered that inverting the order of addition (a solution of 2nd-generation
Hoveyda-Grubbs catalyst in toluene to 34 in refluxing toluene) provided acceptable
yields of 35 on the 5- to 10-g scale. Subsequent global deprotection of 35 with mild
acid, followed by selective acetylation of C7 in 36 via orthoester formation, yielded

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Figure 2. Synthesis of 17S-FD-895 (1), Single-Carbon Isotopically Labeled Materials, and Stereoisomeric Analogs
(A) Stille coupling of side chain 2 and core 3 yield 1 with an effective mass balance. The 17 g batch was prepared by five full-time employees (FTEs) over
the 3 month period; only the effort of 3 FTEs was required the entire period.
(B) Synthesis of 13 C1-17S-FD-895 (Scheme S1) and 13 C30-17S-FD-895 (Scheme S2) were prepared by installing 13 C-containing precursors into the routes
in Schemes 1 and 2. 13 C-NMR returned a single peak, suggestive that a single isomeric material was present within these batches.
(C) SARs in FD-895 were identified through analog development. Red and blue spheres denote stereochemical centers evaluated in this study and those
yet to be explored, respectively.
(D–F) Analogs 1a and 1b were synthesized, and GI 50 values were evaluated in HCT-116 cells. Select regions of 1 H-NMR spectra are provided to illustrate
chemical shift modifications. (D) 17S-FD-895 (1). (E) Replacing dichlorophenylborane and ()-sparteine in the Sammakia aldol addition with TiCl 4 and
diisopropylamine afforded the inverted C3 stereocenter in 1a (Scheme S3). (F) C7 core isomer 1b was synthesized from 34 in six steps (Scheme S4). While
inversion at C17 retained activity, inversion within the macrolactone core at C3 or C7 resulted in marked loss in activity.

core 3. After optimization, we are now able to convert 30 g (0.083 mol) of 22 to 1.8 G
0.2 g (0.0039 mol) of 3 (95+% purity via LC-MS) in less than 2 weeks.

Final Steps and Analyses

At this stage, we were set for the final step (Figure 2A). We opted for a Stille cross-
coupling at C13-C14,37 as alternate installation of the C14-C15 olefin by cross-
metathesis or Julia-Kocienski olefination (Figure 1B)24,28,38 can be complicated by
the formation of undesired cis-olefins. After parallelized reaction screening, we
settled on an olefin coupling using Buchwald’s XPhos Pd G2 catalyst with CuCl
and KF in anhydrous tert-butyl alcohol.37 Under class III safety conditions, we pre-
pared 1 in 80% G 2% yield, with a worker exposure of less than 3 h per 5 g batch
(a key step in deducing risk from this potent agent, MTD at 4 mg/kg).6 Fortunately,
we were able to recover 16% G 3% of 3, which could be recycled, providing an effec-
tive mass balance in the conversion of 3 to 1. Side chain 2 was not recoverable.

To provide materials to assist purity analyses, we introduced 13C isotopic labels in 1

independently at C1 and C30 (Figure 2B). The 13C label at C1 was installed by

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preparing the Sammakia auxiliary with 1-13C acetyl chloride (Scheme S1), relaying it
to the corresponding 13C1-labeled core 3, and coupling it with side chain 2 to afford
1 g of 13C1-17S-FD-895. The 13C tag at C30 was introduced by selective acetylation
of 36 with 1-13C acetic anhydride (Scheme S2). The resulting 13C30-labeled 3 was
coupled to 2 to prepare 100 mg of 13C30-17S-FD-895. 13C-NMR spectroscopy (Fig-
ure 2B) confirmed that batches of 13C1-17S-FD-895 and 13C30-17S-FD-895 were a
single compound with 99% purity. Overall, this improved route has produced
more than 17 g of 17S-FD-895 (1), with all 11 stereocenters installed in high selec-
tivity and reproducibility. Furthermore, the ability to produce gram-scale lots of sta-
ble, isotopically labeled material is especially advantageous for in vivo pharmaco-
logical assessments.

Next, we wanted to expand the stereochemical structure-activity relationship (s-

SAR)39 profile of FD-895 (Figure 2C)2,40–42 by utilizing our route to access non-natu-
ral analogs from late-stage intermediates. The C3-isomer 1a (Figure 2E) and the C7-
isomer 1b (Figure 2F) were synthesized by changes in chiral reagents (1a, Scheme S3
and 1b, Scheme S4) and along with 1 (Figure 2D) were confirmed by NMR peak shift
comparisons (Figure S3). Comparing the activity of 1, 1a, and 1b to the natural prod-
uct, FD-895, in human colorectal tumor HCT-116 cells indicated that inverting the C3
and C7 stereocenters in 1a and 1b, respectively, compromised activity, while the
C17 isomer in 1 retained potency.

DISCUSSION
These results were consistent with established X-ray crystal structure (Figure S1) of
the SF3B core complexed with pladienolide B.14 In this and related structures,18 in-
verting the C3 hydroxyl-group in 1a ablates its interaction with K1071 of the SF3B1
subunit (Figure S1a–f). The lack in activity of the C7 isomer followed a similar
reasoning, as inversion of the C7 acetate in 1b disrupts its interaction with R38 in
PHF5A. These findings support a strict SAR within the 12-membered core, as it
bridges the interface between SF3B1 and PHF5A. Tolerance for inversion of C17
in 1 was also supported structurally. Rotational freedom within the side chain (Fig-
ure S1g and h) permitted pladienolide B and associated analogs to adopt distinct
conformations to access the same binding pocket. Overall, this synthesis has facili-
tated material access to complete preclinical evaluation (setting the stage for devel-
opment of improved GMP manufacturing protocols), delivered isotopic materials,
filled gaps in the SAR data, and contributed to an understanding of structural fea-
tures required to engage small molecule splice modulation.

EXPERIMENTAL PROCEDURES
Resource Availability
Lead Contact
Further information and requests for resources and reagents should be directed to
and will be fulfilled by the Lead Contact, Prof. Michael D. Burkart (mburkart@ucsd.
edu).

Materials Availability
Samples of 17S-FD-895 and the intermediates are available upon request.

Data and Code Availability

Copies of raw NMR data (FIDs) as well as mass spectral data are available on request.
PDF copies of select spectra are provided within the Supplemental Experimental
Procedures.

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Methods
Full procedures for the synthesis of side chain 2, synthesis of core 3, along with all
other synthetic procedures, cell viability studies, and characterization are provided
in the Supplemental Experimental Procedures, Figures S1–S3, and Tables S1–S3.

SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.xcrp.
2020.100277.

ACKNOWLEDGMENTS
This work was funded by a CIRM Therapeutic Translational Research Project entitled,
A Splicing Modulator Targeting Cancer Stem Cells in Acute Myeloid Leukemia, to
C.J. and M.D.B. We thank Peggy Wentworth for program management, Leslie Crews
(UCSD) for critical guidance and discussions, Doug Harvey (UCSD) for assistance
with the development and integration of proper safety protocols, Anthony Mrse
(UCSD) for enabling the NMR studies, and Yongxuan Su (UCSD) for LC-MS and
high-resolution MS (HRMS) analyses.

AUTHOR CONTRIBUTIONS
W.C.C., J.J.L., B.L., K.A.T., C.J., and M.D.B. conceived and designed experiments.
W.C.C. and J.J.L. carried out the synthetic effort. W.C.C., J.J.L.C., B.L., M.L.N.,
K.A.M., and J.S.F. conducted step optimization efforts. K.A.T. performed the bio-
logical analyses. W.C.C., J.J.L.C., M.Q.S., M.T.V., D.U.B., M.T.R., R.M.H., V.L.d.B.,
R.v.d.H., R.B., and P.H. conducted scale-up and methods optimization efforts. All
authors discussed and co-wrote the manuscript.

DECLARATIONS OF INTERESTS
The authors declare no competing interests.

Received: July 22, 2020

Revised: September 8, 2020
Accepted: November 6, 2020
Published: December 23, 2020

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