Sudemycin k: A synthetic antitumor splicing inhibitor variant with improved activity and versatile chemistry

Article abstract of DOI:10.1021/acschembio.6b00562

Important links exist between the process of pre-mRNA splicing and cancer, as illustrated by the frequent mutation of splicing factors in tumors and the emergence of various families of antitumor drugs that target components of the splicing machinery, notably SF3B1, a protein subunit of spliceosomal U2 small nuclear ribonucleoprotein particle (snRNP). Sudemycins are synthetic compounds that harbor a pharmacophore common to various classes of splicing inhibitors. Here, we describe the synthesis and functional characterization of novel sudemycin analogues that functionally probe key chemical groups within this pharmacophore. Our results confirm the importance of a conjugated diene group and in addition reveal significant spatial flexibility in this region of the molecule. Sudemycin K, a derivative that replaces the pharmacophore's oxycarbonyl by an amide group, displays improved potency as an inhibitor of cancer cell proliferation, as a regulator of alternative splicing in cultured cells and as an inhibitor of in vitro spliceosome assembly. Sudemycin K displays higher stability, likely related to the replacement of the oxycarbonyl group, which can be a substrate of esterases, by an amide group. The activity and special reactivity of sudemycin K can pave the way to the synthesis and evaluation of a variety of novel sudemycin derivatives.

Full text of DOI:10.1021/acschembio.6b00562

Subscriber access provided by UniSA Library
Article
Sudemycin K: a synthetic anti-tumor splicing inhibitor
variant with improved activity and versatile chemistry
Kamil Makowski, Luisa Vigevani, Fernando Albericio, Juan Valcárcel, and Mercedes Alvarez
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00562 • Publication Date (Web): 08 Nov 2016
Downloaded from http://pubs.acs.org on November 9, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth
Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 13
ACS Chemical Biology
1
2
3
4
5
6
Sudemycin K: a synthetic anti-tumor splicing inhibitor variant with
improved activity and versatile chemistry
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
§,≠,∫,†
§,‡,∫
≠,#,ǂ,†
*, §, ‡, ¥
Kamil Makowski
Álvarez
, Luisa Vigevani , Fernando Albericio
, Juan Valcárcel
, Mercedes
*
,≠,ǂ,∞
§
.
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barꢀ
celona, Spain
≠
.
Institute for Research in Biomedicine (IRBꢀBarcelona), Baldiri i Reixac 10, 08028, Barcelona, Spain
‡
#
. Universitat Pompeu Fabra (UPF), Dr. Aiguader 88, 08003 Barcelona, Spain
Department of Organic Chemistry, Faculty of Chemistry, University of Barcelona, Martí Franqués 1, 08028 Barceloꢀ
.
na,Spain
¥
.
ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain
ǂ. CIBERꢀBBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona, Spain
∞
. Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain
∫
. Equal contribution
Corresponding authors
*
ABSTRACT: Important links exist between the process of preꢀmRNA splicing and cancer, as illustrated by the frequent mutation
of splicing factors in tumors and the emergence of various families of antiꢀtumor drugs that target components of the splicing maꢀ
chinery, notably SF3B1, a protein subunit of spliceosomal U2 small nuclear Ribonucleoprotein Particle (snRNP). Sudemycins are
synthetic compounds that harbor a pharmacophore common to various classes of splicing inhibitors. Here we describe the synthesis
and functional characterization of novel Sudemycin analogues that functionally probe key functional groups within this pharmacoꢀ
phore. Our results confirm the importance of a conjugated diene group and in addition reveal significant spatial flexibility in this
region of the molecule. Sudemycin K, a derivative that replaces the pharmacophore’s oxycarbonyl by an amide group, displays
improved potency as an inhibitor of cancer cell proliferation, as a regulator of alternative splicing in cultured cells and as an inhibiꢀ
tor of in vitro spliceosome assembly. Sudemycin K displays higher stability, likely related to the replacement of the oxycarbonyl
group, which can be substrate of esterases, by an amide group. The activity and special reactivity of Sudemycin K can pave the
way to the synthesis and evaluation of a variety of novel Sudemycin derivatives.
The high incidence of cancer and severe limitations in curꢀ
rent therapies (e.g. side effects and drug resistance) make the
identification of new drugs and targets an area of intense inꢀ
vestigation in oncology. Several small molecules targeting
components of the RNA splicing machinery have been shown
to display antitumor properties . Of relevance, recent findꢀ
ings indicate that the splicing machinery can indeed be limitꢀ
ing for the proliferation of cancer cells and consequently splicꢀ
mation with the 5’ end of the intron after the first catalytic step
of the splicing reaction), a polypyrimidine tract and a conꢀ
served AG dinucleotide at the 3’ end of the intron. The first
steps of spliceosome assembly include the recognition of the
5’ss by U1 snRNP and of the branch point sequence by U2
snRNP, both involving baseꢀpairing interactions between the
corresponding small RNA components (snRNAs) and the preꢀ
1
ꢀ3
6, 7
mRNA
.
ing inhibition can confer therapeutic vulnerability to Myc
SF3B1 is a protein component of SF3B, a subcomplex withꢀ
in U2 snRNP implicated in branch point recognition. Mutaꢀ
tions in SF3B1, as well as in other 3’ splice siteꢀrecognizing
4, 5
oncogeneꢀdriven cancers
.
RNA splicing is the process by which introns are removed
from messenger RNA precursors (preꢀmRNAs) and is
achieved by the spliceosome, composed of five small nuclear
RiboNucleoProtein complexes (U1, U2, U4, U5 and U6
1, 8
factors, are recurrent in cancer . SF3B1 mutations are particꢀ
ularly frequent in Myelodysplastic Syndromes with Refractory
9, 10
Anemia and Ring Sideroblasts (RARS)
and in Chronic
6, 7
11ꢀ13
snRNPs) and more than 200 additional polypeptides . Introns
Lymphocytic Leukemia (CLL) . In CLL, SF3B1 mutants
correlate with resistance to chemotherapy and poor
prognosis . Notably, SF3B1 was identified as the physical
are recognized via specific sequence signals located at their
boundaries: a short (6ꢀ8 nucleotides) consensus at the 5’ splice
site (5’ss) and three sequence elements at the 3’ splice site
11ꢀ13
target of drugs that display higher cytotoxicity in tumor cells
than in normal cells and are therefore promising therapeutic
(3’ss). The latter include the branch point sequence (containꢀ
1, 14ꢀ18
ing an adenosine involved in 2’ꢀ5’ phosphodiester bond forꢀ
candidates
.
1
ACS Paragon Plus Environment

ACS Chemical Biology
Page 2 of 13
Several natural compounds isolated from bacterial fermentaꢀ
recent studies show that MCL1 is highly sensitive to splicing
inhibition, as depleting several splicing factors induces MCL1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tion products display these properties, including FR901464,
Pladienolides, FDꢀ895, GEX1A, Herboxidiene and Thailanstaꢀ
37ꢀ39
exon 2 skipping . Indeed, MCL1 alternative splicing was
found to be the most affected by SF3B1ꢀtargeting splicing
inhibitors among a panel of alternative splicing events inꢀ
19ꢀ23
tines
. Stabilized derivatives SSA (Spliceostatin A,
FR901464ꢀrelated) and E7107 (Pladienolideꢀrelated) were
shown to inhibit splicing and bind tightly to the SF3B comꢀ
3
9
volved in proliferation and apoptosis control . Indeed,
Spliceostatin A induces apoptosis in chronic lymphocytic
17, 18
15
plex
. Similar results were obtained for Herboxidiene .
2
9
Thus, the SF3B complex has emerged as a target of repreꢀ
sentative drugs in each of the three main classes of natural
compounds (Spliceostatin, Pladienolides and Herboxidienes).
The Spliceostatin analogue Meayamycin was shown to display
leukemia (CLL) cells through MCL1 downregulation and
resistant cell lines reacquire sensitivity to BclꢀXꢀtargeting
drugs when treated with Meayamycin, due to MCL1 regulaꢀ
4
0
tion .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14
antitumor effects at picomolar concentrations .
Despite their very different overall skeletons, SF3Bꢀ
4
1
The drug Spliceostatin A binds SF3B1 and prevents its inꢀ
teraction with the preꢀmRNA, concomitant with interactions of
U2 snRNA with “decoy” sequences upstream of their producꢀ
targeting molecules share a common pharmacophore , which
includes a conjugated diene, an epoxide and an oxycarbonyl
group (Figure 1). While the epoxide group was found not to be
24
19,
tive binding site at the branch point sequence . In addition,
absolutely required for activity, it contributes to increase it
42ꢀ44
the drug E7107 alters the balance between alternative U2
snRNA conformations, also destabilizing U2 snRNP recruitꢀ
. Based in this pharmacophore, a total synthetic compound
series known as Sudemycins has been described . In spite of
45
25
ment . Interestingly, cancerꢀassociated SF3B1 mutations
their simplified structure, containing up to 6 sterocenters less
that natural products, these drugs retain potent anticancer
induce cryptic 3' splice site selection through the use of upꢀ
2
6, 27
45
stream branch points
. Thus, SF3B1 appears to be involved
activity in vitro and in vivo , as well as the ability to target
15
in multiple interactions important for U2 snRNP binding that
are relevant for the control of cell proliferation and apoptosis.
SF3B1 . Previous extensive structureꢀactivity relationship
(SAR) studies reported by the Webb’s group led to the syntheꢀ
sis of stable active derivatives, described as Sudemycin C1
How can drugs targeting a core component of the splicing
machinery not result in general cellular toxicity? Tumor cells
often display an altered balance of alternative isoforms that
4
1, 45, 46
(cyclohexane core) and Sudemycin E (dioxane core)
Figure 1). Challenging synthetic hurdles included the develꢀ
(
28
opment of a synthetic route for the heterocyclic spiro moiety
with two stereocenters, present in all the Sudemycins, and the
diene linker with E,E configuration. The sterocenter in posiꢀ
tion 2 of the pyrane ring was induced by organocatalic reducꢀ
tion of double bond using McMillan catalyst and the spiꢀ
roepoxide was prepared by diastereoselective introduction of
dimethylsulfoxonium methylide to the ketone. The key step
for preparation of diene was the JuliaꢀKocienski olefination. In
prevent apoptosis, promote proliferation and invasion . Tranꢀ
scriptomeꢀwide analyses have identified drugꢀinduced changes
in alternative splicing that particularly affect genes involved in
2
4
cell division, apoptosis and cancer progression , suggesting
that these compounds differentially affect alternative splice
sites. Moreover, recent results indicate that these drugs can
have beneficial therapeutic effects for chronic lymphocytic
leukemia (CLL) and for melanoma cells displaying drug reꢀ
47
2
9, 30
a recent study , the synthetic route was revised and the Juliaꢀ
sistance
. Notably, leukemic cells with spliceosomal mutaꢀ
31,
Kocienski step was optimized by shifting the sulfone and
aldehyde group positions required for the olefination, which in
comparison to the previously described procedures resulted in
better diastereoselectivity and yield. Additionally, new Sudeꢀ
mycin derivatives, mostly with ester moiety modification were
reported, among them Sudemycin D6, which is the most
tions display also increased sensitivity to splicing inhibitors
32
.
A wellꢀcharacterized alternative splicing event relevant for
antiꢀtumor drug function involves inclusion/skipping of exon 2
in the threeꢀexon gene coding for Myeloid Cell Leukemia 1
(
MCL1) proteins. This protein belongs to the Bclꢀ2 family of
47
potent Sudemycin so far, displaying improved solubility ,
apoptosis regulators, displays antiꢀapoptotic functions and is
33ꢀ35
bearing a methylcarbamate group instead of the isobutyric
group present in Sudemycins C1 and E (Figure 1).
overexpressed in several tumors . Due to its rapid turnover
both at protein and RNA levels, MCL1 is highly affected by
transcription and translation inhibitors, causing death of some
tumor cells depending on the levels and activity of BclꢀX,
With the aim of further exploring the chemical space of this
family of drugs, we have designed and synthesized several
novel Sudemycin analogues aimed to probe key chemical
features and exploring possibilities for further derivatization of
the structural frame.
3
5
another alternatively spliced apoptotic regulator . Exon 2
36
skipping leads to the production of a proꢀapoptotic protein
and could therefore facilitate therapeutic effects. Interestingly,
2
ACS Paragon Plus Environment

Page 3 of 13
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Chemical structure of Sudemycins and variants tested in this study. The general feature of each class of modification is
indicated and modifications highlighted in blue. Sudemycin F2 was previously described as compound 19n .
47
RESULTS AND DISCUSSION
gation with Ph P=CHꢀCO Et prepared in situ from correꢀ
3 2
sponding phosphonium salt; reduction of the ester to allylic
alcohol; and oxidation of alcohol to generate aldehyde 10.
Modified JuliaꢀKocienski olefination between the aldehyde 10
We aimed to test: a) the function of the conjugated diene,
which is part of the common pharmacophore of three classes
of splicing inhibitors, b) the function of the oxycarbonyl moieꢀ
ty, another key element of the pharmacophore, and c) the
identity of the ciclohexane ring that links the previous two
moieties. In addition, the most active previously described
47
and sulfone 11 afforded triene 12 with excellent diastereoseꢀ
lectivity (E/Z ratio 96:4). The transformation of compound 12
into 1 required the following steps: chemoselective reduction
of azide functional group of 12 to amine followed by coupling
48
4
7
with (S,Z)ꢀ4ꢀ(tertꢀbutyldimethylsilyloxy)pentꢀ2ꢀenoic acid ;
Sudemycins D6, D1, C1, F1, F2 , and E (Figure 1), were
alcohol deprotection; and formation of ester with isobutyric
anhydride. During the amide formation, isomerisation of Z
double bond in alpha position was produced (Z/E ratio 7:3).
The Z/E mixture was purified by RPꢀHPLC and pure 1 was
obtained.
prepared in parallel, using procedures reported by the Webb’s
47
group and used for biological activity comparison with the
new derivatives.The epoxide group was not modified in our
study because previous work already showed that it contribꢀ
1
9, 42ꢀ44
utes, but is not absolutely required for activity
.
The preparation of 2 with only one double bond between diꢀ
The activity of the compounds was tested in in vitro bioꢀ
chemical assays of spliceosome (complex A) assembly and in
cell culture assays by assessing MCL1 alternative splicing
regulation and cytotoxicity. The structure of the drug variants
and their activities are summarized in Figure 1 and Table 1,
respectively.
46
oxane ring and spiro moiety started from aldehyde 13 and
follows a similar sequence of reactions as described for 1.
However, the generation of olefin 14 by JuliaꢀKocienski was
not diastereoselective. Diasteromeric mixture of E and Z oleꢀ
fins was converted to alcohol 15 in three steps and then both
diastereoisomers were separated using RPꢀHPLC semiꢀ
preparative technique. Stereochemistry of E and Z double was
Conjugated diene modifications
13
Previous studies indicated that the conjugated diene is imꢀ
assigned by C NMR. Sterically compressed carbon nuclei
41
49
portant for the activity of Sudemycins and other drugs . To
test the relevance of this moiety's length, 1, the triene harborꢀ
ing three E conjugate double bonds, and 2, a derivative harborꢀ
ing only one E double bond, were obtained. To evaluate the
importance of the stereochemistry of double bonds for biologꢀ
ical activity, additional compounds 3 and 4, harboring a douꢀ
ble bond in Z configuration, were also prepared.
produce shielding effects , thus, in Z olefin shows 33.7 and
64.6 ppm chemical shift of carbons affected and E olefin 38.3
and 69.1 ppm respectively. Finally, esterification of both alcoꢀ
hols led to corresponding isobutyric esters 2 and 3.
Improved protocol of preparation of diene 16 developed by
Webb produces diasteromeric mixture E,E and E,Z with ratio
9:1. In our hands this mixture as well as TBS protected alcohol
could be separated by column chromatography and a pure
sample of 17 was collected and converted into Sudemycin C1
diastereoisomer, compound 4 (Scheme 1).
The synthesis of triene 1 was performed (Scheme 1) using
4
7
the known aldehyde 9 as starting material. Transformation of
aldehyde 9 into 10 required three synthetic steps: Wittig elonꢀ
3
ACS Paragon Plus Environment

ACS Chemical Biology
Page 4 of 13
When these derivatives were tested for activity, reduction to opposite stereochemistry at the double bond (4, Table 1, Figꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a single double bond (in both E and Z diasteroisomer configuꢀ
rations, 2 and 3, Figure 1) completely suppressed drug's activiꢀ
ty, while the 1 triene displayed highly reduced but still detectꢀ
able activity in splicing assays, but negligible in cytotoxicity
assays (Table 1, Figure 2G and 2H, Figure 3). On the other
hand, the Z,E,Z diastereomer of Sudemycin C1, harboring
ures 1, 2A, 2B and 3) displays lower but still significant activiꢀ
ties (particularly in cytotoxic assays) despite the dramatic
change in spatial orientation of key pharmacophore compoꢀ
nents (Table 1).
Scheme 1. Syntheses of 1, 2, 3 and 4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a) (1ꢀEthoxyꢀ1ꢀoxopropanꢀ2ꢀyl)triphenylphosphonium bromide, tꢀBuOK, CH Cl , 0 °C to rt, 50%; b) DIBALH, CH Cl , ꢀ78 °C, 93%;
2
2
2
2
c) DessꢀMartin periodinane, CH Cl , 0 °C, 70%; d) 11, NaHMDS, THF, −78 °C to rt, 80% for 12 , 42% for 14; e) 1) Ph P, benzene, 55 °C
2
2
3
2
7
) (S,Z)ꢀ4ꢀ(tertꢀbutyldimethylsilyloxy)pentꢀ2ꢀenoic acid, HBTU, NEt , ACN, 0 °C to rt 81% for 15, 75% for 17; f) TBAF, THF, 0 °C to rt,
3
7% for 15, 80% for 4; g) isobutyric anhydride, NEt , 4ꢀDMAP, CH Cl , 0 °C 79% for 2, 76% for 3, 80% for 4; h) RPꢀHPLC semiꢀ
3
2
2
preparative separation. Bt = 2ꢀbenzo[d]thiazole
Cyclohexane substitution by piperazine or 1,3-dioxane.
DIBALH. Confirmation of E stereochemistry was possible
based on lack of signals when performing 1DꢀNOE irradiation
at δ = 6.8 ppm and δ = 1.8 ppm (see the Supporting Inforꢀ
mation). At this point, BOC protecting group was removed
with TFA and free secondary amine was coupled with (S,Z)ꢀ4ꢀ
(tertꢀbutyldimethylsilyloxy)pentꢀ2ꢀenoic acid and then alcohol
was oxidized with DessꢀMartin periodinane, generating 20.
This aldehyde and sulfone 5 were used for JuliaꢀKocienski
olefination, rendering 21 (dr 8:2). Removal of TBS protecting
group and esterification with isobutyric anhydride of 22 proꢀ
duce a mixture of final compounds 5a and 5b that were sepaꢀ
rated by semiꢀpreparative RPꢀHPLC. In order to prepare carꢀ
In previous studies, substitution of the cyclohexane ring by
46
a dioxane improved drug solubility . With the aim of further
increasing aqueous solubility, three novel compounds harborꢀ
ing piperazine rings were synthetized (5a, 5b and 6, Figure 1).
The formation of the Sudemycins derivatives with piperaꢀ
zine core (Scheme 2) started from commercially available tertꢀ
butyl 4ꢀ(2ꢀhydroxyethyl)piperazineꢀ1ꢀcarboxylate (18). Chain
elongation of 18 and transformation into alcohol 19 as unique
E diastereoisomer required: Swern oxidation of 18; Wittig
olefination with Ph P=CHꢀCO Et; and reduction of ester using
3
2
4
ACS Paragon Plus Environment

Page 5 of 13
ACS Chemical Biology
moderate yield was obtained. Acid 30 needed for the preparaꢀ
bamate 6, the diastereomeric mixture of 21 was first purified,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
then TBS group was removed, the alcohol was activated as a
carbonate with nitrophenylcholoroformate, and finally treated
with methylamine to obtain the desired carbamate.
tion of carbamate 8 was obtained in a similar way (Scheme 3)
starting from commercial aldehyde 28. The main advantage of
this synthetic process lies in greater convergence and fewer
synthetic steps than previously described, with a similar yield.
These compounds showed very low activity in splicing or
cell proliferation tests and no detectable activity in vitro asꢀ
says, revealing the requirement of the cyclohexane ring for
Sudemycin’s function (Table 1, Figures 2G, 2H and 3). In
Replacing the oxycarbonyl by an amide group (Sudemycin
K, Figure 1) resulted in a compound with higher activity than
Sudemycin D6, the most potent Sudemycin described so far,
both in biochemical spliceosome assembly assays as well as in
cellular assays for MCL1 alternative splicing and for cytotoxiꢀ
city (Table 1, Figures 2Cꢀ2F and 3). The solubility of Sudeꢀ
mycin K and D6 were found to be comparable (Supplementary
Information). Because the higher activity of Sudemycin K
was observed both in shortꢀterm alternative splicing regulation
of MCL1 and in longꢀterm increased cytotoxicity assays, the
improved activity is likely contributed both by direct effects
on the splicing machinery and by improved cell permeability
or in vivo stability. The replacement of the ester by an amide
group could make Sudemycin K less sensitive to esterases,
4
7
agreement with previous results , substitution of Sudemycin
D6 cyclohexyl group by a dioxane also reduced strongly the
4
7
drug’s activity (Sudemycin F2 , Figure 1 and Table 1, Figure
, Supplementary Figure 1A and 1C).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
Scheme 2. Syntheses of 5a, 5b and 6
50
present for example in plasma and microsomes and believed
to be the main factor responsible for the observed short in vivo
45
halfꢀlife of Sudemycin C1 . Indeed, we observed higher staꢀ
bility of this compound upon incubation in culture medium
containing fetal bovine serum (Figure 4). Replacing the isoꢀ
propyl by a carbamate (8, Figure 1) strongly reduced the comꢀ
pound activity to levels undetectable in in vitro biochemical
assays and very low ꢀbut still detectableꢀ in cytotoxicity and
alternative splicing switching assays (Table 1, Figures 2G, 2H
and 3).
Reagents and conditions: a) (COCl) , DMSO, CH Cl , ꢀ78 °C,
2
2
2
Scheme 3. Synthesis of Sudemycin K and 8
then Et N ꢀ78 °C to rt; b) (1ꢀethoxyꢀ1ꢀoxopropanꢀ2ꢀyl)triphenylꢀ
3
phosphonium bromide, tꢀBuOK, CH Cl , 0 °C to rt 65% (2 steps);
2
2
c) DIBALH, CH Cl , ꢀ78 °C, 78%; d) TFA, CH Cl , 0 °C to rt; e)
2
2
2
2
(
S,Z)ꢀ4ꢀ(tertꢀbutyldimethylsilyloxy)pentꢀ2ꢀenoic acid, HBTU,
NEt , ACN, 0 °C to rt, 90% (2 steps); f) DessꢀMartin periodinane,
3
CH Cl , 0 °C, 78%; g) 11, NaHMDS, THF, −78 °C to rt, 70%; h)
2
2
TBAF, THF, 0 °C to rt, 67%; i) semiꢀpreparative RPꢀHPLC puriꢀ
fication j) isobutyric anhydride, NEt , 4ꢀDMAP, CH Cl , 0 °C,
3
2
2
9
4
8%; k) 4ꢀnitrophenyl chloroformate, NEt , CH Cl , 0 °C to rt,
3 2 2
5%; l) methylamine, ClCH CH Cl, 0 °C to rt, 75%.
2
2
Oxycarbonyl group modifications: Sudemycin K
The oxycarbonyl group is another key element of the comꢀ
46
mon pharmacophore. As reported by Webb and colleagues , a
free alcohol at this position dramatically decreases the activity
compared to ester derivatives. To further explore other chemiꢀ
cal moieties at this position, we prepared compounds with
amide or carbamate groups instead of ester (Sudemycin K or
7
and 8, Figure 1). As amide groups are less susceptible to
hydrolysis than ester groups, these compounds might display
higher stability and efficacy.
Sudemycin K was obtained by reaction of the acid 27 with
the amine previously obtained in the reduction of azide 16
Reagents and conditions a) Isobutyric anhydride, NEt , 4ꢀ
3
DMAP, CH Cl , 0 °C, 99%; b) DIBALH, toluene, ꢀ78 °C, 71%;
2
2
(Scheme 3). The acid 27 was obtained from commercially
c) KHMDS, 18ꢀcrownꢀ6, bis(2,2,2ꢀtrifluoroethyl) (methoxycarꢀ
bonylmethyl)phosphonate, THF, ꢀ78 °C, 82% for 26, 72% for 29 ;
d) Me SnOH, ClCH CH Cl, 85 °C, 50% for 29, 75% for 30; e) 1)
available Lꢀalanine methyl ester hydrochloride by transforꢀ
mation into amide 24 followed by ester reduction to aldehyde
3
2
2
2
5. Olefination of 25 to give Z-26 was achieved using Stillꢀ
16, Ph P, benzene, 55 °C 2) acid 27 or 30, HBTU, NEt , ACN, 0
3
3
Gennari protocol with good diastereoselectivity (dr = 92:8).
The hydrolysis of ester Z-26 using conventional methods
°C to rt, 30% (2 steps) for 7, 50% (2 steps) for 8. The overall yield
of Sudemycin K synthesis was 12%, comparable to previous
45ꢀ47
yields obtained for other Sudemycin variants
.
(
LiOH, NaOH) led to decomposition; however, using milder
reagents like trimethyltin hydroxide, the desired acid with
5
ACS Paragon Plus Environment

ACS Chemical Biology
Page 6 of 13
Structure-function insights
cophore, including Pladienolides, Herboxidienes and
FR901464.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Given that compounds from parallel synthesis of the various
Sudemycin analogs were tested, the higher activity of Sude-
mycin K can be attributed to the amide group and be associatꢀ
ed both with stronger direct effects on the splicing machinery,
e.g. improved affinity for the target, and to higher solubility
and/or stability. As the solubility was found to be comparable
to that of Sudemycin D6, the results of Figure 4 indeed argue
for improved stability, as expected if replacement of the oxꢀ
ycarbonyl by an amide group makes it no longer a substrate of
esterases, believed to be the main factor responsible for the
Webb and colleagues proposed that, despite their structural
variety, natural compounds targeting the SF3B complex share
a common pharmacophore structure . The pharmacophore
was repeated in the synthetic Sudemycins that, in contrast with
natural compounds, are suitable for scalable production, and
display improved stability and solubility
pharmacophore hypothesis is also supported by recent results
arguing that Herboxidiene, Spliceostatin A and Pladienolide B
bind to the same site in the SF3B complex and likely share a
41
4
5, 47
. The common
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
1
common inhibitory mechanism .
45,47
short halfꢀlife of Sudemycin C1 in vivo . Jurica and colꢀ
leagues recently showed that both active compounds and their
inactive analogues compete for binding to the same site, sugꢀ
gesting that the compounds' activities may rely upon a conꢀ
formational change within the SF3B complex induced (or
A conjugated diene is one of the three key features of the
4
1
common pharmacophore . Cyclopropyl modifications in this
moiety were shown to reduce but not suppress the activity of
Meayamycin’s variants, suggesting that the diene needs to be
5
2
in trans configuration . We confirmed that conversion of the
diene to a single double bond suppresses activity. Surprisingly,
we also found that both the compound harboring a triene
moiety and the stereoisomer displaying a Z,E,Z configuration
retained some activity (particularly the latter in cytotoxicity
assays). This result reveals spatial flexibility around the conꢀ
jugated diene moiety, particularly regarding the relative orienꢀ
tation of the oxane ring and its associated epoxi group, as well
as its spatial relationship with the oxycarbonyl moiety (another
key feature of the pharmacophore) at the other end of the
molecule. Interestingly, the Z,E,Z configuration opens the
possibility of versatile modification routes through Dielsꢀ
Alder reactions.
51
prevented) only by the active variants . Therefore the differꢀ
ent activity of Sudemycin variants, including the higher activiꢀ
ty of Sudemycin K, may be also due to more efficient moduꢀ
lation of such conformational changes.
The amide moiety makes Sudemycin K suitable for conjuꢀ
gation with ureas, amides and carbamates, potentially generatꢀ
ing a large variety of chemical derivatives, which once again
might be extrapolable to other families of splicing inhibitors,
like Meayamycin, Spliceostatin and Pladienolides. Future
work will focus on the generation and activity evaluation of
such derivatives.
In summary, in addition to confirming the importance of the
conjugated diene, our studies reveal that changes in the diene
configuration only partially decrease drug activity, while reꢀ
placement of a cyclohexane ring by piperazine abolishes it.
Finally, we obtained a compound with improved activity, at
least partly due to increased stability, Sudemycin K, by reꢀ
placing the oxycarbonyl by an amide group. This variant ofꢀ
fers reactivity possibilities that can potentially expand signifiꢀ
cantly the structural diversity of these drugs.
We also confirmed the need of cyclohexane or dioxane
rings for activity, with cyclohexaneꢀcontaining drugs being
more active. The more planar structure of piperazine disrupts
drug's activity, suggesting that the spatial configuration of this
moiety is essential to display a proper orientation of the oxꢀ
ycarbonyl and conjugated diene groups in the functional
pharmacophore. Substitution of the cyclohexyl group by a
dioxane also reduced strongly the drug’s activity, further supꢀ
porting the importance of this structure.
The introduction of an amide group instead of the ester led
to a compound with improved splicing inhibitory activity and
cytotoxicity. While we only analyzed this variant in the conꢀ
text of Sudemycin molecules, we hypothesize that a similar
modification can have similar enhancing effects on the activity
of the other classes of compounds harboring a similar pharmaꢀ
METHODS. Synthesis methods are summarized in the legꢀ
ends of Schemes 1ꢀ3 and fully detailed, along with the characꢀ
terization of synthetic products by NMR and 2D correlation
spectra, in Supplementary Information. Biochemical and celluꢀ
24, 39
lar assays were described before
Supplementary Information.
, and fully detailed in
6
ACS Paragon Plus Environment

Page 7 of 13
ACS Chemical Biology
1
A
B
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
AA 33 ’’ cc oo mm pp l lee xx f foo r rmm a at it oi on n
150
4
1
A3’ complex
H complex
1
00
50
0
4
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A3’ complex
H complex
100 1000 10000 100000 1×106
nM
1
10
C
E
G
D
AA 33 ’’ cc oo mm pp l lee xx f foo r rmm a at it oi on n
150
Sud D6
A3’ complex
H complex
Sud K
100
Sud D6
Sud K
5
0
0
A3’ complex
H complex
100 1000 10000 100000 1×106
nM
1
10
F
A 3’ complex formation
A3’ complex formation
150
100
A3’ complex
H complex
5
0
0
H
A A3 ’3 c’ oc mo mp lpe l xe xf of or mr ma at i toi on n
1
mM treatments
150
100
A3’
H
5
0
0
Figure 2. In vitro spliceosome (A3’ complex) formation assays. A) Representative Phosphoroimager pictures of electrophoretic separaꢀ
tion of H and A3' complexes assembled upon incubation of a radioactively labeled adenovirus major late promoter RNA (spanning seꢀ
quences corresponding to 3' half of intron 1 and part of the following exon) in HeLa nuclear extracts and fractionation on nonꢀdenaturing
agarose gels. The electrophoretic mobility of A3' and H complexes is indicated, as well as concentrations of the indicated drugs (1 and 4,
conjugate diene variants) or DMSO as control. Only complex H is formed in the absence of ATP. B) Quantification of the percentage of
7
ACS Paragon Plus Environment

ACS Chemical Biology
Page 8 of 13
A3' complex formation for a range of concentrations of the indicated drugs, corresponding to the results reported in A. C) Results equivaꢀ
lent to those in A, for Sudemycins D6 and K. D) Quantification of the results reported in C, as in B. E) Analyses as in A for the indicated
drugs and concentrations. The goal of the experiment was to compare in parallel the different concentrations of various drugs causing 50%
decrease in A3' complex formation. F) Quantifications of results as in E, corresponding to triplicate experiments. Differences between
drugs were not significant (tꢀtest), while they were all significantly different from the control DMSO treatment (pꢀvalue < 0.01). Standard
deviations are indicated. G) Analyses as in E at 1mM drug concentrations (maximal concentrations tested). H) Quantification of results as
in G, corresponding to triplicate experiments. Drug effects were not significantly different from control DMSO treatment (tꢀtest). Standard
deviations are indicated.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A
Sud C1
Sud D1
Sud D6
Sud K
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MCL1
1
2
3
1
3
Sud F1
Sud F2
4
1
Sud E
MCL1
1
2
3
1
3
8
2
3
6
5a
5b
MCL1
1
2
3
1
3
B
M
M
C
C
L 1L 1- e
–
xo
e
n
xo
2
n
in
2
cl ui n
s
c
i ol u
n
sion
M
M
C
C
L
L
1
1
- e
–
x
e
o nx o
2
n
in
2
cl
i
u
n
s
c
i ol u
n
sion
MC xon 2 inclus n
M CL 1L 1- e– exon 2 in ci ol usion
1
50
150
150
Sud E
8
Sud C1
Sud D1
Sud D6
Sud K
Sud F1
2
3
100
100
50
0
Sud F2
100
50
0
4
1
6
5a
5b
5
0
0
100 1000 10000 100000 1×106
nM
100 1000 10000 100000 1×106
nM
100 1000 10000 100000 1×106
nM
1
10
1
10
1
10
C
Viability at 72 h
Viability at 72h
Viability at 72 h
Viability at 72h
Viability at 72 h
Viability at 72h
150
100
50
150
100
50
150
100
50
8
2
3
6
5
5
Sud E
Sud C1
Sud D1
Sud D6
Sud K
Sud F1
Sud F2
4
1
a
b
0
0
0
1
10
100 1000 10000 100000 1×10
nM
6
1
10
100 1000 10000 100000 1×10
nM
6
1
10
100 1000 10000 100000 1×10
nM
6
8
ACS Paragon Plus Environment

Page 9 of 13
ACS Chemical Biology
Figure 3. MCL1 alternative splicing and cytotoxicity assays in HeLa cells. A) Capillary electrophoresis profiles of RTꢀPCR amplificaꢀ
tion of MCL1 alternatively spliced products from RNA isolated 3 h after drug exposure. The positions of the products corresponding to
exon 2 inclusion and skipping are indicated, along with the drug treatment and concentrations (DMSO, control without drug). One repreꢀ
sentative example per condition is shown. B) Quantification of data shown in A for duplicate experiments. Graphs represent % of MCL1
exon 2 inclusion at different drug concentrations, as indicated. Drug treatments were clustered according to the different concentration
ranges at which they induce exon 2 skipping. Standard deviations are shown. Higher activity of Sudemycin K than Sudemycin D6 was
significant (tꢀtest, pꢀvalue < 0.01). C) Cytotoxicity assays. Cell viability was measured using Resazurin assays 72 h after drug exposure.
Graphs indicate fraction of living cells compared to control DMSO treatment. All treatments were performed in triplicate and standard
deviations are shown. Drug treatments were clustered according to the different concentration ranges at which they induce significant
decreases in cell viability. Higher activity of Sudemycin K than Sudemycin D6 was significant (tꢀtest, pꢀvalue < 0.01).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MCL1 - exon 2 skipping regulation
Table 1. Summary of Sudemycin variants’ activities
150
Sud D6
In vitro
A3’
complex
formation
–
MCL1
alternative
splicing
regulation –
IC50 (nM)
Cytotoxicity
in HeLa cells
– IC50 (nM)
Sud K
DRUG
100
5
0
IC50 (nM)
≈250
Sud K (7)
Sud D6
Sud D1
4
≈15
≈250
2,3 ± 0,81
6,3 ± 0,82
109 ±48
≈500
0
≈750
≈630
0
h
24 h
48 h
≈40’000
≈6’300
12’703 ±
16’386
Time of preꢁincubation
in complete medium
1
Sud C1
Sud E
Sud F1
Sud F2
8
≈30’000
≈400
≈10’000
≈12’000
≈50’000
n.d.
≈12’500
≈320
> 30’000
123 ± 154
764 ± 113
646 ± 38
417 ± 0,00
848 ± 200
> 30’000
n.d.
≈2’000
Figure 4. Stability of Sudemycin D6 and Sudemycin K upon
incubation in culture medium with 10% fetal bovine serum.
Complete culture medium containing 1 ꢁM drug or the equivaꢀ
lent volume of DMSO was incubated at 37 °C for the indicated
times and subsequently added to a lawn of HeLa cells. After 3 h
of incubation RNA was isolated and MCL1 alternative splicing
was assessed as a measure of residual drug activity. Exon 2
inclusion levels upon DMSO treatment were used to normalize
values across time points, and the levels of regulation induced at
≈3’500
≈1’200
≈40’000
>100’000
>100’000
>100’000
>100’000
>100’000
2
n.d.
3
n.d.
5a
n.d.
> 30’000
> 30’000
> 30’000
0
h time point by each drug were set at 100%. The reduction of
5
b
n.d.
the effects at each time point is significantly lower for Sudemy-
cin K compared to Sudemycin D6 (pꢀvalue < 0.02 at 24h, p—
value < 0.001 at 48 h by tꢀtest comparison of duplicated treatꢀ
ments).
6
n.d.
Table 1. Summary of activities of the compounds tested in
this study. Biochemical assays to evaluate complex A3' forꢀ
mation, RTꢀPCR assays to evaluate effects on MCL1 alternative
splicing regulation and cytotoxicity assays were carried out and
quantified as described in the Methods section (Supplementary
Information file). Estimates of IC50 values are provided. Sud:
abbreviation for Sudemycin. n.d.: not detected at the maximum
concentration tested (100 ꢁM for cytotoxicity assays, 1 mM for
in vitro spliceosome assembly assay).
ASSOCIATED CONTENT
Supporting Information
Supporting Information 1 (PDF): Material and Methods, Supꢀ
plementary Figure 1.
AUTHOR INFORMATION
9
ACS Paragon Plus Environment

ACS Chemical Biology
Page 10 of 13
isolates reveal a novel viability requirement for PHF5A,
Genes Dev 27, 1032ꢀ1045.
6] Papasaikas, P., and Valcarcel, J. (2016) The Spliceosome: The
Corresponding Authors
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
*
Eꢀmail: mercedesalvarez@ub.edu, juan.valcarcel@crg.eu
[
Ultimate RNA Chaperone and Sculptor, Trends Biochem Sci
1, 33ꢀ45.
Present Addresses
4
†Prof. Fernando Albericio, School of Chemistry and Physics,
University of KwaZuluꢀNatal, Durban , South Africa
[7] Wahl, M. C., Will, C. L., and Luhrmann, R. (2009) The spliceosome:
design principles of a dynamic RNP machine, Cell 136, 701ꢀ
7
18.
†Dr. Kamil Makowski, School of Chemistry, YachayTech
University, Hacienda San Jose SN, San Miguel de Urcuqui
100119 Ecuador
[
8] Yoshida, K., and Ogawa, S. (2014) Splicing factor mutations and
cancer, Wiley Interdiscip Rev RNA 5, 445ꢀ459.
[9] Papaemmanuil, E., Cazzola, M., Boultwood, J., Malcovati, L., Vyas,
P., Bowen, D., Pellagatti, A., Wainscoat, J. S., Hellstromꢀ
Lindberg, E., GambacortiꢀPasserini, C., Godfrey, A. L.,
Rapado, I., Cvejic, A., Rance, R., McGee, C., Ellis, P.,
Mudie, L. J., Stephens, P. J., McLaren, S., Massie, C. E.,
Tarpey, P. S., Varela, I., NikꢀZainal, S., Davies, H. R.,
Shlien, A., Jones, D., Raine, K., Hinton, J., Butler, A. P.,
Teague, J. W., Baxter, E. J., Score, J., Galli, A., Della Porta,
M. G., Travaglino, E., Groves, M., Tauro, S., Munshi, N. C.,
Anderson, K. C., ElꢀNaggar, A., Fischer, A., Mustonen, V.,
Warren, A. J., Cross, N. C., Green, A. R., Futreal, P. A.,
Stratton, M. R., and Campbell, P. J. (2011) Somatic SF3B1
mutation in myelodysplasia with ring sideroblasts, The New
England journal of medicine 365, 1384ꢀ1395.
[10] Yoshida, K., Sanada, M., Shiraishi, Y., Nowak, D., Nagata, Y.,
Yamamoto, R., Sato, Y., SatoꢀOtsubo, A., Kon, A.,
Nagasaki, M., Chalkidis, G., Suzuki, Y., Shiosaka, M.,
Kawahata, R., Yamaguchi, T., Otsu, M., Obara, N., Sakataꢀ
Yanagimoto, M., Ishiyama, K., Mori, H., Nolte, F.,
Hofmann, W. K., Miyawaki, S., Sugano, S., Haferlach, C.,
Koeffler, H. P., Shih, L. Y., Haferlach, T., Chiba, S.,
Nakauchi, H., Miyano, S., and Ogawa, S. (2011) Frequent
pathway mutations of splicing machinery in myelodysplasia,
Nature 478, 64ꢀ69.
11] Quesada, V., Conde, L., Villamor, N., Ordonez, G. R., Jares, P.,
Bassaganyas, L., Ramsay, A. J., Bea, S., Pinyol, M.,
MartinezꢀTrillos, A., LopezꢀGuerra, M., Colomer, D.,
Navarro, A., Baumann, T., Aymerich, M., Rozman, M.,
Delgado, J., Gine, E., Hernandez, J. M., GonzalezꢀDiaz, M.,
Puente, D. A., Velasco, G., Freije, J. M., Tubio, J. M., Royo,
R., Gelpi, J. L., Orozco, M., Pisano, D. G., Zamora, J.,
Vazquez, M., Valencia, A., Himmelbauer, H., Bayes, M.,
Heath, S., Gut, M., Gut, I., Estivill, X., LopezꢀGuillermo, A.,
Puente, X. S., Campo, E., and LopezꢀOtin, C. (2012) Exome
sequencing identifies recurrent mutations of the splicing
factor SF3B1 gene in chronic lymphocytic leukemia, Nat
Genet 44, 47ꢀ52.
[12] Rossi, D., Bruscaggin, A., Spina, V., Rasi, S., Khiabanian, H.,
Messina, M., Fangazio, M., Vaisitti, T., Monti, S., Chiaretti,
S., Guarini, A., Del Giudice, I., Cerri, M., Cresta, S.,
Deambrogi, C., Gargiulo, E., Gattei, V., Forconi, F., Bertoni,
F., Deaglio, S., Rabadan, R., Pasqualucci, L., Foa, R., Dallaꢀ
Favera, R., and Gaidano, G. (2011) Mutations of the SF3B1
splicing factor in chronic lymphocytic leukemia: association
with progression and fludarabineꢀrefractoriness, Blood 118,
6904ꢀ6908.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Author Contributions
∫
These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Work in our laboratories is supported by Fundación Botín,
Banco de Santander through its Santander Universities Global
Division, Consolider RNAREG, AGAUR and the European
Research Council. We acknowledge support of the Spanish
Ministry of Economy and Competitiveness, ‘Centro de Excelenꢀ
cia Severo Ochoa 2013ꢀ2017’. This work was partially funded
by the CICYT (CTQ2015ꢀ67870P) and Generalitat de Catalunya
(2014 SGR 137).
[
ABBREVIATIONS
Sud, Sudemycin; SAR, Structureꢀactivity relationship; 4ꢀ
DMAP, 4ꢀ(Dimethylamino)pyridine; ACN, Acetonitrile;
DIBALH,
Diisobutylaluminium
hydride;
Bt,
2ꢀ
Benzo[d]thiazole; DMSO, Dimethylsulfoxide; NaHMDS, Sodiꢀ
um hexamethyldisilazane, KHMDS, Potassium hexamethyldisiꢀ
lazane; TBAF, Tetrabutylammonium fluoride; THF, Tetrahyꢀ
drofuran
REFERENCES
[
1] Bonnal, S., Vigevani, L., and Valcarcel, J. (2012) The spliceosome
as a target of novel antitumour drugs, Nat Rev Drug Discov
1
1, 847ꢀ859.
[2] Salton, M., and Misteli, T. (2016) Small Molecule Modulators of
PreꢀmRNA Splicing in Cancer Therapy, Trends Mol Med 22,
2
8ꢀ37.
[
[
3] Webb, T. R., Joyner, A. S., and Potter, P. M. (2013) The
development and application of small molecule modulators
of SF3b as therapeutic agents for cancer, Drug discovery
today 18, 43ꢀ49.
4] Hsu, T. Y., Simon, L. M., Neill, N. J., Marcotte, R., Sayad, A.,
Bland, C. S., Echeverria, G. V., Sun, T., Kurley, S. J., Tyagi,
S., Karlin, K. L., DominguezꢀVidana, R., Hartman, J. D.,
Renwick, A., Scorsone, K., Bernardi, R. J., Skinner, S. O.,
Jain, A., Orellana, M., Lagisetti, C., Golding, I., Jung, S. Y.,
Neilson, J. R., Zhang, X. H., Cooper, T. A., Webb, T. R.,
Neel, B. G., Shaw, C. A., and Westbrook, T. F. (2015) The
spliceosome is a therapeutic vulnerability in MYCꢀdriven
cancer, Nature 525, 384ꢀ388.
[13] Wang, L., Lawrence, M. S., Wan, Y., Stojanov, P., Sougnez, C.,
Stevenson, K., Werner, L., Sivachenko, A., DeLuca, D. S.,
Zhang, L., Zhang, W., Vartanov, A. R., Fernandes, S. M.,
Goldstein, N. R., Folco, E. G., Cibulskis, K., Tesar, B.,
Sievers, Q. L., Shefler, E., Gabriel, S., Hacohen, N., Reed,
R., Meyerson, M., Golub, T. R., Lander, E. S., Neuberg, D.,
Brown, J. R., Getz, G., and Wu, C. J. (2011) SF3B1 and
other novel cancer genes in chronic lymphocytic leukemia,
The New England journal of medicine 365, 2497ꢀ2506.
[14] Albert, B. J., McPherson, P. A., O'Brien, K., Czaicki, N. L.,
Destefino, V., Osman, S., Li, M., Day, B. W., Grabowski, P.
J., Moore, M. J., Vogt, A., and Koide, K. (2009)
Meayamycin inhibits preꢀmessenger RNA splicing and
exhibits picomolar activity against multidrugꢀresistant cells,
Mol Cancer Ther 8, 2308ꢀ2318.
[
5] Hubert, C. G., Bradley, R. K., Ding, Y., Toledo, C. M., Herman, J.,
SkuttꢀKakaria, K., Girard, E. J., Davison, J., Berndt, J.,
Corrin, P., Hardcastle, J., Basom, R., Delrow, J. J., Webb, T.,
Pollard, S. M., Lee, J., Olson, J. M., and Paddison, P. J.
[15] Convertini, P., Shen, M., Potter, P. M., Palacios, G., Lagisetti, C.,
de la Grange, P., Horbinski, C., FondufeꢀMittendorf, Y. N.,
Webb, T. R., and Stamm, S. (2014) Sudemycin E influences
(2013) Genomeꢀwide RNAi screens in human brain tumor
10
ACS Paragon Plus Environment

Page 11 of 13
ACS Chemical Biology
alternative splicing and changes chromatin modifications,
Nucleic Acids Res 42, 4947ꢀ4961.
16] Hasegawa, M., Miura, T., Kuzuya, K., Inoue, A., Won Ki, S.,
Horinouchi, S., Yoshida, T., Kunoh, T., Koseki, K., Mino,
K., Sasaki, R., Yoshida, M., and Mizukami, T. (2011)
Identification of SAP155 as the target of GEX1A
lymphocytic leukaemia cells through downregulation of Mclꢀ
1, Leukemia 30, 351ꢀ360.
[30] Salton, M., Kasprzak, W. K., Voss, T., Shapiro, B. A., Poulikakos,
P. I., and Misteli, T. (2015) Inhibition of vemurafenibꢀ
resistant melanoma by interference with preꢀmRNA splicing,
Nature communications 6, 7103.
1
[
[
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(Herboxidiene), an antitumor natural product, ACS Chem
[31] Lee, S. C., Dvinge, H., Kim, E., Cho, H., Micol, J. B., Chung, Y.
R., Durham, B. H., Yoshimi, A., Kim, Y. J., Thomas, M.,
Lobry, C., Chen, C. W., Pastore, A., Taylor, J., Wang, X.,
Krivtsov, A., Armstrong, S. A., Palacino, J., Buonamici, S.,
Smith, P. G., Bradley, R. K., and AbdelꢀWahab, O. (2016)
Modulation of splicing catalysis for therapeutic targeting of
leukemia with mutations in genes encoding spliceosomal
proteins, Nat Med 22, 672ꢀ678.
[32] XargayꢀTorrent, S., LopezꢀGuerra, M., Rosich, L., Montraveta, A.,
Roldan, J., Rodriguez, V., Villamor, N., Aymerich, M.,
Lagisetti, C., Webb, T. R., LopezꢀOtin, C., Campo, E., and
Colomer, D. (2015) The splicing modulator sudemycin
induces a specific antitumor response and cooperates with
ibrutinib in chronic lymphocytic leukemia, Oncotarget 6,
22734ꢀ22749.
Biol 6, 229ꢀ233.
17] Kaida, D., Motoyoshi, H., Tashiro, E., Nojima, T., Hagiwara, M.,
Ishigami, K., Watanabe, H., Kitahara, T., Yoshida, T.,
Nakajima, H., Tani, T., Horinouchi, S., and Yoshida, M.
(
2007) Spliceostatin A targets SF3b and inhibits both
splicing and nuclear retention of preꢀmRNA, Nat Chem Biol
, 576ꢀ583.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
[18] Kotake, Y., Sagane, K., Owa, T., MimoriꢀKiyosue, Y., Shimizu, H.,
Uesugi, M., Ishihama, Y., Iwata, M., and Mizui, Y. (2007)
Splicing factor SF3b as a target of the antitumor natural
product pladienolide, Nat Chem Biol 3, 570ꢀ575.
19] Liu, X., Biswas, S., Tang, G. L., and Cheng, Y. Q. (2013) Isolation
and characterization of spliceostatin B, a new analogue of
FR901464, from Pseudomonas sp. No. 2663, J Antibiot
[
(Tokyo) 66, 555ꢀ558.
[33] Glaser, S. P., Lee, E. F., Trounson, E., Bouillet, P., Wei, A., Fairlie,
W. D., Izon, D. J., Zuber, J., Rappaport, A. R., Herold, M. J.,
Alexander, W. S., Lowe, S. W., Robb, L., and Strasser, A.
(2012) Antiꢀapoptotic Mclꢀ1 is essential for the development
and sustained growth of acute myeloid leukemia, Genes Dev
26, 120ꢀ125.
[
20] Nakajima, H., Sato, B., Fujita, T., Takase, S., Terano, H., and
Okuhara, M. (1996) New antitumor substances, FR901463,
FR901464 and FR901465. I. Taxonomy, fermentation,
isolation, physicoꢀchemical properties and biological
activities, J Antibiot (Tokyo) 49, 1196ꢀ1203.
[
[
21] Sakai, T., Sameshima, T., Matsufuji, M., Kawamura, N., Dobashi,
K., and Mizui, Y. (2004) Pladienolides, new substances from
culture of Streptomyces platensis Merꢀ11107. I. Taxonomy,
fermentation, isolation and screening, J Antibiot (Tokyo) 57,
[34] Tiedemann, R. E., Zhu, Y. X., Schmidt, J., Shi, C. X., Sereduk, C.,
Yin, H., Mousses, S., and Stewart, A. K. (2012)
Identification of molecular vulnerabilities in human multiple
myeloma cells by RNA interference lethality screening of the
druggable genome, Cancer research 72, 757ꢀ768.
1
73ꢀ179.
22] Sakai, Y., Yoshida, T., Ochiai, K., Uosaki, Y., Saitoh, Y., Tanaka,
F., Akiyama, T., Akinaga, S., and Mizukami, T. (2002)
GEX1 compounds, novel antitumor antibiotics related to
herboxidiene, produced by Streptomyces sp. I. Taxonomy,
production, isolation, physicochemical properties and
biological activities, J Antibiot (Tokyo) 55, 855ꢀ862.
[35] Wei, G., Margolin, A. A., Haery, L., Brown, E., Cucolo, L., Julian,
B., Shehata, S., Kung, A. L., Beroukhim, R., and Golub, T.
R. (2012) Chemical genomics identifies smallꢀmolecule
MCL1 repressors and BCLꢀxL as a predictor of MCL1
dependency, Cancer cell 21, 547ꢀ562.
[36] Bae, J., Leo, C. P., Hsu, S. Y., and Hsueh, A. J. (2000) MCLꢀ1S, a
splicing variant of the antiapoptotic BCLꢀ2 family member
MCLꢀ1, encodes a proapoptotic protein possessing only the
BH3 domain, J Biol Chem 275, 25255ꢀ25261.
[37] Laetsch, T. W., Liu, X., Vu, A., Sliozberg, M., Vido, M., Elci, O.
U., Goldsmith, K. C., and Hogarty, M. D. (2014) Multiple
components of the spliceosome regulate Mcl1 activity in
neuroblastoma, Cell Death Dis 5, e1072.
[38] Moore, M. J., Wang, Q., Kennedy, C. J., and Silver, P. A. (2010)
An alternative splicing network links cellꢀcycle control to
apoptosis, Cell 142, 625ꢀ636.
[39] Papasaikas, P., Tejedor, J. R., Vigevani, L., and Valcarcel, J. (2015)
Functional splicing network reveals extensive regulatory
potential of the core spliceosomal machinery, Mol Cell 57, 7ꢀ
22.
[40] Gao, Y., and Koide, K. (2013) Chemical perturbation of Mclꢀ1 preꢀ
mRNA splicing to induce apoptosis in cancer cells, ACS
Chem Biol 8, 895ꢀ900.
[41] Lagisetti, C., Pourpak, A., Jiang, Q., Cui, X., Goronga, T., Morris,
S. W., and Webb, T. R. (2008) Antitumor compounds based
on a natural product consensus pharmacophore, J Med Chem
51, 6220ꢀ6224.
[42] He, H., Ratnayake, A. S., Janso, J. E., He, M., Yang, H. Y.,
Loganzo, F., Shor, B., O'Donnell, C. J., and Koehn, F. E.
(2014) Cytotoxic Spliceostatins from Burkholderia sp. and
Their Semisynthetic Analogues, J Nat Prod 77, 1864ꢀ1870.
[43] Liu, X., Biswas, S., Berg, M. G., Antapli, C. M., Xie, F., Wang, Q.,
Tang, M. C., Tang, G. L., Zhang, L., Dreyfuss, G., and
Cheng, Y. Q. (2013) Genomicsꢀguided discovery of
thailanstatins A, B, and C As preꢀmRNA splicing inhibitors
and antiproliferative agents from Burkholderia thailandensis
MSMB43, J Nat Prod 76, 685ꢀ693.
[44] Villa, R., Kashyap, M. K., Kumar, D., Kipps, T. J., Castro, J. E., La
Clair, J. J., and Burkart, M. D. (2013) Stabilized
cyclopropane analogs of the splicing inhibitor FDꢀ895, J
Med Chem 56, 6576ꢀ6582.
[45] Fan, L., Lagisetti, C., Edwards, C. C., Webb, T. R., and Potter, P.
M. (2011) Sudemycins, novel small molecule analogues of
[23] SekiꢀAsano, M., Okazaki, T., Yamagishi, M., Sakai, N., Takayama,
Y., Hanada, K., Morimoto, S., Takatsuki, A., and Mizoue, K.
(1994) Isolation and characterization of a new 12ꢀmembered
macrolide FDꢀ895, J Antibiot (Tokyo) 47, 1395ꢀ1401.
24] Corrionero, A., Minana, B., and Valcarcel, J. (2011) Reduced
fidelity of branch point recognition and alternative splicing
induced by the antiꢀtumor drug spliceostatin A, Genes Dev
[
[
[
2
5, 445ꢀ459.
25] Folco, E. G., Coil, K. E., and Reed, R. (2011) The antiꢀtumor drug
E7107 reveals an essential role for SF3b in remodeling U2
snRNP to expose the branch pointꢀbinding region, Genes
Dev 25, 440ꢀ444.
26] Alsafadi, S., Houy, A., Battistella, A., Popova, T., Wassef, M.,
Henry, E., Tirode, F., Constantinou, A., PipernoꢀNeumann,
S., RomanꢀRoman, S., Dutertre, M., and Stern, M. H. (2016)
Cancerꢀassociated SF3B1 mutations affect alternative
splicing by promoting alternative branchpoint usage, Nature
communications 7, 10615.
27] Darman, R. B., Seiler, M., Agrawal, A. A., Lim, K. H., Peng, S.,
Aird, D., Bailey, S. L., Bhavsar, E. B., Chan, B., Colla, S.,
Corson, L., Feala, J., Fekkes, P., Ichikawa, K., Keaney, G.
F., Lee, L., Kumar, P., Kunii, K., MacKenzie, C., Matijevic,
M., Mizui, Y., Myint, K., Park, E. S., Puyang, X., Selvaraj,
A., Thomas, M. P., Tsai, J., Wang, J. Y., Warmuth, M.,
Yang, H., Zhu, P., GarciaꢀManero, G., Furman, R. R., Yu,
L., Smith, P. G., and Buonamici, S. (2015) Cancerꢀ
Associated SF3B1 Hotspot Mutations Induce Cryptic 3?
Splice Site Selection through Use of a Different Branch
Point, Cell Rep 13, 1033ꢀ1045.
[
[28] David, C. J., and Manley, J. L. (2010) Alternative preꢀmRNA
splicing regulation in cancer: pathways and programs
unhinged, Genes Dev 24, 2343ꢀ2364.
29] Larrayoz, M., Blakemore, S. J., Dobson, R. C., Blunt, M. D., Roseꢀ
Zerilli, M. J., Walewska, R., Duncombe, A., Oscier, D.,
Koide, K., Forconi, F., Packham, G., Yoshida, M., Cragg, M.
S., Strefford, J. C., and Steele, A. J. (2016) The SF3B1
inhibitor spliceostatin A (SSA) elicits apoptosis in chronic
[
11
ACS Paragon Plus Environment

ACS Chemical Biology
Page 12 of 13
FR901464, induce alternative gene splicing, ACS Chem Biol
, 582ꢀ589.
46] Lagisetti, C., Pourpak, A., Goronga, T., Jiang, Q., Cui, X., Hyle, J.,
Lahti, J. M., Morris, S. W., and Webb, T. R. (2009)
Synthetic mRNA splicing modulator compounds with in vivo
antitumor activity, J Med Chem 52, 6979ꢀ6990.
47] Lagisetti, C., Palacios, G., Goronga, T., Freeman, B., Caufield, W.,
and Webb, T. R. (2013) Optimization of antitumor
[49] Seidl, P. R., Leal, K. Z., Costa, V. E. U., and Mollmann, M. E. S.
6
(1998) Steric effects on carbonꢀ13 NMR shifts: carbon–
hydrogen bond polarization contributions, Magnetic
Resonance in Chemistry 36, 261ꢀ266.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
[
[
[50] Laizure, S. C., Herring, V., Hu, Z., Witbrodt, K., and Parker, R. B.
(2013) The role of human carboxylesterases in drug
metabolism: have we overlooked their importance?,
Pharmacotherapy 33, 210ꢀ222.
[51] Effenberger, K. A., Urabe, V. K., Prichard, B. E., Ghosh, A. K.,
and Jurica, M. S. (2016) Interchangeable SF3B1 inhibitors
interfere with preꢀmRNA splicing at multiple stages, RNA
22, 350ꢀ359.
[52] Osman, S., Albert, B. J., Wang, Y., Li, M., Czaicki, N. L., and
Koide, K. (2011) Structural requirements for the
antiproliferative activity of preꢀmRNA splicing inhibitor
FR901464, Chemistry 17, 895ꢀ904.
modulators of preꢀmRNA splicing, J Med Chem 56, 10033ꢀ
1
0044.
[48] Valverde, S., MartinꢀLomas, M., Herradon, B., and GarciaꢀOchoa,
S. (1987) The reaction of carbohydrateꢀderived
alkoxyaldehydes with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
methoxycarbonylmethylenetriphenylphosphorane:
stereoselective synthesis of βꢀunsaturated esters, Tetrahedron
43, 1895ꢀ1901.
12
ACS Paragon Plus Environment

Page 13 of 13
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Several Sudemycin analogues have been synthetized. Sudemycin K displays improved potency as an
inhibitor of cancer cell proliferation
Sudemycin K
3
59x200mm (143 x 143 DPI)
ACS Paragon Plus Environment