Synthetic mRNA splicing modulator compounds with in vivo antitumor activity

Article abstract of DOI:10.1021/jm901215m

We report our progress on the development of new synthetic anticancer lead compounds that modulate the splicing of mRNA. We also report the synthesis and evaluation of new biologically active ester and carbamate analogues. Further, we describe initial ani

Full text of DOI:10.1021/jm901215m

J. Med. Chem. 2009, 52, 6979–6990 6979
DOI: 10.1021/jm901215m
Synthetic mRNA Splicing Modulator Compounds with in Vivo Antitumor Activity
Chandraiah Lagisetti,†,^ Alan Pourpak,‡,^ Tinopiwa Goronga,† Qin Jiang,‡ Xiaoli Cui,‡ Judith Hyle, Jill M. Lahti,
Stephan W. Morris,‡,§ and Thomas R. Webb*,†
†Department of Chemical Biology and Therapeutics, ‡Department of Pathology and §Department of Oncology, and
Department of Genetics and Tumor Cell Biology, St. Jude Children’s Research Hospital, MS 1000, 262 Danny Thomas Place, Memphis,
Tennessee 38105. ^ C.L. and A.P. are coequal contributors to this study: C.L. performed the majority of the chemistry content of this work,
while A.P. performed the majority of the biology studies described herein.
Received August 14, 2009
We report our progress on the development of new synthetic anticancer lead compounds that modulate
the splicing of mRNA. We also report the synthesis and evaluation of new biologically active ester and
carbamate analogues. Further, we describe initial animal studies demonstrating the antitumor efficacy
of compound 5 in vivo. Additionally, we report the enantioselective and diastereospecific synthesis of a
new 1,3-dioxane series of active analogues. We confirm that compound 5 inhibits the splicing of mRNA
in cell-free nuclear extracts and in a cell-based dual-reporter mRNA splicing assay. In summary, we have
developed totally synthetic novel spliceosome modulators as therapeutic lead compounds for a number
of highly aggressive cancers. Future efforts will be directed toward the more complete optimization of
these compounds as potential human therapeutics.
Introduction
anticancer activity in vivo.5 Recently, a pladienolide B deriva-
tive 2 (E7107)4 entered clinical trials as an anticancer agent (see
Figure 1).4 The natural products 1 and PD both show cyto-
toxicity with IC50 values in the low nanomolar range against
several tumor cell lines and have been reported to cause a
similar distinctive effect on the cell cycle in mammalian cell
lines; cell cycle arrest that can occur at both the G1 and G2/M
phases.5,6 The interaction of these compounds with the spli-
ceosome leads to what has been described as the inhibition of
mRNA splicing, the inhibition of the nuclear retention of pre-
mRNA, and the translation of unspliced mRNA, which
ultimately result in the observed effects on the cell cycle.3,4
The identification of the spliceosome as the target of 1 ana-
logues was made possible through the application of a bioti-
nylated derivative of 1 that was found to interact with the
SAP155, SAP145, and SAP130 subunits of SF3b.3 The bind-
ing of these subunits to streptavidin beads loaded with the
biotinylated 1 derivative was reversible. This result indicates
that no irreversible covalent bond is formed via reaction of the
epoxide group with these proteins, under these conditions,
despite the natural initial expectation to the contrary.3 In the
case of the elucidation of the mechanism of action of PD and
related analogues, a slightly different approach was used: a
doubly labeled affinity probe that cross-linked specifically to
the SAP130 subunit was prepared, and from additional ex-
periments it was concluded that SAP145 was required for the
binding of the probe to SAP130.4
The spliceosome is a critical component of the cellular
machinery in higher organisms and has recently emerged as
an important target for the discovery of selective antitumor
agents.1 The complexity of the spliceosome rivals that of the
ribosome; it is the part of the eukaryotic cell machinery
responsible for the splicing of Pre-mRNA to mRNA and
consists of U1, U2, U4/U6, and U5 small nuclear ribonucleo-
protein particles and over 150 associated proteins.2 An intricate
arrangement of small nuclear RNAs and associated spliceoso-
mal proteins interact with the pre-mRNA during spliceosome
assembly, leading ultimately to intron removal, ligation of
exons, and the release of correctly processed mRNA. Two
natural products have been recently reported to target the
SF3b subunit of the spliceosome.3,4 These naturally occurring
compounds are pladienolide4 (PD,a actually a set of closely
related compounds that have been designated pladienolides
A-G, isolated from Streptomyces platensis)5 and
1
(FR901464)6 isolated from Pseudomonas sp. No. 2663.6-8
The first of these natural products to be discovered (1)
demonstrated significant efficacy in in vivo cancer models but
was chemically unstable and showed a relatively narrow
therapeutic window.6 This prompted development of several
types of partially stabilized compounds by modification of the
hemiketal function of 1.9,10 PD-based compounds (first re-
ported in 2004) show a very promising combination of dra-
matic efficacy and very broad therapeutic window.4 Of seven
naturally occurring PDs, pladienolide B has the most selective
Partially stabilized analogues of 1, such as spliceostatin
(Figure 1), have been used to elucidate the effects of inhibition
of the SF3b subunit on cell function.3 This work led to the
observation that inhibition of SF3b by 1 analogues, or knock-
down of SF3b by siRNA, is associated with the same pheno-
typic changes in cells and results in export from the nucleus
to the cytoplasm and translation of “unspliced” or “incom-
pletely spliced” pre-mRNAs. One of the proteins aberrantly
*To whom correspondence should be addressed. Phone: 901-595-
3928. Fax: 901-595-5715. E-mail: thomas.webb@stjude.org.
a Abbreviations: PD, pladienolide; EDC, 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide); DIAD, diisopropyl azodicarboxylate; DI-
BAL H, diisobutylaluminum hydride; TBAF, tetra-n-butylammonium
fluoride; DMAP, 4-dimethylaminopyridine.
r
2009 American Chemical Society
Published on Web 10/30/2009
pubs.acs.org/jmc

6980 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Lagisetti et al.
Figure 1. Published structures of pladienolide B, 1 (FR901464), 2
(E7107), and spliceostatin.
Figure 3. A 3D representation of an example of an overlay of the
presumed key interaction groups in a low energy conformation of
pladienolide B (shown in yellow; some atoms have been removed for
clarity) and compound 3a, showing that the epoxy group and the
carbonyloxy groups are the same distance in both molecules. This
figure includes a calculated van der Waals interaction surface with the
following color coding: purple = hydrogen-bonding, green = hydro-
phobic, blue = mildly polar.16 This alignment represents the best S
value (S = 167.18) for molecules matching the hypothetical pharma-
cophore and the second best overall of 69 alignments from 500
iterations. The alignment was prepared using the Molecular Operat-
ing System (MOE 2007.09, Chemical Computing Group, Inc.) using
the Flexible Alignment function with both molecules, following a
conformational minimization using MOE default settings.17,18
Figure 2. Overview of some of the representative published data on
SAR for 1 analogues.3,10,12,13 Activity is reported relative to that of 1.
translated as a result of compound treatment (or siRNA
knockdown of SF3b) is p27, resulting in accumulation of a
truncated p27 in treated cells. The truncated p27 retains its
activity as a cell cycle inhibitor but is not targeted for degra-
dation by the proteasome because of deletion of part of its
C-terminus that contains a phosphorylation site at threonine-
187, which normally targets the protein for degradation once
phosphorylated.11 Accumulation of this truncated, function-
ally intact but degradation-deficient p27 in tumor cells con-
tributes to their cell cycle arrest and potentially to the antican-
cer effects of SF3b modulation. It should be emphasized that
these alterations of p27 represent only one of undoubtedly
many (yet-to-be characterized) effects of spliceosome modu-
lation on the expression of oncoproteins, tumor suppressors,
and other proteins that contribute to oncogenesis.
Previous work identified some of the structural require-
ments for activity in 1 and PD analogues, either by modifica-
tion of the natural products,4 or by the total synthesis of some
modified compounds (Figure 2).3,10,12,13 Prior studies have
shown the orientation of the acetyl group and the presence of
the epoxide to be important in conferring activity to 1
analogues10,13 and that the glycosidic hydroxyl group can be
replaced by an alkoxy14 or a methyl group9 to give more stable
compounds, or as in the latter case more stable and more
active compounds9 (Figure 2). Active analogues of PD also
contain functionality similar to that found in 1,4,5 though less
structure-activity information has been reported on these
more recently discovered compounds.
We have recently published the design and enantioselective
synthesis of active concise analogues of 1.15 Our analogue
design was founded on the recognition that 1 and PD have an
apparently identical mechanism of action and present a
similar pharmacophore.15 Molecular overlays of 1 and PD
revealed that despite the gross dissimilarities between the two,
both molecules share certain common key features (known to
be critical to their activity) that can overlap in low energy
conformations in three dimensions (Figure 3).15 Our pub-
lished 3D pharmacophore model prompted our proposal that
compounds of the general structures 3a, 3b, and 3c (see
Figure 4) should share a common pharmacophore with
Figure 4. Synthetic targets that matched a proposed spliceosome
modulation pharmacophore.15
1 and PD and therefore might serve as starting points for
development of both tool and lead compounds with potential
application in human cancer therapy.15 The proposed com-
pound 3a contains the unchanged methylpenta-2,4-diene and
5-oxopent-3-en-2-yl acetate groups along with the modified
deoxyspiroepoxide group, withthe centralpyranring replaced
with a cis-1,4-substituted cyclohexyl group, whereas com-
pounds 3b and 3c include additional polar modifications to
the methylpenta-2,4-diene group that are very synthetically
accessible. These changes (particularly for compound 3a)
were expected to impart similar conformational presentation
of the linker, the epoxy, and the carbonyloxy groups to that
found in 1.
There have been numerous synthetic approaches
to 1;12,14,19-22 however, since these approaches target the
much more complex natural product, they were not highly
instructive for the synthetic targets 3a, 3b, and 3c. Our
approach to these concise analogues provided compounds
3a, 3b, and 3c enantioselectively and diasterospecifically in a
highly step-efficient manner.15 Our recent report included
initial data on the antitumor activity of compounds 3a, 3b,
and 3c against a small panel of human cancer cell lines.15
Compound 3a showed cytotoxic activity against all seven
cancer lines in which it was initially assayed. In particular,
compound 3a exhibited a cytotoxic IC50 of 100 nM against the
JeKo-1 mantle cell lymphoma (MCL) line, which was ∼50-
fold selective for this line compared to the A549 lung cancer
cell line. By contrast, compounds 3b and 3c demonstrated
only modest cytotoxic effects at the highest concentration in

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 6981
Scheme 1. Synthesis of Carbamate and Ester Analogues of Compound 3aa
a Reagents and conditions: (a) 4-nitrophenyl chloroformate, Et3N, dichloromethane; (b) R1-piperazine, 1,2-dichloroethane; (c) isobutyryl chloride,
Et3N, DMAP, CH2Cl2; (d) 4-nitrophenyl chloroformate, pyridine, THF/CH3CN; (e) (1-cycloheptyl)piperazine, 1,2-dichloroethane.
all cell lines tested (IC50 > 5 μM).15 In summary, although
compound 3a is less active than the natural products PD and 1
(that both possess GI50 values in the single-digit nanomolar
range against several tumor lines)5,6 against certain cancer cell
lines tested in common, compound 3a showed considerable
activity (IC50 < 5 μM) against all of the tumor lines examined
and was especially selective and potent in the killing of the two
MCL lines JeKo-1 and JVM-2.15 Additionally, we have
reported that compound 3a has an effect on the cell cycle
that is similar to that of the spliceosome modulator com-
pounds PD and 1,15 which induce an accumulation of cells in
G2/M, with a simultaneous decrease in the fraction of cells in
S phase.3,5,6 In our current studies herein, we describe the
design and synthesis of multiple analogues of compound 3a
together with their biological characterization, including an
initial demonstration of the in vivo antitumor activity of two
of the analogues. Our approach is based on the idea that the
availability of new analogues related to our synthetic scaffolds
will allow for the discovery of drug leads that may circumvent
the liabilities that are present in 1 or PD derivatives.
both of the natural products 1 and PD contain an acetyl
ester group that would be expected to have limited stability
in vivo due to the susceptibility of ester groups to hydrolysis
by ubiquitous plasma and cellular enzymes that exhibit ester
hydrolase activity.23 Indeed, this acetyl has been replaced
with a basic carbamate in the clinical compound 2 (Figure 1)
that has been reported to have better in vivo activity com-
pared to PD.4 The presence of significant esterase metabolic
activity in vivo has been widely studied in the development
of prodrug lead compounds24 and is known to show sig-
nificant species variability.25 Thus, our first experiments
were directed toward experimentally testing the require-
ments for activity of analogues containing modifications of
the O-acetyl and epoxide groups of compound 3a, using
chemistry shown in Scheme 1. We first chose to replace the
acetyl with the more hindered isobutyryl ester (compound 5)
using the alcohol intermediate (compound 4) from our
published synthesis of acetyl derivative 3a, since no informa-
tion regarding such modifications have been published and
because such hindered esters could be more stable in vivo.24
We next prepared carbamate derivatives that contain a basic
amine group, since it is known that in pladienolide analogues
such compounds (e.g., 2, Figure 1) maintain substantial in
vivo activity and indeed have entered clinical trials.4 This
type of modification is expected to impart both improved
solubility and esterase resistance to target compounds.
In addition, good synthetic protocols exist for the prepara-
tion of carbamates of this type.26 The key intermediate in the
preparation of these carbamates is carbonate 6, which was
converted to basic carbamates 7-10 using the published
protocol with minor modifications (Scheme 1).26 Addition-
ally, it is known that the “epoxide-opened” chloro alcohol
1 derivative6-8 and related derivatives also show activity
in tumor growth inhibition like 1. To confirm the impor-
tance of the intact epoxide group for our analogues, we
explored the synthesis of the ring-opened compound (11,
Scheme 1), which is analogous to the known chloro alcohol
analogue of 1.6-8
Results and Discussion
Modification of the ester group of compound 3a yields active
novel compounds. In order to advance novel analogues of 1 as
potential therapeutics for cancer, we have initiated work
directed toward establishing a more robust understanding of
the structure-activity relationships (SAR) of compounds in
this series. We knew that the diene portion of our scaffold
was sensitive to some substitution by heteroatoms (see
compounds 3b and 3c, Figure 4).15 We initiated experiments
designed to test the hypothesis that 1 and PD (and compound
3a) share a common pharmacophore consisting of a carbo-
nyloxy group (2 hydrogen-bond acceptors), a rigid diene
linker (hydrophobic), and a potentially reactive secondary
epoxide (electrophilic hydrogen-bond acceptor) arranged
in a specific three-dimensional orientation constrained
by the possible overlap of 1, 3a, and PD (see Figure 3).15 In
addition to the acid-sensitive electrophilic epoxide group,

6982 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Lagisetti et al.
Table 1. In Vitro Cytotoxicity Screening Data As Determined by XTT for Compounds from Scheme 1, after a 72 h Exposure
IC50 (μM)
cell linea
3a
4
5
7
8
9
10
11
JeKo-1
JVM-2
PC-3
0.10
0.13
2.1
1.6
1.5
10
0.12
0.19
2.2
ND
>0.5
>0.5
>2.5
>1.25
>0.5
>0.5
>2.5
>1.25
>0.5
>0.5
>2.5
>1.25
ND
>1.0
>5.0
4-5
>10.0
>5.0
>1.25
WiDr
2.0
10
0.91
a JeKo-1 and JVM-2, mantle cell lymphoma; PC-3, prostate adenocarcinoma; WiDr, colorectal adenocarcinoma.
Scheme 2. Synthesis of New Ester Analoguesa
We then screened compounds 4, 5, and 7-11 for in vitro
cytotoxicity and obtained the results shown in Table 1.
Acylation of the alcohol formed ester 5, which retained
potency similar to compound 3a. This compound was pre-
pared with the expectation that it might have a longer half-
life in mouse and human plasma, since it would be expected
to be more resistant to esterases present in many biological
fluids (see the section on chemical stability of compounds 3a
and 5 below).24 It is interesting to note that the alcohol 4
shows a dramatic reduction in activity when compared to the
esters 3a and 5 (Table 1); this is very relevant to the in vivo
activity of these compounds, since it is known that unhin-
dered esters have a very limited half-life in mouse plasma in
vivo.24 The more hindered ester 5 demonstrates an in vitro
cytotoxicity profile similar to that of compound 3a in every
tumor line we have examined. Carbamate derivatives
7-10 showed significant reduction in cytotoxic activity by >
5-fold, as in the case of the JVM-2 line (Table 1). The epoxide
ring-opened compound (11) shows an additional reduction
in activity in this case (see Table 1).6 For comparison, the
literature contains only one report of an active carbamate
PD analogue (2), which is 4-fold less active than pladienolide
B.4 It is noted that despite several very high quality total
syntheses,9,13,14,20,22 the complexity of 1 has hindered deve-
lopment of extensive SAR data, and there have been no
reports on the activity of corresponding 1 reference com-
pounds to compare to most of the analogues in Scheme 1.
For example, there are no reports on the synthesis, or
bioactivity, of carbamate analogues of 1 to directly compare
to carbamates 7-10. Our recent results are very useful for the
design of compounds for evaluation in in vivo efficacy
models, since we now know it is relatively easy to prepare
many more new ester derivatives that may have significantly
different pharmacokinetic behavior and yet still retain rea-
sonably potent tumor cytotoxicity in vitro (see Scheme 2
below).
a Reagents and conditions. (a) For compound 12 the corresponding
acid, EDC, and DMAP were used. In all other cases the conditions were
acid chloride/Et3N/DMAP. In vitro cytotoxicity screening data as
determined by XTT assays are shown for the compounds following a
72 h exposure.
line screen.27 This screen identified a number of lines that
appeared to be particularly sensitive to compound 5 (see
Supporting Information Figure S1). Using the NCI-60 cell
line screen as a starting point, we have begun to validate and
determine the IC50 of compound 5 for various adult cancer
types in addition to the cancer cell lines used in Table 1 (see
Table 2, upper panel).
In addition to adult cancers, we were also interested in the
response of the pediatric malignancies, which by and large
are cancers completely different from those that occur in
adult patients, to this class of agents as well. Toward this
goal, we selected a representative panel of cell lines derived
from the more frequent childhood cancers for examination,
with a special focus directed to the inclusion of those tumor
types for which the prognosis remains poor, even following
current best available clinical management (examples in-
clude high-risk neuroblastoma, MLL fusion-positive acute
leukemia, and rhabdoid tumor).28-31 As shown in Table 2,
middle panel, the pediatric cancer cell lines were, in general,
sensitive to compound 5. Importantly, lines representing
several poor-prognosis malignancies such as MLL-AF4-
positive acute myeloid leukemia (MV-4-11) and high-risk
neuroblastoma (NB-1643) were very sensitive to the cyto-
toxic activity of compound 5, with IC50 values of ∼300 and
230 nM, respectively. Certain other pediatric tumor types
that are also very aggressive but for which the prognosis is
less dismal, if the patients undergo aggressive therapies that
are frequently associated with both short- and long-term
morbidity as well as occasional mortality, were quite sensi-
tive. For instance, one example of such a tumor, Burkitt’s
lymphoma, demonstrated marked sensitivity to compound 5
(cell line Ramos; IC50 ≈ 80 nM). Although obviously quite
preliminary, these data are nonetheless promising and sug-
gest a potential role for spliceosome modulator therapy not
Increased Chemical Stability of Compounds 3a and 5
Compared to 1 or Meayamycin. One of the goals of our initial
animal studies is to modify the various pharmacodynamic
properties of our analogues so that we may determine how
this affects the efficacy and toxicity of spliceosome modula-
tors in antitumor efficacy studies. We have noted that the
biologically active compounds 3a and 5 are chemically stable
in physiologic buffer (phosphate buffered saline, pH 7.4;
t
1/2=1200 h at 22 ꢀC) and DMSO (no detectable decomposi-
tion after 2 weeks at 22 ꢀC) but are rapidly degraded in dilute
aqueous acid. This is in marked contrast to the published
stability of 1 (t1/2=4.0 h at 37 ꢀC at pH 7.4)9 and analogues
reported in the literature (the most stable of which has a
t1/2 = 48 h at 37 ꢀC at pH 7.4).9
Tumor Cell Line Screening Results. Compound 5 exhibits
selective antitumor activity against certain adult and pedia-
tric cancer types. Given our initial SAR data, we sought to
explore the sensitivity of tumor lines in the NCI-60 tumor cell

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 6983
Table 2. Compound 5 Cytotoxicity Determinations against Various
Adult and Pediatric Tumor Cell Lines and Cells from Normal Tissuesa
compound is expected to convey additional plasma stability,
as was seen with isobutyryl ester 5.
cell line
cell type
IC50 (μM)
Initial Determination of a Tolerated Dose for Compound 5.
As a prelude to the in vivo efficacy and toxicity studies that
we plan for our spliceosome modulators, we performed
preliminary maximal tolerated dose (MTD) studies using
compound 5 at doses ranging from 0 to 50 mg/kg (physi-
cochemical properties that limited the solubility of the com-
pound using the formulation described below precluded the
administration of higher doses). For this study, NOD/SCID
mice (three per group) were dosed with vehicle, 5, 10, 25, or
50 mg/kg of compound 5 daily for 5 days by both iv and ip
routes. Compound 5 was dissolved in 3.0% DMSO, 6.5%
Tween-80 in 5% glucose solution, which is a modification of
the formulation used by Mizui et al. for the spliceosome
modulator pladienolide B.5 To determine compound-related
toxicities, mice were monitored daily for weight loss, morbid-
ity, and mortality. Following administration of the last dose of
compound 5 on day 5, complete blood counts (CBCs) were
determined along with a full diagnostic chemistry panel mea-
suring albumin, alkaline phosphatase, alanine aminotransfer-
ase (ALT), amylase, blood urea nitrogen (BUN), calcium,
creatinine, globulin, glucose, phosphorus, potassium, sodium,
total bilirubin, and total protein. No significant differences in
blood cell counts were observed in treated versus vehicle-only
mice, indicating that compound 5 is not associated with the
induction of anemia, leukopenia, or thrombocytopenia when
administered under these conditions; likewise, no abnormal-
ities of the blood chemistries were observed in the animals. The
mice were monitored for 1 week following compound adminis-
tration. No significant weight loss was observed in any group
during or after compound administration. There was one
fatality in the ip 25 mg/kg and one in the iv 50 mg/kg groups.
It is unknown whether these deaths were due to compound-
related toxicities or technical complications due to injections
and/or bleeds for the CBCs and diagnostic panels; however, no
fatalities were noted in subsequent efficacy studies employing
these same compound 5 doses (see below), suggesting that
technical complications were the likely cause. Total necropsies
to assess organ histologies of animals from the various treat-
ment and vehicle-only groups in these MTD studies showed no
evidence for significant toxicities in the mice.
Initial in Vivo Efficacy Studies. Following the MTD
studies described above, we sought to determine whether
the doses of compound 5 that we could readily administer
were capable of inhibiting tumor growth in mice (Figure 5).
For these studies, we transplanted JeKo-1 mantle cell lym-
phoma tumors (which we had found to be sensitive to
spliceosome modulator compounds, as shown in Table 1)
to NOD/SCID mice on day 1. Beginning on day 4, the mice
received iv injections of vehicle, 5, 10, 25, or 50 mg/kg of
compound 5 daily for 5 consecutive days. No fatalities or
significant weight loss were observed in any of the groups
(data not shown). Administration of 10, 25, or 50 mg/kg of
compound 5 each led to statistically significant decreases in
tumor volumes compared to vehicle-treated mice (Figure 5).
A widely used criterion for determining antitumor activity is
tumor growth inhibition (T/C).32 This value is calculated by
dividing the median tumor volumes of treated mice by the
median volumes in control mice. According to NCI stan-
dards, a T/C e 42% is the minimum requirement for
activity.32 By performing this calculation, we determined
that the 50 mg/kg compound 5 dose achieved a T/C value of
35%. Although preliminary, these data indicate that even a
Adult Cancer Type
SK-MEL-2
SK-MEL-5
SK-MEL-28
MIA PaCa-2
PANC-1
melanoma
melanoma
0.29 ( 0.04
0.64 ( 0.07
0.55 ( 0.15
1.20 ( 0.12
3.87 ( 0.70
melanoma
pancreatic carcinoma
pancreatic carcinoma
Pediatric Cancer Type
acute lymphoblastic leukemia
Burkitt’s lymphoma
neuroblastoma
MOLT-4
Ramos
0.08 ( 0.004
0.08 ( 0.008
0.23 ( 0.04
0.30 ( 0.01
0.55 ( 0.06
0.66 ( 0.05
0.67 ( 0.14
0.69 ( 0.12
0.70 ( 0.07
0.90 ( 0.09
0.92 ( 0.14
1.34 ( 0.23
2.79 ( 0.12
4.41 ( 0.26
NB-1643
MV-4-11
Rh41
acute myeloid leukemia
rhabdomyosarcoma
neuroblastoma
NB-EBc1
SUP-B15
Rh30
B-cell precursor leukemia
rhabdomyosarcoma
Ewing sarcoma
SK-NEP-1
SJ-GBM2
Karpas-299
BT-14
glioblastoma
anaplastic large-cell lymphoma
medulloblastoma
rhabdoid tumor
BT-12
BT-3
medulloblastoma
Normal Tissue
normal prostate epithelial cell
human foreskin fibroblast
human foreskin fibroblast
(immortalized)
PrEC
HFF
HFF/tert2
7.01 ( 1.1
>10
9.69 ( 0.09
a The cells were treated with compound 5 for 72 h, and cytotoxicity
was determined by XTT assay. Data represent the mean ( SEM
(standard error of the mean) from three independent experiments.
only in the management of certain adult cancers but for
selected pediatric malignancies as well.
To gain preliminary insight into the toxicity of these com-
pounds in normal tissues, we also investigated the cytotoxicity
of compound 5 in normal prostate cells and human foreskin
fibroblasts (Table 2, lower panel). We found that in the PrEC
(normal prostate epithelial cell) line compound 5 had an IC50 of
7.0 ( 1.1 μM and IC50 values of >10 μM and ∼10 μM in the
normal human foreskin fibroblasts (HFF) and HFF/tert2
(HFF immortalized by expression of telomerase), respectively.
These results indicate a significant selectivity of compound 5for
many tumor versus normal cell lines, even under cell culture
conditions (e.g., compound 5 shows an IC50 of 0.08 μM for
MOLT-4 acute T-cell leukemia and Ramos Burkitt’s lympho-
ma cell lines under identical conditions, indicating a selectivity
index of 125 or greater compared to the HFF cells).
Synthesis and in Vitro Cytotoxicity Characterization of
Additional Ester Analogues of Compound 3a. To follow up
on the promising activity of ester analogue 5, we next
prepared a set of diverse ester analogues by acylation of
alcohol 4 (Scheme 2). These ester derivatives were expected
to give analogues with improved plasma stability or solubi-
lity or both. As seen in Scheme 2, though highly polar
modifications were not well tolerated, weakly basic groups
could be abided with only a modest decrease in cytotoxic
activity. Most interesting, additional branching of the hy-
drophobic group gave compound 19, which showed IC50
values of ∼40 and 800 nM for the test cell lines JeKo-1 and
PC-3, respectively (Scheme 2, lower right panel). Although
we have yet to test the plasma half-life of compound 19, the
additional hindrance imparted by the tert-butyl group in this

6984 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Lagisetti et al.
Scheme 3. Synthesis of 1,3-Dioxane Analoguesa
Figure 5. Compound 5 inhibition of tumor growth in JeKo-1
tumor-bearing mice. JeKo-1 mantle cell lymphoma tumors were
transplanted to NOD/SCID mice on day 1, and beginning on day 4,
the mice (4 per group) received iv injections of vehicle, 5, 10, 25, or
50 mg/kg of compound 5 daily for 5 consecutive days. Vehicle alone
did not inhibit tumor growth compared to saline-treated mice (data
not shown). Arrows indicate the dosing schedule. The data are
represented as the mean ( SEM. Tumor volumes were modeled
with treatment, linear and quadratic trends over time, and interac-
tion of treatment with linear and quadratic trends over time in a
repeated measure framework using autoregressive 1 AR(1) varian-
ce-covariance structure and empirical sandwich error estimates for
fixed effect. Significance is reported as the comparison of tumor
volumes in vehicle-treated mice to compound-treated animals. All
analyses were performed using SAS software (SAS Institute, Cary,
NC), Windows, version 9.1: (/) p = 0.01; (//) p < 0.0001.
a Reagents, conditions, and yields: (a) 1H-imidazole-1-sulfonylazi-
de HCl, CuSO4 5H2O, K2CO3, MeOH; (b) CF3COOH/H2O/CHCl3;
3
3
(c) ylide (shown), benzene; (d) (1) DIBAL-H, CH2Cl2, 64% overall; (2)
2-mercaptobenzothiazole, Ph3P, DIAD, THF, 0 ꢀC; (3) (NH4)6Mo7O24
tetrahydrate/H2O2, 78% overall; (e) LiHMDS, then aldehyde 24, 37%;
(f) (1) Ph3P, H2O, benzene, 45 ꢀC; (2) (S,Z)-4-(TBSO)pent-2-enoic acid,
i
HBTU, Pr2EtN, 69% overall; (3) TBAF, THF, 47%; (4) isobutyric
anhydride, Et3N, DMAP, 96%; (g) (1) Ph3P, H2O, benzene, 45 ꢀC; (2)
acetic anhydride/pyridine.
converted into the corresponding azide 20,34 which was then
converted to the aldehyde 21 by selective hydrolysis of the
dimethyl acetal functionality via TFA/H2O (19.5:0.5) in
CHCl3. This aldehyde was further elaborated with the Wittig
ylide (Scheme 3, step c) to give ester 22, which was reduced by
DIBAL-H to provide the alcohol derivative that was con-
verted to the corresponding Julia-Kocienski olefination
reagent35 via a sequential Mitsunobu reaction with the
2-mercaptobenzothiazole anion followed by oxidation of the
resulting sulfide with ammonium molybdate and hydrogen
peroxide to give the sulfone derivative 23 in moderate yields.
The subsequent synthetic steps then utilize and improve
upon the chemistry we have published. The azide sulfone 23
intermediate was then treated with an aldehyde 24 (prepared
as published15) in the presence of LiHMDS (1.1 equiv) to
give the key azide diene intermediate 25. The azide group in
this diene was then reduced to the corresponding amine using
Ph3P and H2O, and this step was followed by amidation with
the protected hydroxyl-cis-pentenoic acid to give the corre-
sponding amide derivative. Cleavage of the TBS group in
diene was accomplished with tetrabutylammonium fluoride
at 0 ꢀC, and the resulting alcohol was then treated with
isobutyric anhydride to afford the final analogue 26. The
overall yield of compound 26 using this approach was similar
to that obtained for the synthesis of 5 (see Scheme 1). This
compound showed in vitro activity similar to that of com-
pound 5 (e.g., JeKo-1 IC50 = 150 nM); furthermore, an in
vivo treatment trial using JeKo-1 tumor-bearing mice re-
vealed compound 26 to possess robust anticancer activity
that was comparable to compound 5 (see Supporting In-
formation Figure S2). As we had hoped, the solubility of this
1,3-dioxane derivative was significantly improved (measured
solubility = 47 μM in PBS, which is >50-fold more soluble
than the cyclohexane derivative 5).
first-generation, minimally optimized compound from this
series of spliceosome modulators could be administered at the
doses necessary to inhibit tumor growth without observable
toxicity. As shown below, we have already designed and pre-
pared additional compounds with the goal of further increasing
in vivo potency, solubility, and selectivity for the spliceosome, in
order to begin to optimize their antitumor potential.
Synthesis of an Active Heteroatom Modified Scaffold
Related to Compound 3a, with Improved Solubility. The
activity of compounds 5 and 19 was promising, but these
compounds showed limited solubility in water or aqueous
buffer (e.g., the measured solubility of 5 was less than 1 μM in
PBS). Since improved solubility is desirable for both the
formulation and in vivo distribution of drugs, we considered
alternative approaches to the introduction of more polar
groups to our lead structure. Since our attempts to obtain
more soluble and potent (IC50 < 100 nM) analogues through
the carbamate (Table 1) or polar ester (Scheme 2) approaches
were not completely successful in the identification of com-
pounds with both improved solubility and stability, we turned
our attention to the introduction of more polar functionality
into the scaffold of our lead compounds, without the addition
of asymmetric centers. We envisioned (Scheme 3) that com-
pounds such as the cyclic acetal 26, in which the cyclohexane
ring is replaced witha 1,3-dioxane ring, would be substantially
more soluble. Though the acetal is acid-sensitive, we surmised
that this would not be an issue, since intravenous infusion will
be the most likely method of dosing in both animal model
experiments and the clinical setting.
The azidosulfone intermediate 23 was prepared from com-
mercially available 1,1,2,2-tetramethoxypropane and 2-ami-
no-1,3-propanediol via a selective transketalization reaction
to give a known aminoacetal,33 which is obtained exclusively
as the cis-product, as shown in Scheme 3. This amine was
Experimental Demonstration of Splicing Modulation by
Compounds 5 and 26. As an initial step in determining whether
thesenewcompounds affect splicing, wetreated HeLa cervical
carcinoma cells with 10 μM compound 26, the most soluble
compound, for 4 h and examined the effects of this drug on the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 6985
(Figure 6).3,4 To confirm that our new compounds were
functioning as spliceosome modulators in a manner similar
to PD and 1, we tested them in a dual-reporter assay that
directly measures the effects on splicing in cells (Figure 7).36,37
Lastly, as an additional means to conclusively demonstrate
that our compounds alter splicing, we also performed an in
vitro cell-free splicing assay thatdemonstrated compound5 to
inhibit the mRNA splicing activity of a HeLa nuclear extract
(see Supporting Information Figure S3).38,39
Conclusions
We have reported our progress on the development of new
synthetic anticancer lead compounds that are concise analo-
gues of the natural product1. Thesenovel compoundscontain
only three chiral centers, which is six less than the natural
product, and were designed on the basis of our recognition of
a common spliceosome modulatory pharmacophore in both 1
and PD. Our design process integrates pharmacophore in-
formation with the simultaneous execution of synthetic step
efficiency and the application of enantioselective and diaster-
eospecific synthetic approaches. This type of design approach
allows for the “molecular concision” of complex natural
products into more approachable synthetic targets. This in
turn makes possible a more robust synthetic examination of
numerous features of the spliceosome pharmacophore, which
have not previously been investigated.15 We present data that
demonstrate these natural product analogues to be substan-
tially more chemically stable than either 1 or the previously
reported stabilized active analogue meayamycin.9 We have
developed active ester and carbamate analogues of our initial
compound 3a, including the equipotent hindered ester 5, that
have new diverse steric and physical properties with a range of
cytotoxic activity. We have explored the scope of selective
antitumor cytotoxic activity of 3a and 5 and discovered a
number of sensitive adult and pediatric tumor lines, which
include leukemia, lymphoma, melanoma, and pancreatic
carcinoma lines, among others. These findings led to some
initial antitumor efficacy studies with compound 5 that clearly
demonstrate significant in vivo inhibition of JeKo-1 tumor
growth in immunocompromised mice following only five
single daily iv doses. Additionally, we have reported the
enantioselective and diastereospecific synthesis of a new
template for active analogues (represented by 26) with sig-
nificant improvements in measured solubility, which drama-
tically facilitates the formulation of these compounds for
intravenous dosing. In vivo efficacy in a JeKo-1 tumor
regression study has also been observed with compound 26
following a minimal 5-day dosing schedule. We also report for
the first time that compound 5 inhibits the splicing of mRNA
in cell-free nuclear extracts. Data are also presented showing
that these compounds selectively inhibit mRNA splicing in a
dual-reporter cell-based splicing assay, and there appears to
be a close correlation between cytotoxicity and the inhibition
of splicing by the compounds tested. In summary, we have
developed a totally synthetic novel spliceosome modulator as
a lead compound as one step in the development of new
treatments for a number of highly aggressive cancers. Future
efforts will be directed toward the more complete optimiza-
tion of these compounds as potential human therapeutics.
Figure 6. Compound 26 causes aggregation of the splicing factor
SC35 and the SF3b protein SAP155. HeLa cells were incubated for 4 h
in culture media containing 10 μM compound 26 in DMSO (B and D)
or an equal volume of DMSO (A and C) and stained with antibodies
recognizing SC35 (A and B) or SAP155 (C and D). The cells were then
stained with fluorescent secondary antibodies and examined with a
fluorescence microscope using an objective with 60ꢀ magnification.
Figure 7. Compounds 5 and 26 selectively inhibit splicing. 293T cells
were transiently transfected with a dual-reporter splicing construct
(see Experimental Section for details) for 48 h and subsequently
treated with or without compound for 5 h. The degree of cell death
was negligible at this time at the above tested concentrations. The
cells were lysed, and equalized amounts of total cellular proteins were
assayed for luciferase and β-galactosidase activity using the Dual-
Light system. The data are expressed as the relative ratio of luciferase
to β-galactosidase activity compared to a control vehicle (DMSO)
treated sample and are presented as the mean ( SEM from multiple
independent experiments. Note that compound 27 (which lacks
cytotoxic activity; data not shown) had no effect in this assay and
that compound 4 (which exhibits only minimal cytotoxic effects at
these concentrations; see Table 1) altered splicing only very slightly at
the highest concentration tested; thus, the cytotoxic effects of our
compounds correlate closely with their effects on splicing.
localization of splicing factors in vivo using immunofluores-
cence analysis and antibodies recognizing the general splicing
factor SC35 and SF3b3 (SAP155). These studies revealed that
compound 26 caused the splicing factors to form large
aggregates, which were similar to those reported in the
literature to be observed in cells treated with either PD or 1
Experimental Section
Chemistry. General Aspects. All materials and reagents were
used as is unless otherwise indicated. Air- or moisture-sensitive

6986 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Lagisetti et al.
1
reactions were carried out under a nitrogen atmosphere. THF,
toluene, acetonitrile, N,N-dimethylformamide, and CH2Cl2
were distilled before use. Flash column chromatography was
performed according to Still’s procedure using 100-700 times
excess 32-64 mm grade silica gel. TLC analysis was performed
using glass TLC plates (0.25 mm, 60 F-254 silica gel). Visualiza-
tion of the developed plates was accomplished by staining with
ethanolic phosphomolybdic acid, ceric ammonium molybdate,
or ethanolic ninhydrin followed by heating on a hot plate
(120 ꢀC). All tested compounds possessed a purity of >95%
as determined by ultrahigh pressure liquid chromatography on a
Waters Acquity UPLC/PDA/ELSD/MS system carried out
with a BEH C18 2.1 mm ꢀ 50 mm column using gradient elution
with stationary phase: BEH C18, 1.7 mm; solvent A, 0.1%
formic acid in water; solvent B, 0.1% formic acid in acetonitrile.
NMR spectra were obtained on Bruker Avance II 400 MHz.
The values δH 7.26 and δC 77.0 ppm were used as references for
NMR spectroscopy in CDCl3. The coupling constants deduced
36 mg (57%) of 6 as a liquid. H NMR (400 MHz, CDCl3)
δ 8.38-8.19 (m, 2H), 7.49-7.33 (m, 2H), 6.32-6.22 (m, 2H),
6.11 (d, J = 7.5 Hz, 1H), 5.97 (dd, J = 11.6, 8.1 Hz, 1H), 5.85
(dd, J = 11.6, 1.0 Hz, 1H), 5.54 (dd, J = 15.7, 6.7 Hz, 1H), 5.45
(t, J = 7.5 Hz, 1H), 4.46 (ddd, J = 11.0, 6.7, 1.9 Hz, 1H), 4.07 (s,
1H), 2.58 (s, 2H), 2.04 (t, J = 6.4 Hz, 2H), 1.98-1.88 (m, 2H),
1.67 (s, 3H), 1.63-1.57 (m, 6H), 1.53 (d, J = 6.5 Hz, 3H), 1.40 (s,
3H), 1.28 (s, 3H), 1.25-1.13 (m, 5H); 13C NMR (101 MHz,
CDCl3) δ 164.12, 155.51, 151.90, 145.36, 140.14, 136.10, 133.94,
131.63, 127.19, 125.27, 124.76, 121.75, 74.38, 73.08, 69.59, 55.69,
51.06, 45.58, 42.46, 38.63, 36.38, 34.14, 31.54, 29.39, 29.32,
27.87, 27.71, 23.79, 20.05, 12.50; IR (neat film) 3316, 2974,
2930, 2856, 1766, 1668, 1631, 1526, 1347, 1256, 1215, 1048 cm-1
;
HRMS (ESI) m/z calcd for C32H43N2O8 (M þ 1)þ 583.3019,
found 583.3028.
(S,Z)-5-((1R,4R)-4-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-di-
oxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)cyclohexyl-
amino)-5-oxopent-3-en-2-yl 4-Cycloheptylpiperazine-1-carboxy-
late (7). A solution of the activated carbonate (6) (4.2 mg,
7.21 μmol) in 1,2-dichloroethane (0.2 mL) was allowed to
stir at room temperature as 1-cycloheptylpiperazine (0.21 mL,
0.02 mmol, 0.1 M stock solution) was added. The resulting
solution was stirred for 75 min, and the solvent was then
removed under vacuum. The crude was purified by flash chro-
matography (2-3% MeOH in CH2Cl2) to give 1.2 mg (27%) of
7 as a viscous oil. 1H NMR (400 MHz, CDCl3) δ 8.22-8.08 (m,
1H), 6.27 (d, J = 15.7 Hz, 1H), 5.82 (d, J = 11.2 Hz, 1H),
5.64-5.47 (m, 4H), 4.55-4.40 (m, 1H), 4.20-4.07 (m, 1H), 3.46
(s, 3H), 2.57 (s, 2H), 2.49 (s, 3H), 2.08 (s, 2H), 2.10-2.03 (m,
2H), 2.02-1.87 (m, 2H), 1.83-1.76 (m, 2H), 1.77 (s, 3H),
1.65-1.50 (m, 14H), 1.47-1.43 (m, 3H), 1.40 (s, 3H), 1.34 (d,
J = 6.0 Hz, 3H), 1.28 (s, 3H), 1.25-1.14 (m, 5H); IR (neat film)
1
in H NMR data cases were obtained by first-order coupling
analysis. Analytical and preparative SFC (supercritical fluid
chromatography) systems (AD-H and OD-H columns) were
used for analysis and purification. IR spectra were collected
using a Nicolet IR 100 (FTIR). FTIR analyses were prepared as
neat and neat films on KBr plates, and the data are reported in
wavenumbers (cm-1) unless specified otherwise. The University
of Illinois Mass Spectroscopy Laboratories collected the high
resolution mass spectral data.
(S,Z)-5-((1R,4R)-4-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-di-
oxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)cyclohexyl-
amino)-5-oxopent-3-en-2-yl Isobutyrate (5). A solution of the
alcohol derivative 4 (215 mg, 0.52 mmol) in CH2Cl2 (5 mL) was
allowed to stir at 0 ꢀC as Et3N (0.36 mL, 2.63 mmol) and DMAP
(12 mg, 0.10 mmol) were sequentially added. After 5 min,
isobutyryl chloride (0.16 mL, 1.58 mmol) was added dropwise.
The resulting solution was allowed to stir for 30 min at this temp.
The reaction mixture was then diluted with water (10 mL) and
CH2Cl2 (30 mL). The organic layer was separated, and the
aqueous layer was again extracted with CH2Cl2 (2 ꢀ 15 mL).
The combined organic layers were washed with saturated aqu-
eous NaHCO3 (20 mL), brine (20 mL) and then dried over
Na2SO4. The solvent was evaporated and the crude residue was
purified by flash chromatography (30% ethyl acetate in hexane)
to give 215 mg (84%) of 5 as a white viscous solid. 1H NMR (400
MHz, CDCl3) δ 7.25 (d, J = 5.2 Hz, 1H), 6.27 (d, J = 15.7 Hz,
1H), 5.78 (d, 11.8 Hz, 1H), 5.78-5.72 (m, 1H), 5.63 (dd, J =
11.8, 9.1 Hz, 1H), 5.55 (dd, J = 15.7, 6.7 Hz, 1H), 5.49 (t, J = 7.5
Hz, 1H), 4.50-4.42 (m, 1H), 4.16-4.09 (m, 1H), 2.57 (s, 2H),
2.58-2.49 (m, 1H), 2.12-2.04 (d, J = 14.2 Hz, 2H), 2.03-1.86
(m, 2H), 1.72 (s, 3H). 1.72-1.66 (m, 1H), 1.64-1.54 (m, 6H),
1.40 (s, 3H), 1.36 (d, J = 6.4 Hz, 3H), 1.28 (s, 3H), 1.25-1.19 (m,
3H) 1.17 (d, J = 7.0 Hz, 6H), 1.14 (d, J = 2.0 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 177.36, 164.87, 137.59, 136.22, 133.77,
132.06, 126.97, 125.62, 73.04, 69.59, 68.97, 55.69, 51.05, 45.31,
42.46, 38.64, 36.60, 34.44, 34.00, 31.55, 29.58, 29.47, 27.84,
27.54, 23.77, 20.30, 18.98, 18.86, 12.49; IR (neat film) 3314,
2973, 2931, 2856, 1733, 1688, 1628, 1533, 1450, 1368, 1241, 1195,
1047 cm-1; HRMS (ESI) m/z calcd for C29H46NO5 (M þ 1)þ
488.3376, found 488.3364
3300, 2926, 2855, 1668, 1535, 1434, 1372, 1244, 1121, 1049 cm-1
;
HRMS (ESI) m/z calcd for C37H60N3O5 (M þ 1)þ 626.4533,
found 626.4546.
(S,Z)-5-((1R,4R)-4-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-di-
oxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)cyclohexyl-
amino)-5-oxopent-3-en-2-yl 1-Methylpiperidine-4-carboxylate
(12). A solution of alcohol derivative 415 (10 mg, 0.024 mmol)
in CH2Cl2 (0.22 mL) was treated with 1-methylpiperidine-4-
carboxylic acid (9.3 mg, 0.065 mmol), diisopropylethylamine
(13 μL, 0.072 mmol), and DMAP (7.3 mg, 0.06 mmol). The
resulting solution was cooled to 0 ꢀC and treated with solid EDC
(11.4 mg, 0.06 mmol). The resulting suspension was allowed to
stir at room temperature overnight. Saturated aqueous NH4Cl
(1 mL) was added, and the product was extracted with CH2Cl2
(2 ꢀ 5 mL). The combined organic layers were washed with
saturated aqueous NaHCO3 (3 mL) and brine (3 mL) and dried
over Na2SO4. This was concentrated and the residue was purified
by flash column (5% MeOH in CHCl3 with 0.2% Et3N) to give 5
mg(39%) of12 as a viscous liquid. 1H NMR (400 MHz, CDCl3) δ
7.04 (bd, 1H), 6.27 (d, J = 15.7 Hz, 1H), 5.90-5.74 (m, 2H),
5.71-5.42 (m, 3H), 4.46 (ddd, J = 11.1, 6.7, 1.8 Hz, 1H), 4.12 (m,
1H), 2.82 (d, J = 11.5 Hz, 2H), 2.57 (s, 2H), 2.28 (s, 4H),
2.12-1.90 (m, 9H), 1.72 (s, 3H), 1.70-1.66 (m, 2H), 1.64-1.54
(m, 6H), 1.40 (s, 3H), 1.34 (d, 3H), 1.27 (s, 3H), 1.26-1.12 (m,
4H); 13C NMR (101 MHz, CDCl3) δ 175.07, 164.78, 138.02,
136.22, 133.80, 132.02, 127.00, 125.50, 73.05, 69.61, 69.17,
55.70, 54.81, 51.06, 46.28, 45.86, 45.37, 42.47, 38.65, 36.60,
34.42, 31.55, 29.55, 29.44, 28.07, 27.87, 27.60, 23.78, 20.27,
12.52; IR (neat film) 3284, 2927, 2853, 1729, 1666, 1628, 1533,
1449, 1374, 1218, 1182, 1047 cm-1; HRMS (ESI) m/z calcd for
C32H51N2O5 (M þ 1)þ 543.3798, found 543.3784.
(S,Z)-5-((1R,4R)-4-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-di-
oxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)cyclohexyl-
amino)-5-oxopent-3-en-2-yl 4-Nitrophenylcarbonate (6). A stir-
red solution of alcohol 4 (45 mg, 0.108 mmol) in anhydrous
CH2Cl2 (2 mL) was treated with Et3N (30 μL, 0.21 mmol) and
4-nitrophenyl chloroformate (32.6 mg, 0.16 mmol) at room
temperature. The resulting light-yellow suspension was allowed
to stir for 4 h at room temperature. This mixture was then dilu-
ted with CH2Cl2 (20 mL), washed with aqueous 1 N HCl (8 mL),
saturated aqueous NaHCO3 (8 mL), and brine (8 mL), and dried
over Na2SO4. Concentration and purification of the residue by
flash chromatography (30-40% ethyl acetate in hexane) gave
General Procedure for the Preparation of Ester Derivatives
from Alcohol 415 Using a Variety of Acid Chlorides. A stirred
solution of the alcohol derivative 415 (10 mg, 0.52 mmol) in
CH2Cl2 (0.5 mL) at 0 ꢀC was sequentially treated with Et3N
(16.6 μL, 0.12 mmol) and DMAP (4.8 μL, 0.10 mmol, 0.5 M in
CH2Cl2). After 5 min at 0 ꢀC, the appropriate acid chloride (0.07
mmol) was added dropwise. The resulting solution was then

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 6987
allowed to stir for 1 h at the same temperature. The reaction
mixture was then diluted with water (3 mL) and CH2Cl2 (6 mL).
The aqueous layer was extracted with CH2Cl2 (2 ꢀ 5 mL). The
combined organic layers were washed with saturated aqueous
NaHCO3 (3 mL) and brine (3 mL) and dried over Na2SO4. The
solvent was evaporated, and the crude residues were purified by
flash chromatography (ethyl acetate in hexane) to give corre-
sponding ester derivatives.
CDCl3) δ 167.80, 134.53, 130.54, 101.12, 69.76, 60.58, 53.12,
34.44, 14.28, 12.71; IR (neat film) 2981, 2860, 2105, 1707, 1654,
1451, 1254, 885; HRMS (ESI) m/z calcd for C11H17N3O4 (M þ
Na) 278.1117, found 278.1106.
(E)-4-((2s,5s)-5-Azido-1,3-dioxan-2-yl)-2-methylbut-2-en-1-ol.
A solution of DIBAL-H (280 mL, 1 M in hexanes, 280 mmol)
was added dropwise to a well stirred solution of ester 22 (28.61 g,
112 mmol) dissolved in CH2Cl2 (500 mL) that had been pre-
cooled to -78 ꢀC in an acetone/dry ice bath for 1 h. After
this time the reaction was quenched successively with MeOH
(200 mL) at -78 ꢀC and saturated aqueous potassium sodium
tartrate tetrahydrate (200 mL). The reaction mixture was then
filtered and extracted with CH2Cl2 (3 ꢀ 300 mL). The combined
organic fractions were dried over MgSO4 and concentrated. The
crude product was chromatographed using 30% EtOAc/hexane
to give 17.32 g (52%, in three steps) of pure alcohol along with 4
g (12%) of 20. 1H NMR (400 MHz, CDCl3) δ 5.50 (s, 1H), 4.67
(t, J = 4.9 Hz, 1H), 4.24 (d, J = 11.1 Hz, 2H), 4.05-3.99 (m,
4H), 2.96 (s, 1H), 2.49-2.41 (m, 2H), 1.67 (s, 3H), 1.46 (bs, 1H);
13C NMR (101 MHz, CDCl3) δ 137.97, 118.29, 102.03, 69.82,
68.56, 53.20, 33.36, 13.94; IR (neat film) 3397, 2917, 2861, 2107,
1393, 1289, 1243, 1142, 1043, 1007; HRMS (ESI) m/z calcd for
C9H15N3O3 (M þ Na) 236.1011, found 236.1004.
(S,Z)-5-((1R,4R)-4-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-di-
oxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)cyclohexyl-
amino)-5-oxopent-3-en-2-yl Tetrahydro-2H-pyran-4-carboxylate
1
(13). Yield: 8 mg (63%) of 13 as a viscous oil. H NMR (400
MHz, CDCl3) δ 7.00 (d, J = 7.7 Hz, 1H), 6.27 (d, J = 15.7 Hz,
1H), 5.93-5.83 (m, 1H), 5.80 (d, J = 11.7 Hz, 1H), 5.65 (dd, J =
11.7, 8.9 Hz, 1H), 5.55 (dd, J = 15.7, 6.7 Hz, 1H), 5.48 (t, J = 7.5
Hz, 1H), 4.52-4.40 (m, 1H), 4.11 (s, 1H), 3.96 (dt, J = 11.6, 3.6
Hz, 2H), 3.43 (td, J = 11.3, 2.9 Hz, 2H), 2.57 (s, 2H), 2.56-2.47
(m, 1H), 2.10-2.04 (m, 2H), 2.02-1.87 (m, 2H), 1.86-1.74 (m,
4H), 1.72 (s, 3H), 1.71-1.65 (m, 1H), 1.64-1.55 (m, 6H), 1.40 (s,
3H), 1.37 (d, J = 6.5 Hz, 3H), 1.28 (s, 3H), 1.26-1.12 (m, 4H);
13C NMR (101 MHz, CDCl3) δ 174.60, 164.71, 138.11, 136.19,
133.81, 131.97, 127.05, 125.45, 73.05, 69.60, 69.30, 67.04, 67.02,
55.69, 51.05, 45.38, 42.46, 40.14, 38.64, 36.56, 34.42, 31.55,
29.72, 29.54, 28.64, 28.59, 27.88, 27.60, 23.78, 20.25, 12.52; IR
(neat film) 3319, 2927, 2853, 1731, 1668, 1629, 1533, 1447, 1378,
1184, 1093, 1046 cm-1; HRMS (ESI) m/z calcd for C31H48NO6
(M þ 1)þ 530.3482, found 530.3481.
2-((E)-4-((2s,5s)-5-Azido-1,3-dioxan-2-yl)-2-methylbut-2-enyl-
thio)benzo[d]thiazole. A solution of 2-mecaptobenzothiazole
(16.1 g, 97 mmol) and Ph3P (24.3 g, 93 mmol) in anhydrous
THF (350 mL) was stirred at 0 ꢀC as DIAD (20.2 mL, 97 mmol)
in toluene (40 mL) was added slowly over 30 min. The resulting
yellow suspension was allowed to stir for 15 min as the alcohol
from above (17.2 g, 81 mmol) in anhydrous THF (80 mL) was
added dropwise over 20 min. The resulting yellow suspension was
allowed to stir for 30 min at 0 ꢀC. The reaction mixture was then
diluted with hexane (200 mL) and ethyl acetate (200 mL), and the
resulting solids were filtered and washed with ethyl acetate (100
mL). This mixture was concentrated, then diluted with ethyl
acetate (400 mL) and washed with saturated aqueous NaHCO3
(100 mL) and water (100 mL), then dried over Na2SO4. This
solution was concentrated and purified by flash chromatography
(30% ethyl acetate in hexane) to give 25 g (84%) of the sulfide as a
viscous oil. 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.2 Hz,
1H), 7.75 (dd, J =8.0, 0.6Hz, 1H), 7.45-7.37(m, 1H), 7.33-7.27
(m, 1H), 5.62 (t, J = 7.1 Hz, 1H), 4.56 (t, J = 5.1 Hz, 1H), 4.18 (d,
J = 11.6 Hz, 2H), 4.00 (s, 2H), 3.93 (dd, J = 12.4 Hz, 2H), 2.95(s,
1H), 2.48-2.37 (m, 2H), 1.80 (s, 3H); 13C NMR (101 MHz,
CDCl3) δ 166.84, 153.22, 135.42, 132.62, 126.00, 124.22, 123.40,
121.62, 120.92, 101.87, 69.64, 53.25, 42.96, 33.97, 15.60; IR (neat
film) 2975, 2917, 2856, 2104, 1457, 1426, 1291, 1239, 1140, 1044,
1004 cm-1; HRMS (ESI) m/z calcd for C16H19N4O2S2 (M þ 1)þ
363.0949, found 363.0942.
(2s,5s)-5-Azido-2-(2,2-dimethoxyethyl)-1,3-dioxane (20). To
a
solution of (2s,5s)-2-(2,2-dimethoxyethyl)-1,3-dioxan-5-
amine33 (59.8 g, 313 mmol) in MeOH (1.5 L) at 0 ꢀC was added
K2CO3 (86 g, 625 mmol), CuSO4 5H2O (780 mg, 3.13 mmol),
and 1H-imidazole-1-sulfonylazide HCl (65.3 g, 375 mmol).
3
3
The mixture was warmed to room temperature and stirred
overnight. The reaction mixture was concentrated and diluted
with water. The resulting mixture was extracted with EtOAc
(3 ꢀ 350 mL). The combined organic fractions were dried over
Na2SO4 and concentrated. The crude product was chromato-
graphed using 30% EtOAc/hexane with 0.5% Et3N to obtain
the azide acetal (20) as a colorless oil. Yield: 33.65 g (50%). 1H
NMR (400 MHz, CDCl3) δ 4.72 (t, J = 5.5 Hz, 1H), 4.56 (t, J =
5.9 Hz, 1H), 4.21 (dd, J = 1.4, 12.5 Hz, 2H), 4.05-3.98 (m, 2H),
3.33 (d, J = 3.7 Hz, 6H), 3.01 (s, 1H), 1.99 (t, J = 5.7 Hz, 2H);
13C NMR (101 MHz, CDCl3) δ 100.90, 99.96, 69.59, 53.38,
53.13, 38.19; IR (neat film) 2950, 2107, 1409, 1123; HRMS (ESI)
m/z calcd for C8H15N3O4 (M þ Na) 240.0960, found 240.0953.
(E)-Ethyl 4-((2s,5s)-5-Azido-1,3-dioxan-2-yl)-2-methylbut-2-
enoate (22). Dioxane 20 (33.65 g, 155 mmol) was dissolved in
CHCl3 (1 L) with water (6.25 mL, 347 mmol) and cooled to 0 ꢀC.
TFA (250 mL, 3.37 mol) was added to the reaction mixture to
give 20% TFA/CHCl3 which was stirred at 0 ꢀC for 40 min. The
reaction was quenched with solid NaHCO3 (400 g, 4.76 mol) at
0 ꢀC. The reaction mixture was filtered and concentrated. The
resulting aldehyde 21 was used in the next step without further
purification and contained 20% of the starting material 20 based
on 1H NMR. Data for 21: 1H NMR (400 MHz, CDCl3) δ 9.79 (t,
J = 2.0 Hz, 1H), 5.10 (t, J = 4.7 Hz, 1H), 4.25 (dd, J = 12.6, 1.4
Hz, 2H), 4.06 (dd, J = 12.6, 1.6 Hz, 2H), 3.05 (s, 1H), 2.76 (dd,
J = 4.7, 2.0 Hz, 2H).
2-((E)-4-((2s,5s)-5-Azido-1,3-dioxan-2-yl)-2-methylbut-2-enyl-
sulfonyl)benzo[d]thiazole (23). A solution of sulfide derivative
from above (0.3 g, 0.82 mmol) in EtOH (6 mL) was treated with
a mixture of ammonium molybdate 4H2O (0.2 g, 0.16 mmol)
3
and 30% H2O2 (1 mL, 8.28 mmol), and the pH was adjusted to
4-5 with 7.0 buffer. This solution was allowed to stir for 4 h at
room temperature and then diluted with water (15 mL) and
extracted with CHCl3 (2 ꢀ 20 mL). The combined organic phase
was washed with brine (25 mL) and dried over Na2SO4. The
solvent was then removed under reduced pressure and the
residue was purified by flash chromatography (40-65% ethyl
acetate in hexane) to give 23 (254 mg, 78%) as a viscous oil. 1H
NMR (400 MHz, CDCl3) δ 8.29-8.21 (m, 1H), 8.05-7.98
(m, 1H), 7.68-7.55 (m, 2H), 5.37 (t, J=7.1 Hz, 1H), 4.30 (t,
J=5.3 Hz, 1H), 4.17 (s, 2H), 4.03-3.95 (m, 2H), 3.77-3.68 (m,
2H), 2.84 (s, 1H), 2.36-2.28 (m, 2H), 1.86 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ 165.57, 152.78, 137.02, 130.65, 127.90,
127.57, 125.60, 125.03, 122.29, 101.14, 69.48, 64.41, 53.01, 34.05,
17.12; IR (neat film) 2974, 2922, 2857, 2105, 1471, 1422, 1327,
1291, 1239, 1136, 1021 cm-1; HRMS (ESI) m/z calcd for
C16H19N4O4S2 (M þ 1)þ 395.0848, found 395.0848.
(Carbethoxyethyldiene)triphenylphosphorane (51.15 g, 141
mmol) was added to a solution of this crude aldehyde 21 (26.59
g, 155 mmol) dissolved in benzene (333 mL) in an ice bath. The
reaction mixture was warmed to room temperature and stirred
for 2 h. TLC was done in 20% EtOAc/hexane. The reaction
mixture was concentrated and the crude product was chroma-
tographed using 20% EtOAc/hexane with 0.5% Et3N to give 22
with 20 as an oil. Yield: 28.61 g. 1H NMR (400 MHz, CDCl3) δ
6.78 (td, J = 7.2, 1.5 Hz, 1H), 4.74 (t, J = 5.1 Hz, 1H), 4.24 (dd,
J = 12.5, 1.4 Hz, 2H), 4.18 (q, J = 7.1 Hz, 2H), 4.07-3.99 (m,
2H), 2.99 (s, 1H), 2.56 (ddd, J = 7.1, 5.2, 1.0 Hz, 2H), 1.84 (d,
J = 1.2 Hz, 3H), 1.27 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz,

6988 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Lagisetti et al.
(3R,7S)-7-((1E,3E)-5-((2s,5R)-5-Azido-1,3-dioxan-2-yl)-3-methyl-
penta-1,3-dienyl)-5,5-dimethyl-1,6-dioxaspiro[2.5]octane (25). A
solution of the sulfone derivative 23 (215 mg, 0.55 mmol) in
anhydrous THF (8 mL) was allowed to stir at -78 ꢀC, while a
solution of lithium bis(trimethylsilyl)amide (0.62 mL, 0.64 mmol,
1.0 M in THF) was added dropwise. After the mixture was stirred
for 30 min at -78 ꢀC, aldehyde 2415 (93 mg, 0.54 mmol) in
anhydrous THF (5 mL) was added dropwise via syringe. The
resulting suspension was stirred at -78 ꢀC for 1.5 h, allowed to
warm to room temperature, and stirred for 12 h. The mixture was
then poured into a saturated aqueous NH4Cl solution (10 mL),
and the product was extractedwith ethyl acetate (3 ꢀ 25 mL). The
combined extractswerewashed withsaturated aqueous NaHCO3
(20 mL) and brine (20 mL) and dried over Na2SO4. Evaporation
of the solvent and purification by flash chromatography (20%
ethyl acetate in hexane) gave 70 mg (37%) of diene 25 (88:12 E/Z)
as a viscous liquid. 1H NMR (400 MHz, CDCl3) δ 6.27 (d, J =
15.8 Hz, 1H), 5.60 (dd, J = 15.7, 6.6 Hz, 1H), 5.51 (t, J = 7.3 Hz,
1H), 4.62 (t, J = 5.2 Hz, 1H), 4.46 (ddd, J = 11.1, 6.7, 1.8 Hz,
1H), 4.27-4.19 (m, 2H), 4.00 (dd, J = 12.4, 2.1 Hz, 2H), 2.98 (s,
1H), 2.59-2.55 (m, 2H), 2.54-2.49 (m, 2H), 2.04-1.85 (m, 2H),
1.74 (s, 3H), 1.40 (s, 3H), 1.28 (s, 3H), 1.25-1.11 (m, 2H); 13C
NMR (101 MHz, CDCl3) δ 135.75, 135.68, 127.95, 125.51,
102.09, 73.03, 69.72, 69.49, 55.68, 53.30, 51.06, 42.46, 38.57,
34.08, 31.54, 23.77, 12.61; IR (neat film) 2974, 2915, 2861, 2105,
1447, 1328, 1289, 1141, 1045 cm-1; HRMS (ESI) m/z calcd for
C18H28N33O4 (M þ 1)þ 350.2080, found 350.2067.
(S,Z)-N-((2R,5R)-2-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-di-
oxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)-1,3-dioxan-5-
yl)-4-hydroxypent-2-enamide. A solution of TBS protected
amide from above (2.1 g, 3.92 mmol) in 20 mL of THF at
-10 ꢀC was stirred as TBAF (5.4 mL, 5.49 mmol, 1.0 M in THF)
was added. The light-yellow solution was allowed to stir for 1.5 h
at 0 ꢀC and allowed to stir at room temperature for 2 h, by which
time TLC and LC/MS showed the starting material had been
consumed. THF was evaporated and diluted with CHCl3
(100 mL) and water (20 mL) and saturated aqueous NaHCO3
(30 mL). The CHCl3 layer was separated, and the aqueous layer
was extracted with CHCl3 (2 ꢀ 75 mL). The combined extracts
were washed with brine (50 mL) and dried over Na2SO4. The
residue was purified by Prep-SFC (OD-H column, 10% MeOH)
to give 780 mg (47%) of the title alcohol as a liquid. 1H NMR
(400 MHz, CDCl3) δ 6.67 (d, J = 7.9 Hz, 1H), 6.28 (d, J = 15.7
Hz, 1H), 6.21 (dd, J = 11.9, 5.6 Hz, 1H), 5.82 (dd, J = 12.0, 1.6
Hz, 1H), 5.61 (dd, J = 15.7, 6.6 Hz, 1H), 5.47 (t, J = 7.2 Hz,
1H), 5.25 (s, 1H), 4.80 (s, 1H), 4.60 (t, J = 5.2 Hz, 1H), 4.46 (ddd,
J = 10.9, 6.6, 1.8 Hz, 1H), 4.02-3.90 (m, 5H), 2.57 (s, 2H),
2.53-2.44 (m, 2H), 2.02-1.85 (m, 2H), 1.74 (s, 3H), 1.40 (s, 3H),
1.35 (d, J = 6.7 Hz, 3H), 1.27 (s, 3H), 1.23-1.13 (m, 2H); 13
C
NMR (101 MHz, CDCl3) δ 165.89, 150.80, 135.82, 135.52,
128.17, 125.28, 122.49, 102.34, 73.08, 70.13, 69.46, 64.48,
55.66, 51.06, 43.87, 42.44, 38.58, 34.11, 31.53, 23.77, 22.67,
12.66; IR (neat film) 3322, 2973, 2912, 2856, 1656, 1627, 1526,
1451, 1377, 1254, 1117, 1077, 1003 cm-1; HRMS (ESI) m/z calcd
for C23H36NO6 (M þ 1)þ 422.2543, found 422.2534.
(S,Z)-4-(tert-Butyldimethylsilyloxy)-N-((2R,5R)-2-((2E,4E)-
5-((3R,5S)-7,7-dimethyl-1,6-dioxaspiro[2.5]octan-5-yl)-3-methyl-
penta-2,4-dienyl)-1,3-dioxan-5-yl)pent-2-enamide. A solution of
the azide derivative 25 (2 g, 5.72 mmol) in benzene (40 mL) at
room temperature under N2 was allowed to stir as Ph3P (4.50 g,
17.17 mmol) was added. The resulting solution was then heated
at 45 ꢀC for 2.5 h, by which time LC/MS showed that the starting
material had been consumed. Water (1 mL) was added and the
mixture was heated at 45 ꢀC for an additional 2 h, by which time
LC/MS indicated that the formation of free amine was com-
plete. The reaction mixture was cooled to room temperature,
diluted with CH2Cl2 (100 mL) and ether (20 mL), and then dried
over Na2SO4. This mixture was then filtered and concentrated.
The residue was dissolved in anhydrous acetonitrile (6 mL) and
used in the next step without further purification.
A stirred solution of (S,Z)-4-(TBSO)pent-2-enoic acid40
(2.6 g, 11.44 mmol) in anhydrous CH3CN (30 mL) at 0 ꢀC was
treated with 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (4.1 g, 10.87) and diisopropylethy-
lamine (6.12 mL, 34.3 mmol) sequentially. The above reaction
solution was added to a solution containing the amine from
above in CH3CN (30 mL) at 0 ꢀC. The resulting solution was
then stirred for another 2 h at room temperature at which time
LC/MC showed the formation of product and disappearance of
all of the starting material. Saturated aqueous NaHCO3 (70 mL)
and ethyl acetate (400 mL) were added. The organic layer was
separated and the aqueous layer was extracted with ethyl acetate
(2 ꢀ 100 mL) and washed with brine (100 mL). Evaporation and
purification of the crude by flash chromatography (25% ethyl
acetate in hexane) gave 2.1 g (69%) of the title compound as a
liquid. 1H NMR (400 MHz, CDCl3) δ 6.41 (d, J = 8.8 Hz, 1H),
6.28 (d, J = 15.7 Hz, 12H), 6.06 (dd, J = 11.6, 7.8 Hz, 1H),
5.64-5.52 (m, 2H), 5.48 (t, J = 7.1 Hz, 1H), 4.59 (t, J = 5.2 Hz,
1H), 4.51-4.42 (m, 1H), 3.98-3.92 (m, 5H), 2.59-2.55 (m, 2H),
2.50-2.46 (m, 2H), 2.04-1.86 (m, 2H), 1.75 (s, 3H), 1.40 (s, 3H),
1.29-1.24 (m, 6H), 1.23-1.14 (m, 2H), 0.88 (s, 9H), 0.05 (s, 3H),
0.04 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 164.98, 151.41,
135.75, 135.55, 128.11, 125.41, 119.06, 102.29, 73.07, 70.44,
70.32, 69.46, 65.43, 55.66, 51.06, 43.52, 42.46, 38.59, 34.17,
31.54, 25.90, 23.78, 18.20, 12.65, -4.68, -4.74; IR (neat film)
3332, 2955, 2930, 2857, 1758, 1715, 1665, 1526, 1471, 1373, 1254,
1117, 1077, 1003 cm-1; HRMS (ESI) m/z calcd for C29H50NO6-
Si (M þ 1)þ 536.3407, found 536.3397.
(S,Z)-5-((2R,5R)-2-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-di-
oxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)-1,3-dioxan-
5-ylamino)-5-oxopent-3-en-2-yl Isobutyrate (26). A solution of
alcohol from above (690 mg, 1.63 mmol) in CH2Cl2 (14 mL) at
0 ꢀC was stirred as Et3N (8.18 mmol, 1.1 mL) and DMAP
(30 mg, 0.246 mmol) were sequentially added. After 5 min,
isobutyric anhydride (0.9 mL, 5.40 mmol) was added. The
solution was allowed to stir for 1 h at the same temperature,
by which time TLC indicated that starting material has com-
pletely been consumed. The reaction mixture was diluted with
water (10 mL) and CH2Cl2 (30 mL). The aqueous layer was
extracted with CH2Cl2 (2 ꢀ 50 mL). The organic layer was
washed with saturated aqueous NaHCO3 (20 mL) and brine and
dried over Na2SO4. The solvent was evaporated and the residue
was purified by flash column (40-50% ethyl acetate in hexane)
to give 770 mg (96%) of 26 as a viscous oil. 1H NMR (400 MHz,
CDCl3) δ 7.16 (d, J = 8.2 Hz, 1H), 6.21 (d, J = 15.7 Hz, 1H),
6.10-5.99 (m, 1H), 5.81-5.72 (m, 2H), 5.53 (dd, J = 15.7, 6.6
Hz, 1H), 5.42 (t, J = 7.1 Hz, 1H), 4.52 (t, J = 5.3 Hz, 1H),
4.45-4.33 (m, 1H), 3.97-3.80 (m, 5H), 2.50 (s, 2H), 2.49-2.39
(m, 3H), 1.97-1.74 (m, 2H), 1.67 (s, 3H), 1.33 (s, 3H), 1.31 (d,
J = 6.7 Hz, 3H), 1.21 (s, 3H), 1.17-1.07 (m, 8H); 13C NMR (101
MHz, CDCl3) δ 176.71, 164.82, 142.32, 135.64, 135.62, 127.99,
125.65, 123.25, 102.19, 73.05, 70.23, 70.20, 69.47, 68.55, 55.66,
51.06, 43.73, 42.46, 38.59, 34.16, 34.02, 31.54, 23.77, 20.04,
18.98, 18.90, 12.63; IR (neat film) 3332, 2974, 2936, 2871,
1730, 1669, 1638, 1525, 1415, 1373, 1266, 1195, 1130 cm-1
;
HRMS (ESI) m/z calcd for C27H42NO7 (M þ 1)þ 492.2961,
found 492.2949.
N-((2s,5R)-2-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-dioxa-
spiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)-1,3-dioxan-5-yl)-
acetamide (27). (2s,5R)-2-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-
1,6-dioxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)-1,3-
dioxan-5-amine (9 mg, 0.029 mmol) which had been dried
azeotropically in CH3CN was dissolved in CH2Cl2 (1.8 mL).
diisopropylethylamine (51 μL, 0.294 mmol) was added to the
reaction mixture followed by Ac2O (14 μL, 0.147 mmol). The
mixture was stirred at room temperature for 28 h. The reaction
mixture was concentrated and chromatographed using 5%
MeOH/CHCl3 to give 1.2 mg (12%) of acetamide 27 as a viscous
liquid. 1H NMR (400 MHz, CDCl3) δ 6.39 (d, J = 8.0 Hz, 1H),
6.28 (d, J = 15.8 Hz, 1H), 5.61 (dd, J = 15.7, 6.6 Hz, 1H), 5.48

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 6989
(t, J = 7.3 Hz, 1H), 4.58 (t, J = 5.3 Hz, 1H), 4.51-4.42 (m, 1H),
3.99-3.87 (m, 5H), 2.57 (s, 2H), 2.52-2.45 (m, 2H), 2.05 (s, 3H),
1.99 (d, J = 14.2 Hz, 1H), 1.91 (dd, J = 13.7, 11.7 Hz, 1H), 1.75
(d, J = 1.0 Hz, 3H), 1.40 (s, 3H), 1.28 (s, 3H), 1.24-1.18 (m,
1H), 1.16 (dd, J = 13.8, 2.0 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 170.25, 136.41, 136.20, 128.77, 126.03, 102.93, 73.72,
71.05, 70.12, 56.31, 51.71, 44.40, 43.10, 39.24, 34.82, 32.18,
24.42, 24.12, 13.31; HRMS (ESI) m/z calcd for C20H32NO5
(M þ 1)þ 366.2280, found 366.2284.
both β-galactosidase and luciferase are expressed. If splicing is
inhibited, the intron remains and only β-galactosidase is ex-
pressed. Assay data are expressed as the relative ratio of
luciferase to β-galactosidase activity compared to control vehi-
cle-treated samples.
In Vitro Splicing Assay. In vitro splicing reactions were
performed as previously described.38 Briefly, standard reactions
contained HeLa nuclear extract (Promega, Madison, WI), 13%
(w/v) polyvinyl alcohol, 33P-labeled β-globin pre-mRNA sub-
strate, 80 mM MgCl2, 12.5 mM ATP/0.5 M creatine phosphate,
and Dignam’s buffer D.39 Reactions were incubated at 30 ꢀC for 1
h with or without spliceosome modulator compound and then
terminated and the RNA products analyzed by autoradiography
following electrophoresis on a 6%polyacrylamide TBE-urea gel.
Immunofluorescence Analysis. HeLa cells were grown on glass
coverslips for 24 h, fixed with cold methanol at -20 ꢀC for 10
min, and rinsed with PBS prior to incubation with SC35 (Sigma)
or SF3B1 (SAP155, MBL International) primary antibodies
(1/100 dilution with PBS) for 1 h at 37 ꢀC in PBS. After the
samples were washed with PBS, the coverslips were incubated
with an antimouse fluorescent secondary antibody (1/200
dilution) labeled with Alexa Fluor 488 (Molecular Probes) as
described above, washed, mounted in Vectashield (Vector
Laboratories), and viewed on an Olympus BX50 fluorescence
microscope equipped with a Cool Snap digital camera using a
60ꢀ magnification objective.
Biology Experimental Section. Cell Culture. The cancer cell
lines were purchased from the American Type Culture Collec-
tion (ATCC, Manassas, VA) or the DSMZ (Brunswick,
Germany) and were maintained in growth media as recom-
mended by the vendor. The PrEC normal prostate epithelial
cells were purchased from Lonza (Basel, Switzerland) and
maintained in prostate epithelial cell growth medium (Lonza).
Human foreskin fibroblast (HFF) cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum, L-glutamine, and penicillin/streptomy-
cin. All cells were grown under standard culture conditions at
37 ꢀC and 5% CO2 in a humidified environment.
In Vitro Cytotoxicity Assay. Cell viabilities were determined
by measuring the cleavage of the tetrazolium salt XTT to a
water-soluble orange formazan by mitochondrial dehydrogen-
ase, following the manufacturer’s instructions (Roche Applied
Science, Indianapolis, IN). Briefly, cell lines were seeded at
various densities depending on the cell type. The adherent cell
lines were allowed to adhere overnight before spliceosome
modulator compound addition, whereas suspension cell lines
were seeded the day of compound addition. Stock concentra-
tions of the compounds were made in DMSO, diluted in culture
media, and subsequently added to the plates at various final
concentrations, then incubated for 72 h. After the incubation
period, the XTT labeling reagent, containing the electron-
coupling reagent, was added to each well and incubated for 4 h.
The absorbance of each well was then determined at 490 nm
with a reference wavelength of 690 nm using a VersaMax
microplate reader (Sunnyvale, CA). The data are expressed as
the percentage of growth compared to vehicle-treated cells, as
calculated from absorbance and corrected for background
absorbance. The IC50 is defined as the drug concentration
that inhibits growth to 50% of the vehicle-treated control;
IC50 values were calculated from sigmoidal analysis of the
dose-response curves using Origin, version 7.5, software
(Northampton, MA).
Acknowledgment. The authors thank the Developmental
Therapeutics Program of the National Cancer Institute,
Bethesda, MD, for providing the 60 cell line in vitro antitumor
screening data for compound 5. This work was supported by
NCI Cancer Center Core Grant CA21765, NIH grant
5R01GM044088 to J.M.L, and by the American Lebanese
Syrian Associated Charities (ALSAC), St. Jude Children’s
Research Hospital.
Supporting Information Available: Procedures for the synth-
esis of compounds 8-11 and 14-19; supplemental Figures
S1-S3. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Rymond, B. Targeting the spliceosome. Nat. Chem. Biol. 2007, 3,
533–535.
In Vivo Tumor Growth Inhibition. JeKo-1 tumors were trans-
planted to female NOD/SCID mice (The Jackson Laboratory,
Bar Harbor, ME) on day 1, and beginning on day 4, the mice
(4 per group) received iv injections of vehicle (3.0% DMSO and
6.5% Tween-80 in 5% glucose solution), 5, 10, 25, or 50 mg/kg
of compound 5 daily for 5 consecutive days. Tumor volume and
mouse weights were measured three times a week. Tumor
volumes were determined on the basis of the following equation:
volume = [length ꢀ (width)2]/2.
(2) Kramer, A. The structure and function of proteins involved in
mammalian pre-mRNA splicing. Annu. Rev. Biochem. 1996, 65,
367–409.
(3) Kaida, D.; Motoyoshi, H.; Tashiro, E.; Nojima, T.; Hagiwara, M.;
Ishigami, K.; Watanabe, H.; Kitahara, T.; Yoshida, T.; Nakajima,
H.; Tani, T.; Horinouchi, S.; Yoshida, M. Spliceostatin A targets
SF3b and inhibits both splicing and nuclear retention of pre-
mRNA. Nat. Chem. Biol. 2007, 3, 576–583.
(4) Kotake, Y.; Sagane, K.; Owa, T.; Mimori-Kiyosue, Y.; Shimizu,
H.; Uesugi, M.; Ishihama, Y.; Iwata, M.; Mizui, Y. Splicing factor
SF3b as a target of the antitumor natural product pladienolide.
Nat. Chem. Biol. 2007, 3, 570–575.
(5) Mizui, Y.; Sakai, T.; Iwata, M.; Uenaka, T.; Okamoto, K.;
Shimizu, H.; Yamori, T.; Yoshimatsu, K.; Asada, M. Pladieno-
lides, new substances from culture of Streptomyces platensis Mer-
11107. III. In vitro and in vivo antitumor activities. J. Antibiot.
(Tokyo) 2004, 57, 188–196.
(6) Nakajima, H.; Hori, Y.; Terano, H.; Okuhara, M.; Manda, T.;
Matsumoto, S.; Shimomura, K. New antitumor substances,
FR901463, FR901464 and FR901465. II. Activities against experi-
mental tumors in mice and mechanism of action. J. Antibiot.
(Tokyo) 1996, 49, 1204–1211.
(7) Nakajima, H.; Sato, B.; Fujita, T.; Takase, S.; Terano, H.;
Okuhara, M. New antitumor substances, FR901463, FR901464
and FR901465. I. Taxonomy, fermentation, isolation, physico-
chemical properties and biological activities. J. Antibiot. (Tokyo)
1996, 49, 1196–1203.
Dual-Reporter Cell-Based Splicing Assay. 293T cells were
transiently transfected using FuGENE 6 (Roche Applied Scien-
ce), as described by the manufacturer, with the pTN24 dual-
reporter splicing construct (gracious gift from Dr. Talat Nasim,
Guy’s Hospital, London, U.K.) for 48 h and subsequently
treated with or without spliceosome modulator compounds
for 5 h.37,38 The degree of cell death was negligible at this time
(data not shown). The cells were lysed, and equal amounts of
protein for each compound concentration were assayed for
luciferase and β-galactosidase activity using the Dual-Light
assay system (Applied Biosystems, Foster City, CA). Each
entire assay was conducted in a single 96-well plate. The
pTN24 dual-reporter construct contains β-galactosidase and
luciferase coding sequences separated by an intronic linker
containing a stop codon.37,38 In the presence of splicing, the
intronic sequence containing the stop codon is removed and
(8) Nakajima, H.; Takase, S.; Terano, H.; Tanaka, H. New antitumor
substances, FR901463, FR901464 and FR901465. III. Structures

6990 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Lagisetti et al.
of FR901463, FR901464 and FR901465. J. Antibiot. (Tokyo) 1997,
50, 96–99.
(9) Albert, B. J.; Sivaramakrishnan, A.; Naka, T.; Czaicki, N. L.;
Koide, K. Total syntheses, fragmentation studies, and antitumor/
antiproliferative activities of FR901464 and its low picomolar
analogue. J. Am. Chem. Soc. 2007, 129, 2648–2659.
(10) Motoyoshi, H.; Horigome, M.; Ishigami, K.; Yoshida, T.;
Horinouchi, S.; Yoshida, M.; Watanabe, H.; Kitahara, T. Struc-
ture-activity relationship for FR901464: a versatile method for the
conversion and preparation of biologically active biotinylated
probes. Biosci., Biotechnol., Biochem. 2004, 68, 2178–2182.
(11) Borriello, A.; Cucciolla, V.; Oliva, A.; Zappia, V.; Della Ragione,
F. p27Kip1 metabolism: a fascinating labyrinth. Cell Cycle 2007, 6,
1053–1061.
(12) Albert, B. J.; Sivaramakrishnan, A.; Naka, T.; Koide, K. Total
synthesis of FR901464, an antitumor agent that regulates the
transcription of oncogenes and tumor suppressor genes. J. Am.
Chem. Soc. 2006, 128, 2792–2793.
(24) Beaumont, K.; Webster, R.; Gardner, I.; Dack, K. Design of ester
prodrugs to enhance oral absorption of poorly permeable com-
pounds: challenges to the discovery scientist. Curr. Drug Metab.
2003, 4, 461–485.
(25) Morikawa, M.; Inoue, M.; Tsuboi, M. Substrate specificity of
carboxylesterase (E.C.3.1.1.1) from several animals. Chem. Pharm.
Bull. (Tokyo) 1976, 24, 1661–1664.
(26) Vaccaro, H. A.; Zhao, Z.; Clader, J. W.; Song, L.; Terracina, G.;
Zhang, L.; Pissarnitski, D. A. Solution-phase parallel synthesis of
carbamates as gamma-secretase inhibitors. J. Comb. Chem. 2008,
10, 56–62.
(27) For more information on the NCI-60 cell-line panel, see the
following: http://dtp.nci.nih.gov/branches/btb/ivclsp.html.
(28) Park, J. R.; Eggert, A.; Caron, H. Neuroblastoma: biology, prog-
nosis, and treatment. Pediatr. Clin. North Am. 2008, 55, 97–120.
(29) Strother, D. Atypical teratoid rhabdoid tumors of childhood:
diagnosis, treatment and challenges. Expert Rev. Anticancer Ther.
2005, 5, 907–915.
(13) Thompson, C. F.; Jamison, T. F.; Jacobsen, E. N. FR901464: total
synthesis, proof of structure, and evaluation of synthetic analo-
gues. J. Am. Chem. Soc. 2001, 123, 9974–9983.
(14) Motoyoshi, H.; Horigome, M.; Watanabe, H.; Kitahara, T. Total
synthesis of FR901464: second generation. Tetrahedron 2006, 62,
1378–1389.
(15) Lagisetti, C.; Pourpak, A.; Jiang, Q.; Cui, X.; Goronga, T.; Morris,
S. W.; Webb, T. R. Antitumor compounds based on a natural pro-
duct consensus pharmacophore. J. Med. Chem. 2008, 51, 6220–6224.
(16) Rantanen, V. V.; Denessiouk, K. A.; Gyllenberg, M.; Koski, T.;
Johnson, M. S. A fragment library based on Gaussian mixtures
predicting favorable molecular interactions. J. Mol. Biol. 2001, 313,
197–214.
(17) Bohm, M.; Sturzebecher, J.; Klebe, G. Three-dimensional quanti-
tative structure-activity relationship analyses using comparative
molecular field analysis and comparative molecular similarity
indices analysis to elucidate selectivity differences of inhibitors
binding to trypsin, thrombin, and factor Xa. J. Med. Chem. 1999,
42, 458–477.
(18) Matter, H.; Schwab, W.; Barbier, D.; Billen, G.; Haase, B.; Neises,
B.; Schudok, M.; Thorwart, W.; Schreuder, H.; Brachvogel, V.;
Lonze, P.; Weithmann, K. U. Quantitative structure-activity
relationship of human neutrophil collagenase (MMP-8) inhibitors
using comparative molecular field analysis and X-ray structure
analysis. J. Med. Chem. 1999, 42, 1908–1920.
(19) Thompson, C. F.; Jamison, T. F.; Jacobsen, E. N. Total synthesis
of FR901464. Convergent assembly of chiral components prepared
by asymmetric catalysis. J. Am. Chem. Soc. 2000, 122, 10482–
10483.
(20) Horigome, M.; Motoyoshi, H.; Watanabe, H.; Kitahara, T. A
synthesis of FR901464. Tetrahedron Lett. 2001, 42, 8207–8210.
(21) Albert, B. J.; Koide, K. Synthesis of a C4-epi-C1-C6 fragment of
FR901464 using a novel bromolactolization. Org. Lett. 2004, 6,
3655–3658.
(30) Bourdeaut, F.; Freneaux, P.; Thuille, B.; Bergeron, C.; Laurence,
V.; Brugieres, L.; Verite, C.; Michon, J.; Delattre, O.; Orbach, D.
Extra-renal non-cerebral rhabdoid tumours. Pediatr. Blood Cancer
2008, 51, 363–368.
(31) Krivtsov, A. V.; Armstrong, S. A. MLL translocations, histone
modifications and leukaemia stem-cell development. Nat. Rev.
Cancer 2007, 7, 823–833.
(32) Bissery, M. C.; Guenard, D.; Gueritte-Voegelein, F.; Lavelle, F.
Experimental antitumor activity of taxotere (RP 56976, NSC
628503), a Taxol analogue. Cancer Res. 1991, 51, 4845–4852.
(33) Gu, K.; Bi, L.; Zhao, M.; Wang, C.; Dolan, C.; Kao, M. C.; Tok,
J. B.; Peng, S. Stereoselective synthesis and anti-inflammatory
activities of 6- and 7-membered dioxacycloalkanes. Bioorg. Med.
Chem. 2006, 14, 1339–1347.
(34) Goddard-Borger, E. D.; Stick, R. V. An efficient, inexpensive, and
shelf-stable diazotransfer reagent: imidazole-1-sulfonyl azide hy-
drochloride. Org. Lett. 2007, 9, 3797–3800.
(35) Blakemore, P. R.; Cole, W. J.; Kocienski, P. J.; Morley, A. A
stereoselective synthesis of trans-1,2-disubstituted alkenes based
on the condensation of aldehydes with metallated 1-phenyl-1H-
tetrazol-5-yl sulfones. Synlett 1998, 26–28.
(36) Nasim, M. T.; Chowdhury, H. M.; Eperon, I. C. A double reporter
assay for detecting changes in the ratio of spliced and unspliced
mRNA in mammalian cells. Nucleic Acids Res. 2002, 30, e109.
(37) Nasim, M. T.; Eperon, I. C. A double-reporter splicing assay for
determining splicing efficiency in mammalian cells. Nat. Protoc.
2006, 1, 1022–1028.
(38) Krainer, A. R.; Maniatis, T.; Ruskin, B.; Green, M. R. Normal and
mutant human beta-globin pre-mRNAs are faithfully and effi-
ciently spliced in vitro. Cell 1984, 36, 993–1005.
(39) Dignam, J. D.; Lebovitz, R. M.; Roeder, R. G. Accurate transcrip-
tion initiation by RNA polymerase II in a soluble extract from
isolated mammalian nuclei. Nucleic Acids Res. 1983, 11, 1475–
1489.
(22) Koide, K.; Albert, B. J. Total syntheses of FR901464. Yuki Gosei
(40) Valverde, S.; Martin-Lomas, M.; Herradon, B.; Garcia-Ochoa, S.
The reaction of carbohydrate derived alkoxyaldehydes with meth-
oxycarbonylmethylenetriphenylphosphorane: stereoselective syn-
thesis of β-unsaturated esters. Tetrahedron 1987, 43, 1895–1901.
Kagaku Kyokaishi 2007, 65, 119–126.
(23) Buchwald, P.; Bodor, N. Physicochemical aspects of the enzymatic
hydrolysis of carboxylic esters. Pharmazie 2002, 57, 87–93.