Structural requirements for the antiproliferative activity of pre-mRNA splicing inhibitor FR901464

Article abstract of DOI:10.1002/chem.201002402

FR901464, a natural product isolated from a bacterium source, activates a reporter gene, inhibits pre-mRNA splicing, and shows antitumor activity. We previously reported the development of a more potent analogue, meayamycin, through the total synthesis of

Full text of DOI:10.1002/chem.201002402

FULL PAPER
DOI: 10.1002/chem.201002402
Structural Requirements for the Antiproliferative Activity of Pre-mRNA
Splicing Inhibitor FR901464
Sami Osman, Brian J. Albert, Yanping Wang, Miaosheng Li, Nancy L. Czaicki, and
Kazunori Koide*[a]
Abstract: FR901464, a natural product
isolated from a bacterium source, acti-
vates a reporter gene, inhibits pre-
mRNA splicing, and shows antitumor
activity. We previously reported the de-
velopment of a more potent analogue,
meayamycin, through the total synthe-
sis of FR901464. Herein, we report de-
tailed structure–activity relationships of
FR901464 that revealed the signifi-
cance of the epoxide, carbon atoms in
the tetrahydropyran ring, the Z geome-
try of the side chain, the 1,3-diene
moiety, the C4-hydroxy group, and the
C2’’-carbonyl group. Importantly, the
methyl group of the acetyl substituent
was found to be inessential, leading to
a new potent analogue. Additionally,
partially based on in vivo data, we syn-
thesized and evaluated potentially
more metabolically stable analogues
for their antiproliferative activity.
These structural insights into FR901464
may contribute to the simplification of
the natural product for further drug de-
velopment.
Keywords: cancer
· FR901464 ·
meayamycin · natural products ·
structure–activity relationships
Introduction
proteasome was not widely accepted as a viable target to
fight cancer until the target of lactacystin was found to be a
proteasome unit.[5] Consequently, Bortezomib has been ap-
proved by the Food and Drug Administration (FDA) as the
first proteasome inhibitor for cancer treatment.[6]
FR901464 (Scheme 1) may contribute to cancer therapy
in a similar fashion. The isolation and antitumor activity of
FR901464 was reported in 1996 by Nakajima and cowork-
ers.[7] Although its biological profile was unique and indica-
tive of a new mode of action, the molecular mechanisms of
this antitumor agent were not known for a long time. In
2007, the Yoshida–Kitahara group reported that FR901464
inhibited pre-mRNA splicing and that its target was splicing
Development of natural product-based anticancer agents:
Development of small molecule ligands for specific protein
targets is a contemporary approach in drug discovery pro-
cesses and chemical biology (reverse chemical genetics).
The success of imatinib (Gleevec) exemplifies this ap-
proach,[1] but the generality of this rational approach re-
mains to be seen,[2] as small molecules often bind to off-tar-
gets. Historically, especially in cancer medicine, major
breakthroughs occurred through the target identifications of
previously known anticancer natural products (forward
chemical genetics). These targets were usually not obvious
prior to these studies. For example, paclitaxel was first iden-
tified as an anticancer compound;[3] however, until Horwitz
reported that this natural product bound to microtubule,
this protein was presumably not viewed as a viable target.[4]
Similarly, although lactacystin was discovered earlier,[5] the
[a] S. Osman, B. J. Albert, Y. Wang, Dr. M. Li, N. L. Czaicki,
Prof. Dr. K. Koide
Department of Chemistry, University of Pittsburgh
219 Parkman Avenue, Pittsburgh, PA, 15260 (USA)
Fax : (+1)412-624-8611
E-mail: koide@pitt.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem201002402.
Scheme 1. Structures of FR901464 and previously prepared analogues.
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factor 3b (SF3b).[8] SF3b, consisting of seven proteins,[9] is re-
quired for pre-mRNA splicing and is part of the spliceo-
some, a complex of approximately 200 proteins and small
nuclear RNAs (snRNAs) that catalyzes RNA splicing.[10] Al-
though the specific molecular target of FR901464 has not
yet been determined, it is likely that it is either spliceosomal
associated protein (SAP)135 or SAP155.[8] The same re-
search group also showed that the splicing inhibition of p27
by FR901464 was linked to cell cycle arrest.[8] These studies
indicate that the anticancer activity of FR901464 is directly
linked to pre-mRNA splicing inhibition. This is potentially a
breakthrough because splicing processes have never been
exploited as therapeutic targets or biomarkers in cancer
medicine. Moreover, post-transcriptional RNA modifica-
tions are an increasingly important theme in biology,[11] for
which FR901464 or its analogues may be used as a chemical
tool. Very recently, the Webb group reported the promising
in vivo antitumor activity of an FR901464 analogue, which
further supports the idea that FR901464 analogues could be
antitumor drugs.[12]
by the Kitahara group, is more active than FR901464 in en-
hancing gene expression of a reporter gene.[23] Unfortunate-
ly, the semi-quantitative description of the activity does not
allow for complete quantitative assessment. Moreover, the
methoxy group at the anomeric center without neighboring
electron-withdrawing groups is acid-sensitive,[24] which raises
the question of whether spliceostatin A is simply an
FR901464-prodrug with enhanced cell permeability. Alter-
natively, the improved activity could be a result of the im-
proved stability of spliceostatin A as compared to
FR901464.[23] The 1-desoxy FR901464, prepared by the Ja-
cobsen group, is slightly more active against Jurkat cells
than FR901464.[17] This analogue shows an important feature
about FR901464: its active form contains a cyclic B-ring. It
is not clear whether the 1-hydroxy group participates in mo-
lecular recognition, because the improved stability of 1-
desoxy FR901464 and loss of the hydroxy group may com-
promise each other, resulting in slightly better anticancer ac-
tivity. We recently substituted the 1-hydroxy group with a
methyl group and found that this analogue, meayamycin,
was 100 times more potent against human breast cancer
MCF-7 cells than FR901464.[22] Moreover, it is more potent
than 1-desoxy FR901464 and should be more stable than
spliceostatin A. Therefore, in this work, all of the analogues
contain the geminal dimethyl group at the C1 position.
Not surprisingly, several pharmaceutical companies re-
cently used reporter assays related to those that the Nakaji-
ma group employed, and discovered a series of new natural
products with biological profiles similar to that of
FR901464.[13,14] The most notable natural products are the
pladienolides,[14] a derivative of which is currently in phase I
trials as the first drug candidate with splicing inhibitory ac-
tivity.[15]
Results and Discussion[25]
In addition to the significance of using splicing inhibitors
as antitumor agents, there is a great need to develop chemi-
cal probes for RNA splicing because the process is not very
tractable with currently available biological methods. As the
first natural product that inhibits pre-mRNA splicing,
FR901464 is now considered a prototype compound for
splicing inhibitors. Given the unique mode of action in con-
junction with its antitumor activity, we envision that
FR901464 or its analogues will be widely used in oncology
and RNA biology. Thus, it is important to understand the
structure–activity relationships (SAR) of FR901464, which
would enable the rational design of more potent analogues
that are compatible with in vivo experiments.
The epoxide moiety: The C3-cyclopropyl analogue
1
(Scheme 1) was prepared by the Jacobsen group and showed
to be inactive even at a concentration of 4 mm (i.e., more
than three orders of magnitude less active than
FR901464).[17] This result implies that the epoxide may be
crucial for the biological activity of FR901464, but it was
not clear whether the oxygen atom or the electrophilicity of
the epoxide was important. If the electrophilicity is impor-
tant, such a notion would be contradictory to the Yoshida–
Kitahara teamꢁs statement that FR901464 does not form a
covalent bond with its target protein.[8] In light of this poten-
tial discrepancy, we set out to prepare non-epoxide ana-
logues that still contain the oxygen atom at the C3 position
(Scheme 2).
Synthetic studies of FR901464: The Jacobsen group accom-
plished the first total synthesis of FR901464[16] and systemat-
ically studied the structure–activity relationship of this natu-
ral product.[17] The results of their SAR studies are de-
scribed throughout this article where they are directly relat-
ed to our studies. The second total synthesis was accom-
plished by the Kitahara group,[18] who later improved their
synthetic scheme.[19] Our group reported the third total syn-
thesis of FR901464 in 2006,[20,21] and later disclosed how the
combination of the epoxide at the C3 position and the hy-
droxy group at the C1 position caused the decomposition of
FR901464.[22]
Compound 2[22] was reduced with LiAlH4 to a diol, which
was protected as an acetal to give 3 in 67% yield for the
two steps (Scheme 2A). This acetal was subjected to olefin
cross metathesis conditions with the previously prepared
diene 4 and Ru-1[26] to give the cross-coupled compound in
41% yield. The acetal of this compound was hydrolyzed
under acidic conditions to give analogue 5 in 66% yield.
To arrive at two similar analogues, alcohol 6[22] was used
as the starting material. The alcohol was protected as its
PMB ether with 74% yield, and its epoxide was subsequent-
ly opened with KOH to give diol
7 in 77% yield
(Scheme 2B). Diol 7 was cleaved by the action of NaIO4,
and the resulting ketone was immediately reduced with l-se-
lectride to give alcohol 8 as a single diastereomer. Alcohol 8
The C1-hydroxy group of FR901464: Spliceostatin A
(Scheme 1), the 1-methoxy analogue of FR901464, prepared
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The Antiproliferative Activity of Pre-mRNA Splicing Inhibitor FR901464
FULL PAPER
Scheme 2. Preparation of the C3 analogues 5, 10, and 13. a) LiAlH4 (5.0 equiv), Et2O, reflux, 80%; b) PMPCHACHTUNTRGNE(UNG OMe)2 (PMP=para-methoxyphenyl,
2.9 equiv), camphorsulfonic acid (CSA, 0.10 equiv), CH2Cl2, 238C, 84%; c) 3 (1.3 equiv), 4 (1.0 equiv), Ru-1 (0.10 equiv), benzoquinone (0.22 equiv), 1,2-
dichlorethane (DCE), 428C, 41%; d) AcOH/THF/H2O (3:1:1), 238C, 66%; e) PMBCl (PMB= para-methoxybenzyl, 2.9 equiv), Ag2O (1.5 equiv), DMF,
608C, 74%; f) KOH (5.0 equiv), DMSO/H2O (1.5:1), 558C, 77%; g) NaIO4 (1.2 equiv), H2O/THF (2:1), 238C; LisBu3BH (3.1 equiv), THF, À788C, 69%
(2 steps); h) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 3.0 equiv), CH2Cl2/saturated aqueous NaHCO3 (9:1), 238C; CSA (0.05 equiv), MeOH,
238C; 5% KOH/H2O, 23 8C, 79% (3 steps); i) PMPCHACHTNUGRTNEUNG(OMe)2 (2.9 equiv), CSA (0.10 equiv), CH2Cl2, 238C, quantitative; j) 4 (1.0 equiv), 9 (1.3 equiv),
Ru-1 (0.10 equiv), benzoquinone (0.22 equiv), DCE, 408C, 48%; k) AcOH/THF/H2O (3:1:1), 238C, 68%; l) MeI (2.0 equiv), NaH (2.0 equiv), THF,
238C, 94%; m) DDQ (2.0 equiv), CH2Cl2/saturated aqueous NaHCO3 (9:1), 238C, 89%; n) 3,4-dihydro-2H-pyran (1.5 equiv), CSA (0.05 equiv), CH2Cl2,
238C, quantitative; o) 4 (1.2 equiv), Ru-1 (0.10 equiv), benzoquinone (0.20 equiv), DCE, 458C, 40%; p) AcOH/THF/H2O (3:1:1), 238C, 63%.
Table 1. Antiproliferative activities against MCF-7 cells.[a]
was treated with DDQ to give a mixture of acetals and
esters (see the Supporting Information), which was treated
with acidic methanol and subsequently with KOH to give a
diol. This diol was converted to acetal 9 in quantitative
yield. Acetal 9 was then subjected to diene 4 and Ru-1 to
give a metathesis adduct in 48% yield, which was deprotect-
ed under acidic conditions to give 10 in 68% yield.
FR901464 analogue
GI50 [nm]
meayamycin
5
10
13
22
28
0.02
>10000
>10000
>10000
0.30
2.5
Alcohol 8 was also methylated by the action of MeI and
NaH to give 11 in 94% yield. The PMB ether 11 was ex-
posed to DDQ to give an alcohol in 89% yield, which was
then protected as its tetrahydropyran (THP) ether 12 in
quantitative yield. Compound 12 was treated with diene 4
and Ru-1 to give the desired cross-coupled product in 40%
yield. Finally, the THP ether of this product was hydrolyzed
to give analogue 13 in 63% yield.
29
30
85
19
0.008
0.48
4.7
5
19
0.38
15
10
meayamycin B
4’-desacetyl meayamycin
4’-epi-desacetyl meayamycin
41
48
50
59
60
None of these non-epoxy analogues (5, 10, and 13) exhib-
ited antiproliferative activity against MCF-7 cells even at a
concentration of 10 mm, meaning that they are at least six
orders of magnitude less active than meayamycin (Table 1).
These data, together with those reported by the Jacobsen
group,[17] suggest that the epoxide of FR901464 plays a cru-
cial role as an electrophile and presumably forms a covalent
bond. Because non-specific epoxide opening by thiols in
vivo may not be a major concern,[27] the potential covalent
bond formation may be specific for the putative target(s).
[a] Each experiment consists of at least twelve data points (twelve differ-
ent concentrations).
cant population (1:3) of this compound existed as conformer
14N-ax despite the bulk of the acetamide group
(Scheme 3A).[28] The somewhat surprising stability of the
axial conformer prompted us to speculate that the more
stable chair conformer of the A-ring of FR901464 might be
FR901464N-ax, provided that the A-ring is indeed in a chair
form (Scheme 3B). This preference can be justified by argu-
ing that in this conformer, if the amide NH group is hydro-
The 12 position: Vasella and co-workers studied the confor-
mations of compound 14 in water and found that a signifi-
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897

K. Koide et al.
phorboxazole analogues,[30] the substitution of the carbon at
position 12 with an oxygen atom looked very appealing
from a synthetic standpoint.
Our synthetic efforts toward analogue 22 began with the
one pot DIBALH reduction–Wittig condensation reaction
sequence of the l-threonine derivative 15 (Scheme 4A).[31]
The resulting enoate was hydrogenated to form the corre-
sponding saturated compound 16 in 87% yield for the two
steps. Treatment of this compound with acetic acid promot-
ed the acetonide cleavage and intramolecular transesterifica-
tion, giving lactone 17 in 73% yield. The allylation of this
lactone followed by the BF3·OEt2–Et3SiH-mediated reduc-
tion of the resulting hemiketal provided tetrahydropyran 18
in 43% yield for the two steps. The same three-step se-
quence as used for our previous synthesis (1. Boc-deprotec-
tion–amidation with acid 19; 2. olefin cross metathesis with
methacrolein; 3. Wittig reaction with Ph3P=CH2)[21] afforded
the conjugated diene 21 in 43% yield over the three steps.
This fragment was then coupled with the compound 6 to
give the designed analogue 22 in 50% yield. The total
number of steps is 25 for this analogue with 11 steps in the
longest linear sequence, which compares favorably to the
synthesis of meayamycin (28 total steps and 13 steps in the
longest linear sequence).
Scheme 3. Chair conformers of A) 14 and B) FR901464.
gen bonded to the oxygen atom of the A-ring, only the ni-
trogen lone pair would point toward the methyl group at the
C12 position, minimizing the 1,3-diaxial interaction. On the
other hand, in conformer FR901464N-eq, there is a substantial
1,3-diaxial interaction between the methyl group at the C15
position and the C10-allylic methylene group. Therefore, we
hypothesized that FR901464 might bind to its target(s)
when it assumed the chair form FR901464N-ax in the A-ring.
With this hypothesis, it appeared to us that the 12-methyl
group in the axial position destabilized this putative binding
conformer. Therefore, removal of this methyl group might
indeed increase the population of the bound conformer,
thereby enhancing the potency of meayamycin. Moreover,
inspired by the work of Wender et al. on a series of bryosta-
tin analogues,[29] which was followed by the Smith group on
The acetal analogue synthesis was even more concise;
treatment of the commercially available compound 23 with
NaBH4 gave the known diol 24 in 87% yield
(Scheme 4B).[32] This diol was treated with 1,2-diethoxy-3-
Scheme 4. Preparation of C12 analogues. a) Diisobutylaluminum hydride (DIBALH, 1.8 equiv), CH2Cl2, À788C; Ph3P=CHCO2Me (1.5 equiv), 458C,
87%; b) H2, Pd/C (1 mol%), EtOAc, 238C, quantitative; c) AcOH, 808C, 73%; d) CH2=CHCH2MgCl (1.9 equiv), THF, À788C; e) Et3SiH (10 equiv),
BF3·OEt2 (4.0 equiv), CF3CH2OH (8.0 equiv), À788C, 43% (2 steps); f) CH2Cl2/trifluoroacetic acid (TFA, 9:1), 238C; 19 (1.2 equiv), O-(7-azabenzotria-
zol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU, 1.2 equiv), iPr2NEt (4.0 equiv), 238C, 84%; g) methacrolein (10 equiv), Ru-1
(2 mol%), 238C, 60%; h) Ph3PCH3Br (2.2 equiv), KOtBu (2.2 equiv), THF, 08C, 86%; i) 6 (1.6 equiv), Ru-1 (0.10 equiv), benzoquinone (0.21 equiv),
DCE, 358C, 50%; j) NaBH4 (2.0 equiv), EtOH, 238C, 87%; k) 1,1-diethoxy-3-butene (1.0 equiv), CSA (1 mol%), CH2Cl2, 08C, 67%; l) CH2Cl2/TFA
(9:1), 238C; 19 (1.2 equiv), HATU (1.2 equiv), iPr2NEt (4.0 equiv), 238C, 51%; m) methacrolein (10 equiv), Ru-1 (2 mol%), 238C, 62%; n) Ph3PCH3Br
(2.2 equiv), KOtBu (2.2 equiv), THF, 08C, 87%; o) 6 (1.6 equiv), Ru-1 (0.10 equiv), benzoquinone (0.22 equiv), DCE, 358C, 53%.
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The Antiproliferative Activity of Pre-mRNA Splicing Inhibitor FR901464
FULL PAPER
butene under acidic conditions to form the cyclic acetal 25
in 67% yield. After the same synthetic procedures described
before, we were able to prepare the target analogue 28 in
15% yield for the 4 steps. The total number of steps is 21
for this analogue with only 11 steps in the longest linear se-
quence.
group and the oxygen atom in the A-ring (pyran or acetal).
An additional reason for the lower potency of 28 may be at-
tributed to the possibility that the additional oxygen is
bound to a water molecule, which may need to be dissociat-
ed to enter the putative hydrophobic binding pocket.
The antiproliferative activity of these two analogues was
evaluated against MCF-7 cells (Table 1). The 12-desmethyl
meayamycin 22 was less potent than meayamycin, but the
GI50 is still in the respectable sub-nanomolar range (0.3 nm).
The acetal analogue 28 was far less active, with the GI50
value at 2.5 nm. These data indicate the significance of hy-
drophobicity at the 12 position. The replacement of the A-
ring with a cyclic acetal provided a means to probe the
impact of substituents at positions 13 and 15. Thus, through
similar synthetic schemes (see the Supporting Information),
analogues 29 and 30 were prepared and evaluated. The addi-
tional methyl group at the 13 position in analogue 29 was
detrimental to the biological activity. The replacement of
the 15-methyl group with a hydrogen atom in analogue 30
also had a negative impact with respect to the biological ac-
tivity. All together, the substituents at positions 12, 13, and
15 are intricately responsible for the biological activity of
FR901464. Although these analogues are more synthetically
accessible, we tentatively decided to exclude these simplifi-
cations because a sufficient quantity of meayamycin could
be produced for the purpose of this study by using tissue-
cultured cells.
The 4’ position: Due to a 1,3-allylic strain, the proton at the
4’ position is nearly in the same plane as the conjugated
amide, as suggested by the H NMR spectrum of FR901464
1
(J3’,4’ =7.8 Hz). Inversion of the stereocenter at the 4’ posi-
tion places its methyl and acetoxy substituents in opposite
faces of the plane defined by the conjugated amide. This an-
alogue, 4’-epi-FR901464, was synthesized by the Jacobsen
group and found to be 15 times less active than
FR901464.[17] The reduced activity could be because either
the acetoxy or the 5’-methyl group is in the wrong orienta-
tion (or both groups). To probe this idea, we prepared 4’-des-
acetyl meayamycin, which began with the methanolysis of
the previously prepared 4[21,22] to give alcohol 31 in 82%
yield (Scheme 5A). This alcohol was then subjected to
olefin cross metathesis conditions with 6 and Ru-1 to give
4’-desacetyl meayamycin in 81% yield. The potency of 4’-
desacetyl meayamycin as compared to meayamycin dropped
by 24-fold (GI50 =0.48 nm; Table 1), further indicating the
significance of the 4’ position.
To understand if the loss in activity of these A-ring ana-
logues was due to a conformational change or a loss of hy-
drophobicity, NMR experiments were undertaken for dienes
4, 21, and 27 in CD3OD. The J14,15 values were 2.1, 1.6, and
From this result, it was not clear whether this reduced ac-
tivity was due to the loss of the acetyl group itself or possi-
bly placing the methyl group in the a face by virtue of the
intramolecular hydrogen bonding between the 4’-hydroxy
group and the amide group. Therefore, we also prepared 4’-
epi-desacetyl meayamycin according to Scheme 5B. The
enantiomer of acid 19 (i.e., epi-19) was prepared by the fol-
lowing route: N-acetyl morpholine was coupled to an in situ
prepared lithium acetylide to give 32 in 90% yield.[33] This
ynone was reduced with HCO2Na and Noyoriꢁs catalyst,[34]
Ru-2, to give an alcohol, which was acetylated to give 33 in
73% yield for the two steps. This material was then exposed
to acidic methanol followed by the Jonesꢁ reagent to give an
ynoic acid in 72%. The ynoic acid was hydrogenated in the
presence of Lindlarꢁs catalyst to give 4’-epi-19 in 68% yield.
Acid 4’-epi-19 was then used to prepare 4’-epi-desacetyl
meayamycin by the same synthetic route previously out-
lined.[21,22] This analogue was found to be one order of mag-
nitude less active (GI50 =4.7 nm, Table 1) than 4’-desacetyl
meayamycin, indicating that the acetyl group is important
for the biological activity.
1.8 Hz for 4, 21, and 27, respectively, indicating similar
chair-like conformers. Therefore, the loss in activities for 12-
desmethyl meayamycin, 22, and 28 is mainly attributed to
the loss of favorable hydrophobic interactions, and, to a
lesser degree, a conformational change in the A-ring.
Why is compound 28 less potent than compound 22? The
lower potency of 28 can be attributed to the additional con-
1
former 28b. From H NMR spectroscopic experiments, the
secondary NH signal was very prominent in CDCl3, which
did not diminish upon the addition of CD3OD. This indi-
cates that there is strong hydrogen bonding between the NH
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K. Koide et al.
Scheme 5. Preparation of 4’-analogues. a) K2CO3 (5.0 equiv), MeOH, 08C, 82%; b) 6 (1.6 equiv), Ru-1 (0.10 equiv), benzoquinone (0.17 equiv), DCE,
ꢀ
328C, 81%; c) nBuLi (2.8 equiv), HC CCH2OTHP (3.0 equiv), THF, À78 to 08C, 90%; d) Ru-2 (2 mol%), HCO2Na (10 equiv), cetyltrimethylammoni-
um bromide (CTAB, 0.2 equiv), H2O, 23 8C, 78%; e) Ac2O (5.0 equiv), pyridine, 238C, 93%; f) pyridinium para-toluenesulfonate (PPTS, 0.2 equiv),
MeOH, 238C, 84%; Na2Cr2O7 (1.5 equiv), acetone, H2SO4, 0 to 238C, 86%; g) H2, Lindlarꢁs catalyst (1 mol%), quinoline (0.10 equiv), EtOH, 238C,
68%; h) 34 (1 equiv), CH2Cl2/TFA (9:1), 238C; then 4’-epi-19 (1.2 equiv), HATU (1.2 equiv), iPr2NEt (4.0 equiv), MeCN, 238C, 71%; i) methacrolein
(10 equiv), Ru-1 (2 mol%), 238C, 62%; j) Ph3PCH3Br (2.2 equiv), KOtBu (2.2 equiv), THF, 08C, 84%; k) K2CO3 (2.0 equiv), MeOH, 08C, 86%; l) 6
(1.5 equiv), Ru-1 (0.10 equiv), DCE, 358C, 34%; m) 1,1’-carbonyldiimidazole (CDI, 1.5 equiv), CH2Cl2, 238C; morpholine (4.1 equiv), 238C, 80%; n) 6
(1.6 equiv), Ru-1 (0.07 equiv), DCE, 428C, 44%.
The correlation between the loss of the acetyl group and
activity was a concern to us because esterases might hydro-
lyze the ester group in tissue culture and in vivo. Conse-
quently, we measured the half-lives of meayamycin in
mouse and human sera at a concentration of 10 mm. As we
expected, meayamycin was hydrolyzed to its desacetyl ana-
logue (t1/2 =2 h in mouse serum, 9 h in human serum), which
was then converted to uncharacterized products. We hy-
pothesized that if we prolong the half-life of meayamycin in
sera, effective concentrations of this compound might be
higher during the assays, thus improving the potency. To test
this hypothesis, we replaced the acetyl group with a morpho-
line-based carbamate (meayamycin B) to enhance the stabil-
ity under biological conditions as carbamates are known to
be more stable than esters under biological conditions.[35]
The synthesis of this analogue was accomplished by the
reaction of 31 with CDI followed by morpholine to give 36
in 80% yield (Scheme 5C). This carbamate was then cou-
pled to 6 in the presence of Ru-1 to give meayamycin B in
44% yield. The half-life of this carbamate analogue was
13 h in mouse serum (Figure 1), significantly longer than
that of meayamycin, presumably due to the esterase-resist-
ant nature of this compound. Encouraged by this result, the
antiproliferative activity of this new analogue was measured;
Figure 1. Degradation of meayamycin (triangles) and meayamycin B
(squares) in mouse serum at 378C (y axis: relative quantity of meayamy-
cin (or meayamycin B) compared to the initial meayamycin (or meaya-
mycin B) quantity). The initial meayamycin concentration was 10 mm.
Meayamycin: y=eÀ0.310x (R2 =0.997). Meayamycin B: y=eÀ0.053x (R2 =
0.954). MAM: meayamycin or meayamycin B.
it was gratifying to find that meayamycin B was even more
potent than the extremely potent meayamycin in MCF-7
cells (Table 2). With this result in hand, we tested meayamy-
cin[36] and meayamycin B with various cancer cell lines.
MCF-7 and MDA-MB-231, both human breast cancer cell
lines, were the most responsive. PC3, HCT116 and H1299
were also very responsive, exhibiting GI50 values in a low pi-
comolar range. A549 and DU145 cells were less responsive,
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The Antiproliferative Activity of Pre-mRNA Splicing Inhibitor FR901464
Table 2. GI50 values of meayamycin and meayamycin B.[a]
FULL PAPER
form. To address this question, we synthesized analogue 41
according to Scheme 6. A key step is the deliberate isomeri-
zation of the cis acid 19 to the activated trans acid derivative
during the amide bond forming reaction. Specifically, we ex-
ploited the facile cis–trans isomerization of the HATU-acti-
vated acid 19 prior to the addition of amine 34 to yield a
ꢁ1:1 cis/trans mixture as illustrated in Scheme 6A. Ana-
logue 41 was assembled in three additional steps as illustrat-
ed. The GI50 of this analogue was three orders of magnitude
higher than that of the corresponding cis compound, indicat-
ing that the cis enamide is the active species.
Cell line
Meayamycin [pm]
Meayamycin B [pm]
MCF-7
MDA-MB-231
A549
DU145
HCT116
H1299
20Æ8.9
71Æ55
8Æ2
15Æ1.4
175Æ77
211Æ23
4Æ2
258Æ162
1230Æ697
157Æ33
841Æ271
196Æ42
146Æ30
PC3
101Æ4.0
[a] Each experiment consists of at least twelve data points (twelve differ-
ent concentrations).
Considering that an a,b-unsaturated amide could serve as
an electrophile,[38] we asked whether the side chain was an
electrophile. To answer this question, analogue 48 was de-
signed and synthesized according to Scheme 6B. The GI50 of
this side chain analogue was found to be 19 nm with MCF-7
cells (Table 1; 93 nm with HCT116, data not shown). The an-
tiproliferative activity of the two analogues 41 and 48 im-
plies that the cis C2’–C3’ enamide is not an electrophile but
rather a structural requirement.
but the GI50 values were still sub-nanomolar. On the aver-
age, meayamycin B is 3.1Æ1.9 times more potent than
meayamycin in these seven cancer cell lines.
After the synthesis and biological evaluation of meayamy-
cin B were completed in our laboratory, the Webb group
published similar carbamate analogues with great loss of ac-
tivity.[12] In retrospect, we were fortunate that we chose mor-
pholine for water solubility, the size of which appeared to
be adequately small to fit in an unknown binding pocket
unlike Webbꢁs larger carbamate groups.
For in vivo studies, the most ideal vehicle is saline; there-
fore, the solubility of drugs in saline is a crucial parameter.
Although meayamycin was not entirely soluble in saline at a
concentration of 0.1 mgmLÀ1, meayamycin B was soluble at
this concentration (presumably soluble at higher concentra-
tions), showing an additional benefit for meayamycin B.[37]
The 4-hydroxy group: 4-O-Alkyl FR901464 analogues were
prepared and evaluated by others.[39] However, it was not
clear to us whether the loss of activity was due to the large
alkyl groups or the loss of a hydrogen bond donor. To mini-
mize the additional sterics, we prepared the 4-O-methyl ana-
logue 50 (Scheme 7). This analogue, meayamycin C, showed
respectable potency with a GI50 of 0.38 nm. The diminished
activity may be explained by the loss of a hydrogen bond
donor or the additional steric bulk. Nonetheless, this ana-
logue may prove to be an important one because in a sepa-
rate study we observed glucuronidation of meayamycin B as
a major metabolite in mice, for which this hydroxy group is
C2’–C3’ Olefin on the side chain: Next, we asked whether
the C2’–C3’ trans isomer was the actual active species after
the potential isomerization of the cis enamide to its trans
Scheme 6. Preparation of C2’–C3’ analogues 41 and 48. a) CH2Cl2/TFA (9:1), 238C; 19 (1.7 equiv), HATU (1.3 equiv), iPr2NEt (5.0 equiv), MeCN, 238C,
43%; b) K2CO3 (2.5 equiv), MeOH, 08C to 238C, 75%; c) CDI (1.2 equiv), CH2Cl2, 238C; morpholine (2–5 equiv), 238C, 97% (38) or 94% (43);
d) methacrolein (13–20 equiv), Ru-1 (2 mol%), CH2Cl2 (39) or PhH (46), 238C or 508C, 74% (39) or 63% (46); e) Ph3PCH3Br (2.4 equiv), KOtBu
(2.3 equiv), THF, 08C, 50% (40) or 76% (47); f) 6 (1.3 equiv), Ru-1 (0.07–0.2 equiv), benzoquinone (0.2–0.5 equiv), DCE, 42–508C, 46% (41) or 36%
(48); g) DIBALH (1.1 equiv), CH2Cl2, À788C; Ph3P=CHCO2Me (1.5 equiv), 238C; h) H2, Pd/C (2 mol%), EtOAc, 238C; i) NaOH (2.1 equiv), MeOH/
H2O (1:1.1), 238C, 46% (over 3 steps); j) 34, CH2Cl2/TFA (9:1), 238C; 44 (1.3 equiv), HATU (1.2 equiv), iPr2NEt (3.4 equiv), 238C, 66%.
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901

K. Koide et al.
yield over two steps. Further transformations yielded alkene
58. Interestingly, this alkene did not undergo cross olefin
metathesis with alkene 6 but did so with alkene 49 to form
59 in 30% yield. By a similar sequence, diastereomer 60 was
prepared (see the Supporting Information).
The GI50 values of analogues 59 and 60 with MCF-7 cells
were found to be 15 and 10 nm, respectively (Table 1). The
potencies of these analogues were approximately one order
of magnitude lower than that of meayamycin C but were
still respectable. These results imply that the C8–C9 olefin
of FR901464 remains trans as an active species in live cells.
In order to fully understand the significance of the C8–C9
olefin, cyclopropyl analogues that would mimic the C8–C9
cis geometry must be prepared and evaluated. Nonetheless,
the similar activity of 59 and 60 compared to meayamycin C
implies that the C8–C9 olefin would remain trans as the
active FR901464 species in cells.
Scheme 7. Preparation of the C4-OMe analogue 50. a) MeI (5.0 equiv),
Ag2O (2.0 equiv), DMF, 238C, 87%; b) 36 (1.0 equiv), 49 (1.5 equiv), Ru-
1 (7 mol%), benzoquinone (0.2 equiv), DCE, 428C, 57%.
the only site. Detailed pharmacokinetic studies of meayamy-
cin B and meayamycin C will be reported by our laboratory
in due course. It is noteworthy that the olefin cross metathe-
sis of 36 with 49 was reproducibly higher yielding than that
with 6 (Scheme 5C). This difference proved to be crucial in
the preparation of cyclopropyl analogues (see below).
The 1,3-diene moiety: We previously observed relatively
facile isomerization of the 1,3-diene moiety under acidic
conditions.[22] The same might occur in live cancer cells, rais-
ing a question of whether the trans,trans-diene moiety is ac-
tually the biologically active species. To examine the signifi-
cance of the diene moiety, we synthesized the cyclopropyl
analogues 59 and 60 (Scheme 8).
Compound 34 was first cross-coupled with methacrolein
in the presence of Ru-1 to give 51 in 84% yield. This alde-
hyde was converted to alcohol 52, which was stereoselec-
tively cyclopropanated by Charetteꢁs method[40] to form 53
in 70% yield with a diastereoselective ratio of 89:11. We
also prepared the epimer at the C8 and C9 position in 88%
with a diastereoselective ratio 87:13 (see the Supporting In-
formation). The allylic cyclopropyl group, after the conver-
sion of the hydroxy group of 53 to an olefin, was not com-
patible with acidic conditions for the Boc removal (data not
shown). These failures prompted us to remove the Boc
group from 53 with TFA before the derivatization of the hy-
droxy group. The resulting amine 54 was condensed with
carboxylic acid 19 to form 55 in 48% yield for the two steps.
The oxidation–olefination sequence gave alkene 57 in 56%
Conclusion
The C3-spiroepoxide (Scheme 9) was shown to be crucial
for the retention of the antiproliferative activity, indicating
that a covalent bond is presumably formed between target
protein(s) and FR901464. The C12 position prefers to be hy-
drophobic, and the C4’-acetate group is important but the
ester is labile, forming a less potent alcohol (4’-desacetyl
meayamycin). The C2’–C3’ side chain olefin needs to be in
its cis form to be active. The replacement of one of the pro-
tons at position 13 with a methyl group diminished biologi-
cal activity. Replacement of the 15-methyl group with a hy-
drogen atom was also somewhat detrimental. The C8–C9
olefin remains in its trans form as the active species of
FR901464. Masking the 4-hydroxy group with a methyl
group reduced the biological activity. Finally, the develop-
ment of the more biologically stable and more saline-soluble
C4’-carbamate analogue, meayamycin B, was shown to be
significantly more potent than meayamycin in seven cancer
Scheme 8. Preparation of the cyclopropyl analogues. a) methacrolein (20 equiv), Ru-1 (3 mol%), 238C, 84%; b) DIBALH (2.0 equiv), CH2Cl2, À788C,
98%; c) (S,S)-dioxaborolane (2.0 equiv), ZnEt2 (5.0 equiv), CH2I2 (7.0 equiv), CH2Cl2, 238C, 70%, d.r.=89:11; d) CH2Cl2/TFA (8:2), 0 to 238C, quantita-
tive; e) 19 (1.2 equiv), HATU (1.0 equiv), iPr2NEt (4.0 equiv), MeCN, 238C, 48%; f) Dess–Martin periodinane (2.0 equiv), CH2Cl2, 08C, 70%;
g) Ph3PCH3Br (6.0 equiv), KOtBu (5.0 equiv), THF, 08C, 80%; h) 1. K2CO3 (2.5 equiv), THF, 08C; i) CDI (2.5 equiv), CH2Cl2, 238C; morpholine
(5.0 equiv), 238C, 94% (over 2 steps); j) 49 (1.7 equiv), Ru-1 (0.1 equiv), benzoquinone (0.2 equiv), toluene, 708C, 30%.
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The Antiproliferative Activity of Pre-mRNA Splicing Inhibitor FR901464
FULL PAPER
Growth inhibition assay: Cells were plated in 96-well plates at an initial
density of 2000 or 4000 cells per well in 100 mL of medium and were incu-
bated for 24–48 h (depending on cell line) prior to compound addition
(plating either 2000 or 4000 cells per well led to similar GI50 values).
Serial two-fold dilutions were used in this experiment for the indicated
ranges. The compounds were added to the cells at 2ꢂ the desired concen-
tration in 100 mL cell culture medium (containing 2% DMSO). The cells
were then incubated for an additional 5–10 days. Cell proliferation was
measured by using the commercial 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)
dye reduction assay with phenazine methosulfate (PMS) as the electron
acceptor (20 mL per well). The absorbance (at 490 nm minus that at
630 nm) was measured by a Spectromax M5 plate reader (Molecular De-
vices) or Modulus II Microplate Multimode Reader (Turner BioSystem).
Evaluation of FR901464 analogues was performed in quadruplicate at
each concentration, and the final numbers were averaged. Microsoft
Excel and GraphPad Prism 5 were used to construct dose–response
curves and calculating the GI50 values. Growth inhibition is calculated as
defined by the National Cancer Institute [GI50 =100ꢂ(TÀT0)/(CÀT0);
U
T0 =cell density at time zero; T=cell density of the test well after period
of exposure to test compound; C=cell density of the vehicle treated.[41]
Vincritsine or cisplatin were used as controls.
Procedure for the half-life determination for the decomposition of
meayamycin and meayamycin B in mouse serum: To a 1 dram glass vial
was added mouse serum (1.0 mL), rhodamine B (10 mm in DMSO,
2.0 mL), and DMSO (7.0 mL) under an open atmosphere and warmed to
378C. To the mixture was added meayamycin or meayamycin B (10 mm
in DMSO, 1.0 mL), and the resulting solution was sealed with a cap, vor-
texed for 15 s, and then placed in a 378C incubator. The decomposition
was monitored by HPLC at the indicated times. The resulting data were
normalized by dividing the ratios of meayamycin/rhodamine B or meaya-
mycin B/rhodamine B by the ratio of the first data point. HPLC monitor-
ing was performed on a Varian Pursuit XRs 5 C18 column, 250ꢂ10.0 cm,
2.5 mLminÀ1, 30% MeOH in H2O (containing 1% HCO2H) to 100%
MeOH linear gradient elution from 0.5 to 22 min, monitored at 230 nm
(for meayamycins) and 550 nm (for rhodamine B).
Scheme 9. Summary of this work.
Acknowledgements
We thank Professors Billy W. Day and Andreas Vogt (University of Pitts-
burgh) for helpful discussions. We thank Professor Stephen G. Weber
(University of Pittsburgh) for allowing us to use his Spectromax M5 plate
reader. B.J.A. and S.O. are recipients of a Graduate Excellence Fellow-
ship from the University of Pittsburgh. N.L.C. is thankful for the Arnold
and Mabel Beckman Scholar Award and the Averill Scholarship. We
thank Dr. Damodaran Krishnan and Dr. John Williams for assisting with
NMR spectroscopic and mass spectrometric (NIH grant S10RR017977-
01) analyses, respectively. This work was supported by the US National
Cancer Institute (R01 CA120792).
cell lines. As FR901464 binds SF3b and inhibits pre-mRNA
splicing, this observation could be very exciting because the
meayamycins should also inhibit the same process and ex-
hibit anticancer activity. Because there is no approved anti-
tumor drug that acts by inhibiting pre-mRNA splicing, the
mode of action, potency, and water solubility of meayamy-
cin B might further trigger interest in both oncology and
RNA biology.
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Received: August 19, 2010
Published online: November 19, 2010
904
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Chem. Eur. J. 2011, 17, 895 – 904