Antitumor compounds based on a natural product consensus pharmacophore

Article abstract of DOI:10.1021/jm8006195

We report the design and highly enantioselective synthesis of a potent analogue of the spliceosome inhibitor FR901464, based on a non-natural product scaffold. The design of this compound was facilitated by a pharmacophore hypothesis that assumed key interaction types that are common to FR901464 and an otherwise unrelated natural product (pladienolide). The synthesis allows for the preparation of numerous novel analogues. We present results on the in vitro activity for this compound against several tumor cell lines.

Full text of DOI:10.1021/jm8006195

6220
J. Med. Chem. 2008, 51, 6220–6224
Antitumor Compounds Based on a Natural Product Consensus Pharmacophore
Chandraiah Lagisetti,† Alan Pourpak,‡ Qin Jiang,‡ Xiaoli Cui,‡ Tinopiwa Goronga,† Stephan W. Morris,‡,§ and
Thomas R. Webb*,†
Department of Chemical Biology and Therapeutics and Departments of Pathology and Oncology, St. Jude Children’s Research Hospital,
MS 1000, 332 North Lauderdale, Memphis, Tennessee 38105
ReceiVed May 27, 2008
We report the design and highly enantioselective synthesis of a potent analogue of the spliceosome inhibitor
FR901464, based on a non-natural product scaffold. The design of this compound was facilitated by a
pharmacophore hypothesis that assumed key interaction types that are common to FR901464 and an otherwise
unrelated natural product (pladienolide). The synthesis allows for the preparation of numerous novel analogues.
We present results on the in vitro activity for this compound against several tumor cell lines.
Introduction
A novel mechanism of antitumor activity was recently
elucidated for two structurally distinct fermentation products
(Figure 1)1 pladienolide2 (a set of closely related compounds
that have been designated pladienolides A-G, isolated from
Streptomyces platensis)3 and FR901464 (isolated from Pseudomo-
Figure 1. Structures of pladienolide B (left) and FR901464 (right).
nas sp. no. 2663).4-6 These natural products have previously
been reported to have a similar novel effect on the cell cycle in
Activeanaloguesofpladienolidealsocontainsimilarfunctionality,2,3
mammalian cell lines, cell cycle arrest at the G1 and G2/M
phases.3,4 This effect on the cell cycle distinguished these
compounds from other antitumor agents that also inhibit
proliferation in tumors and act at previously identified molecular
targets.3-5 This interaction with the spliceosome leads to the
inhibition of mRNA splicing and the translation of unspliced
mRNA, which ultimately results in the observed effects on the
cell cycle.2,7 The first of these natural products to be discovered,
FR901464, showed activity in in vivo cancer animal models
but showed a relatively narrow therapeutic window.4 On the
other hand pladienolide-based compounds (first reported in
2004) have shown a very broad therapeutic window.2 Of the
seven pladienolides, pladienolide B was reported to have the
most promising anticancer activity in vivo, and it has recently
been reported that a derivative of pladienolide B has entered
human clinical trials for cancer.2
though less structure-activity information has been reported
on these more recently discovered compounds. This led us to
propose that the three critical requirements for activity of
FR901464 and pladienolide are an appropriately positioned
epoxy and carbonyloxy groups connected via an appropriately
constrained “linker” group of the right shape and length. We
also assumed that these groups would need to be conforma-
tionally constrained in a way similar to that seen in the natural
products. Information regarding the requirement of the linker
group is not available, but both natural products have significant
similarities in the structure of their diene linker group. In
FR901464, the linking group includes conformational constraints
consisting of two pyran 6-membered rings, whereas in pladi-
enolide the common diene constraint is an unsaturated 12-
membered macrocyclic lactone. Using this information, we
developed a hypothetical 3D pharmacophore model that allowed
us to propose that compounds of the general structures 1-3
(Figure 3) could have similar biological activity and could
therefore serve as starting points for the development of
compounds with potential for human cancer therapy. The
proposed 1 contains the unchanged methylpenta-2,4-diene group
and 5-oxopent-3-en-2-yl acetate group along with the modified
deoxy-spiro-epoxide group, with the pyran ring replaced by a
cis-1,4-substituted cyclohexyl group, whereas 2 and 3 include
additional polar modifications to the methylpenta-2,4-diene
group that are very synthetically assessable. These changes
(particularly for 1) are expected to impart similar conformational
presentation of the linker, the epoxy, and the carbonyloxy groups
as that found in FR901464.
Discussion
Motivated by the recognition that FR901464 and pladienolide
have a very similar (or possibly identical) mechanism of action,
we compared overlays of molecular models of the two natural
products. Upon inspection of these overlays we recognized that
despite the gross dissimilarities between FR901464 and pladi-
enolide, both molecules share certain common key features
(which are known to be critical to their activity) that can overlap
in low energy conformations in three dimensions (Figure 2). It
is known from previous studies that the orientation of the acetyl
group and the presence of the epoxide are important in
conferring activity to FR901464 analogues8,9 but that the
glycosidic hydroxyl group can be replaced by an alkoxy10 or a
methyl group11 to give more stable and more active compounds.
It is noted that these modifications (conversion of the pyran
ring oxygen to carbon, removal of two methyl groups, and
removal of a hydroxyl oxygen) eliminate six chiral centers when
compared to FR901464, which made the synthesis of all of our
target compounds readily achievable. At the outset of our project
it was not clear that compounds containing all of these
substantial modifications would be active, since there have been
no reports providing detailed structural information regarding
* To whom correspondence should be addressed. Phone: 901-495-3928.
Fax: 901-495-5715. E-mail: thomas.webb@stjude.org.
†
Department of Chemical Biology and Therapeutics.
Department of Pathology.
Department of Oncology.
‡
§
10.1021/jm8006195 CCC: $40.75
2008 American Chemical Society
Published on Web 09/13/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6221
Figure 2. A 3D representation of an overlay of the presumed key interaction groups in a low energy conformation of pladienolide and FR901464,
showing that the epoxy group and the carbonyloxy groups are the same distance in both molecules and that the methyl-2,4-pentadienyl groups can
readily adopt a similar positioning. The overlays were prepared using the Molecular Operating System (MOE 2007.09, Chemical Computing Group,
Inc.) using the Flexible Alignment function with both molecules following a conformational minimization using MOE default settings.
Scheme 2. Synthesis of Analogues 2 and 3a
Figure 3. Initial synthetic targets in our study that matched our
hypothetical pharmacophore.
Scheme 1. Synthesis of Analogue 1a
a Reagents and conditions: (a) (i) MsCl, Et3N, 0°C, CH2Cl2; (ii) NaN3,
DMF, 70°C; (b) (i) TFA, CH2Cl2; (ii) (S,Z)-4-(TBSO)pent-2-enoic acid,
HBTU, Hunig’s base, CH3CN; (c) Ph3P, H2O, THF; (d) 7b, 4-nitrophenyl
chloroformate, Et3N, THF; (e) Bu4NF, THF, 0°C; (f) Ac2O, Et3N, CH2Cl2;
(g) 4-nitrophenyl chloroformate, Et3N, THF; (h) 7b, DMAP, CH3CN.
found that application of the appropriate MacMillan conditions13
gave the desired product in good yield (though contaminated
with the starting material 4, which coeluted on silica chroma-
tography) and in high enantiomeric excess (91% based on chiral
SFC chromatography of a derivative; see Supporting Informa-
tion). The absolute stereochemistry of our ketoester product 5
a Reagents and conditions: (a) (2R,5R)-5-benzyl-3-methyl-(5-methylfuran-
2ylimidazolidin-4-one, tert-butyl Hantzsch ester, Cl3CCO2H, Et2O, 4 °C;
(b) NaH, (CH3)3SOI, DMSO; (c) DIBALH, THF, -78°C; (d) BH3 ·THF,
is based on numerous published examples of the stereochemistry
obtained using this organocatalyst on similar substrates.13,14 We
THF; (e) oxalyl chloride, DMSO, Hunig’s base, -78 °C, CH2Cl2; (f) (i)
Ph3PC(CH3)CO2Et, benzene; (ii) DIBALH, -78°C, toluene; (g) 2-mecap-
tobenzothiazole, DIAD, THF; (h) ammonium molybdate, H2O2, EtOH; (i)
(i) TFA, CH2Cl2; (ii) (S,Z)-4-(TBSO)pent-2-enoic acid, HBTU, Hunig’s
base, CH3CN; (j) LHMDS, 7a, THF, -78°C to room temp; (k) Bu4NF,
THF, 0°C; (l) Ac2O, Et3N, CH2Cl2.
then turned our attention to the diastereoselective introduction
of the desired spiroepoxide; we expected that the addition of
dimethylsulfoxonium methylide15 would occur from an axial
approach on the ketone carbonyl of 5 to give primarily the
desired diastereomer (epoxyester 6). We found that this was
indeed the case; in fact, we did not detect any of the undesired
epoxide in the crude reaction mixture, and the product was
obtained in excellent yield (see Supporting Information for
details on the NOE experiments used for assigning the relative
stereochemistry). This approach allowed us to set two of the
three chiral centers in our targets and provided intermediates
that were appropriately functionalized for conversion to our
ultimate targets 1-3.
the interaction of FR901464 and pladienolide with the spliceo-
some SF3b subunit or of the synthesis of compounds that are
similar to 1-3. We believed, however, that there was a
reasonable likelihood that one or more of our compounds could
demonstrate substantial activity, especially given that there is
an example of a FR901464 analogue with a cell-based growth
inhibition IC50 as low as 10 pM.11
There have been numerous synthetic approaches to FR901464;
however, since these synthetic approaches target the much more
complex natural product, these routes were not highly instructive
for the construction of our synthetic targets 1-3. Our approach
to these analogues is shown in Schemes 1 and 2. We found the
readily available inexpensive 4 (which has commercial applica-
tion as an insect repellant) to be an attractive starting material
for the left-hand portion of our synthetic targets 1-3. To
introduce the desired stereochemistry, we explored numerous
transition metal catalysts for the enantioselective reduction of
4 to ketoester 5; for example, we investigated the ruthenium(II)
BINAP complex as a chiral hydrogenation catalyst, using the
published conditions, but found no reduction or at higher
temperatures the undesired reduction of the ketone carbonyl in
preference to the carbon-carbon double bond.12 However, we
The synthesis of the 1 could then be accomplished starting
with the aldehyde 9 derived from the commercially available
cis-cyclohexane derivative 8, followed by homologation to give
trans alcohol 10 cleanly in high overall yield.16 This compound
was converted to the corresponding 2-thiobenzthioazole deriva-
tive 11 using Mitsunobu conditions17 and subsequently oxidized
to the corresponding 2-sulfonylbenzothioazole derivative 12.10
Removal of the BOC group in 12 and coupling of the optically
pure (S,Z)-4-(dimethyl-tert-butylsilyloxy)pent-2-enoic acid (pre-
pared according to the known procedure)18 gave the desired
functionalized amide 13 in excellent overall yield. Coupling of
this reagent with epoxyaldehyde 7a10 to give diene 14 followed
by deprotection and acylation of 15 gave final compound 1
cleanly in acceptable yield (the overall yield of 1 from 4 is
2.7%).

6222 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Brief Articles
Table 1. In Vitro Cytotoxicity of Compound 1 after a 72 h Exposure
published IC50 (µM)
a similar accumulation of cells in the G2/M phases of the cell
cycle, with a simultaneous decrease in the fraction of cells in S
phase.
cell line
pladienolide B2,3
FR9014645
IC50 ( SEM (µM)a
1
A549
0.0014
NAb
0.0013
NAb
4.65 ( 0.37
0.10 ( 0.002
0.13 ( 0.03
2.29 ( 0.38
1.98 ( 0.63
2.12 ( 0.57
2.03 ( 0.20
Conclusion
JeKo-1
JVM-2
MCF-7
OVCAR-3
PC-3
NAb
NAb
We report the enantioselective and diastereospecific synthesis
of 1 that shows an activity profile similar to those of FR901464
and pladienolide but with reduced potency. The design of this
compound is based on insights from the common positioning
of similar interacting groups from these unrelated natural
products. By judicious modification of atom types and the
removal of groups that did not appear to impart interaction
features or conformational constraints, we were able to develop
compounds that showed significant antitumor activity but that
have only three stereocenters (compared to nine in FR901464)
and are therefore much more synthetically attractive. Since 1
displays a similar bioactivity profile, it is quite possible that it
also acts in a manner analogous to FR901464 and pladienolide,
by targeting the SF3b subunit of the spliceosome. The synthetic
scheme that we have developed allows for the introduction of
numerous modifications that will allow us to fully elucidate the
structure-activity relationships in this series and will also allow
us to prepare compounds that have an improved in vivo profile,
in order to develop lead compounds as potential human
therapeutics.
0.0004
0.0004
0.0023
0.00086
0.0018
NAb
NAb
WiDr
NAb
a Experimental IC50 values represent the mean ( SEM from three
independent experiments. b NA ) data not available.
In order to obtain the carbamate 2 and carbonate 3, as shown
in Scheme 2, we used the commercially available alcohol 16 to
prepare the azide 17, which could be deprotected and converted
to the amide azide 18 in excellent yield. This derivative could
be reduced with triphenylphosphine on solid support to cleanly
give amine 19,19 which was then converted to the carbamate 2
in excellent yield20 (the overall yield of 2 from 16 is 12.6%).
In a similar way the alcohol 16 was converted to the p-
nitrophenylcarbonate intermediate 22, which was deprotected
and underwent amidation with (S,Z)-4-(dimethyl-tert-butylsi-
lyloxy)pent-2-enoic acid (prepared according to a published
method)10 to give amide 23 in good yield. This amide could
then be coupled with alcohol 7b (from Scheme 1) to give the
carbonate 3 (the overall yield of 3 from 16 is 14.2%).
Experimental Section
(S)-Butyl 6,6-Dimethyl-4-oxotetrahydro-2H-pyran-2-carboxy-
late (5). A solution of commercially available butopyronoxyl 4 (230
mg, 1.01 mmol) and (2R,5R)-5-butylmethyl-2-(5-methylfuran-2-
yl)imidazolidin-4-one (55 mg, 0.20 mmol) in anhydrous Et2O (4.6
mL) was stirred and cooled at ice bath temperature (0-4 °C) as
di-tert-butyl 2,6-dimethyldicarboxylate (346 mg, 1.11 mmol) and
trichloroacetic acid (TCA) (32 mg, 0.20 mmol) were sequentially
added. The resulting yellow solution was allowed to stir for 3 days
at 4 °C. The reaction mixture was passed through a short pad of
silica gel and eluted with Et2O (60 mL). Concentration of the Et2O
and purification of the residue by flash chromatography (20%
EtOAc in hexane) gave 204 mg (88%) of keto ester 5 as an oil.
[R]2D5 +15.7° (c 2.1 in CHCl3); 1H NMR (400 MHz, CDCl3) δ
4.46 (dd, J ) 4.0, 11.0 Hz, 1H), 4.19 (td, J ) 2.7, 6.7 Hz, 2H),
2.64-2.47 (m, 3H), 2.32 (dd, J ) 1.6, 14.0 Hz, 1H), 1.68-1.59
(m, 2H), 1.46 (s, 3H), 1.43-1.33 (m, 2H), 1.23 (s, 3H), 0.94 (t, J
) 7.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 205.2, 170.1, 75.2,
70.0, 65.4, 52.9, 43.5, 30.6, 30.5, 23.7, 19.0, 13.6; IR (neat film)
2965, 2875, 1734, 1725, 1464, 1379, 1241, 1181 cm-1; HRMS
(FAB) m/z calcd for C12H21O4 (M + 1)+ 229.1440, found 229.1440.
(3R,5S)-Butyl 7,7-Dimethyl-1,6-dioxaspiro[2.5]octane-5-carboxy-
late (6). A suspension of sodium hydride (96 mg, 2.40 mmol, 60%
in mineral oil) in anhydrous DMSO (4.2 mL) was stirred at room
temperature as trimethylsulfoxonium iodide (491 mg, 2.40 mmol)
was added. After 30 min, the keto ester 5 (500 mg, 2.19 mmol) in
DMSO (1.0 mL) was added dropwise. After 1 h at room temper-
ature, the reaction was quenched with ice cold H2O (5 mL). The
product was then extracted with EtOAc (3 × 30 mL), and the
combined extracts were washed with H2O (2 × 15 mL) and brine
(20 mL) and dried over MgSO4. Evaporation of the solvent and
purification by flash chromatography (20% EtOAc in hexane) gave
430 mg (81%) of epoxide derivative 6 as a liquid. [R]2D5 +41.5° (c
Seven human cancer cell lines were then used to evaluate
the cytotoxic potential of 1-3; however, only 1 showed potent
activity. Compound 2 did not show any significant cytotoxic
activity in our initial tumor line screen (in A549, COS-7, MCF-
7, WiDr, and NIH3T3 cells) at concentrations up to 5 µM. In
this screen, 3 inhibited cell growth by only 10-20% at the
highest concentration tested of 5 µM, but only in the A549 and
MCF-7 lines. Compound 1 was then screened in an expanded
tumor panel consisting of the following cell lines: A549 lung
cancer, WiDr colorectal cancer, MCF-7 breast cancer, PC-3
prostate cancer, OVCAR-3 ovarian cancer, and JeKo-1 and
JVM-2 mantle cell lymphoma cell lines. An XTT colorimetric
cytotoxicity assay was employed to estimate the IC50 values
(Table 1). The cytotoxicity profile of 1 against the A549, MCF-
7, OVCAR-3, PC-3, and WiDr cancer cell lines revealed IC50
values of 4.65, 2.29, 1.98, 2.12, and 2.03 µM, respectively
(Table 1). Although 1 is substantially less potent than the natural
products against several of the cell lines tested, it shows potent
and selective killing of two tumor cell lines (JeKo-1 and JVM-
2, IC50 values of 100 and 130 nM, respectively).
To further investigate the biological activity of 1, the effect
of the compound on the cell cycle was evaluated. Following
an 18 h incubation of 1 with the A549 lung cancer, JeKo-1
mantle cell lymphoma, and WiDr colorectal cancer cell lines,
all three lines accumulated in the G2/M phases of the cell
cycle (Figure 4). The degree of G2/M cell cycle arrest
correlated with the sensitivity of the cell lines to the cytotoxic
activity of 1 (JeKo-1 > WiDr > A549). In addition to the
increased fraction of cells in the G2/M phase of the cell cycle,
a concomitant decrease in the S-phase fraction was observed
for all three cell lines following treatment with 1. As reported
in the literature,3,4,7 the spliceosome inhibitor compounds
pladienolide B, FR901464, and spliceostatin A all also induce
1
2.2, CHCl3); H NMR (400 MHz, CDCl3) δ 4.53 (dd, J ) 2.7,
12.1 Hz, 1H), 4.14 (t, J ) 6.7 Hz, 2H), 2.61 (dd, J ) 4.5, 13.9 Hz,
2H), 2.14-2.02 (m, 2H), 1.65-1.58 (m, 2H), 1.47 (ddd, J ) 4.2,
6.7, 10.9 Hz, 1H), 1.39 (s, 3H), 1.38-1.33 (m, 2H), 1.34 (s, 3H),
1.16 (dd, J ) 2.0, 14.0 Hz, 1H), 0.93 (t, J ) 7.3 Hz, 3H); 13C
NMR (CDCl3, 100 MHz) δ 171.5, 74.0, 68.3, 64.9, 55.2, 51.1, 42.2,
35.2, 31.0, 30.5, 23.3, 19.0, 13.7; IR (neat film) 2966, 2874, 1756,
1465, 1378, 1218; 1180 cm-1; HRMS (FAB) m/z calcd for
C13H23O4 (M + 1)+ 243.1596, found 243.1595.

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6223
Figure 4. Cell cycle analysis following treatment with 1. The (A) A549, (B) JeKo-1, and (C) WiDr cancer cell lines were treated with 1 for 18 h,
then permeabilized and stained with propidium iodide for cell cycle analysis by flow cytometry.
(3R,5S)-7,7-Dimethyl-1,6-dioxaspiro[2.5]octane-5-carbalde-
hyde (7a). To a stirred solution of ester 6 (1 g, 4.1 mmol) in
anhydrous hexane (15 mL) and THF (9 mL) was added DIBALH
(4.7 mL, 4.75 mmol, 1.0 M in hexane) at -78 °C. The resulting
solution was allowed to stir for 30 min at -78 °C, by which time
TLC indicated that the alcohol has been consumed. MeOH (1.5
mL) was added, and the mixture was diluted with saturated aqueous
sodium/potassium tartarate (30 mL) and EtOAc (80 mL). The
solution was stirred for 15 min at room temperature to get a clear
solution. The organic layer was separated, and the aqueous layer
was extracted with EtOAc (2 × 100 mL). The combined organic
layers were washed with brine (45 mL) and dried over Na2SO4.
Evaporation of the solvent and purification of the residue by flash
chromatography (50% EtOAc in hexane) gave 390 mg (56%) of
with brine (40 mL) and dried over MgSO4. Evaporation of the
solvent and purification of the residue by flash chromatography
(25% EtOAc in hexane) gave 70 mg of diene 14 as a liquid. H
1
NMR (400 MHz, CDCl3) δ 6.27 (d, J ) 15.7 Hz, 1H), 5.99 (dd, J
) 7.8, 11.6 Hz, 1H), 5.61-5.45 (m, 5H), 4.48-4.44 (m, 1H), 4.03
(s, 1H), 2.57 (s, 2H), 2.08 (t, J ) 7.2 Hz, 2H), 2.01-1.89 (m, 2H),
1.72 (s, 3H), 1.57-1.53 (m, 6H), 1.46-1.36 (m, 1H), 1.40 (s, 3H),
1.29-1.27 (m, 6H), 1.18-1.08 (m, 4H), 0.87 (s, 9H), -0.01 (d, J
) 5.3 Hz, 6H); IR (neat film) 3394, 2926, 2856, 1735, 1666, 1629,
1532, 1448, 1320, 1244, 1049 cm-1; HRMS (ESI) m/z calcd for
C31H54NO4Si (M + 1)+ 532.3822, found 532.3825.
(S,Z)-N-((1R,4R)-4-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-
dioxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)cyclohexyl)-4-
hydroxypent-2-enamide (15). A solution of TBS protected diene
14 (13 mg, 0.02 mmol) in THF (1.0 mL) was stirred and cooled at
0 °C as TBAF (49 µL, 0.04 mmol, 1.0 M in THF) was added. The
resulting yellow solution was allowed to stir for 2 h at room
temperature, by which time the TLC showed that all of starting
material has been consumed. Evaporation of THF and purification
of the residue by flash chromatography (60-80% EtOAc in hexane)
gave 9.5 mg (95%) of 15 as a liquid. [R]2D5 +78° (c 0.86, CHCl3);
1H NMR (400 MHz, CDCl3) δ 6.27 (d, J ) 15.7 Hz, 1H), 6.15
(dd, J ) 5.4, 11.9 Hz, 1H), 5.87 (bd, 1H), 5.75 (dd, J ) 1.6, 12.0
Hz, 1H), 5.56 (dd, J ) 6.7, 15.6 Hz, 1H), 5.47 (t, J ) 7.7 Hz, 1H),
4.80-4.73 (m, 1H), 4.50-4.43 (m, 1H), 4.09-4.02 (m, 1H), 2.57
(s, 2H), 2.09 (t, J ) 7.3 Hz, 2H), 2.04-1.88 (m, 2H), 1.72 (s, 3H),
1.69-1.57 (m, 6H), 1.52-1.44 (m, 1H), 1.40 (s, 3H), 1.34 (d, J )
6.7 Hz, 3H), 1.28 (s, 3H), 1.26-1.23 (m, 2H), 1.18-1.11 (m, 2H);
13C NMR (100 MHz, CDCl3) δ 165.9, 150.0, 136.1, 134.1, 131.6,
127.1, 123.1, 73.0, 69.6, 64.6, 55.7, 51.0, 45.8, 42.4, 38.6, 36.4,
34.1, 31.5, 29.2, 27.8, 27.7, 23.8, 22.8, 12.5; IR (neat film) 3307,
1
aldehyde as a liquid. H NMR (400 MHz, CDCl3) δ 9.65 (s, 1H),
4.35 (dd, J ) 3.0, 12.0 Hz, 1H), 2.60 (q, J ) 4.6 Hz, 2H),
2.04-1.86 (m, 2H), 1.46-1.40 (m, 1H), 1.39 (s, 3H), 1.33 (s, 3H),
1.20 (dd, J ) 2.0, 14.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
201.7, 74.0, 73.6, 54.7, 51.1, 42.5, 32.6, 30.6, 30.9, 23.3; IR (neat
film) 2970, 2913, 1732, 1184, 1084 cm-1; HRMS (FAB) m/z calcd
for C9H14O3 (M+) 170.0943, found 170.0943.
7,7-Dimethyl-1,6-dioxaspiro[2.5]octan-5-yl)methanol (7b). A so-
lution of epoxy ester 6 (320 mg, 1.32 mmol) in anhydrous THF
(5.0 mL) was stirred and cooled at -78 °C as DIBALH (4.6 mL,
1.0 M in hexane) was slowly added dropwise. The solution was
stirred at the same temperature for 3 h. Saturated aqueous NH4Cl
(4 mL) was introduced to destroy excess reagent at -78 °C. This
mixture was allowed to warm to room temperature and was stirred
for 30 min to get a clear mixture. The organic layer was then
separated, and the aqueous layer was extracted with EtOAc (3 ×
30 mL). The combined layers were washed with saturated aqueous
NaHCO3 (15 mL) and brine (15 mL) and dried over Na2SO4.
Concentration and purification by flash chromatography (50%
EtOAc in hexane) gave the alcohol 7b in quantitative yield as a
liquid. 1H NMR (400 MHz, CDCl3) δ 4.07-3.98 (m, 1H),
3.70-3.61 (m, 1H), 3.53-3.43 (m, 1H), 2.58 (q, J ) 4.6 Hz, 2H),
2.15 (t, J ) 5.8 Hz, 1H), 1.99-1.88 (m, 2H), 1.38 (s, 3H), 1.25 (s,
3H), 1.17 (dd, J ) 2.0, 13.9 Hz, 1H), 1.08-1.01(m, 1H); 13C NMR
(100 MHz, CDCl3) δ 73.1, 68.7, 65.9, 55.5, 51.0, 42.7, 33.7, 31.3,
23.8; IR (neat film) 3435, 2973, 2914, 1376, 1250, 1221, 1186,
1093, 1052 cm-1; HRMS (FAB) m/z calcd for C9H16O3 (M)+
172.1100, found 172.1099.
(S,Z)-4-(tert-Butyldimethylsilyloxy)-N-((1R,4R)-4-((2E,4E)-5-
((3R,5S)-7,7-dimethyl-1,6-dioxaspiro[2.5]octan-5-yl)-3-methylpenta-
2,4-dienyl)cyclohexyl)pent-2-enamide (14). A solution of sulfone
derivative 13 (430 mg, 0.75 mmol) in anhydrous THF (3 mL) was
stirred and cooled at -78 °C as LHMDS (0.92 mL, 0.92 mmol,
1.0 M in THF) was added dropwise. The solution turned orange
during the addition. After the mixture was stirred for 10 min at
-78 °C, aldehyde 7a (75 mg, 0.75 mmol) in anhydrous THF (2
mL) was introduced dropwise. The resulting suspension was again
stirred for 1 h at -78 °C. The mixture was then slowly allowed to
warm to room temperature and stirred for 2 h. Saturated aqueous
NH4Cl solution (15 mL) was added, and the product was extracted
with EtOAc (3 × 35 mL). The combined extracts were washed
2926, 2857, 1656, 1622, 1536, 1447, 1372, 1258, 1183, 1108 cm-1
;
HRMS (ESI) m/z calcd for C25H40NO4 (M + 1)+ 418.2957, found
418.2948.
(S,Z)-5-((1R,4R)-4-((2E,4E)-5-((3R,5S)-7,7-Dimethyl-1,6-
dioxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dienyl)cyclohexylami-
no)-5-oxopent-3-en-2-yl Acetate (1). A solution of alcohol 15 (7
mg, 0.01 mmol) in CH2Cl2 (0.9 mL) was allowed to stir at 0 °C as
Et3N (16 µL, 0.11 mmol) and DMAP (2.0 mg, 0.01 mmol) were
sequentially added. After 5 min, acetic anhydride (5.4 µL, 0.05
mmol) was added using a micropipet. The solution was stirred for
1 h at 0 °C. Evaporation of the solvent and purification of the
residue by flash chromatography (50% EtOAc in hexane) gave 6.1
1
mg (79%) of 1 as a viscous oil. [R]2D5 -1.0° (c 0.21, CHCl3); H
NMR (400 MHz, CDCl3) δ 7.04 (br d, 1H), 6.27 (d, J ) 15.7 Hz,
1H), 5.81 (m, 2H), 5.66 (dd, J ) 9.2, 11.6 Hz, 1H), 5.55 (dd, J )
6.7, 15.7 Hz, 1H), 5.49 (t, J ) 7.5 Hz, 1H), 4.50-4.43 (m, 1H),
4.12-4.06 (m, 1H), 2.57 (s, 2H), 2.12-2.06 (m, 2H), 2.07 (s, 3H),
2.01-1.88 (m, 2H), 1.72 (s, 3H), 1.72-1.66 (m, 1H), 1.64-1.57
(m, 6H), 1.40 (s, 3H), 1.36 (d, J ) 6.4 Hz, 3H), 1.28 (s, 3H),
1.27-1.19 (m, 3H), 1.15 (dd, J ) 2.0, 13.8 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 171.1, 164.8, 137.9, 136.2, 133.8, 131.9,
126.9, 125.4, 73.0, 69.6, 69.2, 55.6, 51.0, 45.5, 42.4, 38.6, 36.4,
34.2, 31.5, 29.5, 29.4, 27.8, 27.6, 23.7, 21.2, 20.3, 12.5;21 IR (neat
film) 3310, 2928, 2856, 1660, 1626, 1534, 1445, 1365, 1252, 1219,

6224 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Brief Articles
1185, 1116, 1072 cm-1; HRMS (ESI) m/z calcd for C27H42NO5
(M + 1)+ 460.3063, found 460.3049.
(6) Nakajima, H.; Takase, S.; Terano, H.; Tanaka, H. New antitumor
substances, FR901463, FR901464 and FR901465. III. Structures of
FR901463, FR901464 and FR901465. J. Antibiot. 1997, 50, 96–99.
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Tani, T.; Horinouchi, S.; Yoshida, M. Spliceostatin A targets SF3b
and inhibits both splicing and nuclear retention of pre-mRNA. Nat.
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J. Am. Chem. Soc. 2001, 123, 9974–9983.
(9) Motoyoshi, H.; Horigome, M.; Ishigami, K.; Yoshida, T.; Horinouchi,
S.; Yoshida, M.; Watanabe, H.; Kitahara, T. Structure-activity
relationship for FR901464: a versatile method for the conversion and
preparation of biologically active biotinylated probes. Biosci. Bio-
technol. Biochem. 2004, 68, 2178–2182.
(10) Motoyoshi, H.; Horigome, M.; Watanabe, H.; Kitahara, T. Total
synthesis of FR901464: second generation. Tetrahedron 2006, 62,
1378–1389.
(11) Albert Brian, J.; Sivaramakrishnan, A.; Naka, T.; Czaicki Nancy, 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.
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(2S,Z)-5-((1s,4R)-4-((((7,7-Dimethyl-1,6-dioxaspiro[2.5]octan-5-
yl)methoxy)carbonylamino)methyl)cyclohexylamino)-5-oxopent-3-
en-2-yl Acetate (2). Acetylation of 21 (6 mg, 0.01 mmol) used the
same procedure as for conversion of 15 to the corresponding acetate
1, to give 5.9 mg (90%) of 2 as an oil. 1H NMR (400 MHz, CDCl3)
δ 7.19 (br d, 1H), 5.82-5.74 (m, 2H), 5.65 (dd, J ) 9.1, 11.8 Hz,
1H), 4.89 (br t, 1H), 4.17-4.04 (m, 4H), 3.16-3.08 (m, 2H), 2.57
(q, J ) 4.6 Hz, 2H), 2.09 (s, 3H), 1.97 (d, J ) 13.9 Hz 1H), 1.84
(dd, J ) 11.6, 13.5 Hz, 1H), 1.77-1.69 (m, 2H), 1.68-1.59 (m,
4H), 1.37-1.34 (m, 6H), 1.28-1.24 (m, 6H), 1.18-1.11 (m, 2H);
13C NMR (100 MHz, CDCl3) δ 164.9, 152.6, 137.3, 125.8, 73.1,
69.1, 67.1, 55.4, 50.9, 45.8, 45.2, 42.5, 36.2, 31.3, 29.7, 29.1, 25.4,
25.1, 23.6, 21.3;21 IR (neat film) 3334, 2928, 2859, 1717, 1666,
1533, 1452, 1371, 1247, 1122, 1047 cm-1; HRMS (ESI) m/z calcd
for C24H39N2O7 (M + 1)+ 467.2757, found 467.2759.
(2S,Z)-5-((1S,4R)-4-((((7,7-Dimethyl-1,6-dioxaspiro[2.5]octan-5-
yl)methoxy)carbonyloxy)methyl)cyclohexylamino)-5-oxopent-3-en-
2-yl Acetate (3). Acetylation of 25 (7 mg, 0.01 mmol) was achieved
following the same procedure used for conversion of 15 to the
1
corresponding acetate 1, to give 6 mg (78%) of 3 as an oil. H
NMR (400 MHz, CDCl3) δ 7.20 (br d, 1H), 5.84-5.74 (m, 2H),
5.65 (dd, J ) 9.1, 11.8 Hz, 1H), 4.20-4.08 (m, 4H), 4.02 (d, J )
6.9 Hz, 2H), 2.57 (q, J ) 4.6 Hz, 2H), 2.06 (s, 3H), 2.04-1.85 (m,
2H), 1.84-1.80 (m, 1H), 1.78-1.63 (m, 6H), 1.44-1.35 (m, 8H),
1.25 (s, 3H), 1.20-1.12 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
171.4, 164.9, 155.3, 137.4, 125.6, 73.3, 71.7, 70.4, 69.1, 66.4, 55.4,
50.9, 45.3, 42.4, 35.1, 34.3, 31.2, 28.9, 28.8, 24.4, 24.2, 23.5, 21.2,
20.3;21 IR (neat film) 3326, 2932, 1742, 1668, 1631, 1532, 1451,
1371, 1252, 1119, 1048 cm-1; HRMS (ESI) m/z calcd for
C24H38NO8 (M + 1)+ 468.2597, found 426.2584.
Acknowledgment. This work was supported by NCI Cancer
Center Core Grant CA21765 and by the American Lebanese
Syrian Associated Charities (ALSAC), St. Jude Children’s
Research Hospital.
Supporting Information Available: General experimental
methods, procedures for the synthesis of 9-13 and 17-25, and
full experimental details for the cell cycle analysis and the in vitro
cytotoxicity assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
(20) Pessah, N.; Reznik, M.; Shamis, M.; Yantiri, F.; Xin, H.; Bowdish,
K.; Shomron, N.; Ast, G.; Shabat, D. Bioactivation of carbamate-based
20(S)-camptothecin prodrugs. Bioorg. Med. Chem. 2004, 12, 1859–
1866.
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JM8006195