Total synthesis and selective activity of a new class of conformationally restrained epothilones

Article abstract of DOI:10.1002/chem.200701143

Stereoselective total syntheses of two novel conformationally restrained epothilone analogues are described. Evans asymmetric alkylation, Brown allylation, and a diastereoselective aldol reaction served as the key steps in the stereoselective synthesis of

Full text of DOI:10.1002/chem.200701143

DOI: 10.1002/chem.200701143
Total Synthesis and Selective Activity of a NewClass of Conformationally
Restrained Epothilones
Mamoun M. Alhamadsheh,[a, c] Shuchi Gupta,[a, c] Richard A. Hudson,[a]
Lalith Perera,[b] and L. M. Viranga Tillekeratne*[a]
Abstract: Stereoselective total synthe-
ses of two novel conformationally re-
strained epothilone analogues are de-
scribed. Evans asymmetric alkylation,
Brown allylation, and a diastereoselec-
tive aldol reaction served as the key
steps in the stereoselective synthesis of
one of the two key fragments of the
Enzyme resolution was employed to
obtain the second fragment as a single
enantiomer. The molecules were as-
sembled by esterification, followed by
ring-closing metathesis. In preliminary
cytotoxicity studies, one of the ana-
logues showed strong and selective
growth inhibitory activity against two
leukemia cell lines over solid human
tumor cell lines. The precise biological
mechanism of action and high degree
of selectivity of this analogue remain
to be examined.
Keywords: antitumor
agents
·
epothilone analogues · macrolide ·
natural products · total synthesis
convergent
synthetic
approach.
Introduction
The clinically used anticancer drug paclitaxel (taxol) exerts
its cytotoxic activity by a mechanism invoking the stabiliza-
tion of microtubules.[1,2] However, the susceptibility of the
drug to multiple drug resistance has stimulated extensive re-
search on molecules with a paclitaxel-like mechanism of
action.[3,4] The most extensively investigated of the new mo-
lecular entities discovered are the epothilones, a class of
macrolide natural products initially isolated from a soil bac-
terium.[3,5,6] Some epothilones exhibit more potent anticanc-
er activity than paclitaxel and have favorable attributes that
may improve their pharmacological profile.[7,8]
Replacing the thiazole ring in epothilone B by methylpyr-
idine rings, in which the nitrogen atom is ortho to the pyri-
dine-vinyl linker attachment, produced analogues with
higher potencies against drug-resistant tumor cells.[9] The
role of the N atom at an ortho-position in the heterocycle
has been attributed to a hydrogen-bond interaction between
the thiazole N atom and a protonated His residue in tubu-
lin.[10–13] The 16-Me and 19-H adopt a syn orientation, liber-
ating the thiazole N atom from the steric bulk of the 16-Me,
making it more accessible for potential hydrogen bonding.
Rigidification of the aromatic side chain through incorpora-
tion of the C-16–C-17 olefinic double bond into a fused het-
eroaromatic ring system such as benzothiazole resulted in
analogues more potent than parent epothilone B and D.[14,15]
Interestingly, in contrast to the pyridine analogues, there
was no dependence of the tubulin-polymerizing activity of
these rigidified analogues on the position of the nitrogen
atom in the heterocycles.[16] Antiproliferative activity, on the
other hand, showed a strong dependence on the position of
[a] Dr. M. M. Alhamadsheh, Dr. S. Gupta, Dr. R. A. Hudson,
Dr. L. M. V. Tillekeratne
Department of Medicinal and Biological Chemistry
College of Pharmacy, The University of Toledo
Toledo, OH 43606 (USA)
Fax : (+1)419-530-7946
E-mail: ltillek@utnet.utoledo.edu
[b] Dr. L. Perera
Laboratory of Structural Biology
National Institute of Environmental Health Sciences
Research Triangle Park, NC 27709 (USA)
[c] Dr. M. M. Alhamadsheh, Dr. S. Gupta
Current address: Department of Chemistry
Portland State University, P.O. Box 751
Portland, OR 97207 (USA)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author: General meth-
ods, syntheses of intermediates 3 and 4, spectral data for all com-
pounds, and cytotoxicity data for compound 1b.
570
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FULL PAPER
the nitrogen atom, as in the pyridine analogues. Attempts to
rigidify the southern C-1–C-8 sector of the molecule have
resulted in loss of biological activity.[17,18] Analogues made
by rigidification of some parts of the northern C-9–C-13
sector have retained biological activity.[19–21] A series of con-
formationally rigidified analogues bridging the northern and
southern sectors across the macrocycle were prepared re-
cently. However, their biological activities have not been re-
ported.[22]
The extensive database on SAR for epothilone has led to
the discovery of potent epothilones.[23,24] At least five ana-
logues are currently in different stages of clinical develop-
ment.[7] Most of the current potent epothilone analogues
bear close structural resemblance to the parent compounds.
While largely uninvestigated, compounds with more strin-
gent structural modifications of the epothilone scaffold may
produce new lead structures with altered pharmacological
profiles for drug discovery.[25,26]
Figure 1. Energy-minimized epothilone D (gray); X-ray crystal structure
of 14-(S)-methyl-epothilone D (cyan); compound 1b (green); and C-14-
(R)-1b (magenta). The nitrogen atoms are shown in blue, the oxygen
atoms are in red, and the sulfur atoms are in yellow. Main-ring heavy
atoms were used in the alignment and the energy-minimized epothilone
D was used as the reference structure. The figure was created by using
Sybyl 7.3 (Tripos, St. Louis, MO).
Results and Discussion
We designed a new class of conformationally restrained ana-
logues of epothilones (compounds 1a and 1b) by restricting
14-(R) epimer are aligned with the minimum-energy struc-
ture of epothilone D by using the heavy atoms in the large
ring and the root-mean-square deviations were found to be
0.287 and 0.283 Š for 1b and its C-14-(R) epimer, respec-
tively. In comparison, the root mean deviation of the heavy-
atom alignment for epothilone D and the X-ray crystal
structure of 14-(S) -methyl-epothilone D was found to be
0.186 Š. As can be seen from Figure 1, the side chain heter-
oaromatic ring segment in 1b, compared to its C-14-(R)
epimer, can adopt an overall configuration close to those of
epothilone D and 14-(S)-methyl-epothilone D. The confor-
mational flexibility of the heteroaromatic ring-containing
side chains of these analogues permit the N atoms in the
heteroaromatic rings to come within 1 Š of each other. In
contrast, the heteroaromatic ring-containing side chain of
the C-14-(R) epimer of 1b is virtually perpendicular to the
plane of the macrocycle.
A convergent synthetic approach with ring-closing meta-
thesis (RCM) of 2a and 2b in the final step was adapted
(Scheme 1). Retrosynthetic disconnection of 2a and 2b led
to the three key synthons 3, 4, and either 5a or 5b. The ste-
reoselective aldol coupling product between aldehyde 3 and
ketone 4 would be converted to the corresponding carboxyl-
ic acid and then esterified with alcohols 5a and 5b to give
2a and 2b. This intermediate would then undergo RCM to
yield the desired macrolide skeleton.
the mobility of the aromatic side chain. In doing so, we re-
tained the C-1–C-8 sector unchanged, in view of its seeming-
ly crucial role in biological activity. A single methylene
bridge was introduced between C-14 and C-17. The resulting
cyclopentene moiety, which incorporated the C-16–C-17
double bond, was designed to rigidify the side chain while
still permitting sufficient mobility of the pyridine ring to
allow for the preferred N-atom orientation for a hydrogen
bonding interaction with the receptor.[11]
The choice of a S configuration at the new chiral center at
C-14 in the designed analogues was based on molecular
modeling studies, and is consistent with the observations of
Taylor et al.,[27,28] whose extensive studies on the bioactive
conformation of epothilones have shown that the (S)-C-14-
Me analogue of epothilone D is far more active than the
corresponding R epimer. We performed energy minimiza-
tions for 1b (C-14-S) as well as for its C-14-(R) epimer and
for epothilone D by using Gaussian 03[29] at the B3LYP/6—
Aldehyde 3 was made by using the Evans asymmetric al-
kylation protocol (Scheme 2).[30] Wittig olefination of the
keto acid 6 gave the alkene 7. It was converted to the alde-
hyde 3 via the imide 9, following Schinzerꢁs procedure.[31]
The bis-silyl ether ketone 4 was made as shown in
Scheme 3. Compound 10 was synthesized as reported earli-
er.[32] Selective reduction of the aldehyde group of 10 with
311++G(d,p) level (Figure 1). Compound 1b was found to
A
be more stable than the C-14-(R) epimer by 8.8 kcalmolÀ1.
The main ring configuration in the minimized structure of
epothilone D remained very similar to the X-ray crystal
structure of 14-(S)-methyl-epothilone D (Figure 1).[27] In
Figure 1, the energy-minimized structures of 1b and its C-
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571

L. M. V. Tillekeratne et al.
Scheme 4. a) LDA, THF, À788C, 83%; b) TrocCl, pyridine, CH2Cl2, 08C,
1 h, 93%; c) CSA, CH2Cl2/MeOH, 08C, 7 h, 87%; d) DMP, CH2Cl2, RT,
15 min; e) NaClO2, NaH2PO4, H2O/tBuOH, 2-methyl-2-butene, RT, 1 h,
90% (2steps). TrocCl =2,2,2-trichloroethyl chloroformate, CSA=(1S)-
(+)-10-camphorsulfonic acid, LDA=lithium diisopropylamide, DMP=
Dess–Martin periodinane.
Scheme 1. Retrosynthetic analysis of the designed epothilones.
lective desilylation of 14, followed by sequential DMP and
Pinnickꢁs oxidations gave the carboxylic acid 17.
The synthesis of enantiomerically pure b-ketoalcohols 22a
and 22b by means of diastereoselective reduction of the cor-
responding b-ketoesters 21a and 21b seemed a logical and
convenient approach to this moiety as the racemate of these
b-ketoesters could be very conveniently prepared by aldol
cyclization of the corresponding g-aryl-substituted b-ketoest-
ers 20a and 20b (Scheme 5). We investigated the conversion
of 21a and 21b to 22a and 22b in high enantiomeric purity.
Scheme 2. a) MeP+(Ph)3BrÀ, nBuLi, DMSO/THF, RT, 48 h, 78%;
b) (COCl)2, benzene; c) (S)-4-isopropyl-2-oxazolidonone, nBuLi, THF,
À788C.
Scheme 3. a) NaBH4, CH2Cl2, EtOH, À788C; b) TBSCl, imidazole,
CH2Cl2, 08C. TBSCl=tert-butyldimethylsilyl chloride.
NaBH4 gave the primary alcohol 11 as a mixture with its
hemiacetal 12, and was converted to the bis-silyl ether 4
with TBSCl and imidazole.
A highly diastereoselective aldol reaction between the al-
dehyde 3 and ketone 4 under kinetic control generated aldol
13 (Scheme 4). The desired syn aldol 13 was formed togeth-
er with the unwanted syn diastereomer (10:1) without any
detectable formation of the anti product.[33] The syn diaste-
reomers were separated by column chromatography. The S
stereochemistry at C-7 in 13 was confirmed by Mosherꢁs
ester analysis.[34] The hydroxyl function was protected with a
Troc group to give compound 14. The use of the Troc
group[35] in this context was based on an earlier failed se-
quence. We had initially approached the synthesis of 1a by
employing Suzuki coupling for making the C-12–C-13
double bond and Yamaguchi macrolactonization for final
ring closure and with a TBS (and later TES) protecting
group at this position instead of Troc. However, desilylation
of these groups in the final step proved problematic. Milder
desilylating agents were ineffective and harsher conditions
gave decomposition products. Thus, the present protocol
was adopted with olefin metathesis for final ring closure. Se-
Scheme 5. a) NaH, THF, 08C, 82% ( 20a), 76% (20b); b) NaOH, anhy-
drous EtOH, 78% (21a), 73% (21b).
The diketoesters 20a and 20b were synthesized from
ethyl propionylacetate 18 and a-bromoacetoaromatic deriv-
atives 19a and 19b (Scheme 5). Direct intramolecular aldol
condensation led to the cyclopentenones 21a and 21b. The
2-bromoketone 19b was prepared as shown in Scheme 6.[36]
Bromination of 25 to 19b was carried out conveniently and
reproducibly by using commercially available polymer-sup-
À
ported tribromide (Amberlyst A26-Br3 ).[37]
After a number of unsuccessful attempts at stereoselective
reduction of the ketone 21a,[38,39] including reduction with
high catalyst loading of Coreyꢁs oxazaborolidine CBS re-
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Conformationally Restrained Epothilones
FULL PAPER
Scheme 6. a) nBuLi, Et2O, À788C; b) N,N-dimethyl-acetamide, Et2O,
À788C, 72% (2steps); c) polymer-supported Amberlyst A26-Br À, THF,
3
91%.
agent,[40] the desired trans product 22a and its enantiomer
ent-22a were finally obtained in equal amounts by reduction
under chelation control by using Zn
(BH4)2 (Scheme 7).[41]
A
Scheme 8. a) i) TFA, CH2Cl2, solvent evaporated, ii) residue in MeOH,
NaBH4, 08C, 30 min; b) PS-D lipase, vinyl acetate, 4 Š MS, pentane, RT,
3 d, 48% (ent-22b), 49% (28); c) anhydrous K2CO3, anhydrous EtOH,
RT, 12h, 94%. TFA =trifluoroacetic acid.
tween the two protons at the two stereogenic centers, and
no such NOE was observed between the two corresponding
protons in 22b or ent-22b.
Reduction of the esters 29a and 29b with DIBAL-H gave
the corresponding aldehydes 30a and 30b, which were sub-
jected to Wittig olefination to obtain the olefins 31a and
31b (Scheme 9). Desilylation with TBAF gave the desired
Scheme 7. a) Zn(BH4)2/ether, 48C, 75%; b) PS-D lipase, vinyl acetate,
4 Š MS, pentane, RT, 4 d, 48% (ent-22a), 49% (26); c) anhydrous
K2CO3, anhydrous EtOH, RT, 12h, 92%. MS =molecular sieves.
A
The trans racemate mixture 22a/ent-22a was efficiently
separated by enzymatic resolution by using Amano PS-D
Lipase enzyme (Scheme 7). Alcohol 22a was acetylated to
form 26, whilst ent-22a remained unchanged. They were
separated by column chromatography (49% 26 and 48%
ent-22a). The stereochemistry at the secondary hydroxyl
carbon atom of the ent-22a isomer and its enantiomeric
purity were determined by Mosherꢁs ester analysis (R,
99% ee).[34] Ethanolysis of 26 gave the desired alcohol 22a
(S, 98% ee by Mosherꢁs ester analysis).[34] The trans configu-
ration of 22a was confirmed by NOE experiment (vide
infra).
Compound 21b was found to be much more resistant to
reducing agents, including Zn(BH4)2. Indeed, using even an
A
Scheme 9. a) TESCl, imidazole, CH2Cl2, 08C, 2h, 93% ( 29a), 92%
(29b); b) DIBAL-H, toluene, À788C, 1 h; c) MeP+(Ph)3BrÀ, nBuLi,
THF, 08C, 30 min, 71% (31a), 73% (31b); d) TBAF, THF, 08C, 30 min,
80% (5a), 83% (5b). TESCl=triethylsilyl chloride, DIBAL-H = diiso-
butylaluminum hydride, TBAF=tetrabutylammonium fluoride.
excess of NaBH4 resulted in only partial reduction. We
speculated that altering electronic effects exerted by the
basic nitrogen atom could render the molecule susceptible
to reduction. Gratifyingly, prior conversion of 21b to the
corresponding trifluoroacetate salt by treatment with two
equivalents of trifluoroacetic acid (TFA) allowed rapid
NaBH4 reduction producing a mixture of all four diastereo-
mers (Scheme 8). The product mixture was separated into
the cis and trans enantiomeric pairs by column chromatogra-
phy (trans/cis 2:1, determined by 1H NMR spectroscopy).
The fraction containing the pair of trans enantiomers (22b
and ent-22b) was resolved by using Amano PS-D Lipase
enzyme as described earlier for the phenyl analogue.
alcohols 5a and 5b. At this stage, we confirmed the relative
trans stereochemistry of the protons H-1 and H-5 in the cy-
clopentene moiety 5a and the absolute stereochemistry of
the molecule as shown based on NOE correlations
(Figure 2) in conjunction with the already-established S con-
figuration of the secondary hydroxyl carbon atom.[34]
As with the phenyl analogue, the desired enantiomer 22b
was acetylated to 28 while the enantiomer ent-22b remained
unchanged. They were separated by column chromatogra-
phy and 28 was subjected to ethanolysis as before to obtain
22b (S, 95% ee by Mosherꢁs ester analysis).[34] NOE experi-
ments confirmed the trans configuration of the molecule.
The cis isomers 27 and ent-27, isolated by a similar enzyme-
mediated resolution protocol, showed a strong NOE be-
Figure 2. NOE correlations of (1S,5S)-2-methyl-3-phenyl-5-vinylcyclo-
pent-2-enol (5a).
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573

L. M. V. Tillekeratne et al.
The carboxylic acid 17 was esterified to 32a with alcohol
5a by using DCC/DMAP (Scheme 10). The Troc and TBS
protecting groups were sequentially removed with zinc dust/
ammonium chloride in dry ethanol and TAS-F,[42] respective-
Scheme 11. a) DCC, DMAP, CH2Cl2, 08C (15 min), RT (16 h), 85%;
b) TAS-F, DMF, 2d; c) Zn, NH 4Cl, anhydrous EtOH, RT, 45 min, 62%
(2steps); d) CH 2Cl2, 508C, 16 h, 55%.
Scheme 10. a) DCC, DMAP, CH2Cl2, 08C (15 min), RT (16 h), 64%;
b) Zn, NH4Cl, anhydrous EtOH, RT, 45 min; c) TAS-F, DMF, 2d, 62%
(2steps); d) CH 2Cl2, 508C, 16 h, 50% (Z+E). DCC=1,3-dicyclohexyl-
carbodiimide, DMAP=4-dimethylaminopyridine, TAS-F=tris(dimethyl-
amino)sulfur (trimethylsilyl)difluoride.
human tumor screen, compound 1b showed strong growth
inhibitory activity on CCRF-CEM and SR leukemia cell
lines with GI50 values of 2.7 and 2.9 nm, respectively (Table 1
and Figure 3 A). Surprisingly, it showed no significant activi-
ty on any of the other cell lines, including those derived
from breast (MCF-7) and ovarian (SK-OV-3) cancers
(Table 1 and Figure 3B,C). Although activity data of natural
epothilone D against the NCI-60 cell lines is not available,
Danishefsky et al. reported a potent and nonselective cyto-
toxic activity of epothilone D against these cell lines
(Table 1).[43] It is also significant to note that the mechanisti-
cally analogous cancer drug paclitaxel (taxol) is slightly less
effective (GI50 of 12.6 nm for CCRF-CEM and 15.8 nm for
SR) and less selective against these cell lines (NCI-60 cell
line screen data). The reason for the highly selective inhibi-
tion of compound 1b against leukemia cell growth over
solid tumor cell lines is still under investigation. We also
tested the open-chain analogue 35, which was isolated as a
byproduct of the RCM reaction along with 1b, and it
showed no significant activity on any of the NCI-60 cell
lines (Table 1). This is in accordance with our previous data
on acyclic epothilone analogues which showed weak cyto-
toxic activity.[44] So far we have subjected analogue 1a only
to a preliminary growth inhibition assay on MCF-7 breast
cancer cells, in which it showed no activity. We are currently
generating more material for testing in the NCI-60 cell line
tumor screen.
ly, to give intermediate 2a. Finally, RCM of 2a by using
second-generation Grubbs catalyst gave the desired Z-
alkene 1a, accompanied by what appeared to be the E
isomer as a minor product. A strong NOE correlation be-
tween the C-12-methyl protons and the C-13 olefinic proton
confirmed the Z stereochemistry of the double bond of 1a.
A similar approach to synthesize the pyridine analogue
1b by starting from the carboxylic acid 17 and the alcohol
5b was complicated by an unexpected retroaldol decoupling
of the alcohol obtained by the removal of the Troc protect-
ing group upon treatment with TAS-F. Gratifyingly, this was
easily overcome by changing the order of removal of the
two protecting groups (Scheme 11). Thus, the TBS protect-
ing group was removed first with TAS-F[42] to give 34 which
was then treated directly with zinc dust and ammonium
chloride in dry ethanol to give intermediate 2b. Finally,
RCM of 2b by using second-generation Grubbs catalyst
gave the desired product 1b. The Z configuration of the
double bond was confirmed by NOESY. The phenyl ana-
logue 35 and a dimerlike product from 2b were isolated as
byproducts. A large coupling constant between the two ole-
finic protons and the absence of NOE correlations estab-
lished the E configuration of
the nonterminal double bond
Table 1. In vitro cytotoxicities (GI50 and IC50) against human tumor cell lines.[a]
of 35. Formation of 35 can be
attributed to high catalyst
loading required to overcome
the sluggishness of the RCM
reaction.
GI50 [mm]
IC50 [mm]
Cell line
1b
1a
35
Paclitaxel
Epothilone D[b]
CCRF-CEM
SR
0.0027
0.0029
>15
nd
nd
192
nd
>12.5
0.0126
0.0158
0.0100
0.0251
0.0095
nd
0.0029
0.0069
> 12.5
>12.5
>12.5
MCF-7
SK-OV-3
>15
Cytotoxic activity: In prelimi-
nary in vitro cytotoxicity stud-
ies in the NCI-60 cell line
[a] In vitro cell growth inhibition was measured by using the NCI-60 cell line screen (SRB assay). CCRF-CEM
and SR are leukemia cell lines, MCF-7 is breast cancer cell line, and SK-OV-3 is ovarian cancer cell line.
[b] Data from reference [43] (measured by SRB assay). nd: Not Determined.
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Conformationally Restrained Epothilones
FULL PAPER
cidate the biological mechanism in relation to selective ac-
tivity.
Experimental Section
General methods: NMR spectra were recorded on Varian INOVA 600,
Varian VXRS-400, Bruker AC-F 300 MHz, or Nicolet NM-500 MHz
(modified with a Tecmag Libra interface) instruments and calibrated
using residual undeuterated solvent as internal reference. Optical rota-
tions were recorded on an AUTOPOL III 589/546 polarimeter. High-res-
olution mass spectra (HRMS) were recorded on a Micromass LCT Elec-
trospray mass spectrometer performed at the Mass Spectrometry and
Proteomics Facility, The Ohio State University.
(3S,6R,7S,8S)-1,3-Bis(tert-butyldimethylsilanyloxy)-7-hydroxy-4,4,6,8,12-
pentamethyltridec-12-en-5-one (13):
A solution of ketone 4 (1.8 g,
4.48 mmol, 2.3 equiv) in THF (5 mL) was added dropwise to a solution
of freshly prepared LDA in THF (prepared by adding nBuLi (2.92 mL of
1.6m solution in hexanes, 4.67 mmol) to diisopropylamine (4.67 mmol,
0.655 mL) in THF (5 mL) at À788C, then warming the solution to 08C
for 20 min, and finally cooling back to À788C). The reaction mixture was
stirred at À788C for 1 h and at À408C for 30 min and was then cooled
back to À788C. A precooled (À788C) solution of aldehyde 3 (0.272 g,
1.95 mmol, 1 equiv) in THF (10 mL) was added by the use of a cannula
to the mixture over 2min. The reaction mixture was stirred at À788C for
15 min before it was quenched rapidly by injection of a solution of acetic
acid (0.55 mL) in THF (1.64 mL). The mixture was stirred at À788C for
5 min and brought to room temperature. Saturated aqueous ammonium
chloride (20 mL) and Et2O (25 mL) were added and the layers were sep-
arated. The aqueous layer was extracted with Et2O (3”25 mL) and the
organic extracts were combined, dried over anhydrous sodium sulfate,
and concentrated in vacuo. Flash column chromatography (4–20% Et2O/
hexanes) gave recovered ketone 4 (0.78 g) followed by syn aldol 13
(0.87 g, 83%) as the pure diastereomer along with the other syn aldol
diastereomer (74 mg, 7%) as colorless oils.
Data for 13: [a]2D2 =À39.6 (c=0.7 in CHCl3); Rf =0.57 (silica gel, 20%
Et2O/hexanes); 1H NMR (600 MHz, CDCl3): d=4.66 (s, 1H), 4.65 (s,
1H), 3.88 (dd, J=2.4, 7.8 Hz, 1H), 3.67–3.63 (m, 1H), 3.60–3.55 (m, 1H),
3.30–3.27 (m, 2H), 2.05–1.95 (m, 2H), 1.69 (s, 3H), 1.76–1.26 (m, 7H),
1.19 (s, 3H), 1.07 (s, 3H), 1.01 (d, 3J=6.6 Hz, 3H), 0.88 (s, 9H), 0.87 (s,
9H), 0.82(d, J=7.2Hz, 3H), 0.09 (s, 3H), 0.06 (s, 3H), 0.02ppm (s,
6H); 13C NMR (100 MHz, CDCl3): d=222.7, 146.5, 109.9, 75.1, 74.3, 60.7,
54.2, 41.5, 38.4, 35.7, 32.8, 26.4, 26.3, 26.2, 25.0, 23.1, 22.6, 20.7, 18.6, 18.5,
15.6, 9.8, À3.5, À3.8, À5.0 ppm; HRMS (ESI): m/z: calcd for
C30H62O4Si2 +Na+: 565.4084 [M+Na+]; found: 565.4067.
Figure 3. NCI in vitro 60 cell line human tumor screen (dose response
~
&
^
curves for A) leukemia ( : CCRF-CEM, : HL-60(TB), : K-562, ”:
!
~
:
&
^
RPMI-8226, : SR) B) breast cancer ( : MCF7, : NCI/ADR-RES,
!
:
*
:
MDA-MB-231/, ”: HS 578T,
MDA-MB-435,
BT-549), and
~
:
&
^
:
C) ovarian cancer
(
IGROV1,
:
OVCAR-3,
OVCAR-4, ”:
!
*
OVCAR-5, : OVCAR-8, : SK-OV-3.
(3S,6R,7S,8S)-Carbonic
acid-1-[4,6-bis(tert-butyldimethylsilanyloxy)-
1,3,3-trimethyl-2-oxohexyl]-2,6-dimethyl-1-hept-6-enyl ester 2,2,2-trichloro-
ethyl ester (14): To a solution of aldol 13 (0.80 g, 1.48 mmol) in methyl-
ene chloride (30 mL) at 08C was added pyridine (0.96 mL, 11.84 mmol,
8 equiv) followed by 2,2,2-trichloroethyl chloroformate (0.8 mL,
5.92mmol, 4 equiv), and the reaction mixture was stirred at 0 8C for 1 h.
Saturated aqueous sodium bicarbonate (50 mL) was added and the or-
ganic layer was separated. The aqueous layer was extracted with methyl-
ene chloride (3”50 mL), and the combined organic layers were dried
over anhydrous sodium sulfate and concentrated in vacuo. Purification
by flash column chromatography (2% EtOAc/hexanes) afforded protect-
ed aldol 14 (0.98 g, 93%) as a colorless oil. [a]2D2 =À51.5 (c=1.6 in
CHCl3); Rf =0.72(silica gel, 17% EtOAc/hexanes); 1H NMR (600 MHz,
CDCl3): d=4.85 (d, J=12.0 Hz, 1H), 4.78 (dd, J=4.2, 7.8 Hz, 1H), 4.66
Conclusion
Two novel conformationally restrained epothilones 1a and
1b were synthesized. The strategy developed should be ap-
plicable to the synthesis of other important analogues of the
series. The strong and selective growth inhibitory effect ex-
hibited by analogue 1b on two leukemia cell lines, while not
suppressing the proliferation of breast cancer and ovarian
cancer cells which are very sensitive to natural epothilones,
indicates that it may be possible to also develop new lead
molecules of varying pharmacological profile by substantial
modification of the epothilone scaffold. Our future efforts
will focus on a more detailed investigation of this new class
of conformationally restrained epothilone analogues to elu-
(d, J=12.0 Hz, 1H), 4.66 (s, 1H), 4.62(s, 1H), 3.72(dd,
J=2.4, 7.8 Hz,
1H), 3.63–3.59 (m, 1H), 3.58–3.53 (m, 1H), 3.50–3.45 (m, 1H), 1.98–1.91
(m, 2H), 1.72–1.61 (m, 2H), 1.67 (s, 3H), 1.51–1.42 (m, 2H), 1.34 (s, 3H),
1.31–1.24 (m, 3H), 1.04 (d, J=6.6 Hz, 3H), 0.99 (s, 3H), 0.94 (d, J=
6.6 Hz, 3H), 0.88 (s, 9H), 0.85 (s, 9H), 0.082ppm (s, 6H), 0.001 (s, 6H);
13C NMR (100 MHz, CDCl3): d=215.8, 154.5, 145.8, 110.3, 95.0, 83.1,
76.8, 75.8, 60.5, 53.8, 42.7, 38.2, 34.9, 31.5, 26.4, 26.1, 24.9, 23.7, 22.6, 21.2,
Chem. Eur. J. 2008, 14, 570 – 581
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575

L. M. V. Tillekeratne et al.
18.6, 16.3, 11.3, À3.3, À4.1, À5.0, À5.1 ppm; HRMS (ESI): m/z: calcd for
C33H63Cl3O6Si2 +Na+ 739.3126; found: 739.3163 [M+Na+].
3.53 (dd, J=5.2, 18.4 Hz, 1H), 2.91–2.70 (m, 2H), 1.28 (t, J=7.2Hz,
3H), 1.12ppm (t, J=7.2Hz, 3H); 13C NMR (75 MHz, CDCl3): d=205.4,
197.4, 169.3, 136.3, 133.6, 128.8, 128.3, 61.9, 53.2, 37.7, 36.6, 14.2, 7.8 ppm;
HRMS (ESI): m/z: calcd for C15H18O4 +Na+ 285.1103; found: 285.1093
[M+Na+].
(3S,6R,7S,8S)-Carbonic
acid-1-[4-(tert-butyldimethylsilanyloxy)-6-hy-
droxy-1,3,3-trimethyl-2-oxohexyl]-2,6-dimethyl-1-hept-6-enyl ester 2,2,2-
trichloroethyl ester (15): A solution of bis-silyl compound 14 (0.8 g,
1.11 mmol, 1 equiv) in methylene chloride (30 mL) and methanol
(20 mL) was cooled to 08C. A solution of (1S)-(+)-10-camphorsulfonic
acid (77 mg, 0.33 mmol, 0.3 equiv) in methanol (10 mL) was added to this
solution. The reaction mixture was stirred at 08C for 7 h before it was
quenched with saturated aqueous sodium bicarbonate (10 mL). The solid
precipitate was filtered and the filtrate was concentrated in vacuo. The
residue was diluted with Et2O (100 mL) and washed with brine. The or-
ganic layer was separated and the aqueous layer was extracted with Et2O
(3”20 mL). The combined organic phases were dried over anhydrous
sodium sulfate and concentrated in vacuo. Purification by flash column
chromatography (10% EtOAc/hexanes) afforded the unstable alcohol 15
(0.583 g, 87%). It was used directly in the next step. Rf =0.57 (silica gel,
33% EtOAc/hexanes); 1H NMR (600 MHz, CDCl3): d=4.87–4.80 (m,
2H), 4.69–4.61 (m, 3H), 3.92 (dd, J=3.0 Hz, 7.8 Hz, 1H), 3.67–3.59 (m,
2H), 3.45–3.40 (m, 1H), 1.98–1.90 (m, 2H), 1.66 (s, 3H), 1.70–1.42 (m,
8H), 1.26 (s, 3H), 1.07–1.06 (m, 6H), 0.95 (d, J=6.6 Hz, 3H), 0.89 (s,
9H), 0.10 (s, 3H), 0.08 ppm (s, 3H); HRMS (ESI): m/z: calcd for
C27H49Cl3O6Si+Na+: 625.2262 [M+Na+]; found: 625.2260.
3-Methyl-2-oxo-4-phenylcyclopent-3-enecarboxylic acid ethyl ester (21a):
A solution of the diketoester 20a (12g, 45.8 mmol) in dry ethanol
(150 mL) was added dropwise to a solution of sodium hydroxide (1.83 g,
45.8 mmol) in dry ethanol (75 mL) with vigorous stirring. The solution
was heated to 508C and stirred overnight at that temperature. Et2O
(1.5 L) was added and the organic phase was washed with HCl (2n, 3”
300 mL) and dried over anhydrous sodium sulfate. The solvent was re-
moved in vacuo to give an oil that was purified by flash column chroma-
tography (6% EtOAc/hexane) to give the cyclic b-ketoester 21a (8.7 g,
1
78%) as a yellow oil. Rf =0.40 (silica gel, 25% EtOAc/hexane); H NMR
(400 MHz, CDCl3): d=7.56–7.52(m, 2H), 7.50–7.42(m, 3H), 4.26 (q,
J=
7.2Hz, 2H), 3.58 (dd, J=3.2, 7.6 Hz, 1H), 3.38–3.31 (m, 1H), 3.15–3.07
(m, 1H), 1.99 (s, 3H), 1.32ppm (t, J=7.2Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=202.6, 169.6, 166.5, 135.8, 134.9, 130.2, 128.9, 127.9, 61.9, 51.3,
33.7, 14.5, 10.5 ppm; HRMS (ESI): m/z: calcd for C15H16O3 +Na+:
267.0997 [M+Na+]; found: 267.0974.
1-(5-Methylpyridin-2-yl)ethanone (25): nBuLi (6.25 mL of 1.6m solution
in hexanes, 10 mmol, 1 equiv) was added dropwise to a solution of 2-
bromo-5-methyl pyridine 23 (1.73 g, 10 mmol) in dry Et2O (20 mL),
cooled to À788C. The reaction mixture was allowed to warm to À408C
for 15 min, then cooled back to À788C again. N,N-Dimethylacetamide
(1.023 mL, 11 mmol, 1.1 equiv) was added dropwise and the mixture was
stirred at À788C for 2h. Saturated aqueous ammonium chloride (10 mL)
was added and the organic layer was separated. The aqueous layer was
extracted with Et2O (3”10 mL) and the combined organic layers were
dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo
to give an oily residue that was subjected to flash column chromatogra-
phy by using (5% methanol/methylene chloride) to give compound 25
(0.977 g, 72%) as a yellow oil. Rf =0.48 (silica gel, 25% EtOAc/hexanes);
1H NMR (400 MHz, CDCl3): d=8.50–8.48 (brs, 1H), 7.94 (d, J=8.0 Hz,
1H), 7.63–7.60 (m, 1H), 2.70 (s, 3H), 2.41 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3): d=200.2, 151.7, 149.7, 137.8, 137.4, 121.7, 26.0,
18.9 ppm; HRMS (ESI): m/z: calcd for C8H9NO+Na+: 158.0582
[M+Na+]; found: 158.0580.
(3S,6R,7S,8S)-3-(tert-Butyldimethylsilanyloxy)-4,4,6,8,12-pentamethyl-5-
oxo-7-(2,2,2-trichloroethoxycarbonyloxy)tridec-12-enoic acid (17): Dess–
Martin periodinane (0.545 g, 1.28 mmol, 1.4 equiv) was added to a solu-
tion of alcohol 15 (0.550 g, 0.914 mmol) in methylene chloride (4 mL).
The mixture was stirred at room temperature for 15 min. An additional
amount of Dess–Martin reagent (0.23 g, 0.6 equiv) was added and the re-
action mixture was stirred for 15 min and was then subjected to flash
column chromatography (10% EtOAc/hexanes) to furnish crude alde-
hyde 16 which was used directly in the next step. A solution of sodium di-
hydrogenphosphate (270 mg, 2.25 mmol, 2.46 equiv) and sodium chlorite
(270 mg, 3 mmol, 3.27 equiv) in distilled water (5 mL) was added to the
crude aldehyde 16 (ca. 0.914 mmol) in tert-butanol (25 mL) and 2-methyl-
2-butene (6 mL). The mixture was stirred at room temperature for 1 h
and quenched by the addition of saturated aqueous ammonium chloride
(50 mL) and water (50 mL). The mixture was extracted with ethyl acetate
(3”60 mL) and the combined organic extracts were dried over anhydrous
sodium sulfate and concentrated in vacuo. Purification by flash column
chromatography (10% EtOAc/hexanes) afforded the pure carboxylic
acid 17 (0.505 g, 90% over two steps) as a colorless oil. [a]2D2 =À53.7 (c=
0.8 in CHCl3); Rf =0.58 (silica gel, 40% EtOAc/hexanes); 1H NMR
(600 MHz, CDCl3): d=4.85 (d, J=12.0 Hz, 1H), 4.76 (dd, J=4.2, 7.2 Hz,
1H), 4.66 (d, J=12.0 Hz, 1H), 4.66 (s, 1H), 4.62 (s, 1H), 4.22 (dd, J=3.6,
6.6 Hz, 1H), 3.47–3.41 (m, 1H), 2.60 (dd, J=3.6, 17.4 Hz, 1H), 2.21 (dd,
J=6.6, 16.8 Hz, 1H), 1.96–1.91 (m, 2H), 1.74 À1.68 (m, 1H), 1.66 (s,
3H), 1.52–1.42 (m, 2H), 1.31 (s, 3H), 1.28 (d, J=16.8 Hz, 3H), 1.06 (s,
6H), 0.94 (d, J=7.2Hz, 3H), 0.86 (s, 9H), 0.1 (s, 3H), 0.03 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=215.5, 177.7, 154.5, 145.8, 110.3, 94.9,
82.7, 76.9, 75.0, 53.8, 42.4, 39.9, 38.2, 34.9, 31.5, 26.3, 26.2, 24.8, 23.0, 22.6,
20.3, 18.4, 16.3, 11.5, À4.3 ppm; HRMS (ESI): m/z: calcd for
C27H47Cl3O7Si+Na+: 639.2054 [M+Na+]; found: 639.2079.
À
2-Bromo-1-(5-methylpyridin-2-yl)ethanone (19b): Amberlyst A26-Br3
(Aldrich, 1.26 mmol Br3 per g; 4.76 g, 6 mmol, 0.9 equiv) was added in
one portion to a solution of compound 25 (0.9 g, 6.67 mmol) in THF
(25 mL). The mixture was stirred at 508C for 10 h and the decolored
resin was filtered off and washed with ethyl acetate. The organic solution
was washed with water, dried over anhydrous sodium sulfate, and con-
centrated in vacuo to give an oily residue that was chromatographed on
silica gel with (10–30% methylene chloride/hexanes) to give compound
19b (1.29 g, 91%) as a yellow oil. Rf =0.62(silica gel, 02% EtOAc/
hexane); 1H NMR (400 MHz, CDCl3): d=8.50 (brs, 1H), 8.01 (d, J=
8.0 Hz, 1H), 7.66 (dd, J=1.6, 8.0 Hz, 1H), 4.84 (s, 2H), 2.44 ppm (s, 3H);
13C NMR (75 MHz, CDCl3): d=192.5, 149.8, 149.3, 138.7, 137.6, 122.6,
32.5, 18.9 ppm; HRMS (ESI): m/z: calcd for C8H8BrNO+Na+: 235.9687
[M+Na+]; found: 235.9676.
3-Oxo-2-(2-oxo-2-phenylethyl)pentanoic acid ethyl ester (20a): Ethyl
propionylacetate 18 (10 g, 69.4 mmol) was added slowly to a stirred sus-
pension of sodium hydride (60% dispersion in mineral oil, 3.33 g,
83.3 mmol, 1.2equiv) in THF (100 mL) at 0 8C and the mixture was
stirred for 30 min. 2-Bromoacetophenone 19a (15.2g, 76.34 mmol,
1.1 equiv) in THF (10 mL) was added dropwise and the reaction mixture
was stirred at room temperature for 16 h. Saturated aqueous ammonium
chloride (60 mL) was added and the mixture was subsequently extracted
with Et2O (3”70 mL). The combined organic layers were washed with
brine and dried over anhydrous sodium sulfate. The solvent was removed
in vacuo to give a dark-yellow oil that was purified by flash column chro-
matography (10% EtOAc/hexane) to give the diketoester 20a (14.8 g,
3-Oxo-2-(2-oxo-2-pyridin-2-ylethyl)pentanoic acid ethyl ester (20b):
Ethyl propionylacetate 18 (5 g, 34.7 mmol) was added slowly to a stirred
suspension of sodium hydride (60% dispersion in mineral oil, 1.665 g,
41.65 mmol, 1.2equiv) in THF (50 mL) at 0 8C and the mixture was
stirred for 30 min. Compound 19b (8.092g, 38.17 mmol, 1.1 equiv) in
THF (5 mL) was added dropwise and the reaction mixture was stirred at
room temperature for 16 h. Saturated aqueous ammonium chloride
(30 mL) was added and the mixture was subsequently extracted with
Et2O (3”30 mL). The combined organic layers were washed with brine
and dried over anhydrous sodium sulfate. The solvent was removed in
vacuo to give a dark-yellow oil that was purified by flash column chroma-
tography (10% EtOAc/hexanes) to give the diketoester 20b (7.3 g, 76%)
as a yellow oil: TLC Rf =0.33 (silica gel, 20% EtOAc/hexane); 1H NMR
(400 MHz, CDCl3): d=8.50 (brs, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.66 (dd,
J=1.6, 8.0 Hz, 1H), 4.20 (q, J=7.2Hz, 2H), 4.15 (dd, J=6.0, 8.0 Hz,
82%) as
a yellow oil. Rf =0.50 (silica gel, 25% EtOAc/hexanes);
1H NMR (400 MHz, CDCl3): d=7.98 (d, J=8.4 Hz, 2H), 7.58 (m, 1H),
7.46 (m, 2H), 4.22 (q, J=7.2Hz, 3H), 3.74 (dd, J=8.4, 18.4 Hz, 1H),
576
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Chem. Eur. J. 2008, 14, 570 – 581

Conformationally Restrained Epothilones
FULL PAPER
1H), 3.92(dd, J=8.4, 18.8 Hz, 1H), 3.74 (dd, J=6.0, 18.8 Hz, 1H), 2.85–
2.66 (m, 2H), 2.41 (s, 3H), 1.27 (t, J=7.2Hz, 3H), 1.10 ppm (t, J=
7.2Hz, 3H); 13C NMR (75 MHz, CDCl3): d=205.5, 198.9, 169.6, 150.7,
149.8, 138.1, 137.3, 121.7, 61.8, 53.5, 37.1, 36.2, 18.9, 14.2, 7.9 ppm; HRMS
(ESI): m/z: calcd for C15H19NO4 +Na+: 300.1212; found: 300.1200
[M+Na+].
nol was removed under reduced pressure and the residue was dissolved
in methylene chloride and washed with a saturated aqueous solution of
ammonium chloride. The organic phase was dried over anhydrous
sodium sulfate and the solvent was removed in vacuo. The residue was
purified by flash column chromatography (25% EtOAc/hexane) to give
compound 22a (393 mg, 92%, 99% ee) as a slightly yellow oil. [a]2D2 =+
14.8 (c=0.8 in CHCl3); Rf =0.17 (silica gel, 25% EtOAc/hexane);
1H NMR (400 MHz, CDCl3): d=7.37–7.25 (m, 5H), 4.99 (s, 1H), 4.23 (q,
J=7.2 Hz, 2H), 3.05–2.94 (m, 3H), 2.33 (d, J=5.6 Hz, 1H), 1.90 (s, 3H),
1.32ppm (t, J=7.2Hz, 3H); 13C NMR (100 MHz, CDCl3): d=174.9,
137.2, 135.8, 135.1, 128.4, 127.9, 127.3, 84.0, 61.1, 51.7, 37.1, 14.5,
12.9 ppm; HRMS (ESI): m/z: calcd for C15H18O3 +Na+ 269.1154
[M+Na+]; found: 269.1148.
3-Methyl-2-oxo-4-pyridin-2-ylcyclopent-3-ene carboxylic acid ethyl ester
(21b): A solution of the diketoester 20b (3.1 g, 11.19 mmol) in dry etha-
nol (35 mL) was added dropwise to a solution of sodium hydroxide
(0.447 g, 11.19 mmol) in dry ethanol (15 mL) with vigorous stirring. The
solution was stirred at room temperature overnight. Et2O (200 mL) was
added and the organic phase was washed with HCl (2N, 3”100 mL). The
aqueous layer was cooled to 08C and made slightly basic by the addition
of sodium bicarbonate. The aqueous layer was then extracted with ethyl
acetate (3”300 mL) and the organic phase was dried over anhydrous
sodium sulfate. The solvent was removed in vacuo to give an oil that was
purified by flash column chromatography (10% EtOAc/hexanes) to give
the cyclic b-ketoester 21b (2.115 g, 73%) as a yellow oil. Rf =0.35 (silica
(1R*,2S*,3E)-2-Hydroxy-3-methyl-4-pyridin-2-ylcyclopent-3-ene carbox-
ylic acid ethyl ester (22b/ent-22b) and (27/ent-27): Trifluoroacetic acid
(0.297 mL, 3.86 mmol) was added to a stirred solution of compound 21b
(0.5 g, 1.93 mmol) in methylene chloride (6 mL). The solvent was re-
moved in vacuo and the resulting salt was dissolved in methanol (7 mL)
and cooled to 08C. Sodium borohydride (0.73 g, 19.3 mmol) was added
rapidly at once to this solution at 08C. After stirring at the same temper-
ature for 30 min, chloroform was added and the mixture was washed with
saturated aqueous sodium bicarbonate solution and brine. The organic
layer was dried over anhydrous sodium sulfate and concentrated. The
residue (0.459 g, 91%, cis/trans 1:2, determined by 1H NMR spectrosco-
py) was separated by using silica-gel chromatography (16–25% EtOAc/
hexane) to obtain 22b/ent-22b (0.306 g, 67%) and 27/ent-27 (0.153 g,
33%).
Data for 27/ent-27: Rf =0.29 (silica gel, 20% EtOAc/hexane); 1H NMR
(C6D6, 600 MHz): d=8.38 (s, 1H), 6.88–6.87 (m, 2H), 4.61 (s, 1H), 3.99–
3.89 (m, 2H), 3.54–3.49 (m, 1H), 3.05–3.01 (m, 1H), 2.88–2.84 (m, 1H),
2.33 (d, J=7.8 Hz, 1H), 2.13 (s, 3H), 1.77 (s, 3H), 0.90 ppm (t, J=14.4,
3H); 13C NMR (C6D6, 400 MHz): d=172.9, 153.5, 149.9, 138.6, 136.2,
135.9, 130.7, 121.9, 82.0, 60.4, 46.5, 36.2, 17.8, 14.1, 13.8 ppm; HRMS
(ESI): m/z: calcd for C15H19NO3 +Na+: 284.1263 [M+Na+]; found:
284.1248.
1
gel, 33% EtOAc/hexanes); H NMR (400 MHz, CDCl3): d=8.59 (s, 1H),
7.62–7.60 (m, 1H), 7.54–7.52 (m, 1H), 4.24 (q, J=7.2Hz, 2H), 3.56 (dd,
J=2.8, 7.2 Hz, 1H), 3.42–3.25 (m, 2H), 2.41 (s, 3H), 2.12 (s, 3H),
1.31 ppm (t, J=7.2Hz, 3H); 13C NMR (75 MHz, CDCl3): d=203.2,
169.5, 164.4, 151.4, 150.6, 136.8, 136.5, 134.2, 123.3, 61.7, 51.1, 32.5, 18.6,
14.3, 10.6 ppm; HRMS (ESI): m/z: calcd for C15H17NO3 +Na+: 282.1106
[M+Na+]; found: 282.1109.
(1R*,2S*,3E)-2-Hydroxy-3-methyl-4-phenylcyclopent-3-enecarboxylic
acid ethyl ester (22a) and (ent-22a): A solution of zinc borohydride
(150 mL of 0.3m solution in Et2O, 45 mmol, 4 equiv) was added dropwise
at 08C to a stirred solution of b-ketoester (+/-)-21a (2.75 g, 11.25 mmol)
in THF (5 mL). The mixture was stirred overnight at 48C, quenched by
slow addition of water and stirred for an additional 1 h. Anhydrous
sodium sulfate was added and the resulting suspension was filtered and
the filtrate was concentrated. The residue was dissolved in methylene
chloride and filtered again and dried. Purification by flash column chro-
matography (8% EtOAc/hexanes) gave recovered starting material
(0.27 g, 10%) and racemic b-hydroxyesters 22a and ent-22a (2.1 g, 75%)
as a yellow oil. Rf =0.17 (silica gel, 25% EtOAc/hexanes); 1H NMR
(400 MHz, CDCl3): d=7.37–7.24 (m, 5H), 4.98 (s, 1H), 4.22 (q, J=
7.2 Hz, 2H), 3.03–2.94 (m, 3H), 2.32 (d, J=5.6 Hz, 1H), 1.90 (s, 3H),
1.31 ppm (t, J=7.2Hz, 3H); 13C NMR (100 MHz, CDCl3): d=174.9,
137.2, 135.7, 135.1, 128.4, 127.9, 127.3, 83.9, 61.1, 51.8, 37.1, 14.5,
12.8 ppm; HRMS (ESI): m/z: calcd for C15H18O3 +Na+: 269.1154
[M+Na+]; found: 269.1143.
A
acid ethyl ester (28) and (1R,2R,3E)-2-hydroxy-3-methyl-4-pyridin-2-yl-
cyclopent-3-ene carboxylic acid ethyl ester (ent-22b): Vinyl acetate
(0.95 mL, 10.3 mmol) was added to a solution of 22b/ent-22b (0.27 g,
1.03 mmol) in anhydrous pentane (3 mL). Amano PS-D lipase (0.27 g)
and 4 Š MS (0.27 g) were added and the suspension was stirred at room
1
temperature. The reaction was monitored by TLC and H NMR spectros-
copy and after 3 d, 49–50% conversion of 22b/ent-22b to acetate 28 was
achieved. The sieves and lipase were filtered off and washed with Et2O.
The solvent was removed and the crude product was purified by column
chromatography (17% EtOAc/hexane) to give 28 (0.154 g, 49%) as a
pale-yellow oil and ent-22b (0.130 g, 48%).
Data for 28: [a]2D2 =À40.6 (c=1.0 in CHCl3); Rf =0.26 (silica gel, 75%
EtOAc/hexanes); 1H NMR (CDCl3, 600 MHz): d=8.44 (s, 1H), 7.47 (d,
J=7.8 Hz, 1H), 7.20 (d, J=7.8 Hz, 1H), 6.07 (s, 1H), 4.19 (q, J=14.4 Hz,
2H), 3.22–3.16 (m, 1H), 3.05–3.01 (m, 2H), 2.32 (s, 3H), 2.09 (s, 3H),
1.94 (s, 3H), 1.26 ppm (t, J=7.2Hz, 3H); 13C NMR (CDCl3, 400 MHz):
d=174.1, 170.9, 152.5, 150.0, 137.2, 136.7, 135.0, 137.8, 122.5, 85.9, 61.1,
48.5, 37.5, 21.3, 18.5, 14.4, 13.2 ppm; HRMS (ESI): m/z: calcd for
C17H21NO4 +Na+: 326.1368 [M+Na+]; found: 326.1354.
A
ethyl ester (26) and (1R,2R,3E)-2-hydroxy-3-methyl-4-phenyl-cyclopent-
3-ene carboxylic acid ethyl ester (ent-22a): Vinyl acetate (7.5 mL,
81.4 mmol) was added to a solution of 22a and ent-22a (2g, 8.13 mmol)
in anhydrous pentane (30 mL). Amano PS-D lipase (2.0 g) and 4 Š MS
(2.0 g) were added and the suspension was stirred at room temperature.
The reaction was monitored by TLC and 1H NMR spectroscopy and
after 4 d, 48–50% conversion of 22a to the acetate was achieved. The
sieves and lipase were filtered and washed with Et2O. The solvent was re-
moved and crude product was purified by flash column chromatography
(20% Et2O/hexanes) to give 26 (1.15 g, 49%) as a slightly yellow oil and
ent-22a (0.96 g, 48%, 98% ee).
Data for 26: [a]2D2 =À50.5 (c=0.6 in CHCl3); Rf =0.38 (silica gel, 25%
EtOAc/hexanes); 1H NMR (600 MHz, CDCl3): d=7.40–7.25 (m, 5H),
6.06 (s, 1H), 4.20 (q, J=7.2 Hz, 2H), 3.08–2.95 (m, 3H), 2.11 (s, 3H),
1.80 (s, 3H), 1.28 ppm (t, J=7.2Hz, 3H); 13C NMR (100 MHz, CDCl3):
d=174.2, 170.9, 138.4, 136.7, 131.8, 128.5, 128.0, 127.7, 85.9, 61.2, 48.7,
38.6, 21.3, 14.4, 12.9 ppm; HRMS (ESI): m/z: calcd for C17H20O4 +Na+:
311.1259 [M+Na+]; found: 311.1240.
Data for ent-22b: [a]2D2 =+7.8 (c=1.0 in CHCl3). See 22b below for 1H
and 13C NMR spectroscopic data.
A
acid (22b): Anhydrous potassium carbonate (45.6 mg, 0.33 mmol) was
added at 08C to a solution of 28 (100 mg, 0.33 mmol) in dry ethanol
(2.8 mL) and the solution was stirred at room temperature for 12 h. Etha-
nol was removed under reduced pressure and the residue was dissolved
in methylene chloride and washed with a saturated aqueous solution of
ammonium chloride. The organic phase was dried over anhydrous
sodium sulfate and the solvent was removed in vacuo. The residue was
purified by flash column chromatography (25% EtOAc/hexane) to give
compound 22b (81 mg, 94%, 95% ee) as a pale-yellow oil. [a]2D0 =À8.1
(c=0.26 in CHCl3); TLC Rf =0.27 (silica gel, 75% EtOAc/hexanes);
Data for ent-22a: [a]2D2 =À15.2( c=0.6 in CHCl3); see 22a below for 1H
and 13C NMR spectroscopic data.
A
ethyl ester (22a): Anhydrous potassium carbonate (240 mg, 1.74 mmol)
was added at 08C to a solution of 26 (500 mg, 1.74 mmol) in dry ethanol
(15 mL) and the solution was stirred at room temperature for 12h. Etha-
Chem. Eur. J. 2008, 14, 570 – 581
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
577

L. M. V. Tillekeratne et al.
1H NMR (C6D6, 600 MHz): d=8.48 (s, 1H), 6.97–6.92(m, 2H), 5.03 (s,
1H), 4.04–4.00 (m, 2H), 3.22–3.21 (m, 2H), 3.02–2.98 (m, 1H), 2.18 (s,
3H), 1.87 (s, 3H), 0.98 ppm (t, J=14.4 Hz, 3H); 13C NMR (CDCl3,
400 MHz): d=174.6, 153.1, 149.9, 138.4, 136.7, 134.9, 131.4, 122.4, 84.1,
61.0, 51.7, 35.9, 18.5, 14.5, 13.1 ppm; HRMS (ESI): m/z: calcd for
C15H19NO3 +Na+: 284.1263 [M+Na+]; found: 284.1279.
chromatography (10% EtOAc/hexanes) afforded pure alcohol 5a
(0.245 g, 80%) as a white solid. [a]2D2 =À14.17 (c=1.2in CHCl 3); Rf =
0.45 (silica gel, 33% EtOAc/hexanes); 1H NMR (600 MHz, CDCl3): d=
7.36–7.23 (m, 5H), 6.00–5.94 (m, 1H), 5.18 (dd, J=1.2, 16.8 Hz, 1H),
5.08 (dd, J=0.6, 10.2Hz, 1H), 4.48 (d, J=6.6 Hz, 1H), 2.85–2.81 (m,
1H), 2.71–2.65 (m, 1H), 2.59–2.54 (m, 1H), 1.89 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=140.4, 137.8, 136.3, 135.9, 128.4, 127.9, 127.2,
115.4, 86.4, 52.4, 39.8, 12.9 ppm; HRMS (ESI): m/z: calcd for C14H16O+
Na+: 223.1099 [M+Na+]; found: 223.1100.
A
car-
boxylic acid ethyl ester (29a): Triethylsilyl chloride (876 mL, 5.22 mmol,
1.5 equiv) was added dropwise to a solution of alcohol 22a (856 mg,
3.48 mmol) and imidazole (711 mg, 10.44 mmol, 3 equiv) in methylene
chloride (25 mL) at 08C. After stirring at 08C for 2h, the reaction was
quenched with water (15 mL) and extracted with ethyl acetate (3”
30 mL). The organic extracts were washed with brine (20 mL) and dried
over anhydrous sodium sulfate. Filtration and evaporation of the solvents
in vacuo furnished an oily crude product which was purified by flash
column chromatography (2% EtOAc/hexanes) to give TES ether 29a
(1.16 g, 93%) as a yellow oil. [a]2D2 =À34.2( c=0.6 in CHCl3); Rf =0.53
(silica gel, 10% EtOAc/hexanes); 1H NMR (400 MHz, CDCl3): d=7.36–
7.22 (m, 5H), 5.11 (s, 1H), 4.21 (q, J=7.2Hz, 2H), 3.08–2.82 (m, 3H),
1.83 (s, 3H), 1.32(t, J=7.2Hz, 3H), 1.00 (t, J=8.0 Hz, 9H), 0.68 ppm (q,
J=8.0 Hz, 6H); 13C NMR (75 MHz, CDCl3): d=175.5, 137.5, 135.7,
134.9, 128.3, 128.0, 127.2, 84.5, 60.9, 51.8, 38.6, 14.5, 12.9, 7.1, 5.1,
0.2ppm; HRMS (ESI): m/z: calcd for C21H32O3Si+Na+: 383.2018
[M+Na+]; found: 383.2019.
A
3-ene carboxylic acid ethyl ester (29b): Triethylsilyl chloride (145 mL,
0.863 mmol, 1.5 equiv) was added dropwise to a solution of alcohol 22b
(150 mg, 0.575 mmol) and imidazole (17 mg, 1.72mmol, 3 equiv) in meth-
ylene chloride (4 mL) at 08C. After stirring at 08C for 2h, the reaction
was quenched with water (2.5 mL) and extracted with ethyl acetate (3”
5 mL). The organic extracts were washed with brine (3.5 mL) and dried
over anhydrous sodium sulfate. Filtration and evaporation of the solvents
in vacuo furnished an oily crude product which was purified by flash
column chromatography on silica (2–7% EtOAc/hexanes) to give 29b
(198 mg, 92%) as a yellow oil. [a]2D2 =À51.04 (c=0.7 in CHCl3); Rf =0.29
(silica gel, 25% EtOAc/hexanes); 1H NMR (600 MHz, CDCl3): d=8.41
(s, 1H), 7.44 (dd, J=1.8, 8.4 Hz, 1H), 7.19 (d, J=7.8 Hz, 1H), 5.12(d,
J=4.8 Hz, 1H), 4.18 (dd, J=7.2, 14.4 Hz, 2H), 3.17–3.12 (m, 1H), 3.00–
2.89 (m, 2H), 2.30 (s, 3H), 1.97 (s, 3H), 1.28 (t, J=14.4 Hz, 3H), 0.96 (t,
J=7.8 Hz, 9H), 0.65 ppm (q, J=7.8 Hz, 6H); 13C NMR (100 MHz,
CDCl3): d=175.3, 153.3, 149.9, 139.1, 136.5, 134.1, 131.1, 122.4, 84.5, 60.8,
51.6, 37.4, 18.4, 14.5, 13.3, 7.0, 5.2ppm; HRMS (ESI): m/z: calcd for
C21H33NO3Si+H+: 376.2308 [M+H+]; found: 376.2302.
ACHTREUNG
A
2.2 mmol, 1.1 equiv) was added dropwise to a solution of silyl ether 29a
(720 mg, 2 mmol) in toluene (10 mL) at À788C. The reaction was stirred
at that temperature for 1 h and quenched by dropwise addition of satu-
rated NH4Cl (1.0 mL). The reaction was allowed to reach room tempera-
ture and a saturated aqueous solution of Rochelle salt (3.0 mL) and
brine (2.0 mL) were added. The mixture was extracted with ethyl acetate
(3”7 mL) and the combined organic extracts were dried over anhydrous
sodium sulfate, filtered, and evaporated in vacuo to give crude aldehyde
30a as a colorless liquid which was used in the next step without further
purification; 1H NMR (400 MHz, CDCl3): d=9.86 (d, J=2.0 Hz, 1H),
7.38–7.25 (m, 5H), 5.08 (s, 1H), 3.12–2.98 (m, 2H), 2.90–2.84 (m, 1H),
1.84 (s, 3H), 1.00 (t, J=8.0 Hz, 9H), 0.68 ppm (q, J=8.0 Hz, 6H);
13C NMR (75 MHz, CDCl3): d=200.2, 135.5, 133.9, 133.7, 126.6, 126.3,
125.6, 79.6, 57.4, 33.1, 11.3, 5.3, 3.4 ppm.
(3S,4S)-5-Methyl-2-(2-methyl-3-triethylsilanyloxy-4-vinylcyclopent-1-
enyl)pyridine (31b): A solution of DIBAL-H (0.44 mL, 1.0m in hexane,
0.44 mmol, 1.1 equiv) was added dropwise to a solution of silyl ether 29b
(150 mg, 0.4 mmol) in toluene (2mL) at À788C. The reaction mixture
was stirred at that temperature for 1 h and quenched by dropwise addi-
tion of saturated aqueous ammonium chloride solution (0.2mL). The re-
action mixture was allowed to reach room temperature and a saturated
aqueous solution of Rochelle salt (1 mL) and brine (0.4 mL) were added.
The mixture was extracted with ethyl acetate (3”2mL) and the com-
bined organic extracts were dried over anhydrous sodium sulfate, filtered,
and evaporated in vacuo to give crude aldehyde 30b as a yellow liquid
which was used in the next step without further purification. nBuLi
(0.49 mL of 1.6m solution in hexanes, 0.78 mmol, 1.95 equiv) was added
dropwise to a precooled (08C) solution of methyltriphenylphosphonium
bromide (286 mg, 0.8 mmol, 2 equiv) in THF (3 mL). The reaction mix-
ture was stirred at 08C for 30 min. A solution of crude aldehyde 30b (ca.
0.4 mmol) in THF (1.5 mL) was added and the mixture was stirred at
08C for 30 min and then quenched with saturated aqueous ammonium
chloride solution (3 mL). The solvent was removed under reduced pres-
sure and the aqueous residue was extracted with ethyl acetate (3”3 mL).
The combined organic extracts were dried over anhydrous sodium sulfate
and concentrated in vacuo. Purification by flash column chromatography
on silica (7% EtOAc/hexanes) afforded pure olefin 31b (96 mg, 73%) as
a yellowish oil. [a]2D2 =À21.29 (c=0.7 in CHCl3); Rf =0.66 (silica gel,
33% EtOAc/hexanes); 1H NMR (600 MHz, CDCl3): d=8.42(s, 1H),
7.44 (dd, J=1.8, 7.8 Hz, 1H), 7.18 (d, J=7.8 Hz, 1H), 5.95–5.88 (m, 1H),
5.12(d, J=16.8 Hz, 1H), 5.04 (d, J=10.8 Hz, 1H), 4.54 (d, J=5.4 Hz,
1H), 2.97–2.93 (m, 1H), 2.74–2.68 (m, 1H), 2.60–2.56 (m, 1H), 2.30 (s,
3H), 1.96 (s, 3H), 0.98 (t, J=7.8 Hz, 9H), 0.64 ppm (q, J=7.8 Hz, 6H);
13C NMR (100 MHz, CDCl3): d=153.9, 149.8, 140.9, 139.8, 136.5, 135.0,
130.9, 122.4, 115.2, 87.2, 51.8, 39.2, 18.4, 13.4, 7.2, 5.5 ppm; HRMS (ESI):
m/z: calcd for C20H31NOSi+H+: 330.2253 [M+H+]; found: 330.2258.
A
(31a): nBuLi (0.775 mL of 1.6m solution in hexanes, 1.24 mmol,
1.95 equiv) was added dropwise to a precooled (08C) solution of methyl-
triphenylphosphonium bromide (455 mg, 1.27 mmol, 2 equiv) in THF
(5 mL). The reaction mixture was stirred at 08C for 30 min. A solution of
aldehyde 30a (0.2g, 0.633 mmol) in THF (2mL) was added and the mix-
ture was stirred at 08C for 30 min and quenched with saturated aqueous
ammonium chloride (5 mL). The solvent was removed under reduced
pressure and the aqueous residue was extracted with ethyl acetate (3”
5 mL). The combined organic extract was dried over anhydrous sodium
sulfate and concentrated in vacuo. Purification by flash column chroma-
tography (5% Et2O/hexanes) afforded pure olefin 31a (0.14 g, 71%) as a
colorless oil. [a]2D2 =À14.3 (c=0.4 in CHCl3); Rf =0.73 (silica gel, 10%
1
Et2O/hexanes); H NMR (600 MHz, CDCl3): d=7.34–7.21 (m, 5H), 5.94–
5.88 (m, 1H), 5.12(d, J=16.8 Hz, 1H), 5.04 (dd, J=1.8, 10.2Hz, 1H),
4.52(d, J=4.8 Hz, 1H), 2.88–2.82 (m, 1H), 2.74–2.68 (m, 1H), 2.52–2.47
(m, 1H), 1.82(s, 3H), 0.98 (t, J=7.8 Hz, 9H), 0.66 ppm (q, J=7.8 Hz,
6H); 13C NMR (100 MHz, CDCl3): d=141.1, 138.1, 136.3, 135.7, 128.3,
127.9, 126.9, 115.2, 87.0, 52.1, 40.4, 13.1, 7.2, 5.5 ppm; HRMS (ESI): m/z:
calcd for C20H30OSi+Na+: 337.1964 [M+Na+]; found: 337.1970.
A
G
Tetrabutylammonium fluoride (0.152mL of 1 m solution in THF,
0.152mmol) was added dropwise to a solution of compound 31b (50 mg,
0.152mmol) in THF (1 mL) at 0 8C. The reaction mixture was stirred at
08C for 30 min and then water (1 mL) and ethyl acetate (2mL) were
added. The layers were separated and the aqueous layer was extracted
with ethyl acetate (2”2mL). The combined organic layers were dried
over anhydrous sodium sulfate and concentrated in vacuo. Purification
ammonium fluoride (1.53 mL of 1m solution in THF, 1.53 mmol) was
added dropwise to a solution of compound 31a (0.48 g, 1.53 mmol) in
THF (7 mL) at 08C. The reaction mixture was stirred at 08C for 30 min
and then water (6 mL) and ethyl acetate (10 mL) were added. The layers
were separated and the aqueous layer was extracted with ethyl acetate
(2”10 m). The combined organic layers were dried over anhydrous
sodium sulfate and concentrated in vacuo. Purification by flash column
578
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 570 – 581

Conformationally Restrained Epothilones
FULL PAPER
by flash column chromatography on silica (16–50% EtOAc/hexanes) af-
forded pure alcohol 5b (28 mg, 83%) as a white solid. [a]2D2 =À26.0 (c=
0.3 in CHCl3); Rf =0.25 (silica gel, 50% EtOAc/hexanes); 1H NMR
(600 MHz, CDCl3): d=8.43 (s, 1H), 7.46 (dd, J=1.8 Hz, 7.8 Hz, 1H),
7.18 (d, J=7.8 Hz, 1H), 6.0–5.95 (m, 1H), 5.14 (d, J=16.8 Hz, 1H), 5.08
(d, J=10.2 Hz, 1H), 4.48 (s, 1H), 2.96–2.91 (m, 1H), 2.69–2.60 (m, 2H),
2.32 (s, 3H), 2.03 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=153.6,
149.9, 140.2, 139.4, 136.7, 135.6, 131.2, 122.3, 115.4, 86.4, 52.3, 38.5, 18.5,
13.2ppm; HRMS (ESI): m/z: calcd for C14H17NO+Na+: 238.1208
[M+Na+]; found: 238.1191.
2E,4S,7S,10R,11S,12S,16Z,18S)-7,11-Dihydroxy-3,8,8,10,12,16-hexameth-
yl-2-phenyl-3a,7,8,10,11,12,13,14,15,17a-decahydro-1H,6H-4-oxacyclo-
pentacyclohexadecene-5,9-dione (1a): A solution of the second-genera-
tion Grubbs catalyst (1.5 mg, 0.0018 mmol; weighed under argon) in
methylene chloride (1.5 mL) was added to a solution of compound 2a
(1.5 mg, 0.0032mmol) in methylene chloride (0.5 mL). The reaction mix-
ture was heated at 508C for 16 h and applied directly to a preparative
TLC plate (25% EtOAc/hexanes) to give the target (Z)-1a and slightly
impure (E)-1a (0.2mg) separately ( ꢀ50% overall yield). The Z isomer
was purified by a second preparative TLC by using (3% methanol/meth-
ylene chloride) to remove the last traces of the catalyst and to furnish
the desired target molecule (Z)-1a (0.5 mg). 1H NMR (600 MHz,
CDCl3): d=7.36–7.33 (m, 2H), 7.27–7.24 (m, 3H), 5.82 (d, J=7.2Hz,
1H), 5.24 (d, J=10.2Hz, 1H), 4.2–4.17 (m, 1H), 3.69–3.66 (m, 1H),
3.22–3.15 (m, 2H), 2.81 (dd, J=8.4, 15.6 Hz, 1H), 2.66 (d, J=4.2Hz,
1H), 2.58 (dd, J=10.8, 16.8 Hz, 2H), 2.52–2.47 (m, 1H), 2.39–2.31 (m,
2H), 1.80–1.75 (s overlapping with m, 4H), 1.68 (s, 3H), 1.66–1.62 (m,
3H), 1.36 (s, 3H), 1.18 (d, J=6.6 Hz, 3H), 1.07 (s, 3H), 0.98 ppm (d, J=
7.2Hz, 3H); HRMS (ESI): m/z: calcd for C30H42O5 +Na+: 505.2930
[M+Na+]; found: 505.2893.
(3S,6R,7S,8S)-3-(tert-Butyldimethylsilanyloxy)-4,4,6,8,12-pentamethyl-5-
oxo-7-(2,2,2-trichloroethoxycarbonyloxy)tridec-12-enoic acid (1S,2E,5S)-
2-methyl-3-phenyl-5-vinylcyclopent-2-enyl ester (32a): DCC (0.027 mL of
1m solution in CH2Cl2, 0.027 mmol, 1.3 equiv) was added dropwise to a
solution of acid 17 (13 mg, 0.021 mmol), alcohol 5a (4.6 mg, 0.023 mmol,
1.1 equiv), and DMAP (1 mg, 0.008 mmol, 0.4 equiv) in methylene chlo-
ride (0.5 mL) at 08C. The reaction mixture was stirred for 15 min at 08C
and for 16 h at room temperature. The solid precipitate was filtered off
and the filtrate was concentrated in vacuo. Purification by flash column
chromatography (5% Et2O/hexanes) afforded ester 32a (11 mg, 64%) as
a colorless oil. [a]2D2 =À53.2( c=0.85 in CHCl3); TLC Rf =0.67 (silica gel,
25% EtOAc/hexanes); 1H NMR (600 MHz, CDCl3): d=7.36–7.25 (m,
5H), 5.98–5.91 (m, 1H), 5.70 (d, J=4.2Hz, 1H), 5.10 (d, J=17.4 Hz,
1H), 5.03 (d, J=10.2Hz, 1H), 4.88–4.85 (m, 1H), 4.70 (dd, J=3.0,
8.4 Hz, 1H), 4.66–4.60 (m, 3H), 4.29 (t, J=4.2Hz, 1H), 3.50–3.46 (m,
1H), 2.96–2.92 (m, 1H), 2.86–2.81 (m, 1H), 2.68 (dd, J=3.6, 17.4 Hz,
1H), 2.59–2.55 (m, 1H), 2.23 (dd, J=5.4, 17.4 Hz, 1H), 1.95–1.87 (m,
2H), 1.75 (s, 3H), 1.66 (s, 3H), 1.54–1.53 (m, 6H), 1.33–1.27 (m, 5H),
1.06–1.04 (m, 3H), 0.96 (d, J=6.6 Hz, 3H), 0.87 (s, 9H), 0.13 (s, 3H),
0.05 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=215.4, 172.3, 154.4,
145.8, 139.3, 137.3, 132.4, 128.4, 127.9, 127.4, 115.3, 110.3, 95.0, 88.2, 82.4,
76.9, 75.0, 56.0, 54.0, 47.6, 42.2, 40.5, 40.3, 38.2, 35.2, 34.9, 31.8, 31.2, 26.2,
25.7, 24.9, 24.8, 22.7, 22.6, 20.6, 18.4, 16.1, 13.1, 11.1, À4.1, À4.5 ppm;
HRMS (ESI): m/z: calcd for C41H61Cl3O7Si+Na+: 821.3150 [M+Na+];
found: 821.3178.
(3S,6R,7S,8S,1’S,5’S)-3-(tert-Butyldimethylsilanyloxy)-4,4,6,8,12-penta-
methyl-5-oxo-7-(2,2,2-trichloroethoxycarbonyloxy)tridec-12-enoic acid 2-
methyl-3-(5-methylpyridin-2-yl)-5-vinylcyclopent-2-enyl
ester
(32b):
DCC (0.017 mL of 1m solution in CH2Cl2, 0.017 mmol, 1.3 equiv) was
added dropwise to a solution of acid 17 (8.0 mg, 0.013 mmol), alcohol 5b
(3.0 mg, 0.014 mmol, 1.1 equiv), and DMAP (1 mg, 0.008 mmol,
0.6 equiv) in methylene chloride (0.5 mL) at 08C. The reaction mixture
was stirred for 15 min at 08C and for 16 h at room temperature. After
this time, the solid precipitate was filtered off and the filtrate was concen-
trated in vacuo. Purification by flash column chromatography on silica
(5% EtOAc/hexanes) afforded ester 32b (9 mg, 85%) as a colorless oil.
[a]2D2 =À29.8 (c=1 in CHCl3); Rf =0.48 (silica gel, 25% EtOAc/hexanes);
1H NMR (600 MHz, CDCl3): d=8.43 (s, 1H), 7.46 (dd, J=1.8, 7.8 Hz,
1H), 7.18 (d, J=7.8 Hz, 1H), 5.98–5.91 (m, 1H), 5.73 (d, J=4.8 Hz, 1H),
5.11 (d, J=16.8 Hz, 1H), 5.02(d, J=10.2Hz, 1H), 4.86 (d, J=12.0 Hz,
1H), 4.73–4.70 (m, 1H), 4.67–4.61 (m, 3H), 4.30–4.28 (m, 1H), 3.50–3.45
(m, 1H), 3.06–3.02 (m, 1H), 2.88–2.83 (m, 1H), 2.70–2.65 (m, 1H), 2.33
(s, 3H), 2.23 (dd, J=5.4, 17.4 Hz, 1H), 1.98–1.91 (m, 2H), 1.95 (s, 3H),
1.76–1.74 (m, 1H), 1.66 (s, 3H), 1.50–1.22 (m, 5H), 1.10–1.04 (m. 6H),
0.96 (d, J=7.2Hz, 3H), 0.87 (m, 12H), 0.13 (s, 3H), 0.05 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=215.4, 172.3, 154.4, 153.1, 149.9, 145.8,
139.5, 138.3, 136.7, 135.7, 131.5, 125.5, 122.5, 115.4, 110.3, 95.0, 88.2, 82.4,
75.0, 54.0. 47.5, 42.2, 40.2, 39.2, 38.2, 34.9, 31.8, 29.9, 26.3, 24.8, 22.6, 22.5,
20.7, 18.5, 18.4, 16.1, 13.4, 11.1, À4.1, À4.5 ppm; HRMS (ESI): m/z: calcd
for C41H62Cl3NO7Si+H+: 814.3434 [M+H+]; found: 814.3481.
(3S,6R,7S,8S)-3,7-Dihydroxy-4,4,6,8,12-pentamethyl-5-oxotridec-12-enoic
acid (1S,2E,5S)-2-methyl-3-phenyl-5-vinylcyclopent-2-enyl ester (2a): An-
hydrous ammonium chloride (75 mg) followed by zinc dust (75 mg) was
added to a solution of ester 32a (12mg, 0.015 mmol) in dry ethanol
(1.5 mL). The reaction mixture was stirred at room temperature for
45 min before it was diluted with ethyl acetate (5 mL) and filtered
though a plug of Celite. The solution was concentrated and passed
though a small plug of silica gel to give compound 33 which was used in
the next step without further purification. To the crude solution of com-
pound 33 was added a solution of tris(dimethylamino)sulfur (trimethylsi-
lyl)difluoride (TAS-F) (5 mg, 0.187 mmol, 1 equiv) in N,N-dimethylform-
amide (0.2mL). After 24 h, another 5 mg of TAS-F were added and the
mixture was stirred for an additional 24 h after which it was diluted with
ethyl acetate (5 mL) and washed with phosphate buffer pH 7 (5 mL).
The aqueous layer was extracted with ethyl acetate (3”5 mL) and the
combined organic layers were dried over anhydrous sodium sulfate, fil-
tered, and concentrated in vacuo. The crude oil was purified by silica gel
chromatography (10% EtOAc/hexanes) to give pure compound 2a
(4.7 mg, 62% over two steps) as a colorless material. [a]2D2 =À77.5 (c=
0.2in CHCl 3); Rf =0.74 (silica gel, 50% EtOAc/hexanes); 1H NMR
(600 MHz, CDCl3): d=7.37–7.25 (m, 5H), 6.0–5.92 (m, 1H), 5.76 (d, J=
3.6 Hz, 1H), 5.13 (d, J=16.8 Hz, 1H), 5.06 (d, J=10.2Hz, 1H), 4.66 (d,
J=10.2 Hz, 2H), 4.29–4.26 (m, 1H), 3.39–3.35 (m, 2H), 3.27–3.23 (m,
2H), 2.99–2.93 (m, 1H), 2.88–2.83 (m, 1H), 2.61–2.57 (m, 1H), 2.52 (dd,
J=1.8, 16.2Hz, 1H), 2.44 (dd, J=10.2, 16.2 Hz, 1H), 2.04–1.96 (m, 2H),
1.79 (s, 3H), 1.75–1.72(m, 1H), 1.69 (s, 3H), 1.54–1.52(m, 1H), 1.36–
1.30 (m, 1H), 1.20–1.18 (s overlapping with m, 4H), 1.15 (s, 3H), 1.06 (d,
J=7.2Hz, 3H), 0.84 ppm (d, J=7.2Hz, 3H); 13C NMR (100 MHz,
CDCl3): d=222.5, 173.2, 146.4, 139.8, 139.4, 137.1, 131.8, 128.5, 127.9,
127.6, 115.5, 109.9, 88.6, 75.0, 72.7, 52.3, 47.8, 41.1, 40.4, 38.4, 36.9, 35.7,
32.7, 25.0, 22.6, 21.5, 19.2, 15.7, 13.1, 10.2 ppm; HRMS (ESI): m/z: calcd
for C32H46O5 +Na+: 533.3243 [M+Na+]; found: 533.3213.
(3S,6R,7S,8S,1’S,5’S)-3,7-Dihydroxy-4,4,6,8,12-pentamethyl-5-oxotridec-
12-enoic acid 2-methyl-3-(5-methyl-pyridin-2-yl)-5-vinylcyclopent-2-enyl
ester (2b): A solution of tris(dimethylamino)sulfur (trimethylsilyl)difluor-
ide (TAS-F) (1.7 mg, 0.008 mmol, 1 equiv) in N,N-dimethylformamide
(0.2mL) was added to a solution of ester 32b (5 mg, 0.006 mmol) in
DMF (0.2mL). After 24 h, another 1.7 mg of TAS-F were added and the
mixture was diluted with ethyl acetate (3 mL) and washed with phos-
phate buffer pH 7 (5 mL). The aqueous layer was extracted with ethyl
acetate (3”5 mL) and the combined organic layers were dried over anhy-
drous sodium sulfate, filtered, and concentrated in vacuo. The crude oil
was purified by silica-gel chromatography (10% EtOAc/hexanes) to give
compound 34 which was dissolved in dry ethanol (1.2mL) and treated
with anhydrous ammonium chloride (62mg) followed by zinc dust
(62mg). The reaction mixture was stirred at room temperature for
45 min before it was diluted with ethyl acetate (3 mL) and filtered
though a plug of Celite. The solution was concentrated and purified by
silica-gel chromatography (20% EtOAc/hexanes) to give pure compound
2b (2mg, 62% over two steps) as a colorless material. Rf =0.36 (silica
1
gel, 33% EtOAc/hexanes); H NMR (500 MHz, CDCl3): d=8.47 (s, 1H),
7.50 (d, J=8.0 Hz, 1H), 7.21 (d, J=8.5 Hz, 1H), 6.02–5.95 (m, 1H), 5.81
(d, J=4.5 Hz, 1H), 5.16 (d, J=17.5 Hz, 1H), 5.07 (d, J=10.5 Hz, 1H),
4.68 (d, J=9.0 Hz, 2H), 4.30 (d, J=10.5 Hz, 1H), 3.41–3.37 (m, 2H),
3.30–3.27 (m, 1H), 3.25 (d, J=3.5 Hz, 1H), 3.10–3.3.06 (m, 1H), 2.91–
2.88 (m, 1H), 2.71–2.69 (m, 1H), 2.54 (dd, J=2.5, 16.5 Hz, 1H), 2.46 (dd,
Chem. Eur. J. 2008, 14, 570 – 581
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L. M. V. Tillekeratne et al.
J=10.5, 16.5 Hz, 1H), 2.35 (s, 3H), 2.06–2.02 (m, 2H), 1.98 (s, 3H), 1.78–
1.76 (m, 1H), 1.72(s, 3H), 1.33–1.31 (m, 2H), 1.22(s, 3H), 1.17 (s, 3H),
1.08 (d, J=6.5 Hz, 3H), 0.87 ppm (d, J=7.0 Hz, 3H); 13C NMR
(125 MHz, CDCl3): d=222.1, 172.9, 152.8, 149.8, 146.2, 139.1, 136.5,
135.0, 131.4, 122.2, 115.3, 109.7, 88.3, 74.9, 72.5, 52.1, 47.5, 41.0, 38.9, 38.2,
36.8, 35.5, 32.5, 29.7, 24.9, 22.4, 21.3, 19.0, 18.3, 15.6, 13.2, 10.0 ppm;
HRMS (ESI): m/z: calcd for C32H47NO5 +Na+: 548.3352[ M+Na+];
found: 548.3331.
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(3S,6R,7S,8S,1’S,5’S)-7,11-Dihydroxy-3,8,8,10,12,16-hexamethyl-2-(5-
methylpyridin-2-yl)-3a,7,8,10,11,12,13,14,15,17a-decahydro-1H,6H-4-oxa-
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(2.5 mg, 0.0052 mmol) in methylene chloride (0.5 mL). The reaction mix-
ture was heated at 508C for 16 h and applied directly to a preparative
TLC plate and developed (25% EtOAc/hexanes) to give the target (Z)-
1b (0.5 mg) along with phenyl analogue 35 and the dimer 36 (ꢀ0.5 mg
each; ꢀ55 overall yield).
Data for compound 1b: Rf =0.23 (silica gel, 50% EtOAc/hexanes);
1H NMR (500 MHz, CDCl3): d=8.60 (s, 1H), 8.16 (d, J=8.0 Hz, 1H),
7.54 (d, J=8.0 Hz, 1H), 5.84 (d, J=7.0 Hz, 1H), 5.25 (d, J=10.0 Hz,
1H), 4.53 (d, J=10.0 Hz, 1H), 3.64–3.62(m, 1H), 3.47–3.40 (m, 2H),
3.26–3.21 (m, 2H), 2.81 (dd, J=2.5, 16.5 Hz, 1H), 2.65 (dd, J=10.5,
16.5 Hz, 1H), 2.59 (s, 3H), 2.38–2.31 (m, 1H), 2.00 (s overlapping with
m, 5H), 1.84–1.79 (m, 2H), 1.72 (s, 3H), 1.42 (s, 3H), 1.35–1.31 (m, 2H),
1.16 (d, J=6.5 Hz, 3H), 1.06 (s, 3H), 1.02ppm (d, J=7.0 Hz, 3H);
HRMS (ESI): m/z: calcd for C30H43NO5 +H+: 498.3219 [M+H+]; found:
498.3231.
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3,7-Dihydroxy-4,4,6,8,12-pentamethyl-5-oxotridec-12-enoic acid 2-methyl-
3-(5-methylpyridin-2-yl)-5-styrylcyclopent-2-enyl ester (35): Rf =0.61
(silica gel, 50% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3): d=8.48
(d, J=2.0 Hz, 1H), 7.51 (dd, J=2.0, 8.0 Hz, 1H), 7.37–7.30 (m, 4H),
7.25–7.21 (m, 2H), 6.52 (d, J=16.0 Hz, 1H), 6.34 (dd, J=8.0, 16.0 Hz,
1H), 5.87 (d, J=4.5 Hz, 1H), 4.68 (d, J=9.0 Hz, 2H), 4.31–4.28 (m, 1H),
3.42–3.36 (m, 2H), 3.30–3.26 (m, 1H), 3.24 (d, J=4.0 Hz, 1H), 3.19–3.04
(m, 2H), 2.81–2.76 (m, 1H), 2.55 (dd, J=2.5, 16.0 Hz, 1H), 2.46 (dd, J=
10.5, 16.0 Hz, 1H), 2.36 (s, 3H), 2.01 (s overlapping with m, 5H), 1.72
(m, 2H), 1.71 (s, 3H), 1.35 (m, 2H), 1.22 (s, 3H), 1.17 (s, 3H), 1.07 (d,
J=7.0 Hz, 3H), 0.86 (d, J=7.0 Hz, 3H); HRMS (ESI): m/z: calcd for
C38H51NO5 +Na+: 624.3665 [M+Na+]; found: 624.3713.
Dimer 36: Rf =0.37 (silica gel, 50% EtOAc/hexanes); 1H NMR
(500 MHz, CDCl3): d=8.45 (d, J=2.0 Hz, 1H), 7.48 (dd, J=2.0, 8.0 Hz,
1H), 7.17 (d, J=8.0 Hz, 1H), 5.81–5.78 (m, 1H), 5.66 (dd, J=2.5, 5.5 Hz,
1H), 4.66 (d, J=7.5 Hz, 2H), 4.45–4.41 (m, 1H), 4.20–4.17 (m, 1H), 3.55
(s, 1H), 3.40 (d, J=9.0 Hz, 1H), 3.35 (dd, J=6.5, 13.5 Hz, 1H), 2.98–2.93
(m, 1H), 2.87–2.83 (m, 1H), 2.65–2.60 (m, 1H), 2.54–2.51 (m, 2H), 2.34
(s, 3H), 2.05–1.94 (m, 3H), 1.91 (s, 3H), 1.70 (s, 3H), 1.26 (s overlapping
with m, 5H), 1.18 (s, 3H), 1.09 (d, J=7.0 Hz, 3H), 0.87 ppm (d, J=
7.0 Hz, 3H); HRMS (ESI): m/z: calcd for C62H90N2O10 +Na+: 1045.6493
[M+Na+]; found: 1045.6522.
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Acknowledgements
This research was initiated with seed funding provided by the Elsa U.
Pardee Foundation. Support from the Intramural Research Program of
the NIH, NIEHS for molecular modeling studies (to L.P.) is gratefully ac-
knowledged. The authors thank the Developmental Therapeutics Pro-
gram of the National Cancer Institute and Professor A. A. L. Gunatilaka,
Director of the Southwest Center for Natural Products Research of the
University of Arizona, for assistance with cytotoxicity studies.
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