Total synthesis of epothilone B, epothilone D, and cis- and trans-9,10-dehydroepothilone D

Article abstract of DOI:10.1021/ja010454b

The phosphonium salt 35, representing one of the two principal subunits of the epothilones, was prepared from propargyl alcohol via heptenone 22. A Wittig reaction of the phosphorane from 35 with aldehyde 33, obtained from aldol condensation of ketone 27 with aldehyde 28, afforded 37. Seco acid 42 derived from 37 underwent lactonization to give cis-9,10-dehydroepothilone D (43) which was selectively reduced with diimide to yield epothilone D (4) and, after epoxidation, epothilone B (2). An alternative route to epothilone D employed alkyne 39, obtained from 33, in a Castro-Stephens reaction with allylic bromide 34 to furnish enyne 40. The latter was semi-hydrogenated to provide 37. Alkyne 46, prepared from alcohol 45, was converted to trans-vinylstannane 47 which, in a Stille coupling with allylic chloride 50, gave 51. Seco acid 52 derived from 51 underwent lactonization to give trans-9,10-dehydroepothilone D (54). Bioassay data comparing the antiproliferative activity and tubulin polymerization of 43 and 54 with epothilone B (2), epothilone D (4), and paclitaxel (7) showed that the synthetic analogues were less potent than their natural counterparts, although both retain full antiproliferative activity against a paclitaxel-resistant cell line. No significant difference in potency was noted between cis analogue 43 and its trans isomer 54.

Full text of DOI:10.1021/ja010454b

J. Am. Chem. Soc. 2001, 123, 5407-5413
5407
Total Synthesis of Epothilone B, Epothilone D, and cis- and
trans-9,10-Dehydroepothilone D
James D. White,*† Rich G. Carter,† Kurt F. Sundermann,† and Markus Wartmann‡
Contribution from the Department of Chemistry, Oregon State UniVersity, CorVallis, Oregon 97331-4003,
and NoVartis Pharma AG, CH-4002 Basel, Switzerland
ReceiVed February 20, 2001
Abstract: The phosphonium salt 35, representing one of the two principal subunits of the epothilones, was
prepared from propargyl alcohol via heptenone 22. A Wittig reaction of the phosphorane from 35 with aldehyde
33, obtained from aldol condensation of ketone 27 with aldehyde 28, afforded 37. Seco acid 42 derived from
37 underwent lactonization to give cis-9,10-dehydroepothilone D (43) which was selectively reduced with
diimide to yield epothilone D (4) and, after epoxidation, epothilone B (2). An alternative route to epothilone
D employed alkyne 39, obtained from 33, in a Castro-Stephens reaction with allylic bromide 34 to furnish
enyne 40. The latter was semi-hydrogenated to provide 37. Alkyne 46, prepared from alcohol 45, was converted
to trans-vinylstannane 47 which, in a Stille coupling with allylic chloride 50, gave 51. Seco acid 52 derived
from 51 underwent lactonization to give trans-9,10-dehydroepothilone D (54). Bioassay data comparing the
antiproliferative activity and tubulin polymerization of 43 and 54 with epothilone B (2), epothilone D (4), and
paclitaxel (7) showed that the synthetic analogues were less potent than their natural counterparts, although
both retain full antiproliferative activity against a paclitaxel-resistant cell line. No significant difference in
potency was noted between cis analogue 43 and its trans isomer 54.
Introduction
The isolation and structural elucidation of epothilones A-F
(1-6) by Ho¨fle and Reichenbach from the soil myxobacterium
Sorangium cellulosum1 brought to light a remarkable new family
of macrolides which has been the source of a large body of
research directed toward the anticancer properties of these
substances.
and their effectiveness against tumor cells which exhibit multiple
drug resistance.4 Epothilone B (2), which is approximately 50
times more active than paclitaxel and which is available in
kilogram quantities from fermentation of S. cellulosum, along
with epothilone D (4), now appears to be the optimal candidates
for drug development from among the naturally occurring
epothilones and a large ensemble of synthetic analogues.5 It was
these substances which initially provided the focus for our
synthetic efforts directed toward the epothilones.6
At the outset, our primary goal was the development of a
synthetic route to 2 which would embody the highest possible
degree of stereocontrol. We considered it especially important
to establish clean Z geometry of the 12,13-trisubstituted double
bond and to set in place the stereocenters at C6, C7, and C15
with complete stereochemical accuracy. As our plan for a
convergent synthesis unfolded, it became evident that an
attractive means for connecting two major subunits such as A
and B would be via C-C bond formation at C9-C10 (Scheme
1). This plan would not only allow the incorporation of
As microtubule-stabilizing agents with a mechanism of action
similar to that of paclitaxel (Taxol, 7),2 the epothilones exhibit
exceptional promise as a new lead in cancer chemotherapy,
especially where conventional treatments are ineffective.3
The advantages shown by the epothilones over paclitaxel include
their increased water solubility, their more rapid action in vitro,
† Oregon State University
‡ Novartis Pharma AG
(1) (a) Ho¨fle, G.; Bedorf, N.; Gerth, H.; Reichenbach (GBF), DE-B
4138042, 1993; Chem. Abstr. 1993, 120, 52841. (b) Ho¨fle, G.; Bedorf, N.;
Steinmeth, H.; Schomburg, D.; Gerth. H.; Reichenbach, H. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1567.
(2) (a) Bollag, D. M.; McQueney, P. A.; Zhu, J.; Hensens, O.; Koupal,
L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Cancer Res. 1995,
55, 2325. (b) For a recent review, see: Altmann, K.-H.; Wartmann, M.;
O’Reilly, T. Biochim. Biophys. Acta 2000, 1470, M79.
(3) Harris, C. R.; Danishefsky, S. J. J. Org. Chem. 1999, 64, 8434 and
references therein.
(4) For a review of the chemical biology of epothilones, see: Nicolaou,
K. C.; Roschangar, F.; Vourloumis, F. Angew. Chem., Int. Ed. 1998, 37, 7,
2014.
(5) Altmann, K.-H.; Bold, G.; Caravatti, G.; End, N.; Flo¨rsheimer, A.;
Guagnano, V.; O’Reilly, T.; Wartmann, M. Chimia 2000, 54, 612.
10.1021/ja010454b CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/16/2001

5408 J. Am. Chem. Soc., Vol. 123, No. 23, 2001
White et al.
Scheme 1
of this structure to that of paclitaxel. The solid-state structure
of 2 revealed by X-ray crystallographic1b analysis shows that
the carbon atoms attached to C9 and C10 are antiperiplanar,
resulting in an almost perfect zigzag alignment of the C7-C12
segment. Structural studies of epothilone A (1) using NMR
techniques suggest that the major conformer in solution is similar
to that of 2 in the crystal, although the C6-C11 segment of the
perimeter shows flexibility, and ca. 20% of a minor conformer
can be seen.8 One current view of the pharmacophoric relation-
ship between paclitaxel (7) and the epothilones overlays C6-
C9 of 7, where a trans antiperiplanar orientation is enforced by
the fusion of the B and C rings, with C11-C8 of 2.9
Incorporation of a cis double bond at C9-C10 of 4 would
rigidify this segment of the macrolide perimeter and would
remove its conformational homology with 7 by enforcing a syn
coplanar arrangement of the four carbons C8-C11. It might
therefore be expected that the conformational change induced
by a cis 9,10 double bond would impact the biological activity
of 4. By contrast, a trans double bond at C9-C10 of 4 would
constrain the C8-C11 domain of the macrolide perimeter to
an antiperiplanar orientation, thereby restoring conformational
homology with paclitaxel.10
Two approaches were projected for installing a cis-olefin at
C9-C10 of 4 and, hence, 2. The first envisioned a Wittig
olefination which would connect phosphorane 8 with aldehyde
9, while the second option postulated alkylation of the anion
from terminal alkyne 11 with allylic bromide 10. The latter route
would generate a seco acid 12 which could be semi-
hydrogenated to obtain the substance produced from 8 and 9.
Alternatively, the alkyne 11 could be used to produce a trans-
vinylstannane, and Stille coupling with 10 would then lead to
a trans-9,10-dehydroepothilone in which the antiperiplanar
orientation of atoms around C9-10 was fixed and immutable.
The versatility offered by these approaches to epothilone
synthesis appeared inviting, especially since 8 and 11 could in
principle be acquired from 10 and 9, respectively, through
straightforward transformations. Efforts therefore began toward
synthesis of the coupling partners in the set 8-11.
functionality in a domain of the macrolide, which has not been
extensively explored from the viewpoint of analogue synthesis,
but would also offer an opportunity to constrain a region of the
perimeter thought to be somewhat flexible.7 The latter point
impinges upon an important issue relevant to the three-
dimensional structure of the epothilones, and to the relationship
Results and Discussion
(6) Total synthesis of epothilone B: (a) Su, D.-S.; Meng, D.; Bertinato,
P.; Balog, A.; Sorensen, E. J.; Danishefsky, S. J.; Zheng, Y.-H.; Chou, T.-
C.; He, L.; Horwitz, S. B. Angew. Chem. 1997, 109, 775; Angew. Chem.,
Int. Ed. Engl. 1997, 36, 757. (b) Meng, D.; Bertinato, P.; Balog, A.; Su,
D.-S.; Komenecka, T.; Sorensen, E. J.; Danishefsky, S. J. J. Am. Chem.
Soc. 1997, 119, 10073. (c) Nicolaou, K. C.; Winssinger, N.; Pastor, J. A.;
Ninkovic, S.; Sarabia, F.; He, Y.; Vourloumis, D.; Yang, Z.; Li, T.;
Giannakakou, P.; Hamel, E. Nature 1997, 387, 268. (d) Nicolaou, K. C.;
Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay,
M. R. V.; Yang, Z. J. Am. Chem. Soc. 1997, 119, 7974. (e) Balog, A.;
Harris, C.; Savin, K.; Zhang, X.-G.; Chou, T.-C.; Danishefsky, S. J. Angew.
Chem. 1998, 110, 2821; Angew. Chem., Int. Ed. 1998, 37, 2675. (f) Schinzer,
D.; Bauer, A.; Schieber, J. Synlett 1998, 861. (g) May, S. A.; Grieco, P.
Chem. Commun. 1998, 1597. (h) Mulzer, J.; Mantoulidis, A.; O¨ hler, E.
Tetrahedron Lett. 1998, 39, 8633. (i) White, J. D.; Carter, R. G.;
Sundermann, K. F. J. Org. Chem. 1999, 64, 684. (j) White, J. D.;
Sundermann, K. S.; Carter, R. G. Org. Lett. 1999, 1, 1431. (k) Nicolaou,
K. C.; Hepworth, D.; Finley, M. R. V.; King, N. P.; Werschkun, B.; Bigot,
A. Chem. Commun. 1999, 519. (l) Schinzer, D.; Bauer, A.; Schieber, J.
Chem. Eur. J. 1999, 5, 2492. (m) Harris, C. R.; Kuduk, S. D.; Savin, K.;
Balog, A.; Danishefsky, S. J. Tetrahedron Lett. 1999, 40, 2263. (n) Harris,
C. R.; Kuduk, S. D.; Balog, A.; Savin, K.; Danishefsky, S. J. J. Am. Chem.
Soc. 1999, 121, 7050. (o) Martin, H. J.; Drescher, M.; Mulzer, J. Angew.
Chem., Int. Ed. 2000, 39, 581. (p) Sawada, D.; Kanai, M.; Shibasaki, M. J.
Am. Chem. Soc. 2000, 122, 10521. (q) Mulzer, J.; Mantoulidis, A.; O¨ hler,
E. J. Org. Chem. 2000, 65, 7456. (r) For a recent review of synthetic efforts
directed towards epothilones, see: Mulzer, J. Chem. Mon. 2000, 131, 205.
(7) For other modifcations to this region of the epothilone perimeter,
see: (a)Winkler, J. D.; Holland, J. M.; Kasparec, J.; Axelsen, P. H.
Tetrahedron 1999, 55, 8199. (b) Taylor, R. E.; Galvin, G. M.; Hilfiker, K.
A.; Chen, Y. 217th ACS National Meeting, Anaheim, CA, March 21-25,
1999.
Synthesis of Subunits A and B. Since early installation of
the Z trisubstituted C12-C13 double bond of 4 was judged to
be of pivotal importance, our route began with construction of
this moiety in a form which would permit extension from each
terminus in succession toward subunit A. Carbocupration of
propargyl alcohol has been shown to proceed with clean regio-
and stereoselectivity to give iodo alcohol 13,11 and although
the yield of this conversion is low, the minimal cost of reagents
makes this an acceptable process for constructing a function-
alized trisubstituted alkene suitable for our purpose. After
protection of 13 as its THP ether 14, the latter was subjected to
halogen-metal exchange with tert-butyllithium. Transmetalation
with cuprous cyanide then gave an alkenylcopper species which
underwent conjugate addition to (S)-3-acryloyl-4-benzyloxazo-
(8) Taylor R. E.; Zajicek, J. J. Org. Chem. 1999, 64, 7224.
(9) Giannakakou, P.; Gussio, R.; Nogales, E.; Downing, K. H.; Zaha-
revitz, D.; Bollbuck, B.; Poy, G.; Sackett, D.; Nicolaou, K. C.; Fojo, T.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2904.
(10) For a different view of the pharmacophoric relationship between
epothilones and Taxol, see: (a) Ojima, I.; Chakravarty, S.; Inoue, T.; Lin,
S.; He, L.; Horwitz, S. B.; Kuduk, S. D.; Danishefsky, S. J. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 4256. (b) Wang, M.; Xia, X.; Kim, Y.; Hwang,
D.; Jansen, J. M.; Botta, M.; Liotta, D. C.; Snyder, J. P. Org. Lett. 1999, 1,
43.
(11) Duboudin, J. G.; Jousseaume, B.; Bonakdar, A. J. Organomet. Chem.
1979, 168, 227.

Synthesis of Epothilones
J. Am. Chem. Soc., Vol. 123, No. 23, 2001 5409
Scheme 2
Scheme 4
Scheme 3
with regard to installation of the stereocenters at C6 and C7.
Although an aldol construction is an attractive option for creating
the C6-C7 linkage of B and has been employed in many
approaches to this segment of the epothilones,5 the double
stereodifferentiating anti-Felkin pathway which is required to
generate the correct stereochemical triad at C6, 7, 8 has only
recently been achieved in a stereocontrolled fashion. Thus,
Nicolaou, after obtaining an initially disappointing 3:1 stereo-
selectivity,6d was eventually able to accomplish this aldol
connection in a process which favored the anti-Felkin mode by
10:1.6k Mulzer improved this ratio to 95:5,6o and in his synthesis
of 2 Schinzer obtained a stereoselectivity of 9:1.6l Our own
studies with double stereodifferentiating aldol reactions have
demonstrated that seemingly minor structural variation in the
substrates or in the reaction conditions can exert a profound
influence on the stereochemical outcome. In particular, the
presence of a strongly ligating substituent such as a p-
methoxybenzyl ether adjacent to the aldehyde component can
play a pivotal role in steering an enolate toward one face of the
carbonyl through the operation of a chelation effect.18 This
effect, which was noted during our synthesis of the polypropi-
onate segment of rutamycin B,19 has also been recognized by
Mulzer in the context of a chelation-controlled aldol reaction
that furnished the same anti,syn stereotriad seen in 29.20
Application of this principle to our plan for the synthesis of
subunit B led to the proposal that the enolate of ketone 27,
previously synthesized by Nicolaou from keto aldehyde 26,6d
should be reacted with (S)-aldehyde 28 bearing a p-methoxy-
benzyloxy (PMBO) substituent for secondary chelation with the
aldehyde carbonyl. Our previous experience suggested that an
ether that ligates less strongly to an alkali metal cation than
PMBO would result in diminished stereoselectivity,19 and this
indeed proved to be the case (vide infra).
lidin-2-one (15).12 The sodium enolate of the resulting oxazo-
lidinone 16 was hydroxylated13 using Davis' oxaziridine14 17
to afford alcohol 18 with diastereoselectivity >98:2 (Scheme
2). The (S) configuration of the hydroxyl substituent in 18 was
confirmed by its ozonolytic degradation to dimethyl (S)-maleate
(19) (Scheme 3).
After protection of 18 as its silyl ether 20, the chiral auxiliary
was removed with ethanethiol containing a catalytic quantity
of potassium ethanethiolate.15 The resulting thioester 21,
obtained in nearly quantitative yield, proved to be an excellent
substrate for introduction of the requisite methyl ketone 22
through reaction with lithium dimethylcuprate. A Wadsworth-
Emmons olefination of this ketone with the lithium anion of
the known phosphonate 2316 furnished the E,Z diene 24,
accompanied by a trace (<5%) of the Z,Z isomer. The latter
was easily removed from the mixture by rapid chromatography,
leading to a fully stereocontrolled preparation of 24. Since we
next intended to elaborate this substance at the primary ether
terminus, a means had to be found for removing the THP block
without unmasking the silyl ether. Magnesium bromide in ether17
served well for this purpose and afforded primary alcohol 25
in an overall yield of 36% for the seven steps from 14 (Scheme
4). The stable alcohol 25, representing the subunit A required
for assembly of 12 and thus 4, provided a convenient staging
point for access to 10 and 8 and was readily acquired in
quantities as large as 10 g by the foregoing route.
The second subunit B needed for the convergent plan outlined
in Scheme 1 presents a more demanding exercise, particularly
(12) Evans, D. A.; Chapman, K. T.; Bisaha, J. J. Am. Chem. Soc. 1998,
110, 1238.
(13) Evans, D. A.; Morrissey, M. M.; Dorow, R. L. J. Am. Chem. Soc.
1985, 107, 4346
(14) Davis, F. A.; Chaltophadhyay, S.; Towson, J. C.; Lol, S.; Reddy,
T. J. Org. Chem. 1988, 53, 2087.
(15) Evans, D. A.; Ripin, D. H. B.; Johnson, J. S.; Shaughnessy, E. A.
Angew. Chem., Int. Ed. Engl. 1997, 36, 2117.
(16) Schinzer, D.; Limberg, A.; Bo¨hm, O. M. Chem. Eur. J. 1996, 2,
1477.
(18) For a different view of mechanistic details underlying this aldol
construction, see Wu, Z.; Zhang, F.; Danishefsky, S. J. Angew. Chem., Int.
Ed. 2000, 39, 4505.
(19) White, J. D.; Porter, W. J.; Tiller, T. Synlett 1993, 535.
(20) Mulzer, J.; Mantoulidis, A. Tetrahedron Lett. 1996, 37, 9179.
(17) Kim, S.; Park, J. H. Tetrahedron Lett. 1987, 28, 439.

5410 J. Am. Chem. Soc., Vol. 123, No. 23, 2001
White et al.
secondary chelation between the benzyl ether and aldehyde
carbonyl which directs the addition of the Z enolate of 27 toward
the re face of 28. Whatever the true explanation for the
stereoselectivity observed in the coupling of 27 and 28, our result
was most welcome since it avoided the need to remove
stereoisomers of 29 from the reaction.
Figure 1. Possible transition state for the anti-Felkin aldol reaction of
27 with 28 invoking double chelation.
After protection of alcohol 29 as its silyl ether 30, a
degradative sequence was implemented which transformed the
terminal vinyl group of 30 by oxidative cleavage to carboxylic
acid 31 and then to methyl ester 32. Hydrogenolysis of the
p-methoxybenzyl ether of 32 gave a primary alcohol which was
oxidized directly to aldehyde 33 with a catalytic quantity of
Ley’s reagent in the presence of N-methylmorpholine-N-oxide.23
Aldehyde 33 represents subunit B in our strategic plan (Scheme
1) and can be viewed either as a masked version of 9 or the
direct precursor of terminal alkyne 11.
Scheme 5
Assembly of Subunits. Our initial tactic for connecting
subunits A and B envisioned the Wittig olefination shown in
Scheme 1 which would set in place a C9-C10 cis double bond.
It was anticipated that a triene such as 12 would provide access
to a seco acid precursor of epothilone D (4) by selective
hydrogenation of the disubstituted alkene, but it was also our
intention to advance 12 toward a cis-9,10-dehydroepothilone
by leaving the double bond in place so that this analogue could
be evaluated against its congeners for antimitotic activity.
In preparation for the Wittig coupling leading to 12,
alcohol 25 was first converted to bromide 34 by reaction with
methanesulfonic anhydride followed by displacement of the
allylic mesylate with lithium bromide. The allylic bromide 34
was then homologated to phosphonium bromide 35 by displace-
ment with the phosphorane 36 obtained from methyltriph-
enylphosphonium bromide and n-butyllithium. The Wittig
reaction of 33 with the phosphorane prepared from 35 with
lithium hexamethyldisilylazide, when carried out under carefully
controlled conditions, afforded the pure E,Z,Z-triene 37 in
excellent yield (Scheme 6).
Although the Wittig coupling of 33 with 35 was in most
respects satisfactory, the strongly basic conditions of the reaction
along with the fact that stoichiometry was difficult to control
on a small scale, prompted investigation of an alternative method
for linking subunits A and B. A protocol which effected direct
coupling of A with B rather than preliminary homologation of
the bromide 34 to 35 would confer obvious advantage, but this
implied that a one-carbon homologation of aldehyde 33 would
be needed. In practice, homologation of 33 was readily achieved
through its condensation with dimethyl diazomethylphospho-
nate24 (38), leading to terminal alkyne 39. The latter was now
poised for displacement of bromide from 34, which was
accomplished via a modified Castro-Stephens25 coupling using
cuprous iodide in the presence of triethylamine.26 This reaction
yielded dienyne 40 along with a small amount of the conjugated
diyne resulting from oxidative self-coupling of 39. Semihydro-
genation of 40 over Lindlar’s catalyst in hexane27 gave 37
(Scheme 7), identical with the product obtained from Wittig
coupling of 33 and 35.
Aldol condensation of 27 with 28, the latter prepared from
methyl (S)-3-hydroxy-2-methylpropionate, yielded anti-Felkin
product 29 as the sole stereoisomer (Scheme 5).21 The impor-
tance of the PMB ether in 28 is without question here since its
replacement by a TBS ether resulted in much lower stereose-
lectivity in this aldol reaction. Results published by Schinzer6l
imply that there are structural component(s) of the enolate which
participate in chelation to the metal cation and thereby reinforce
the anti-Felkin course of this aldol reaction, but our observations
are not in accord with this view since structural variation in the
ketone had little effect on the stereoselectivity of this reaction.
The explanation proposed by Mulzer for the stereochemical
outcome of this aldol reaction envisions a boatlike transition
state in which a single lithium cation is coordinated to the benzyl
ether, the aldehyde carbonyl, and the enolate oxygen.20 An
alternative model for the transition state of the reaction of 27
with 28 is presented in Figure 1, where double metal chelation
involving both lone pairs of the aldehyde oxygen is invoked in
a chairlike transition state. There is ample precedent in the
literature,22 including crystal structures of bis chelated carbonyl
compounds, to support the concept of double chelation as
proposed in Figure 1. In the present instance, it would be
The three secondary silyl ethers of 37 are in sufficiently
similar stereochemical environments to make selective unmask-
ing of the ether at C15 seem implausible. Indeed, a variety of
(23) Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, S. P. Synthesis
1994, 639.
(24) Gilbert, J. C.; Weerasooriya, V. J. Org. Chem. 1982, 47, 1837.
(25) Stephens R. D.; Castro, C. E. J. Org. Chem. 1963, 28, 3313
(26) Mignani, G.; Chevalier, C.; Grass, F.; Allmang, G.; Morel, D.
Tetrahedron Lett. 1990, 31, 5161.
(27) Freifelder, M. Practical Catalytic Hydrogenation, Techniques and
Applications; Wiley-Interscience: New York, 1971; p 99.
(21) For a similar result, see Claus, E.; Pabl, A.; Jones, P. G.; Meyer,
H. M.; Kalesse, M. Tetrahedron Lett. 1997, 38, 1359.
(22) (a) Sharma, V.; Simard, M.; Wuest, J. D. J. Am. Chem. Soc. 1992,
114, 7931. (b) Tschinkl, M.; Schier, A.; Riede, J.; Gabbai, F. P. Organo-
metallics 1999, 18, 1747.

Synthesis of Epothilones
J. Am. Chem. Soc., Vol. 123, No. 23, 2001 5411
Scheme 6
Scheme 8
Scheme 7
Scheme 9
seco acid 42. Under Yamaguchi conditions,28 42 gave the bis
tert-butyldimethylsilyl ether of 9,10-dehydroepothilone D in a
yield virtually identical with that obtained by others6 for
macrolactonization of the dihydro analogue of the seco acid.
Removal of the two remaining silyl ethers from the macrolactone
with trifluoroacetic acid gave 9,10-dehydroepothilone D (43).
Attempts to selectively hydrogenate the disubstituted olefin
of 43 using Wilkinson’s catalyst were unsuccessful. However,
reduction of 43 with diimide,29 generated from dipotassium
azodicarboxylate, was moderately selective and resulted in
epothilone D (4) in acceptable yield (Scheme 9). Final epoxi-
dation of 4 with dimethyldioxirane under conditions described
by Danishefsky6a afforded epothilone B (2), whose 1H and 13
C
NMR spectra exactly matched those of an authentic sample.
reagents known to cleave silyl ethers displayed little or no
selectivity with 37. However, after saponification of 37, the
resultant carboxylic acid 41 underwent selective desilylation at
C15 to afford hydroxy acid 42 in excellent yield (Scheme 8).
Similarly selective desilylation was noted by Nicolaou in the
Stille Coupling Route. The synthesis of cis-9,10-dehydroe-
pothilone D (43) via Castro-Stephens coupling of alkyne 39
with bromide 34 provides only modest advantage over the Wittig
pathway employing 33 and 35, but it does offer an opportunity
for replacing the cis double bond at the C9-C10 position in
the macrolide perimeter by a trans-olefin. Stille coupling30 of
a trans-vinylstannane derived from 39 with an allylic halide
corresponding to 34 was initially envisioned as the means of
access to trans-9,10-dehydroepothilone D. However, the modest
(66%) yield of 41 from the saponification of 37 indicated that
a further modification to this plan would be desirable, namely
case of the 9,10 dihydro version of carboxylic acid 41.6d
A
possible explanation for the markedly different results obtained
in the deprotection of ester 37 and acid 41 may involve
intramolecular silyl transfer from the C15 oxygen to an
intermediate carboxylate anion; however, no evidence for a
transient silyl ester was seen in the reaction with 41. Neverthe-
less, this result suggested that the hydroxyl and carboxyl centers
can achieve proximity and augured well for the ensuing
macrolactonization. It is possible based on conformational
analysis of 41 that the cis-9,10-olefin encourages this proximity,
but a “folding” effect was not manifest in the lactonization of
(28) Inanaga, J.; Kirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. Jpn. 1979, 52, 1989.
(29) Pasto, D. J.; Taylor, R. T. Org. React. 1991, 40, 91.
(30) Farina, V.; Krishnamurthy, V.; Scott, W. J. Org. React. 1997, 50,
1.

5412 J. Am. Chem. Soc., Vol. 123, No. 23, 2001
White et al.
Scheme 10
Scheme 11
Scheme 12
installation of an ester which could be removed efficiently and
simultaneously with the C15 silyl-protecting group. An obvious
choice for this purpose was a (trimethylsilyl)ethyl ester, and
31 was therefore esterified with 2-(trimethylsilyl)ethanol using
Mitsunobu conditions to provide 44. Hydrogenolysis removed
the p-methoxybenzyl ether from 44, and oxidation of alcohol
45 afforded an aldehyde which was reacted with Bestmann’s
reagent31 to give terminal alkyne 46. Hydrostannylation of the
latter in the presence of a palladium dichloride catalyst furnished
vinylstannane 47 (Scheme 10).
Although removal of the TBS ether at C15 of carboxylic acid
41 had led in high yield to seco acid 42 in which silyl ethers at
C3 and C7 were retained, it was not clear that similar selectivity
would pertain to deprotection at C15 of a tris(silyl) ether bearing
both a trans double bond and a (trimethylsilyl)ethyl ester. For
this reason, it was decided to replace the TBS protection of 25
with the corresponding triethylsilyl group (TES) so that
unmasking of a seco ester carrying different silyl ethers would
now be unambiguous. This modification necessitated a return
to 18, which was protected as TES ether 48 with triethylsilyl
triflate. The latter was advanced to alcohol 49 by a four-step
sequence exactly analogous to that used for the conversion of
20 to 25 (see Scheme 4). For Stille coupling purposes, the allylic
chloride 50 derived from 49 was found to be more effective
than the corresponding bromide (Scheme 11).
heptadecanoate 51 (Scheme 12). As expected, exposure of 51
to tetra-n-butylammonium fluoride cleaved both the (trimeth-
ylsilyl)ethyl ester and the triethylsilyl ether but left tert-
butyldimethylsilyl ethers at C3 and C7 intact. The resulting seco
acid 52 underwent facile macrolactonization to 53, and subse-
quent removal of the remaining pair of TBS ethers with
trifluoroacetic acid furnished trans-9,10-dehydroepothilone D
(54).
Coupling of 47 with 50 in the presence of catalytic dipalla-
dium tris(dibenzylideneacetone)chloroform complex and triph-
enylarsine32 proceeded in high yield and gave the 9E,12Z,16E-
Biological Data
The antiproliferative activity of 43 and 54 was assessed in
vitro using a panel of human cancer cell lines. As illustrated in
Table 1, 43 was 20- to 30-fold less potent than natural epothilone
(31) Mu¨ller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J. Synlett 1996,
521.
(32) Farina, V.; Krishnan, B. J. Am. Chem. Soc. 1991, 113, 9585.

Synthesis of Epothilones
J. Am. Chem. Soc., Vol. 123, No. 23, 2001 5413
Table 1. Comparison of Antiproliferative Activity of 43 and 53 with Natural Epothilones and Paclitaxel
a
IC50 values
compound
KB-31b
KB-8511c
A549d
HCT-116e
PC-3Mf
MCF-7g
epothilone B (2)
0.17
1.94
0.16
1.00
841.80
28.54
70.37
0.16
4.62
0.34
4.48
0.32
7.40
0.29
2.31
epothilone D (4)
paclitaxel (7)
cis-9,10-dehydroepothilone D (43)
trans-9,10-dehydroepothilone D (54)
2.67
5.19
4.88
6.62
3.26
59.39
103.70
109.03
108.27
101.83
109.97
146.47
146.80
72.00
95.03
b
c
a Values are expressed in nM and represent the mean of three independent experiments. Epidermoid cancer cell line. Multidrug-resistant
d
e
f
g
subline of KB-31 overexpressing P-glycoprotein. Lung cancer cell line. Colon cancer cell line. Prostate cancer cell line. Breast cancer cell
line
Table 2. Tubulin Polymerization by Epothilones and Paclitaxel.a
carcinoma PC-3M was obtained from Dr. I. J. Fidler (MD Anderson
Cancer Center, Houston, TX). The human KB-31 (drug-sensitive) and
KB-8511 (P-gp overexpressing, multidrug-resistant) epidermoid car-
cinoma cell lines were obtained from Dr. R. M. Baker, Roswell Park
Memorial Institute (Buffalo, NY) and have been previously described.36
In Vitro Tubulin Polymerization Assay. Induction of tubulin
polymerization was determined using a modified version of a previously
described microtubule protein centrifugation assay.37 Briefly, MAP-
associated porcine brain tubulin was incubated with 5 µM compound
for 20 min at room temperature. The samples were then centrifuged
for 15 min at 14 000 rpm to separate polymerized from nonpolymerized
microtubule protein. The protein concentration of the supernatant
containing the remainder of nonpolymerized, soluble microtubule
protein was determined by the Lowry method (DC Assay Kit, Bio-
Rad Laboratories, Hercules, CA) using a SpectraMaxPlus photometer
(Molecular Devices, Sunnyvale, CA). The reduction in optical density
at 750 nm induced by the test compound was compared to that for 25
µM epothilone B (2), which served as a positive control.
Determination of Antiproliferative Activity. Antiproliferative
assays were performed as previously described.38 Cells were seeded at
1.5 × 103 cells/well into 96-well microtiter plates and incubated
overnight. Compounds were added in serial dilutions on day 1.
Subsequently, the cells were incubated for 3 or 4 days (allowing for at
least 2 population doublings), and then were fixed with 3.3% v/v
glutaraldehyde, washed with water, and stained with 0.05% w/v
methylene blue. After the cells were washed, the dye was eluted with
3% HCl, and the optical density was measured at 665 nm with a
SpectraMax 340 photometer (Molecular Devices, Sunnyvale, CA). IC50
values were determined by mathematical curve-fitting using SoftPro3.0
software (Molecular Devices, Sunnyvale, CA) employing the formula
(ODtreated - ODstart)/(ODcontrol - ODstart) × 100. The IC50 was defined
as the drug concentration which resulted in 50% net cell growth
compared to control cultures at the end of the incubation period.
compoundb
polymerization (%)c
epothilone A (1)
71
95
88
53
56
36
epothilone B (2)
epothilone D (4)
paclitaxel (7)
cis-9,10-dehydroepothilone D (43)
trans-9,10-dehydroepothilone D (54)
b
a Porcine microtubule protein was used in this assay. Concentration
is 5 µM. c Extent of tubulin polymerization is expressed relative to that
induced by 25 µM 2, which was defined as 100%.
D (4), and 330- to 670-fold less potent than epothilone B (2).
Interestingly, trans-9,10-dehydroepothilone D (54) showed
biological activity very similar to that of its cis isomer 43 despite
an apparent difference in the conformation of these two macro-
lactones.33 Thus, the average IC50 of 54 for growth inhibition
in the cell line panel used in this study was only 1.36-fold higher
than that observed for 43. As noted for epothilones B and D,
43 and 54 retain anti-proliferative activity against KB-8511 cells,
a paclitaxel-resistant cell line overexpressing P-glycoprotein
(Table 1). While the tubulin polymerization activity of 43 and
54 was lower than of 4 (56, 36, and 88%, respectively) (Table
2), it is conceivable that decreased cellular penetration may
contribute to the marked reduction in antiproliferative potency
observed for 43 and 54. The absence of a clear difference in
the biological profiles of cis-43 and trans-54 analogues of 9,-
10-dehydroepothilone D observed here has a parallel in results
previously reported for other epothilone analogues. Thus,
epothilones incorporating a trans-epoxide or trans-olefin at
C12-C13 have been shown to possess biological activity
comparable to their cis isomer.34,35 Taken together, these data
support the proposition that the C8-C13 region of the epothilone
perimeter is relative tolerant of structural modification and
suggest that the interaction of this segment of the molecule with
tubulin is less stringently defined. Further evaluation of
analogues with more extreme deformation in this portion of the
structure is planned to define the limit of this tolerance.
Acknowledgment. We are grateful to Professor K. C.
Nicolaou, Scripps Research Institute, and Professor Paul A.
Grieco, Montana State University, for the NMR spectra of
epothilone B, and to Dr. Karl-Heinz Altmann, Jacqueline
Loretan, and Robert Reuter, Novartis Pharma AG, for assistance
with the biological studies. One of us (R.G.C.) is indebted to
the National Institutes of Health for a Postdoctoral Fellowship
(F32 CA 76743). This research was assisted by financial support
from the National Institutes of Health (GM50574), Pfizer Global
Research and Development, and DuPont Pharmaceutical Com-
pany.
Materials and Methods
The following human cancer cell lines were obtained from the
American Type Culture Collection (ATCC, Rockville, MD): non-small
cell lung adenocarcinoma A549 (CCL 185), human colon carcinoma
HCT116 (ATCC CCL 247), and estrogen-dependent breast carcinoma
cell line MCF-7 (ATCC HTB 22). The human metastatic prostate
Supporting Information Available: Experimental proce-
1
dures, characterization data, and copies of H and 13C NMR
spectra for new compounds, epothilone B (2), and epothilone
D (4) (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
(33) A conformational study of 43 and 53 is currently in progress and
will be reported in due course.
(34) (a) Nicolaou, K. C.; Vourloumis, D.; Li, T.; Pastor, J.; Winssinger,
N.; He, Y.; Ninkovic, S.; Sarabia, F.; Vallberg, H.; Roschangar, F.; King,
N. P.; Finlay, M. R. V.; Giannakakou, P.; Verdier-Pinard, P.; Hamel, E.
Angew. Chem. 1997, 109, 2181; Angew. Chem., Int. Ed. Engl. 1997, 36,
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Zheng, Y.-H.; Chou, T.-C.; He, L.; Horwitz, S. B. Angew. Chem. 1997,
109, 2178; Angew. Chem., Int. Ed. Engl. 1997, 36, 2093.
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