Conformation-activity relationships in polyketide natural products. Towards the biologically active conformation of epothilone

Article abstract of DOI:10.1039/b312213c

The conformation-activity relationships for the biologically active polyketide, epothilone, have been determined. Computer-based molecular modeling and high field NMR techniques have provided the solution preferences for epothilones 1 and 2. For the C1-C8

Full text of DOI:10.1039/b312213c

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Conformation–activity relationships in polyketide natural products.
Towards the biologically active conformation of epothilone
Richard E. Taylor,* Yue Chen,† Gabriel M. Galvin‡ and Praveen K. Pabba§
Department of Chemistry & Biochemistry and the Walther Cancer Research Center,
251 Nieuwland Science Hall, University of Notre Dame, Notre Dame, IN, 46556-5670, USA.
E-mail: taylor.61@nd.edu
Received (in Pittsburgh, PA, USA) 1st October 2003, Accepted 10th November 2003
First published as an Advance Article on the web 20th November 2003
The conformation–activity relationships for the biologically active polyketide, epothilone, have been determined.
Computer-based molecular modeling and high field NMR techniques have provided the solution preferences for
epothilones 1 and 2. For the C1–C8 polypropionate region, two conformational families, conformers 1 and 2, have
been identified as having significant populations in polar and non-polar solvents. In the C11–C15 region, additional
flexibility was observed and two local conformations have been identified as important, conformers 3 and 4.
Epothilone analogues with altered conformational profiles have been designed and synthesized. Conformational
analysis and the results of biological assays have been correlated to provide increased understanding of the
biologically active conformation for the epothilone class of natural product. Conformation–activity relationships
have been shown to be an important complement to structure–activity data.
C15. The overall goal of this project was the identification of
Introduction
the biologically active conformation of epothilone. Critical to
The epothilones represent an important lead in the search for
the success of this novel approach would be the rational
improved chemotherapeutic agents, which has led to intense
incorporation of functionality capable of affecting conform-
interest from the synthetic, medicinal, and biological com-
ational preferences with minimal structural alteration. In this
munities.1 Biological experiments have shown that epothilones
report, we demonstrate several examples of analogue design.
One set of analogues probes the biologically active conform-
ation in the C1–C8 polypropionate region. An additional
four analogues were prepared to investigate the conform-
ational flexibility of the C11–C15 region. From a general per-
spective this approach demonstrates that conformation–activity
relationships are an important complement to classic SAR.
interact with β-tubulin at the paclitaxel-binding site.2 However,
despite enormous efforts over the past several decades, only a
low resolution (4 Å) electron diffraction structure of the tubulin
dimer has been obtained.3 The lack of detailed structural
information with regards to the interaction of paclitaxel (and
other microtubule-stabilizing natural products)4 with tubulin at
the molecular level has made rational design of analogues more
difficult.5 Epothilone’s relatively simple structure and accessi-
Results and discussion
bility from biosynthetic expression has allowed the preparation
of hundreds of synthetic or semi-synthetic analogues. In fact,
several compounds in the epothilone class are in the early stages
of Phase I and Phase II clinical trials.6
Conformational preferences in the C1–C8 region
Several years ago, we reported a study on the conformational
properties of the epothilones, which utilized a combination of
computational methods and high field NMR experiments.7 Our
report concentrated on the critical polypropionate region and
concluded that, in solution, the C1–C11 of epothilones pre-
ferred to exist in at least two conformational families controlled
by syn-pentane interactions and intramolecular hydrogen
bonds.8 Since the epothilone conformation while bound to
microtubules may not be similar to the solution structure, we
felt it important to use computational modeling methods to
gain insight into the available conformational space. This was
accomplished by using internal coordinate Monte Carlo search-
ing available in MacroModel (ver. 5.0 and 7.0).9 We determined
that 10000 structures are sufficient to reproduce search con-
vergence from different starting geometries. The conformations
were minimized using the MM2* force field with both the
generalized Born/surface area (GB/SA) water and chloroform
models. However, we found that the chloroform model over-
estimated the importance of internal hydrogen bonds and chose
to use the water model on a routine basis. Analysis of the out-
put file showed that the conformation of the C1–C11 region
could be clustered into two low energy conformers within 2 kcal
molϪ1 of the global minimum (conformers 1 and 2, Fig. 1).
Conformer 1 is characterized by the C3–OH residing above
the plane of the macrolide, a C5–C8 gaucheϪ torsion, and a
series of anti torsions across the C8–C12 region. In contrast,
Many epothilone analogues that have been prepared were
accessible due to their isolation as intermediates in total syn-
thesis or as undesired by-products from unselective reactions.
Most significantly, modifications to the C3–C8 polypropionate
region, particularly stereochemical changes, drastically reduce
their cytotoxicity and tubulin polymerization ability. Therefore,
we proposed that the conformation of the macrolide ring along
with its spatial relationship to the thiazole side chain are key
elements, essential for the biological activity of epothilones.
Moreover, these conformational requirements are controlled by
the C3–C8 polypropionate region and the stereochemistry at
† Current address: Kosan Biosciences, Hayward, CA.
‡ Current address: Bristol-Myers Squibb, Syracuse NY.
§ Current address: Lexicon Pharmaceuticals, Princeton, NJ.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 7 – 1 3 2
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an alternative synthetic route to this compound through
manipulation of the epothilone PKS gene cluster.12 Finally, the
S-stereochemistry was chosen to induce a destabilizing syn-
pentane interaction with the methyl group at C8. Through this
indirect manner we hoped to influence the conformational pref-
erences of the polyketide region by minimal and distal struc-
tural modifications. In fact, computer-based conformational
analysis of 3 showed a reversal of preference and conformer 2
became the global minimum. Thus, it was planned that solution
conformational analysis and the biological activity of analogue
3, accessible via total synthesis, would provide critical inform-
ation with regards to the tubulin-bound conformation of
epothilone’s C1–C8 region.
The strategy we chose for the synthesis of analogue 3 was
related to our early efforts in the total synthesis of epothilone A
and C, and highlighted by a ring-closing metathesis to form a
C12–C13 olefin. While several groups have used a similar dis-
connection strategy for the total synthesis of epothilone A, we
reported original and independent approaches to each of three
fragments.13 For the synthesis of analogue 3, aldehyde 7 was
prepared as shown in Scheme 1. The methyl-substituted stereo-
genic centers were created using Evans’ oxazolidinone chem-
istry.14 Each alkylation proceeded in good yield with excellent
stereocontrol. The chiral auxiliary was removed by hydrolysis
and the resulting carboxylic acid reduced to the primary alco-
hol, which proved to be a convenient point for storage. When
appropriate, oxidation with TPAP provided the aldehyde 7 in
good yield.
Fig. 1 Calculated conformational preferences of the C1–C8 region.
conformer 2 has the C3-hydoxyl roughly in the plane of
the macrolide ring. An anti C5–C8 torsion is compensated by a
C8–C11 gaucheϪ torsion.
The computationally predicted conformer 1 is quite similar
to the conformation of epothilone B in the solid state.10 How-
ever, proton NMR coupling constants as well as distinctive
NOE data supported the existence of both conformers 1 and 2
in both polar (DMSO/D2O) and non-polar solvents. A strong
NOE was observed between H3 and H6, which supports a
significant population of conformations where the C3-hydroxyl
resides in the plane of the macrolide. Thus, a putative hydrogen
bond between the C3-hydroxyl and the C5 carbonyl (or C1
carbonyl) is not a controlling feature. In fact, it was recently
reported that C3-cyano epothilones, which lack the ability to
hydrogen bond, retain significant biological activity.11 A second
important marker of conformational preference in solution is
the H6–H7 coupling constant. Computational models sug-
gested a H–C6–C7–H dihedral angle of Ϫ174Њ (Jcalc = 10.4 Hz)
in conformer 1 and 66Њ (Jcalc = 1.0 Hz) in conformer 2. The
Boltzman-weighted coupling constant for H6–H7 was 7.5 Hz.
In excellent agreement with calculated predictions, the H6–H7
coupling constants of epothilones 1 and 2 have values of J6–7
=
2–5 Hz in chlorinated solvents and J6–7 = 8–10 Hz in more polar
solvent such as DMSO-d6. These intermediate values support
significant population of both conformers 1 and 2 with an
increased population of conformations related to conformer 2
in non-polar solvents. This is to be expected due to the presence
of an intramolecular hydrogen bond between the C7-hydroxyl
and the C5 carbonyl, which is more likely in non-polar solvents.
Scheme 1
The coupling of the three fragments is summarized in
Scheme 2. Aldol reaction between the lithium enolate of
ketone13a 8 and aldehyde 7 provided the desired 6,7-syn-7,8-anti
stereochemistry of the aldol product 9 in 70% yield with 5 : 1
diastereoselectivity. A comparison of proton NMR data
between 9 and related compounds prepared in our laboratory
as well as compounds prepared by others in the field supported
the expected anti-Felkin selectivity. The rationalization of the
Destabilization of conformer 1 through a designed syn-pentane
interaction
Our approach to the identification of the conformational con-
straints to epothilone’s biological activity would be based on
the preparation of analogues with altered conformational pref-
erences. Consideration of an appropriately positioned methyl
group had a number of positive attributes and (S)-10-methyl-
epothilone C 3 was selected as an initial target, Fig. 2.
Fig. 2 Conformer 2—stabilized analogue.
This designed molecule 3 is “natural product-like” as it
retains a polyketide pattern. In fact, methyl substitution at C10
represents an acetate
propionate switch in the biogenetic
pattern. This has two interesting implications. During the
evolution of the organism that produces the epothilones it is
possible that this substitution pattern was previously bio-
synthesized and abandoned for lack of beneficial activity.
Alternatively, if found to be active, the recent advances in
genetic engineering of polyketide synthases (PKS) may provide
Scheme 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 7 – 1 3 2
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Table 1 Biological assays conducted at Bristol-Myers Squibb [IC50/
nM]
A2780
SKBR3
HCT116
LX-1
K562
1a
1b
2a
2b
3
7.36
0.57
71.0
17.3
>1012
>1012
6.57
1.65
46.4
22.5
>1012
>1012
6.88
1.33
47.9
10.8
>1012
>1012
5.30
0.25
47.8
8.4
>1012
>1012
5.92
0.38
56.9
12.3
>1012
>1012
Fig. 4 Biologically active epothilone analogues.
Stabilization of conformer 1 through torsional rigidity (C8–C11)
E-3
There is additional evidence that conformations related to con-
former 1 are important for biological activity. The Danishefsky
group reported the synthesis of 9,10-dehydroepothilone D 15,
which was prepared via a ring-closing metathesis as an altern-
ative route to the natural series through selective hydrogenation
of the 9,10-olefin, Fig. 5.18 Biological assays of the intermediate
unsaturated analogue 15 showed increased cytotoxicity relative
to epothilone D. More recently, epoxide 16 was shown to be one
of the most active epothilone derivatives prepared to date
with >3× the activity of epothilone B itself.19 Our contributed
analysis of NMR and computational studies concluded that
while the presence of the 9,10-trans-olefin stabilized conformer
1 in the polypropionate region (C1–C8), it also increased the
flexibility of the C11–C15 olefin region.
facial selectivity is based upon the detailed analysis of the
aldol reaction presented by Roush15a and explored in detail by
Danishefsky15b as applied to the epothilone system. After the
aldol and protecting group manipulation, the primary alcohol
was oxidized to carboxylic acid 10, which was necessary for
coupling to thiazole13b 11. The sequence to (S)-10-methyl-
epothilone C was then completed in 3 steps. Thiazole 11 was
coupled to acid 10 under Yamaguchi conditions.16 Ring-closing
olefin metathesis of the diene 12 provided the 16-membered
macrolide as a 1.5 : 1 mixture of Z- and E-isomers. The
designed analogue 3 was then generated by deprotection of
the silyl ethers with TFA. Compound 3 and its E-12,13-olefin
isomer were easily separated by chromatography.
With analogue 3 in hand, solution NMR experiments were
used to determine its conformational preferences. As high-
lighted in Fig. 3 a number of key transannular NOEs were
observed to support the presence of conformations related to
conformer 2. The observed H6–H7 coupling constant of 0 Hz
matched calculated predictions for conformer 2 (1.0 Hz). In
contrast, conformer 1 has a calculated H6–H7 coupling con-
stant of 10.0 Hz. NMR experiments at varied temperatures
(10–60 ЊC) showed key differences between the C3 and C7
hydroxyl protons. The change in chemical shift was much
greater for C3–OH (∆δ = 0.7 ppm) than for C7–OH (∆δ =
0.3 ppm). This supports a strong hydrogen bond between the
C7–OH and the C5 carbonyl. Attempts to obtain crystals
suitable for X-ray crystallographic analysis were unsuccessful.
Despite the lack of information with regard to the solid-state
conformation, the solution data do support the population of
conformations related to conformer 2.
Fig.
5
Structure and preferred conformation of 9,10-dehydro-
epothilones.
Conformational flexibility of the C11–C15 region
Exploration of the conformational properties of the C11–C15
region of this molecule was a critical next phase in the
analysis of the epothilone pharmacophore.20 Coupled with our
understanding of the polypropionate region, this would
complete our studies and, hopefully, allow the design of struc-
turally simplified analogues, which could mimic the biologically
active conformation of epothilone and still retain significant
activity. Conformational analysis of the C11–C15 region of
epothilones B and D suggested conformational flexibility. The
two extreme conformations, conformer 3 and conformer 4, are
represented in Fig. 6. Conformer 3 is characterized by an anti
relationship between C13 and C16 while, on the other hand,
conformer 4 contains a gauche relationship. Computational
and NMR studies suggested the presence of each, in solution,
but that conformer 3 is more highly populated than conformer
4. This is also supported by experimental findings where
epoxidation of epothilone D gave good β-selectivity providing
direct access to epothilone B.21 Several groups have reported
models of the epothilone pharmacophore and its relationship
to other classes of microtubule-stabilizing natural products
based on computational methods and reported structure–
Fig. 3 Solution conformation of (S)-10-methyl epothilone C.
Purified samples of (S)-10-methylepothilone C 3, as well as
the 12,13-E isomer, were submitted to Bristol-Myers Squibb for
biological evaluation. As shown in Table 1, neither compound
showed any cytotoxicity in the explored range of tumor cell
lines. As with a classic SAR profile much can be learned from
inactive compounds. The significant decrease in activity in the
C10-methyl series may not be attributed to the minimal struc-
tural alteration provided by the C10-methyl. A significant low-
ering of the population of conformations related to conformer
1 is more likely responsible for the attenuation of cytotoxicity.
In fact, cyclopropane-containing analogue 13, Fig. 4, has been
recently shown to possess significant activity despite having
C10-subsitution.17 In contrast to 3 the C10-alkyl substituent of
13 is tied back relieving the syn-pentane interaction with the
methyl group at C8 and repopulating conformer 1. Moreover,
the trans-cyclopropane ring rigidifies the C9–C10–C11–C12
torsion to ∼180Њ similar to active analogue epo 490 (14).17
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 7 – 1 3 2
129

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tert-butoxydiphenylsilyl ether which is more easily removed
under basic conditions. Oxidative cleavage provided 22 and 23
in good yield.
As detailed in Scheme 4, intermolecular Ni/Cr coupling27 of
vinyl iodide 24 with the thiazole aldehyde 22 (2 equiv.) provided
the desired allylic alcohol 25 in 75% yield as a single dia-
stereomer. The relative stereochemistry at this position was
inconsequential since exposure to thionyl chloride in ether–pen-
tane provided the desired primary chloride 26 in 85% yield with
excellent stereochemical control. LiEt3BH not only reductively
cleaved the chiral auxiliary but also efficiently reduced the
allylic chloride in 72% yield providing the primary alcohol.
Oxidation with Dess–Martin periodinane28 then provided alde-
hyde 27. The tandem Ni/Cr coupling followed by thionyl chlor-
ide rearrangement has become a powerful method for natural
product fragment coupling and the control of trisubstituted
olefin geometry.
Fig. 6 Conformation of the C11–C14 region of epothilones.
activity relationships.22 In fact, two of these studies have
proposed that the bioactive conformation of the epothilone
epoxide region is similar to conformer 4.22a,b,23,24
In an effort to distinguish conformers 3 and 4 and determine
which is more closely related to the biologically active conform-
ation of the epothilones, we designed and prepared simple C14-
methyl substituted compounds 17–20, Fig. 7. We rationalized
that 17/18 and 19/20 would preferentially exist in conformers 3
and 4, respectively. In addition, the presence of the C14-methyl
substituent would destabilize the alternative conformer. These
preferences were supported by computer modeling techniques
similar to our previous analysis of the natural products. It is
important to again point out that the methyl analogues 17–20
represent an acetate
propionate switch in the biogenetic
pathway to these compounds. Thus, they could represent com-
pounds accessible to the microorganism in its evolutionary
history or by genetic manipulation of its polyketide synthase
machinery in the future.
Scheme 4
We had previously reported an efficient route to the
chiral ketone necessary for the preparation of epothilone con-
geners.13a However, the synthesis of the chiral starting material
for that route was dependent on an enzymatic resolution of a
secondary alcohol which was found to make large-scale produc-
tion tedious. Scheme 5 describes an alternative route, which
exploits the elegant chemistry of Kiyooka.29 The reaction of
2-methyl-1-phenoxy-1-trimethylsilyloxy-1-propene with readily
available aldehyde 28 under the influence of Kiyooka’s chiral
boron reagent provided aldol adduct 29 in 71% yield (96% ee by
chiral GC analysis). The use of the phenoxy-ketene acetal not
only provides higher enantioselectivity than the commercially
available methoxy-ketene acetal but also allows for a one-step
ester-to-ketone conversion. Protection of the secondary alcohol
was followed by exposure to ethylmagnesium bromide in the
presence of triethylamine which provided the desired ethyl
ketone 30.
Fig. 7 Conformer 3 and 4 stabilized analogues.
The four analogues were prepared using the identical
methodology we developed and successfully applied to the total
synthesis of epothilones B and D.25 Thiazole aldehyde 21 is the
point of divergence for the synthesis of 22 and 23, Scheme 3.
Brown asymmetric crotylboration26 efficiently controlled the
enantio- and diastereoselectivity of the C14, C15 stereogenic
centers. Model systems suggested that the steric hindrance pro-
vided by the additional methyl substituent at C14 would cause
selectivity issues with the late stage deprotection of a C15 TBS
ether. Therefore, we protected the secondary hydroxyl as a
Scheme 5
Completion of the route to (S)-14-methylepothilone D 17
was accomplished by aldol condensation, functional group
manipulation, and lactonization as detailed in Scheme 6.
Scheme 3
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 7 – 1 3 2
130

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Scheme 9
Scheme 6
175Њ (Jcalc = 10.9 Hz) in 17 (conformer 3) and 62Њ (Jcalc = 3.1 Hz)
in 19 (conformer 4). A strong NOE enhancement was observed
between the H14 and one of the C11 protons in both 17 and 19.
In addition a strong NOE enhancement was observed between
H15 and H13 in epoxide 18 but not in epoxide 36. Attempts to
obtain single crystals of 19 and 36 were unsuccessful. However,
additional NMR evidence for these conformational differences
was observed in ROESY experiments on all four analogues. An
NOE enhancement between H13 and the C16–CH3 was only
observed in analogues 19 and 36 but not in 17 and 18. These
data not only support the preference for conformer 4 but also
the configuration of epoxide 36.
The conversion of aldehyde 23 to (R)-14-methyl epo D 19
preceeded with similar efficiency through an identical synthetic
sequence as outlined in Schemes 7 and 8.
As we previously reported, the biological activity of epothil-
one analogues 17, 18, 19, and 36 were evaluated against a panel
of human tumor cell lines by collaborators at Kosan Bio-
sciences.20 Compounds 17 and 18 (conformer 3 preference)
maintain significant cytotoxicity similar to epothilone D. In
marked contrast, analogues 19 and 36 (conformer 4 preference)
showed no measurable cytotoxicity. This study is thus another
clear example that conformation has a significant impact on the
biological activity.
Scheme 7
The conformation–activity relationship study carried out on
the C11–C15 region of epothilones strongly supports the
importance of conformer 3 as the bioactive conformation of
the 12,13-epoxide (olefin) region of the epothilones. Moreover,
investigation of the polypropionate region found strong
evidence for the activity of conformers closely related to
conformer 1 despite alternative conformations, with significant
population, found in solution. It is clear from this study that the
conformation originally observed in the solid state is closely
related to the bound structure. Recently, the Novartis group
reported the conformation of epothilone A while bound to
tubulin through elegant use of transfer NOE and cross corre-
lation relaxation data.30 In fact, their reported structure is
consistent with conformers 1 and 3 although their proposed
orientation of the C3-hydroxyl is in the plane of the macrolide
as in conformer 2. Of additional significance is their proposed
orientation of the thiazole side chain, a region previously
shown to be flexible in solution.7
Overall, the approach presented here offers a new perspective
on rational design of modified biologically active polyketide
macrolides which complements transfer NOE studies and can
directly provide new leads for further investigation. In addition,
the conformational analogues such as those presented here are
designed such that the recent advances in genetic engineering of
polyketide synthases (PKS) may provide an alternative syn-
thetic route through manipulation of the PKS gene cluster. This
work has demonstrated that the successful design of polyketide
analogues is reliant on an understanding of the structural and
conformational constraints of the pharmacophore.
Scheme 8
(S)-14-Methylepothilone D 17 underwent a highly selective
epoxidation with mCPBA to provide (R)-14-methylepothilone
B 18 in 57% yield, Scheme 9. Epoxidation of (R)-14-methyl-
epothilone D 19 also proceeded in a highly selective fashion.
However, the C11–C15 region of 19 exists primarily in con-
former 4 due to the conformational constraints imposed by
the C14-methyl substituent. Therefore, the stereochemistry of
the epoxide 36 is epimeric to epothilone B and analogue 18.
X-Ray diffraction studies of single crystals of 17 and 18ؒH2O
showed that the conformation of the C11–C15 region of each
in the solid-state was quite similar to that reported for epothil-
one B and thus conformer 3.20 Proton NMR coupling con-
stants (J14–15 = 9.9, 10.5 Hz, respectively) also supported this
preference in solution. In contrast, proton NMR coupling con-
stants of analogues 19 and 36 had the expected values for con-
former 4 (J14–15 = 3.3, < 2.0 Hz, respectively). Computational
models, Fig. 6, suggested a H–C14–C15–H dihedral angle of
Acknowledgements
Dr. Jaroslav Zajicek is thanked for his assistance with NMR
experiments. Dr. Craig Fairchild and Dr. Barry Long (Bristol-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 7 – 1 3 2
131

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16 J. Inanaga, K. Hirata, H. Saeki, T. Katsuki and M. Yamaguchi, Bull.
Chem. Soc. Jpn., 1979, 52, 1989.
17 K. Biswas, H. Lin, J. T. Njardarson, M. D. Chappell, T.-C. Chou,
Y. Guan, W. P. Tong, L. He, S. B. Horwitz and S. J. Danishefsky,
J. Am. Chem. Soc., 2002, 124, 9825.
18 A. Rivkin, F. Yoshimura, A. E. Gabarda, T.-C. Chou, H. Dong,
W. P. Tong and S. J. Danishefsky, J. Am. Chem. Soc., 2003, 125, 2899.
19 F. Yoshimura, A. Rivkin, A. E. Gabarda, T.-C. Chou, H. Dong,
G. Sukenick, F. F. Morel, R. E. Taylor and S. J. Danishefsky,
Angew. Chem., Int. Ed., 2003, 42, 2518.
Myers Squibb) are acknowledged for the biological activity
data on compound 3. Support provided by the National Cancer
Institute/National Institutes of Health (CA85499). YC thanks
the University of Notre Dame for support through a Reilly
Fellowship. RET acknowledges support from Bristol-Myers
Squibb, Kosan Biosciences, and Eli Lilly through a Lilly
Grantee award.
20 R. E. Taylor, Y. Chen, A. Beatty, D. C. Myles and Y. Zhou, J. Am.
Chem. Soc., 2003, 125, 26.
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 7 – 1 3 2
132