Design and synthesis of tubulin ligands based on epothilones: A preliminary study

Article abstract of DOI:10.1016/S0040-4039(02)02242-6

Starting from the X-ray structure of epothilone B, an original, non macrocyclic, structure has been designed to mimic the key structural features of this potent anticancer agent; the preparation of a key intermediate incorporating four stereogenic centers is described from cyclohexane-1,3-dione.

Full text of DOI:10.1016/S0040-4039(02)02242-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 9213–9216
Design and synthesis of tubulin ligands based on epothilones:
a preliminary study
Sophie Vielle,a Eric Raimbaud,b Philippe Bertrand,a Delphine Quintard,a Pierre Renard,c
Bruno Pfeifferc and Jean-Pierre Gessona,*
aUMR 6514, Universite´ de Poitiers et CNRS, 40, Avenue du Recteur Pineau, F-86022 Poitiers, France
bInstitut de Recherches Servier, 11 rue des Moulineaux, F-92150 Suresnes, France
cADIR, 1 rue Carle He´bert, F-92415 Courbevoie, France
Received 14 March 2002; revised 30 September 2002; accepted 4 October 2002
Abstract—Starting from the X-ray structure of epothilone B, an original, non macrocyclic, structure has been designed to mimic
the key structural features of this potent anticancer agent; the preparation of a key intermediate incorporating four stereogenic
centers is described from cyclohexane-1,3-dione. © 2002 Elsevier Science Ltd. All rights reserved.
Epothilones A 1 and B 2 are the first members of a new
class of fascinating and biologically active natural prod-
ucts isolated by Ho¨fle from the myxobacterium
Sorangium cellulosum1 (Fig. 1). The current high inter-
est for these 16-membered macrocyclic lactones arise
from their potent microtubule binding property leading
to significant in vivo cytotoxic activity against various
cancer cell lines including multidrug-resistant ones.2
syntheses since 19964 allowing to gather interesting
structure–activity relationships.
Indeed, few efforts have been made to design analogues
based on the X-ray structure and conformational analy-
sis of 2 alone5 and/or of paclitaxel either alone6 or
binded to tubulin.7 Thus, Winckler and Axelsen have
designed simpler analogs based on the introduction of
an ethano bridge between C2 and C10 and synthetized
compounds such as 3 which do not bind to tubulin.8
These compounds share the same tubulin binding site
as paclitaxel3 but have the advantage over the latter to
be more water soluble. These properties and their inter-
esting molecular structure have prompted numerous
Starting from the X-ray structure of 2, we searched for
compounds having the same spatial arrangement of its
key features: the C6ꢀC8 aldol moiety, the C5 gem-
dimethyl group, the C2ꢀC4 b-hydroxyester group, the
side chain with a thiazole ring and the C12ꢀC13 epox-
ide or alkene residue (epothilone D which only differs
from epothilone B by the presence of an alkene group
instead of the epoxide has demonstrated a superior
therapeutic index9). Selection of potential candidates
was based on their conformational analysis and super-
position with 2 and on their synthetic feasability. This
study led to the design of a cyclohexane analogue which
fulfill the above requirements (Fig. 2).1
Retrosynthetic analysis of 4 (possessing an alkene
group insteed of an epoxide moiety) is based on the
preparation of a cyclohexane derivative bearing 4
stereogenic centers and on the introduction at a proper
stage the thiazole side chain (Scheme 1). We now
disclose the diastereoselective synthesis of the racemic
compound 4. Starting from 2,2-dimethyl-1,3-cyclohex-
Figure 1.
* Corresponding author. Tel.: +33-5-4945-3862; fax: +33-5-4945-3501;
e-mail: jean-pierre.gesson@univ-poitiers.fr
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02242-6

9214
S. Vielle et al. / Tetrahedron Letters 43 (2002) 9213–9216
Scheme 3.
Figure 2. Dark grey: epothilone; light grey: analogue.
Figure 3.
the former group (JH1ꢀH2=1.7 Hz). The exclusive for-
mation of 9 is best explained by a retro-aldol reaction
followed by recyclisation to afford the axial isomer.13
Since molecular modelisation at the AM110 level reveals
that 9 is slightly less stable than its equatorial epimer
(DE=1.1 kcal/mol), its formation is best explained by
the more favorable chair–chair bicyclo[3,3,1]nonane-
type transition state A compared to B (Scheme 3).
Then the hydroxyl group of 9 was protected as a THP
ether to give 10 (74%). Although a 70/30 mixture of
diastereoisomers is produced, no other protecting group
could be introduced due to steric hindrance (silyl
ethers) or decomposition under basic conditions
(MOM). At this stage, NOESY experiments confirmed
the proposed stereochemistry with strong correlations
between H1 and one C1 Me group (1,3 diaxial interac-
tion), the methine proton of the OTHP moiety and the
other C1 Me and H2 with both C1 Me groups (Fig. 3).
Then, 10 was converted to acid 11 (93%) upon
hydrogenolysis (H2, Pd/C, EtOAc).
Scheme 1.
anedione 5,11 alkoxycarbonylation at C4 followed by
reduction, alcohol protection, esterification and aldoli-
sation should afford 4.
Since alkoxycarbonylation was unsuccessful on 5 under
different conditions, mono ketalisation to 6 (81%) was
first carried out followed by condensation with benzyl-
cyanoformate according to the procedure of Mander12
(Scheme 2). The desired keto ester 7 (68%, 1/1 ketone–
enol tautomeric mixture) was then reduced with NaBH4
to give a 90% mixture of the two epimers 8a and 8b in
variable ratios depending on experiments. Then, ketone
deprotection was carried out on this mixture and, sur-
Compound 11 (2 equiv.) and the racemic alcohol 12 (1
equiv.) in presence of DCC (2 equiv.) and 4-DMAP (2
equiv.) in CH2Cl2 afforded esters 13a and 13b in a 7/3
1
ratio (68% isolated yield) (Scheme 4). H and 13C NMR
1
prisingly, only 9 was isolated (97%). H NMR of 9 is in
agreement with a cis relationship of the hydroxyl and
ester groups leading to a pseudo-axial conformation of
for both esters revealed that they only differ by the
configuration of the chiral stereogenic center of the
THP protecting group (acid catalysed hydrolysis
Scheme 2.

S. Vielle et al. / Tetrahedron Letters 43 (2002) 9213–9216
9215
Scheme 4.
afforded the same compound) and that only one
diastereoisomer is formed during the esterification step,
although its configuration could not be determined.
This is in agreement with a kinetic resolution which
may arise from a diastereofacial selectivity in the nucle-
ophilic attack of 12 on the bulky intermediate resulting
from the addition of acid 11 to DCC. Similar kinetic
resolutions have been reported in the literature.14
Finally, condensation of the lithium enolate of 13a
(LDA, THF, −78°C) with 1 equiv. of acetaldehyde
Acknowledgements
We thank ADIR and La Ligue Nationale contre le
Cancer (Comite´ de Charente-Maritime) for financial
support.
References
1. Ho¨fle, G.; Bedorf, N.; Steinemetz, H.; Schomburg, D.;
Gerth, K.; 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) Bollag, D.
M. Exp. Opin. Invest. Drugs 1997, 6, 867; (c) Kowalski,
R. J.; Giannakakou, P.; Hamel, E. J. Biol. Chem. 1997,
272, 2534.
3. (a) ter Har, E.; Kowalski, R. J.; Hamel, E.; Lin, C. M.;
Longley, R. E.; Gunasakera, S. P.; Rosenkranz, H. S.;
Day, B. W. Biochemistry 1996, 35, 243; (b) Hamel, E.;
Sackett, D. L.; Vourloumis, D.; Nicolaou, K. C. Bio-
chemistry 1999, 38, 5490.
4. (a) Biswas, K.; Lin, H.; Njardarson, J. T.; Chappell, M.
D.; Chou, T.-C.; Guan, Y.; Tong, W. P.; He, L.; Hor-
witz, S. B.; Danishefsky, S. J. J. Am. Chem. Soc. 2002,
124, 9825; (b) Nicolaou, K. C.; Roschangar, F.; Vourlou-
mis, D. Angew. Chem., Int. Ed. Engl. 1998, 37, 2014; (c)
Mulzer, J. Chem. Mon. 2000, 131, 205.
afforded aldols 14 and 15 in a 59/41 ratio (36%).11 1
H
NMR data for the first aldol is in agreement with a cis
relationship between the CH3CHOH and COOMe
groups, the observed coupling constants between H5,
H6ax (J=13 Hz) and H6eq (J=6.8 Hz) confirming the
axial orientation of H5. In the case of 14, larger modifi-
1
cations (with respect to 13a) are noticed in the H and
13C NMR spectra (H5 appears as a quadruplet (J=9.1
Hz)). Since it has already been shown that cyclohex-
anone enolates afford anti-aldol with aliphatic alde-
hydes,15 it may be assumed that 15 results from the
other possible diastereofacial addition. The lack of
selectivity may arise from 1,4 (with the OTHP group)
and 1,3 (with a C1 Me) steric interactions in both
transition states. Upon treatment with cat. PTSA in
acetone, diols 4 (53%) and 16 (56%) were obtained from
14 and 15, respectively.
In summary, comparison of a cyclohexane derivative
(4) with the X-ray structure of epothilone B was found
to result in similar spatial arrangement of the key
features of this potent new anticancer agent. Synthesis
of racemic compound 4 has been completed in eight
steps from 1,3-cyclohexanedione. Indeed, no cytotoxic-
ity was observed for 4 (and 16) against L1210 leukemia
cells.16 In order to explain this result, it will be crucial
to determine the relative configuration of the ester side
chain resulting from the kinetic resolution step. Since
all intermediates and the final compounds are oils,
further work is under way to search for cristalline
derivatives in order to get an X-ray analysis.
5. Taylor, R. E.; Zajicek, J. J. Org. Chem. 1999, 64, 7224.
6. (a) Wang, M.; Xia, X.; Kim, Y.; Hwang, D.; Jansen, J.
M.; Botta, M.; Liotta, D. C.; Snyder, J. P. Org. Lett.
1999, 1, 43; (b) Ojima, I.; Chakravarty, S.; Inoue, T.; Lin,
S.; He, L.; Horwitz, S. B.; Kuduk, S. D.; Danishefsky, S.
J. Proc. Natl. Acad. Sci. 1999, 96, 4256; (c) Giannakakou,
P.; Gussio, R.; Nogales, E.; Downing, K. H.; Zaharewitz,
D.; Bollbuck, B.; Poy, G.; Sackett, D.; Nicolaou, K. C.;
Fojo, T. Proc. Natl. Acad. Sci. 2000, 97, 2904; (d) He, L.;
Jagtap, P. G.; Kingston, D. G. I.; Shen, H.-J.; Orr, G. A.;
Horwitz, S. B. Biochemistry 2000, 39, 3972.
7. Snyder, J. P.; Nettles, J. H.; Cornett, B.; Downing, K. H.;
Nogales, E. Proc. Natl. Acad. Sci. 2001, 98, 5312.

9216
S. Vielle et al. / Tetrahedron Letters 43 (2002) 9213–9216
8. Winckler, J. D.; Holland, J. M.; Kasparec, J.; Axelsen, P.
NMR (75 MHz, CDCl3): l 14.08, 19.12, 19.68, 20.67,
24.27, 26.34, 37.69, 42.20, 50.14, 50.90, 67.54, 77.97,
78.64, 116.41, 118.13, 120.89, 133.15, 136.84, 152.02,
164.99, 173.39, 216.97; MS (m/z) (CI): MS (m/z)
422.1999 [M+H]+. Compound 16: 1H NMR (300 MHz,
CDCl3): l 1.13 (s, 3H), 1.15 (s, 3H), 1.25 (s, 3H),
1.60–1.68 (m, 1H), 2.10 (s, 3H), 2.40–2.59 (m, 3H), 2.71
(s, 3H), 2.85–2.95 (m, H),3.53 (d, 1H, J=3.5 Hz), 3.85–
3.98 (m, 2H), 5.10 (dd, 1H, J=1.7, 10.1 Hz), 5.15 (dd,
1H, J=1.7, 17.2 Hz), 5.43 (t, 1H, J=6.5 Hz), 5.73 (ddt,
1H, J=6.9, 10.1, 17.2 Hz), 6.55 (s, 1H), 6.99 (s, 1H); 13C
NMR (75 MHz, CDCl3): l 14.61, 19.31, 19.85, 21.83,
22.95, 24.33, 37.64, 42.28, 49.80, 50.89, 68.10, 77.98,
79.08, 116.29, 117.83, 123.12, 133.45, 136.81, 152.44,
164.70, 171.60, 219.20; MS (m/z) (CI): 346 [M+H]+, MS
(m/z) 422.2005.
H. Tetrahedron 1999, 55, 8199.
9. (a) Chou, T.-C.; Zhang, X.-G.; Balog, A.; Su, D.-S.;
Meng, D.; Savin, K. A.; Bertino, J. R.; Danishefsky, S. J.
Proc. Natl. Acad. Sci. 1998, 95, 9642; (b) Chou, T.-C.;
Zhang, X.-G.; Harris, C. R.; Kuduk, S. K.; Balog, A.;
Savin, K. A.; Bertino, J. R.; Danishefsky, S. J. Proc.
Natl. Acad. Sci. 1998, 95, 15798.
10. Structures discussed in this study were modeled using
standard bond lengths and angles, using the molecular
modeling software SYBYL® 6.41, Tripos Inc., 1699
South Hanley Road, Saint-Louis, Missouri 63144, USA,
running on an Indigo2 R4400 Extreme Silicon Graphics
workstation. Geometries and energies were obtained by
AM1 semi-empirical calculations (Dewar M. J. S.; Zoe-
bish E. G.; Healy, E. F.; Stewart, J. P. J. Am. Chem. Soc.
1985, 107, 3902–3909) with the Gaussian92® package
(Gaussian 92/DFT, Revision F.3; Frisch, M. J.; Trucks,
G. W.; Schlegel, H. B.; Gill, P. M. W.; Johnson, B. G.;
Wong, M. W.; Foresman, J. B.; Robb, M. A.; Head-Gor-
don, M.; Replogle, E. S.; Gomperts, R.; Andres, J. L.;
Raghavachari, K.; Binkley, J. S.; Gonzalez, C.; Martin,
R. L.; Fox, D. J.; Defrees, D. J.; Baker, J.; Stewart, J. J.
P.; Pople, J. A. Gaussian, Inc.: Pittsburgh, PA, 1993).
12. Mander, L. N.; Sethi, S. P. Tetrahedron Lett. 1983, 24,
5425.
13. (a) Quesnel, Y.; Toupet, L.; Duhamel, L.; Duhamel, P.;
Poirier, J.-M. Tetrahedron: Asymmetry 1999, 10, 1015
and references cited therein; (b) Yang, W.; Digits, C. A.;
Hatada, M.; Narula, S.; Rozamus, L. W.; Huestis, C. M.;
Wong, J.; Dalgarno, D.; Holt, D. A. Org. Lett. 1999, 1,
2033.
14. Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochemistry
of Organic Compounds; John Wiley: New York, 1994; pp.
395–405.
15. (a) Dubois, J.-E.; Dubois, M. Bull. Soc. Chim. 1969,
3120; (b) Gesson, J.-P.; Renoux, B.; Schulten, E. Tetra-
hedron Lett. 1997, 38, 5811.
16. Rodier, S.; Le Hue´rou, Y.; Renoux, B.; Doyon, J.;
Renard, P.; Pierre, A.; Gesson, J.-P.; Gree´, R. Anti-Can-
cer Drug Des. 2001, 16, 109–117.
1
11. All new compounds have been characterized by H, 13C
NMR and HRMS. Selected data: Compound 4: 1H
NMR (300 MHz, CDCl3): l 1.18 (s, 3H), 1.21 (d, 3H,
J=6.8 Hz), 1.25 (s, 3H), 1.95–2.04 (m, 5H), 2.50–2.65 (m,
3H), 2.71 (s, 3H), 3.24 (ddd, 1H, J=1.8, 4.5, 12.9 Hz),
3.36 (sl, 1H), 3.79 (sl, 1H), 3.99 (quint., 1H, J=6.8 Hz),
4.10 (s, 1H), 5.09 (dd, 1H, J=1.7, 10.1 Hz), 5.13 (dd, 1H,
J=1.7, 17.0 Hz), 5.37 (t, 1H, J=6.4 Hz), 5.74 (ddt, 1H,
J=6.9, 10.1, 17.0 Hz), 6.56 (s, 1H), 6.98 (s, 1H); 13C