Total synthesis and evaluation of a key series of C5-substituted vinblastine derivatives

Article abstract of DOI:10.1021/ja1027748

A remarkably concise seven- to eight-step total synthesis of a systematic series of key vinblastine derivatives is detailed and used to characterize the importance and probe the role of the C5 ethyl substituent (R = H, Me, Pr, CH=CH₂, C≡CH, CH₂OH, and CHO vs Et). The analogues, which bear deep-seated structural changes accessible only by total synthesis, were prepared using a powerful intramolecular [4 + 2]/[3 + 2] cycloaddition cascade of 1,3,4-oxadiazoles ideally suited for use in the assemblage of the vindoline-derived lower subunit followed by their incorporation into the vinblastine analogues through the use of a single-step biomimetic coupling with catharanthine. The evaluation of the series revealed that the tubulin binding site surrounding this C5 substituent is exquisitely sensitive to the presence (Et > H, 10-fold), size (Me ≤Et > Pr, 10-fold), shape (Et > CH=CH₂ and C≡CH, >4-fold), and polarity (Et > CHO > CH₂OH, >10-20-fold) of this substituent and that on selected occasions only a C5 methyl group may provide analogues that approach the activity observed with the naturally occurring C5 ethyl group.

Full text of DOI:10.1021/ja1027748

Published on Web 06/02/2010
Total Synthesis and Evaluation of a Key Series of
C5-Substituted Vinblastine Derivatives
Porino Va, Erica L. Campbell, William M. Robertson, and Dale L. Boger*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received April 1, 2010; E-mail: boger@scripps.edu
Abstract: A remarkably concise seven- to eight-step total synthesis of a systematic series of key vinblastine
derivatives is detailed and used to characterize the importance and probe the role of the C5 ethyl substituent
(R ) H, Me, Pr, CHdCH2, CtCH, CH2OH, and CHO vs Et). The analogues, which bear deep-seated
structural changes accessible only by total synthesis, were prepared using a powerful intramolecular [4 +
2]/[3 + 2] cycloaddition cascade of 1,3,4-oxadiazoles ideally suited for use in the assemblage of the vindoline-
derived lower subunit followed by their incorporation into the vinblastine analogues through the use of a
single-step biomimetic coupling with catharanthine. The evaluation of the series revealed that the tubulin
binding site surrounding this C5 substituent is exquisitely sensitive to the presence (Et > H, 10-fold), size
(Me e Et > Pr, 10-fold), shape (Et > CHdCH2 and CtCH, >4-fold), and polarity (Et > CHO > CH2OH,
>10-20-fold) of this substituent and that on selected occasions only a C5 methyl group may provide
analogues that approach the activity observed with the naturally occurring C5 ethyl group.
Introduction
Vinblastine (1)1 and vincristine (2) are the most widely
recognized members of the class of vinca alkaloids as a result
of their clinical use as antitumor drugs (Figure 1). They were
originally isolated in trace quantities from the leaves of
Catharanthus roseus (L.) G. Don,2 and their biological proper-
ties were among the first to be shown to arise from inhibition
of microtubule formation and mitosis that today is still regarded
as one of the more successful targets for cancer therapeutic
intervention.3 Vinblastine and vincristine possess an identical
velbanamine upper subunit and nearly identical vindoline-
derived lower subunits differing only in the dihydroindole
N-substituent. Despite this small structural distinction, vinblas-
tine and vincristine differ in their antitumor properties and dose-
limiting toxicities.1,3 The major limitation on the use of the vinca
alkaloids is the emergence of drug resistance derived principally
from overexpression of phosphoglycoprotein (Pgp), an efflux
pump that transports many of the major drugs out of the cell.4
Thus, in addition to the identification of vinblastine and
vincristine analogues that may address their dose-limiting
toxicities, the development of a modified vinca alkaloid that is
not a substrate for Pgp efflux and is efficacious against
multidrug-resistant (MDR) tumors would constitute a major
advance.
Figure 1. Natural products.
total synthesis of vindoline7 enlisting a tandem [4 + 2]/[3 + 2]
cycloaddition cascade of 1,3,4-oxadiazoles8 that is applicable
to the preparation of structural analogues9 and the use of a
single-pot, two-step biomimetic Fe(III)-promoted coupling with
catharanthine and subsequent oxidation for their incorporation
into vinblastine and its analogues.10,11 Significantly, the approach
(2) (a) Noble, R. L.; Beer, C. T.; Cutts, J. H. Ann. N.Y. Acad. Sci. 1958,
76, 882. (b) Noble, R. L. Lloydia 1964, 27, 280. (c) Svoboda, G. H.;
Nuess, N.; Gorman, M. J. Am. Pharm. Assoc. Sci. Ed. 1959, 48, 659.
For related naturally occurring bisindole alkaloids, see: (d) Leurosidine:
Svoboda, G. H. Lloydia 1961, 24, 173. (e) Deoxyvinblastine: De
Bruyn, A.; Sleechecker, J.; De Jonghe, J. P.; Hannart, J. Bull. Soc.
Chim. Belg. 1983, 92, 485. Neuss, N.; Gorman, M.; Cone, N. J.;
Huckstep, L. L. Tetrahedron Lett. 1968, 783. (f) N-Desmethylvin-
blastine: Simonds, R.; De Bruyn, A.; De Taeye, L.; Verzele, M.; De
Pauw, C. Planta Med. 1984, 50, 274. (g) Desacetoxyvinblastine: Neuss,
N.; Barnes, A. J., Jr.; Huckstep, L. L. Experientia 1975, 31, 18. (h)
Desacetylvinblastine: Svoboda, G. H.; Barnes, A. J., Jr. J. Pharm.
Sci. 1964, 53, 1227. (i) Anhydrovinblastine: Goodbody, A. E.; Watson,
C. D.; Chapple, C. C. S.; Vukovic, J.; Misawa, M. Phytochemistry
1988, 27, 1713.
Although extensive derivatization of vinblastine has been
conducted in the exploration of semisynthetic analogues of the
natural products,3,5 a more limited series of synthetic analogues
that contain more deep-seated changes in the structure have
been examined.3,6 This reflects the structural complexity of the
natural product and the intrinsic challenge in preparing such
analogues. Recently, we reported the development of a concise
(1) Neuss, N.; Neuss, M. N. In The Alkaloids; Brossi, A., Suffness, M.,
Eds.; Academic Press: San Diego, CA, 1990; Vol. 37, p 229.
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A R T I C L E S
Va et al.
group and the incorporation of other unnatural functional groups
at C5 (e.g., alkyne, hydroxymethyl, diol) but also to examine
the incorporation of both polar and hydrophobic functional
groups at C5 to further define the subtle features of this binding
region. In addition, such changes (e.g., large hydrophobic
groups) could impact Pgp export in resistant cell lines, address-
ing the one significant clinical limitation to the class. Access to
the targeted series was anticipated to be provided through use
of the [4 + 2]/[3 + 2] cascade recently introduced for the
synthesis of vindoline (Figure 2).
Results and Discussion
The initial modifications were conducted on the 4-desacetoxy-
6,7-dihydrovindoline scaffold, which is accessible in four steps
from oxadiazole 5 and the corresponding 4-substituted 4-pen-
tenoic acids 6 and in turn may be coupled with catharanthine
in a single step to provide the synthetic vinblastine analogue in
(6) For reviews, see: (a) Kuehne, M. E.; Marko, I. In The Alkaloids; Brossi,
A., Suffness, M., Eds.; Academic Press: San Diego, CA, 1990; Vol.
37, p 77. (b) Potier, P. J. Nat. Prod. 1980, 43, 72. (c) Kutney, J. P.
Nat. Prod. Rep. 1990, 7, 85. (d) Kutney, J. P. Synlett 1991, 11. (e)
Kutney, J. P. Acc. Chem. Res. 1993, 26, 559. For recent studies, see:
(f) Kuehne, M. E.; Bornmann, W. G.; Marko, I.; Qin, Y.; Le Boulluec,
K. L.; Frasier, D. A.; Xu, F.; Malamba, T.; Ensinger, C. L.; Borman,
L. S.; Huot, A. E.; Exon, C.; Bizzarro, F. T.; Cheung, J. B.; Bane,
S. L. Org. Biomol. Chem. 2003, 1, 2120. (g) Miyazaki, T.; Yokoshima,
S.; Simizu, S.; Osada, H.; Tokuyama, H.; Fukuyama, T. Org. Lett.
2007, 9, 4737.
(7) (a) Ishikawa, H.; Elliott, G. I.; Velcicky, J.; Choi, Y.; Boger, D. L.
J. Am. Chem. Soc. 2006, 128, 10596. (b) Choi, Y.; Ishikawa, H.;
Velcicky, J.; Elliott, G. I.; Miller, M. M.; Boger, D. L. Org. Lett.
2005, 7, 4539. (c) Kato, D.; Sasaki, Y.; Boger, D. L. J. Am. Chem.
Soc. 2010, 132, 3685.
(8) (a) Wilkie, G. D.; Elliott, G. I.; Blagg, B. S. J.; Wolkenberg, S. E.;
Soenen, D. B.; Miller, M. M.; Pollack, S.; Boger, D. L. J. Am. Chem.
Soc. 2002, 124, 11292. (b) Elliott, G. I.; Fuchs, J. R.; Blagg, B. S. J.;
Ishikawa, H.; Tao, H.; Yuan, Z.; Boger, D. L. J. Am. Chem. Soc. 2006,
128, 10589. For reviews of heterocyclic azadiene cycloaddition
reactions, see: (c) Boger, D. L. Tetrahedron 1983, 39, 2869. (d) Boger,
D. L. Chem. ReV. 1986, 86, 781. (e) Boger, D. L.; Weinreb, S. M.
Hetero Diels-Alder Methodology in Organic Synthesis; Academic
Press: San Diego, CA, 1987.
Figure 2. (a) Synthetic strategy for C5-substituted analogues. (b) X-ray
crystal structure of vinblastine bound to tubulin12 (an additional view is
provided in the Supporting Information).
(9) (a) Ishikawa, H.; Boger, D. L. Heterocycles 2007, 72, 95. (b) Elliott,
G. I.; Velcicky, J.; Ishikawa, H.; Li, Y.; Boger, D. L. Angew. Chem.,
Int. Ed. 2006, 45, 620. (c) Yuan, Z.; Ishikawa, H.; Boger, D. L. Org.
Lett. 2005, 7, 741. (d) Campbell, E. L.; Zuhl, A. M.; Liu, C. M.; Boger,
D. L. J. Am. Chem. Soc. 2010, 132, 3009.
proved to be sufficiently concise that systematic explorations
of each vinblastine structural feature not accessible by semi-
synthetic derivatization can be envisioned. One such site that
remains unexplored is the C5 ethyl group of the lower vindoline
subunit (Figure 2a). The recent X-ray crystal structure of
vinblastine bound to tubulin12 indicates that the tubulin binding
pocket surrounding this ethyl substituent is composed of
hydrophobic residues (i.e., Leu, Ala, Val) and yet potentially
partially exposes the C5 ethyl group to solvent (Figure 2b). As
a result, we sought not only to explore the removal (R ) H),
contraction (R ) Me), or extension (R ) n-Pr) of the C5 ethyl
(10) (a) Ishikawa, H.; Colby, D. A.; Boger, D. L. J. Am. Chem. Soc. 2008,
130, 420. (b) Ishikawa, H.; Colby, D. A.; Seto, S.; Va, P.; Tam, A.;
Kakei, H.; Rayl, T. J.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc.
2009, 131, 4904.
(11) For studies of the coupling reaction, see: (a) Potier, P.; Langlois, N.;
Langlois, Y.; Gueritte, F. J. Chem. Soc., Chem. Commun. 1975, 670.
(b) Langlois, N.; Gueritte, F.; Langlois, Y.; Potier, P. J. Am. Chem.
Soc. 1976, 98, 7017. (c) Kutney, J. P.; Ratcliffe, A. H.; Treasurywala,
A. M.; Wunderly, S. Heterocycles 1975, 3, 639. (d) Kutney, J. P.;
Hibino, T.; Jahngen, E.; Okutani, T.; Ratcliffe, A. H.; Treasurywala,
A. M.; Wunderly, S. HelV. Chim. Acta 1976, 59, 2858. (e) Kutney,
J. P.; Choi, L. S. L.; Nakano, J.; Tsukamoto, H.; McHugh, M.; Boulet,
C. A. Heterocycles 1988, 27, 1845. (f) Magnus, P.; Stamford, A.;
Ladlow, M. J. Am. Chem. Soc. 1990, 112, 8210. (g) Magnus, P.;
Mendoza, J. S.; Stamford, A.; Ladlow, M.; Willis, P. J. Am. Chem.
Soc. 1992, 114, 10232. (h) Kuehne, M. E.; Matson, P. A.; Bornmann,
W. G. J. Org. Chem. 1991, 56, 513. (i) Bornmann, W. G.; Kuehne,
M. E. J. Org. Chem. 1992, 57, 1752. (j) Kuehne, M. E.; Zebovitz,
T. C.; Bornmann, W. G.; Marko, I. J. Org. Chem. 1987, 52, 4340. (k)
Schill, G.; Priester, C. U.; Windhovel, U. F.; Fritz, H. Tetrahedron
1987, 43, 3765. (l) Yokoshima, S.; Ueda, T.; Kobayashi, S.; Sato, A.;
Kuboyama, T.; Tokuyama, H.; Fukuyama, T. J. Am. Chem. Soc. 2002,
124, 2137. (m) Kuboyama, T.; Yokoshima, S.; Tokuyama, H.;
Fukuyama, T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11966. (n)
Szantay, C., Jr.; Balazs, J.; Bolcskei, J.; Szantay, C. Tetrahedron 1991,
47, 1265. (o) Sundberg, R. J.; Hong, J.; Smith, S. Q.; Sabato, M.;
Tabakovic, I. Tetrahedron 1998, 54, 6259. (p) Sundberg, R. J.;
Gadamsetti, K.; Hunt, P. J. Tetrahedron 1992, 48, 277.
(3) (a) Owellen, R. I.; Hartke, C. A.; Dickerson, R. M.; Haines, F. O.
Cancer Res. 1976, 36, 1499. (b) Pearce, H. L. In The Alkaloids; Brossi,
A., Suffness, M., Eds.; Academic Press: San Diego, CA, 1990; Vol.
37, p 145. (c) Borman, L. S.; Kuehne, M. E. In The Alkaloids; Brossi,
A., Suffness, M., Eds.; Academic Press: San Diego, CA, 1990; Vol.
37, p 133. (d) Fahy, J. Curr. Pharm. Des. 2001, 7, 1181.
(4) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.;
Gottesman, M. M. Nat. ReV. Drug DiscoVery 2006, 5, 219.
(5) For recent examples, see: (a) Voss, M. E.; et al. Bioorg. Med. Chem.
Lett. 2009, 19, 1245. (b) Shao, Y.; Zhang, H.; Ding, H.; Quan, H.;
Lou, L.; Hu, L. J. Nat. Prod. 2009, 72, 1170. (c) Sheng, L. X.; Da,
Y. X.; Long, Y.; Hong, L. Z.; Cho, T. P. Bioorg. Med. Chem. Lett.
2008, 18, 4602. (d) Passarella, D.; Giardini, A.; Peretto, B.; Fontana,
G.; Sacchetti, A.; Silvani, A.; Ronchi, C.; Cappelletti, G.; Cartelli,
D.; Borlak, J.; Danieli, B. Bioorg. Med. Chem. 2008, 16, 6269.
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Synthesis and Evaluation of Vinblastine Derivatives
A R T I C L E S
Scheme 1
Scheme 2
) alkynyl) or after treatment with Lawessons’ reagent for
thioamide 20 (R ) Pr).14 Treatment of thioamides 17-20 with
Meerwin’s salt followed by sodium borohydride reduction of the
resulting S-methyliminium ion served to reductively remove the
thioamide with concomitant diastereoselective opening of the oxido
bridge, affording the C5-substituted vindoline analogues 21-24
(R ) H, Me, Et, Pr). Interestingly, treatment of thioamide 16 (R
) alkynyl) with Lawesson’s reagent afforded the corresponding
thioamide in only 25% yield. In this case, a higher yield (67%)
was obtained utilizing P4S10 (Scheme 2), and subsequent
treatment with Meerwin’s salt and then sodium borohydride
gave the C5 alkyne 25. The corresponding vindoline analogue
26 containing a C5 vinyl substituent was also obtained from
16 utilizing an analogous synthetic sequence after an initial
Lindlar reduction of the C5 alkyne (Scheme 2).
Without efforts to optimize the conversions, the vindoline
analogues 21-24 were coupled with catharanthine (3) through
Fe(III)-promoted coupling and subsequent oxidation10 to afford
the vinblastine analogues 27, 30, 33, and 36 representing the
series with H, Me, Et, and Pr substituents, respectively, at C5
(Scheme 3). In addition to the vinblastine analogues shown,
the corresponding C20′ epimeric leurosidine analogues (28, 31,
34, 37) were generated with the now characteristic ∼2:1 ꢀ/R
diastereoselectivity for the introduction of the C20′ alcohol, and
the intermediate anhydrovinblastines (29, 32, 35, and 38) were
also isolated in yields ranging from 6 to 22%.
a remarkably concise five-step synthesis. When the synthesis
of the common oxadiazole 5 (in two or three steps from
N-methyl-6-methoxytryptamine) and a direct chromatographic
enantiomer resolution of the intermediate cycloadducts are
included, this approach provides the vinblastine analogues in
an overall seven- to eight-step synthetic sequence that is
especially suitable for systematic examination of the importance
of the vindoline C5 substitutent and highlighting the generality
of the synthetic methodology.
The requisite 4-substituted 4-pentenoic acids 6 were coupled
to oxadiazole 5 to afford cycloaddition substrates 7-11,
representing the series with R ) H,9a Me, Et,7a Pr, and ethynyl,
respectively (Scheme 1). Warming solutions of the substrates
7-11 in 1,2-dichlorobenzene (DCB) afforded single diastere-
omers of the corresponding [4 + 2]/[3 + 2] cycloadducts
12-16. Consistent with prior studies, the exclusive formation
of a single diastereomer of the cascade cycloadduct is a result
of indole endo [3 + 2] cycloaddition in which the intermediate
1,3-dipole is directed to the sterically less encumbered face of
the stabilized carbonyl ylide opposite the newly formed lactam.
The reaction of the unsubstituted substrate 7 (R ) H) was
considerably faster than those of 8-11 (6 vs 24 h), reflecting
the relative ease of the rate-determining [4 + 2] cycloaddition
initiated by the tethered dienophile. Although this was not
examined in detail, all of the reactions were conducted in
refluxing DCB under dilute conditions (1-4 mM) to preclude
competitive intermolecular reactions and, in the cases examined,
displayed a modest concentration dependence within the narrow
range examined (see the Supporting Information). Finally, it is
notable that in substrate 11, which also bears a pendant alkyne
on the dienophile tether, only the olefin served to undergo the
[4 + 2] cycloaddition with the oxadiazole.13 Treatment of
cycloadducts 12-15 with Lawessons’ reagent afforded thio-
amides 17-20. The racemic intermediates were resolved by
chiral-phase HPLC (2 × 25 cm ChiralCel OD, R ) 1.2-1.6)
after the cycloaddition for 12-14 (R ) H, Me, Et) and 16 (R
Attempts to prepare the C5 alkynyl (39) and vinyl (40)
vinblastine analogues by direct coupling of the C5 alkynyl (25)
and vinyl (26) vindoline analogues, respectively, were not
successful, in part because of competitive oxidation of the C5
unsaturation during the oxidative stage of the biomimetic
coupling (Scheme 4). However, omitting the oxidant [air,
Fe2(ox)3] and the second step of the direct coupling afforded
the C5 vinyl anhydrovinblastine analogue 41 in good yield
(12) Gigant, B.; Wang, C.; Ravelli, R. B. G.; Roussi, F.; Steinmetz, M. O.;
Curmi, P. A.; Sobel, A.; Knossow, M. Nature 2005, 435, 519.
(13) A single-crystal X-ray structure determination of 16 (CCDC 755749)
confirmed the structural and stereochemical assignment.
(14) In addition to the confirmation of the structural and relative stereo-
chemical assignments, the absolute configurations were assigned by
X-ray structure determinations of a heavy-atom derivative of a
synthetic intermediate (CCDC 609613) leading to 4-desacetoxy-4-
desethylvindoline (R ) H),9a a heavy-atom (S) derivative disclosed
earlier (R ) Et, natural enantiomer of 19, CCDC 295590),7a and the
heavy-atom derivatives prepared herein: R ) Me (unnatural enantiomer
of 18, CCDC 755748) and R ) Pr (unnatural enantiomer of 20, CCDC
755747). The absolute configurations of 25 (R ) CtCH), 26 (R )
CHdCH2), and their derivatives were established by conversion of
the unnatural enantiomer of 16 (R ) CtCH) into 14 (R ) Et) by
reduction with Raney Ni (THF, 25 °C, 15 h, 60%) and correlation of
the optical rotation.
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A R T I C L E S
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Scheme 3
Scheme 5
Scheme 6
Scheme 4
single diastereomer (Scheme 6). To explore the size of the
tubulin binding site and because the incorporation of hydro-
phobic groups has been shown to overcome Pgp-mediated
multidrug resistance with other classes of antitumor drugs,15
diol 46 was also converted to the dibutyrate 48 for incorporation
into a vinblastine analogue. Alternatively, treatment of C5 diol
46 with sodium periodate gave aldehyde 49, and its reduction
afforded the primary alcohol 50 (Scheme 7). The transformation
of 49 and 50 into the corresponding vinblastine analogues
bearing C5 formyl and hydroxymethyl substituents (R ) CHO
or CH2OH, respectively, vs Et) was anticipated to address
directly the ability of the tubulin C5 ethyl binding site to
accommodate polar functionalities and the potential accessibility
of solvent to this site. Just as interesting, the primary alcohol
50 could be converted to lactone 51 by a facile base-catalyzed
intramolecular transesterification with the C3 methyl ester. This
unique lactonization alters the conformational state of the
vindoline subunit. The central six-membered ring characteristi-
cally adopts a boat conformation stabilized by a transannular
H bond from the C3 hydroxyl group to N9, placing the C5 ethyl
(54%). Alternatively, protection of the alkyne of 25 as the tert-
butyldimethylsilyl (TBS) alkyne 42 enabled the two-step
coupling with catharanthine and subsequent oxidation to afford
the corresponding analogue 43 (Scheme 5). Treatment of 43
with Bu4NF dried over 4 Å molecular sieves afforded the C5
alkyne vinblastine analogue 39.
The vinyl substituent of the vindoline analogue 26 was
converted into a series of additional key functional groups with
which we could probe the introduction of polar functionalities
and explore a unique series of conformational constraints.
Treatment of 26 with osmium tetroxide afforded diol 46 as a
(15) (a) Lampidis, T. J.; Kolonias, D.; Podona, T.; Israel, M.; Safa, A. R.;
Lothstein, L.; Savaraj, N.; Tapiero, H.; Priebe, W. Biochemistry 1997,
36, 2679. (b) Perego, P.; De Cesare, M.; De Isabella, P.; Carenini,
N.; Beggiolin, G.; Pezzoni, G.; Palumbo, M.; Tartaglia, L.; Pratesi,
G.; Pisano, C.; Carminati, P.; Scheffer, G. L.; Zunino, F. Cancer Res.
2001, 61, 6034.
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Synthesis and Evaluation of Vinblastine Derivatives
A R T I C L E S
Scheme 7
Scheme 9
Scheme 8
group axial and the C3 methyl ester equatorial on this ring. This
not only potentially attenuates the basicity of N9 but also
strategically places the C3 methyl ester and C4 acetate of
vinblastine at the interface of the solvent with the tubulin/
vinblastine complex (Figure 2b). The lactonization to provide
51 requires the central six-membered ring of vindoline to adopt
a chair (vs boat) conformation in which both the C3 and C5
substituents are axial, disrupting the C3 alcohol/N9 transannular
H bond, and this would be expected to alter only the confor-
mational features of vinblastine at the C3/C4 sites forming the
interface with the solvent in the complex with tubulin without
perturbing the relative location of the C5 substituent. Comple-
mentary to analogue 51, the C5 diol 46 could also be closed to
1
give lactone 52 (Scheme 8). Moreover, the detailed H NMR
characterization of 52 was utilized to confidently assign the C5
side-chain secondary alcohol stereochemistry obtained through
1
OsO4-catalyzed dihydroxylation of olefin 26. The 2D H-1H
rotating-frame Overhauser spectroscopy (ROESY) data revealed
diagnostic nuclear Overhauser effects (NOEs) between Ha and
Hb and between Hc and Hd, defining the stereochemistry shown.
Significantly, this stereochemistry rigidly places the lactone
hydroxymethyl substituent in a position to extend toward the
solvent interface in the vinblastine/tubulin complex.
The biological activity of the vinblastine analogues was
examined in three cytotoxic assays including the matched colon
cancer cell lines HCT116 and HCT116/VM46, the latter of
which is multidrug-resistant by virtue of Pgp overexpression.15
Consistent with expectations based on past observations, the
vinblastine analogues (C20′ ꢀ-OH) proved more potent (∼10-
fold) than the corresponding anhydrovinblastine derivatives
(C15′/C20′ double bond), which in turn were more potent than
the corresponding leurosidine analogues (C20′ R-OH). The
complete set of test results is provided in Table S1 in the
Supporting Information and highlights such comparisons for
the analogues prepared herein. A summary of the key results
focusing on the impact of the C5 substituent is provided in
The vindoline analogues 46, 48, 49, 51, and 52 were coupled
to catharanthine using the biomimetic Fe(III) coupling and
oxidation10 to afford the corresponding vinblastine analogues
without optimization (Scheme 9). Coupling of the C5 aldehyde
49 occurred with concomitant reduction to afford the C5
hydroxymethyl analogue 56, subsequent oxidation of which
afforded the C5 aldehyde analogue 58. Coupling of the
conformationally constrained vindoline analogues 51 and 52
gave the corresponding vinblastine analogues 59 and 60 but
interestingly failed to provide isolable quantities of the corre-
sponding leurosidine (C20′ R-OH) products.
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Va et al.
Scheme 10
solvent interface in the bound complex. More subtly, the
introduction of unsaturation into the C5 substituent also led to
significant losses in activity. The alkyne 39 was found to be
>5-fold less active than 33, indicating that either the rigidity of
this altered C5 substituent or its π unsaturation reduces the
tubulin binding affinity. Similarly, although we did not prepare
the vinblastine analogue bearing a C5 vinyl substituent, comparison
of the corresponding anhydrovinblastine analogues 35 and 41
indicates that even the substitution of a vinyl group for the C5
ethyl group reduces the activity, despite the fact that the two
hydrophobic substituents would be expected to embody analogous
size and conformational characteristics. Finally, the vinblastine
analogues possessing even larger C5 substituents (43, 53, and 55)
were inactive, as were the lactone derivatives 59 and 60 with altered
conformational characteristics at C3/C4.
As a result of these observations, an analogous but smaller
series of comparisons of the effect of replacing the C5
substituent on a more potent vinblastine scaffold was conducted.
4-Desacetoxyvinblastine (61) is a naturally occurring vinca
alkaloid, but it is an even more minor (∼10-fold) constituent
of C. roseus than 1 itself (0.00025% of dry leaf weight).2g It
has been reported to possess equally efficacious antitumor
activity as 1, albeit at higher doses, but was not pursued at the
time of its isolation because of its even lower natural abun-
dance.16 Along with the efforts that provided 1 itself,10 we
recently reported the total synthesis of 61 and its extension to
provide the vinblastine analogue 62 lacking the C5 ethyl
substituent (R ) H).9a,10b The preparation of the corresponding
analogue 70 bearing a C5 methyl substituent was undertaken
and required the additional steps needed to introduce the C6/
C7 double bond utilizing the cycloadduct 13 (Scheme 10).7
Thus, R-hydroxylation of lactam 13 and subsequent triisopro-
pylsilyl (TIPS) ether protection of the free alcohol 63 provided
64. Thiolactam formation and its reductive removal with Raney
Figure 3. Cytotoxic activity.
Figure 3. The activity proved to be remarkably sensitive to the
presence, size, and polar nature of the C5 substituent. Whereas
the analogues bearing the C5 methyl group (30 and 32) matched
the potency of the corresponding analogues incorporating the
natural C5 ethyl substituent (33 and 35), the analogues lacking
a C5 substituent or bearing a C5 propyl group (36), which
extends the length of the substituent by just one carbon relative
to the natural ethyl substituent, led to ∼10-fold losses in activity.
Even more significant, the introduction of polar functionality
with the C5 aldehyde group (58) or C5 hydroxymethyl group
(56), substituents whose sizes closely approximate that of the
ethyl group, led to progressively larger losses in activity of ∼10-
fold and >20-fold, respectively. This clearly indicates that the
tubulin ethyl binding site is exclusively hydrophobic in nature
and does not benefit from or permit an interaction with the
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8494 J. AM. CHEM. SOC. VOL. 132, NO. 24, 2010

Synthesis and Evaluation of Vinblastine Derivatives
A R T I C L E S
Because of the unusual nature of these results, the activities of
both 70 and 30 were examined on multiple occasions with
independently prepared materials to ensure the accuracy of the
results. As a result, we are confident that the comparisons
indicate that while on occasion a methyl group may substitute
effectively for a C5 ethyl group (e.g., 30 vs 33), its incorporation
may also lead to substantial (10-fold, 70 vs 61) losses in activity,
especially within the more potent analogue scaffolds. Thus, not
only is the C5 ethyl group uniquely effective, but the magnitude
of its effects on the properties of vinblastine is also surprisingly
large (10-fold).
Conclusions
A concise total synthesis of a series of key vinblastine
analogues that systematically probe and define the importance
of the C5 ethyl substituent has been detailed. The requisite deep-
seated structural changes, which were accessible only by total
synthesis, were accomplished by enlisting an intramolecular
tandem [4 + 2]/[3 + 2] cycloaddition cascade of 1,3,4-
oxadiazoles that is ideally suited for use in the preparation of
the vindoline-derived lower subunit followed by its incorporation
into the vinblastine analogues using a single-step biomimetic
coupling with catharanthine. The evaluation of the key series
of analogues revealed that this site is exquisitely sensitive to
the presence (Et > H, 10-fold), size (Me e Et > Pr, 10-fold),
shape (Et > CHdCH2 and CtCH, >4-fold), and polarity (Et >
CHO > CH2OH, >10-fold) of the C5 substituent, with the
corresponding analogues experiencing pronounced losses in
activity even with such minor structural changes. The only
exception to these observations is the selected equipotent activity
observed with a C5 methyl analogue (30 vs 33 but not 70 vs
61), indicating that the C5 ethyl group is surprisingly important
to the properties of vinblastine (g10-fold) and that the tubulin
binding site surrounding this ethyl substituent is remarkably
important, exclusively hydrophobic in nature, and restricted in
size and does not access the adjacent solvent interface of the
complex. Studies targeting additional sites for single systematic
changes enlisting the cycloaddition cascade synthetic strategy
are in progress and will be disclosed in due course.
Figure 4. Cytotoxic activity.
Ni followed by diastereoselective cleavage of the oxido bridge
provided 67. TIPS ether deprotection and subsequent regiose-
lective alcohol elimination7 provided the key 4-desacetoxyvin-
doline analogue 69 bearing the C5 methyl substituent. Without
optimization, single-step Fe(III)-mediated coupling of 69 with
3 and its subsequent in situ oxidation provided vinblastine
analogue 70 (36%), its leurosidine C20′ isomer 71 (15%), and
the corresponding anhydrovinblastine analogue 72 (28%).
Notably, this preparation of the synthetic analogue 70 of the
complex natural product 61 containing a deep-seated single-
site modification required only 10 steps from oxadiazole 5 with
a resolution of intermediate 13.
The results of the examination of 70 are presented in Figure
4 alongside those for the natural product 61. Analogous to
observations made with 27 versus 33, removal of the C5 ethyl
substituent (62 vs 61) resulted in a 10-fold loss in biological
activity. However, and surprisingly distinct from the observa-
tions made with 30 versus 33, the incorporation of a C5 methyl
substituent also provided an analogue that was 10-fold less
potent (70 vs 61) and roughly equipotent with 62 (R ) H).
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA115526 and
CA042056) and the Skaggs Institute for Chemical Biology. We
thank Dr. Raj Chadha for the X-ray structures disclosed herein,
and we are especially grateful to Dr. Paul Hellier of Pierre Fabre
for the generous gift of catharanthine. W.M.R. is a Skaggs Fellow.
Supporting Information Available: Full experimental details,
complete ref 5a, and crystallographic data (CIF). This material
is available free of charge via the Internet at http://pubs.acs.org.
(16) Barnett, C. J.; Cullinan, G. J.; Gerzon, K.; Hoying, B. C.; Jones, W. E.;
Newlon, W. M.; Poore, G. A.; Robison, R. L.; Sweeney, M. J.; Todd,
G. C.; Dyke, R. W.; Nelson, R. L. J. Med. Chem. 1978, 21, 88.
JA1027748
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