Synthesis and biological evaluation of (-)-16-normethyldictyostatin: A potent analogue of (-)-dictyostatin

Article abstract of DOI:10.1021/ol050808u

(Chemical Equation Presented) (-)-16-Normethyldictyostatin has been made by total synthesis and is a potent antitumor agent in cells expressing wild-type tubulin and in one mutant cell line that is resistant to paclitaxel, but it is much less active than

Full text of DOI:10.1021/ol050808u

ORGANIC
LETTERS
2005
Vol. 7, No. 14
2873-2876
Synthesis and Biological Evaluation of
)-16-Normethyldictyostatin: A Potent
(−
Analogue of (−
)-Dictyostatin†
Youseung Shin,‡ Jean-Hugues Fournier,‡ Raghavan Balachandran,§
|
Charitha Madiraju,§ Brianne S. Raccor,‡ Guangyu Zhu,‡ Michael C. Edler,
|
Ernest Hamel, Billy W. Day,*,‡,§ and Dennis P. Curran*,‡
Department of Chemistry and Department of Pharmaceutical Sciences, UniVersity of
Pittsburgh, Pittsburgh, PennsylVania 15261, and Screening Technologies Branch,
DeVelopmental Therapeutics Program, DiVision of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick,
Maryland 21702
curran@pitt.edu
Received April 13, 2005
ABSTRACT
(−)-16-Normethyldictyostatin has been made by total synthesis and is a potent antitumor agent in cells expressing wild-type tubulin and in
one mutant cell line that is resistant to paclitaxel, but it is much less active than dictyostatin in another paclitaxel-resistant cell line where Val
is substituted for Phe270. This provides strong evidence that the C16 methyl group of the dictyostatins is oriented toward Phe270 in the
paclitaxel-binding site on
â-tubulin.
Although it was first described by Pettit and co-workers in
1994,1 the potent anticancer agent (-)-dictyostatin2 has
advanced slowly because it was only available in tiny
quantities and because its complete stereostructure was not
known. In 2004, Paterson and Wright proposed structure 1a
for dictyostatin.3 Concurrent total syntheses of 1a by our
group and Paterson’s confirmed the structure and have begun
to address the supply issue.4,5 Detailed in vitro and cell assays
of dictyostatin 1a suggest that it is as active asspossibly
even more active thansits much more well studied cousin
discodermolide 2a (Figure 1).6
The stage is now set for study of structure/activity
relationship in the dictyostatin series, and we have already
described several active analogues that were made before
the full structure of dictyostatin was known and that, in
† Dedicated to Prof. Iwao Ojima on his 60th birthday.
‡ Department of Chemistry, University of Pittsburgh.
§ Department of Pharmaceutical Sciences, University of Pittsburgh.
| National Cancer Institute.
(1) (a) Pettit, G. R.; Cichacz, Z. A.; Gao, F.; Boyd, M. R.; Schmidt, J.
M. J. Chem. Soc., Chem. Commun. 1994, 1111-1112. (b) Pettit, G. R.;
Cichacz, Z. A. US patent 5,430,053, 1995. (c) Isbrucker, R. A.; Cummins,
J.; Pomponi, S. A.; Longley, R. E.; Wright, A. E. Biochem. Pharm. 2003,
66, 75-82.
(2) Originally called dictyostatin-1, the compound is now commonly
called more simply dictyostatin.
(3) Paterson, I.; Britton, R.; Delgado, O.; Wright, A. E. Chem. Commun.
2004, 632-633.
(4) (a) Paterson, I.; Britton, R.; Delgado, O.; Meyer, A.; Poullennec, K.
G. Angew. Chem., Int. Ed. 2004, 43, 4629-4633. (b) Shin, Y.; Fournier,
J.-H.; Fukui, Y.; Bru¨ckner, A. M.; Curran, D. P. Angew. Chem., Int. Ed.
2004, 43, 4634-4637.
(5) Model and fragment studies: (a) O’Neil, G. W.; Phillips, A. J.
Tetrahedron Lett. 2004, 45, 4253-4256. (b) Kangani, C. O.; Bru¨ckner, A.
M.; Curran, D. P. Org. Lett. 2005, 7, 379-382.
(6) Madiraju, C.; Edler, M. C.; Hamel, E.; Raccor, B. S.; Balachandran,
R.; Zhu, G.; Giuliano, K. A.; Vogt, A.; Shin, Y.; Fournier, J.-H.; Fukui,
Y.; Bru¨ckner, A. M.; Curran, D. P.; Day, B. W. Submitted for publication.
10.1021/ol050808u CCC: $30.25
© 2005 American Chemical Society
Published on Web 06/15/2005

and bottom (C3-C9) fragments 3 along with small ole-
finating reagents 6 and 7.
The synthesis of the middle fragment 4 is simpler than
that of its counterpart in the dictyostatin synthesis4b and is
summarized in Scheme 1. Starting from the popular disco-
Scheme 1. Synthesis of the Middle Fragment 4
Figure 1. Structures of dictyostatin, discodermolide, and their
normethyl analogues.
hindsight, are closely related.7 Among the many possible
analogues to target, we focused on 16-normethyldictyostatin
1b because Smith has already shown that the analogous
discodermolide analogue 2b is highly active8,9 and especially
because the isolated stereocenter at C16 is the most difficult
one to introduce. If this stereocenter were not needed for
activity, then synthesis of analogues for SAR and drug
development would be facilitated. We report herein the
syntheses and biological characterization of (-)-16-
normethyldictyostatin 1b. This compound proves to be highly
potent, with very interesting features.
The strategy to make 16-normethyldictyostatin 1b is
similar to that used for the parent 1a4b and is summarized in
Figure 2. The molecule is cut into large fragments at the
dermolide and dictyostatin intermediate 8,10 oxidation and
Horner-Wadsworth-Emmons (HWE) reaction provided
unsaturated ester 9. This was reduced with nickel boride to
saturate the alkene, and then the ester was reduced to give
10. Protection of the free alcohol as a TBS ether followed
by removal of the PMB group gave 11. Oxidation, Corey-
Fuchs olefination, and subsequent elimination then provided
key alkyne 4 in 26% overall yield (unoptimized) from 8.
The fragment coupling and completion of the synthesis
of 1b follow the parent synthesis4b with comparable or better
yields (Scheme 2). Briefly, fragment coupling of 3 and 4
followed by Noyori and Lindlar reductions provided 12 ready
(7) Shin, Y.; Choy, N.; Turner, T. R.; Balachandran, R.; Madiraju, C.;
Day, B. W.; Curran, D. P. Org. Lett. 2002, 4, 4443-4446.
(8) (a) Martello, L. A.; LaMarche, M. J.; He, L.; Beauchamp, T. J.; Smith,
A. B.; Horwitz, S. B. Chem. Biol. 2001, 8, 843-55. (b) Smith, A. B.; Freeze,
B. S.; LaMarche, M. J.; Hirose, T.; Brouard, I.; Rucker, P. V.; Xian, M.;
Sundermann, K. F.; Shaw, S. J.; Burlingame, M. A.; Horwitz, S. B.; Myles,
D. C. Org. Lett. 2005, 7, 311-314. (c) Smith, A. B.; Freeze, S. B.;
LaMarche, M. J.; Hirose, T.; Brouard, I.; Xian, M.; Sundermann, K. F.;
Shaw, S. J.; Burlingame, M. A.; Horwitz, S. B.; Myles, D. C. Org. Lett.
2005, 7, 315-318.
Figure 2. Synthetic strategy for 16-normethyldictyostatin.
(9) The backbone of dictyostatin has two more carbons than discoder-
molide, so C16 of 1b corresponds to C14 of 2b.
C-O bond of the macrolactone, C9-C10 and C17-C18 (see
wavy lines), and then the dienes are truncated (see arrows).
This leaves large top 5 (C18-C23), middle 4 (C10-C17),
(10) Mickel, S. J.; Sedelmeier, G. H.; Niederer, D.; Daeffler, R.; Osmani,
A.; Schreiner, K.; Seeger-Weibel, M.; Berod, B.; Schaer, K.; Gamboni, R.
Org. Process Res. DeV. 2004, 8, 92-100.
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Org. Lett., Vol. 7, No. 14, 2005

Scheme 2. Fragment Coupling and Synthesis of (-)-16-Normethyldictyostatina
a Key: (a) n-BuLi, THF, 86%; (b) (S,S)-Noyori catalyst (20 mol %), i-PrOH, 95%; (c) Lindlar catalyst, H2 (balloon), toluene, 91%; (d)
TBSOTf, 2,6-lutidine, CH2Cl2, 96%; (e) HF-pyridine, pyridine, THF, 0 °C, 1 day, 71%; (f) (i) Dess-Martin oxidation, (ii) Ba(OH)2, 5,
THF-H2O, 83% (two steps); (g) NiCl2, NaBH4, MeOH-THF, 65%; (h) Li(OtBu)3H,THF, 95% (â), 5% (R); (i) TBSOTf, 2,6-lutidine,
CH2Cl2, 92%; (j) DIBALH, CH2Cl2, 90%; (k) (i) Dess-Martin oxidation, (ii) CH2dCHCH(TMS)Br, CrCl2, THF, (iii) NaH, THF, 82%
(three steps); (l) ZnBr2, CH2Cl2-MeOH, 89%; (m) (i) Dess-Martin oxidation, (ii) (CF3CH2O)2P(O)CH2CO2Me, KHMDS, 18-crown-6,
THF, 88% (two steps); (n) DDQ, CH2Cl2-H2O, 82%; (o) 1 N KOH, EtOH-THF; (p) 2,4,6-trichlorobenzoyl chloride, Et3N, THF then
4-DMAP (10 equiv), toluene, 76% (two steps); (q) 3 N HCl-MeOH, THF, 24%.
for addition of the top fragment. This was accomplished by
deprotection, oxidation, and HWE olefination with 5 to give
13. Reduction of 13 with nickel boride was followed by
ketone reduction with Li(OtBu)3H11 to give 14 in 95% yield;
a small amount of the minor R-isomer was easily removed
by flash chromatography. Elaboration of the top diene to
give 15 was followed by completion of the bottom diene to
give 16. Deprotection of the PMB group and ester hydrolysis,
followed by Yamaguchi lactonization,7 provided the pro-
tected macrolactone. Global deprotection followed by careful
purification resulted in some material loss but provided 25
mg of 1b (24% yield) in excellent purity.
been discovered in clinical cancer samples, but they are
important cell biology tools to establish orientations of
ligands within the taxoid binding site.
Paclitaxel (Ptx) showed subnanomolar potency against the
parental 1A9 cells, but as expected the mutant cells showed
about 90- and 70-fold resistance to the drug. Discodermolide
2a was 2- to 4-fold less potent than 1a. Normethyldictyostatin
1b appeared to be essentially equipotent to its parent 1a
against the parental 1A9 cells and the 1A9PTX22 cells, but
Table 1. Biological Test Results of 1a, 1b, 2a, and Paclitaxel
(Ptx)
16-Normethyldictyostatin 1b was thoroughly characterized
spectroscopically by the usual means (see the Supporting
Information). Essentially all the resonances in the 1H NMR
spectrum of 1b could be assigned by 2D NMR experiments,
allowing a direct comparison with 1a. Interestingly, while
the proton-proton coupling constants of the two molecules
are very similar, the chemical shifts of a number of protons,
including several remote from C16, are quite different.
Biological analyses of 1b provided exciting results. The
antiproliferative activity of the compound was examined in
human ovarian carcinoma 1A9 cells and in clones 1A9PTX10
and 1A9PTX22 of this line resistant to paclitaxel due to
mutations in â-tubulin at the paclitaxel binding site (Table
1).11 The 1A9PTX10 line expresses â-tubulin with a
Phe270fVal mutation, while the 1A9PTX22 line expresses
Ala364fThr â-tubulin. Neither of these mutations has ever
% inhib
of [3H]Ptx assembly
tubulin
antiproliferative GI50 ( SD, nMa
(fold resistance relative to 1A9)
binding
( SDb
EC50 (
SDc (µM)
compd
1A9
1A9PTX10 1A9PTX22
1b
0.41 ( 0.52 470 ( 70
(1146)
5.6 ( 4.7
(14)
48 ( 3
78 ( 2
80 ( 2
20 ( 1
14 ( 7
1a
0.69 ( 0.80 3.2 ( 2.4
(4.6)
1.3 ( 1.0
(1.9)
3.1 ( 0.2
3.6 ( 0.4
25 ( 3
2a
1.7 ( 1.2
6.2 ( 3.6
(3.6)
7.0 ( 8.4
(4.1)
Ptx
0.71 ( 0.11 64 ( 8
51 ( 9
(72)
(90)
a Concentration of agent that caused 50% growth inhibition of cultures
after 72 h (N ) 8). b Percent inhibition by 4 µM test agent of binding of 2
µM [3H]paclitaxel to a stoichiometric amount of microtubule polymer (N
) 9). c Bovine brain tubulin (10 µM) in 0.2 M monosodium glutamate, 15
min at 20 °C, centrifugation, and Lowry determination of remaining soluble
tubulin. The EC50 is the drug concentration required to polymerize 50% of
the tubulin (N ) 3).
(11) Hung, D. T.; Nerenberg, J. B.; Schreiber, S. L. J. Am. Chem. Soc.
1996, 118, 11054-11080.
Org. Lett., Vol. 7, No. 14, 2005
2875

experienced over 1000-fold resistance from the Phe270fVal
â-tubulin mutant 1A9PTX10 cells. This remarkable result
strongly suggests that the presence of a â-C16-methyl group
in dictyostatin 1a yields favorable interactions with either a
phenylalanine or a valine at postion 270 in â-tubulin but
that its absence in 1b is tolerated only by the Phe270 wild
type and not the Val270 mutant.
Examination of effects against isolated bovine brain tubulin
showed 1b to have about 60% of the ability of 1a to inhibit
the binding of [3H]paclitaxel to microtubules,6 and it was
only 4.5-fold less active than 1a in its ability to cause
formation of tubulin polymer.13 In each case, 1b was more
potent than paclitaxel in these activities found with isolated
tubulin.
exhibits clear and powerful discodermolide/dictyostatin
behavior. It is a potent antitumor agent in cells expressing
wild-type tubulin and in one mutant cell line that is resistant
to paclitaxel, but is much less active than dictyostatin in
another paclitaxel resistant cell line. This provides strong
evidence that the C16 methyl group of the dictyostatins, and
by inference the C14 methyl group of the discodermolides,
is oriented toward Phe270 in the paclitaxel binding site on
â-tubulin.
Acknowledgment. We thank the NIH for partial funding
(CA078039) of this work, the National Cancer Institute’s
Drug Synthesis Branch for paclitaxel and its tritiated form,
Dr. Kenneth Bair for a sample of discodermolide, and Drs.
Tito Fojo and Paraskevi Giannakakou for the ovarian
carcinoma cell lines.
These results show that 16-normethyldictyostatin is an
important member of the dictyostatin family. The middle
fragment of this molecule is easier to make than that of
dictyostatin, and this simplifies its synthesis. This analogue
Supporting Information Available: Characterization
data and copies of spectra for 16-normethyldictyostatin. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(12) Giannakakou, P.; Sackett, D. L.; Kang, Y. K.; Zhan, Z.; Buters, J.
T.; Fojo, T.; Poruchynsky, M. S. J. Biol. Chem. 1997, 272, 17118-17125.
(13) Lin, C. M.; Jiang, Y. Q.; Chaudhary, A. G.; Rimoldi. J. M.; Kingston,
D. G. I.; Hamel, E. Cancer Chemother. Pharmacol. 1996, 38, 136-140.
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