Improved synthesis of 6-epi-dictyostatin and antitumor efficacy in mice bearing MDA-MB231 human breast cancer xenografts

Article abstract of DOI:10.1021/jm800979v

Structure-activity studies centered on the naturally occurring antitumor agent dictyostatin have recently identified several highly active epimers and analogues. From these compounds, 6-epi-dictyostatin was selected for scaleup preparation and evaluation in animals. Here we describe a new total synthesis that produced more than 30 mg of 6-epi-dictyostatin. The compound was found to have potent antitumor activity in SCID mice bearing MDA-MB231 human breast cancer xenografts.

Full text of DOI:10.1021/jm800979v

6650
J. Med. Chem. 2008, 51, 6650–6653
Improved Synthesis of 6-epi-Dictyostatin and
Antitumor Efficacy in Mice Bearing
MDA-MB231 Human Breast Cancer
Xenografts
Julie L. Eiseman,†,‡,* Lihua Bai,†,‡ Won-Hyuk Jung,§
Gustavo Moura-Letts,§ Billy W. Day,§,| and
Dennis P. Curran§,*
Department of Pharmacology & Chemical Biology and UniVersity
of Pittsburgh Cancer Institute, G.27.b Research PaVilion, Hillman
Cancer Center, 5117 Centre AVenue, Pittsburgh, PennsylVania
15213-1862, Department of Chemistry, UniVersity of Pittsburgh,
1101 CheVron Science Center, 219 Parkman AVenue, UniVersity of
Pittsburgh, Pittsburgh, PennsylVania 15260-3900, Department of
Pharmaceutical Sciences, UniVersity of Pittsburgh, 10017
Biomedical Sciences Tower, 3501 Fifth AVenue,
we report a new synthetic approach to the dictyostatin family
that produced more than 30 mg of 6-epi-dictyostatin 1. In the
first animal tests of any dictyostatin, epimer 1 showed high
antitumor activity in SCIDa mice bearing xenografts of the
MDA-MB231 human estrogen receptor negative breast cancer
cell line. 6-epi-Dictyostatin 1 was significantly better than
paclitaxel in inhibiting tumor growth.
We adopted the retrosynthesis plan for 6-epi-dictyostatin
shown in Figure 1. Compared to the original fluorous mixture
synthesis of 1,12 the new route is more convergent because it
features fully elaborated bottom 5, middle 6, and top 3
fragments. It also introduces a new strategy for uniting the
bottom and middle fragments by a silicon-tethered ring-closing
metathesis (RCM) reaction14 of 4 to form the critical C10-C11
Z-alkene.
Pittsburgh, PennsylVania 15213-3301
ReceiVed August 1, 2008
Abstract: Structure-activity studies centered on the naturally occurring
antitumor agent dictyostatin have recently identified several highly
active epimers and analogues. From these compounds, 6-epi-dictyostatin
was selected for scaleup preparation and evaluation in animals. Here
we describe a new total synthesis that produced more than 30 mg of
6-epi-dictyostatin. The compound was found to have potent antitumor
activity in SCID mice bearing MDA-MB231 human breast cancer
xenografts.
Since the approval of paclitaxel in the early 1990s and
docetaxel in the early 2000s, interest in compounds that bind
to and stabilize microtubules has continued to grow.1 Recently,
a semisynthetic derivative of epothilone B, ixabepilone, was
approved by the FDA for use either alone or in combination to
combat certain types of breast tumors.2 Other potent microtubule
stabilizing agents, including members of the discodermolide/
dictyostatin class, have also generated significant interest as drug
candidates.3
Dictyostatin is a rare macrocyclic lactone isolated from marine
sponges4,5 that has recently become more available through total
synthesis.6-10 Members of the dictyostatin family strongly
inhibit cancer cell growth, have high affinities for the taxoid
binding site, and competitively inhibit the binding of paclitaxel
and epothilone B to microtubules.4,9,11-13 Among several potent
analogues, including 7-epi-dictyostatin, 16-normethydictyostatin
and 15(Z),16-normethyldictyostatin, 6-epi-dictyostatin 1 was the
most potent inhibitor of binding of epothilone B to microtubules.
It was also the most potent inhibitor of HeLa cell growth, and
its activity was not affected by mutations that induce paclitaxel
resistance in the taxoid binding site.9,13
Figure 1. Retrosynthetic analysis of 6-epi-dictyostatin.
Accordingly, 6-epi-dictyostatin 1 was selected for a scaleup
synthesis and in vivo evaluation of its antitumor properties. Here
The C1-C10 fragment 5 was synthesized through five steps,
starting from readily available intermediate 7,15 as shown below.
Deprotection of TBS ether 7 with HF·pyr and Dess-Martin
* To whom correspondence should be addressed. For J.L.E.: phone,
1-412-623-3239; fax, 1-412-623-1212; E-mail, eisemanj@msx.upmc.edu.
For D.P.C.: phone, 1-412-624-8240; fax, 1-412-624-9861; E-mail, curran@
pitt.edu.
a Abbreviations: CBS, Corey-Bakshi-Shibata; DDQ, dichlorodicyano-
quinone; GH-II, second-generation Grubbs-Hoveyda catalyst; HWE,
Horner-Wadsworth-Emmons reaction; PMB, p-methoxybenzyl; RCM,
ring-closing metathesis; SCID, severe combined immunodeficient; T/C,
tumor volume of test agent-treated group divided by tumor volume of
untreated control group; T/V, tumor volume of test agent-treated group
divided by tumor volume of vehicle-treated group.
†
Department of Pharmacology & Chemical Biology, University of
Pittsburgh Cancer Institute.
‡
University of Pittsburgh Cancer Institute.
Department of Chemistry, University of Pittsburgh.
Department of Pharmaceutical Sciences, University of Pittsburgh.
§
|
10.1021/jm800979v CCC: $40.75
2008 American Chemical Society
Published on Web 10/08/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6651
Scheme 1. Fragment Couplings and Macrolactonization
oxidation16 of the resulting alcohol provided the aldehyde 8 in
91% yield over two steps. Addition of vinyl magnesium
bromide, Dess-Martin oxidation, and Corey-Bakshi-Shibata
(CBS)17 reduction gave an inseparable mixture of two C9-
epimers 5 (R) and 9 (ꢀ) in a 5:1 ratio favoring the needed alcohol
5 (74% yield, three steps).
the ꢀ-hydroxyketone 15 in 68% over two steps. The 1,3-syn-
reduction of the hydroxyketone 15 with NaBH4 and Et2BOMe19
and then selective protection of the C19 hydroxy group of the
resulting diol with TBSOTf furnished the alcohol 16 (74% yield
over two steps).8,20
In the usual end game, the C1 methyl ester was hydrolyzed
by using KOH to produce a seco-acid, which was used for the
next reaction without further purification. The macrolactoniza-
tion of the seco-acid using the Yamaguchi reagent (2,4,6-
trichlorobenzoyl chloride)21 gave a mixture of the C2 E/Z
isomers of the TBS-protected macrolactone in a varying ratio,
but the use of the Shiina reagent (2,6-methylnitrobenzoyl
anhydride)22 in toluene suppressed the isomerization of the C2Z-
alkene (1:13, E/Z). Finally, global deprotection with HCl
provided 33 mg of 6-epi-dictyostatin in 45% yield over three
steps.23
The sequence of fragment couplings and macrolactonization
is summarized in Scheme 1. The C11-C17 alcohol 6 was
treated with BuLi/dimethyldichlorosilane, followed by addition
of imidazole and the C1-C10 alcohol epimer mixture of 5 and
9. This provided another inseparable C9 mixture of the silyl
acetals 4 and 10 in 85% yield, again in about a 5:1 ratio. The
RCM reaction of this silyl acetal mixture was mediated by the
Grubbs-Hoveyda II catalyst to provide a third inseparable C9
mixture of the eight-membered disiloxanes in 57-69% yield.
This mixture was deprotected with dichloroacetic acid, and the
crude product was purified by silica gel chromatography. This
time, the C9 epimers separated and the target diol 11 (C9S)
was isolated in 45% yield alongside 7% of the C9R-epimer 12.
Protection of the diol 11 as a TBS ether followed by treatment
with DDQ gave the alcohol 13 in 62% yield over two steps.
The alcohol 13 was oxidized by using the Dess-Martin
periodinane to the aldehyde 2, which was coupled with the
phosphonate 3 to provide the enone 14 possessing the full
C1-C26 carbon skeleton of 6-epi-dictyostatin (89% yield over
two steps).
6-epi-Dictyostatin 1 and paclitaxel were each administered
intravenously in three doses of 20 mg/kg/dose spaced 7 days
apart to 10 CB-17 SCID female mice bearing established MDA-
MB231 human breast cancer xenografts. Mean tumor volumes
(Figure 2) and body weight loss (Figure 3) were periodically
measured. One of the mice receiving 6-epi-dictyostatin 1 was
accidentally injured then euthanized between days 14 and 17,
while the other nine mice in that group continued to be followed.
Mean tumor volumes in the mice treated with 6-epi-
dictyostatin 1 were significantly smaller than those in the control
and vehicle-treated groups beginning on day 7 of treatment. And
beginning on day 10, they were also smaller than those in the
paclitaxel-treated group (Figure 2). Tumor regression was
observed in six of the nine 6-epi-dictyostatin 1-treated mice on
day 14 of study, and these tumors continued to regress until
day 28 of study. In the remaining three 6-epi-dictyostatin
1-treated mice, tumor volumes did not increase until day 28,
when tumor regrowth was observed. Tumors continued to grow
in all the paclitaxel-treated mice, albeit at a slower rate than
that observed for the tumors in the control and vehicle-treated
groups. Paclitaxel-treated mice were euthanized between days
24 and 28 (Figure 2).
The C17-C18 alkene of the enone 14 was reduced using
the Stryker reagent ([Ph3PCuH]6)18 to give a saturated ketone,
then the C21 PMB group was removed with DDQ to provide

6652 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Letters
Table 1. Tumor Doubling Times, Median Optimal %T/C, and Median
Optimal %T/V in CB-17 SCID Mice Bearing MDA-MB231 Xenograftsa
treatment group days to one tumor days to two tumor %T/C %T/V
N ) 10
paclitaxelb
doubling
doublings
day 14 day 17
33e 31e
7.2 ( 2.9d
19.6 ( 7.4d
(7.9)e
(19.0)e
6-epi-dictyostatin 1b
vehiclec
27.4 ( 18.7d
(30.9)e,f
4.9 ( 3.2
(4.0)
38.1 ( 12.1d
(35.0)e,f
9.8 ( 3.1
(8.9)
14e,f 13e,f
61
100
control
2.8 ( 1.7
(2.5)
7.7 ( 2.4
(8.6)
100
a Tumors were implanted in CB-17 SCID female mice as 25 mg
fragments and mice were stratified on day 17 postimplantation, when tumors
reached 101-463 mm3. Treatment began at day 0 (day 17 postimplantation).
Results of time to one doubling and time to two doublings are presented as
the mean ( std (median). b 20 mg/kg iv q7dx3. c Cremophor EL-ethanol-
saline 1:1:8 (v/v/v) 0.01 mL/g body weight iv q7dx3. d Value significantly
different from control and vehicle, p e 0.05 by Dunnett’s test. e Value
significantly different from control and vehicle, p e 0.05 by Mann-Whitney
test.f Valuesignificantlydifferentfrompaclitaxel,pe0.05byMann-Whitney
test.
Figure 2. Antitumor activity in MDA-MB231 human breast cancer
xenograft-bearing CB-17 SCID female mice (n ) 10 animals per group)
treated with 20 mg/kg iv q7dx3 6-epi-dictyostatin 1 (O) or paclitaxel
(4), vehicle (2), or no treatment (3). Arrows inidicate the days of
dosing.
Acknowledgment. We thank the NIH for grant CA78039
to support this work. We dedicate this paper to Dr. Ernest Hamel
on the occasion of his 65th birthday.
Supporting Information Available: Experimental procedures
and characterization data for all new compounds and experimental
procedures for biological testing. This material is available free of
charge via the Internet at http://pubs.acs.org.
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