SCALED-UP SYNTHESIS OF DISCODERMOLIDE
Multigram quantities of anticancer marine natural product synthesized by
Some 60 g of (+)-discodermolide, a potent
inhibitor of tumor cell growth, has been prepared in a 39-step synthesis
by the Novartis Chemical & Analytical
Development Group in Basel, Switzerland. The synthetic material is now
undergoing Phase I clinical trials for pancreatic cancer at the Cancer
Therapy & Research Center in San Antonio, Texas.
"The large-scale total synthesis of such a complex natural
product in such quantities was a first for Novartis and probably the entire
pharmaceutical industry," says Novartis principal scientist Stuart J. Mickel,
who played a significant part in the large-scale synthesis effort.
The synthesis is spectacular, according to Steven
V. Ley, chemistry professor at the University of Cambridge, England.
"It's probably the best piece of synthetic work to come out from an industrial
company," he comments. "The ability to make something at this level of
complexity as opposed to extracting it from natural product sources illustrates
the power of modern synthetic chemistry."
Novartis team, led by Mickel, took 20 months to produce 60 g of (+)-discodermolide.
(+)-Discodermolide is a novel polyketide lactone natural
product that was first isolated in 1990 from extracts of the rare Caribbean
marine sponge Discodermia dissoluta by chemistry group leader Sarath P.
Gunasekera and coworkers at Harbor Branch Oceanographic Institution, Fort
Pierce, Fla. [J. Org. Chem., 55, 4912 (1990)].
"The sponge that yielded the first sample of (+)-discodermolide
was collected by hand using scuba at Lucay, Grand Bahamas Island, at a
depth of 33 meters," Gunasekera says. "The gross structure of (+)-discodermolide
was determined by extensive NMR studies, and the relative stereochemistry
was assigned by single-crystal X-ray crystallography."
The molecule's 24-member polyketide carbon skeleton is
made up of eight polypropionate and four acetate units. The structure has
13 stereocenters, lactone and carbamate moieties, and three Z-configured
alkenes, one of which is part of a terminal diene unit. It also features
a stereo triad--methyl, hydroxyl, methyl--that is repeated three times.
COMPOUND has been shown to inhibit the proliferation
of human and mouse cells by arresting the gap 2 (G2) and mitosis (M) stages
of the cell cycle. It does so by binding to and stabilizing microtubules--hollow
filaments consisting of tubulin protein subunits. Microtubules play an
important role in cell division.
(+)-Discodermolide is one of a small, but structurally
diverse, collection of naturally occurring microtubule-stabilizing agents
discovered over the past decade. They include epothilones, eleutherobin,
"The compound belongs to the class of antimitotic agents
known to act by microtubule stabilization whose clinically used member
is Taxol," Gunasekera says. "(+)-Discodermolide is more potent in stabilizing
microtubules and more water soluble than Taxol. In addition, it is active
in Taxol-resistant human cancer cell lines that overexpress P-glycoprotein,
the multidrug-resistant transporter."
(+)-Discodermolide has been shown to be a promising candidate
for clinical development as a drug for colon, ovarian, and breast cancers.
It also, unusually, exhibits synergy with Taxol.
"Due to its strong microtubule-stabilizing properties and
its strong activity against multiple drug-resistant tumors, Harbor Branch
Oceanographic Institution licensed (+)-discodermolide to Novartis Pharmaceutical
Corp. in early 1998 for development as an anticancer drug," Gunasekera
Mickel points out that the marine sponge cannot provide
the quantities of discodermolide needed for drug development. "The sponge
has to be harvested using manned submersibles, and the compound accounts
for only 0.002% by weight of the dried material," he says. "Some 3,000
kg of the sponge--a quantity that probably does not exist--would have been
needed to deliver 60 g of (+)-discodermolide.
"Attempts to reproducibly isolate a discodermolide-producing
microorganism for fermentation have not been successful to date," he continues.
"Therefore, all discodermolide used for preclinical R&D activities
as well as for the ongoing clinical trial has been supplied by total synthesis."
Chemistry professor Stuart
L. Schreiber and coworkers at Harvard University reported the first
total syntheses of the nonnatural (–)-antipode of discodermolide
in 1993 [J. Am. Chem. Soc., 115, 12621 (1993)]
and the natural (+)-antipode the following year [Chem. Biol.,
1, 67 (1994)].
SYNTHESES were reported together with the discovery
of a cellular (+)-discodermolide binding activity, which was shown in 1996
[Chem. Biol., 3,
2878, (1996)] to be the Taxol-binding site of microtubules," Schreiber notes.
was prepared by combining three structural segments, each containing
the same stereo triad (marked by three asterisks), derived from a common
FULL SIZE IMAGE)
Since the publication of the syntheses by Schreiber's group,
several other total syntheses and preparations of various discodermolide
fragments have been reported. According to Gunasekera, there are 24 U.S.
patents and more than 300 scientific papers on discodermolide and its analogs
in the literature.
The Novartis team evaluated all the available literature
syntheses of the molecule with respect to yield, ease of reactions with
regard to scale-up, availability of reagents, safety, and other requirements.
The synthetic route employed by the group was a hybrid
of two literature approaches. The early stages used the 1-g synthesis of
(+)-discodermolide achieved by chemistry professor Amos
B. Smith III and coworkers at the University of Pennsylvania, Philadelphia
Am. Chem. Soc.,
122, 8654 (2000)]. The latter stages, leading to the natural product itself, employed chemistry reported by a group at the University of Cambridge led by chemistry professor Ian Paterson [Angew. Chem. Int. Ed., 39, 377 (2000)].
"Novartis licensed our patented synthesis from the University
of Pennsylvania," Smith notes. "They had access to all of our information
as well as the patented material."
The Novartis-Smith-Paterson synthetic route, which took
about 20 months to complete, was reported in a series of five papers earlier
this year [Org. Process Res. Dev.,
The first paper describes a multistep process, based on
Smith's procedure, to convert the commercially available starting material
acid methyl ester, known as Roche ester, to a Weinreb amide (an N-methoxy-N-methylamide).
Weinreb amides are versatile compounds that are commonly used to prepare
aldehydes and ketones. The one prepared by the Smith and Novartis groups
contains the methyl-hydroxyl-methyl stereo triad that is repeated in three
discodermolide structural segments--the C16, C914, and C1521
The Novartis team modified Smith's procedure to facilitate
large-scale production of the amide in a pilot plant. Modifications included
switching a reducing agent from lithium aluminum hydride to lithium borohydride
to avoid the accumulation of large quantities of aluminum salts that do
not allow for efficient filtration.
The large-scale preparations of the C16, C914,
and C1521 discodermolide fragments from the common Weinreb amide
precursor using Smith's approach are reported in the second and third papers.
The fourth paper describes the preparation of the C724
intermediate--the "Paterson aldehyde"--that involves coupling the C914
and C1521 fragments. In the course of this synthetic sequence, the
transition is made from the Smith to the Paterson chemistry.
"Although the original Smith synthesis was attractive,
it contained a step requiring a high-pressure reaction for the formation
of a phosphonium salt from an alcohol intermediate for a late-stage Wittig
reaction," Mickel says. "This step was not an option for us on any sort
of scale. We then rationalized that this intermediate could be fed into
the attractive end-game approach of Paterson."
The final paper describes the linkage of the C16
fragment (a methyl ketone) and the C724 aldehyde fragment using a
boron-mediated aldol coupling procedure developed by Paterson's group.
The total synthesis required 17 large-scale chromatographic
purifications, and many complex scale-up issues had to be solved along
the way, according to Mickel.
after 30-odd steps and 18 months' work, you come to a crucial fragment
coupling," Mickel says. "It has to work first time on a large scale. You
can't quickly go back to the beginning and bring more material through.
You have little material for a proper laboratory investigation, in contrast
to a normal development project, so things have to work, and you have to
take a calculated risk."
As an example of the challenges faced in scaling up the
synthesis, Mickel cites the final step, which involved removing discodermolide's
protecting groups to produce the final product.
"Easy chemistry, one might think," Mickel remarks. "We
chromatographed the material and isolated it by evaporation from ethyl
acetate. Now the HPLC [high-performance liquid chromatography] of the ethyl
acetate solution revealed a material with a purity of more than 99%. After
evaporation, we obtained a nice crystalline material. HPLC showed the presence
of around 8% impurity. Panic broke out! After we calmed down, we realized
that the lactone ring had probably opened and we had an equilibrium mixture.
We produced the pure material again by adding a trace of acid."
The scale-up process progressed in three stages: proof of synthesis,
preparation of a 6-g batch, and finally the production of 60 g of (+)-discodermolide.
More than 43 chemists were involved in the synthesis concept, experimental
design, and execution. They included chemists in the Process Research &
Development Group in the Chemical & Analytical Development Department
at Novartis Pharma, Basel; the Novartis Institutes for Biomedical Research,
East Hanover, N.J; and the University of Cambridge.
Gunasekera examines model of (+)-discodermolide.
option of optimizing the present synthesis further or replacing it with
a better one is a topic of our ongoing studies, and we are confident of
climbing this mountain as the situation demands," the authors note in the
fifth paper of the series.
Paterson, who is one of the authors of parts four and five
of the five-paper series, believes that the Novartis large-scale synthesis
is a significant achievement. He is delighted that the chemistry developed
by his group over a number of years has reached maturity.
"The work indicates that the challenging total synthesis
of complex natural products, like discodermolide, can be performed successfully
within the strict timelines set by the pharmaceutical industry, delivering
sufficient material for human clinical trials that may ultimately generate
new medicines," he comments. "It leads the way for the large-scale synthesis
and clinical development of many other intriguing biologically active natural
products, with even more complex structures, that are only available in
very low natural abundance."
Smith's group has since overcome the high-pressure problem
in his synthesis by replacing a large protecting group with a sterically
less demanding one in the preparation of the Wittig salt during the final
stages of the synthesis [Org. Lett., 5, 4405
Smith refers to the syntheses developed by his group in
terms of generations. "Our first-generation synthesis was done shortly
after the Schreiber first synthesis wherein we also synthesized the nonnatural
enantiomer," he explains. "Our second-generation synthesis was the 1-g
synthesis, and the third-generation synthesis was the work removing the
high-pressure requirement in conjunction with other as yet unpublished
improvements to our 1-g synthesis."
The longest linear sequence in the second-generation synthesis
is 24 steps in a total of 34 steps, he points out, compared with 26 and
39 steps, respectively, in the Novartis large-scale synthesis. The overall
yield of the Smith 1-g synthesis is 6.0%, whereas the Novartis yield is
"We anticipate that our new third-generation approach will
further simplify the synthesis and reduce the cost of the clinical material,"
Smith remarks. "Clearly, the Novartis synthesis is a wonderful accomplishment,
demonstrating that if a new drug candidate is sufficiently valuable, synthetic
chemists will rise to the challenge of developing a viable synthetic approach
no matter how complex the structure."