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Meeting the Supply Needs of MarineNatural Products 26David J. Newman and Gordon M. Cragg
Contents
26.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286
26.2 Case Study 1: Didemnin B and Dehydrodidemnin B (Aplidine) . . . . . . . . . . . . . . . . . . . . . 1287
26.2.1 Didemnin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1287
26.2.2 Dehydrodidemnin B (Aplidine) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1289
26.2.3 Lessons from Case Study 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1290
26.3 Case Study 2: Bryostatin 1 and Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1291
26.3.1 Bryostatin 1 from Natural Sources (Wild Collections and Aquaculture) . . . 1291
26.3.2 Bryostatins by Chemical Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1292
26.3.3 Bryostatin Analogues/Mimics by Chemical Synthesis . . . . . . . . . . . . . . . . . . . . . . 1293
26.3.4 Putative Microbial Source(s) of Bryostatins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293
26.3.5 Lessons from Case Study 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1294
26.4 Case Study 3: Dolastatin 10 and Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1295
26.4.1 Dolastatin 10, Original Source and Syntheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1295
26.4.2 Auristatin PE/TZT-1027; Dolastatin 10 Derivatives . . . . . . . . . . . . . . . . . . . . . . . . 1295
26.4.3 Actual Source(s) of the Dolastatins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296
26.4.4 Lessons from Case Study 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296
26.5 Case Study 4: Halichondrin B and Eribulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1297
26.5.1 Halichondrin B from Natural Sources (Wild Collections and
Aquaculture) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1297
26.5.2 Synthetic Production of Halichondrin B and Derivatives . . . . . . . . . . . . . . . . . . . 1299
26.5.3 Lessons from Case Study 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
26.6 Case Study 5: Ecteinascidin 743 (Yondelis®) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1301
26.6.1 Ecteinascidin 743 from Natural Sources (Wild Collections and
Aquaculture) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1301
26.6.2 Ecteinascidin 743 from Chemical Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1302
26.6.3 Lessons from Case Study 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1302
26.7 Case Study 6: Salinosporamide A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1303
26.7.1 Discovery of Salinosporamide A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1303
D.J. Newman (*) • G.M. Cragg
Natural Products Branch, Developmental Therapeutics Program, National Cancer Institute,
NCI-Frederick, Frederick, MD, USA
e-mail: [email protected], [email protected]
E. Fattorusso, W. H. Gerwick, O. Taglialatela-Scafati (eds.),
Handbook of Marine Natural Products, DOI 10.1007/978-90-481-3834-0_26,# Springer Science+Business Media B.V. 2012
1285
26.7.2 Large-Scale Fermentation/Isolation of cGMP Product . . . . . . . . . . . . . . . . . . . . . . 1303
26.7.3 Synthetic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1304
26.7.4 Genomic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1304
26.7.5 Lessons from Case Study 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305
26.8 In Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305
26.9 Study Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1306
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1306
Abstract
Perhaps the major perception both in the general medical and scientific commu-
nities, and also to some extent in the natural products community, is that marine
natural products are of interest but not directly feasible for further development.
This perception was fundamentally correct up through roughly the early 1990s,
due to major physical difficulties in obtaining enough raw materials from which
to isolate the compound of interest. In this chapter, we will show how application
of novel techniques in growth and isolation processes, increased knowledge of
genomics, and major advances in chemical synthesis have made observers alter
their perceptions to the extent that, nowadays, production of marine natural
product molecules is a combination of many techniques, each adding their
quota of experience. We will use a case study mechanism to demonstrate how
these methods evolved and have now enabled marine natural products to be
serious contenders as drug candidates.
26.1 Introduction
Marine natural products with significant activities in many pharmacologic areas have
beenwell documented over the years, particularly in the extensive reviews initiated by
the late Dr. John Faulkner and now continued by the New Zealand marine group [1].
However, therewas a perception, based on reality prior to themiddle to late 1990s, that
the development ofmarine-based compounds would require immense efforts from the
aspect of recovery and processing of large amounts of biomass in order to obtain even
enoughmaterial for initial in vivo screening.Added to thiswas that very large amounts
would be necessary for further drug development processes, where requirements
might well be in the hundreds of grams to multikilogram levels of pure compound
produced under current good manufacturing practices (cGMP).
These perceptions were not aided by the fact that most of the agents reported
from marine sources were exquisitely potent, often with IC50 values in the
nanomolar range and present at very low levels in the invertebrate. For example,
when reports indicated that 18 g of bryostatin 1 was isolated from over 13 metric
tons of Bugula neritina or 300 mg of halichondrin B from 1 metric ton of a deep
water Lyssodendoryx sp., it did not help sell the concept of “drugs from the sea” to
pharmaceutical houses. Although these figures were true at the time of publication,
nowadays thanks to advances in biology and chemistry over the last 10 plus years
the situation has undergone a “sea change,” with combination methods and routes
1286 D.J. Newman and G.M. Cragg
such as microbial biosynthesis, aquaculture (in sea or on land) coupled to potential
chemical syntheses, now being considered as part and parcel of the initial discov-
ery/development process.
In order to put the recent advances in perspective, we will discuss a series of
marine-sourced agents being evaluated in anticancer therapies, mainly due to the
initial funding coming from the National Cancer Institute (NCI) or its European
equivalents. These case studies in general have been chosen in order to demonstrate
how the initial large-scale harvesting methods, necessary in some cases due to the
time frame of the initial discovery, have been transformed as knowledge from many
sources were applied to the situations as time progressed.
26.2 Case Study 1: Didemnin B and DehydrodidemninB (Aplidine)
26.2.1 Didemnin B
The first compound entered into human clinical trials directly from a marine source
was the cyclic depsipeptide, didemnin B (1), isolated by the Rinehart group from
the encrusting tunicate Trididemnum solidum [2, 3]. This was one of a number of
very similar compounds isolated from the same organism and included a very close
analogue differing by only two hydrogens, dehydrodidemnin B (DDB) now known
as aplidine (2). This compound subsequently followed DB into human clinical trials
[4] approximately 15 years later.
1 Didemnin B; R =
2 Aplidine; R =
3 Tamendarin A; R =
O OH
O OH
O O
NO
N
O
O
O
NH
O
O
HOO
O
NH
O
O
NH
O
N
O
N
R
NO
N
O
O
O
NH
O
O
HOO
O
HN
NH
O
N
O
O
26 Meeting the Supply Needs of Marine Natural Products 1287
O O
O
O
H
O
O
O
OHO
O
O
OHO
O
H
OHHOH
O
6 Ring Expanded Bryostatin 1
N
OO
HN
OO
N
N S
OHN
O
N
H
7 Dolastatin 10
8 TZT-1027
N
OO
HN
OO
N
OHN
O
N
H
O
O
O
O O
H H
H
OHOOH
OHO
OH15C7
5 Wender's Bryolog
O
O
O O
O
O
H
O
O
O
OHO
O
O
H
OHO
O
H
OHHOH
4 Bryostatin 1
O
Due to the paucity of the producing organism, the level of didemnin B and the
multiple derivatives coproduced by the invertebrate, plus the requirement for the
production of materials for clinical trials under “current good manufacturing
practice (cGMP)” for human trials, synthetic methods were the only viable route
at that time for production of compound. In 1987, the Rinehart group published
a synthetic method to three of the didemnins, A, B, and C [5] followed the same
year with a publication from the Joullie group demonstrating how to synthesize
further derivatives of the unusual amino acid “statine” found in the didemnins [6].
1288 D.J. Newman and G.M. Cragg
In 1991, the Jouin group in France reported on synthesized derivatives that had
the linear lipophilic peptide side chain modified. They showed that except for
a derivative where palmitic acid was substituted for the lactyl group of didemnin
B, the biological activities both in vitro and in vivo were not significantly changed
when compared with the natural product [7]. However, the palmityl derivative lost
effectively all cytotoxic activity, though no immunosuppressive assays were
reported as didemnin B and other congeners exhibited significant immunosuppres-
sive activities [8].
Seven further syntheses by the Schmidt, Shioiri, Joullie, and Jou groups were
later discussed in detail in a 2002 review by Vera and Joullie [9]. These, together
with the 1996 paper from the Rinehart group referred to above [8], yielded a very
significant number of enantiomerically pure analogues together with the required
didemnin B. As an example of what can be done, using modern techniques once the
materials are available in purity is the report in 2004 by Marco et al., where the
probable target for cytotoxicity of the didemnins was shown to be elongation factor
eLF1A [10].
Didemnin B was placed into a significant number of clinical trials by the NCI
working through a variety of groups but was taken off trials in the middle 1990s
due to (cardiac) toxicities [11], with the last published example being the report
from a phase II trial in brain carcinomas by Mittelman et al. in 1999 [12]. What is
of potential significance, however, were the retrospective comments made by Vera
and Joullie (2002) on the methods of delivery used in these early trials – a single
bolus dose at close to the maximum tolerated dose (MTD). From later work with
aplidine [4], the use of different dosing schedules and a modicum of other
supportive medications avoided the significant toxicities seen previously with
didemnin B.
26.2.2 Dehydrodidemnin B (Aplidine)
This compound was reported as being isolated from the Mediterranean tunicate
Aplidium album in a review on the 1990 USA-Japan Seminar on Bioorganic Marine
Chemistry by Schmitz and Yasumoto [13], and it was also reported in a US patent
in 1994 [14] and in a citation to an application for a UK patent in 1989 in the
synthetic paper by Sakai et al. [8] with the initial report of formal activity of
dehydrodidemnin B (DDB or aplidine) in animals being the paper in 1996 by
Urdiales et al. [15].
Just as in the case of its very close analogue, didemnin B, the amounts required
for both initial preclinical studies and then for any future studies precluded
the use of wild collections; thus total synthesis was the only viable route to
production of this agent, even though there had been reports in some of the very
early papers on these agents implying production by a symbiotic interaction
between the blue-green alga Synechocystis trididemni and the invertebrate
[7, 16, 17].
26 Meeting the Supply Needs of Marine Natural Products 1289
The method used for the production of aplidine was a route based upon the liquid
phase peptide synthesis described in 1997 by Jou et al. [18] in which synthetic
didemnin A was coupled with the side chain “pyruvate-proline-OH” to give DDB.
Two routes to produce a protected didemnin A were investigated by Jou et al. The
first used macrocyclization of a linear seven-membered peptide containing the first
extracyclic amino acid of the didemnin side chain. This gave an overall yield of 4% of
the protected didemnin A. The other approach, initial formation of the basic didemnin
macrocycle followed by coupling of the first extracyclic amino acid, used a six-
membered linear peptide that led to the didemnin macrocycle on cyclization.
Following addition of the first amino acid of the side chain, the overall yield of the
protected didemnin A was 27%. Irrespective of the methods used to derive didemnin
A, the subsequent coupling step to yield DDB proceeded with a 62% yield.
These methods will have been further optimized in order to produce materials
under cGMP conditions for the clinical development of the agent but as is usual, the
information that is given in the chemical and manufacturing controls (CMC)
section for an investigational new drug application (INDA) for the FDA, or its
equivalent section in any submission to the EMEA in the European Union, is not
available for public dissemination nor have any publications with such information
been published as far as can be determined to date.
Aplidine (DDB or plitidepsin) is currently in phase I/II clinical trials predom-
inately in blood-based carcinomas, including multiple myeloma where a phase II
trial (NCT00229203) has just completed and a phase II trial (NCT00884286) has
just commenced studying its activity in non-Hodgkin’s lymphoma, with a new
phase III trial (NCT01102426) beginning to recruit patients for a comparison of
aplidine plus dexamethasone versus dexamethasone in relapsed/refractory mye-
loma (ADMYRE trial). In addition, over the years, it has been in trials against
a variety of cancers in the EU, Canada, and the USA with a significant number of
publications listed through the middle of 2005 [4, 11], and eight more reports
from phase I/II shown in a current search of the Scopus database from late 2005
to date.
26.2.3 Lessons from Case Study 1
As a result of the very low yields of material from the natural sources (encrusting
didemnin invertebrates), though the possibility of a symbiotic production from
a cyanobacterium (blue-green alga) of metabolites was suggested as early as 1979
[16], the only viable method of production was total chemical synthesis. The initial
work of the Rinehart group led to the very successful application of liquid-phase
methods by the Jou group, leading to the current clinical trials of DDB.
The lessons learned from these compounds were aptly demonstrated by two
other investigators involved in the didemnin story, and led to a report in 2007 of
variations in the synthetic approaches to the didemnin analogues, the tamandarins
(3) [19]. These synthetic variations compared to those originally reported [20, 21]
1290 D.J. Newman and G.M. Cragg
led to significantly increased synthetic yields and production of analogues with
in vitro antitumor activity, thus, extending the structure activity relationships in this
very active series.
26.3 Case Study 2: Bryostatin 1 and Derivatives
26.3.1 Bryostatin 1 from Natural Sources (Wild Collections andAquaculture)
After didemnin B, the next “direct from the sea” compound to go into clinical
trials was bryostatin 1 (4); currently it has been in over 80 clinical trials, with six
phase I/II trials in cancer still being listed in the NIH clinical trials database, of
which five are still active but not recruiting further patients, and the remaining one
is a phase I trial involving bryostatin 1 in concert with temsirolimus. In addition,
a phase II trial in Alzheimer’s is listed but not yet recruiting patients.
That this compound ever made it from a biological discovery to human clinical
trials can be ascribed to the determination of G. Robert (Bob) Pettit, who first
reported this class of compounds in 1970 [22]. The subsequent work, leading to the
identification of 19 variations on the same basic structure, mainly as a result of
Pettit’s group at Arizona State University (ASU) and collaborators in multiple other
institutions has been reported in a large number of papers and reviews [23, 24].
The latest “variation” on the basic structure from a natural source was the new
bryostatin derivative (bryostatin 20) reported from an Atlantic Bugula neritina by
Lopanik et al. in 2004 [25], and on comparison with the known 19 earlier mole-
cules, its structure is effectively the ring-closed version of bryostatin 10.
As a result of the initial preclinical and clinical (phase I) responses to bryostatin 1
[26, 27], it was necessary to produce gram quantities of the compound for initial trials.
No syntheticmethodwas available so theNCI contracted a small California company,
Marinus Inc., in Long Beach to collect close to 13 metric tonnes of wet Bugulaneritina from depths between 3 and 30 m off the coast of Southern California.
Since this particular animal is a large-scale fouling organism and is ubiquitous in
the waters off California, no significant effect on the environment was anticipated.
Following shipment of the 40,000 L of wet organism in isopropanol to
NCI-Frederick, the US Government contractor who ran the NCI-Frederick Cancer
Research Center, then Program Resources Incorporated together with scientists
from NCI and Bristol Myers Squibb (who had licensed the bryostatin patent from
ASU), produced 18 g of cGMP quality bryostatin 1 in 10 months of work [28].
A later small-scale rework of the process using supercritical fluid extraction rather
than conventional liquid–liquid extraction, followed by large-scale chromatogra-
phy reduced the processing time from 10 months to under a month assuming scaling
would be relatively linear [29].
From the raw material supply problems that arose in the case of Taxol® [30, 31],
NCI decided in the early 1990s to “plan for success” when natural product-sourced
26 Meeting the Supply Needs of Marine Natural Products 1291
compounds entered preclinical/clinical trials. Therefore, as a result of the volumes
of wild-collected material that would be involved if bryostatin 1 was successful as
an antitumor drug, a very large amount of raw material would be required in order
to produce enough of the compound for clinical use, NCI funded a competitive
SBIR phase I and a subsequent phase II contract with the small California biotech-
nology company, CalBioMarine.
The aim of this initial contract (phase I), and its subsequent extension (phase II),
was to grow Bugula neritina using both in-sea and on-land aquaculture in order to
avoid the problems of seasonal variation in both levels of this bryozoan and, as
learned later, in the levels of bryostatin in the animal. The goals of the contract were
successfully met, demonstrating that the organism could be produced by either in-
tank aquaculture or via seeded screens suspended in the Pacific Ocean off La Jolla,
California. The levels of bryostatin were high enough to obtain a useful supply of
the compound, though significant optimization of feeding protocols and recovery
methods would have been necessary. The work involved was subsequently
described by one of the CalBioMarine principals, Dominick Mendola [32].
26.3.2 Bryostatins by Chemical Synthesis
From the initial reports on the structures of the bryostatins, chemists recognized the
base molecule as a challenge for their synthetic skills. A significant number of
syntheses of parts of the bryostatin 1 molecule and total syntheses of various
members of the complex were reported, with the first being the enantiomeric
synthesis of bryostatin 7 in 1990 by Masamune’s group [33], followed by papers
from the Evans’ group giving details of an enantiomeric synthesis of bryostatin 2 in
the 1998–1999 time frame [34, 35]. The third in the initial series was the synthesis
of bryostatin 3 by Nishiyama and Yamamura in 2000 [36]. These earlier syntheses
together with partial syntheses of other bryostatins were reviewed in detail through
2002 by Hale et al. [37]. Following a break of 8 years, in 2008, Trost and Dong
published [38] their relatively simple and quite elegant synthesis of bryostatin
16 involving some novel metal-linked catalysis steps [39], including a ruthenium
tandem alkyne-enone coupling and then a palladium catalyzed alkyne-ynoate
macrocyclization to give the cyclized precursor of bryostatin 16.
In spite of all of these elegant methods, to date, no de novo synthesis of
bryostatin 1 has been published, though in the early days of studying the
bryostatins, Pettit et al. demonstrated that bryostatin 2 could be converted to
bryostatin 1 and bryostatin 12 [40]. Thus, one could argue that a formal synthesis
of bryostatin 1 has been achieved by using the Evans et al. bryostatin 2 asym-
metric synthesis [34, 35] and then applying the Pettit conversion method. Very
recently, an excellent review of the synthetic chemistry surrounding the
bryostatins was published by Hale and Manaviazar [41], which should be
consulted by those interested in the specific methodological differences between
the various syntheses.
1292 D.J. Newman and G.M. Cragg
26.3.3 Bryostatin Analogues/Mimics by Chemical Synthesis
From the early days of the bryostatins, Wender’s group postulated that if one
could synthesize a simpler analogue with comparable activity, then chemical
production of such an agent might well be a viable option. Over the years,
Wender and his colleagues have successfully synthesized a significant number
of what they have named “bryologs”; nominally simpler molecules that have
pharmacologic characteristics similar to bryostatin 1. These were originally
designed by modeling bryostatin 1 on to the binding site of the phorbol esters
on protein kinase C and over the years, a significant number have been published
with the latest variations being those reported in 2008 [42, 43], with
a representative molecule being shown in (5). Other synthetic groups have also
produced synthetic modifications that are different from the Wender bryologs,
but still have significant biological activities. One example is the ring-expanded
molecule (6) where five extra carbon atoms have been inserted between the C16
and C17 carbon atoms of bryostatin 1 [44] while maintaining bryostatin-like
activity. However, other more subtle modifications where only four changes
were made on the periphery of the bryostatin 1 molecule, converted the
bryostatin 1 structure to a molecule that had biological activities that mimicked
those of the phorbol ester tumor promoters rather than those of the parent
molecule [45]. Thus, one has to be very careful in assessing the actual activities
of what appear to be small modifications to the base molecule.
26.3.4 Putative Microbial Source(s) of Bryostatins
One of the discoveries made from the collections made by Calbiomarine of wild
Bugula neritina when they were collaborating with the Haygood group at the
Scripps Institution of Oceanography was that there appeared to be two potential
subspecies, one from deep (below �10 m) and one from shallow (above �7 m).
However, the bacterial symbionts of the invertebrate were investigated [46], and
suggested that the actual producer of the bryostatins was a previously unrecognized
and, as yet, noncultured bacterium.
Over the next 10 plus years, in conjunction with experts in gene identifica-
tion and cloning, Haygood and her collaborators have been able to identify and
clone, but not yet express in a heterologous host, the putative biosynthetic
cluster that should produce what they are calling bryostatin 0, that would then
be further elaborated by as yet unidentified tailoring enzymes. Thus, Haygood
and her collaborators investigated the potential sources of bryostatins in both
the adult animal and in the larvae. In a series of excellent investigations, they
demonstrated that within the larvae of bryostatin-producing animals there were
as yet uncultured bacteria (given the name of Candidatus Endobugula sertula)in the first report by Davidson and Haygood in 1999 [47] and extended in
2001 [48].
26 Meeting the Supply Needs of Marine Natural Products 1293
A series of later papers included a review of the possibilities of identification and
expression of genes from symbionts [49], the identification of different symbionts
from other Bugula species that produced bryostatins and contained similar gene
constructs [50], together with the identification of a putative bryA cluster [51].
Further work by Lopanik et al. on Atlantic-sourced, rather than Pacific or Gulf
Coast-sourced Bugula neritina, has confirmed the presence of bryA-like fragments
from this geographic area’s organisms as well [52].
It should be emphasized that in none of the cases described does the identifi-
cation of the bryA cluster in these organisms prove the absolute production of
bryostatins by the symbiont, but the evidence is highly suggestive. Currently
the complete putative genomic sequence has been identified and sequenced but
not yet expressed [53] including the recognition of an unusual transacylase [54].
Finally, in addition to the genomic work related to the biosynthesis in a variety of
Bugula species and locations, work from an ecological perspective has also been
reported by Haygood and collaborators, demonstrating that bryostatins are
important molecules from an ecological as well as a pharmacological perspective
[54, 55].
26.3.5 Lessons from Case Study 2
Just as in the case of didemnin B, bryostatin 1 was present in vanishingly small
amounts in what was thought to be the producing organism, the encrusting and
fouling bryozoan, Bugula neritina. Unlike didemnin B, due to the massive amounts
of organism available off the California coast, it proved ecologically feasible to
isolate 18 g of cGMP quality material from 13 metric tonnes of wet organism, and
methods were also developed on a small scale for a much faster isolation process
using supercritical fluid extraction.
Due to the obvious problems of very low levels and massive collection programs
if bryostatin became a potential drug, NCI, who had learned their lesson from the
problems of sourcing enough Taxus bark for clinical trials of Taxol®, commis-
sioned aquacultural methods and directly and indirectly through the NIH grants
programs, aided in funding synthetic approaches to both bryostatins and their
“simpler” modifications, the “bryologs.”
Basic ecological studies coupled to the massive advances in the study of
biosynthetic gene processes led to the recognition that an as yet uncultured symbi-
otic microbe may be a potential source of bryostatin precursors; these may be
modified by either chemistry or genomic methods to produce the desired chemical
skeletons for further development.
Finally, although bryostatin is not a particularly viable antitumor agent at the
present time, it may well have utility in Alzheimer’s disease as shown in the work
by researchers at the University of West Virginia [56–58] and the approval from the
FDA for a phase II clinical trial of bryostatin 1 in patients with Alzheimer’s under
the ClinicalTrials.gov identifier number NCT00606164.
1294 D.J. Newman and G.M. Cragg
26.4 Case Study 3: Dolastatin 10 and Derivatives
26.4.1 Dolastatin 10, Original Source and Syntheses
In the same time frame as the bryostatins, the Pettit group also reported on the
dolastatins, a series of cytotoxic linear and cyclic peptides that were originally
isolated in very low yield from the Indian Ocean gastropod mollusk Dolabellaauricularia. The history and work-up of the dolastatins, in particular the tubulin
interactive linear peptide dolastatin 10 (7) through early 2005, were reported in
detail by Flahive and Srirangam in a 2005 book chapter [59].
Similarly to the case of didemnin B, the original discoverers realized from
the beginning that only total chemical synthesis would yield the necessary amounts
of material to even pursue advanced preclinical and hopefully clinical studies. Thus,
the Pettit group devised synthetic methods for the large-scale enantiomeric synthe-
ses of the unusual g-amino-b-methoxy-acid residues dolaproline and dolaisoleucine,
each of which contained three chiral centers, and the novel C-terminal residue,
dolaphenine, which contains a thiazole and is derived from phenylalanine. Their
approach was described in a paper in 1989 [60] and fuller details of this and other
syntheses were reported in an excellent review by Pettit in 1997 [61].
Due to the potency, at the time it was the most potent agent tested by the NCI,
and mechanism of action as a tubulin-interactive agent binding close to the Vincadomain [62–64], dolastatin 10 entered phase I clinical trials in the 1990s under the
auspices of the NCI. It progressed through to phase II trials as a single agent,
but although tolerated at the doses used, which were high enough to give the
expected levels in vivo to inhibit cell growth, it did not demonstrate significant
antitumor activity in a phase II trial against prostate cancer [65], nor in metastatic
melanoma [66].
26.4.2 Auristatin PE/TZT-1027; Dolastatin 10 Derivatives
Although the natural products did not succeed in demonstrating activity in humans,
the syntheses described by Pettit and others led the way to a series of other
molecules based upon the natural product but with modifications to the base linear
peptide. Numbers of these have been discussed in the literature including
a compound simultaneously synthesized by both the Pettit group under the name
of auristatin PE [67] and a group from the Japanese company Teikoku Hormone (8)under the name TZT-1027 [68]. This compound under the names, TZT-1027,
auristatin PE, and soblidotin, depending on the particular organization sponsoring
the compound, went into phase I/II trials with mixed results and is not currently
listed as being in active trials in the NIH clinical trials site.
However, demonstrating how compounds that exhibit interesting activities can
be further optimized by clever chemistry and biology, the compound with a small
linker was used as a warhead by Seattle Genetics. Two variations differing in the
26 Meeting the Supply Needs of Marine Natural Products 1295
antibody and the specific linker are currently in phase II (glembatumumab
vedotin) and phase III (brentuximab vedotin) with another (anti-CD19-vcMMAE)
in preclinical trials. Using the auristatin PE derivative, auristatin F (where the
C-terminal amino acid is now phenylalanine), a third monoclonal antibody-
warhead combination (1 F6-MMAF) is currently in phase I clinical trials and
unlike the other two, it does not have a cleavable linker. Up-to-date discussions of
the rationale behind the use of such methods with both cleavable and
noncleavable linkers are presented by Senter’s group from Seattle Genetics
[69, 70] and by Teicher from Genzyme [71].
In addition to the linked molecules referred to above, auristatin PE itself was
shown to be a vascular disruption agent with three relatively recent papers
discussing the possibilities of such agents as antitumor treatments as they cause
the internal vasculature of the tumor to collapse [72–74]
26.4.3 Actual Source(s) of the Dolastatins
Similarly to the situation with the bryostatins and the didemnins, there was always
a potential question with the dolastatins as to whether or not they were microbial
in origin, as peptides with unusual amino acids have been well documented in the
literature as coming from the Cyanobacteria. In the last few years, this supposition
has been shown to be a fact. Thus, in 1998, workers at the Universities of Guam and
Hawaii reported the isolation and purification of simplostatin 1 [75] from the
marine cyanobacterium Simploca hynoides. This molecule differed from
dolastatin 10 by the addition of a methyl group on the first N, N-dimethylated
amino acid. Subsequently, in 2001, the same groups reported the direct isolation of
dolastatin 10 from another marine cyanobacterium that was known to be grazed on
by D. auricularia [76]. Dolastatin 10 was in fact isolated from the opistobranch
following feeding of the cyanobacterium, thus confirming the original hypothesis
(Paul 2006, #145).
26.4.4 Lessons from Case Study 3
As with didemnin B, recognition that collections of the raw material source was not
a feasible method led to the derivation by Pettit’s group of synthetic methods on
a large scale (for an academic institution) for the intermediates. These individual
syntheses then allowed the synthesis of dolastatin 10 with its multiple chiral centers
and three very unusual amino acids in its short peptide structure. From these initial
studies came the capability to produce novel molecules such as auristatin PE.
Modifications around that structure combined with clever utilization of monoclonal
antibody technologies have led to currently active agents in phases I to III clinical
trials.
The work in the late 1990s by the Hawaii and Guam groups demonstrating
that, as suspected for many years, the agents were linked to cyanobacterial diets of
the opistobranchs, leads to the possibility that in due course the biosynthetic gene
1296 D.J. Newman and G.M. Cragg
clusters may be identified. If this can be done, then there is the possibility that
the metabolites might be expressed in a heterologous host. Examples of this
technique were reported simultaneously by two groups with Prochloron peptide
metabolites using the heterologous host E. coli. One used shotgun cloning of the
gene clusters [77] and the other identification of the cluster(s) followed by cloning
and subsequent expression [78].
26.5 Case Study 4: Halichondrin B and Eribulin
26.5.1 Halichondrin B from Natural Sources (Wild Collections andAquaculture)
Halichondrin B (9) was first reported from the Japanese sponge Halichondriaoakdai by Uemura’s group in 1985 [79] and 1986 [80], and, subsequently,
halichondrin B or congeners were reported over the next few years from
a variety of other sponge sources. These initial reports were then followed by
two papers that made this series of agents very interesting for development as
antitumor agents.
O
O
O
O OO
H
H
O
OO
H
HO
O
O
O
O
O
O
H
H
H
HOH
HOOH
9 Halichondrin B
Lactone
12 Ecteinascidin 743
N
N
OO
O
O
H
OH
HO
O
H
H
NH
OO
O
HO
S
10 E7389; R = NH211 E7390; R = OH
O
O
O
OOH
RO O
O
H
H
O
OO
H
Ketone
26 Meeting the Supply Needs of Marine Natural Products 1297
13 Phthalascidin
N
N
OO
O
O
H
CN
HO
O
H
H
N
O
ON
N
O
OH
CN
HO
O
H
HO
NH
O NH2
14 Cyanosafracin B
15 Salinosporamide A
16 Omuralide
HN
O
OHO
O
HN
O
OOH
O
H
Cl
The first, in 1991, showed that halichondrin B acted as a tubulin interactive agent
(TIA) potentially binding close to the Vinca site [81]. The other, in 1992 from
Kishi’s group at Harvard (funded by NCI), presented a synthetic route to
halichondrin B [82]. As a result of the first of these reports and other work at
NCI, the NCI’s Decision Network Committee decided in early 1992 to recommend
further preclinical development of halichondrin B.
Although recommended for development, there was no natural source of the
material reported to that time that had the potential to provide enough material for
even initial preclinical studies. Thus, the NCI’s Natural Products Branch (NPB) put
out a general request to the natural products worldwide community asking for
potential sources. The New Zealand groups at the University of Canterbury
(Munro and Blunt) and the New Zealand Government’s National Institute of
Water and Atmospheric Research (Battershill) responded with information that
had not been published on the presence of halichondrins in a deepwater sponge
identified as a species of Lissodendoryx that had been collected by dredging off the
Kaikoura peninsula at a depth of �100 m.
The NCI first funded an environmental assessment of the extent of the sponge
field and using this data, were able to obtain a permit from the New Zealand
government that permitted up to 1 tonnes of sponge to be dredged, extracted,
and processed in a joint operation between the New Zealand collaborators and
the NCI. Direct funding was provided by the NCI with (predominately) payment in
kind by the New Zealand operations. The raw materials were harvested over the
next 4 years and processed in New Zealand to yield 300 mg of 98% pure
halichondrin B.
Contemporaneously, NCI financed a series of significant in-sea aquaculture
experiments in New Zealand waters that demonstrated for the first time that
1298 D.J. Newman and G.M. Cragg
a deepwater sponge could be cultured in shallow water and still produce
a significant amount of the desired secondary metabolite. The initial reports were
given in abstract format, covering the experiments in mussel farms in South Island,
New Zealand at depths up to 30 m and also in waters as shallow as 10 m in
Wellington harbor. These results were then used as the basis for a larger series of
experiments, and the results were published as a case study by the New Zealand
scientists who were directly involved [83].
By the end of 1997, NCI had samples from a very significant supply of pure
halichondrin B and used it to compare activities of the pure halichondrin B versus
materials that had only been isolated in very small amounts (3–5 mg) from other
sources. These were reported in abstract format at the American Association for
Cancer Research (AACR). Thus, the stage was set to use the sponge-derived
materials to perform early preclinical studies and optimistically, to be able to
produce material via in-sea aquaculture in order to provide material for later
development [83].
26.5.2 Synthetic Production of Halichondrin B and Derivatives
In 1992, at the same time as the decision by NCI to develop halichondrin B, Kishi’s
group at Harvard reported the total synthesis of halichondrin B [82], a study funded
via an NCI grant. Because of the relationship between the Eisai Research Institute
(ERI) in the USA and Professor Kishi, this organization investigated the potential of
the Kishi synthesis, having licensed the essential patent from Harvard [84].
Kishi’s group had shown, in patent form, that the required portion of the
molecule for maintenance of bioactivity was the macrolide ring, whereas other
groups had been modifying the “tail” part of the molecule. The Eisai chemical
group realized that they could convert the macrolide ring into a ketone by changing
the oxygen to a methylene group while still maintaining the basic activity. In
addition, truncation of the “tail” and changes to the oxygen heterocycle next to
the macrocyclic ring led to two very similar compounds E7389 and E7390.
Fortuitously, the head of ERI’s oncology group happened to see the work that
NCI’s Developmental Therapeutics Program (DTP) scientists reported in abstract
form on the in vitro activity of pure halichondrin B at the 1998 AACRmeeting. This
led to a collaborative project between ERI/Kishi and DTP, comparing the NCI’s
pure halichondrin B with two truncated, fully synthetic molecules that they had
made, E7389 (10) and E7390 (11). This preliminary work demonstrated that E7389
was more active in vivo and with a better therapeutic index than either halichondrin
B or its chemical precursor, E7390. ERI then provided sufficient E7389 synthesized
under cGMP conditions that permitted NCI to perform preclinical studies including
INDA-directed toxicology and led to approval in 2001 by the then NCI Decision
Network Committee for human clinical trials.
The compound was approved by the NCI for clinical development in 2001,
entered clinical trials soon thereafter and as of January 2010, was in phase III
clinical trials against refractory breast carcinoma under the Eisai Metastatic Breast
26 Meeting the Supply Needs of Marine Natural Products 1299
Cancer Study Assessing Physician’s Choice versus E-7389 (the EMBRACE stud-
ies, ClinicalTrials.gov Identifier NCT00388726). In addition to the global studies
under the EMBRACE consortium, eribulin is in other trials at the phase II level in
different carcinomas. Applications were made to both Switzerland and Singapore
for marketing approval in 2009 on the preliminary results from the global studies
and were followed in early 2010 by simultaneous submissions to the US FDA, the
EU’s EMEA, and the Japanese Ministry of Health, Labor and Welfare (MHLW) for
approval for the treatment of inoperable or recurrent breast cancer. As of mid 2010,
the FDA and the MHLW have given priority review status to these submissions.
A short paper presenting the basic chemistry was published by the ERI group in
2004 [85], whereas a thorough discussion of the details of the synthetic and
base biological information was published by the leaders of the studies at ERI
in 2005 [86]. In 2007, a short article covering the basic details but with later clinical
citations was published by Wang [87], and recently Kishi’s group have published
novel and simpler routes to the macrocyclic ring of halichondrin and eribulin [88, 89].
Themolecule, like its parent, is a tubulin-interactive agent with very potent activity
at the nanomolar level in in vitro studies [90] and binding at or close to the Vinca sitefrom modeling studies [91]. In a recent paper from Jordan’s group, the mechanism of
action is defined as: “The strong correlation between suppression of kinetochore-
microtubule dynamics and mitotic arrest indicates that the primary mechanism by
which eribulin blocks mitosis is suppression of spindle microtubule dynamics” [92].
26.5.3 Lessons from Case Study 4
This is an example of where the base molecule was the initial active agent, and
a source had to be identified with the political agreements necessary for collections
of the deepwater sponge negotiated at government to government levels. Once
permission had been obtained, then large-scale collection and extraction processes
had to be developed. At the same time, methods of large-scale production had to
be considered, even before the first large-scale deepwater collection had been made,
because if the compound was successful, multiple grams of material would be
required. Thus, aquaculture methods ran in concert with the large-scale recovery
and purification.
NCI had funded, via a competitive grant mechanism quite distinct from the
NPB-directed work, a total synthesis of the base halichondrin B molecule which
was reported at the same time as the initial decision to go forward with the
compound in preclinical studies at NCI. The synthesis was not judged by DTP to
be feasible under their restricted chemical synthetic capabilities as a substitute for
the natural product. What was not known by DTP/NCI at the time was the
relationship between Professor Kishi and the ERI. ERI licensed the Harvard patents
that demonstrated the viability of the truncated halichondrins (realized from bio-
logical assays of the intermediates in the synthesis at ERI).
Once DTP reported their in vitro work with the pure halichondrin B at the 1998
AACR meeting, discussions were held between the ERI scientists, Prof. Kishi, and
1300 D.J. Newman and G.M. Cragg
DTP in the late summer of 1998, at which time the two compounds ultimately
known as E7389 and E7390 were tested by DTP biologists against a series of cell
lines in vitro and in vivo. The compounds tested were the last samples that ERI had
as their synthetic tour de force (over 200 variations had been synthesized at ERI)
was winding down. When the DTP data was reported to ERI, they began a synthesis
of the most active compound with the highest therapeutic index and produced
enough cGMP grade E7389 by total synthesis for NCI to proceed with the necessary
pre-IND directed studies in toxicology, culminating in the entry into phase I clinical
trials in early 2002, 10 years after the Decision Network Committee had approved
further development of the natural product.
Thus, this particular example demonstrates how modern synthetic chemistry can
build extremely complex molecules that have clinical potential, based upon the
natural product structure, and in quantities sufficient for clinical use. However, it
must not be forgotten that eribulin or its congeners would not have entered clinical
trials in the absence of the massive effort across the globe to obtain enough of the
natural product to use as both a standard and as a comparator. Another beneficial
spin-off was the knowledge gained in how to aquaculture deepwater sponges in
shallow water and maintain metabolite production.
26.6 Case Study 5: Ecteinascidin 743 (Yondelis®)
26.6.1 Ecteinascidin 743 from Natural Sources (Wild Collections andAquaculture)
The antitumor activity of extracts of Ecteinascidia turbinata was first reported in
1969 [93], and the isolation and structure of ET743 was independently reported by
the groups of Wright [94] and Rinehart [95] in back to back papers in 1990.
Evaluation of the structures showed that they contained the essential aspects of
the saframycins, known terrestrial microbial tetrahydroisoquinoline antitumor anti-
biotics, and that there were other marine natural products of a similar basic structure
in the literature. For further information on this general class of molecules, the 2002
review by Scott and Williams should be consulted [96].
ET743 (12) was licensed in the very early 1990s to PharmaMar for development,
and over the next 10 or so years, a whole variety of methods were utilized in order
to obtain enough biomass to isolate the compound of interest. These included wild
collections in the Caribbean where three production periods were usually seen [97],
and the Mediterranean sea where temporal variation due to temperature meant that
only in the summer could the Mediterranean stocks be sampled [98, 99], as well
as in-sea and lake-based aquaculture [100]. Using multiple sites in addition to
major operations in the Spanish Balearic island Formentera and surrounding
areas, PharmaMar in the years between 1998 and 2004 produced approximately
100 tonnes of biomass, from which enough material was purified to carry preclin-
ical and clinical trials to phase II [101]. The overall approximate yield to purifica-
tion was �1.0 mg.g�1, or 100 g from 100 metric tonnes.
26 Meeting the Supply Needs of Marine Natural Products 1301
26.6.2 Ecteinascidin 743 from Chemical Synthesis
In order to provide enough material for advanced clinical trials and then commer-
cial production, it became apparent that none of the then-current methods would
suffice. Although Corey et al. had reported an enantioselective synthesis inspired by
the proposed biosynthesis [102] of the natural product in 1996 [103], it was not
amenable to large-scale production. Martinez and Corey then published a revised
synthetic method that was designed to increase yields significantly, but it was still
not adequate for industrial-scale production [104]. However, it did demonstrate
a method of production of a simpler analogue named phthalascidin (13) that hadsimilar activity to ET-743 [105].
Since these methods were not satisfactory for multigram production under
cGMP conditions, the PharmaMar group published [106] a semisynthetic method
starting with a microbial product, cyanosafracin B (14), produced by large-scale
fermentation of Pseudomonas fluorescens [107, 108]. The capabilities of this
particular method were demonstrated by the preparation of a large number of
natural ecteinascidins [109], together with a process that also permitted production
of “unnatural” analogues [101]. A more detailed discussion of the developments
through early 2005 was reported by Henriquez et al. in 2005 [4] and extended
recently by Cuevas and Francesch in an excellent review in 2009 [101].
Et743 was approved by the EMEA in September 2007 for treatment of sarcoma
and was launched in Sweden, Germany, and the UK by the end of 2007, thus
becoming the first marine natural product used as an antitumor drug. Currently,
mid-2010, there are 25 clinical trials listed in the NIH clinical trials database, with
three recruiting at the phase III level, and recently it was approved in the EU for
ovarian carcinoma treatment in conjunction with liposomal doxorubicin.
26.6.3 Lessons from Case Study 5
Just as in the case of bryostatin, enough material was obtained from a combination
of wild collections and very extensive aquaculture in a number of in-sea and on-
land operations to produce enough material for preclinical and clinical trials up
through some early phase II trials. Although academic groups had produced
enantiomeric syntheses of both the natural product and an active derivative that
was similar in concept to the bryologs, it was realized that these were not amenable
to the large-scale production necessary for further clinical trials and hoped for
commercialization.
PharmaMar then decided to use a semisynthetic method based upon modification
of a known microbial metabolite, cyanosafracin B, which could be obtained by
fermentation on a very large scale, and thus a low overall yield could be tolerated
[110]. This method was optimized and modified permitting the synthesis of unnat-
ural derivatives by a relatively simple modification of one of the synthetic steps
[101]. Thus, the methods developed were designed to increase the number of
potential derivatives to “expand the franchise” where or when necessary.
1302 D.J. Newman and G.M. Cragg
26.7 Case Study 6: Salinosporamide A
In this particular case, the paradigm is quite different, in that rather than wild
collections, syntheses and other methods of large-scale production, including
identification of the actual rather than the potential producer, the producing organ-
ism was known at the beginning of the work as a prokaryote, in this case a marine-
sourced actinomycete. Though we will use the same format in the previous five case
studies, this one will be a simpler story as it has taken only 3–4 years from
recognition of the molecule and its potential mechanism of action (MOA) to
a first use in human phase I clinical trial.
26.7.1 Discovery of Salinosporamide A
In the late 1980s, Fenical and Jensen at Scripps Institution of Oceanography began
their systematic investigation of the microbial diversity of marine habitats and its
potential relationship to marine natural products. In late 1989, the SIO group
investigated the distribution of actinomycetes in near-shore sediments in the Baha-
mas and discovered a number of organisms that required seawater for growth, and
that the majority of such organisms were possibly new genera within the family
Micromonosporaceae [111]. Although some cursory inspection of the secondary
metabolites was performed at that time, no further work was done with these
samples until in 1999, a reassessment of their phylogeny at the molecular level
suggested that these were a new genus, originally named Salinospora within the
Micromonsporaceae [112]. Further work confirmed this postulate and the genus
was renamed Salinispora [113].
A reinvestigation of the secondary metabolite profile of these organisms led
rapidly to an organism known as CNB-440, which when fermented on a 20-L
scale, yielded a very potent cytotoxin named as salinosporamide A (15), whosestructure was reminiscent of omuralide (16) but with significant variations,
including a chlorine substituent that was required for the activity [114]. The
final confirmation of the structure and proof of its MOA as a proteasome inhibitor
was obtained via X-ray crystallographic techniques as described in detail by
Groll et al. [115].
26.7.2 Large-Scale Fermentation/Isolation of cGMP Product
Salinosporamide A and other variants were licensed to San Diego Nereus Pharma-
ceuticals for further development as a cytotoxin. Nereus scientists, in concert with the
now defunct fermentation group at Industrial Research Limited (IRL) in Lower Hutt,
North Island, New Zealand, were able to produce the necessary cGMP product for
clinical trials by fermentation in a saline environment, the first time that this task had
been successfully performed on any significant (1,000 L) scale with a marine-sourced
microbe. This work included the proof required for the production of a Drug Master
26 Meeting the Supply Needs of Marine Natural Products 1303
File (DMF), that such fermentation processes could be performed in a saline/alkali
metal ion environment. The reason for concern was that such media would “pit”
stainless steel fermentation vessels and, therefore, would need quite different
cleaning/validation techniques in order to maintain the FDA requirements for
cGMP production of the active pharmaceutical intermediates (APIs). This was
successful, and the material obtained was satisfactory for advanced toxicology and
then clinical trials in man. A series of reports on the fermentation media and on some
of the other metabolites produced during the large scale isolation processes have
begun to appear in print in the last few years [116–120], and fermentation modifica-
tions demonstrating that differing formulations with lower sodium ion concentration
[121] or substitution by sodium sulfate can still maintain production but reduce
corrosiveness of the medium [122], and in a later paper the same year, the same
authors demonstrated that the ionic strength is a major determinant in growth and
production [123]. In addition, an excellent discussion of the entire salinosporamide
A story was published by Fenical et al. in 2009 [124].
26.7.3 Synthetic Processes
Even though the supply of material for clinical trials is from fermentation
(a replacement for IRL was identified by Nereus in Eastern Canada so the supply
of material was assured), chemists rose to the challenge and a number of groups
including Nereus chemists have published total syntheses of salinosporamide A and
analogues [125–134], thus demonstrating if one has a novel structure, chemists
will devise syntheses for it as rapidly as possible. As mentioned earlier, the method
of production is fermentation for the clinical candidate, but it is possible that
derivative production may utilize some form of semisynthesis via the chemistries
described above in due course.
26.7.4 Genomic Methods
Although some of the possibilities for genomic modifications/production were
alluded to in the discussions on bryostatin (Sect. 26.3, Case 2) and dolastatin
(Sect. 26.4, Case 3) where the producing microbe has been “identified,”
salinosporamide, in addition to all of its other “firsts,” is the first antitumor agent
whose biosynthetic pathway has been described and worked on contemporaneously
with the late preclinical and early clinical studies. The genomic sequence of the
producing actinomycete was investigated and 17 (plus) genomic clusters, one of
which was the salinosporamide cluster, were reported by the SIO group of Moore in
collaboration with the Fenical/Jensen group in 2007 [135]. The impact of this
information from just the biosynthetic control of salinosporamide microbial syn-
thesis can be seen by the papers that have been published using this basic informa-
tion in the last 2 or so years. Thus, the biosynthetic route to the very unusual
chlorinase that is essential for salinosporamide A’s activity has been reported [136]
1304 D.J. Newman and G.M. Cragg
together with methods of extending the polyketide synthons to give “unnatural
derivatives” [137, 138] and a discussion on more general aspects of bioengineering
from the organism itself [139, 140].
26.7.5 Lessons from Case Study 6
This example is quite different from the previous five in that the time frame has
been very significantly compressed, from 18 to 20 plus years to less than 3 from
realization that the microbes were a new marine-sourced genus, to identification of
the metabolites and their potential. Thus, there was no large-scale method necessary
except for development of fermentation processes to proceed with this compound.
It should also be realized that the fermentation of marine-based microbes had never
been attempted in regular fermentation systems prior to this work, let alone produce
cGMP quality materials from such a process.
What also needs to be borne in mind is that there have been massive advances in
genomic analyses in the last 5 plus years with the first actinomycete genomic
sequence being published in 2002. The rapid development of recognition methods
by earlier workers for defining the biosynthetic potential of gene clusters enabled
the SIO discoverers and their collaborators in Nereus Pharma and SIO, in particular
Moore’s group, to be able to use chemistry, knowledge of biosynthetic processes
and the fermentation capabilities of Nereus to explore the producing microbe(s)
extremely rapidly.
It is obvious from this example that the essential factors, aside from timing, are
the dramatic interplay of scientists from many areas of chemistry and biology.
Thus, a true multidisciplinary effort is absolutely essential for success.
26.8 In Conclusion
The examples used here for teaching purposes were chosen to show how scientists
had to adapt to what was feasible rather than what might make sense using the
knowledge of today. Thus, except for case study 6, a wide variety of collection and
production methods were used as the time and funds permitted with recognition that
at times, chemical synthesis was the only feasible route. Thus, the dolastatins were
the product of total synthesis, as was aplidine. Initially, PharmaMar had developed
a series of productive aquacultural methods that were used to produce Yondelis®
for some of the earlier clinical trials and then moved to semisynthesis from
a microbial secondary metabolite for large-scale clinical production.
With eribulin, Eisai used a modification of the natural product, halichondrin B,
that relied upon an academic exercise in total synthesis of the natural product,
during which the active pharmacophore was identified, although published only in
a patent. It required a head-to-head comparison with the wild-collected natural
product by NCI before Eisai was willing to commit to full scale synthetic
production.
26 Meeting the Supply Needs of Marine Natural Products 1305
In case study 6, we have shown how the dramatic scientific advances in geno-
mics and the influence of these techniques coalesced around a marine microbial
metabolite that is currently the fastest natural product agent (and maybe even the
fastest compound irrespective source) to go from discovery to clinical trials in
under 4 years.
Thus, in the second decade of the twenty-first century, once an “active” agent
has been identified as a potential drug lead, all methods for obtaining large-scale
production are “on the table” for discussion and if/when the necessity arises for the
production of significant amounts, the examples given above show what is currently
available and investigators will mix and match as necessary, now that the
pioneering work has been performed and evaluated. However, one can predict
that new, currently unimagined technologies will come into being, and assist in
the ever problematic issue of “meeting the supply needs of marine natural
products.”
26.9 Study Questions
1. You have discovered a novel agent from a mixture of marine invertebrates.
How would you determine if it was the product of an individual invertebrate?
If it is, what are your next obvious steps? If it is not, what could be your best
options?
2. You think that you have a small peptide as your active agent. What could be the
various methodologies that could be used to produce larger quantities of the
material in question, and why?
3. You have an active but quite toxic agent that you want to consider as a potential
drug. How might you deliver this agent and why might one method be superior
to another?
4. You have a very potent but complex structure as your discovery. What possible
method(s) could you use to optimize the discovery for investigation as
a potential drug?
5. You think that you have the producing microbe identified. How would you
utilize current knowledge to prove that this is the case?
Acknowledgments The opinions expressed in this chapter are those of the authors, not neces-
sarily those of the US Government.
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