handbook of marine natural products volume 426 || meeting the supply needs of marine natural...

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Meeting the Supply Needs of Marine Natural Products 26 David 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

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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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