The influence of natural products upon drug discov

David J. Newman,*a† Gordon M. Cragg a and Kenneth M. Snader b

a Natural Products Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville MD 20852, USA

b Pharmaceutical Resources Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville MD 20852, USA

Received (in Cambridge) 20th December 1999

Covering: Antiquity to late 1999.

1Introduction

2General role of traditional medicine in drug

discovery

3Role of natural products in treatment of diseases

4Antiinfective agents (including antimalarials)

4.1Antibacterials: b-lactams

4.2Antibacterials: aminoglycosides

4.3Antibacterials: tetracyclines

4.4Antibacterials: macrolides

4.5Antibacterials: synergistic mixtures of

streptogramins

4.6Antifungals: general

4.7Antifungals: lipopeptides

4.8Antifungals: non-lipopeptides

4.9Antimalarials

4.10Antiviral area: general

4.11Antiviral area: nucleoside analogues

4.12Antiviral area: HIV protease inhibitors

5Cardiovascular: general

5.1Cardiovascular: the b-adrenergic amines

5.2Cardiovascular: cholesterol lowering agents

5.3Cardiovascular: angiotensin converting enzyme

inhibitors (ACE inhibitors)

6Pain/central nervous system: history

6.1Pain/central nervous system: the opiates

6.2Pain/central nervous system: the conotoxins

6.3Pain/central nervous system: the epibatidines

7Antineoplastics: general

7.1Antineoplastics: plant sources

7.2Antineoplastics: microbial sources

7.3Antineoplastics: marine sources

8Future trends: general

8.1Future trends: production of ‘previously unknown

structures’

8.2Future trends: the optimization of lead structures 8.3Future trends: novel delivery systems for ‘old

compounds’

9References

1Introduction

Throughout the ages humans have relied on nature for their basic needs for the production of foodstuffs, shelters, clothing, means of transportation, fertilizers, flavors and fragrances, and, not least, medicines. Plants have formed the basis of sophisti-cated traditional medicine systems that have been in existence for thousands of years.1The first records, written on hundreds of clay tablets in cuneiform, are from Mesopotamia and date from about 2600 BC; amongst the approximately 1000 plant derived substances which they used were oils of Cedrus species (cedar) and Cupressus sempevirens(cypress), Glycyrrhiza glabra(licorice), Commiphora species (myrrh), and Papaver somniferum(poppy juice), all of which are still in use today for the treatment of ailments ranging from coughs and colds to parasitic infections and inflammation.

Egyptian medicine dates from about 2900 BC, but the best known Egyptian pharmaceutical record is the “Ebers Papyrus”dating from 1500 BC; this documents over 700 drugs (mostly plants, though animal organs were included together with some minerals), and includes formulae such as gargles, snuffs, poultices, infusions, pills and ointments, with beer, milk, wine and honey being commonly used as vehicles.

The Chinese Materia Medica has been extensively docu-mented over the centuries, with the first record dating from about 1100 BC(Wu Shi Er Bing Fang, containing 52 prescriptions), followed by works such as the Shennong Herbal (~100 BC; 365 drugs) and the Tang Herbal (659 AD; 850 drugs). Likewise, documentation of the Indian Ayurvedic system dates from about 1000 BC(Charaka; Sushruta and Samhitas with 341 and 516 drugs respectively), and this system formed the basis for the primary text of Tibetan medicine, Gyu-zhi (Four Tantras) translated from Sanskrit during the eighth century AD.2,3

In the ancient Western world, the Greeks contributed substantially to the rational development of the use of herbal drugs. The philosopher and natural scientist, Theophrastus (~300 BC), in his ‘History of Plants’, dealt with the medicinal qualities of herbs, and noted the ability to change their characteristics through cultivation. Dioscorides, a Greek physi-cian (100 AD), during his travels with Roman armies throughout the then ‘known world’, accurately recorded the collection, storage, and use of medicinal herbs, and is considered by many to be the most important representative of the science of herbal drugs in ‘ancient times’. Galen (130–200 AD), who practiced and taught pharmacy and medicine in Rome, and published no less than 30 books on these subjects, is well known for his complex prescriptions and formulae used in compounding drugs. These were based on the Hippocratic theory that all illnesses were based on an imbalance of four primary ‘humours’and were extremely complex at times containing dozens of ingredients (the so-called ‘galenicals’).

During the Dark and Middle Ages, from the fifth to the twelfth centuries, the monasteries in countries such as England, Ireland, France and Germany preserved the remnants of this Western knowledge. However, it was the Arabs who were

†Address for correspondence: Natural Products Branch,NCI, PO Box B,

Frederick, MD, 21702, USA. E-mail: dn22a@nih.gov

This journal is © The Royal Society of Chemistry 2000MILLENNIUM REVIEW

DOI: 10.1039/a902202c Nat. Prod. Rep., 2000, 17, 215–234215

responsible for the preservation of much of the Greco-Roman expertise, and for expanding it to include the use of their own resources, together with Chinese and Indian herbs unknown to the Greco-Roman world. The Arabs were the first to establish privately owned drug stores in the eighth century, and the Persian pharmacist, physician, philosopher and poet Avicenna contributed much to the sciences of pharmacy and medicine through works, such as Canon Medicinae , regarded as ‘the final codification of all Greco-Roman medicine ’. This was subse-quently superceded by the comprehensive compilation known as the Corpus of Simples by Ibn al-Baitar who practiced in Malaga during the Moorish occupation of Spain. This document combined the data of Dioscorides with works from the Middle and Far East.

These, and many other works, were formally codified at least in the UK by the publication in 1618 of the London Pharmacopoeia and the idea of ‘pure ’ compounds as drugs may be traced to the isolation of the active principles of commonly used plants and herbs such as strychnine, morphine, atropine and colchicine in the early 1800s. These isolations were then followed by what can be considered the first commercial pure natural product, morphine, by E. Merck in 1826 and the first semi-synthetic pure drug based on a natural product, aspirin, by Bayer in 19.4

David Newman was born in Grays, Essex, UK. His initial training was as an analyst (GRIC) followed by an MSc in Organic Chemistry (University of Liverpool) and then after some time in the UK chemical industry, a DPhil in Microbial Chemistry from the University of Sussex in 1968. Following two years of postdoctoral studies on the structure of electron transport proteins at the University of Georgia, USA, he worked with Smith Kline and French in Philadelphia, PA, as a biological chemist predominantly in the area of antibiotic discovery. During this time period, he obtained an MLS in Information Sciences in 1977 from Drexel University, Phil-adelphia, PA. Following the discontinuance of antibiotic discovery programs at SKF, he worked for a number of US-based pharmaceutical companies in natural-products based discovery programs in anti-infectives and cancer treatments,and joined the Natural Products Branch of the NCI in 1991. He is responsible for the marine and microbial collection programs of the NCI and in concert with Gordon Cragg, for the NCI’s Open and Active Repository programs. His scientific interests are in the discovery and history of novel natural products as drug leads in the anti-infective and cancer areas, in novel delivery methods for such agents and in the application of information technologies to drug discovery. In conjunction with Gordon Cragg, he has established collaborations between the National Cancer Institute and organizations in many countries promoting drug discovery from their natural resources. He has published over 50 papers and patents that are related to these interests and is both an UK Chartered Chemist and an UK Chartered Biologist.

Gordon Cragg was born in Cape Town, South Africa, and obtained his undergraduate training in Chemistry at Rhodes University before proceeding to Oxford University where he obtained his DPhil in Organic Chemistry in 1963. After two years of postdoctoral research in natural products chemistry at the University of California, Los Angeles, he returned to South Africa to join the Council for Scientific and Industrial Research.In 1966, he was appointed to the staff of the Department of

Chemistry at the University of South Africa, and transferred to the University of Cape Town in 1972. In 1979, he returned to the United States to join the Cancer Research Institute at Arizona State University, working with Professor G. Robert Pettit on the isolation of potential anticancer agents from plant and marine invertebrate sources. In 1985, he moved to the National Cancer Institute in Bethesda, Maryland, and was appointed Chief of the Natural Products Branch in 19. His major interests lie in the discovery of novel natural product agents for the treatment of cancer and AIDS. In 1991 he was awarded the National Institutes of Health Merit Award for his contributions to the development of the drug, Taxol ®, and in 1998 he was elected President of the American Society of Pharmacognosy. He has established collaborations between the National Cancer In-stitute and organizations in many countries promoting drug discovery from their natural resources. He has published over 100 papers related to these interests.

Kenneth Snader was born in Harrisburg, PA, USA. He obtained his undergraduate training at the Philadelphia College of Pharmacy and Science and after some years in the pharmaceu-tical industry returned to obtain his PhD in Organic Chemistry from the Massachusetts Institute of Technology as a Walter G.Karr Fellow. He went directly to Smith Kline and French Laboratories where, after a brief period in the study of anti-inflammatory compounds he returned to natural products to oversee the antibiotics discovery chemistry group. Following the discontinuance of the antibiotic discovery program at SKF,he briefly supervised the marine natural products chemistry group at SeaPharm before he joined the Natural Products Branch of the National Cancer Institute in 1987. After a significant effort at producing enough of the drug Taxol ®to complete the clinical trials of that natural product, for which he was awarded the National Institutes of Health Merit Award, he moved from the Natural Products Branch to the Pharmaceutical Resources Branch of the NCI, taking on his current responsibil-ities for production of GMP bulk drugs for NCI clinical trials.His scientific interests are in the discovery, identification, and

large scale production of natu-ral products for medicinal use and together with Gordon Cragg and members of the Nat-ural Products Branch has pro moted and encouraged the dis-covery of new chemotherapeu-tic agents from natural sources.He has published over 50 pa-pers and patents related to these interests.

David J. Newman Gordon M. Cragg Kenneth M. Snader

216

Nat. Prod. Rep., 2000, 17, 215–234

2General role of traditional medicine in drug discovery

As mentioned above, plants have formed the basis for traditional medicine systems which have been used for thousands of years in countries such as China 5and India.2,6The use of plants in the traditional medicine systems of many other cultures has been extensively documented.7These plant-based systems continue to play an essential role in health care, and it has been estimated by the World Health Organization that approximately 80% of the world ’s inhabitants rely mainly on traditional medicines for their primary health care.8,9

Plant products also play an important role in the health care systems of the remaining 20% of the population, mainly residing in developed countries. Analysis of data on prescrip-tions dispensed from community pharmacies in the United States from 1959 to 1980 indicated that about 25% contained plant extracts or active principles derived from higher plants.Currently, at least 119 chemical substances, derived from 90plant species, can be considered as important drugs that are in use in one or more countries.8Of these 119 drugs, 74% were discovered as a result of chemical studies directed at the isolation of the active substances from plants used in traditional medicine.

A more recent study using US-based prescription data from 1993, demonstrated that natural products still play a major role in drug treatment, as over 50% of the most-prescribed drugs in the US had a natural product either as the drug, or as a ‘forebear ’in the synthesis or design of the agent.10

3Role of natural products in treatment of diseases

This review will cover the following general areas of disease:microbial and some parasitic areas; neoplastic; cardiovascular and hypertension; pain/CNS and will be discussed from the aspect of the disease rather than the more customary division by source of organism. This approach was chosen because of the cross-over that is now being seen where agents originally isolated and purified as a result of their ability to inhibit one type of screen are now being utilized in other entirely different areas.The review will finish with a short section on ‘Future Trends ’.

Perhaps the best example of this cross-over is the work over the last 30 or so years on the microbial product, rapamycin.Originally isolated as an antifungal agent, it has just been approved as an immunosuppressive drug and is also being tested (as a derivative) as an anti-neoplastic agent. Thus the use of a disease area permits agents from all sources to be discussed in a group.

For earlier coverage of the role of natural products in drug discovery, the excellent 1995 review by Cordell should be consulted.11

4Antiinfective agents (including antimalarials)

We have not attempted to make the following discussion comprehensive in its coverage of the various anti-infective classes. The guiding principle has been to show areas where the natural product has led to ‘improved materials by semi-synthetic modifications of a base molecule that a medicinal chemist would not even dream of making ’. Thus the glycopep-tides such as vancomycin, or the simple but very effective agent,chloramphenicol, are not further considered.

Although there were anecdotal stories of the use of poultices of rotten bread on puncture or slash wounds from the Roman wars, and from Arab medicinal treatments used during the many conflicts that the Western ‘navigators ’ had with the Arabs from the fifteenth century onwards, no systematic method of treatment appeared in the literature for suppurating wounds caused by bacterial infections until Lister used P. glaucum for

“curing ” a large non-healing abscess in 1884 (cf . p. 124 in Mann 12).4.1

Antibacterials: b -lactams

The ‘Golden Age of Antibiotics ’, as the time period from the 1940s to the 1970s has been christened, can be considered to have its beginning in the serendipitous discovery of penicillin by Fleming in 1928, reported in the British Medical Literature in 1929. The history of the discovery of penicillin has been told and retold and we do not intend to go over this story in detail again. An excellent reprint has just been issued of the ‘History of Penicillin Production ’13and that publication goes into the story in detail from the aspect of large scale production of penicillin G (1) and penicillin V (2). It is rather ironic that the

major impetus to the development and production of the initial b -lactams was World War II and the requirement for penicillin to be available for D-Day. In the application of the resources of the UK and the US to the problem, coupled to its importance, it can rightly be considered to be the microbiological equivalent of the Manhattan Project.

The base structure of the penicillin molecule was new to science when it was elucidated and since it acted by a mechanism that targeted a structure unique to the bacterial cell wall, it immediately became a structure ‘ripe for semi-synthesis ’. With the almost immediate realization that ‘mi-crobes were smarter than man ’, as they produced protective enzymes, penicillinases or b -lactamases that degraded the penicillins by opening the b -lactam ring, came the requirement to produce/discover other, non-lactam antibiotics, and/or chem-ically modify the basic penicillin nucleus to reduce or eliminate the enzymic activity.

Almost all of the penicillins that have been made have started from the simple fermentation-derived product 6-amino-pen-icillanic acid (6-APA, 3), which is produced by a simple

chemical or biochemical deacetylation from penicillin G itself.The actual number of 6APA-based structures that have been made and tested by the pharmaceutical industry and associated academic laboratories is unknown, as substantial numbers of inactive materials or compounds with only minor increases in potency were not reported in the literature, but it was well in excess of 10000 in the late 1970s.14

In 1948, Brotzu 15reported on the ring-expanded molecule,cephalosporin C (4), which was discovered from an isolate of a ‘pseudo ’-marine fungus, Cephalosporium acremonium , taken from a sewer outfall in Sardinia. Some of the same Oxford group that had originally worked on penicillin G, and who had seen but not isolated and purified the material at roughly the

Nat. Prod. Rep., 2000, 17, 215–234

217

same time as Brotzu, now isolated a weakly active agent from the fungal extract and determined the structure of cephalosporin C.16,17Although very weakly antibacterial, it did not appear to be susceptible to the then known b -lactamases and this finding,coupled to the demonstration by chemists at Lilly that penicillin G could be chemically expanded to give the cephalosporin C nucleus, led to the same type of semi-synthetic effort in producing modified cephalosporins as had been seen in the case of the penicillins. An example is the first orally-available cephalosporin, cephalexin (5). Originally, cephalosporins were used to treat infections resistant to penicillins, but with the advent of b -lactamases that were either specific for ceph-alosporins, or attacked both ring systems, the search for more resistant analogues continued.

A large effort was mounted in the late 1960s and early 1970s,predominately by Beecham and Pfizer to find molecules that would have pharmacokinetic properties similar to those of penicillins but that would inhibit some or all of the common b -lactamases. Beecham were successful in producing the mole-cules known as clavulanates (6) from natural sources, and Pfizer produced the semi-synthetic molecules known as the sulbac-tams where the thiazole sulfur was oxidized to the sulfone 7. In

both cases, combination therapies were devised where mixtures,usually equimolar in nature, were administered using a common penicillin such as ampicillin or the more potent amoxicillin plus the b -lactamase inhibitor. To give an idea of the value of these patented combinations, in 1997 SKB sold over US$ 1.5 billion of Augmentin ®worldwide and the US patent does not expire until 2002.18As yet, a corresponding cephalosporin combina-tion has not been marketed, though from the late 1980s workers at Roche had reported on some interesting combination antibiotics where two different nuclei (b -lactams and quino-lones) were linked by esters,19carbamates 20or via a tertiary amine function 21and reported on their stability to three common Gram-negative b -lactamases,22showing that only the compounds that contained the cefotaxime structure were stable to two of the three enzymes used.

The search for a ‘simple b -lactam ’ (i.e., an isolated single ring) went on for many years, predominately in synthetic chemistry laboratories. Again, Mother Nature proved to be the superior chemist when in early 1981, Imada reported on the sulfazecins 238and, within two months, Sykes et al.at Squibb reported that after herculean efforts, involving over 1000000small-scale fermentations, they successfully isolated the mono-bactams including sulfazecin from aquatic organisms collected almost in their own ‘backyard ’.24This was an entertaining repetition of the search by the NRRL scientists for a high-producing P. chrysogenum strain that they eventually found on a rotting melon in a local fruit market in Peoria across from the laboratory.13With the identification of the naturally occurring monobactam compounds, it became ‘chemically obvious ’ why Nature was the best chemist. Of all the synthetic modifications made by medicinal chemists from Squibb, SmithKline and other pharmaceutical houses, none had considered that an N-sulfonic acid substituent would stabilize the b -lactam system. In a clever move, the Squibb researchers then proceeded to synthesize and patent all of the side-chain modifications that had been reported to give improved activities in the penicillin and cephalosporin series; an early compound in this series, aztreonam (Azactam ®,9) is currently used clinically.4.2

Antibacterials: aminoglycosides

Concomitantly with the search for the penicillins, Waksman at Rutgers University commenced a search for microbial metabo-lites that would be active against the then (and perhaps now)major scourge, tuberculosis. In 1943, the aminoglycoside antibiotic streptomycin was isolated from Streptomyces griseus and, in addition to being active against Mycobacterium tuberculosis , it was also active against a wide range of other bacterial infections. Further work, predominately by screening metabolites from soil microbes of the Actinomycetales , led to the identification and isolation of a large number of antibiotics of similar structural types known generically as the aminoglyco-sides. These materials are mainly used against Gram-negative organisms, but due to their mechanism of action (inhibition of protein synthesis), they also exhibit true synergy with pen-icillins in vivo . Their major drawback in clinical use is the fairly rapid build-up of resistance (to a large extent, plasmid-mediated phosphorylases and acetylases) and their innate oto- and nephro-toxicity, which means that treatments have to be very carefully regulated. There have been some successful chemical modifications to produce more resistant molecules (e.g.amika-cin, 10), but in most cases, the isolated metabolite is used as the drug.

4.3Antibacterials: tetracyclines

The next major class of metabolites to be discovered and used extensively were the tetracyclines. These were produced by various Streptomyces sp. and had the then unique skeleton of four linear fused rings. Although the parent molecule was not used to any great extent as an antibacterial, the chlorinated derivative, named Aureomycin ®(11) because of its color, was.

218

Nat. Prod. Rep., 2000, 17, 215–234

A large number of semi-synthetic tetracyclines have been made

and a significant number are still in use as first line therapy,

even though the parent molecule is almost fifty years old.

Amongst these are doxycycline (12) and minocycline (13).

Tetracycline has another ‘claim to fame’ in antibiotic therapy,

as it was this molecule that gave its name to the ‘tet resistance

pump’. In eubacteria, and in its homologous form in eukaryotes

(P130 glycoprotein), this pump gives rise to the phenomenon of

multidrug resistance (or MDR), where the toxic agent is

pumped out as fast as it is transported in (either by diffusion or

by an active ingress mechanism). Molecules based on the

tetracycline molecule have since been synthesized that will

block the tet pump, thus possibly permitting antibiotic therapy

with relatively cheap tetracyclines to be used again.25–27

4.4Antibacterials: macrolides

The other extremely important Streptomyces metabolites are the

14-membered macrolides exemplified by erythromycin (14).

There are relatively large numbers of compounds with various

sizes of macrolide rings that have been isolated as a result of

screening campaigns in the antibiotics industry, but, of all of

them, those based on the erythronolide ring system have been

the most used, even though they have to have complex salts

made in order to generate oral activity. In a fairly recent

development, a chemical modification has been made to the

basic structure to overcome the resistance exhibited by strains

from most of the important pathogens to the base molecule.

These are the ‘aza-macrolides’ exemplified by Azithromycin®

(15), where a nitrogen has been chemically inserted into the

base macrolide ring, overcoming most if not all of the resistance

shown to erythromycin.28The downside to such a molecule is

the expense of the synthetic process, thus pricing such

treatments beyond both developing and a number of developed

countries.

4.5Antibacterials: synergistic mixtures of

streptogramins

In the early days of antibiotic discovery, a series of synergistic

mixtures were isolated from Streptomyces sp. and identified as

being extremely potent agents against Gram-positive organ-

isms. However, they had a major drawback, they were quite

toxic in comparison to the tetracyclines and erythromycin. They

were, however, developed as animal feed supplements and used

extensively in the US and in Europe (virginiamycins A and B

being an example).

With the advent of epidemics of methicillin-resistant Staphy-

lococcus aureus(MRSA), and the potential for larger outbreaks

of the glycopeptide-resistant entero- and staphylococci, came

the realization that we are very close to not having any

antibiotics left that will kill such resistant organisms. To make

matters worse, most of the big antibiotic-discovery companies

had down-sized their operations in the mid-1980s to early 1990s

time frame. Rhone-Poulenc in France had had a ‘franchise’ on

the streptogramin Pristinamycin, and in a series of elegant semi-

syntheses, modified the ‘A’ and ‘B’ components to produce the

water-soluble compounds, dalfopristin (16) (RP54476) and

quinuprisitin (17) (RP57669). The synergistic mixture (70+30

ratio) has now been approved by the US FDA under the name of

Synercid®(RP59500) for use against methicillin-resistant

Staphylococcus aureus(MRSA), vancomycin resistant entero-

and staphylococci and drug-resistant S. pneumoniae.29–31

4.6Antifungals: general

Unlike the antibacterial arena where, in relative terms, bacterial-

specific targets abound, fungi, being eukaryotic in nature, have

a metabolism similar to those of mammals, with differences in

the t-RNA-AA-acyltransferases (only found from proteoge-

Nat. Prod. Rep., 2000, 17, 215–234219

nomics studies),32portions of their steroid synthetase systems

and their carbohydrate-based cell walls. As a result, there are

few clinically viable antifungal agents, with the well known

amphotericin B (aka‘ampho-terrible’ because of its side

effects) still being the gold standard against which other

antifungal agents are measured.

To date, no clinically effective antibiotic directed towards the

chitin synthetases has yet survived the testing process though a

lot of work was performed, starting in the late 1970s, in attempts

to chemically convert the peptidic antibiotics of the Nikkomy-

cin class into viable clinical candidates against C. albicans.

With these and similar agents, problems still arise with the

specificity of this organism’s peptide transport systems (cf.

McCarthy et al.33for a discussion of these processes), thus the

following discussions will be of agents directed against other

targets.

4.7Antifungals: lipopeptides

Though there have been many agents reported to have

antifungal activity in vitro, when they were tested in relevant

animal models, their ADME (absorption, distribution, metabo-

lism and excretion) characteristics were such that significant

chemistry had to be performed to obtain reasonable pharma-

cology.

Such operations were performed by Lilly on echinocandin in

order to produce cilofungin. This compound reached Phase II

clinical trials, and, following abandonment due to toxicity,

further modification of the structure produced LY303366,

culminating in the synthesis of its more soluble prodrug

LY307853 (18) by converting the phenolic hydroxy to a sodium

phosphate ester.34,35

Using a similar type of compound as the starting material,

pneumocandin A0, chemists at Merck have produced MK-0991

(19) which likewise demonstrates good activity against the

major human pathogen, C. albicans and other pathogenic fungi

from diverse genera.36

4.8Antifungals: non-lipopeptides

Using a high throughput screen directed against protein

synthesis in Candida, Glaxo-Wellcome discovered that an

analogue of the previously known molecule sordarin was an

effective in vitro inhibitor, with apparent selectivity for fungal

protein synthesis. Following a long program of mutation and

medicinal chemistry, semi-synthetic derivatives of sordarin

were produced with the analogue GM237354 (20) entering

preclinical development.37

4.9Antimalarials

The quintessential antimalarial lead was quinine, originally

isolated from Cinchona bark, acting as the template for the

synthetic agents of the chloroquine/mefloquine type. With the

rise of parasites resistant to these agents, came the search for

other synthetic and natural product-based agents. Inspection of

the data from Chinese herbal remedies led to the investigation of

extracts of Artemisia annua(Wormwood). This particular plant

had been used for centuries in China as an antimalarial38

(known as ‘Quinghaosu’) and in 1972, the active agent was

isolated and identified as a sesquiterpene endoperoxide named

artemisinin (21). In a recent review, Tan et al.39reported that

this particular agent is not limited to the species annua, but is

found in at least two others. Using the base structure of

artemisinin, many semi-synthetic compounds were made with

the aim of optimizing the pharmacology of the base molecule,

leading to the identification of artemether, 22, (a relatively

simple modification of the actual active metabolite, dihy-

droartemisinin, 23) as a potent antimalarial agent that is now in

widespread use throughout the world. Other simple modifica-

tions have been made to improve solubility and distribution but,

in all cases, the active constituent ends up to be the same

molecule, so these can all be considered to be ‘prodrugs’ of

dihydroartemisinin (cf. Table IV in De Smet).40

It has been suggested from many in vitro and in vivo studies

that the active principle of artemisinin resides in the peroxide

bridge and that an iron-catalyzed conversion occurs to give an

electrophilic free radical composed of the majority of the

molecule. This compound then acylates proteins that are present

in the parasite but not, to any extent, in the host.41–43With the

recognition that the peroxy bridge is essential for activity, came

the syntheses by many groups in both China and the West, of

compounds containing the basic artemisinin structure. The 220Nat. Prod. Rep., 2000, 17, 215–234

major group in the USA working on these compounds has been associated with the Walter Reed Army Institute of Research and the papers by Avery et al.should be consulted for details.44–46As time progressed, much simpler structures containing the required peroxy bridge were synthesized (e.g . 24, 25) and they retained nanomolar activity. Recent reviews of the work leading to such molecules have been published by Posner et al.47,48

Natural products from marine sources also contain the peroxy-bridge found in the artemisins, examples being the norsesterterpenes sigmosceptrellin 49(26) and muqubilin 50(27)from the Red Sea sponges Sigmosceptrella and Prianos . Other

similar molecules have also been reported by the Kashman group 51from a South African Plakortis . At that time, their potential as leads to antimalarials was not appreciated, though later work by Hamann’s group at the University of Mississippi 52and Konig’s group at Braunschwieg 53have shown that a variety of marine-derived structures may have potential as leads to other antimalarial structures. It will be interesting to see how these molecules act as leads versus those from plant sources.In a very recent paper, workers in Germany 54have demon-strated that a very simple series of microbial metabolites, the fosmidomycins (28), originally reported by workers at Fuji-

sawa 55,56as Streptomyces metabolites, have antimalarial activ-ity both in vitro and in vivo in rodent models. These simple molecules appear to inhibit isoprenoid biosynthesis that is dependent upon the DOXP pathway (from 1-deoxy-D -xylulose 5-phosphate) rather than the more usual HMG-CoA reductase route. This discovery of a non-mammalian isoprenoid bio-synthetic route in the parasite and the identification of an inhibitor may well open up an entirely new route to anti-malarials, particularly as synthetic routes to this type of molecule have been published in detail 57and they appear to lend themselves to parallel syntheses to produce a wide variety of congeners.4.10

Antiviral area: general

If one thinks that there is a paucity of effective antifungal agents, then the antiviral area is positively barren by compar-ison, in spite of the vast number of chemical compounds,derived from natural sources, semi- or total synthesis, that have been tested for their efficacy as antiviral agents. One major reason for this is the very nature of a viral disease, in that the

virus, irrespective of type, effectively takes over an infected cell and hence there are very few specific viral targets for small molecules to interact with. With the advent of molecular cloning techniques, however, the situation is changing as it is now possible to identify specific viral-related proteins, clone them,express them and then use in rapid screening systems, looking for specific interactions in the absence of the host proteins.Rather than deal with each type of viral infection, we have elected to show how a serendipitous discovery of a series of natural products has led to the plethora of similar compounds that are now in preclincal or clinical evaluation, and in some cases, in clinical use.4.11

Antiviral area: nucleoside analogues

From 1950 to 1956, Bergmann et al.58–60reported on two compounds that they had isolated from marine sponges,spongouridine (29) and spongothymidine (30). What was significant about these materials was that they demonstrated, for the first time, that naturally occurring nucleosides could be found using sugars other than ribose or deoxyribose.

These two compounds can be thought of as the prototypes of all of the modified nucleoside analogues made by chemists that have crossed the antiviral and anti-tumor stages since then.Once it was realized that biological systems would recognize the base and not pay too much attention to the sugar moiety,chemists began to substitute the ‘regular pentoses’ with acyclic entities, and with cyclic sugars with unusual substituents.

These experiments led to a vast number of derivatives that were tested extensively as antiviral and anti-tumor agents over the next thirty plus years. Suckling, in a 1991 review 61showed how such structures evolved in the (then) Wellcome laborato-ries, leading to AZT and, incidently, to Nobel Prizes for Hitchens and Elion, though no direct mention was made of the original arabinose-containing leads from natural sources.

Showing that ‘Mother Nature’ may follow chemists rather than the reverse, or conversely that it was always there but the natural products chemists were ‘slow off the mark’, arabinosy-ladenine (Ara-A or Vidarabine ®, 31) was synthesized in 1960 as a potential anti-tumor agent,62but was later produced by fermentation 63of S. griseus and isolated, together with spongouridine,from a Mediterranean gorgonian (Eunicella cavolini ) in 1984.

Of the many compounds derived from these early discov-eries, some, such as Ara-A, Ara-C, Acyclovir (32) and later

Nat. Prod. Rep., 2000, 17, 215–234

221

AZT and DDI, have gone into clinical use, but most have simply

become entries in chemical catalogues.

4.12Antiviral area: HIV protease inhibitors

As mentioned above, there are few targets that are ‘virus-only’

in nature when it comes to screening, but in the case of HIV 1,

a specific target is the aspartic protease that is an essential part

of the processing of the viral proteins pol and gag that permit the

virus to replicate in the host cell.

The initial work on this protease by the Merck group

demonstrated that it was an aspartic proteinase and could be

inhibited by the microbial pepsin inhibitor, pepstatin. In fact,

inhibition by this peptide could be seen in both isolated enzymic

and in whole cell assays.65Pepstatin (33) contains an unusual

hydroxy-amino acid, statine, which can be thought of as a

mimic of a putative transition state intermediate, where the

hydroxy group takes the place of a water molecule that is the

second substrate for the hydrolytic reaction.66Using this

hypothesis, plus the idea that statine is acting as a dipeptide

replacement, two groups, one at Wisconsin and the other at

Merck, collaborated to produce renin inhibitors containing a

hydroxyethylene isostere that gave activity comparable to that

of pepstatin as a pepsin inhibitor.66Concomitantly, studies with

replacement of amino acids in aspartic-proteinase substrates (in

general, 6 to 8 residues in length) with statine or isosteres, led to

the production of potent inhibitors of the aspartic proteinases,

renin and elastase.66

Once the investigators realized from the pepstatin results that

the activity of HIV 1 protease was due to the presence of the

same (or similar) catalytic site to that of renin, then the

collection of renin inhibitors was tested, leading to identifica-

tion of potential HIV 1 inhibitors. Using similar techniques to

those that proved fruitful with the renin and elastase systems,

variations around those inhibitors, and/or others based on a

short peptide that was the consensus substrate of HIV 1

protease, were synthesized using isosteric replacements for a

variety of the amino acids, but, in most cases, keeping the

‘statine-mimic’ aligned with the geometry of the active site.

These synthetic exercises based on a natural product model

have led to successful drug entities67,68that are now available

for the clinical treatment of HIV infections, though the

molecules used, such as Crixivan®(34), show no formal

structural relationship to the original natural product inhibitor,

pepstatin.69–72

5Cardiovascular: general

In our usage, cardiovascular diseases will cover the following

general areas. Control of the b-adrenergic nervous system

which will include some aspects of respiratory function, control

of cholesterol/lipid metabolism and control of angiotensin

levels. The underlying physiological principle that is addressed

in these areas is the homeostatic control of blood pressure.

5.1Cardiovascular: the b-adrenergic amines

Although epinephrine (adrenaline, 35) was discovered from

natural sources (extraction of sheep adrenal glands) around the

turn of the 20th century and was marketed as a pharmaceutical,

it was not until the investigations by Chinese scientists in

Peking in the early 1920s that a reasonable source of what

eventually became known as the sympathomimetic amines was

identified.

The Chinese had known of the potential of the plants Ephedra

sinaica and E. equisetina73for millenia as treatments for

asthmatic and other bronchial conditions and then, in 1923,

Chen obtained pure ephedrine (36) from E. sinaica and

demonstrated that its physiological actions were very similar to

adrenaline, causing elevation of blood pressure, plus inotropic

and chronotropic actions on the heart. Following regulatory

approval, it became the first in a very long line of bronchodila-

tors/CV agents.

Work between the world wars led to the identification of

other amino compounds based on the ephedrine basic structure,

with benzedrine and methamphetamine being widely issued

during WWII as stimulants. Following WWII, these compounds

became tightly regulated in most jurisdictions because of their

abuse potential. The next major development was the synthesis

of the molecule isoprenaline (Isoprel®, 37) by combining the o-

catechol ring with a modified amphetamine side-chain. This

molecule showed excellent activity as a bronchodilator without

significant action on blood pressure but it had significant

cardiac stimulant effects.

It was the subsequent work of Black (part of the studies for

which he ultimately received the Nobel Prize) that demonstrated

that there were two basic types of b-receptor, the b1, which is

predominately cardiac, and the b2which is predominately

tracheal/lung. He, together with co-workers at ICI Pharmaceuti-

cals developed the first true b-blocker, propranolol74(38). To

add to the ‘confusion’, the compounds that were being 222Nat. Prod. Rep., 2000, 17, 215–234

developed at that time (the middle to late 1960s through the

1980s) could have both agonist and antagonist activities and, in

some cases, these activities could ‘partially cross-over’ and

show mixed agonist/antagonist activities. Chemical manipula-

tion around the basic structure, coupled to use of isolated

receptor assay techniques has led to compounds with much

better separation of the b-blocking activities such as atenolol

(39) and metaprolol (40) that have no detectable intrinsic

sympathomimetic activities.

Thus, starting with an agonist structure (ephedrine) that

significantly affected blood pressure due to its effect on cardiac

output and on release of other sympathomimetic amines, one

now has available related structures that are specific blockers of

such activities on cardiac tissue and are excellent ‘reducers of

hypertension’.

5.2Cardiovascular: cholesterol lowering agents

Another major cause of elevated blood pressure is due to the

physical blockage of the arteries by plaques of cholesterol/

lipoproteins (atherosclerotic plaque). Since the human synthe-

sizes about 50% of its requirement for cholesterol, if the

synthesis can be inhibited, then a reduction in overall choles-

terol may reduce the deleterious effects of this steroid.

A potential site for inhibition of cholesterol biosynthesis in

eukaryotes is at the rate-limiting step in the system, the

reduction of hydroxymethylglutaryl coenzyme A by HMG-

CoA reductase to produce mevalonic acid (41). By following

inhibition of sterol production and using fungal fermentation

broths as the source of natural products, Sankyo discovered

compactin (42) from a fermentation of Penicillium brevi-

compactum and patented it in 1975.75,76Compactin was also

reported at the same time as an antifungal agent by Brown

et al.77This material was shown to be a competitive inhibitor of

the enzyme with K i s in the nanomolar range, but it was not

developed further.

Using a similar assay, a homologue of compactin, mevinolin

(43) or 7-methylcompactin, was isolated by Sankyo from M.

ruber and reported in 197978,79with a submission by Endo,

under the name Monacolin K, to the Japanese Patent Office for

the activity, but without a structure. Concomitantly, Merck

discovered the same material from A. terreus,using an isolated

HMG-CoA reductase assay and microbial broths as the source

of test agents. Merck reported the mevinolin discovery in 1980

in a communication in the Proceedings of the National

Academy of Science, USA80and after submission of both

structure and findings to the Patent Office, a US Patent was

issued in late 1980.81Following a very significant volume of

work, mevinolin became the first commercialized HMG-CoA-

reductase inhibitor in 1987.82,83

Further work using either chemical modification of the basic

structure (a la b-lactam modifications based on 6APA) or use of

biotransformation techniques led to two further compounds by

converting the 2-methylbutanoate side-chain into 2,2-dime-

thylbutanoate, giving simvastin (44), or opening of the

exocyclic lactone to give the free hydroxy acid, pravastatin

(45).

Data from 1997, the last year for which full sales information

is available, shows that these three natural-product derived

drugs are the second best-selling compounds in the world, with

combined sales of US$7.53 billion.18

Comparison of the ring-opened lactone structure common to

all of the ‘statins’ shows the resemblance to mevalonic acid and

this recognition led to the synthesis of the three synthetic

clinical products fluvastatin (46), cerivastatin (47) and atorvas-

tatin (48). The first two have the dihydroxy-heptenoic acid side-

chain of the fungal-derived products linked to a lipophilic ring

structure whilst the third uses the reduced form of the acid

Nat. Prod. Rep., 2000, 17, 215–234223

directly linked to the heteroatom of a pyrrole ring. In the

nomenclature of Cragg et al.,84these would be classified as S*

compounds; synthetic in nature but derived from a natural

product prototype.

5.3Cardiovascular: angiotensin converting enzyme

inhibitors (ACE inhibitors)

The angiotensinogen to angiotensin I to angiotensin II cascade

is an essential mechanism in the maintenance of blood pressure

in humans and it was realized that if one could inhibit the

conversion of the decapeptide (angio I) into the biologically

potent octapeptide (angio II), then it might be possible to control

blood pressure by such compounds. In 1965, Ferreira85reported

that fractions from the venom of the pit viper, Bothrops

jararaca, inhibited the degradation of the mammalian non-

apeptide, bradykinin. The enzyme that degraded this peptide, a

dipeptidyl carboxypeptidase, was subsequently identified as

having ACE activity and was, in fact, the same enzyme working

with two different substrates.

The role of ACE in hypertension was identified as a result of

the work of Ondetti et al.86demonstrating that the active

principle in the viper venom was a simple nonapetide, teprotide

(49). This material had specific activity as an ACE inhibitor and

also had hypotensive efficacy in clinical trials, though, due to a

lack of oral availability, it was a good lead but not a particularly

good drug candidate.

With the recognition that ACE was a metallo-enzyme came

the utilization of a similar carboxypeptidase (carboxypeptidase

A; a monopeptidyl-carboxypeptidase; CpdA) as a surrogate

model. Previous work by the Squibb group had shown that all of

the peptidic inhibitors of ACE had a C-terminal proline, and by

using this information, plus the fact that benzyl-succinic acid

was a specific inhibitor of CpdA, they derived a series of

carboxy- and mercapto-alkanoyl esters of proline that demon-

strated good to excellent inhibition of ACE. One of the

compounds (SQ14225), 50, subsequently became the proto-

typical ACE drug, Captopril®.86,87

Further development of the concepts shown in this work has

led to more potent ACE inhibitors as information as to ADME

and the specific spatial requirements of the ACE active site(s)

were further delineated. In some cases, the drugs that are used

are formally prodrugs, with cleavage of carboxylic esters in the

cases of Enalapril®(51) and Quinapril®(52), or of a

phosphinate ester in the case of Fosinopril®(53), to give the

active drug once in the circulation.

6Pain/central nervous system: history

The conquest of pain was one of the major uses of medicinal

plants in the Ancient World, with the possible use of the crude

extract of the opium poppy (Papaver somniferum) dating from

around 6000 years ago in Sumeria. However, the earliest

undisputed reference to the use of the ‘juice of the poppy’ is

from Theophrastus approximately 2300 years ago. The use of

opium, or laudanum as it was named by Paracelsus in the

sixteenth century, was popularized in both Europe and partic-

ularly in the Orient, even leading to the so-called “Opium Wars”

between the UK and China in the 1840s.

6.1Pain/central nervous system: the opiates

Dioscorides first described the method of production from the

seeds of the poppy (cf.p. 175 in Mann12), and a similar method

is still used today. Crude ‘Opium’, the dried exudate, contains

about a quarter of its weight as opium alkaloids with morphine

and codeine being the major components with up to 20 more

distinct alkaloids including thebaine, papaverine and noscapine.

Morphine (54) was first isolated by Serturner in 1806, followed

by codeine (55) in 1832 by Robiquet and then the non-morphine

alkaloid papaverine by Merck in 1848. With the invention of the

hypodermic needle and the availability of the purified alkaloids,

the benefits and problems associated with widespread use of

these alkaloids rapidly became apparent88,leading to the

search for potent drugs without the abuse potential. Ironically,

heroin, the compound that has probably caused the most human

anguish since its preparation in the UK from morphine in 1874,

was marketed by Bayer from 18 predominately as a cough

suppressant. A close relative, dextromethorphan (56), is in fact

used in most cough syrups today, but lacks the abuse potential

of its chemical cousin. Interestingly, there is now evidence that

morphinans (codeine and morphine at least) may be synthesized

in mammalian tissues as well.90,91

224Nat. Prod. Rep., 2000, 17, 215–234

With the recognition of the abuse potential of the opiates came the search to make compounds that mimicked morphine in pain control but lacked the abuse potential. With the exception of the semi-synthetic compound buprenorphine (57), which is

approximately 25–50 times more potent than morphine and has a lower addiction potential, none of the compounds made to date from modifications around the phenanthrene structure of morphine have exceeded the pain control properties without a concomitant addiction potential. Another interesting compound whose structure is based on that of morphine, is pentazocine (Talwin ®, 58). This is about 30% as effective as morphine, but with a much lower incidence of abuse and, in fact, will cause withdrawal symptoms in morphine addicts at high dosage due to antagonism at the morphine-active m -receptor.

Perhaps the major advance in the understanding (at least to some molecular level) came from the work of investigators who made the then novel assumption that the complex interactions amongst this class of drugs could best be described by suggesting that there were multiple opiod receptors for these drugs.92Following this, in 1973 three independent groups (those of Terenius;93Pert and Snyder;94Simon, Hiller and Edelman 95) described receptor binding sites in the mammalian nervous system. These sites were stereospecific and saturable by varying opioid drugs. Following these discoveries and confirming the suspicion that the body had a method of quelling pain, came the isolation from porcine brain by Hughes and Kosterlitz in Aberdeen in 1975 of two pentapeptides that demonstrated opioid-like effects on guinea pig ileal strips.These were named enkephalins and, following work by others reported the same year, it was realized that there are at least three distinct families of peptides that exhibit opioid-like activities, the enkephalins, the endorphins and the dynorphins.96Although a substantial amount of work was performed with small peptide compounds based on these relatively simple structures, none have succeeded as drugs to replace opioids as yet (though see below under conotoxins). However, these small peptides and synthetic derivatives derived from them have seen extensive use as delineators of opioid receptor sub-types in many tissues, not just those of the central nervous system.Examples of some structures are given in the review by Sibinga and Goldstein 97and in the paper by Kramer et al.986.2Pain/central nervous system: the conotoxins

The cone snails (phylum Mollusca, genus Conus ) with 500species known are venomous marine animals that are predom-inately found in the Western and South-western Pacific, though there are some species found in Californian waters. Their particular ‘expertise ’ is that they stun/kill their prey through the use of a disposable hollow tooth that contains a multitude of peptidic toxins. One might even claim that Mother Nature invented the disposable hypodermic needle, as their method of delivery in most cases is via a harpoon-like hollow tooth that breaks off once in the prey.

These animals would have simply been ‘just another venomous marine organism ’ but for the work of Olivera et al.99,100who demonstrated that the venom from each of these animals contained over thirty polypeptides ranging in size from

10 to 35 amino acids and they demonstrated that specific peptides gave specific physiologic responses in mammalian systems (and by inference, in the piscatorial arena where they were originally designed to work). From these original discoveries has come the identification of a novel class of analgesics that appear to specifically target a voltage gated Ca 2+channel, are very potent and have low probabilities for abuse.Olivera 101has used the term ‘Janus-ligand ’ for these peptidic ligands as they appear to have both a ‘docking face ’ and a ‘locking face ’, thus giving exquisite selectivity and sensitivity in the same molecule. The original material that has undergone clinical development, and is currently in Phase III trial/awaiting approval from the FDA, is SNX-111 (59) from Neurex

Corporation (now a part of Elan Pharmaceuticals). Though made synthetically, it is in fact identical to MVIIA from Conus geographicus .102Many other synthetic variants have been made and tested 102and will probably follow SNX-111, but for other indications.

The potential for advances in other physiological areas as the properties of the myriad of other peptides are investigated is very high, showing the advantages of Nature ’s combinatorial chemistry approach with these molecules.6.3

Pain/central nervous system: the epibatidines

In 1974, John Daly at the NIH isolated a small amount of a novel alkaloid from the skin of an Ecuadorian poison frog (Epipedo-bates tricolor ) and demonstrated that it had excellent analgesic effects in a particular mouse model. The material could not be recollected and, without a structure, no further work was done.With the advent of new NMR techniques in the late 1980s, the 750 micrograms remaining was enough to show that the structure was a substituted chloronicotine, that was named epibatidine 103(60). This molecule was synthesized and demon-

strated excellent inhibition of the nicotinic receptors in neurons and neuromuscular junctions but with a lack of specificity exemplified by its very low therapeutic index as referenced by Ellis from UCB Research.104In addition to Ellis ’ group at UCB Research, the Abbott group led by Decker 105–107have shown the potential of this base molecule, and the manifold modifica-tions that have been made have shown analgesic effects mediated via one or more of the nAChR (nicotinic acetylcholine receptor) subtypes in neuronal tissues. The structures shown in these papers demonstrate the relative simplicity of these agents,being predominately based on the nicotine/epibatidine locus. To date, one compound from the Abbott group, ABT594 (61), has

reached advanced preclinical status. If it moves into Phase I, it will be the first epibatidine derivative to cross that hurdle and may demonstrate that specificity as to subtype will work in man as well as mouse.

Nat. Prod. Rep., 2000, 17, 215–234

225

7Antineoplastics: general

It is in the treatment of cancers and in anti-infective areas that natural products have made their major impact as templates or direct treatments. In the cancer area, of the 92 drugs commer-cially available prior to 1983 in the United States, or approved worldwide between 1983 and 1994, approximately 62% can be related to a natural product origin, ignoring those of biological origin such as interferon or recombinately produced cyto-kines.84

A common feature with many of the natural product-derived anticancer drugs now in clinical use is that the original natural product was too toxic. Chemists therefore, had to make semi-synthetic compounds based on the natural product structures that had the more deleterious problems of the natural product diminished by selective modification. In some cases, notably that of mitoxantrone (62), a totally synthetic product evolved

but with the attributes of two or more parent natural product structures in one molecule.847.1

Antineoplastics: plant sources

Although plants have a long history of use in the treatment of cancer,108many, if not all, of the claims for the efficacy of such treatment should be viewed with some skepticism because cancer, as a specific disease entity, is poorly defined in terms of folklore and traditional medicine.109

Amongst the best known are the so-called vinca alkaloids,vinblastine (63) and vincristine (), isolated from the Mada-gascan periwinkle, Catharanthus roseus. C. roseus was used by various cultures for the treatment of diabetes, and these compounds, together with two other related active alkaloids,vinleurosine and vinrosidine, were isolated during an investiga-tion of the plant as a source of potential oral hypoglycemic agents. Therefore, the discovery of the initial two compounds may be indirectly attributed to the observation of an unrelated medicinal use of the source plant.109Selective chemical modifications of these two molecules have led to two semi-synthetic compounds (from many) being approved in Europe for cancer treatment, vinorelbine (65) and vindesine (66). The former was recently approved in the US and the latter is in US clinical trials.110

The parent of the two clinically-active agents etoposide (67)and teniposide (68) is epipodophyllotoxin. This is the naturally occurring epimer of podophyllotoxin (69) which was isolated as the active anti-tumor agent from the roots of various species of the genus Podophyllum . These plants possess a long history of medicinal use by early American and Asian cultures, including the treatment of skin cancers and warts.109Although podo-phyllotoxin was investigated at length by the NCI as a potential anti-tumor agent it was shelved due to intractable toxicity problems. Subsequent elegant work by Sandoz, prior to its merger with Ciba-Geigy, led to the synthesis from epipodo-phyllotoxin of etoposide and teniposide, both of which are in general clinical use. Further work by Nippon Kayaku has led to a water-soluble derivative of etoposide, NK-611 (70), where a dimethylamino group was placed into the sugar ring, thus giving significant water solubility; the material is now in clinical trials (cf . references 60 to 62 in the recent review by Wang 111). Two recent reviews covering work at the University of North Carolina, give information on the semi-synthetic derivatives based on the podophyllotoxins 112and the earlier review covers inhibitors of topoisomerase I and II either from natural sources or based upon natural products.113These reviews should be read

226

Nat. Prod. Rep., 2000, 17, 215–234

in conjunction with this article as further examples of where

natural product-derived templates for anti-tumor agents have

led to in a synthetic sense.

Camptothecin (71) was isolated from the Chinese ornamental

tree Camptotheca acuminata by Wani and Wall114contempora-

neously with the initial discovery of Taxol®. As the sodium salt,

camptothecin was advanced to clinical trials by NCI in the

1970s, but was dropped because of severe bladder toxicity. It

was resurrected as a result of its very specific biochemical

activity as an inhibitor of topoisomerase I and the efforts of one

of the earliest NCI National Cooperative Drug Discovery

Groups (NCDDGs) involving Johns Hopkins University and the

then SmithKline Beckman. From these studies eventually came

the modified camptothecin, topotecan (Hycamptin®) (72),

which was approved for use in the USA in 1996. Other groups

modified the basic structure in slightly different ways,114

leading to the approved agent irinotecan (Camptosar®) (73), and

two others awaiting approval, 9-amino- (74) and 9-nitro-

camptothecin (75).

The complex diterpene Taxol®76(Paclitaxel) initially was

isolated from the bark of Taxus brevifolia, collected in

Washington State as part of a random collection program by the

U.S. Department of Agriculture for the National Cancer

Institute.115Historically, parts of the yew tree (T. brevifolia and

other Taxus species) had been used by several Native American

tribes for the treatment of some non-cancerous conditions,108

and leaves of T. baccata are used in the traditional Asiatic

Indian (Ayurvedic) medicine system,6with one reported use in

the treatment of ‘cancer’108

Paclitaxel, along with several key precursors (the baccatins),

occurs in the leaves of various Taxus species, and the ready

semi-synthetic conversion of the relatively abundant baccatins

into paclitaxel, as well as to active paclitaxel analogs, such as

docetaxel116(77), has provided a major, renewable natural

source of this important class of drugs. Using the baccatins as

starting materials, many semi-synthetic taxanes have been

synthesized in an effort to obtain materials that have better

solubility in aqueous environments. A recent paper by Kingston

et al.demonstrates the methods used to prepare more active

2-acyl analogues of paclitaxel117and covers, in its references,

the many modifications that workers have made to the basic

taxane skeleton in efforts to better understand the SAR of

tubulin binding and activity.

The flavone, flavopiridol (78), is currently in Phase I/II

clinical trials against a broad range of tumors.118While

flavopiridol is totally synthetic, the basis for its novel structure

is the natural product rohitukine (79) isolated from Dysoxylum

binectariferum.119Flavopiridol has a very interesting mecha-

nism of action in that it is an inhibitor of cyclin dependent

kinases (the regulators of the G2to M transition in the cell

cycle). This discovery has led to a series of compounds (the

paullones as exemplified by structure 80) from the NCI open

compound database that, though not natural products, would not

have been discovered except for the use of the natural product-

derived agent as a ‘seed compound’. This story is given in detail

in a recent paper by Sausville et al.120Other recent papers

dealing with discovery and modification around the paullone

structure are those of Schultz et al.121and Zaharevitz et al.122

Flavopiridol is not the only CDK inhibitor discovered from a

natural source and then extended to give semi-synthetic and

synthetic compounds with a variety of potencies and specific-

ities. Workers associated with Meijer’s group in France have

published extensively on the potential of the highly related

purine analogues, olomoucine (81) and roscovitine (82) as CDK

inhibitors.123–125These naturally occurring purine analogues

are competitive inhibitors with ATP and were thus thought to

bind at the ATP site in the protein. This was confirmed when the

Nat. Prod. Rep., 2000, 17, 215–234227

structures of the protein inhibitor complexes were solved at high

resolution126,127confirming the results from the enzyme

inhibition studies. The two natural products were not of

sufficient potency for use in in vivo experiments and these

findings led groups to use combinatorial chemistry techniques

to optimize the ‘natural product backbones’ of these two

substituted purines. Work from Schultz’ group at Berkeley was

reported in 1998128and then more complete reviews from the

same group were published in 1999.129,130Comparison of the

structures of purvalanols A (83) and B (84) with olomoucine

and roscovitine show that only very small changes in the side-

chains are enough to alter the IC50figures by three orders of

magnitude. The interesting aspect, however, is that the base

structure is effectively that of the natural products, thus

demonstrating the value of using combinatorial chemistry

techniques to optimize an existing active structure rather than

attempting to synthesize de novo.

For a more general review of the history of CDK inhibitors

and a discussion of structures and derivatives in addition to

those mentioned above, a majority of which are natural product

based, the reader should consult the recent review by Web-

ster,131though, because this is such a fast-moving area, any

review will be outdated by the time it is published.

7.2Antineoplastics: microbial sources

Anti-tumor antibiotics are amongst the most important of the

cancer chemotherapeutic agents, which include members of the

anthracycline, bleomycin, actinomycin, mitomycin and aureolic

acid families.132Clinically useful agents from these families are

the daunomycin-related agents, daunomycin itself, doxorubicin

(85), the semi-synthetic derivatives idarubicin and epirubicin;

the glycopeptidic bleomycins A2and B2(blenoxane); the

peptolides exemplified by dactinomycin (86); the mitosanes

such as mitomycin C (87); and the glycosylated anthracenone,

mithramycin. Except for the semi-synthetic compounds, all

were isolated from various Streptomyces species.

That there is still a lot to be learned from old molecules was

demonstrated by Hecht from the University of Virginia, who in

a plenary lecture133at the November, 1999 AACR-NCI-

EORTC meeting on Molecular Targets and Cancer Ther-

apeutics demonstrated that bleomycin might well be targeting

amino acid t-RNA’s. He subsequently outlined a simple

polymer bead-based split and pool technique that his group is

using in order to produce 105bleomycins within the foreseeable

future in order to search for a molecule that is only t-RNA

specific.

A current notable series of microbially-derived anti-tumor

compounds are those directed around the staurosporin nucleus

88.These include UCN-01 (7-hydroxystaurosporine, ),

isolated from a Streptomyces species, though now made by

chemical modification of staurosporin itself and many other

derivatives, with the Novartis agent CGP41251134(90) and

UCN-01 being in clinical trials. Novartis scientists have also

recently reported that this analogue may have multiple modes of

action, inhibiting angiogenesis in vivo in addition to its PKC

inhibitory activity. If this translates into inhibition of human

angiogenesis and subsequent anti-tumor activity then it distin-

guishes this agent from other staurosporin congeners.135

Perhaps the most interesting discovery from the microbial

world over the last few years is the series of compounds known

as the epothilones. These were originally isolated from species

of myxobacteria by Hofle in Germany136,137and simultane-228Nat. Prod. Rep., 2000, 17, 215–234

ously by workers at Merck in the USA.138Various problems

over patents and biological activities prevented epothilones A

(91) and B (92) from being developed by companies; this

despite the fact that the Merck group had demonstrated that they

mimicked paclitaxel in their interactions with tubulin in their

initial paper. Once the German group was able to assign the

absolute stereochemistry,139then multiple groups began at-

tempts to synthesize the epothilones.

Three groups have made major contributions to the synthesis

of this class of compounds, those of Danishefsky at Sloan-

Kettering who were the first to synthesize epothilone A, the

second by Nicolaou at the Scripps Research Institute and the

third by Schinzer in Braunschweig, close to the birthplace of the

natural epothilones. Rather than go into the details of each

individual group’s synthetic strategies, the reader should

consult the excellent recent review by Nicolaou et al.140on the

epothilones. This review shows the synthetic strategies of the

groups but, more importantly, shows the classical synthetic and

combinatorial efforts around the structures that have led to third

and fourth generation compounds now heading towards clinical

development. The activity data (cf.Tables 7 and 8 in the

Nicolaou review140) that are shown in these tables for some of

the classical synthetically and combinatorially derived deriva-

tives against paclitaxel-resistant cell lines are demonstrative of

the power of modern synthetic chemistry when applied to an

active natural product structure.

Recently, Ojima et al.141showed that a non-aromatic mimic

of paclitaxel, nonataxel (93) was more effective than paclitaxel

in vitro and was amenable to solution NMR analyses in order to

determine solution conformation. Using this information to-

gether with the published data for three other quite diverse anti-

mitotic agents, discodermolide, eleutherobin and epothilone B,

they were able to suggest a common pharmacophore that could

account for the activities seen against tubulin by these agents

and paclitaxel. A presentation by the NCI group and their

collaborators142at the AACR-NCI-EORTC Conference on

Molecular Targets and Cancer Therapeutics in November, 1999

proposed a modified pharmacophore. Their structure differed

from the Ojima model by requiring the baccatin and the

epothilone skeletons to maintain similar conformations permit-

ting the required phenyl and ester linkages to be in their correct

spatial relationships for maximal reaction with the binding sites.

This model explained the tubulin cross-resistance profiles seen

for paclitaxel-resistant cell lines.

7.3Antineoplastics: marine sources

The first notable discovery of biologically-active compounds

from marine sources was the serendipitous isolation of the C-

nucleosides spongouridine and spongothymidine (cf.Antiviral

section for further discussion) from the Caribbean sponge

Cryptotheca crypta in the early 1950s. These compounds were

found to possess antiviral activity, and synthetic analog studies

eventually led to the development of cytosine arabinoside (Ara-

C) (94) as a clinically useful anticancer agent approximately 15

years later.143The systematic investigation of marine environ-

ments as sources of novel biologically active agents only began

in earnest in the mid-1970s. During the decade from

1977–1987, about 2500 new metabolites were reported from a

variety of marine organisms. These studies have clearly

demonstrated that the marine environment is a rich source of

bioactive compounds, many of which belong to totally novel

chemical classes not found in terrestrial sources.144

As yet, no compound isolated from a marine source has

advanced to commercial use as a chemotherapeutic agent,

though several are in various phases of clinical development as

potential anticancer agents. One of these is bryostatin 1 (95),

isolated from the bryozoan Bugula neritina.145,146This agent

exerts a range of biological effects, thought to occur through

modulation of protein kinase C, and has shown some promising

activity against melanoma in Phase I studies,147and showed

some stabilization of disease progression148in Non-Hodgkin’s

Lymphoma (NHL). Further Phase I/II trials are either in

progress or are planned against a variety of tumors, including

ovarian carcinoma and NHL, with bryostatin as both mono-

therapy or as an adjunct to other compounds (further informa-

tion is on the NCI’s web site at cancernet.nci.nih.gov).

Although bryostatin is still produced by isolation from the

bryozoan, whether by aquaculture or by wild collection, work

has progressed on the synthesis of simpler analogs with

comparable activity. In 1988, Wender et al.149suggested a

simplified pharmacophore model to explain the binding charac-

teristics of the bryostatins to PKC. They followed this proposal

up by synthesizing a series of simplified macrolide structures

that they demonstrated had both PKC binding and cytotoxic

activities of the same order as the bryostatins. Wender’s group

have reported at length on these compounds in a series of recent

communications150–154and the structure of their most active

compound (96) from the first series is shown.

Another marine compound that is also in Phase I/II clinical

trials but under the auspices of the Spanish company Pharma-

Nat. Prod. Rep., 2000, 17, 215–234229

Mar rather than the NCI, is ecteinascidin 743 (97) from the shallow water tunicate Ecteinascidea turbinata . Two groups,those of Rinehart et al.155and Wright et al.,156reported simultaneously on this compound from similar sources. An interesting speculation from the structure of Et743 (the usual abbreviation for ecteinascidin 743) is that the actual producing organism is a commensal microbe as the structures of these molecules resemble the saframycins 157,158(98) from Strepto-myces lavendulae and renieramycins 159(99) from the Pacific sponge, Reniera sp.

Due to the very low levels of Et743 in the tunicate and the problems of amassing enough material for large clinical trials and/or commercial use, Corey developed a synthesis of Et743and reported it in 1996.160As mentioned by Corey in the discussion in this publication, Et 743 was made in ‘relatively ’high yield but, perhaps more importantly, the synthetic methods used opened up the potential for synthetic approaches to derivatives or to simpler compounds such as the saframycins.That this was a prescient comment was demonstrated by the publication in 1999 of simpler structures related to Et 743 made from a common intermediate in the original synthetic schema.The best compound of those reported was named phthalascidin (100), which demonstrated comparable activity to Et 743 in all the in vitro systems that it was tested in, including the NCI 60cell line panel.161

The last marine examples to be discussed are materials that are still in preclinical stages of development. What is important about these compounds, however, is that they have spawned both total syntheses and then a number of combinatorial libraries that have succeeded in optimizing the in vitro activity of one of them. Secondly, the original compounds (in their synthetic versions) and their derivatives have been used to further delineate the binding characteristics of compounds to tubulin.

The compounds are from two series of related compounds,each containing the eunicellane carbon skeleton, a diterpene first reported in 1968 from the gorgonian octocoral Eunicella stricta .162One series was glycosylated, the eleutherobin/eleuthosides, the other being effectively their aglycone, the sarcodicytins.

The sarcodicytins (101) were first reported as compounds by Pietra ’s group, following their isolation from the Mediterranean stoloniferan coral Sarcodictyon roseum in 1987163and in 1988.1These reports were followed almost ten years later by an abstract from workers at Pharmacia-Upjohn presented at the 1997 American Association for Cancer Research Meeting.165This report demonstrated that the sarcodicytins had a paclitaxel-like action on tubulin.

In 1994, during the intervening ten years from the original report on the sarcodicytins to that of their activity, workers in Fenical ’s group at The Scripps Institute of Oceanography discovered a related compound from the Western Australian coral, Eleutherobia sp. They named the compound eleutherobin (102) and showed that it contained the eunicellane skeleton but,unlike the sarcodictyins, it was glycosylated in a manner similar to that of the eleuthosides. What was unique, at that time, was its activity against tubulin where it mimicked pacilitaxel. The compound, and its activity, were first reported in the patent literature 166followed by the chemical 167and biological 168publications.

In a prime example of what the late British historian of science Derek De Solla-Price termed the ‘invisible college ’, two excellent synthetic chemists, Nicolaou at the Scripps Research Institute and Danishevsky at Memorial Sloan Kettering suc-

230

Nat. Prod. Rep., 2000, 17, 215–234

ceeded in synthesizing both the sarcodictyins and eleutherobin.In a series of recent papers both groups described their respective synthetic methods, with Nicolaou ’s group being the first to press with a total synthesis of sarcodictyin A and the core of eleutherobin.169This publication was then followed by a very rapid series of papers from both groups.170–174In the middle of this series of papers, Lindel, who was one of the discoverers of eleutherobin, published a short historical review of the discovery and the relative synthetic methods used by the groups.175

The crux of the synthetic efforts from a drug discovery aspect, however, was the paper by Nicolaou ’s group that described the derivation of combinatorial libraries built around the solid and solution phase synthetic methods that they developed for the sarcodictyins.176In an elegant biochemical paper by Hamel et al.177the authors demonstrated the effects of one of these compounds, a compound that has some aspects of eleutherobin but without the sugar moiety (compound 1, 103),

upon binding to tubulin. In their conclusions they make two telling points:

‘Like sarcodictyin A compound 1 interacts poorly with tubulin,but like eleutherobin compound 1 has substantial antiprolifer-ative activity.’

‘The activity of this agent (compound 1) in cells suggests that further synthetic efforts with the sarcodictyin class would yield compounds with activities greater than those of paclitaxel in cells and perhaps with tubulin as well.’

Both of these quotes show the efficacy of using combinatorial chemistry techniques around the skeleton of an active natural product in an attempt to optimize both molecular recognition sites and transport across cell membranes.

It is tempting to speculate that similar biochemical and chemical experiments are going on with other active com-pounds in this area; we will, however, have to await publication of results in either the patent or general literature to see if these speculations are correct.8

Future trends: general

Rather than discuss each disease area from the viewpoint of future trends, it is tempting to suggest that there are areas of natural product research that are ‘blooming ’ in-so-far as derivation of unusual or even ‘un-natural ’ structures are concerned, and that, in the not too distant future, they will become major sources of novel agents with pharmacological promise.

8.1Future trends: production of ‘previously unknown structures’

What we are referring to is the marriage of genetic information on biochemical pathways with the manifold techniques of ‘gene manipulation ’. This work was pioneered by Hopwood 178in the 1970s with initial attempts to produce hybrid antibiotics using protoplast fusion techniques in the Streptomyces . With the advent of recombinant DNA technology, the identification of biosynthetic pathways to antibiotics and the ability to screen large number of clones for expression of particular activities at low expression levels, these technologies have the potential to produce very unusual compounds that are true hybrids of different biosynthetic pathways.179–183Recently McDaniel et al.184reported on the ability to interchange modules in different type I polyketide synthases (PKSs) and Shen et al.185were able to express three genes from the other type (type II) of PKS systems. In a very recent paper Zhao et al.186reported the ‘combinatorial biosynthesis ’ of methymycin or pikromycin aglycones coupled to the sugar from a totally unrelated antibiotic, calcheamicin. A recent review article by Hutch-inson,187from the University of Wisconsin, who is also a pioneer in this field, should be consulted for further information on these techniques and their import in drug discovery.

In addition to the ‘directed techniques ’ referred to above,where the biosynthetic genes are identified and then ‘mixed and matched ’, there is another source of unusual/unknown struc-tures that is currently being actively investigated, the ‘as yet uncultured microbes ’. Pioneers in this field are Colwell,188who described the recognition of visible but uncultured bacteria in marine environments and Handelsman and her collaborators for similar discoveries with terrestrial bacteria.1By isolating gross DNA from these sources, followed by clean-up, separa-tion into eukaryotic and prokaryotic DNA pools, cloning by a variety of means in suitable hosts and then screening of the hosts for expression of novel activities and/or chemical entities a vast new area of chemical diversity may be opened up.

That these techniques are viable as sources of new structures and agents can be seen from the companies that have been set up to study these techniques and their academic linkages. Exam-ples are Kosan Biosciences with Chaitan Khosla (University of California, Berkeley) and TerraGen Bidiversity (Julian Davies,University of British Columbia), plus two others that have changed names and affiliations over the years, Diversa and ChromoXome.8.2

Future trends: the optimization of lead structures

In this, bioactive natural product lead structures would be identified and then new agents with improved pharmacokinetics and/or toxicology synthesized. This is ‘optimization of a lead structure ’, but instead of using the classical medicinal chemistry techniques, rapid combinatorial/parallel synthesis methods would be applied.190That these are viable strategies for deriving ‘new and improved ’ molecules can be seen from the earlier discussions under anti-tumor agents, in particular the work around paclitaxel, epothilones and sarcodictyins. What is also becoming evident is that the synthesis of complex natural products is no longer an end in itself. The molecules that are used are themselves becoming substrates for combinatorial approaches or as sources of simplified molecules, as shown by the work on bryostatins and on ecteinascidins, where simpler but biologically active molecules have been made as a result of synthetic endeavors.

8.3Future trends: novel delivery systems for ‘old compounds’

As a result of the massive screening that has been carried out in antibiotic and anti-tumor discovery programs, there are sig-Nat. Prod. Rep., 2000, 17, 215–234

231

There are a variety of methods that can be used for such delivery systems; the two that have been studied most extensively in the past are use of liposome-encapsulated toxic agents191and antibody-conjugates with natural toxins192(i.e. ricin) or pure compounds such as calcheamicin and doxor-ubicin. A further modification would be to use a prodrug and an enzyme-coupled antibody to release the material at the site.193 Over the last few years, another novel strategy for delivery of anticancer drugs to the tumor site has been proven clinically. This is the use of a water-soluble copolymer with a warhead attached. A driving force in this area has been the group now at the University of London’s School of Pharmacy.194They, originally in conjunction with a polymer group at Prague and then with Farmitalia (now part of Pharmacia-Upjohn), success-fully coupled doxorubicin to an N-(2-hydroxypropyl)methacry-lamide (HPMA) copolymer to produce the construct known as PK1. By adding a targeting sugar moiety to PK1, a construct (PK2) that targeted the liver was made and some preliminary imaging results from the Phase I trials have been reported.195 PK1 is now in Phase II trials in the UK, and PK2 has just completed its first Phase I trial, also in the UK.195–198This work has now been extended to cover platinum complexes as well, with the first in vivo preclinical results being reported.199 Two final points:

(1)an analysis of the compounds mentioned in the 694 posters

presented at the AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics meeting in November 1999, showed that of the 433 molecular entities mentioned, 2 fell into the N/ND or S* categorizations of Cragg et al.84

(2)In an ASAP paper in Organic Letters, Smith et al.200

published their gram-scale synthesis of the tubulin-active marine natural product, discodermolide. This synthesis may well open the way to combinatorial development of a large number of related compounds for this agent, which is currently in preclinical development as an anti-tumor agent.

Thus, in closing, as can be seen from the above discussions, and as referred to by other reviewers,201,202‘the notification of the demise of natural products as leads to drug entities is highly premature’ (with apologies to Mark Twain).

9References

1Anon, Herbalgram, 1998, 33.

2S. Dev, Environ. Health Perspect., 1999, 107, 783.

3M. Fallarino, Herbalgram, 1994, 31, 38.

4S. Grabley and R. Thiericke, Adv. Biochem. Eng./ Biotech., 1999, , 104.

5H.-M. Chang and P. P.-H. But, Pharmacology and Applications of Chinese Materia Medica, World Scientific Publishing, Singapore, 1986, vols 1 & 2.

6L. D. Kapoor, CRC Handbook of Ayurvedic Medicinal Plants, CRC Press, Boca Raton, 1990.

7R. E. Schultes and R. F. Raffauf, The Healing Forest, Dioscorides Press, Portland, 1990.

8R. Arvigo and M. Balick, Rainforest Remedies, Lotus Press, Twin Lakes, 1993.

9N. R. Farnsworth, O. Akerele, A. S. Bingel, D. D. Soejarto and Z. Guo, Bull. WHO, 1985, 63, 965.

10F. Grifo, D. Newman, A. S. Fairfield, B. Bhattacharya and J. T.

Grupenhoff, in The Origins of Prescription Drugs, ed. F. Grifo and J.

Rosenthal, Island Press, Washington, D.C., 1997, p. 131.

11G. A. Cordell, Phytochemistry, 1995, 40, 1585.12J. Mann, Murder, Magic and Medicine, Oxford University Press, Oxford, 1992.

13R. I. Mateles, Penicillin: A Paradigm for Biotechnology, Candida Corporation, Chicago, 1998.

14J. R. E. Hoover, personal communication, 1980.

15G. Brotzu, Lav. Ist. Igiene Cagliari, 1948.

16G. G. F. Newton and E. P. Abraham, Biochem. J., 1956, 62, 651.

17E. P. Abraham and G. G. F. Newton, Biochem. J., 1961, 79, 377.

18D. J. Newman and S. A. Laird, The Commercial Use of Biodiversity, ed. K. ten Kate and S. A. Laird, Earthscan Publications, London, 1999, p. 333.

19H. A. Albrecht, G. Beskid, K. K. Chan, J. G. Christenson, R. Cleeland, K. H. Deitcher, N. H. Georopapadakou, D. D. Keith, D. L. Pruess and J. Sepinwall, J. Med. Chem., 1990, 33, 77.

20A. J. Corraz, S. L. Dax, N. K. Dunlap, N. H. Georgopapadakou, D. D.

Keith, D. L. Pruess, P. L. Rossman, R. Then, J. Unowsky and C. C.

Wei, J. Med. Chem., 1992, 35, 1828.

21H. A. Albrecht, G. Beskid, J. G. Christenson, K. H. Deitcher, N. H.

Georgopapadakou, D. D. Keith, F. M. Konzelmann, D. L. Pruess and

C. C. Wei, J. Med. Chem., 1994, 37, 400.

22N. H. Georgopapadakou and C. McCaffrey, Antimicrob. Agents Chemother., 1994, 38, 959.

23A. Imada, K. Kitano, K. Kintaka, M. Muroi and M. Asai, Nature (London), 1981, 2, 590.

24R. B. Sykes, C. M. Cimarusti, D. P. Bonner, K. Bush, D. M. Floyd, N.

H. Georgopapadakou, W. H. Koster, W. C. Liu, W. L. Parker, P. A.

Principe, M. L. Rathnum, W. A. Slusarchyk, W. H. Trejo and J. S.

Wells, Nature (London), 1981, 291, 4.

25T. C. Barden, B. L. Buckwalter, R. T. Testa, P. J. Petersen and V. J.

Lee, J. Med. Chem., 1994, 37, 3205.

26P. E. Sum, V. J. Lee, R. T. Testa, J. J. Hlavka, G. A. Ellestad, J. D.

Bloom, Y. Gluzman and F. P. Tally, J. Med. Chem., 1994, 37, 184. 27R. Wise and J. M. Andrews, Antimicrob. Agents Chemother., 1994, 38, 1096.

28G. Bright, A. Nagel, J. Bordner, K. Desai, J. Dibrino, J. Nowakowska, L. Vincent, R. Watrous, F. Sciavolina, A. English, J. Retsema, M.

Anderson, L. Brennan, R. Borovoy, C. Cimchowsji, J. Faiella, A.

Girard, D. Gorard, C. Herbert, M. Manousos and R. Mason, J.

Antibiot., 1988, 41, 1029.

29R. G. Finch, Drugs, 1996, 51, 31.

30R. C. Moellering, P. K. Linden, J. Reinhardt, E. A. Blumberg, F.

Bompart and G. H. Talbot, J. Antimicrob. Chemother., 1999, 44, 251.

31R. L. Nichols, D. R. Graham, S. L. Barriere, A. Rodgers, S. E. Wilson, M. Zervos, D. L. Dunn and B. Kreter, J. Antimicrob. Chemother., 1999, 44, 263.

32F. P. Tally, personal communication, 1997.

33P. J. McCarthy, D. J. Newman, L. J. Nisbet and W. D. Kingsbury, Antimicrob. Agents Chemother., 1985, 28, 494.

34R. Lucas, K. DeSante, B. Hatcher, J. Hemingway, R. Lachno, S.

Brooks and M. Turik, 36th Intersci. Conf. Antimicrob. Agents Chemother., 1996, LA, USA, Abstract no. F50.

35D. W. Denning, J. Antimicrob. Chemother., 1997, 40, 611.

36Anon, Scrip, 1997, 6.

37O. S. Kinsman, P. A. Chalk, H. C. Jackson, R. F. Middleton, A.

Shuttleworth, B. A. M. Rudd, C. A. Jones, H. M. Noble, H. G.

Wildman, M. J. Dawson, C. Stylli, P. J. Sidebottom, B. Lamont, S.

Lynn and M. V. Hayes, J. Antibiot., 1998, 51, 41.

38A. M. Clark, Pharm. Res., 1996, 13, 1133.

39R. X. Tan, W. F. Zheng and H. Q. Tang, Planta Med., 1998, , 295.

40P. A. G. M. De Smet, Drugs, 1997, 54, 801.

41W. Asawamahasakda, I. Ittarat, Y.-M. Pu, H. Ziffer and S. R.

Meshnick, Antimicrob. Agents Chemother., 1994, 38, 1854.

42S. R. Meshnick, Trans. R. Soc. Trop. Med. Hyg., 1994, 88 (supplement 1), S31.

43S. R. Meshnick, T. E. Taylor and S. Kamchonwongpaisan, Microbiol.

Rev., 1996, 60, 301.

44M. A. Avery, P. Fan, J. M. Karle, J. D. Bonk, R. Miller and D. K.

Goins, J. Med. Chem., 1996, 39, 1885.

45M. A. Avery, S. Mehrota, J. D. Bonk, J. A. Vroman, D. K. Goins and R. Miller, J. Med. Chem., 1996, 39, 2900.

46M. A. Avery, S. Mehrota, T. L. Johnson, J. D. Bonk, J. A. Vroman and

D. K. Goins, J. Med. Chem., 1996, 39, 4149.

47G. H. Posner, X. Tao, J. N. Cumming, D. Klinedinst and T. A. Shapiro, Tetrahedron Lett., 1996, 37, 7225.

48G. H. Posner, H. O’Dowd, P. Ploypradith, J. N. Cumming, S. Xie and T. A. Shapiro, J. Med. Chem., 1998, 41, 21.

49M. Albericci, J. C. Braekman, D. Daloze and B. Tursch, Tetrahedron, 1982, 38, 1881.

50Y. Kashman and M. Rotem, Tetrahedron Lett., 1979, 20, 1707.

232Nat. Prod. Rep., 2000, 17, 215–234

51A. Rudi, R. Talpir, Y. Kashman, Y. Benayahu and M. Schleyer, J. Nat.

Prod., 1993, 56, 2178.

52K. A. El Sayed, D. C. Dunbar, D. K. Goins, C. R. Cordova, T. L. Perry, K. J. Wesson, S. C. Sanders, S. A. Janus and M. T. Hamann, J. Nat.

Toxins, 1996, 5, 261.

53G. M. Konig and A. D. Wright, Planta Med., 1996, 62, 193.

54H. Jomaa, J. Wiesner, S. Sanderbrand, B. Altincicek, C. Weidemeyer, M. Hintz, I. Turbachova, M. Eberl, J. Zeidler, H. K. Lichtenthaler, D.

Soldata and E. Beck, Science, 1999, 285, 1573.

55M. Okuhara, Y. Kuroda, K. Goto, M. Okamoto, H. Terano, M.

Kohsaka, H. Aoki and H. Imanaka, J. Antibiot., 1980, 33, 24.

56E. Iguchi, M. Okuhara, M. Kohsaka, H. Aoki and H. Imanaka, J.

Antibiot., 1980, 33, 18.

57E. Ohler and S. Kanzler, Synthesis, 1995, 539.

58W. Bergmann and R. J. Feeney, J. Am. Chem. Soc., 1950, 72, 2809. 59W. Bergmann and R. J. Feeney, J. Org. Chem., 1951, 16, 981.

60W. Bergmann and D. C. Burke, J. Org. Chem., 1956, 21, 226.

61C. J. Suckling, Sci. Prog. Edin., 1991, 75, 323.

62W. W. Lee, A. Benitez, L. Goodman and B. R. Baker, J. Am. Chem.

Soc., 1960, 82, 28.

63Parke Davis & Co., UK Pat.1151290, 1969.

G. Cimino, S. De Rosa and S. De Sttefano, Experientia, 1984, 40, 339.

65P. L. Darke, C. T. Leu, L. J. Davis, J. C. Heimbach, R. E. Diehl, W. S.

Hill, R. A. Dixon and I. S. Sigal, J. Biol. Chem., 19, 2, 2307. 66R. A. Wiley and D. H. Rich, Med. Res. Rev., 1993, 13, 327.

67F. Gago, IDrugs, 1999, 2, 309.

68K. Z. Rana and M. N. Dudley, Pharmacotheropy, 1999, 19, 35.

69B. D. Dorsey, J. P. Vacca and J. R. Huff, in Anti-infectives: Recent Advances in Chemistry and Structure–Activity Relationships, Royal Society of Chemistry, London, 1997, p. 238.

70A. H. Fray, R. L. Kaye and E. F. Kleinman, J. Org. Chem., 1986, 51, 4828.

71T. A. Lyle, C. M. Wiscount, J. P. Guare, W. J. Thompson, P. S.

Anderson, P. L. Darke, J. A. Zugay, E. M. Ermini, W. A. Schlief, J. C.

Quintero, R. A. F. Dixon, I. S. Sigal and J. R. Huff, J. Med. Chem., 1991, 34, 1228.

72J. P. Vacca, J. P. Guare, S. J. deSoloms, W. M. Sanders, E. A. Giuliani, S. D. Young, P. L. Darke, J. Zugay, I. S. Sigal, W. A. Scheif, J. C.

Quintero, E. A. Emini, P. S. Anderson and J. R. Huff, J. Med. Chem., 1991, 34, 1225.

73K. K. Chen and C. F. Schmidt, Medicine, 1930, 9, 1.

74J. W. Black and J. S. Stephenson, Lancet, 1962, 2, 311.

75A. Endo, M. Kuroda, Y. Tsujita, A. Terahara and C. Tamura, Ger. Pat.

2524355, 1975.

76A. Endo, J. Med. Chem., 1985, 28, 401.

77A. G. Brown, T. C. Smale, T. J. King, R. Hasenkamp and R. H.

Thompson, J. Chem. Soc., Perkin Trans. 1, 1976, 1165.

78A. Endo, J. Antibiot., 1979, 32, 852.

79A. Endo, J. Antibiot., 1980, 33, 334.

80A. W. Alberts, J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoffman, J.

Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monaghan, S. Currie, E. Stapley, G. Albers-Schonberg, O. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch and J. Springer, Proc. Natl. Acad. Sci. USA, 1980, 77, 3957.

81R. L. Monaghan, A. W. Alberts, C. G. Hoffman and G. Albers-Schonberg, US Pat.4231938, 1980.

82A. W. Alberts, J. S. MacDonald, A. E. Till and J. A. Tobert, Cardiol.

Drug. Dev., 19, 7, .

83P. R. Vagelos, Science, 1991, 252, 1080.

84G. M. Cragg, D. J. Newman and K. M. Snader, J. Nat. Prod., 1997, 60,

52.

85S. H. Ferreira, Br. J. Pharmacol., 1965, 24, 163.

86M. A. Ondetti, B. Rubin and D. W. Cushman, Science, 1977, 196, 441.

87D. W. Cushman, H. S. Cheung, E. F. Sabo and M. A. Ondetti, Biochemistry, 1977, 16, 5484.

88C. E. Terry and M. Pellens, The Opium Problem, Bureau of Social Hygiene, New York, 1928.

D. F. Musto, The American Disease, Yale University Press, New Haven, 1973.

90C. J. Weitz, K. F. Faull and A. Goldstein, Nature (London), 1987, 330, 674.

91J. Donnerer, G. Cardinale, J. Coffrey, C. A. Lisek, I. Jardine and S.

Spector, J. Pharmacol. Exp. Ther., 1987, 242, 583.

92W. R. Martin, Pharmacol. Rev., 1967, 19, 463.

93L. Terenius, Annu. Rev. Pharmacol. Toxicol., 1978, 18, 1.

94S. H. Snyder, Science, 1984, 224, 22.

95E. J. Simon and J. M. Hiller, Annu. Rev. Pharmacol. Toxicol., 1978, 18, 371.

96A. Goldstein, in Opioids: Past, Present and Future, ed. H. O. J. Collier, J. Hughes, M. J. Rance and M. B. Tyers, Taylor and Frances, London, 1984, p. 127.

97N. E. S. Sibinga and A. Goldstein, Annu. Rev. Immunol., 1988, 6, 219.

98T. H. Kramer, J. E. Shook, W. Kazmierski, E. A. Ayres, W. S. Wire, V. J. Hruby and T. F. Burks, J. Pharmacol. Exp. Ther., 19, 249, 544.

99B. M. Olivera, C. Walker, G. E. Cartier, D. Hooper, A. D. Santos, R.

Schenfeld, R. Shetty, M. Watkins, P. Bandyopadhyay and D. R.

Hillyard, Ann. N.Y. Acad. Sci., 1999, 870, 223.

100C. S. Walker, D. Steel, R. B. Jacobsen, M. B. Lirazan, L. J. Cruz, D.

Hooper, R. Shetty, R. C. DelaCruz, J. S. Nielsen, L. M. Zhou, P.

Bandyopadhyay, A. G. Craig and B. M. Olivera, J. Biol. Chem., 1999, 274, 306.

101B. M. Olivera, Mol. Biol. Cell, 1997, 8, 2101.

102A. Justice, T. Singh, K. C. Gohil, K. L. Valentino and G. P. Miljanich, in Omega Conopeptide Compositions, USP, 5587454/1996.

103T. P. Spande, H. M. Garrafo, M. W. Edwards, H. J. C. Yeh, L. Pannel and J. W. Daly, J. Am. Chem. Soc., 1992, 114, 3475.

104J. L. Ellis, D. Harman, J. Gonzalez, M. L. Spera, R. Liu, T. Y. Shen,

D. M. Wypij and F. Zuo, J. Pharmacol. Exp. Ther., 1999, 288,

1143.

105A. W. Bannon, M. W. Decker, M. W. Holladay, P. Curzon, D.

Donnelly-Roberts, P. S. Puttfarcken, R. S. Bitner, A. Diaz, A. H.

Dickenson, R. D. Porsolt, M. Williams and S. P. Arneric, Science, 1998, 279, 77.

106M. W. Decker and M. D. Meyer, Biochem. Pharmacol., 1999, 58, 917.

107P. Curzon, A. L. Nikkel, A. W. Bannon, S. P. Arneric and M. W.

Decker, J. Pharmacol. Exp. Ther., 1998, 287, 847.

108J. L. Hartwell, Plants Used Against Cancer, Quarterman, Lawrence, 1982.

109G. M. Cragg, M. R. Boyd, J. H. Cardellina II, D. J. Newman, K. M.

Snader and T. G. McCloud, in Ethnobotany and the Search for New Drugs, Ciba Symposium No. 185, 1994, p. 178.

110J. M. Pezzuto, Biochem. Pharmacol., 1997, 53, 121.

111H.-K. Wang, IDrugs, 1998, 1, 92.

112K.-H. Lee, J. Biomed. Sci., 1999, 6, 236.

113H.-K. Wang, S. L. Morris-Natschke and K.-H. Lee, Med. Res. Rev., 1997, 17, 367.

114M. Potmeisel and H. Pinedo, Camptothecins: New Anticancer Agents, CRC Press, Boca Raton, 1995.

115G. M. Cragg, S. A. Schepartz, M. Suffness and M. R. Grever, J. Nat.

Prod., 1993, 56, 1657.

116J. E. Cortes and R. Pazdur, J. Clin. Oncol., 1995, 13, 23.

117D. G. I. Kingston, A. G. Chaudhary, M. D. Chordia, M. Gharpure,

A. A. I. Gunatilaka, P. I. Higgs, J. M. Rimoldi, L. Samala, P. G. Jaktap,

P. Giannakakou, Y. Q. Jiang, C. M. Lin, E. Hamel, B. H. Long, C. R.

Fairchild and K. A. Johnston, J. Med. Chem., 1998, 41, 3715.

118M. C. Christian, J. M. Pluda, T. C. Ho, S. G. Arbuck, A. J. Murgo and

E. A. Sausville, Semin. Oncol., 1997, 24, 219.

119R. G. Naik, S. L. Kattige, S. V. Bhat, B. Alreja, N. J. de Sousa and R. H.

Rupp, Tetrahedron, 1988, 44, 2081.

120E. A. Sausville, D. Zaharevitz, R. Gussio, L. Meijer, M. Louarn-Leost,

C. Kunick, R. Schultz, T. Lahusen,

D. Headlee, S. Stinson, S. G.

Arbuck and A. Senderowicz, Pharmacol. Ther., 1999, 82, 285.

121C. Schultz, A. Link, M. Leost, D. W. Zaharevitz, R. Gussio, E. A.

Sausville, L. Meijer and C. Kunick, J. Med. Chem., 1999, 42, 2909. 122D. W. Zaharevitz, R. Gussio, M. Leost, A. M. Senderowicz, T.

Lahusen, C. Kunick, L. Meijer and E. A. Sausville, Cancer Res., 1999, 59, 2566.

123L. Havlicek, J. Hanus, J. Vesely, S. Leclerc, L. Meijer, G. Shaw and M.

Strnad, J. Med. Chem., 1997, 40, 408.

124J. Vesley, L. Havlicek, M. Strnad, J. J. Blow, A. Donelladeana, L.

Pinna, D. S. Letham, J. Kato, L. Detivaud, S. Leclerc and L. Meijer, Eur. J. Biochem., 1994, 224, 771.

125L. Meijer, A. Borgne, O. Mulner, J. P. J. Chong, J. J. Blow, N. Inagaki, M. Inagaki, J. G. Delcros and J. P. Moulinoux, Eur. J. Biochem., 1997, 243, 527.

126U. Schulze-Gahmen, J. Brandsen, H. D. Jones, D. O. Morgan, L.

Meijer, J. Vesley and S. H. Kim, Proteins, 1995, 22, 378.

127W. F. De Azevedo, S. Leclerc, L. Meijer, L. Havlicek, M. Strnad and S. H. Kim, Eur. J. Biochem., 1997, 243, 518.

128N. S. Gray, L. Wodicka, A.-M. W. H. Thunnissen, T. C. Norman, S.

Kwon, F. H. Espinosa, D. O. Morgan, G. Barnes, S. LeClerc, L. Meijer, S.-H. Kim, D. J. Lockhart and P. G. Schultz, Science, 1998, 281, 533.

129Y. T. Chang, N. S. Gray, G. R. Rosania, D. P. Sutherlin, S. Kwon, T. C.

Norman, R. Sarohia, M. Leost, L. Meijer and P. G. Schultz, Chem.

Biol., 1999, 6, 361.

Nat. Prod. Rep., 2000, 17, 215–234233130N. Gray, L. Detivaud, C. Doerig and L. Meijer, Curr. Med. Chem., 1999, 6, 859.

131K. R. Webster, Exp. Opin. Invest. Drugs, 1998, 7, 865.

132W. O. Foye, Cancer Chemotherapeutic Agents, American Chemical Society, Washington, DC, 1995.

133S. M. Hecht, Proc. AACR-NCI-EORTC Meeting, 1999, Abs. P3. 134P. Thavasu, D. Propper, A. McDonald, N. Dobbs, T. Ganesan, D.

Talbot, J. Braybrook, F. Caponigro, C. Hutchison, C. Twelves, A.

Man, D. Fabbro, A. Harris and F. Balkwill, Cancer Res., 1999, 59, 3980.

135D. Fabbro, E. Buchdunger, J. Wood, F. Hofmann, S. Ferrari, H. Mett, T. O’Reilly and T. Meyer, Pharmacol. Ther., 1999, 82, 293.

136G. Hofle, N. Bedorf, H. Gerth and H. Reichenbach, Ger. Pat.4138042, 1993.

137K. Gerth, N. Bedorf, G. Hofle, H. Irschik and H. Reichenbach, J. Antibiot., 1996, 49, 560.

138D. M. Bollag, P. A. McQueney, J. Zhu, O. Hensens, L. Koupal, J.

Liesch, M. Goetz, E. Lazarides and C. M. Woods, Cancer Res., 1995, 55, 2325.

139G. Hofle, N. Bedorf, H. Steinmetz, D. Schomberg, K. Gerth and H.

Reichenbach, Angew. Chem., Int. Ed. Engl., 1996, 35, 1567.

140K. C. Nicolaou, F. Roschangar and D. Vourloumis, Angew. Chem., Int.

Ed. Engl., 1998, 37, 2014.

141I. Ojima, S. Chakravarty, T. Inoue, S. Lin, He, S. B. Horowitz, S. D.

Kuduk and S. J. Danishefsky, Proc. Natl. Acad. Sci. USA, 1999, 96, 4256.

142R. Gussio, G. Paraskevi, E. Nogales, K. H. Downing, D. Zaharevitz, D.

Sackett, K. C. Nicolau and T. Fojo, Proc. AACR-NCI-EORTC Meeting, 1999, 130 Abs 0.

143O. J. McConnell, R. E. Longley and F. E. Koehn, in The Discovery of Natural Products with Therapeutic Potential, ed. V. P. Gullo, Butterworth-Heinemann, Boston, 1994, p. 109.

144B. K. Carte, BioScience, 1996, 271.

145G. R. Pettit, C. L. Herald, D. L. Doubek, D. L. Herald, E. Arnold and J. Clardy, J. Am. Chem. Soc., 1982, 104, 6846.

146D. J. Newman, in Bryozoans in Space and Time, Proceedings of the 10th International Bryozoology Conference, NIWA, Wellington, NZ, 1996, p. 9.

147P. A. Philip, D. Rea, P. Thavasu, J. Carmichel, N. S. A. Stuart, H.

Rockett, D. C. Talbot, T. Ganesan, G. R. Pettit, F. Balkwill and A. L.

Harris, J. Natl. Cancer Inst., 1993, 85, 1812.

148M. L. Varterasian, R. M. Mohammad, D. S. Eilender, K. Hulburd, D.

M. Rodriguez, P. A. Pemberton, J. M. Pluda, M. D. Dan, G. R. Pettit,

B. D. M. Chen and A. M. Al-Katib, J. Clin. Oncol., 1998, 16, 56. 149P. A. Wender,

C. M. Cribbs, K. F. Koehler, N. A. Sharkey, C. L.

Herald, Y. Kamano, G. R. Pettit and G. M. Blumberg, Proc. Natl.

Acad. Sci. USA, 1988, 85, 7197.

150P. A. Wender, J. De Brabander, P. G. Harran, J.-M. Jimenez, M. F. T.

Koehler, B. Lippa, C.-M. Park and M. Shiozaki, J. Am. Chem. Soc., 1998, 120, 4534.

151P. A. Wender, J. De Brabander, P. G. Harran, J.-M. Jimenez, M. F. T.

Koehler, B. Lippa, C.-M. Park, C. Siedenbiedel and G. R. Pettit, Proc.

Natl. Acad. Sci. USA, 1998, 95, 6624.

152P. A. Wender, J. De Brabander, P. G. Harran, K. W. Hinkle, B. Lippa and G. R. Pettit, Tetrahedron Lett., 1998, 39, 8625.

153P. A. Wender, B. Lippa, C.-M. Park, K. Irie, A. Nakahara and H.

Ohigashi, Bioorg. Med. Chem. Lett., 1999, 9, 1687.

154P. A. Wender, K. W. Hinkle, M. F. Koehler and B. Lippa, Med. Res.

Rev., 1999, 19, 388.

155K. L. Rinehart, T. G. Holt, N. L. Fregeau, J. G. Stroh, P. A. Keifer, F.

Sun, L. H. Li and D. G. Martin, J. Org. Chem., 1990, 55, 4512.

156A. E. Wright, D. A. Forleo, G. P. Gunawardana, S. P. Gunasekera, F. E.

Koehn and O. J. McConnell, J. Org. Chem., 1990, 55, 4508.

157T. Arai, K. Takahashi, S. Nakahara and A. Kubo, Experientia, 1980, 36, 1025.

158T. Arai, K. Takahashi, A. Kubo, S. Nakahara, S. Sato, K. Aiba and C.

Tamura, Tetrahedron Lett., 1979, 2355.

159J. M. Frincke and D. J. Faulkner, J. Am. Chem. Soc., 1982, 104, 265.

160E. J. Corey, D. Y. Gin and R. S. Kania, J. Am. Chem. Soc., 1996, 118, 9202.

161E. J. Martinez, T. Owa, S. L. Schreiber and E. J. Corey, Proc. Natl.

Acad. Sci. USA, 1999, 96, 3496.

162O. Kennard, W. D. G. L. Riva de Sanserverine, B. Tursch and R.

Bosmans, Tetrahedron Lett., 1968, 9, 2879.

163M. D’Ambrosio, A. Guerriero and F. Pietra, Helv. Chim. Acta, 1987, 70, 2019.1M. D’Ambrosio, A. Guerrerio and F. Pietra, Helv. Chim. Acta, 1988, 71, 9.

165M. Ciomei, C. Albanese, W. Pastori, M. Grandi, F. Pietra, M.

D’Ambrosio, A. Guerrerio and C. Battistini, Proc. Am. Assoc. Cancer Res., 1997, 38, 5 (Abstract 30).

166W. Fenical, P. R. Jensen and T. Lindel, US Pat.5473057, 1995. 167T. Lindel, P. R. Jensen, W. Fenical, B. H. Long, A. M. Casazza, J.

Carboni and C. R. Fairchild, J. Am. Chem. Soc., 1997, 119, 8744. 168B. H. Long, J. M. Carboni, A. J. Wasserman, L. A. Cornell, A. M.

Casazza, P. R. Jensen, T. Lindel, W. Fenical and C. R. Fairchild, Cancer Res., 1998, 58, 1111.

169K. C. Nicolaou, J.-Y. Xu, S. Kim, T. Ohshima, S. Hosokawa and J.

Pfefferkorn, J. Am. Chem. Soc., 1997, 119, 11353.

170X.-T. Chen, B. Zhou, S. K. Bhattacharya, C. E. Gutteridge, T. R. R.

Pettus and S. J. Danishevsky, Angew. Chem., Int. Ed. Engl., 1998, 37, 7.

171K. C. Nicolaou, S. Kim, J. Pfefferkorn, J.-Y. Xu, T. Ohshima, S.

Hosokawa, D. Vourloumis and T. Li, Angew. Chem., Int. Ed. Engl., 1998, 37, 1418.

172K. C. Nicolaou, J.-Y. Xu, S. Kim, J. Pfefferkorn, T. Ohshima, D.

Vourloumis and S. Hosokawa, J. Am. Chem. Soc., 1998, 120, 8061. 173K. C. Nicolaou, T. Ohshima, S. Hosokawa, F. L. van Delft, D.

Vourloumis, J. Y. Xu, J. Pfefferkorn and S. Kim, J. Am. Chem. Soc., 1998, 120, 8674.

174X.-T. Chen, S. M. Bhattacharya, B. Zhou, C. E. Gutteridge, T. R. R.

Pettus and S. J. Danishevsky, J. Am. Chem. Soc., 1999, 121, 6563. 175T. Lindel, Angew. Chem., Int. Ed. Engl., 1998, 37, 774.

176K. C. Nicolaou, N. Winssinger, D. Vouloumis, T. Ohshima, S. Kim, J.

Pfefferkorn, J.-Y. Xu and T. Li, J. Am. Chem. Soc., 1998, 120, 10814.

177E. Hamel, D. L. Sackett, D. Vourlanis and K. C. Nicolaou, Biochemistry, 1999, 38, 5490.

178D. A. Hopwood, Chem. Rev., 1997, 97, 2465.

179D. E. Cane, C. T. Walsh and C. Khosla, Science, 1998, 282, 63. 180L. Katz, Chem. Rev., 1997, 97, 2557.

181C. Khosla, Chem. Rev., 1997, 97, 2577.

182J. Staunton and B. Wilkinson, Chem. Rev., 1997, 97, 2611.

183C. R. Hutchinson, Curr. Opin. Microbiol., 1998, 1, 319.

184R. M. McDaniel, A. Thamchaipenet, C. Gustafsson, H. Fu, M. Betlach and G. Ashley, Proc. Natl. Acad. Sci. USA, 1999, 96, 1846.

185Y. Shen, P. Yoon, T.-W. Yu, H. G. Floss, D. A. Hopwood and B. S.

Moore, Proc. Natl. Acad. Sci. USA, 1999, 96, 3622.

186L. Zhao, J. Ahlert, Y. Xue, J. S. Thorson, D. H. Sherman and H.-W.

Liu, J. Am. Chem. Soc., 1999, 121, 9881.

187C. R. Hutchinson, Proc. Natl. Acad. Sci. USA, 1999, 96, 3336.

188R. R. Colwell, J. Ind. Microbiol., 1997, 18, 302.

1M. R. Rondon, R. M. Goodman and J. Handelsman, Tibtech, 1999, 17, 403.

190L. H. Caporale, Proc. Natl. Acad. Sci. USA, 1995, 92, 75.

191L. D. Mayer, Cancer. Met. Rev., 1998, 17, 211.

192E. S. Vitetta, P. E. Thorpe and J. U. Uhr, Immunol. Today, 1993, 14, 252.

193R. F. Sherwood, Adv. Drug Del. Rev., 1996, 22, 269.

194R. Duncan, Pharm. Sci. Technol. Today, 1999, 2, 441.

195P. J. Julyan, L. W. Seymour, D. R. Ferry, S. Daryani, C. M. Boivin, J.

Doran, M. David, D. Anderson, C. Christodolou, A. M. Young, S.

Hesslewood and D. J. Kerr, J. Controlled. Release, 1999, 57, 281. 196V. Omelyanenko, P. Kopeckova, C. Gentry and J. Kopecek, J.

Controlled Release, 1998, 53, 25.

197P. A. Vasey, S. B. Kaye, R. Morrison, C. Twelves, P. Wilson, R.

Duncan, A. H. Thomson, L. S. Murray, T. E. Hilditch, T. Murray, S.

Burtles, D. Fraier, E. Frigerio and J. Cassidy, Clin. Cancer Res., 1999, 5, 83.

198A. H. Thomson, P. A. Vasey, L. S. Murray, J. Cassidy, D. Fraier, E.

Frigerio and C. Twelves, Br. J. Cancer, 1999, 81, 99.

199E. Gianasi, M. Wasil, E. G. Evagorou and A. Keddle, Eur. J. Cancer, 1999, 35, 994.

200A. B. Smith III, M. D. Kaufman, T. J. Beauchamp, M. J. LaMarche and

H. Arimoto, Org. Lett., 1999, 1, 1823.

201A. L. Harvey, Trends Pharmacol. Sci., 1999, 20, 196.

202A. L. Harvey and P. G. Waterman, Curr. Op. Drug Discov. Develop., 1998, 1, 71.

234Nat. Prod. Rep., 2000, 17, 215–234