Protocols Natural Products as Foundation for Drug Discoveryng 2009

UNIT 9.11Natural Products as a Foundationfor Drug Discovery

John A. Beutler1

1National Cancer Institute, Frederick, Maryland

ABSTRACT

Natural products have provided chemical leads for the development of many drugs for diverseindications. While most U.S. pharmaceutical firms have reduced or eliminated their in-housenatural product groups, there is a renewed interest in this source of new chemical entities. Manyof the reasons for the past decline in popularity of natural products are being addressed by thedevelopment of new techniques for screening and production. The aim of this unit is to reviewcurrent strategies and techniques that increase the value of natural products as a source for noveldrug candidates. Curr. Protoc. Pharmacol. 46:9.11.1-9.11.21. C© 2009 by John Wiley & Sons,Inc.

Keywords: natural products � drugs discovery � HTS

HISTORY

Early Natural Product DrugsHumans have long used naturally occur-

ring substances for medical purposes. Plants,in particular, have played a leading role asmedicinals in most cultures. With the devel-opment of chemistry at the dawn of the 19thcentury, plants were examined more carefullyto understand their therapeutic utility. In 1804,Serturner purified morphine from opium andfound that it largely reproduced the analgesicand sedative effects of opium (Lockemann,1951). His success led others to seek “activeprinciples” of medicinal plants. Throughoutthe century, purified bioactive natural productswere extracted from cinchona (quinine), coca(cocaine; Gay et al., 1975), and many otherplants. The ability to determine the chemi-cal structure of these compounds developedmore slowly, with the planar structure of mor-phine determined only in 1923 by Gullandand Robinson (1923), and the structures ofquinine and cocaine in 1908 and 1898, re-spectively (Willstatter and Muller, 1898; Rabe,1908). It was not until 1956 that a methodfor synthesizing morphine was reported (Gatesand Tschudi, 1956). While the active principletheory does not explain all biological activi-ties of natural substances, its validity is firmlyestablished.

The antibiotic eraThe identification of the antibacterial activ-

ity of penicillin by Fleming (Fleming, 1929),

and its isolation by Chain and Florey (Chainet al., 1940), revolutionized medicine and ledto extensive screening of microbes, particu-larly soil actinomycetes and fungi, to identifyother antimicrobials. Using simple bioassays,organisms from soil samples were culturedand identified, resulting in the isolation andcharacterization of dozens of classes of antibi-otics. Many of these were ultimately commer-cialized, with some still employed clinically(Wenzel, 2004). While drug resistance limitsthe use of many antibiotics, their discoveryand clinical development laid the scientific andfinancial foundation for the pharmaceuticalindustry after World War II.

TaxolTo stimulate the development of cancer

therapies, in the 1960s the US NationalCancer Institute supported the establishmentof an extensive academic network examin-ing plant sources of potential anti-canceragents. To date, taxol (Wani et al., 1971)and camptothecin analogs (Wani and Wall,1969; Lerchen, 2002) are the most prominentdrugs resulting from this program. Unfortu-nately, neither of these drugs reached the mar-ket until the early 1990s. Difficulty in obtain-ing commercial quantities of taxol slowed itsadvancement, while camptothecin proved tohave poor solubility, requiring modificationsto its structure to improve clinical activity.Once launched, however, the use of taxol grewquickly, and it continues to be a mainstay ofcancer chemotherapy.

Current Protocols in Pharmacology 9.11.1-9.11.21, September 2009Published online September 2009 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471141755.ph0911s46Copyright C© 2009 John Wiley & Sons, Inc.

Drug DiscoveryTechnologies

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Supplement 46

Natural Productsin Drug Discovery

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Supplement 46 Current Protocols in Pharmacology

WHY HAS PHARMA MINIMIZEDTHE USE OF NATURAL PRODUCTSAS A SOURCE FOR NEW DRUGS?

Over the past decade, pharmaceutical com-panies have reduced their investment in nat-ural product research, with companies suchas Merck (Mullin, 2008) and Bristol MyersSquibb cutting staff and closing in-house pro-grams in this area. The waning interest innatural products has been most visible inthe United States, with some European andJapanese companies continuing to support thefield. Several reasons have been given for thistrend and are discussed below.

Discovery and Development of NaturalProducts is Perceived as a SlowProcess. It Does Not Match the Pace ofHigh-Throughput Screening (HTS)

Current HTS programs attempt to compressthe testing and prioritization of chemical hitsinto a period of several months. Even if naturalproduct extracts are tested first, the pace ofnatural product isolation makes it difficult tokeep up with the demand for hit structures inthis time frame. As detailed below, a numberof strategies have been developed to addressthis problem.

Natural product samples have most oftenbeen tested as whole fermentation broths or ascrude extracts of plants and marine organisms.Once a hit has been confirmed in the biolog-ical screen, the extract must be fractionatedto isolate the active compound(s), which typ-ically requires that bioassays be conducted ateach level of purification. Thus, the length oftime required to conduct the bioassay and re-port the results, and the number of separationcycles needed to obtain pure compounds, areimportant factors in dictating the time neces-sary to process and identify a natural producthit. Even when cycles are made on a weeklybasis using a rapid bioassay, rarely is the pu-rified compound isolated and identified in lessthan a month. Other factors affecting the speedof compound identification are instability ofthe active agent, difficult separations, and theunreliability of bioassays.

All of the Easy Natural Product DrugDiscoveries Have Been Made

This perception is sometimes expressed bythe sentiment “that pond’s all fished out.”While it is true that the number of species isfinite, it is also true that only a very small frac-tion of all species have undergone chemicalanalysis, let alone been examined in a broadpanel of bioassays. The number of higher plant

species is estimated to be between 300,000 and400,000. The largest plant-screening programof the 1960s was conducted by Smith Kline& French, with ∼19,000 species screenedfor alkaloid content using a simple color test(Raffauf, 1996). For over 20 years, the U.S.National Cancer Institute has collected higherplants for screening, with the current collec-tion composed of ∼30,000 species.

There is no simple way to tally the numberof microbial samples that have been screenedfor biological activity, as little effort is gener-ally made to identify the species before initi-ating tests in the selected bioassays. Certainly,the number of microbial samples screenedover the decades has been enormous, althoughthe taxonomic diversity of these samples islimited by the predilection for soil samples andthe difficulty in growing all but a small frac-tion of microbes in culture. Recent advances inenvironmental microbiology have shown thatthere is an enormous unsampled microbiota(Epstein and Lopez-Garcia, 2008). In view ofthese limitations, it is more accurate to statethat the pond has not been fished out, but ratherthat new types of bait, or new fishing strate-gies, must be employed to fully exploit it.

Marine invertebrates have been heavilysampled in the last two decades, and they haveprovided abundant new chemistry and biol-ogy (Blunt et al., 2004). However, the extentof biodiversity among marine invertebrates isunknown as there are too few taxonomists toidentify and classify new species, and becauseonly SCUBA-accessible, shallow, warm ma-rine waters have been thoroughly explored.

The argument that there is little more tobe found by natural product research is rem-iniscent of the claim by some 19th centuryphysicists that their field was nearing comple-tion. While this was perhaps true of Newtonianphysics, events of the last century have clearlyshown how short sighted those scientists were.Even if the new developments in natural prod-ucts consist of incremental advances in tech-niques, it seems clear that many “fish” remainin the pond.

The Synthesis of Natural Products istoo Difficult—the Structures are tooComplex

Natural product structures span the rangefrom very simple to extremely complex(Fig. 9.11.1). With improvements in struc-ture elucidation capability, it has been possi-ble to determine the complete stereostructureof natural compounds as complex as the pa-lytoxins (Moore and Bartolini, 1981; Uemura


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Current Protocols in Pharmacology Supplement 46

A

B

Figure 9.11.1 Extremes of complexity in natural product structures: (A) Palytoxin and (B) pinene.

et al., 1985a), compounds of molecular weight>2650 Da incorporating >60 chiral centers.While such agents are unlikely to ever be suit-able for commercial total synthesis, the vastmajority of natural products that have to datebeen isolated and elucidated are <1000 Da.In many cases, drugs have been developed bysynthetic modification of a naturally producedprecursor not requiring chemical synthesis.

Alternatively, structure-activity studiesconnected with total synthesis may make itpossible to drastically reduce the size and chi-rality of a bioactive natural product. Examplesof this are bryostatin (Wender et al., 2005) andhalichondrin (Dabydeen et al., 2006).

Resupply is DifficultObtaining large quantities of a natural com-

pound for preclinical studies can be a chal-lenge. If derived from a plant that grows in aremote tropical location, physical access fora recollection may be difficult, or permissionto collect and ship the material may be hard to

obtain. In addition, the plant may only producequantities of the desired compound under cer-tain environmental or ecological conditions.A marine organism may require an expensiveexpedition, especially if the animal grows indeep waters or in regions with strong or un-predictable currents. Even when one has a mi-crobial culture in hand, the factors that induceproduction of the metabolite may not be under-stood. Pharmaceutical companies clearly pre-fer predictable, controllable sources, and forcommercial viability, solutions to the prob-lems associated with natural product produc-tion must be found (see Sourcing below).

Combinatorial Chemistry is Viewed asSuperior to Natural Products Researchin Identifying New Chemical Entities

Parallel synthesis techniques make it possi-ble to create synthetic libraries of hundredsof thousands of distinct compounds. How-ever, such rapid synthetic techniques have notyet appeared to have accelerated the drug


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discovery process. Early combinatorial li-braries were composed of compounds withpoor solubility and yielded few useful hits. Insome cases, the quantities of compound pro-duced were very small and the purity poorlycontrolled. Although more recently, smaller,focused libraries have yielded some usefuldrug leads, the most powerful role of paral-lel synthesis appears to be in expanding anexisting lead, rather than creating screeninglibraries.

WHY NATURAL PRODUCTS AREATTRACTIVE FOR DRUGDISCOVERY

Secondary Metabolites Have Evolvedto be Bioactive

The metabolic energy and the genetic costof making a small molecule requires that it im-part some benefit to the organism, such as de-fending it against predators or providing someadvantage over competing organisms. Whilethe functions of most natural products in theirproducing organism are not currently known,opinion has shifted markedly since the dayswhen these chemicals were generally viewedas waste products. Whatever the precise role,it is becoming clear that many natural prod-ucts must be able to reach receptor sites withincells, just as a drug does. The large number ofpure natural products found to interact withspecific mammalian receptors testifies to theinherent bioactivity in these substances. Forexample, at the GABA receptor, known natu-ral product ligands include muscimol (Brehmet al., 1972), bicuculline (Johnston et al.,1972), securinine (Beutler et al., 1985), andpicrotoxin (Akaike et al., 1985).

Structures are Not Limited by theChemist’s Imagination

While chemists may be as creative as nat-ural systems, the natural systems have beenat it for a much longer time. The most im-portant and visible value of natural productchemistry is the introduction of novel molecu-lar skeletons and functionalities. Examples ofthis include mitomycin (Stevens et al., 1965),bleomycin (Umezawa, 1976), and esperamicin(Golik et al., 1987).

The Lipinski Rules of Five Do NotApply to Natural Products

These rules were developed to assist syn-thetic chemists in preparing compounds withbiophysical properties generally needed fororal bioavailability and appropriate pharma-cokinetics. Thus, compounds should be under

the molecular weight of 500 Da, possess <5hydrogen bond donors, <10 hydrogen bondacceptors, and have log P<5 (Lipinski et al.,1997). What is not well appreciated is thatLipinski explicitly excluded natural productsfrom the rules, primarily for the reasons setforth above (see Secondary Metabolites HaveEvolved to be Bioactive), and because theyoften utilize transporters rather than passivediffusion to enter cells (Lipinski et al., 1997).

HIGH-THROUGHPUT SCREENINGAND NATURAL PRODUCTS

Miniaturization and ReductionismHigh-throughput screening for new chemi-

cal entities grew out of automated clinical an-alyzer technology and miniaturization in thelate 1980s, as drug screeners sought methodsto increase the pace of testing and lower thecosts per sample. Robotic methods of samplemanipulation and specialized detectors capa-ble of reading 96-well microtiter plates weredeveloped. At the same time, the emphasis ofscreening shifted from empirical measures ofcell growth or function to molecular targets.This was driven by an increasing knowledgeof genes and receptor biology.

Cell-Free or Cellular Assays?In its most extreme reductionist forms, tar-

geted screening started with detection of theinteraction of test compounds with a purified,naked protein. Hits from that experimentalmodel were then tested in a functional as-say before progression to a cellular, and, ul-timately, tissue and organ system. Because thehighest level of reductionism provides the low-est barrier to successfully identifying activecompounds, the large number of hits has to befiltered by secondary, tertiary, and even qua-ternary assays. Abundant and common naturalproducts such as tannins (see Tanninsbelow) overwhelmed reductionist assay strate-gies with their high hit rates.

There has been a major shift in the lastdecade to assays conducted in cells, and thosein which biological function is directly mea-sured. These typically can be tuned to higherstringency, and therefore lower hit rates, whiledelivering samples with the desired biologicalproperties.

Change the Assay or Change theSample?

Natural product samples are not alwayswell behaved even in cellular assays, orfunctional cell-free assays. The question arises


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as to whether it is better to adapt the assay tothe sample or vice versa. Both tactics haveshown success, with the choice dependingupon the relative availability of resources inchemistry and biology.

A common problem with natural productextracts is that a substantial proportion fluo-resce in the fluorescein range (emission maxi-mum 521 nm). This leads to a high number offalse positives in a screen with a direct fluores-cent endpoint. If the endpoint is changed to afluorescent label emitting at >560 nm (Cy3B,for example), much less autofluorescence isseen. Alternatively, use of a time-resolved flu-orescence label also substantially decreasessample interference. Most sample autofluo-rescence has a short half-life (i.e., 10 nsec),while europium fluorescence labels, for exam-ple, have a much longer half-life (∼700 msec).Thus, by gating the photodetector to record thesignal after a 1-msec delay, the majority of thesample autofluorescence is filtered out, whilethe label is sensitively detected (Hemmila andWebb, 1997).

Prefractionation of ExtractsOne approach to sample modification that

has attracted significant interest is “prefrac-tionating” the crude extract. In its mostcomplex forms, this means isolating purecompounds and partially characterizing thembefore testing them in bioassays. Several com-panies have embraced this as a business model,with mixed success (Bindseil et al., 2001;Eldridge et al., 2002). Simpler, lower coststrategies, which separate the crude extractinto 5 to 15 samples based on a single chro-matography step, followed by solvent evap-oration, may provide much of the benefit ata reduced cost (Bugni et al., 2008; Wage-naar, 2008). All of these approaches requirea capital investment, with automated weigh-ing capability, flexible programmable liquidhandling, and low-cost separation media beingneeded.

There are several benefits to this approach(1) cytotoxic compounds, which might maskactivity of another compound in a cellu-lar assay may be separated; (2) minor con-stituents are concentrated and can be testedat higher effective concentration; (3) very po-lar or lipophilic constituents of an extractcan be ignored or discarded entirely. The ini-tial testing results from several laboratoriesthat adopted prefractionation strategies sup-port their use and they have demonstratedhigher hit rates in screening assays.

SOURCING

Natural Sources for Drug DiscoveryAs noted above, plants have historically

played the leading role in providing drugsor drug leads, with microbes following inthe antibiotic era. Screeners have more re-cently examined marine sources, once the in-vention of SCUBA made it easier to collectand study algae and marine invertebrates. Onlya few marine natural products have reachedthe clinic, although many marine compoundsare active in screens and quite a few havebeen evaluated preclinically. Compound sup-ply has been a major hurdle in the advancementfrom marine invertebrate sources. For exam-ple, bryostatin 1 was initially produced from itsmarine source organism, Bugula neritina, un-der Good Manufacturing Practices (Schaufel-berger et al., 1991). However, a mere 18 g ofmaterial was purified from 14,000 kg of theproducing bryozoan. Mariculture of the sameanimal has since been accomplished with suc-cessful production of bryostatin 1 (Mendola,2003).

A few programs have used insects as ascreening source, notably in a collaborationbetween the Merck and InBio in Costa Rica(Sittenfeld et al., 1999), and in the Eisner lab-oratory at Cornell University (Schroder et al.,1998). Also notable is the work of John Dalyusing amphibians as a rich source of bioactivecompounds (Daly et al., 2005). Epibatidine, afrog alkaloid (Badio and Daly, 1994), servedas the stimulus for design of the analgesic drugcandidate ABT-594 (Arneric et al., 2007).

Microbial or Dietary Origin of Marineand Plant Metabolites

Natural product scientists often encounterdifficulties in obtaining reliable productionof desired compounds from their produc-ing organism. For example, it is common inmicrobial screening to confirm bioactivity byre-growing the microbe under the same condi-tions as those that produced the initial screen-ing sample. In these cases, a success rate of50% is common. Likewise, when a plant iscollected for re-isolation, it is not unusual tofind lower amounts of the desired metabolite,or no compound at all. With marine inverte-brates, this occurrence is quite common.

The reasons for these problems are poorlyunderstood, but clearly there are a variety ofcauses. With microbes, obtaining good pro-duction of a desired metabolite is often a mat-ter of studying the culture conditions (e.g., me-dia, time, temperature, and oxygenation) and


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defining the best conditions for reliable pro-duction. With plants, the problem may be apoor understanding of taxonomy, with carefulbotanical field studies possibly revealing sev-eral closely related species, only one of whichproduces the compound in question (McKeeet al., 1998a). Dependence of metabolite pro-duction on environmental factors (climate,season, herbivore pressure) also often plays animportant role for plants and requires study.For marine invertebrates, a poor understand-ing of taxonomy plays a role but, additionally,vectoring of metabolites from one organismto its predator and sequestration in the secondorganism is important in some cases (Thomset al., 2006; Paul and Ritson-Williams, 2008).Dietary sources of bioactive compounds havealso been identified in amphibians, which con-sume arthropods and other small leaf litter an-imals (Saporito et al., 2003; 2004; 2007).

A final reason for erratic production may bethat the higher organism is not the source ofthe compound at all. Rather, the desired chemi-cal may be produced by a microbial symbiont.In many cases, marine invertebrates containcompounds that look suspiciously like micro-bial metabolites (Simmons, et al., 2008). Insome cases, similar compounds have been iso-lated from both a marine invertebrate and amicrobe (Suzumura et al., 1997; McKee et al.,1998b). If the microbe is an obligate symbiont,proof of the relationship may be difficult to ob-tain. A very good case was made by Haygoodet al. that bryostatins are produced by a sym-biont, although the details of the symbiosisare yet to be completely defined (Hildebrandet al., 2004). Similar reports for some plant-derived compounds are also intriguing, as inthe isolation of taxol from an endophytic fun-gal associate of the Pacific yew (Stierle et al.,1993).

Synthesis of Natural Products versusBiological Production

Organic chemists have made great stridesin their ability to synthesize complex, chiralmolecules such as natural products. While dif-ficulty and cost still are largely a function ofthe number of chiral centers and molecularweight, total synthetic approaches to naturalproducts are becoming more commercially vi-able as a sourcing option. Given sufficient re-sources, it is possible to reduce the numberof synthetic steps to the target molecule andimprove the yield at each step, while usinginexpensive starting materials. Bryostatin isa good example of this advance. Bryostatin2 has been synthesized in 40 steps (Evanset al., 1999), and although a total synthesis ofbryostatin 1 has not yet been reported, it canbe converted from bryostatin 2 (Pettit et al.,1991b). Wender’s group, bypassing synthesisof the natural product, has developed syntheticroutes to “bryologs” (Fig. 9.11.2), which dis-play potent activity similar to bryostatin 1 buthave simplified structures. One recent, highlyactive bryolog was prepared in 10 steps inan overall yield of 30 percent (Wender et al.,2008).

A second example of synthetic success witha complex natural product is that of hali-chondrin B. Wild collection of the produc-ing sponge gave poor yields (Uemura et al.,1985b; Pettit et al., 1991a), and elaborate mar-iculture in New Zealand yielded similar levelsof this agent (Munro et al., 1999). Total syn-thesis by the Kishi group was accomplished(Aicher et al., 1992) and, in the process, severalfragments half the size of the natural productwere identified that possess all of the bioactiv-ity (Wang et al., 2000; Dabydeen et al., 2006).This led to the clinical development of eribulin(Fig. 9.11.3; Newman, 2007).

BA

Figure 9.11.2 (A) Bryostatin 1 and a (B) bryolog (Wender et al., 2008).


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A

B

Figure 9.11.3 (A) Halichondrin B and its simplified analog (B) eribulin (Newman, 2007).

Biosynthesis in HeterologousOrganisms

Investigation of the biosynthetic path-ways that lead to secondary natural productshas gained momentum as DNA sequencingtools have improved (Galm and Shen, 2007).The biosynthesis of polyketide natural prod-ucts has attracted the most attention becausemany commercial antibiotics are largely de-rived from this pathway. Non-ribosomal pep-tide synthesis, terpenoid biosynthesis, andflavonoid pathways have also been elucidatedin many organisms. A key observation hasbeen that many of these pathways consist ofmodular gene clusters that can be manipulatedas a whole unit (Donadio et al., 1991). Polyke-tide synthase modules share enough homologythat they can be isolated from relatively dis-tantly related organisms by lowering the strin-gency of hybridization reactions. In fact, suchmodules may be detected in uncultivatable mi-crobes (Piel, 2002).

This opens up the possibility of express-ing the module in a convenient heterologousorganism and obtaining the desired secondarymetabolite, if appropriate precursors are avail-able and other cellular machinery is compat-ible with the metabolite’s production (Zhanget al., 2008). In addition, by altering the mod-ule, analogous metabolites may also be pro-

duced (Xu et al., 2009). It has even been pos-sible to predict the biosynthetic product fromthe sequence of a polyketide module (Ban-skota et al., 2006).

ETHNOBOTANYKnowledge of the medicinal effects of

plants is certainly not limited to European cul-tural traditions. Botanists trained in anthro-pology have studied many non-western cul-tures to inventory their use of plants and othernatural substances for medical and other pur-poses. Chemical and pharmacologic investiga-tion of ethnobotanical information is a viablealternate pathway to high-throughput screen-ing for drug discovery, although it has its ownlimitations.

First, cultural concepts of disease are notperfectly aligned. While most cultures readilyrecognize a superficial fungal infection or di-abetes in the same way that western medicinedoes, disease concepts such as cancer are notinterpreted in the same way in different cul-tures (Hartwell, 1967), although some haveclaimed that plants used for medicinal pur-poses yield a higher fraction of anti-canceractivity than unselected plants (Spjut, 2005).Secondly, the medicinal effects of many plantsin traditional cultures may be less specificthan that desired by western medicine. For


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example, tannins are often found in herbalpreparations and they may play a role in theirbiological activity; however, they are not wellsuited to drug development. SP-303, devel-oped by Shaman Pharmaceuticals (Holodniyet al., 1999), was a carefully defined tanninpreparation from Croton lechleri, a Peruvianethnobotanical (Williams, 2001), which wastested for several prescription indications be-fore finally being launched as an over-the-counter anti-diarrheal agent.

A third issue is exemplified by bothChinese traditional medicine and the IndianAyurvedic system. Both of these ancient tra-ditions utilize polyherbal preparations for themajority of prescriptions. Each component isthought to play a particular role, in somecases by modulating the toxicity of anothercomponent. This complexity makes activeprinciple analysis difficult, and reductionistapproaches to Chinese and Ayurvedic prepa-rations have been largely unsuccessful in val-idating their traditional uses, although manybioactive molecules have been isolated fromboth pharmacopeias (Tang and Eisenbrand,1992; Deocaris et al., 2008). The use of mi-croarrays to study the in vivo effects of suchcomplex preparations may hold some promisefor a better understanding of their potentialapplications (Yin et al., 2004).

CHEMICAL ECOLOGYWhile it is now appreciated that natural sub-

stances are generally not waste products of theproducing organism, the purpose they servefor their host is rarely similar to their potentialuse in clinical medicine. However, most drugsact through interactions with protein receptors,and protein domains, though not their precisefunction, are widely conserved (Rompler et al.,2007). Thus, ligands targeted to a particulardomain may also have activity in an orthol-ogous or paralogous receptor. C. elegans, forexample, has been proposed as a model organ-ism for anti-Parkinson drug screening; manyof the compounds, which affect dopaminer-gic systems in humans, also have more or lessparallel effects in worms (Nass et al., 2008).

Investigation of the ecological function ofnatural products is a field unto itself, with elu-cidation of the role a compound plays beingextremely difficult to ascertain experimentally.Roles that have been successfully addressedinclude insect antifeedant effects (Lidert et al.,1987), allelopathy (interference with growthof competitors; Tseng et al., 2001), and en-docrine disruption (Dinan and Lafont, 2006).

NATIONAL CANCER INSTITUTE(NCI) LETTER OF COLLECTION

In the late 1980s, contracts were awardedby the U.S. National Cancer Institute for thecollection of large numbers of plant, micro-bial, and marine samples worldwide. Permitswere required for collecting in many differ-ent countries, and assurances were needed thatthe rights of the source country would be re-spected in the drug development process. Tothat end, the NCI developed a standard Let-ter of Collection, which could be signed byboth parties (Cragg and Newman, 2005a). Thisletter states the NCI’s willingness to collabo-rate with source country scientists, to depositvoucher specimens in source country reposi-tories, and to develop benefit-sharing arrange-ments when patents were filed. In addition,Memoranda of Understanding could also bedeveloped to frame direct collaborations.

CONVENTION ON BIODIVERSITYThe NCI agreements predated and presaged

the 1992 Rio Convention on Biological Diver-sity (CBD). While the U.S. has not signed thetreaty, U.S. Department of State policy callsfor following the principles of the treaty. TheCBD is aimed at preserving biological diver-sity, protecting the source country genetic re-sources from exploitation, equitable sharingof the benefits of technology, and facilitatingtechnology transfer to the source country.

While it is generally perceived that the CBDhas made access to natural products resourcesmore difficult, it has interrupted the worstabuses of source countries by the developedworld. However, the CBD has not resolved thepolitical issue of how benefits should be dis-tributed within the source country. See for ex-ample, the case of Hoodia, a weight-loss prod-uct from the San people in South Africa. Theactive constituents of Hoodia were patentedby government scientists at the South AfricanCSIR and licensed to Phytopharm plc andUnilever (Wynberg, 2004; Anonymous, 2006;Bladt and Wagner, 2007).

TECHNIQUES IN NATURALPRODUCTS DRUG DISCOVERY

ExtractionBefore organisms or their tissues can be

tested, they must undergo an initial extractionto separate the desired small molecules fromthe biopolymers (proteins, cellulose, chitin,nucleic acids) that comprise the bulk of thesample. In the case of plants, it is common todry the plant parts thoroughly in the field at


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the point of collection, before extraction, sothat the material does not decompose en routeto the laboratory. To accelerate extraction, thedry tissue is ground using any of several mills(e.g., a Wiley mill or a hammer mill). Alter-natively, tissues may be frozen, although thisis expensive and cumbersome in many cases.Frozen material may be lyophilized. If DNA ormRNA is desired for cloning of proteins, flashfreezing the freshly collected tissue into liquidnitrogen is required to obtain useful material.

There are very few standard techniquesfor extraction, as the choice of solvent andconditions depends on the spectrum of smallmolecules desired. For extraction of drug-likemolecules of intermediate polarity, percolationat room temperature with a 1:1 (v/v) mixtureof dichloromethane and methanol is useful.Extraction techniques that involve heating thesolvent and extracted compounds, as in aSoxhlet apparatus, are generally avoided un-less the desired compounds are known to beheat stable. When preparing samples to beused in biological screening, heating shouldbe avoided.

Tissues of marine invertebrates presentunique problems in extraction, because of highwater and salt content. A solution adopted atthe NCI has proven generally applicable toa wide variety of marine specimens. Frozensamples are broken into pieces small enoughto be fed into a commercial hamburger grinderwith CO2 pellets. The resulting powdered ma-terial is stored frozen long enough for the CO2

to sublime, then thawed briefly, and stirredwith water as a slurry. Filtration through pa-per in a low-speed centrifuge removes the mu-cilaginous tissue, and the resulting aqueousextract is freeze-dried. The marc (remainingsolid residue) is also lyophilized and then ex-tracted with the methylene chloride-methanol1:1(v/v) mixture.

The solvent must then be removed fromthe extraction solutions. This is done to obtaina weight for the extracted material, as wellas to avoid reactions in solution, which mayalter the constituents. Aqueous solutions arelyophilized, while organic solvent mixtures aredried using rotary evaporators. A final finish-ing under high vacuum removes most solventtraces. Materials should be stored in borosili-cate glass bottles or vials at −20◦C to ensurestability.

For high-throughput screening (HTS) ap-plications, it is common to store libraries in aDMSO solution. DMSO is an extraordinarilygood solvent for most natural product sam-

ples, including extracts. Organic extracts canoften be entirely dissolved at concentrationsof 10 to 100 mg/ml in 100% DMSO, while50% DMSO solutions of aqueous extracts arepossible. It should be noted that DMSO con-centrations >25% generally suppress bacte-rial growth in aqueous extract solutions. Thebulk extract material should not be stored inDMSO, however, since this solvent can facili-tate a number of oxidation reactions. In addi-tion, the hygroscopic nature of DMSO leads tomoisture absorption even in nominally sealedmicroplates in the freezer (Ellson et al., 2005).Extract plates should be reconstituted frombulk stocks on an annual basis to avoid de-terioration of the samples.

Each bioassay in which these extracts aretested will have a limit to tolerance of DMSO.With cellular assays, this is usually 0.5% to 1%of assay volume. For biochemical assays, it isoften as high as 5% to 10% of assay volume.The limit should be defined for the particularassay in advance and DMSO controls run ineach experiment.

SeparationsOnce an extract has been confirmed as a

hit in a biological assay, the active compoundsin the extract must be identified. This is ac-complished in an iterative process of sepa-ration and bioassay termed bioassay-guidedfractionation. An extract is separated into sev-eral fractions and the parent extract and frac-tions are tested in the assay. Several outcomesare possible. One is that all activity may belost in the daughter fractions, in which casethe separation method is deemed unsuitable.Loss of biological activity may be due to irre-versible binding to the separation media, or toinstability of the active compound. A secondoutcome would be for all, or most, daughterfractions to have some low amount of activity.This too is undesirable and simply indicatesthat the separation mode is not suitable. Thethird and most desirable outcome is that one orseveral daughter fractions contain substantialbioactivity, and that the mass of active frac-tions has been reduced from the parent witha corresponding increase in potency. A usefultechnique in monitoring separations is to cal-culate both mass and activity recoveries for theprocess. Thus, if 5 g of a parent extract wereseparated, yielding a summed fraction mass of4.5 g, the mass recovery would be 90 percent.If dose-response curves are available for theassay, bioactivity recovery can be calculatedby the equation


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(M /I )

M /I

i i

p p

∑

where Mi are the masses of the fractions, Ii

are the IC50 values for each fraction, and Mp

and Ip the respective values for the parent ex-tract. If a fraction has no activity, the term canbe ignored. This calculation is limited by theprecision of the bioassay, but can be useful injudging the success of a trial separation.

The invocation of synergism to explain lossof activity on fractionation has only rarelybeen substantiated experimentally. If activityis lost, most commonly it is attributable tocompound instability or irreversible bindingto chromatography media. Given a suitablyprecise assay, calculation of mass and activ-ity recovery can often yield clues to the sourceof the problem.

A single separation step is rarely sufficientto obtain purified active compounds. While useof high-performance chromatography can of-ten yield a superb separation of complex mate-rials, it is more cost-effective to save the highperformance step for last, since crude extractscan often wreak havoc on expensive prepar-ative HPLC columns. The most useful firstseparation process is one based on polarity.For example, the so-called Kupchan partitionuses a series of two-phase mixtures in a sepa-ratory funnel to sort components by partitioncoefficient. While simple, the technique suf-fers from a propensity to form emulsions, andfrom difficulties associated with evaporatingwater-saturated organic layers to dryness. Amore convenient approach for organic plantextracts uses solid-phase extraction with diol-bonded phase media, with increasingly po-lar solvents used to elute successive fractions(Beutler et al., 1990). The procedure can bescaled over a wide range of volumes, and it in-troduces no water into the samples. For marinesamples, a wide-pore C4 bonded phase schemecan be used with methanol-water mixtures toseparate the large amount of salts and otherpolar material from the more drug-like inter-mediate polarity fractions (Cardellina et al.,1993).

Intermediate resolution techniques such asflash chromatography or gel permeation chro-matography are useful once the polarity cutshave been made. Open column systems us-ing Sephadex LH-20 with a variety of solventsseparate based on both size exclusion and ad-sorption mechanisms, and can be very useful.

Final purification is most often accom-plished by preparative HPLC. A wide variety

of bonded phases are available (e.g., cyano,C18, phenyl, diol, amino) for this purpose.These can be operated in reversed-phase ornormal phase modes, as well as by ion ex-change or hydrophilic interaction chromatog-raphy. Pilot thin-layer chromatography exper-iments can provide useful hints as to the bestchoice of column packing and elution con-ditions. Then, analytical scale HPLC may beused to define precise flow and solvent strengthparameters. Even with relatively purified frac-tions, it is often useful to use gradient elutionto obtain an optimum separation. While C18bonded phases dominate the analytical chem-istry market, they are only one of the tools inthe HPLC column drawer of a natural productsisolation laboratory.

It is also important to pay attention to peakdetection. It is common practice to use UVdetection at 254 nm, which is useful for analy-sis of many drugs with suitable chromophores.However, many constituents of natural materi-als lack absorbance in this range. The most ef-fective strategy is to use lower wavelengths fordetection. For example, for acetonitrile-watersystems it is possible to use wavelengths aslow as 200 nm to observe compounds withpoor UV absorbance. An alternative is to useevaporative light scattering detection or re-fractive index detection, although neither ofthese modes is very well suited for larger scaleseparations.

Next, the separation must be scaled upto semi-preparative or preparative scale usinglarger diameter HPLC columns with the samelength, column chemistry, particle size, andporosity. Loading studies with increasing in-jections of material establish how much masscan be effectively separated in one run. Thehigh cost of larger columns is readily offsetby the shorter time required to run the separa-tion, with columns as large as 41-mm diameterbeing used with laboratory-scale pumping sys-tems capable of delivering 50 to 100 ml/minof solvent to the column. If flow rates andinjection volumes are scaled proportionately,preparative separations can be obtained withthe same reproducibility and resolution as an-alytical separations. The sample injected onan expensive preparative scale column mustbe carefully filtered and the solvent conditionsmust be chosen to elute virtually all of theapplied sample, otherwise particles and otheruneluted material will rapidly degrade columnperformance.

An excellent overview of preparative chro-matographic techniques applied to naturalproduct isolation is recommended for those


9.11.11


seeking additional information on this topic(Hostettmann et al., 1998).

Structure ElucidationOnce the active compounds are obtained

in pure form, their chemical structures canbe elucidated. The key technique for thispurpose is NMR. Specifically, a series oftwo-dimensional experiments (COSY, HSQC,HMBC, NOESY) are performed that make itpossible to establish the connectivity of allhydrogen and carbon atoms in a molecule.Serving a very important complementary roleis high-resolution mass spectrometry (MS),which is capable of providing precise massmeasurements that identify the molecular for-mula of the compound. It is often possible tofully elucidate the structure of an unknownmolecule using these two techniques alone.Other spectroscopic techniques such as UV,IR, and optical rotation serve ancillary roles,although they may be critical in specific cases.As the number of atoms in a molecule in-creases, structure elucidation becomes moredifficult due to the exponential increase inpossible structures for a given formula. It iscurrently routine to determine structures ofcompounds under a molecular weight of 500Da, while structural elucidation of compoundsover 2000 Da nearly always requires exten-sive chemical transformations. Exceptions aresmaller biopolymers such as peptides that canbe routinely sequenced if all of the constituentrepeating components are known.

The ability of NMR and MS to provideuseful information from smaller amounts ofcompound has increased dramatically in re-cent years. Advances in NMR probe design,especially gradient probes, flow probes, andcryoprobes, have greatly increased sensitiv-ity (Reynolds and Enriquez, 2002). Higher-field-strength magnets have increased NMRspectral dispersion so that more peaks canbe resolved in a spectrum. Improved NMRpulse sequences have reduced experiment timeand resolution. Similar improvements havebeen made in MS, with electrospray ion-ization and matrix-assisted laser desorptionbeing two ionization techniques that haveproven their value in natural product char-acterization. Cutting edge techniques suchas Fourier-transform ion cyclotron resonancemass spectrometry (FTICR-MS) have been ap-plied in industrial settings with utility in struc-ture elucidation, but the cost of the equipmenthas kept it from being widely applied (Fengand Siegel, 2007).

An alternative technique for structure elu-cidation is X-ray crystallography, which has along history in natural product structure eluci-dation. It is still an important technique, espe-cially for determining the absolute configura-tion of complex chiral molecules. The majorlimitation of this approach is that the com-pound must exist in a crystalline form. If thenative compound cannot be persuaded to crys-tallize, it can be derivatized with a variety ofmodifiers to attempt to improve its ability toform crystals. Application of robotics to auto-matically generate many small-scale crystal-lization experiments has increased the abilityto find workable crystallization conditions.

Hyphenated TechniquesHyphenated techniques such as HPLC-MS,

HPLC-UV, and HPLC-NMR are useful analyt-ical platforms for detection, identification, andquantification of compounds in extracts. Thus,they serve as important tools for determiningthe compounds in a sample, and may informpreparative separation methods. They form animportant part of chemical dereplication (seebelow). In addition to coupling several dif-ferent detection methods, HTS bioassays maybe conducted on individual fractions to com-plement the physicochemical data. One of thedrawbacks of using hyphenated techniques isthe large datasets that are generated for eachrun. Managing, analyzing, and interpreting theresults can be a daunting task.

Dereplication

Biological and chemicalWith over 150,000 known small molecules

characterized from natural sources, previouslyknown natural products are often re-isolated inthe course of bioassay-guided fractionation.While this may be acceptable if the biolog-ical activity is new, it is frustrating to wasteresources on the de novo structure elucida-tion of known compounds. This problem firstemerged in the antibiotic industry, where mi-crobial cultures were generally not identifiedprior to screening. Methods intended to avoidinvesting resources in the elucidation of knowncompounds go by the general term of derepli-cation (Corley and Durley, 1994). In all itsforms, this process attempts to shift the iden-tification of known compounds to an earlierpoint in the discovery process, either beforea pure active substance is isolated, or beforea complete NMR data set is acquired andanalyzed.


9.11.12


Most effective is a combination of biologi-cal and chemical methods. If the source organ-ism has been identified, reference to databasesof known compounds such as the CRC PressDictionary of Natural Products (Buckinghamand Thompson, 1997) can suggest candidatestructures. Physicochemical data, in particularultraviolet spectra and mass spectra, if avail-able, can rapidly limit the scope of possiblecompounds, especially when combined withanalytical HPLC (Lang et al., 2008).

Direct physical comparison with standardcompounds can be a very effective tactic, al-though amassing a library of known com-pounds is a huge task for most laboratories.

Nuisance compoundsNot all of the compounds contained in a nat-

ural product extract are desirable as drug leads.It is generally desirable to dereplicate samplescontaining nuisance compounds so that effortscan be focused on compounds with greaterdrug development potential. Several classes ofsuch undesirables are described below.

TanninsTannins are polyphenolic plant metabolites,

which were initially discovered as the princi-ples responsible for tanning leather. Oak barkand many other plant materials contain sub-stantial quantities of tannins, complex molec-ular structures that incorporate gallate esters(hydrolysable tannins, e.g., Fig. 9.11.4A) orflavanol polymers (condensed tannins, e.g.,Fig. 9.11.4B; Khanbabaee and Ree, 2001).Phlorotannins are a third class found in brownalgae, which have similar properties. Tanninsplay an important ecological role in deterringfeeding by herbivores, and they may be pro-duced in response to tissue injury. Many tan-nins are antinutritional, that is, they reduce thedigestibility of protein in foods (Butler, 1992).The mechanism for both the tanning effect andthe antifeedant/antinutritional roles is nonco-valent binding to proteins. Since this is a rela-tively nonspecific effect—a given tannin is ca-pable of binding to many different proteins—they are generally considered to be poor drugcandidates. A cautionary tale of what can hap-pen when this is ignored is the case of SP-303, a highly characterized but nonspecifictannin mixture from the Amazonian plant Cro-ton lechleri (Holodniy et al., 1999). Originallyput forward by Shaman Pharmaceuticals asan antiviral agent against respiratory syncytialvirus (Wyde et al., 1993) and herpes simplexvirus (Safrin et al., 1994), it was unsuccess-ful in initial human trials. It was then studied

for treatment of AIDS-related (Holodniy et al.,1999) and travelers’ diarrhea (DiCesare et al.,2002) with somewhat better success, althoughits antidiarrheal activity did not appear to belinked to its direct antiviral activity (Fischeret al., 2004). In 1999, the FDA denied approvalfor SP-303’s antidiarrheal indications, and thecompany soon reformulated itself as ShamanBotanicals, marketing SP-303 as a botanicalsupplement (Clapp and Crook, 2001). Essen-tially, the nonspecific activity of the SP-303tannin was misinterpreted, and the product wasnever able to demonstrate clinical activity suf-ficient for FDA approval. Much effort has beenexpended over the years in removing tanninsfrom natural product screening samples, sincethey can be active in a wide variety of cell-freeand cell-based assays (Cardellina et al., 1993;Wall et al., 1996).

Phorbol estersPhorbol esters are diterpenes produced ex-

clusively by plants in the Euphorbiaceae andThymelaeaceae families (e.g., Fig. 9.11.4C).Many compounds of the class are skin irri-tants and tumor promoters, and act in cellsthrough binding to protein kinase C (PKC;Nishizuka, 1984). Since many cellular func-tions are dependant on PKC, phorbol estersare considered to be pleiotropic agents that canmodulate many cellular pathways. Hence, theyappear as hits in many cellular screens, but areundesirable due to their potential toxicity andtumor-promoting properties. The general dis-tribution of phorbol esters in different specieshas been described (Beutler et al., 1989; 1990;1995; 1996).

SaponinsSaponins are glycosides of triterpenes or

sterols produced by many plants (Hostettmannand Marston, 1995). The number of sugarresidues may vary from one to a dozen, andother chemical functionalities may be ap-pended in various ways (e.g., Fig. 9.11.4D).Their ability to act as detergents and formfoams in water solution is related to the useof saponin-containing plants as soaps and tokill fish. These same properties in the contextof biomedical screening assays lead to celllysis, which can be either a false positive oran interference, depending on the assay end-point. In addition, some saponins cause hemol-ysis, an undesirable property in a drug candi-date. A diagnostic feature of saponins in a cellgrowth assay is that cell lysis is an extremelyrapid process, on the order of several min-utes, whereas other cell-killing mechanismsgenerally require several hours to take effect.


9.11.13


BA

DC

FE

Figure 9.11.4 Structures of some common nuisance compounds. (A) A common condensed tannin, proanthocyanin C1;(B) a hydolyzable tannin; (C) a phorbol ester, phorbol 12-tigliate 13-decanoate; and (D) a saponin, ginsenoside Rb2. (E)General repeating structure of a marine anionic polysaccharide. (F) General repeating structure of a cationic polymericalkylpyridine, halitoxin.


9.11.14


Thus, time-course studies can help distinguishsaponins from other types of hits. It is impor-tant to note that not all saponins are detergentsor hemolytic, and that some could be usefuldrug leads (Bento et al., 2003; Tang et al.,2007).

Anionic polysaccharidesThe primary structural material of plant

tissues is cellulose, a neutral polysaccharide.For animals, cartilage plays a similar role andis composed of collagen and proteoglycan.The carbohydrate portion of proteoglycan iscomposed of N-acetylglucosamine and hex-uronic acid units that are heavily sulfated (e.g.,Fig. 9.11.4E). These materials are often foundin marine invertebrate aqueous extracts, are ofhigh molecular weight, and they carry a sub-stantial negative charge (Beutler et al., 1993).Anionic polysaccharides are highly active incellular HIV assays (Beutler et al., 1993), al-though their high molecular weight and hetero-geneity make them unlikely drug candidates.Sulfated cyclodextrins have substantially thesame antiviral activity without some of the lia-bilities (Moriya et al., 1993). Sulfated polysac-charides are encountered as hits in a variety ofcellular screens. They may be removed fromextracts by precipitation from ethanol solutionat low temperatures. Plants also produce an-ionic polysaccharides, which generally haveweaker activity.

Cationic polymeric alkylpyridinesLess often, cationic polymers are found as

nonspecific hits from natural product extracts.Marine sponges are the usual sources of theseagents (e.g., Fig. 9.11.4F; Schmitz et al., 1978;Davies-Coleman et al., 1993).

Pattern matchingAnother approach to identifying or elim-

inating known natural products without in-vesting resources in their re-isolation andcharacterization is to compare biological andchemical “fingerprints” with standard com-pounds. By using the results of multiple bi-ological and chromatographic experiments inwhich the standard compounds have previ-ously been tested, one can group similarsamples together, and pose a dereplicationhypothesis for the samples whose resultsmatch those of a known compound.

The most data-rich environment in whichthis has been done is for the NCI 60-cell panel.Since thousands of natural product compoundshave been tested, these can be used as ref-erence points in data analysis in comparison

with the results for crude extracts of fractions.A variety of mathematical approaches havebeen used for the analysis, including calcula-tion of Pearson correlation coefficients (Paullet al., 1989), neural networks (Weinstein et al.,1992), and self-organizing maps (Keskin et al.,2000). Often, if the mechanism of action ofthe reference compound is known, the corre-lated test samples can be rapidly tested to con-firm similar mechanisms (Paull et al., 1992;Weinstein et al., 1997). This has been demon-strated in the case of agents affecting tubulin(Paull et al., 1992), epidermal growth factorpathways (Wosikowski et al., 1997), and vac-uolar ATPase inhibitors (Boyd et al., 2001),among others.

There is no a priori reason why patternmatching must be limited to cell growth inhibi-tion data. In fact, any type of data can, in princi-ple, be mixed, even chromatographic, spectro-scopic, and taxonomic information. The utilityof pattern matching depends primarily on thenumber of dimensions present in the data ma-trix. While redundant dimensions (i.e., cellsthat respond identically) do not contribute,scattered missing data are only a minor is-sue if the appropriate analytical techniques areapplied.

RECENT NATURAL PRODUCTDRUG INTRODUCTIONS

Natural products and their relatives con-tinue to be approved as new drugs. The listshown in Table 9.11.1 is not comprehen-sive, since it excludes peptide drugs and otheragents that could arguably be considered asderivatives of natural products. For more com-prehensive discussions of natural productsdrugs currently on the market or in clinicaltesting, see the reviews of Cragg and Newman(Newman and Cragg, 2004, 2006, 2007; Craggand Newman, 2005b; Newman, 2008) and ofButler (2008).

WHICH COMPANIES ARE STILLCONDUCTING NATURALPRODUCTS DISCOVERY?

Natural products groups have been elimi-nated in most large pharmaceutical companiesin the United States; however, this trend has notpenetrated as deeply in Europe and in Japan.Of the companies listed in Table 9.11.1, BristolMyers Squibb, Merck, Johnson & Johnson,Pfizer, GlaxoSmithKline, and Lilly no longermaintain internal natural products discoverygroups. Up until its recent merger with Pfizer,Wyeth had an active natural products group


9.11.15


Table 9.11.1 Recent Natural Product Drug Introductions

Year Drug name Company Indication NP template Location

2007 Ixabepilone Bristol MyersSquibb

Cancer Epothilone U.S.

2007 Temsirolimus Wyeth Cancer Rapamycin U.S.

2007 Retapamulin GSK Impetigo Pleuromutilin U.S.

2007 Trabectedin PharmaMar/J&J Cancer Ecteinascidin Europe

2006 Anidulafungin Pfizer/Lilly Antifungal Echinocandin U.S.

2005 Tigecycline Wyeth Antibacterial Tetracycline U.S.

2005 Micafungin Astellas Antifungal Echinocandin U.S.

2004 Everolimus Novartis Immunosuppressant Rapamycin Europe

2003 Daptomycin Cubist Antibacterial Lipopeptide U.S.

2002 Pimecrolimus Novartis Immunosuppressant Rapamycin U.S.

2001 Acemannan Carrington Labs Wound healing Polysaccharide U.S.

2001 Caspofungin acetate Merck Antifungal Echinocandin U.S.

2001 Telithromycin Aventis Antibiotic Erythromycin Europe

2000 Arteether Artecef BV Antimalarial Artemisinin Europe

2000 Dosmalfate Faes Antiulcer Diosmin Europe

2000 Egualen sodium Kotobuki Seiyaku Antiulcer Guaiazulene Japan

at its Pearl River facility, bucking the trend,at least for the time being. In Europe, Novar-tis has been notable in maintaining its naturalproducts pipeline.

A corresponding trend is the developmentof smaller companies as “boutique” naturalproducts operations, which can license natu-ral products leads at various stages of devel-opment to larger entities (Gullo and Hughes,2005). Pharmamar is one example of a smallcompany that has had success in bringing anatural product drug candidate (Yondelis) for-ward in recent years. Nereus Pharmaceuticalshas advanced a marine microbial proteasomeinhibitor (NP-0052) into phase Ib combinationtrials in cancer. Kosan Biosciences, which hasdeveloped epothilone analogs using biosyn-thetic technology, was acquired by BristolMyers Squibb in 2008 on the strength of its de-velopment pipeline. Alternatively, small com-panies can serve as screening contractors, orprovide the natural product libraries and ex-pertise for pharma screening (e.g., AlbanyMolecular Research).

Thus, it is clear that the landscape of nat-ural products research and drug developmentis changing rapidly. It is a major challengeto maintain the knowledge base and resourcesthat have been developed in large companiesin natural products research.

DIVERSITY-ORIENTEDSYNTHESIS

The new field of diversity-oriented synthe-sis aims to take its structural cues from na-ture. As a daughter of combinatorial chem-istry, the field seeks to meld parallel synthesiswith chiral synthesis technologies. Thus, nat-ural product scaffolds are designated as priv-ileged structures and then functionalized byparallel synthesis (Hu et al., 2001; Sternsonet al., 2001; Kulkarni et al., 2002; Burke et al.,2003). While attractive in concept, for thesame reasons that natural products are desir-able for drug leads (see Why natural productsare attractive for drug discovery above), it re-mains to be seen how efficient the strategy willbe. After all, the choices of functionalizationare still up to the chemist.

SPECIALIST JOURNALS INNATURAL PRODUCT SCIENCE

Table 9.11.2 provides a listing of specialistjournals that are important in natural productsresearch.

ACKNOWLEDGEMENTSThis research was supported by the Intra-

mural Research Program of the NIH, NationalCancer Institute, Center for Cancer Research.The content of this publication does not


9.11.16


Table 9.11.2 Specialist Journals in Natural Products Research

Journal Name Publisher

Economic Botany NY Botanical Garden Press

Fitoterapia Elsevier

Journal of Antibiotics Japan Antibiotics Research Association

Journal of Chemical Ecology Springer

Journal of Ethnopharmacology Elsevier

Journal of Natural Products American Society of Pharmacognosy/American Chemical Society

Marine Drugs Molecular Diversity PreservationInternational

Natural Product Reports Royal Society of Chemistry

Natural Product Research Taylor & Francis

Pharmaceutical Biology Informa Healthcare

Phytochemical Analysis Wiley Interscience

Phytochemistry Elsevier

Phytochemistry Reviews Springer

Phytomedicine Elsevier

Phytotherapy Research Wiley Interscience

Planta Medica Thieme

Toxicon Elsevier

necessarily reflect the views or policies of theDepartment of Health and Human Services,nor does mention of trade names, commercialproducts, or organizations imply endorsementby the U.S. Government.

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