[CANCERRESEARCH55, 753-760,February15, 19951

Special Lecture

Camptothecin and Taxol: Discovery to Clinic—Thirteenth Bruce F. CainMemorial Award Lecture'

Monroe E Wall2 and Mansukh C. Warn

Research Triangle institute, Research Triangle Park, North Carolina 27709-2194

Abstract

Camptothecin and taxol are secondary metabolites found, respectively,in the wood bark of Cainptotheca acuminata, a native of China, and Taxusbrevifolia, found In the northwest Pacific coastal region of the UnitedStates. The compounds were isolated guided by bioassay on variousextracts and chromatographic fractions. Their unique and hitherto maknown structures were elucidated by nuclear magnetic resonance, mass

spectrometry, and X-ray analysis. Both compounds have unique mechanisms of antltumor activity; camptothecin uniquely inhibits an enzyme,topoisomerase I, involved in DNA replication. Taxol binds to a protein,tubulin, thus inhibiting cell division. Taxol has been called the best newanticancer agent developed from natural products, showing particularefficacy against ovarian cancer. Camptothecin and analogues singly orcombined with cisplatin show efficacy against solid tumors, breast, lung,and colorectal, which hitherto have been unaffected by most cancerchemotherapeutic agents.

Introduction

Natural products chemists and phytochemists have always beenimpressed by the fact that compounds found in nature display analmostunbelievablerangeof diversityin termsof their structuresandphysical and biological properties. Most of these compoundsaresecondarymetaboliteswith functions in plants, fungi, and marineorganismsthatarestill notwidely understood.Currently,it isbelievedthatmany of thesecompoundsact in defenseof the harmful effectsoftoxins,carcinogens,or mutagensfoundin the plant (1, 2) or attackbyexternalpredators(3).

Phytochemical Screening for Cortisone Precursors

During the period 1950—1959,M. E. Wall was the director of alarge programat the EasternRegionalResearchLaboratory,USDA,3Philadelphia,PA, which involved a screeningstudyof thousandsofplants, searchingfor steroidalsapogeninswhich would be cortisoneprecursors.The plant collectionswere conductedby botanistsunderthe auspices of the Plant Introduction Division of the USDA.Thousands of plants were collected and meticulously identified beforeshipping to the Eastern Regional Research Laboratory. This jointeffort of chemists and botanists proved to be a good model for futureprograms. Indeed, it firmly established the fact that the closecooperationbetweenchemistsand botanistswas requiredfor a successfulnaturalproductsprogram.The surveyincludednot only quantitative data for steroidalsapogeninsbut also qualitative analysisfor

Received1217/94,accepted12/12/94.I Presented at the 85th Annual Meeting of the American Association for Cancer

Research, April 13, 1994, San Francisco, CA. The discovery, isolation, and structureelucidationof campothecinandtaxol werecarriedout underContractSA-43-ph-4322.

2 To whom requests for reprints should be addressed, at Chemistry and Life Sciences,ResearchTriangle Institute,P.O.Box 12194,3040CornwalhisRoad,ResearchTrianglePark,NC 27709-2194.

3 The abbreviations used are: USDA, United States Department of Agriculture; RTI,ResearchTriangleInstitute;NC!,NationalCancerInstitute;CPT,camptothecin;TIC,mean tumor weight of treated animals divided by mean tumor weights of controlanimalsX 100;NMR, nuclearmagneticresonance;T-I, topoisomeraseI; P-4, leukemiaP1534.

sterols, alkaloids, tannins, and flavonoids (4—7).We saved thousands(but not all) of the plant ethanolicextracts,particularly of the moreunusual plants.

One of the extractsthus savedand storedwas preparedfrom theleavesof Camptothecaacuminata,Nyssaceae,a treewhich is a nativeof China, growing in relatively warm areasof Southeasternprovincesof China suchasSzechwan,Yunnan, andKwangsi (8). This plantwasintroduced several times into the United States, and eventually a fewtrees were growing at a USDA Plant Introduction Garden in Chico,CA, from which this sample was procured (see Ref. 8 for a detailedaccountofthe introductionof C. acuminatainto the United StatesandCalifornia).

Discovery of Antitumor Activity in Extracts of C. acuminata

In addition to screeningthe plantscollected for various chemicalconstituents,we beganto testsomeextractsfor antibiotic,antitumor,and antiviral activity. In 1957, after a visit by the late Dr. JonathanHartwell, Cancer Chemotherapy National Service Center, who isconsidered by most natural products scientists to be the pioneerworker in the field of plant antitumor constituents, we agreed to sendhim 1000 ethanolicplant extractsfor testing for antitumor activity.Almost 1 year later came the astonishing result that the Camptotheca

extracts were the only ones to have high activity in the CA-755 assaythen used as one of the standard test systems.

Discovery of Camptothecin

Wall becameintenselyinterestedin discoveringthe natureof thecompound(s)responsiblefor the antitumor activity of C. acuminataextracts.For administrativereasonsit wasnotpossibleto do thisin theUSDA; hencein July 1960, he left theUSDA andestablisheda naturalproductsgroup at the RTI with support from the NC!. By 1963 asizable sample of approximately 20 kg of the wood and bark of thetree becameavailable to him, and in 1964 Dr. M. C. Wani joinedWall's group commencing a fruitful 30-year collaboration.

Use of L1210 Assay for Fractionation

We commenced a typical biodirected isolation in which the fractionation of the sample would be directed entirely by the antitumorassay. By this time it was known that the crude extracts of C.acuminata were very active in the L1210 mouse leukemia life prolongation assay.This was, and still is, highly unusual.Most of thehundredsof plant extractswith which we and others subsequentlyworked were never sufficiently active in L1210 to conductfractionationsin thissystem.However, thiswasthecasefor C. acuminataandhence it arousedvery high interest on the part of the NC!. In general,after conducting a particular phaseof the isolation, it would often takeus 3 months or more before the results were received. This wasprimarily due to the fact that the L1210 assay is based on lifeprolongation.In the case of highly active extractsthe study wouldhave to be carried out for 30 days or more. Although it was a slow

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TableI Craigcountercurrentdistribution of thechloroform extractfromFig.1NSC

No.TubeWt. fraction(g)Dose(mg/kg)@'T/C9KBF098a125153<1.0F099121.52501795.3FO10028.762.52010.8FOlOl37.262.52010.5F010246.931.22060.09F010357.631.21560.3F010467.231.21980.5F010579.331.2160<1.0F0106811.52501720.5F0107916.12501484.3F01081019.22509633.0F01091162.7250100>100.0K128

(camptothecin)0.51630.7

CAMFIOTHECIN AND TAXOL IN PRECLINICALAND CLINiCAL RESEARCH

process, we persisted, working at the same time on other plantbioassay-directed fractionations, one of which resulted in the isolationof taxol (Compound 17) (see below).

Fractionation of C. acuminata

Fig. 1 showsthe resultsobtainedfrom the extractionof almost20kg of dry plant materialwhich consistedof the wood pluswood barkof the tree. It shouldbe notedthat this samplewas obtainedfrom thePlant IntroductionStationin Chico, CA, or in somecasesfrom treesthat were made available growing nearby in California. Our procedureinvolveda continuoushot extractionwith heptane.The residualplantmaterialwasextractedwith hot 95%ethanol;andafterconcentration,the aqueousethanolic residue was extracted with chloroform asshownin Fig. 1. The fractions shown in Fig. 1 were given an L1210 assay.Only the chloroformfractionswere active.

Craig Countercurrent Partition

Severalproceduresinvolving chromatography,particularlyon alumina, were tested on a small scale. They were all unsuccessful forreasonsunknownto us at the time, althoughwe now know that CPT(Compound1) is adsorbedvery tightly on aluminaandthat this is notan appropriatechromatographicagent for CPT. We then testedtheCraig countercurrentpartitionprocedure.This methodologywas designed and brought to a high pitch of perfection by the late L. C. Craigwho receivedthe Nobel Prize for his work (9, 10). Various typesofcountercurrent equipment were available in the early 1960s. Most ofthis equipment have now become museum pieces, because moreconvenient and far less space-taking equipment is now available. Thebeauty of the Craig partition technique was that it involved liquidliquid partition and avoided harsh treatments which could causechangesin a substanceof unknownconstitution.

Most of the chloroform phase (Fig. 1) after concentrationwassubjectedto an 11-stagepreparativeCraig countercurrentdistributionasshownin Table 1. In thisvery simpleuseof theCraig methodology,the partition was carried out in large separatoryfunnels using achloroform-carbon tetrachloride-methanol-water partition system. Allof the fractions were analyzed both by the in vivo L1210 mouse life

PkaMater@ll9kg(thynaieriI@

Heptam@ (Hot)

A@@oas

Coriiiioi@sCHC1,narn

ClJk@rm Aqtr.L32.OgF125 F126HW@yActhe S@1lyActhe

Fig. 1. Fractionationof C. acunainata.F123T/C, <125 from 200 to 25 mg/kg.F127T/C, <125from 200 to 25 mg/kg.F124T/C = 209at 50 mg/kg; 163at 25 mg/kg.F125T/C = 214at 50 mg/kg; 151at 25 mg/kg.F125T/C = 128at 200mg/kg;<125at 100-25mg/kg.

a S@ing material.

b@ at which maximum T/C in L1210 was observed.

prolongationassayand by a 9KB in vitro cytotoxicity assay.Therewas reasonablecorrelationbetweenthe two assays.Tubes 2—6werejudged to containthe mostactive material. It shouldbe kept in mindthat, in the in vivolife prolongationassay,it is the combinationof thelowest dose with the largest T/C activity which is the basis forselectingactive fractions. In the cytotoxicity assayit is simply thelowestED50thatwill inhibit thegrowthof the9KB cells.We hadthusremovedabout80% of the total weight while concentratingthe mostactive fractions in 20% of the original weight of the chloroformextracts.By comparisonwith a puresampleobtainedlater but againgiven the standardL1210 assay(Table 1, K128), it can be estimatedthat the most active fractionscontainedbetween 1 and 2% of theactivecompound.It shouldbenotedthat9KB activity wasreasonablycorrelatedwith the L1210 assay.

When fractions 2—6were combined and the solvent was partiallyconcentrated,a yellow precipitatewasformedwhich wascollectedbyfiltration. This material was subsequently further purified by chromatographyon a silica gel columnand crystallization.As statedabove,thepurecompoundisvery activein L1210. Dosesaslow as0.5 mg/kggave an appreciablelife prolongation.A dose of 4 mg/kg was themaximum doseprior to occurrenceof toxicity.

Physical Properties of Camptothecin

Someof themajorphysicalpropertiesof camptothecin(Compound1) are listedhere.The compoundwas a high melting substance,witha molecular weight of 348.111, obtained by high resolution massspectrometry, corresponding to the formula CI@H16N2O4.It gives @iintenseblue fluorescenceunder UV and is optically active ([a] D,+31.3°).The compoundgives the following qualitative reactions:negativephenol (FeCl3); negativeindole tests;negativeDragondorfandMayer tests.No crystallinesaltscouldbe obtainedwith a varietyof acids.The compoundcannotbe methylatedwith diazomethaneordimethyl sulfateundera variety of conditions.The compounddoesnot react with bicarbonateor carbonatebut can be quantitativelyconvertedto the sodiumsaltwith sodiumhydroxideat room temperature. On acidification, the sodium salt regeneratescamptothecin.Other physicalpropertiesare given in detail in Ref. 11.

Structure of Camptothecin

It was found that camptothecin(Compound 1) could be readilyconvertedto anesteracetateor a chloroacetate.The chioroacetatewasconverted to the corresponding iodoacetate by treatment with sodiumiodide-acetone.The iodoacetatecrystallizedin orthorhombiccrystalssuitablefor X-ray analysis.The X-ray studieswere carriedoutby Drs.A. T. McPhail and G. A. Sim, then in the Departmentof Chemistry,University of Illinois. The structuresof camptothecin(Compound1)andthecorrespondingsodiumsalt(Compound2) areshownin Fig. 2.

HoptareLtr@t

Concentrate

1'rec@ Mod@r[à@uor10gF127 468gFI23th@dw b@üw

Pk±@

F—(Coi@iira@)

@fleatwIh4 w,kincsd*tro*@nn+1/4vokice5%a@taol@edanol

Chbm@nn222g Fl24

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CAMPTOTI{ECIN AND TAXOL IN PRECLINICAL AND CUNICAL RESEARCH

Encouragedby the broad scope of animal antitumor activity ofCPT, a decision was made by the NC! to go to clinical trial with thesodiumsalt (Compound2). In contrastto camptothecin,the sodiumsalt was water soluble and hence was easily formulated for i.v.administration.In a PhaseI trial a studyof 18 patientswas conducted,and it was found that there were partial responses in 5 of them (15).These responseswere primarily in gastrointestinal tumors and of shortduration.Toxicity that involved mainly dose-limiting hematologicaldepressionwas noted, along with some vomiting and diarrhea. Inanother Phase I trial only two partial responseswere found in tenevaluablepatients(16). Becauseof the somewhatencouragingresultsobtained in an earlier Phase I study (15), a Phase II study wasundertaken in 61 patients with adenocarcinomasof the gastrointestinaltract,but only 2 patientsshowedobjectivepartial responses(17). Thedrug has also been under study as a sodium salt in the People'sRepublic of China. Up to 1000 patientswere entered into trial, andeffective resultswere reported in gastric cancer, intestinal cancer,headand neck tumors,and bladdercarcinoma(18). These resultsaremore promisingthan thosein the United Statestrial but may be dueto the fact that in the United States only patients who have beentreated with many other drugs previously and have become resistantwere tested.

Structure-Activity Relationship

Although our work on camptothecindiscoverymay be consideredto have nominally ceased by 1970, our interest in CPT and/or itsanaloguesdid not wane, particularlywith regardto structure-activityrelationships (Fig. 3). In 1969, we reported the isolation oflO-hydroxy- (Compound3) and 10-methoxy-CPT (Compound4) asminor components of C. acuminata (12). Later we found that10-hydroxy-CPT (Compound 3) was much more active than CPT in a

@(1)L121O.T@22O.t2ntgPS.TA@197at4n@tg

o1o@cQ@10.H@drox@vanpIotheth1

L1210.DV144a2n@tg L121O.TaV23OatOin@tgPS, T/C 314 at4 @kg

CamptothecinSodium(2)Cançtothecin(1), RnR'=R2=R3=H10-Hy&oxycan@*othecia(3), R=R1=R3=}L R2=OH10-Methxycaniptothecin (4), R=R1=R3=H, R2=OMe9-Nirocamptothecin(13),R@=R2=R3=H,R1=NO@9-Aininocançtotl@cin(11), RnR2=R3=H, R1=NB@

10,1l-Methyhnedioxycan@@tothecin(12), R=R'=H, R2,R3=OOI@O7-Ethyl-10-(@(l-p,erilino)-l-pipeniuioJcathonykatycan@@tothecin(14),

(cPT-l1), R'=R3=H,R=C@H@,R2=

9-Dirmthyhmiaomathyl-10@hydroxycaipptothecii(15) @FopoIecan),R=R3=H, R2=OH@R1=CH2-N(CH@.HQ7-(4-Mtsperazinomathyhne)-10,11.ethyhned@cançtotbscin(16)

(G 147211C), R1=K R2,R3=OCH@CH@O,R@ a@—(@J@4M.sHa

Fig. 2. Camptothecin,camptothecinsodium,and analogues.

This structure was completely in accord with UV-IR spectra, NMRspectra, and mass spectral data (11). The structure is unique. Camptothecinhasbeenshownto be relatedto the indole alkaloids,andthe6-memberedring B and a 5-memberedring C are formed by a ringexpansion/ringcontraction sequenceof reactions.The pentacyclicring structure is highly unsaturated. Some of the unique structuralfeaturesinvolve thepresencein ring E of an a-hydroxylactonesystemand in ring D of an unsaturated conjugated pyridone moiety. Ontreatmentwith alkali, the compoundreadily opensforming an openlactone sodium salt shown in Fig. 2 (11). On acidification, theextremely water-solublesodiumsalt is relactonizedreadily. The parent compound,however, is extremelywater insolubleand, indeed,isinsoluble in virtually all organic compounds except dimethylsulfoxide in which it exhibits moderate solubility.

Biological Activity of Camptothecin and Analogues

The isolationandstructureproof for camptothecinandsomeof theanalogues obtained at an early stage, lO-hydroxy (Compound 3) andlO-methoxycamptothecin (Compound 4) (12) enabled extensive studies to be conductedon CPT and a few of its analogues.As statedpreviously, CPT (Compound 1) was remarkably active in the lifeprolongation of mice treated with L1210 leukemia cells, showingactivity in dosesbetween0.5 and4.0 mg/kg in this L1210 mouselifeprolongationassay(11). It showedactivity of asimilar orderin thelifeprolongationassayfor P388 leukemia (13). The compoundwas alsovery active in the inhibition of solid tumors that were being studied atthis early stage, including the Walker WM tumor which was completely inhibitedby CPT. 10-Methoxy-CPT (Compound4) was foundto be to be active but somewhat less active than CPT, whereas10-hydroxy-CPT (Compound3) was themostactivecompoundin theseries and was more active than CPT in both L1210 and P388leukemia life prolongation assays(12—14).Unfortunately, lO-hydroxy-CPT (Compound 3) is found in nature in only very smallquantities, probably amounting to about 10% of the CPT content. Allof thesepreviouscompoundswere extremely insolublein water, butit will be recalled that the lactone could be opened under mildconditionswith sodium hydroxideor sodiummethoxide,and it wasfound that this sodiumsalt of CPT was very solublein water. It wasnot until much later that it was shownby definitive studiesthat thiscompoundwasonly one-tenthasactiveasCPTin theP388assay(13).

755

CançsodaciiSodbn(2)L1210.TR 209at3 nVka

PS.T't2l2ai4Onvtg

L1210

Fig. 3. Structure-activityrelationshipin the camptothecinseries.

Early Clinical Trial

@cHv

N C—N--Ui,

L1210.TR 1flat3.5n@tg

S@i@,Acthca25 n@tgL1210.T@125ii*g

8,5.R@H

L1210

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Table2 Comparisonof in vivoandin vitroactivityof camptothecinanditsanaloguesInhibition

:@ DNA scission (%) Antitumor activitya (1 + C X 100) ED@ (mg/mi)

super-coiledCamptothecinderivative DNA (%) Supercoiled―@ L1210 P388 9KB'9PSS

52 48 41 197(8) 197(4) 10_2 102R 20 12 9 <125 10'10°R.S

30 25 15 222(8)10'20-Deoxy-(R,S)0 0 1 <125

10-OH-(S) 73 71 40 348(20) 297 (3)10_211-OH-(R.S)74 35 31 357(60)

10-OCH3-(R,S) 167(2) 1O_210,11-(CH3O)2-(R,S) 0 0 2 <125 <125 10@10,11-OCH2O-(RS) 62 82 40 325(2) 1O_210-N02-(R,S) 19 23 22 219(16) 10@11-N02-(R,S) 0 11 11147(80)12-N02-(S)

0 10 3 151(40) 151(40)9-N02-(5) 40 50 22 348(10) 10_29-NH2-(S) 91 35 31 348 (2.5) 10210-NH2-(R.S) 16 16 18 325(8) 1O@11NH2@(R,S) 33 7 14 147(40) 10_i12-NH2-(S) 0 0 3 <125 <12521-L.actam-(S) 3 0 2 178(40)10_iTncyclic-(R,S)

0 0<125aMo@ leukemias L1210 and P388, inoculated i.p.; drug administered i.p. days 1 and 5. Numbers in parentheses, dose (mg/kg) giving the greatest reported T/C value.Antitumordata

arefrom thework of Wani et aL (13, 21, 22).b Effect of 10 mM test compound as a percentage of the effect of the control; cf Jaxel et aL(24).C

Inhibition of 9KB cell growth expressed as ED@ (mg/mi).

CAMPTOTHECINAND TAXOL IN PRECLINICALAND CLINICAL RESEARCH

numberof assays(13), andthis mayhavestimulatedsyntheticeffortsby both the SmithKline Beechamgroup and a Japanesepharmaceutical company, Daiichi, to prepare water-soluble 10-hydroxy-CPTanalogues(Fig. 2). CPT reactedreadily with aminessuchasmethylamine to give the hydroxyamide (Compound 5), resulting in ringopening and lower activity. Oxidation of the ring B nitrogenwithchloroperbenzoic acid led to considerable loss of activity. Of greatinterestwas the fact that hydroxylationin ring A, at leastat position10, was compatible with activity and indeed this compound, 10-hydroxy-CPT (Compound 3), has greater activity than CPT. Majorreduction of antineoplastic activity was noted as a result of reactions involving the hydroxyl or lactone moiety in ring E. Afteracetylation of CPT, the resultingacetate(Compound 7) is virtuallyinactive. Other reactionsalso point to the absoluterequirementofthe a-hydroxy group, as shown by the fact that after replacementof this group by chlorine, both the resultant chloro analogue(Compound 8) and the correspondingreduction product, deoxycamptothecin (Compound 9), are inactive in L1210 leukemia.Reduction of the lactone under mild conditions to give the lactol(Compound 10) also results in complete loss of activity (14).

Inhibition of Topoisomerase I

Interest in CPT and analoguesremainedat a low ebb until 1985when it was discoveredthat camptothecin,by a unique mechanism,inhibited T-I (19). The enzymehasbeenimplicatedin variousDNAtransactionssuch as replication, transcription,and recombination.CPT and analoguesbind to a complex formed by DNA with T-I. Withthe mechanism now understood,there opened the possibility forclinical use of camptothecin and analogues by virtue of their inhibition of 1-I which might be found in cancercells and hencewouldinhibit tumor growth.

Many new CPT analogues were synthesized at R'fl (20—23)andwere tested for their topoisomeraseI inhibition. The inhibition oftopoisomerase I activity closely parallels in vivo mouse leukemiaassays,as is shown in Table 2 (24, 25). Finally, we were able toseparatethe racemicsynthonby which we synthesizedvariouscamptothecinanaloguesinto its corresponding20-(S)and20-(R)analogues(23) and have been able to demonstrate that the 20-(R) form isinactive,bothin thetopoisomeraseinhibitionandin the in vivoassays(Table 2) (24).

Anotherstudyconductedcooperativelyinvolved the StehlinFoundation, New York University Medical School, the Johns HopkinsUniversity, and the ResearchTriangle Institute. In these studies,utilizing nude mice, both 9-amino- (Compound 11) and 10,11-methylenedioxy-CVF(Compound12) hadgreatpotencyin inhibiting human colon cancerxenograftsin nude mice (26). In these studiesalargenumberof compoundscommonlyusedin cancerchemotherapywere totally ineffective.

Clinical Trials

Camptothecinand a numberof its analoguesare now in activeclinical trial (Fig. 2). The compounds include CPT (Compound 1),9-nitro (Compound13), and 9-amino-CPT(Compound11), all firstdiscovered at RTI (11, 21, 22). These are all water insoluble and areadministered p.o. Two water-soluble analogues of 10-hydroxy-CPT(Compound 3) also discovered at RTI (12) are in very active clinicalstudy.TheseareCPT-11 (Compound14)(27), a productof a Japanesepharmaceutical company, Daiichi, and topotecan (15) (28), a productof the American pharmaceutical company, SmithKline Beecham. Finally, a camptothecin analogue of novel structure, 01 147211C (29),@which originated at Glaxo, also water soluble, is in initial trials.Basically,camptothecinandanaloguesareshowingpromisingactivityagainsta variety of solid tumorswhich have been refractoryto anysort of treatment.

More clinical studiesinvolving dosage,administrationforms, andsingle or combined therapy are under way. Nonetheless, it is alreadyapparent that significant objective responses have been found ontreatment of many hitherto resistant solid tumors with CPT andanalogues: 20-30% response rates with single agent trials with patients with non-small cell lung, cervical, ovarian, colorectal, andbreastcancers;30-40% with patientswith small cell lung and acuteT-cell leukemia/lymphoma.As high as40-50% responsesin PhaseItrialsof CPT analoguepluscisplatinhavebeenfoundin patientswithnon-small cell lung cancer.

Recently some new CVF analogues prepared at RTI have beenshown to have potent in vitro activity in antiparasitic studies with

4 M. J. Luzzio, J. M. Besterman, M. 0. Evans, and P. L Myers. Water soluble

camptothecin derivatives. European Patent application 0540, 0099A1, May 1993.

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CAMPTOTHECINAND TAXOL IN PRECLINICAL AND CLINICAL RESEARCH

idue betweenwater and chloroform; followed by a large numberofCraig countercurrentdistribution treatments,the last of which involved a 400-tube Craig countercurrentdistribution(Fig. 6). In thismanner, approximately 0.5 g of taxol was isolated starting with 12 kgof air-dried stem and bark. The yield was about 0.004%. All thevarious stepswere monitoredby an in vivo bioassaywhich, at thattime, involved the inhibition of the Walker WM solid tumor. As isshown in Fig. 5, increased purification is accompanied by lower T/Canddosevalues.The isolationhencewas carriedout laboriously,butin a mannerin which no lossesby the treatmentor no changesin theunknown chemical constitution of the eventual product occurredbecauseof the mild countercurrentdistributionmethodology.Muchsimpler procedureshave beensubsequentlydevelopedboth at RTIand elsewhere.

Biological Activity of Crude and Purified Taxol

The name“taxol―was assignedto this compoundbeforewe reallyknew its complete structure, but it was evident that it did contain somehydroxyl groups, and the name had a nice ring to it. Apart from theactualisolationof the purematerial, the crudeextractswere subjectedto a large number of tests. In early work we found that the crude

StemBark- 12kilos,airdr@d

Extract9@%Ethanol

PSfl@@I:flc::t::eH2Oandchktroform- 1(4:1)

Fig.5. Isolationof taxolfromT. brevjfolia.5WM,solidtumorknownasWalkerNo.256i.m.ratcarcinosarcoma.

Qik*@oftzin(146 g solkis)

11Tie craigccDSystem: hexane,acetono,t-butanol, water (5:4:42)

Tubes 6, 7, 841gsoids,activity5WM

T@ 30%at45mg/kg

30TubecraigCCDSystem@methanol,water,CCJ@CHCl@(827:3)

Tubes8-18l4gsolkls

Ti@30@at23mg/kg

400TUt!ecraigcCDSystem@mathanol,wat&,CCL@,CHCl@(8273)

Tube1170.1892.4gso&ls,TiC 16%at15mg/kg

Tritumtin with benzeneO.5gTaxol@TK l6%at 10mg/kg

24%at5 mg/kgYmkl circa 0.004%

Fig. 6. Purification of crude taxol extract.CCD, countercurrentdistribution; 5WM,Walker No. 256 i.m. rat carcinosarcoma.

Aqueouslayer(d@&d)

chloroform(146 g sobds)— 5WMT@=31%at 100mg/kg

757

0

óH@,,o

@ 12 ::@@ : :

IsH3

cell5

000CH30C1H5

Tmd(17),R=Ac,R1aC5H@Teictere (23). R@H,R1@t-Bt@

Fig.4. Structuresof taxolandtaxotere.

trypanosomes and the organisms responsible for malaria. In both thesecases,this is due to the inhibition of T-I in theseorganisms.

Discovery of Taxol

Since 1988, the remarkableclinical efficacy of taxol (Compound17) (Fig. 4), resulting in numerous observations of partial and complete remission of advanced ovarian cancer in women, and morerecently, reports of the efficacy of the drug in breast, lung, andprostatecancer have arousedgreat interest in this antitumor compoundwhich was discoveredat RTI many yearsago(30, 31). Like somany other investigationsof this type, a combinationof serendipityfollowed by much hardwork lead to the discoveryof thisvery activeantitumoragent.

Initial Procurement and Isolation

A screeningprogramfor antitumoragentsin the PlantKingdomwas initiated in 1960 under Dr. JonathanL Hartwell, NCI. In thisprogram, plant samples collected at random were supplied by theUSDA under an interagency agreement with NC!. In August 1962,USDA botanist,ArthurS. Barclay,and3 collegestudentfield assistants collected 650 plant samples in California, Washington, andOregon,includingthebark, twigs, leaves,andfruit of Taxusbrevifoliain Washingtonstate(32).

T. brevifolia is a slowly growing tree which is found only primarilylocalized in the coastal areas of the above-mentioned West Coaststates. It had never received any chemical investigation until it wasassignedto our laboratoryby Dr. Hartwell. The assignmentof theplant was not entirely serendipitous.Someof thesesampleshadbeenshown to have9KB cytotoxicity. At that time therewere only threegroupsworking undercontractto NC! in the laboratoriesof Dr. JackCole, University of Arizona; the late Dr. S. Morris Kupchan,then atthe University of Wisconsin;andWall's laboratoryat RTI. The othergroupswere not particularly interestedin plantswith 9KB activity.We hadnotedan excellentcorrelationin our camptothecinstudiesbetween L1210 in vivo activity and the 9KB cytotoxicity. Accordingly, we had requestedDr. Hartwell to assignas many 9KB activesas possibleto us, and from this arosethe assignmentto RTI of T.brevifolia. A number of other plants also highly active in 9KB werealsoassignedto us, and severalhighly active novel compoundswerefoundin thesecasesalso,includingcolubrinol,a maytansineanalogue(33); carminomycin,relatedto daunomycin(34); andanactivequassinoid (35). As discussedpreviously,all of thesecompoundsaswell ascamptothecin(Compound1) andtaxol (Compound17)aresecondarymetaboliteswhich were isolatedguidedby bioactivitydeterminations.

Initial samples of plant material arrived in our laboratory in 1964.The isolation procedure finally adopted after several unsuccessfultrials is shown in Figs. 5 and 6. Extraction was carried out by ourstandardprocedure:ethanolextraction;partitionof the ethanolicres

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SystemtestedAdministrationActivity (%T/C)i.p.

P388leukemiai.p.+(164)i.p.

B16 melanomai.p.++(283)i.p.

Ll210 leukemiai.p.+(139)SRC―

CX-1 colon xenografts.c.++(3)SRC

LX-1 lung xenografts.c.+(8)SRC

MX-1mammaryxenografts.c.++(—77)a

SRC, subrenal capsule.

.- . .@@ _@@ CAMPTOTHECIN AND TAXOL IN PRECLINICAL AND CUNICAL RESEARCH

Table3 Antitwnor activity of taxol Final Structure of Taxol

The final structureof Compound17 requiresthe placementof thetwo hydrolyzed ester functions of Compound 17 on the tetraol(Compound 19). Taxol could not be oxidized by neutral, activatedmanganesedioxide, indicating that the two esterswere located at theallylic positions 10 and 13. The chemical shifts of the protons at C-tOand C-13 were also in accord with this observation.Oxidation ofCompound 17 with activated manganesedioxide under mild basicconditions(pH of the aqueoussuspension8.0) in acetoneyieldedthe7f3-hydroxy-conjugated ketone, Compound 22 (Fig. 7). The molecularcompositionby highresolutionmassspectrometrywas in accordwiththe formula 3361@ suggestingthat it was formed by the lossofthe nitrogen-containinga-hydroxy esterfunctionandoxidationof theliberatedallylic a-hydroxyl group.Severalindependentlines indicatethat the hydrolyzedesterfunctionwas at C-13. Th@UV (Amaxmethanol, 272 nm; €4800) and IR (Vm,@CHC131680 cm') spectraare incompleteaccordwith this structureand rule out the alternative @ti9,10-dioxoformulation. In addition, the ‘H-NMRspectrumof Compound 22 clearly showsthe presenceof a singlet due to the C-1Oprotonat@ 6.46 as requiredby Formulation22.

Prior to the isolation of taxol (Compound 17), no natural taxanederivative was reported to have antitumor activity. There are manyinteresting features of the molecule, particularly the 4-memberedoxide ring 4, which is not found in any of the other natural taxanes.The estermoiety itself is of interest,containingtwo phenyl groups,one of which is attachedas part of an amide function. The largenumberof asymmetricalcarbonatomsrenderstotal synthesisof taxolmostdifficult.

For antitumoractivity, it isessentialthattheestermoietyat C-13 bepresent.We haveshownthat the esterandthe tetraol formedby lowtemperaturecleavageof taxol are eachessentiallyinactive (31).

Subsequent Developments

Our initial discoveryof taxol essentiallyendedwith thepublicationof our paperon the structurein 1971 (31). We maderepeatedeffortsto interest the NC! administratorswho were then involved in thisprogramto obtain largeramountsof the bark andwood of T. brevifolia so that advanced animal studies, toxicology, and eventuallyclinical trial could ensue. The response was that the compound waspresentin too limited a quantity,that the extractionand isolationwasdifficult, and that the supplyof the treewas limited.

OCOC6H5

lO-Deacetyl-baccatin ifi (19), R4-OH, R@=H@R@=0H

7,lO.Bitiockacetate (21), R=@3-OCOCH@l,R1@OCH2l, R@=OH7@.HydroxyConjugatedKetone(22),R1=Ac,R@=O

C@H@CONH0

C@1('@.0Me

Methyl Ester (18), R=Hp-Bromobenzoate (20), R@0CJL@Br

Fig. 7. Structuresof relatedtaxanesandtaxol estersidechain.

extracts were not only active in the Walker tumor inhibition assaybutalso had shown modest activity in Ll210 leukemia and particularlyhigh activity in the 1534 (P4) leukemia assay.The latter assaywas alife prolongation assay in mice, and it had been used some yearsbefore by scientists at Eli Lilly to isolate the Vinca alkaloids which

showedvery great activity in life prolongationin this system.Thesamewas notedby us for taxol with T/C valuesin the P4 systeminexcessof 300, evenwith crudeextracts.The activity of pure taxol ina number of in vivo rodent assays is shown in Table 3. Particularlyhigh activity wasshownin the B-l6 melanomaassay.Years later thiswas one criterion responsible for placing taxol in clinical trial (36).

Structure Determination of Taxol

As soonas we had isolatedtaxol (Compound 17) in pure form agreat deal of work on the structure of the compound was carried outby available spectroscopic methods. Although methods for UV, IR,and massspectrometry were at a reasonably advancedstagein the late1960s, NMR was relatively primitive compared to the very sophisti

cated instrumentation now available.As soonaswe had isolatedtaxol (Compound17) in pureform, the

structureof the compoundwas investigatedusing availablespectroscopic methods. Although methods for UV, !R, and mass spectrometry were at a reasonably advanced stage in the late 1960s, NMRspectroscopywas relatively primitive comparedto the sophisticatedinstrumentationand proceduresnow available.

The determination of the structure of taxol proved to be an cx

tremelydifficult task.The molecularformulaof taxol wasdeterminedto be C47H51N014by a combination of mass spectrometry and dcmental analysis. Data from 1H-NMR spectrum and biogenetic considerations suggestedthat taxol was a diterpenoid possessinga taxaneskeletonto which several esterswere attached.Becauseof the cxtremely limited quantity of taxol available at that time and its evidentstructural complexity, attempts were made to prepare derivativessuitable for X-ray analysis. Although a number of crystalline, halogenated compounds were obtained, none had properties suitable forX-ray analysis.

Taxol was thereforesubjectedto a mild base-catalyzedmethanolysis at 0°C yielding a nitrogen containing a-hydroxy ester (Com

pound 18), C17H17N04, a tetraol (Compound 19), C@9H36O10,andmethyl acetateas shown:

C47H51NO14 + 2CH3OH-@C17H17NO,,+@ + CH3CO2CH317 18 19

Compound18 was convertedto a p-bromobenzoate(Compound20)and Compound 19 to a 7,10-bisiodoacetate(Compound 21) (Fig. 7) andthe full structuresofthe halogenatedderivativesofCompounds 18and 19were determinedby X-ray analysis.For full detailsof the X-ray analysisand physical constantsof Compounds 18—21seeRef. 31.

The structuresof the methyl ester(Compound 18) and the tetraol(Compound 19; 10-deacetylbaccatinIII) (Fig. 4) were derived fromthe X-ray structures of Compounds 20 and 21, respectively.Compound18 is the methyl esterof N-benzoyl-f3-phenylisoserine.

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Table 4 Chronologyof taxoldevelopmentIsolation

andstructurefP388andL1210activity1971Activityin a panelof tumorsystems1975—1976Preclinical

development1977Mechanismofaction1979Animal

toxicology1982PhaseI clinicaltrials1983—1984PhaseII clinical trials/activity in ovariancancer1985—1986Synthesis

of taxol sidechain1986Semisynthesisoftaxol1988Improved

synthesesof taxolsidechain1990—1993Bristol-MyersSquibbreceivesCRADA'@from N@January1991NDA

filed with FDADecember1992Totalsynthesisof taxollHolton-Nicolaou1994

Award; NDA, New Drug

CAMPTOTHECIN AND TAXOL IN PRECLINICAL AND CLINICAL RESEARCH

Cancer Institute issued a Cooperative Researchand DevelopmentAward which was opento competition.The awardwas thenissuedtoBristol-Myers Squibb in 1991. The company moved rapidly to obtainFood and Drug Administration approval for the marketing. A NewDrug Application was filed in July 1992and approvedin December1992. The initial shortageof taxol for generalclinical usehasbeenatthis time (October 1994) greatly alleviated.

Chemkal Developments

Structure Activity. There has been great interest in carrying outvarious chemical transformations,with both taxol and taxotere, ananalogue. Such studies have been reviewed in detail by Kingston(45), in whose laboratory extensive SAR studies on taxol havebeen conducted.Recently, Chaudhary et a!. (46) have reported thediscovery of a very active analogue with considerably greaterpotency than taxol. This analogue is formed by removing the2-benzoate group of taxol and subsequently reacylating withcertain meta-substitutedbenzoic acids.

Synthesisofthe Taxol SideChain. During theperiod 1990—1994,extensiveprogresshasbeenmadeon the synthesisof the estermoietyof taxol (and theseresearchershavebeenreviewed in detail) (36, 41).Moreover, the side chain has been synthesizedin a manner whichpermits facile esterification of the taxane portion of taxol so that thereis now a practical semisynthesis of taxol available. As a consequence,Bristol-Myers Squibb has been quoted as stating that in the relativelynear future, taxol will not be producedfrom the bark of T. brevifoliabut from baccatinIII or 1O-deacetylbaccatinIII which occur in otherTaxus species in much greater quantity. Moreover, these are smaller

shrub-likeplantssuchas Taxusbaccata,a Europeanyew. This plantis a renewable source. The component used in the semisynthesis isfound in the needles of T. baccata and other Taxus species. As aconsequence,the availability of taxol in a continuingsupply is nowassured.

Total Synthesis.Becauseof the enormousinterestin taxol, anumberof groups,particularlythoseof Wender at Stanford,Holton atFlorida State,andNicolaou at ScrippsInstitute,havemadeintensiveefforts to synthesize taxol. Recently, both the Holton and Nicolaou

groupshave announcedthe total synthesisof taxol (47, 48), truly anepochal event. Although this will not be a practical supply source, theavailability of synthetic methodology may lead to important newinformation.

Conclusion

The authorshave been delighted that their initial discovery morethan twenty-five years ago of a novel natural product with excellent activity in a number of animal models has presently reachedthe stage where taxol is now available in adequate quantity fortherapeutic use. Undoubtedly, there are other highly active naturalproducts from plant, marine, and fungal sourcesas yet unknownwhich, when discovered,will have therapeuticutility. Cancer is notone, but several hundred diseasesand will require many differenttypes of agents.

Acknowledgments

We are appreciative of the NC! for continuous support of our work.

References

1. Mitscher, L A., Drake, S., Gollapudi, S. R., Harris, J. A., and Shankel, D. M.Isolationandidentificationof higherplantagentsactiveinmutagenicassaysystems:Glycyrrhiza glabra@In: D. A. Shankel, P. E. Hartman, T. Kada, and A. Hollaender(eds.),AntimutagenesisandAnticarcinogenesisMechanisms,pp. 153—168.NewYork: PlenumPublishingCorp., 1986.

2. Williams, D. H., Stone, M. I., Hauck, P. R., and Rahman,S. K. Why are secondary

a CRADA, Cooperative Research and Development

Application; FDA, Food and Drug Administration.

Fortunately, two major developments occurred. As shown in Table3, it was noted years later that taxol had very high activity in the B-16melanomaassay(36). Then during the next severalyears,studiesbyDr. SusanHorwitz at theAlbert EinsteinMedical Centerin New Yorkshoweddramaticallythat the modeof activity of taxol was due to itsbinding with tubulin in a novel manner.

Tubulin Binding

For some time there had been some interestin the mechanismofactionof taxol. Was the uniquestructureaccompaniedby an unusualmechanism of action? Initial studies by Fuchs and Johnson (37)indicatedthat taxol inhibited proliferation at the G2-M phasein thecell cycle and blocked mitosis. Thus it appearedto be one in a seriesof naturallyoccurringspindlepoisonssuchasvincristineandvinblastine, colchicine,podophyllotoxin,maytansine,andothers.The findingthat taxol was a spindle poison was not necessarilyencouraging.Shortly thereaftera more detailedinvestigationby the Horwitz group(38) established that, while taxol was a mitotic inhibitor, the mechanism was unique in that it stabilized microtubules and inhibiteddepolymerizationback to tubulin; this was the oppositeeffect of theotherantimitoticagentscited above,which all bind to solubletubulinandinhibit thepolymerizationof tubulinto form microtubules(36, 39,40). This informationwas importantin making the argumentthat byvirtue of uniqueness of both structure and mechanism, taxol (Compound 17) was a worthy candidatefor development.

Our early observationsthat taxol extracts and pure taxol werehighly activein P-4 assaynow becameexplicable.Tubulin is a proteininvolved in the processof mitosis.The Vinca alkaloids were noteworthy for their activity against P-4. Taxol is also highly active

againstP-4. Both compoundsbind to tubulinbut, asdescribedabove,by completelydifferent mechanisms.

Era of Rapid Progress, 1982—1994

Rapid progressboth in chemicalsynthesisof taxol and in clinicaldevelopmentsoccurredin 1982—1994(Table 4). The events in thisdecadehave been coveredin detail by a numberof reviews (36, 41,42). Hence this section will present only a brief outline of thetremendousprogressand extensive researchconductedduring thisperiod.

Animal toxicology and formulation were in place by 1982—1984.

Clinical PhaseI and PhaseII trials were conductedover the period1983 to 1986. Great interestwas generatedin taxol, both in scientificcircles and by the general public, by the announcement of the remarkable efficacy of taxol againstovarian cancer(43, 44). Subsequently,taxol and an analogue, taxotere (Compound 23) (Fig. 4), are beingstudiedfor efficacy againstmany solid tumors(breast,lung) (36, 41).

In an effort to obtain adequate supplies of taxol, the National759

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CAMPTOTHECINAND TAxOL IN PRECLINICALAND CUNICAL RESEARCH

metabolites (natural products) biosynthesized?J. Nat. Prod. (Lloydia), 52:1189—1208,1989.

3. Woodbury, A. M., Wall, M. E., and Willaman, J. 3. Steroidal sapogenins.LVI!!.Steroidalsapogeninsfrom theJoshuatree.J. Econ.Bot., 15: 79—86,1961.

4. Wall, M. E., Krider, M. M., Krewson,C. F., Eddy,C. R., Willaman,J. J., Correll,D. S., and Gentry, H. S. Steroidal sapogenins. VI!. Survey of plants for steroidalsapogeninsandotherconstituentsJ.Am. Pharm.Mane. (Sci.Ed.),43: 1-7, 1954.

5. Wall, M. E., Eddy, C. R., Willaman, J. I., Correll, D. S., Schubert,B. G., andGentry,H. S. Steroidalsapogenins.XII. Surveyof plantsforsteroidalsapogeninsandotherconstituents.J. Am. Pharm.Assoc.(Sci. Ed.), 43: 503—SOS,1954.

6. Wail,M.E.,Fenske,C.S.,Kenney,H.E.,Wilaman,J.J.,Correll,D.S.,Schubert,B. G., andGentry,H. S. Steroidalsapogenins.XLII!. Surveyof plantsfor steroidalsapogeninsandother constituents.J. Am. Pharm.Assoc.(Sci. Ed.), 46: 653-684,1957.

7. Wail, M. E., GaMe, J. W., Willaman, J. J., Jones,0., and Schubert,B. G. Steroidalsapogenins. LX. Survey of plants for steroidal sapogenins and other constituents.J. Pharm.Sci. 50: 1001—1034,1961.

8. Perdue,R. E.,Jr.,Smith,R. L, Wall,M. E.,Hartwell,J. L, andAbbott,B. J.Camptothecaacuminata decaisne(nyssaceae).Sourceof camptothecin,an antileukemic alkaloid. U. S. Agric. Res. Serv. Tehn. Bull., No. 1415, 1970.

9. Craig,L C. Identificationof smallamountsof organiccompoundsby distributionstudies.II. Separationby countercurrentdistribution.J. Biol.Chem.,155:519—534,1944.

10.Craig,L C.,andPost,0. Apparatusforcountercurrentdistribution.Anal.(1cm.,21:500—504,1949.

11. Wall,M. E.,Wani,M. C.,Cook,C. E.,Palmer,K. H., McPhail,H. T., andSins,0. A.Plantantitumoragents.I. The isolationand structureof camptothecin,a novelalkaloidalleukemiaandtumorinhibitor from Camptothecaaewninata.J.Am. Chem.Soc.,88:3888—3890,1966.

12. Wani, M. C., and Wall, M. E. Plant antitumoragents.I!. The structureof two newalkaloidsfrom Camptothecaacuminata.J. Org. Chem.,51: 1364-1367,1969.

13. Wani,M. C., Ronman,P.E., Lindley,J. T., andWall, M. E. Plantantitumoragents.18.Synthesisandbiologicalactivityof camptothecinanalogs.J.Med.(1cm.,23:554—560,1980.

14. Wall,M. E.Alkaloidswithantitumoractivity.In: K. Mothes,K@Schreiber,andH. R.Schutte(eds.), InternationalSymposiumon Biochemistryand Physiologyof theAlkaloids, pp. 77-87. Berlin: Academic-Verlag,1969.

15. Gottheb,J.A., andLice, J.K. Treatmentof malignantmelanomawithcamptothecin(NSC-100880).CancerChemother.Rep.PartI, 56: 103—105,1972.

16. Muggia,F.M., Creaven,P.J.,Hanson,H. H., Cohen,M. C.,andSelawry,0. S.PhaseI clinicaltrial of weeklyanddaily treatmentwith camptothecin(NSC-100880):correlationwith preclinicalstudies.Biochemistry,56: 515—521,1972.

17. Moertel, C. 0., Schuu, A. J., Reitemerer,B..G., and Hahn, R. 0. PhaseH studyofcamptothecin(NSC-100880)in the treatmentof advancedgastrointestinalcancer.CancerChemother.Rep.PartI, 56: 95—101,1972.

18.Bin,X. Newresultsin pharmacologicresearchof someanticanceragents.In: J.J.Burns and P. J. Tsuchitani (eds.), U.SiChina Pharmacology Symposium, pp.151-188.Washington,DC: NationalAcademyof Sciences,1980.

19. Hsiang,Y-H., Hertzbcrg,R., Hecht,S., andLiu, L Camptothecininducesproteinlinkeddnabreaksvia mammalianDNA topoisomeraseI. J. Biol. Chem.,260:14873-14878,1985.

20.Wall,M. E.,Wani,M. C.,Natschke,S.M., andNicholas,A. W. Plantantitunioragents.22. Isolationof 11-hydroxycamptothecinfrom CamptothecaacuminataDecne:total synthesisandbiologicalactivity. J. Med.Chem.,29: 1553—1555,1986.

21.Wani,M.C.,Nicholas,A.W.,andWall,M.E.Plantantitumoragents.23.Synthesisandantileukemicactivityofcamptothecinanaingues@J.Med.Chem.,29:2358-2363,1986.

22. Wani, M. C., Nicholas, A. W., Manikumar, 0., and Wall, M. E. Plant antitumoragents.25. Totalsynthesisandantileukemicactivityof ringA-substitutedcamptothecinanalogs,structureactivity. J. Med.Chem.,30: 1774—1779,1987.

23. Want, M. C., Nicholas, A. W., and Wall, M. E. Plant antitumor agents.28. Resolutionof a key tricyclic synthon,5'(R@1,5-dioxo-(5'-ethyl-5'-hydmxy-2'-H,5'-H, 6'-H-6-oxopyrano)[3',4'-J]-6,8-tetrahydroindolizine: total synthesis and antitumor activityof 20(5)-and20(R)-camptothecin.J.Med.(1cm.,30:2317-2319,1987.

24. Jaxel,C., Kohn, K. W., Wani, M. C., Wall, M. E., andPommier,Y. Structureactivitystudyof theactionsof camptothecinderivativeson mammaliantopoisomeraseI,evidencefor a specificreceptorsite andfor a relationto antitumoractivity. CancerRes.,49: 1465—1469,1989.

25. Hsiang, Y-H., Liu, L F., Wall, M. E., Want, M. C., Kirschenbaum, S., Silber, R., andPotmesil,M. DNA topoisomeraseI-mediatedDNA cleavageand cytotoxicity of

camptothecinanalogs.CancerRes.,49: 4385—4389,1989.26. Giovanclla, B. C., Wall, M. E., Wani, M. C., Nicholas, A. W., Liii, L F., Silber, R.,

andPotmesil,M. HighlyeffectiveDNAtopoisomerase-itargetedchemotherapyofhumancoloncancerin xenografts.Science(WashingtonDC),246: 1046—1048,1989.

27. Sawada,S., Okajima, S., Aiyama, R., Nokata, K., Furuta,T., Yokokura, T., Sugino,E., Yamaguchi,K. and Miyasaka,T. SynthesisandantitumoraCtivityof 20(5)-camptothecin derivatives: carbamate-linked water-soluble derivatives of 7-ethyl-lOhydmxycamptothecm Chem. Pharm. Bull., 39: 1446-1454, 1991.

28. Kingsbury, W. D., Boehm, J. C., Jakas,D. IL Holden, K. G., Hecht, S. M., Gallagher,G., Caranfa,M. J., McCabe,F. L, Faucette,L R., Johnson,R. K., andHertzberg,R. P. Synthesisof water-soluble(aminoalkyl)camptothecinanalogues:inhibitionoftopoisomeraseI andantitumoractivity.J.Med.Chem.,34:98—107,1991.

29. Luzzio, M. J., Besterman,J., Evans,M. G., Lackey,K. E., Leitner,P., Peel,M. R.,Sisco,J. M., Sternbach,D. D., Tong, T., and Vehling, D. E. Novel water-solublecamptothecinanalogsasinhibitorsoftopoisomerasel.Proc.Am. Assoc.CancerRes,34:A1980, 1993.

30. Wall, M. E., and Wan!, M. C. Recent progress in plant anti-tumor agents. PaperM-006. in: 153rd National Meeting ofthe American Chemical Society, Miami Beach,FL, 1967.

31. Wani, M. C., Taylor, H. L, Wall, M. E., Coggon,P., and McPhail, A. T. Plantantitumoragents.VI. Theisolationandstructureof taxol,anovelantileukemicandantitumoragentfromTaxusbrevifolia.J.Am.Chem.Soc.,93:2325—2327,1971.

32. Persinos,E. (ed.).WashingtonInsight,September15, 1990.33.Wani,M.C.,Taylor,H.L, andWall,M.E.Plantantitumoragents:colubrinolacetate

andcolubrinol,antileukaemicANSAmacrolidesfromColubrinatexensis.them.Commun. Chem. Soc. Sect. D, 470: 390, 1973.

34. Wani, M. C., Taylor, H. L, Wall, M. E., McPhail, A. T., and Onan, K. D. Antitumoragents.XII!. Isolationandabsoluteconfigurationof carminomycinfrom Streptosporangium sp. J. Am. Chem. Soc., 97: 5955-5956, 1975.

35. Wani,M. C.,Taylor,H. L, Thompson,J.B., andWall, M. E. Plantantitunioragents@XVI. 6a-Senecioyloxychaparrinone, a new antileukemic quassinoid from Simabamultiflora. Uoydia (Chin.), 41: 578—583,1978.

36.Suffness,M.Taxol:fromdiscoverytotherapeuticuse.in: J.A. Bristol(ed.),AnnualReportsin Medicinal Chemistry,pp. 305-314. San Diego, CA: AcademicPress,1993.

37. Fuchs, D. A., and Johnson,R. K. Cytologic evidence that taxol, an antineoplasticagentfrom Taxusbrev4folia,actsasa mitotic spindlepoison.CancerTreat.Rep.,62:1219—1222,1978.

38. Schiff@P.B.,Fain,J.,andHorwitz,S.B. Promotionof microtubuleassemblyin vitroby taxol.Nature(Land.),22: 665—667,1979.

39. Horwitz,S. B., Parness,J.,Schiff,P. B., andManfredi,J.J.Taxol:a newprobeforstudying the structureand function of microtubules.Cold Spring Harbor Symp.Quant.Biol.,46:219—226,1982.

40. Horwitz, S. B. Mechanism of action of taxol. Trends Pharmacol. Sci., 13: 134—136,19fl

41.Wall,M. E.,andWani,M. C. Taxol:discoveryto clinic.in: H. WagnerandN.Farnsworth(eds.),Economicand Medicinal Plant Research,Vol 6, pp. 299-322,NewYork:AcademicPress,1994.

42.Wall,M. E. CamptOtheCinandtaxol.in: D. Lednicer(ed.),ChroniclesOf DrugDiscovery, Washington DC: pp. 153—165.American Chemical Society, 1993.

43.MCGUIIC,W. P.,Rowinsky,E. K., Rosenshein,N. B., Grumbine,F. C.,Ettinger,D. S.,Armstrong,D. K., andDonehower,R. C. Taxol:a uniqueantineoplasticagentwith significant activity in advanced ovarian epithelial neoplasms.Ann. Intern. Med.,111:273—279,1989.

44. Slichenmyer, W. J., Rowinsky, E. K., Donehower, R. D., and Kaufmann, S. H. Thecurrentstatusof camptothecinanaloguesasantitumoragents.I. NatL CancerInst.,85: 271—291,1993.

45. Kingston,D. G. I. The chemistryof taxol. Pharmacol.Ther.,52: 1—34,1991.46. Chaudhary, A. G., Gharpure, M. M., Rimoldi, J. M., Chordia, M. D., Gunatilaka,

A. A. L, andKingston,D. G. I. Unexpectedlyfacilehydrolysisof the2.benzoategroup oftaxol and synthesesof analogs with increased activities@J. Am. Chem. Soc.,116:4097—4098,1994.

47. Holton,R. A., Somoza,C., Kim, H-B., L4ang,F., Biediger,R. J., Boatman,P. D.,Nadizadeh, M., Suzuki, Y., Tao, C., Vu, P., Tang, S., Thang, P., Murthi, K. K.,Gentile,L N.,andLiu,J.H.FirsttotalsynthesisoftaxoL1.Functionalizationof theB ring. J. Am. Chem.Soc.,116: 1597—1598,1994.

48. Nicolaou, K. C., Yang, Z., Liu, J. J., Ueno, H., Nantermet, P. G., Guy, R. K.,a@ib@me,C.F.,Renaud,J.,Couladouros,E.A.,andPaulvannan,K.Totalsynthesisof taxol. Nature(Lond.),367: 630—634,1994.

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1995;55:753-760. Cancer Res   Monroe E. Wall and Mansukh C. Wani  F. Cain Memorial Award Lecture

Thirteenth Bruce−−Camptothecin and Taxol: Discovery to Clinic

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