recent studies on natural products as anticancer agents

8/2/2019 Recent Studies on Natural Products as Anticancer Agents

1/27

Current Topics in Medicinal Chemistry 2004, 4 , 241-265 241

1568-0266/04 $45.00+.00 2004 Bentham Science Publishers Ltd.

Recent Studies on Natural Products as Anticancer Agents

ngel G. Ravelo*, Ana Estvez-Braun, Haydee Chvez-Orellana, Elisa Prez-Sacau, Dulce MesSiverio

Instituto Universitario de Bio-Orgnica Antonio Gonzlez, Universidad de La Laguna, Avda. Astrofsico Fco.Snchez 2. 38206. La Laguna, Tenerife, Spain

Abstract: Cancer will be the major cause of death in the 21st century and natural products should provide novel andmore effective anticancer agents. This review deals with new natural molecules liable to become anticancer drugwell as recent specific strategies for a selective treatment of cancer. The introduction presents the current state oart on anticancer research. Beside, in the following subheadings we summarize our research on cytotoxic natquinone methide-triperpenes and their analogues. We also discuss our results on the anti-tumour promoactivity of natural naphthoquinones and their derivatives.

1. INTRODUCTION

The traditional medicine systems, based on plants, havebeen used all over the world for thousands of years [1-4].

These plant based systems continue to play an essential rolein health care. The WHO (World Health Organization)estimates that approximately 80% of the worlds inhabitantsrely mainly on traditional medicine for their primary healthcare [5-6]. To a large extent, the use of natural products indrug design represents the natural evolution of this oldtradition. Natural products have had a major impact astemplates or direct treatments for cancers and infectivediseases. In the cancer area, of the 92 drugs commerciallyavailable prior to 1983 in the United States, or approvedworldwide between 1983 and 1994, approximately 62% canbe related to a natural product origin. This figure ignoresdrugs of biological origin such as interferon, or recombinatelyproduced cytokines [7]. The influence of natural products in

drug discovery has been recently reviewed in two excellentpapers by Cordell [8] and Newman [9], with sectionsdevoted to antineoplastics from natural sources.

Natural products also play an important role in chemicalbiology. In fact, natural products have been extensively usedto elucidate complex cellular mechanisms, including signaltransduction and cell cycle regulation, leading to theidentification of important targets for therapeutic intervention[10-11]. As a result of recent advances in biology, thereexists now an increasing demand for new natural products, ornew natural products-like small molecules. Specifically, thefields of genomic and proteomics promise the rapididentification of large numbers of gene products for whichsmall molecule modulators will be of both biological andmedicinal interest [12-16]. Moreover, the combination of cellbiology and high throughput technology has led to thedevelopment of various cellular assays in which small

*Address correspondence to this author at Instituto Universitario de Bio-Orgnica Antonio Gonzlez, Universidad de La Laguna, Avda.Astrof sico Fco. Snchez 2. 38206. La Laguna, Tenerife, Spain; Fax:922318571; E-mail: [email protected]

molecule libraries can be used to identify and previously unknown targets [17-18].

Despite the ever-increasing demand of new n

products, their isolation and structure elucidation still rea labor-intensive process [8, 19-20]. As an alternchemists are now enlisting the tools of solid-phase cnatorial synthesis to construct libraries of natural panalogues and natural product like compounds [21-23]are examples of this approach based upon the nproducts paclitaxel [24] and epothilones [25-26]. Ni[27-29] introduced this strategy for the constructinatural productlike libraries using the concept of privstructures, a term first proposed by Evanset al. [30-31] describe select structural types that bind to muunrelated classes of protein receptors as high affinity liGiven the success of privileged structures in medchemistry, the approach of Nicolaou, and the solid

synthesis that it entails, are now leading this area.Waldmannet al. [32-33] have developed the dom

concept for a more efficient search for drug candidateconcept aims at the generation of compound librariesvery high hit rate. The domain concept is basestructurally conserved yet genetically mobile pdomains and the corresponding natural products seduring evolution. The biologically active natural prcan be regarded as chemical entities that were evolutioselected and validated for binding to particular pdomains. Therefore, they are already biologically valand the underlying structural architectures of such n

products may provide powerful guiding principledevelopment of new bioactive molecules. The dconcept regards a natural product class as a starting pothe drug finding process and the development of comlibraries rather than attempting to synthesizede novo.

The two approaches mentioned above, privstructures and domain concept, are already obtasignificant results and one can easily foresee a future sin the area of cancer treatment.


2/27

242 Current Topics in Medicinal Chemistry, 2004 , Vol. 4, No. 2 Ravelo et al

Although plants have a long record of use in the cancertreatment [34], many if no all of the claims on the efficacy of such treatments should be viewed with some skepticism.Cancer, as an specific disease, is poorly defined in terms of folk and traditional medicines [35]. Even more, there iscontroversy on the evaluation of the potential cancerchemotherapeutic efficacy of natural products and someresults seem to depend on specific tests [36].

From now on we shall focus on anticancer agents;specifically, we consider those appearing after the review byNewman [9]. A first set of studies are listed as refereences[37-52]. But rather than going quickly through all of them,we would mention in some detail a few additional worksthat illustrate the most recent advances.

-lapachone (1) is a naturally occurring quinone obtainedfrom the lapacho tree (Tabebuia avellaneda). This compoundhas a broad spectrum of biological activities and preclinicalstudies as anticancer drugs are in a developmental phase. Thepharmacokinetics of -lapachone shows to be biexponentialwith a rapid distribution phase after 40mg/kg IP adminis-tration in nude mice. Maximum liver concentration for-

lapachone was obtained at 5 minutes post-injection [53]. Invitro -lapachone was cytotoxic against a variety of drug-sensitive and drug resistant tumour cell lines, includingMDR1-overexpressing cell lines, camptothecin-resistant(CPT-K5 and U93/CR), and the atypical multidrug-resistantCEM/V-1 cell line [54].-lapachone induced apoptosis inhuman promyelocytic leukaemia cells (HL-60), humanprostate cancer cells (DU-145, PC-3 and LNCaP) [55,56],and MCF-7:WS8 breast cancer cells [57]. The combinationof -lapachone and paclitaxel was highly effective ininhibiting tumour survivalin vitro [58]. CoPharma haslicensed worldwide rights to-lapachone (year 2001).Isoasterriquinone (2) is one of a series of asterriquinoneanalogues isolated from the fungal species Aspergilluscandidus . The potential use of these compounds [59] in thetreatment of cancer is being evaluated in preclinical studiesin the USA (year 2000).

Other quinones studied as anticancer agents aregeldanamycin (3), isolated from a strain of Streptomyceshygroscopicus [60],17-allylamino-17-demethoxy geldanamy-cin (4) [ 61-62], which act as tyrosine kinase inhibitors, andirisquinone (5), isolated from the seeds of Iris latea pallasii,commonly used in traditional Chinese cancer therapies [63-64]. (5) acts as radiosensitizer and may also have chemo-sensitising activity. Other kinds of natural products likeflavonoids baicalein (6) and baicalin (7) isolated fromScutellaria baicalensis [65-66] are under preclinicalinvestigation as anticancer agents. These compounds havebeen shown to inhibit the replication of the human T cellleukaemia virus. The preclinical studies on human ovariancancer cells of sylibin (8) continues [67]. Quercetin (9), alsoknown as Prosta-Q [68-69], has been evaluated in patientswith prostatitis and it showed antineoplastic effects, althoughnephrotoxicity was found as adverse events [70-71]. Thecoumarin esculetin (10) isolated from Artemisia scoparia[72], continues preclinical studies as a potential anticancerdrug in Taiwan (2001).The diterpene isolated from Nocardiabrasiliensis strain IFM 0406, called brasilicardin A (11), hasalso potential use as an anticancer agent [73-74]. The

triterpenes have recovered attention after the last finThe ursolic acid (12), isolated from many kinds of mediplants, has in vitro anti-tumour activity, possibly attributo matrix metalloprotease inhibition [75-76]. Ursolic currently undergoing preclinical investigation as a pochemopreventive and/or anti-inflammatory agent Uncarinic acid A (13) and its isomer, uncarinic acid(14), both with oleane skeleton, isolated fromUncariarhynchophylla, are under preclinical investigation in Korpotential anticancer agents [78].The diterpene dysid(15) is the first naturally occurring inhibitor of the specificity cdc25 protein phosphatase family, which pcrucial role in the regulation of the cell cycle [79]. Bof this property and the resulting antitumor acdysidiolide has been of interest to chemists, biologisphamacologists [32, 80-88].

A recent paper reports on the preparation of potent,active, heterocycle-based combrestastin A-4 analogueCombrestatin A-4 (16), is a natural product isolated fromSouth African treeCombretum caffrum . This compouexhibits strong antitubulin activity by binding tcolchicines binding site of tubulin [90]. CA-4 does noin vivo efficacy against murine colon 26 adenocarcinoma,part, due to its poor pharmacokinetics resulting from ilipophilicity and low aqueous solubility. The replacemthe cis double bond by a 1,2-disubstituted five-memheterocycle such as imidazole, oxazole or pyrazole, prthe formation of analogues which show potent oral antactivity in vivo .

Tryprostatin A (17) and B (18) [91] have been isolatedsecondary metabolites from the fermentation brothmarine fungal strain of Aspergillus fumigatus BM939. Thecompounds completely inhibited cell cycle progresstsFT210 cells in the G2/M phase at a final concentra50 g/mL of (17) and 12.5g/mL of (18), respectively.

Other specific strategies for a selective treatment of (angiogenesis, antibody-directed enzyme prodrug thproteosome inhibition, dual topoisomerase I and II inhibitioetc) have also benefited from the discovery and use of products. These will be considered next.

The angiogenesis, i.e. the development of new vessels from pre-existing ones, is crucial to wound rinflammation, and embryonic development. Aberrant genesis is believed to be a key step in tumor growth, sand metastasis [92-95]. Vascular development depenendothelium-specific receptor tyrosine kinases, in parthe vascular endothelial growth factor receptors(VEGFR1-3) and the Tie-2 receptor [96-97]. All receptors have been implicated in tumor angiogenesi102], and antagonization of Tie-2, VEGFR-2 or VEGligand of VEFGR-3) inhibits tumor growth and metastasis in vivo [101,103-104]. The finding of molecular weight inhibitors of these receptor tyrosine kis one of the most promising approaches to the develoof new alternative antitumor drugs. Several inhibitVEGFR-2 are in clinical trials [103-104]. The combiof VEGFR-2 inhibitors with Tie-2 antagonists spotentiate their anti-angiogenic effects [100]. Furtheinhibitors of VEGFR-3 would suppress the metasta


3/27

Recent Studies on Natural Products as Anticancer Agents Current Topics in Medicinal Chemistry, 2004 , Vol. 4, No. 2 243

lymphogenic tumors. To date, however, only a few cases of small-molecule inhibitors of the Tie-2 and VEGFR-3receptors have been reported [105-107]. Waldmannet al.[33,110] synthesized a library of 56 nakijiquinone analogues(19), the only natural product known to be inhibitors of theHer-2/Neu receptor tyrosine kinases. Studies are underway totest these analogues as possible inhibitors of the tyrosinekinase receptors involved in angiogenesis.

The antibody-directed enzyme prodrug therapy (ADEPT)[111-112], first described by Bagshawe [113], is a newstrategy for a selective treatment of cancer. A non-toxicprodrug is enzymatically converted into a citotoxic compoundselectively at the surface of malignant cells by employing anenzyme-immuno conjugate. As a proposed requirement forthe prodrug, the corresponding cytotoxic compound shouldposses an IC50 < 10nM, [114] (IC50: drug concentrationrequired for 50% inhibition of target cells), and the quotientof the IC50 of the prodrug in the presence of the relatedenzyme (QIC50) should be above 1000 [115]. Tietzeet al.[116], knowing that the antibiotic CC-1065 (20) isolatedfromSteptomyces zelensis is a good target [117], synthesizedthe compound (21). This new potent prodrug meets all therequirements for a successful use in ADEPT and first preclin-ical investigations on mice showed promising results [116].

The proteasome is an intracellular multicatalytic proteasecomplex which in combination with the ubiquitin pathwayplays a central role in major cellular processes, such as antigenpresentation, cell proliferation and differentiation, andapoptosis [118-121]. Proteolysis occurs in a barrel-shapedcore structure known as 20S proteasome, which consist of four stacked rings arrayed in an 7 7 7 7 mode [122]. Ineukaryotic proteasome three subunits of each-ring areenzymatically active with an N-terminal threonine residue asthe active nucleophile involved in proteolysis [123], withthree more or less distinct substrate specifities, that ischymotrypsin-like (CL), trypsin-like (TL), and peptidyl-glutamyl-peptide hydrolase (PGPH) activities [124]. Becauseof the physiological role of proteasome in critical intracellularprocesses, this enzyme represents a promising target for drugdevelopment in inflammatory and autoimmune diseases aswell as in tumor therapy [125-127]. Consequently, attentionhas been paid to the discovery of potent and selectiveproteasome inhibitors by structure-based design or naturalproduct screening approaches. Most of the synthetic inhibitors(consisting of peptide aldehydes, boronates and vinyl-sulfones), as well as the natural products lactacystin andepoxymicins, inhibit in a more or less selective manner theproteasome by reaction with the N-terminal threonineresidue. For a recent review on the topic see [128].

A notable exception is the highly selective andcompetitive proteasome inhibitor TMC-95A (22), which wasisolated from the fermentation broth of Apiospora montagneiSaac TC 1093,127 and its synthesis has attractedconsiderable interest [130-135]. Moroderet al. [136]synthezised the first TMC-95A analogue (23), and the resultsobtained suggest that not all of the complex structuralelements of the natural product are required for the inhibitionof proteasome, thus markedly facilitating the synthesis of TMC-95A related compounds.

The topoisomerases are essential enzymes inregulation of DNA topology which are required for cdivide and proliferate [137]. They represent important cetargets for a number of successful chemotherapeutic [137]. Charltonet al. [138-139] undertook a programdiscover orally active dual inhibitors of topoisomeraseII that avoid MDR. They succeeded with the novel achiral benzophenazine (24), a compound selected as a candida

for further evaluation. This compound is structurally to a remarkable family of natural products, the cephalostfirst isolated from the marine tube-inhabiting inverwormCephalodiscus gilchristi [140].

Finally, in an effort to enhance access to informavailable in the National Cancer Institutes (NCI) antidrug-screening database, a new web site is accessible spheroid.ncifcrf.gov) where computational tools havassembled for self-organizing map-based (SOM) analysis and data visualization. These analyses iderelationships between chemotypes of the screened agentheir effects on four major classes of cellular actimitosis, nucleic acid synthesis, membrane transporintegrity, and phosphatase and kinase-mediated cellregulation [141].

All the examples cited above illustrate the centraplayed by natural products in drug development fprevention and treatment of cancer. In the forthcsections we shall describe our own efforts in this direWe elaborate on the isolation and chemistry of two knatural products, namely, quinone-methide-triterpeneprenyl naphthoquinones. The triterpenic quinonesalready shown interesting cytotoxic activities against vtumour cell lines. We carried out chemical modificaimed at obtaining derivatives more active and/or selas well as to establish structure-activity relationshipprenyl naphthoquinones constitute yet another impgroup of natural products studied in cancer research. Bthe quinones related to lapachol have a notable preventive potential. We shall present the inhibitory efvarious natural prenyl naphthoquinones, as well asderivatives of lapachol, on the Epstein-Barr virusantigen (EBV-EA) activation induced by 1Otetradecanoylphorbol-13-acetate (TPA) as the promoter.

2. NOR -QUINONE-METHIDE-TRITERPENES AND NOR -CATECHOL-TRITERPENES WITH ANTI-TUMOUR ACTIVITY.

The Celastraceae species have a long tradition of traditional medicine, specially in Asia and Latin A[142-143]. In a recent review on plants used against cbased on the results of a query of the NAPRALERT dafor plants species, numerous Celastraceae species belto Maytenus , Celastrus and Tripterygium genera have bereferenced [144] .

The antitumoral properties are mainly attributearomatic and quinoid triterpenoids which constitrelatively small group of unsaturated and oxygenatedfriedo nor -oleananes. Thesenor- triterpenoid pigments restricted to the family of the Celastraceae (inclHippocrataceae), although the extended quinone charac


4/27


O

OO

O

OOH

N

NO

O

O

MeOOHH3CO

R

OCONH2

O

OH3CO

OO

O

OCH3

OH

OH

O

HO

OH

OH

O

R5R6

R1O

R4

R3R2

OCOOH

OHOHO

HOO

HO

HO O

O

H

OH

OOCH3

NH2HO

O

O

OO

OHO

HO

HOHO

NHCOCH3HO

HO

H COOH

H

HO

H COOH

HR

O

O

OH

HO

OMeOMeMeO

MeO

HO

(1)

-lapachone

(2)

Isoasterriquinone

(3) R=OCH3, Geldanamycin(4) R=NHCH

2CH=CH

2, 17-

(allylamino)-17-demethoxGeldanamycin

(5) Irisquinone

(8) Sylibin

(6) R1=R5=R6=H; R2=R3=R4=OH Baicalein(7) R1=R5=R6=H; R2=R4=OH; R3=Baicalin(9) R1=R2=R4=R5=R6=OH; R3=HQuercetin (10) Esculetin

(11) Brasilicardin A (12) Ursolic acid

(13) R= trans -ferulic esterUncarinic acid A(14) R= cis -ferulic esterUncarinic acid B (15) Dysidiolide (16) Combrestatin A-4 (CA-4)


5/27


Scheme 1. (contd.)

Scheme 1. Natural milecules liable to become anticancer drugs.

of nor-quinone methide triterpenes is also present in anunusual tetracyclic triterpene isolated from Russula flavida(Agaricales) [145]. For this reason, Brning and Wagner[146] coined the general name celastroloids for this class of compounds, which are isolated from the roots and aretherefore regarded as taxonomic indicators. The basic skeletonof these compounds is represented in Fig. (1), whichinvariably contains oxygenated functionalities at C-2 and C-3, the only exception being celastranhydride [147] in whichring A has undergone oxidative cleavage. Other known sitesof oxidation of the quinonemethide system are C-6 and C-7.Oxidation at C-6 affords the class of 6-oxophenolic nor-triterpenoids whereas oxidation at C-7 results in 7-oxoquinon-emethides. On the othe hand, triterpenoid quinonemethideswith an additional unsaturation at C-14(15) containing arearranged methyl group are also known and these are.

Fig. (1). Basic skeleton.

classified under 14(15)-ene quinonemethides, respeThe most common site of oxidation outside the quemethide system is C-29. This methyl group is freq

HC

A

2930

E

12 10

11

27 20

22

2815

2625

13

23

N

N

O

O

H

HR

ON

OH

ROH

O

O

H

OOH

OHHO

OH

(17) R=OMe; Tryprostatin A(18) R=H; Tryprostatin B (19) Nakijiquinones (21)

N

Cl

Ind2

O

HN

N O

N

NO

NH

N O

NH2

OH

OCH3

O

O

NH

ONH

O

HO

NH

O

HO O

NH

O

NH

CONH2

OH

O

NH

ONH

O

H3CONH

O

H O

NH

O

NH

CONH2 N

N

CONH C CH2 N(Me)2

HMeO

(20) CC-1065 (22) TMC-95A

(23) TMC-95A analogueZ=N-(benzyloxy-carboxyloxy) succinimide

(24) Chiral benzophenazine


6/27


found oxidized to CH2OH, CO2H or CO2Me; subsequentdecarboxylation yielding 29-nor analogs is also common.Other known sites of oxidation are C-15, C-21, C-22, C-23and C-28. 23- Nor analogs resulting from decarboxylation of C-23 oxidized 6-oxophenolic triterpenoids are also known.The major classes of celastroids are depicted in Fig. (2).

Although no experimental proof has so far been providedfor the biosynthetic origin of celastroloids, their co-occurrencewith friedelanes in several plants and in cell cultures hasprompted postulation of biosynthetic pathways implicatingpolpunonic acid, zeylanol, salaspermic acid and orthosphenicacid as possible precursors [148].

Driven by our interest in bioactive metabolites present inCelastraceae species used in South American folk medicine,we carried out phytochemical studies on diverse species mostof them of the Maytenus genus ( M. horrida , M. amazonica ,

M. macrocarpa, M. magellanica, M. disticha, M.chubutensis , M. boaria , M. blepharodes ). The best results toisolate these compounds were obtained using the root bark,and employing a soxhlet apparatus withn-hexanes: Et2O 1:1for extraction. The purification of the metabolites was

realized using common chromatographic techniques such assilica gel column, sephadex LH-20 column, preparativeTLC, and HPLC. From these phytochemical studies we haveelucidated several tens of celastroids [149-171], most of theirstructures being new to the literature. The determination of their structures has been carried out on the basis of exhaustive and rigorous spectroscopic studies, includingNMR bidimensional COSY, ROESY, HSQC and HMBCexperiments. The absolute configuration of these compoundshas been determined by means of X-rays, DC studies and/orchemical correlations with othernor -triterpenoids of knownabsolute stereochemistry.

In addition to the antitumoral activity, some celashave also shown antimicrobial activity [172-174]. Althougno celastroids has so far been prepared by total synthepartial synthesis has been attempted for the purpounambiguous structure confirmation [165, 175].

In the next sections our results achieved using ncelastroids as well as a library of 63 analogues inantitumour activity evaluation are presented. These analhave been obtained by acylation, oxidation, extensionstructure, modifications on the E-ring, and treatmentDDQ, NBS and morpholine. Whenever possible, comon the structure-activity relationships that can be infrom the available data are included.

2.1. Natural Triterpenequinones

The quinones 22-hydroxy tingenone (25), pristimeri(26), 21-hydroxy-pristimerine (27) and tingenone (28), usualare the main secondary metabolites present in the ro

Maytenus species. The relative abundances depend specifion the species. For instance 22-hydroxi-tingenone is the masecondary metabolite present in M. amazonica and M. macro-carpa [156-158], while pristimerine is the main compof M. blepharodes [159] and M. scutioides [160]. The resulof cytotoxic activity of these quinonemethide triterptogether with other naturally occurring 7-oxo and 7-hyquinone methide triterpenes are shown in Table1.

From the data shown in Table1, it is easy to concluthat compounds type 7-oxo, or 7-hydroxyquinonemethidesless active than the corresponding quinonemethides. Prothis can be explained as a lost of conjugation throuring. The comparison of the activities of compounds (25-28shows that the different functionalities in E ringinfluenced their activity and selectivity [151-152].

Fig. (2). Major classes of celastroids.

O

HO

Quinonemethides

O

HO O

7-Oxoquinonemethides

O

HO

14 (15)-Enequinonemethides

HO

HO

Phenolic

HO

HO

O

6-Oxophenolic


7/27


2.2. Library of Analogues of Triterpenequinones

The design of the triterpenequinone analogues was basedon the modular structure of the natural products. The

triterpenoquinones consist of two hydrophilic parts centredon the A and E rings, which contains H-bond acceptor andH-bond-donor groups, respectively. They also have anhydrophobic part, located on the B,C and D rings. CarbonC-6 at B ring may act as a Michael acceptor.

Considering this reactivity scheme, we decided to studythe influence on the antitumour activity of some modificationson the triterpenemethide skeleton, like the introduction the

acyl groups, oxidations, extension of the structure, adof nucleophiles, etc.. In the next subheadings we presresults obtained when carrying out these transformation

2.2.1- Acylation ReactionWhen performing this reaction, and depending o

conditions followed, two selective different behavioursquinonemethides were found. We obtained selacylat ions in C-3 using 1.5 eq of the correspondinchloride, three eq of dry Et3N in dry CH2Cl2 at 0 C. Usithese conditions, different acyl derivatives with sacylating agents of different nature and lipophilic cha

Fig. (3). Naturally occuriring Quinonementhide Triterpenoids.

Table 1. Cytotoxic Activity Against Cultured Cell Lines (IC 50 M) of (25-32).

(25) (26) (27) (28) (29) (30) (31) (32)

P-388 0.22 0.22 0.26 0.24 5.73 11.41 1.10 5.50

A-549 0.54 0.27 0.26 0.60 11.47 11.41 2.21 11.0

HT-29 1.10 0.27 0.26 0.60 11.47 11.41 2.21 11.0

MEL-28 1.10 0.27 0.26 0.60 11.47 11.41 2.21 11.0

P-388: mouse lymphoma (ATCC:CCL 46);A-549: human lung carcinoma (ATCC:CL85);HT-28: human colon carcinoma (ATCC: HTB38);MEL-28: human melanoma (ATCC:HTB72).

Fig. (4). Modular structure of triterpenequinones.

R1

O

HO

R2

R2

(25) R1=H; R2==O; R3=OH(26) R1=COOMe; R2=R3=H(27) R1=COOMe; R2=OH ; R3=H(28) R1=R3=H; R2= =O

R1

O

HO

R2

R3

R4

(29) R1=R3=H; R2=R4= =O(30) R1=R3=H; R2==O; R4=OH(31) R1=H; R2=R4==O; R3=OH(32) R1=H; R2= =O; R3=R4=OH

7

O

HO

O

B

C D

EOH

A

H-bond acceptor

H-bond donor

H-bond acceptor

H-bond donor

Michael Acceptor

Hydrophobic part

Hydrophilic part

Hydrophilic part

Hydrophobic part


8/27


were prepared (Fig. (5)). Table 2 shows the correspondingvalues of cytotoxic activity.

Acylation of the hydroxyl group located on C-3 with thefollowing acyl groups acetyl (33), lauroyl (36), anddimethylcarbamoyl (37) produces only a marginal decrease of activity (2-3 fold). In contrast, the rest of derivatives lost thecytotoxicyty. The extent to which functionality is tolerateddepends on the size and direction of the electronic effect. Adramatic lost of activity was observed for the isobutyrylderivative (35), indicating a steric constraint in this region.This conjecture is supported by the lost of cytotoxicity of benzoyl analogues [176].

The treatment of natural 22--hydroxy-tingenone (25) inpyridine with an excess of Ac2O afforded three compounds(42-44) (Fig. (6)). The acetylated mixture was purified bypreparative TLC and the structural elucidation of thesecompounds was accomplished by spectroscopic studies.These studies suggested the aromatisation of ring A in (42)with the formation of a catechol system, and the presence inring B of a vinyl pro ton and a hydrogen geminal to anoxygenated function. The disposition of these groups was

established by HMBC experiments while the stereochemistryof the hydroxyl group on C-6 was determined as in (42)because of the NOE effect detected between H-6 and Me-25.Compound (43) showed the same molecular formula inHRMS than (42) and similar1HRMN spectrum to those of (42). The main difference was in the signal attributable to H-6. The NOE effect between H-6 and Me-25 was not observedin ROESY experiments and thus (42) and (43) must be

epimers in C-6.The third compound (44) showed simi1HRMN than compound (42), including the signal assigto H-6 and the surprising presence of an ethoxy group.

The formation of these compounds can be explainenucleophilic attack of the acetate group to the C-6, produces the aromatisation of the ring A (Fig. (7)). This faagrees with the observed reaction mixture colour changdark orange (characteristic of quinones) to pale y(phenolic form). This mechanism also explains the formof the two epimers as the result of the two possible athrough the or faces of the molecule. Compou(42) and (43) do not present an acetate group on C-6thus it has to be assumed an additional hydrolysistrans-esterification of the acetate group to yield corresponding alcohols.

The formation of the ethoxy group was attributed presence of EtOH traces existing in the CHCl3 used with tmixture of reaction. When the reaction was repeatedanother solvent such as AcOEt the corresponding derivative was not found. The observed stereochemistry the ethoxy group was ratified by ROESY experiments

showed NOE effect between the OCH2CH3 and Me-25. Thstereochemistry agrees with a nucleophilic attack to thproduct through a typical SN2 process. Similar results wother natural quinones such as pristimerine (26), 21 -hydroxpristimerine (27), and tingenone (28) were also obtained.

Table 3 shows the antitumoral activity for the phederivatives (42-52), together with the antitumoral activi

Fig. (5). Preparation of C-3-acyl-derivatives.

Tabla 2. Cytotoxic activity against cultured cell lines (IC 50 M) of (33-41).

P-388 A-549 HT-29 Mel-28 P-388 A-549 HT-29 Mel-28

25 0.22 0.54 1.10 1.10 37 0.20 0.99 0.99 0.99

33 0.52 1.04 1.04 1.04 38 1.84 1.84 1.84 1.84

34 ---- 2.03 2.03 ---- 39 ---- 1.59 7.94 ----

35 ---- 10.2 10.2 ---- 40 17.0 17.0 17.0 17.0

36 0.40 1.62 1.62 1.62 41 0.85 1.69 1.69 1.69


O

HO

O

OH O

RO

O

OH

(25)

1.5 eq RCOCl3 eq Et3N

CH2Cl2 / 0o C

(33) R=COCH3(34) R=CO(CH2)2CH3(35) R=COCH(CH3)2(36) R=CO(CH2)1 0CH3(37) R=CON(CH3)2(38) R=nicotinoyl oxi(39) R=CO-Ph(OMe)3(40) R=CO p-NO2Ph(41) R=SO2PHMe


9/27


cis platinum, etoposide, vinblastine and taxol, four of thecommon world-wide anti-cancer drugs.

Comparing the activity of compounds (42), (43) and (44)with the starting product (25), it was observed that theactivity improved for the derivative possessing a hydroxygroup in -disposition on C-6 (42), while it drasticallydecreased for the corresponding epimer (43). For the etoxyderivative (44) the activity was similar to that of (25). Weobtained the best results with the pristimerine derivatives.Thus, compound (45) exhibited the highest antitumoralactivity against the P-388 cell lines with an IC50=0.044M,5-fold more potent than (26). The selectivity for this cell linewas also improved, being much larger than that of thestarting product (26). This result was better than thoserecorded forcis-platinum, etoposide and taxol, and regardingselectivity it was even better than vinblastine. Comparison

of the activity of the four starting products (25), (26), (27and (28), which have only structural differences in ring, showed that this E ring has a significant relatiwith the activity. The same conclusion was reached froactivity data of (42), (45), (48) and (51).

In general, the stereochemistry of the hydroxy glocated on C-6 produces a lost of antitumoral activit stereochemistry leads to activities higher than thosecorresponding starting materials. Only in the case of p(45) (from pristimerine), the cell line P-388 selectivitalso increased. The results of the etoxy derivatives depthe starting material. Compound (44), derived from (25)showed improved activities for the cell lines P-388, and MEL-28, but not for A-549. Compound (47), fropristimerine (26), exhibited an improvement in selecfor P-388 vs. MEL-28. This selectivity was one fo26)

Fig. (6). Phenolic derivatives obtained under treatment with Ac2O/Py.

Fig. (7). Probable formation of phenolic derivatives.

AcO

AcO

R3

R1

H

R2

OH

O

HO

R3

R1

H

R2

AcO

AcO

R3

R1

H

R2

OH

AcO

AcO

R3

R1

H

R2

OCH2CH3

(25) R1=H; R2= =O; R3=OH(26) R1=COOMe; R2=R3=H(27) R1=COOMe; R2=OH; R3=H(28) R1=R3=H; R2= =O

(44) R1=H; R2= =O; R3=OH(47) R1=COOMe; R2=R3=H(50) R1=COOMe; R2=OH; R3=H

(43) R1=H; R2= =O; R3=OH(46) R1=COOMe; R2=R3=H(49) R1=COOMe; R2=OH; R3=H(52) R1=R3=H; R2= =O

(42) R1=H; R2= =O; R3=OAc(45) R1=COOMe; R2=R3=H(48) R1=COOMe; R2=OAc; R3=H(51) R1=R3=H; R2= =O

HO

O

AcO

AcO

OAcAcO

AcO

OHOAc

trans -esterification


10/27


while it became four for compound (47), and eight if comparing P-388 vs. HT-29; Similarly compound (50) alsoshowed an activity improvement.

Table 3. Cytotoxic Activity in Cultured Cell Lines (IC 50 M)of derivatives (42-52).

Compounds P-388 A-549 HT-29 MEL-28

25 0.22 0.54 1.10 1.10

26 0.22 0.27 0.27 0.27

27 0.26 0.26 0.26 0.26

28 0.24 0.60 0.60 0.60

42 0.17 0.43 0.86 0.86

43 17.2 17.2 17.2 17.2

44 0.16 0.82 0.82 0.82

45 0.044 0.18 0.18 0.18

46 2.22 2.22 2.22 2.2247 0.21 0.84 1.68 0.84

48 0.19 0.19 0.19 16.0

49 16.0 16.0 16.0 16.0

50 0.15 0.15 0.15 0.19

51 0.23 0.23 0.23 0.23

52 19.0 19.0 19.0 19.0

Cis-platinum 8.33 16.67 33.33 33.33

Etoposide 0.17 0.17 1.70 0.85

Vinblastine 0.022 0.022 0.022 0.022Taxol 0.59 0.012 0.012 0.012

P-388D 1: Mouse lymphoma (ATCC CCL-46);AT-549 : Human lung carcinoma(ATCC CCL-185);HT-28 : Human colon carcinoma (ATCC HTB-38);SK-MEL-28 : Human melanoma(ATCC HTB-72).

We also studied the reaction with other acylating agents,such as benzoyl chloride, p-nitrobenzoyl chloride, and p-

bromobenzoyl chloride, obtaining the correspondingvatives mixture (C6-OH ; C6-OH and C6-OEt), whicwere evaluated for activity. None of them was activthey exhibited an IC50 >10 g/ml. For these type of catecderivatives, the size of the acyl groups located on riand E played an important role in the activity. There wa significant electronic effect of the substituents on aof the benzoylated derivatives, since we obtained the

activity with different substituents in the aromatic ring 2.2.2- Modifications on the Carbonyl and Hydroxyl Group Located on Ring E

Due to our interest in finding structure-activity relships, modifications of the type and number of hydrbond donors and acceptors present in ring E of 22-hydroxtingenone (25) were conducted. Modification of the Ccarbonyl group with the formation of the oxime deri(62) and its reduction to hydroxy group (64) (see Fig. (9)and transformation of the 22-hydroxy group into the esacid derivative (65), under treatment with succinic anhywere performed. Then we altered the carboxylic grorender derivatives (66) and (67). The substitution of carbonyl group with an oxime function (62) increased tactivity against the cell lines A-549, HT-29 and Mel-fold for A-549 and 4-fold for the other two cell lHowever, the selectivity of the P-388 cell line vs Mwas lost. A slight improvement was also obtained wacetylation of (62) to give (63). This tendency was aobserved for the dihydroxyl derivative (64), which improvits activity but lost its corresponding selectivity. Whhydroxyl group located on C-22 was transformed inderivatives (65), (66) and (67) the results were not even. derivative (65) displayed an attractive activity and selecfor the cell line Mel-28. The activity for the P-388 ceof the ethyl ester notably improved with respect to (65) (12fold), and also with respect the starting triterpene (25) (3fold). Likewise derivative (67) yielded the same IC50 valufor the four cell lines. In short, we have obtained anawith higher activity and/or selectivity than those of etopor cis-platinum (see Table3). It is thus shown that with ominor changes, the activity and the selectivity for thlines P-388, (65), and Mel-28, (66) were largely improved

The relative cytotoxic activities obtained by the chadifferent functional groups on ring E (see also the valu

Fig. (8). Benzoylated derivatives.

O

ORHRO

RO

OH

O

ORHRO

ROOH

O

ORHRO

RO

OCH2CH3

(53) R= p-Br-Bz(56) R= p-NO2-Bz(59) R=Bz




11/27


the natural nor -triterpenequinones (26-28) in Table 4)together with the fact that modifications of the A/B ringsaffect the cytotoxic activity, seem to suggest that the primarypharmacophore resides on the A/B rings, whereas the C-,D-,and E rings stabilize the quinone methide system of thisclass of cytotoxic compounds.

2.2.3. Oxidation Reactions

Several methods of oxidation that allowed us to obtain1,2-dicarbonyl compounds with an hydroxyl or methoxygroup on C-4, keeping the extended conjugation of doublebonds present in ring B were performed. We could not

oxidize it with the common reagents for this type of funcalization: H2O2 in alkaline medium,tert -butyl hydroperoxor sodium perborate, which have been successfully uthe epoxidation of benzoquinones. We succeededDMDO, CAN, NCS and O2 /K2CO3 /MeOH [179,180]. Thighest yields were obtained with DMDO, and the hselectivity with O2 /K2CO3 /MeOH.

Compound (25) was treated with ceric ammonium n(CAN) in aqueous acetonitrile and yielded a mixtcompounds (68) and (69) in a 3:1 ratio. The stereochemof the hydroxyl group on C-4 in (68) was solved byROESY experiment showing a NOE effect between th

Fig. (9). Transformation on E ring.

Table 4. Cytotoxic Activity in Cultured Cell Lines (IC 50 M) of derivatives (62-67).

P-388 A-549 HT-29 Mel-28 P-388 A-549 HT-29 Mel-28

25 0.22 0.54 1.10 1.10 63 0.16 0.16 0.16 0.16

26 0.22 0.27 0.27 0.27 64 0.23 0.23 0.23 0.23

27 0.26 0.26 0.26 0.26 65 0.93 1.87 0.93 0.22

28 0.24 0.60 0.60 0.60 66 0.08 0.44 0.44 0.44

62 0.22 0.27 0.27 0.27 67 0.13 0.13 0.13 0.33

P-388D 1: Mouse lymphoma (ATCC CCL-46);AT-549 : Human lung carcinoma (ATCC CCL-185);HT-28 : Human colon carcinoma (ATCC HTB-38);SK-MEL-28 : Human melanoma (ATCC HTB-72).

OH

OHH

OHH

NHOH

NOH

OHHO

OH

O

O

O

OH O

O

N

NH

O

O

HO

OEt

O

NaBH4

O

HO

OOH

NH2CH3HClO

O

O

AcO

AcO

NOAc

OAcH

OH

(64) E

Traces

DCC, Py, EtOH

(67)

(66)

(63)

(25)

(65)

(62)


12/27


23 and Me-25. Similar spectroscopic results were obtainedfor the minor diastereomer (69) which is the correspondingepimer in C-4 of (68). The new hydroxy group in this case islocated in the more congested-face of the molecule.

Similar results were obtained when (25) was treated withNCS, the yield and the ratio of compounds (68):(69) beinghigher than those obtained with CAN. The ratio of these

diastereomers was also increased to 9:1 using DMDO.The reactions with O2 and K2CO3 /MeOH were stereo-

specific, because just one diastereomer was produced (70).The presence of oxygen was necessary because when thereaction was repeated under inert atmosphere and, after 24 h,formation of the diketone compound was not detected, andthe starting material remained unaltered. The stereochemistryof the methoxy group was established as by ROESYexperiments, which also showed NOE effects between the

Me-23 and Me-25. The products with an OH group lon C-4 turned out to be less stable than the correspoones with OMe group.

With the rest of the natural quinones (26-28), wobtained similar products.

Table 5 shows the antitumoral activity of compo(25-28), (68), (70), (72), (75) and (78), together with antitumoral activity of etoposide. The rest of the derivcould not be evaluated because they resulted very unrapidly degrading with time. All these compounds (68) have activities similar to that of etoposide.antitumoral activity of dicarbonyl compounds (70), (72) an(75) for the cell lines P-388, HT-29 and MEL-28 reshigher than those of the corresponding starting nproducts. The best result was obtained for compoun72)which showed an IC50=60 nM for the cell line P-3

Fig. (10). Oxidation of quinonemethide triterpenes.

Table 5. Cytotoxic Activity in Cultured Cell Lines (IC 50 M).

P-388 A-549 HT-29 MEL-28 P-388 A-549 HT-29 MEL28

Etp 0.17 0.17 1.70 0.85 68 2.21 2.77 2.77 2.77

25 0.22 0.54 1.10 1.10 70 0.11 0.54 0.54 0.54

26 0.22 0.27 0.27 0.27 72 0.06 0.56 0.56 0.56

27 0.26 0.26 0.26 0.26 75 0.10 0.20 0.20 0.20

28 0.24 0.60 0.60 0.60 78 0.24 0.98 0.98 0.98

P-388D 1: Mouse lymphoma (ATCC CCL-46);AT549: Human lung carcinoma (ATCC CCL-185);HT-29: Human colon carcinoma (ATCC HTB-38);SK-MEL-28: Human melanoma (ATCC HTB-72).

C

O

OR3

OR

R1R2

H

A

HO

OR3

R1R2

HC

C

O

O

R3

HO

R1R2

H

23

25

NOE

(68) R1=R=H; R2= =O; R3=OH(70) R1=H; R2= =O; R3=OH; R=Me(71) R1=R3=R=H; R2= =O(72) R1=R3=H; R2= =O; R=Me(74) R1=COOMe; R2=R3=R=H(75) R1=COOMe; R2=R3=H; R=Me(78) R1=COOMe; R2=OH; R3=H; R=Me

E

(25) R1=H; R2= =O; R3=OH(28) R1=R3=H; R2= =O(26) R1=COOMe; R2=R3=H(27) R1=COOMe; R2=OH; R3=H

25

NOE

(69) R1=H; R2= =O; R3=OH(73) R1=R3=H; R2= =O(76) R1=COOMe; R2=R3=H


13/27


Transformation of the quinone methides (25, 28, 26 and 27)in the 1,2-diketones (70, 72, 75, 78) had only a slightinfluence in the selectivity for the cell lines P-388 vs. MEL-28, so the average selectivity was two-fold higher than thatfor the quinone methides. The largest improvement in thisselectivity was that from compound (28) (selectivity P-388vs. MEL-28 equals 2) to compound (72) (selectivity P-388 vs.MEL-28 equals 10). The importance of the methoxy groupin C-4 appeared after comparison of the activity of compounds(68) and (70); the activities for (70) (with OMe) are between5 and 21-fold higher than the activities for (2) (with OH).

These data allow to draw the following conclusion: themodifications on the carbons C-3 and C-4 of the ring A,keeping the extended conjugation of double bonds presentin ring B, do not provoke a dramatic lost of cytotoxicity.This fact supports the key role of the B ring, whichpossibly acts as a Michael acceptor in the interaction with

the target molecule. 2.2.4. Extension of the Structure

Starting with the 1,2-dicarbonyl compounds, we extendedthe structure from ring A by condensation with diamino oraminoalcohols. These reactions introduce 1,4-diaza or 1,4-oxaza systems which characterize several bioactive compoundslike the antitumoral antibiotic dactinomycin or clofazimine.

Fig. (11) shows the reactions of diketone (70) with 1,2-diaminoanthraquinone, 4, 5 -dimethyl - bencene - diamine,o-aminophenol and aminoethanol. In the case of the reactions

with aminoalcohols, we just obtained the regioisome81and (82). The structure of compounds (79-82) was thoroughdetermined by one and two-dimensional NMR experim

Derivatives (79-82) were evaluated and the corresponresults are shown in Table6, these compounds resulted active than (70). The introduction of the oxaza systemmore effective (81 and 82) than the introduction of the dring (79 and 80). Compounds (81) and (82) maintained tsame selectivity for the cell line P-388 vs. Mel-28 original (70). From this result we conclude that the incin size and the absence of carbonyl groups in ring A pimportant role in the activity.

Other reactions to extend the structure of a nor tritemethyilenquinone, namely the reactions of 22-hydroxtingenone (25) and pristimerine (26) with o-bromoanil wetested. The reaction of 76 mg (0.17 mmol) of hydroxytingenone with 1.5 equiv of o-bromoanil in 20 mof dry bencene at room temperature yielded the mixtadducts showed in Fig. (12).

These adducts are presumably formed by a hetero Alder reaction between the rich electron double bond and the dicarbonyls present ino-bromoanil. The macompound (83) is formed in the less congested face, i.e face. The stereochemistry of this adduct was estabby ROESY experiments, which showed a NOE effect betwMe-23 and Me-25. Similar results were obtained pristimerine was used. These reactions were also perfat 0 C to see if the selectivity was improved, but it d

Fig. (11). Extension of the structure.

N

N

OMe

O

O

H

O

N

OMeOH

O

N

OMeOH

N

N

OMeNH2

NH2

O

O

O

OHH

OMe

NH2NH2

O

ONH2

NH2H2N OH

NOE

(79)

(80)

(70)

(82)

(81)


14/27


change. Table 6 shows the antitumoral activity of compounds(83-85). There was not much gain in activity with respect tothe starting products (compare83 and 84 with 25, and also85 with 26); however, the adducts become a little moreselective against P-388 and A-549. The almost identical degreeof activity of (83) and (84) proved that the activity was notrelated to the adduct stereochemistry. This result reinforcesthe importance of the carbonyl group on C-2 and the extendedconjugation in ring B since the extension of the structurefrom the carbons C-3 and C-4 do not imply a lost of activity.

2.2.5- Additional Reactions

The formation of the enamine of the C-21 carbo(25) using morpholine, toluene and Molecular Sieveattempted, but we obtained compound (87). This compouwas presumably formed by an initial nucleophilic adon the carbonyl group on C-2, followed by a prototroinvolves the hydrogen-11, the rearrangement of Me-2the lost of hydroxy group on C-2. (25) under treatment wNBS afforded the stereoselective formation of jus

Fig. (12). Reaction witho-bromoanil.

Fig. (13). Other transformations.

R1R2

R3H

OH

O 1

3

O

O

O

BrBr

Br

Br

HO

(83) R1=H; R2= =O; R3=OH(85) R1=COOMe; R2=R3=H

OO

O

BrBr

Br

Br

HO



O

O

Br

Br

Br

Br

O

HO

Br

HO

HO

SCH2Ph

O

O

CN

CN

NO

HOO

N

OH

O

HO

ODDQ

NBS

SHCH2Ph

(87)

, C7H8

MolecularSieves

(89)

(90) (88)


15/27


derivative (88). The stereochemistry of the bromine atomwas established as because of the NOE effect between H-11and Me-27. Another type of transformation that was triedwas the extension of the conjugation under treatment with

DDQ. We did not suceed but, instead, the derivativ89was obtained. A sound hypothesis on how this mayhappened can not be suggested. Compounds (87), (88) an(89) showed values of IC50 > 10g/ml.

Table 6. Cytotoxic Activity in Cultured Cell Lines (IC 50 M) of (79-85 and 90).

P-388 A-549 HT-29 MEL-28 P-388 A-549 HT-29 MEL28

79 17.6 17.6 17.6 17.6 83 0.14 0.14 1.16 0.58

80 1.50 1.50 1.50 1.50 84 0.14 0.14 1.16 0.58

81 0.20 0.98 0.98 0.98 85 0.14 0.28 0.56 0.56

82 0.17 0.90 0.90 0.90 90 0.22 0.22 1.79 0.90


Fig. (14). Naturally occurring catechol triterpenes.

HO

HO

O

COOMe

HO

HO

CHO O

COOMe

HO

HO

O

O

HO

HOCHO

O

HO

HOCHO O

O

OHHO

HO

COOHO

O

OH

HO

HO

CHO

O

OHHO

MeO

COOHO

O

OH

HO

MeO

O

O

OH

HO

HO

O

O

OH

H

HO

HO

O

O

OHHO

MeO

O

O

OH

HO

(91) (92) (93)

(94) (95) (96)

(97) (98) (99)

(100) (101) (102)


16/27


Yet a following reaction was the treatment with phenyl-methanethiol as nucleophilic agent, the expected product (90)was obtained, which appears to be due to a Michael additionon C-6. The addition occurs in the less hindered facesi-si of

the C5-C6 double bond, probably due to the large size of thephenyl-methanethiol. The cytotoxic activities of (90) and itsprecursor (25) were similar (Table6). In a similar way to theformation of (90) discussed above, the quinone methidecould act as a Michael acceptor of the nucleophilic groupspresent in the molecular target(s) in the cell. On the otherhand, the high level of activity of derivatives with structuretype (90) and a substituent at C-6 (as obtained in acylationreactions) let us point out a second possibility; thesecompounds could also react with the nucleophilic groupspresent in the target butvia nucleophilic substitution on C-6.

2.3. Natural Catechol Triterpenes

This type of compounds also appears in the roots of Maytenus species, but in lesser quantities than thequinonemethides. Fig. (14) shows the structure of severalrepresentative natural phenolic triterpenes and Table7contains the corresponding cytotoxic activities.

From the results presented in Table7, it can be seen thatthe phenolic triterpenes are less active than the correspondingquinonemethide triterpenes. In this type of compounds, thepresence of a carbonyl group on C-6 seems to be critical forthe activity. Compounds without a 6-oxo function presentedlow activity (e.g.94 and 96). In the case of 6-oxo-phenolictriterpenes, the absence of the conjugated double bond C7-

C8 implies a reduction of activity which was also obin those compounds that have lost Me-23 and prehydrogen instead, e.g. (100). Yet another interestconclusion on the activity structure relationship ap

from the comparison of derivatives with and withoumethoxy group on C-3 (e.g.96 and 98). The activdecreased with the presence of the methoxy grouphighest activity was achieved with the 6-oxo-catriterpenes that present a C7-C8 double bond and whMe-23 was oxidized to aldehyde. A loss of activitobserved when a carboxylic acid function was present of the aldehyde.

2.3.1. Modifications on Phenolic Triterpenes

In an attempt to establish the role of the different glocated in the aromatic ring as well as the carbonyl fuat C-6 in 6-oxo phenolic triterpenes, we carried outsimple modifications on the most active natural tritisolated so far (95).These reactions are illustrated in Fig 15)From these results we observed how the activity decwith the introduction of methoxy groups, while it incwith the introduction of acetyl groups. In all these caslipophilic character was greater than that of compoun95)Analysing these two different function-alities, it seemthe activity was affected by the electronic effect of thsubstituents on ring A. The replacement of a six-ring B seven-ring B resulted in a dramatic cytotoxicity likewise an activity loss was observed with the heteroderivative (107).

Tabla 7. Cytotoxic activity in Cultured Cell Lines (IC 50 M) of (91-102).

P-388 A-549 HT-29 Mel-28 P-388 A-549 HT-29 Mel-28

91 10.37 10.37 10.37 10.37 97 5.55 11.11 11.11 11.11

92 1.00 0.20 2.02 1.00 98 20.16 20.16 20.16 20.16

93 11.42 11.42 22.83 22.83 99 2.15 2.15 2.15 2.15

94 23.04 23.04 23.04 23.04 100 5.71 5.71 5.71 5.71

95 0.54 0.54 1.07 1.07 101 5.51 5.51 5.51 5.51

96 5.19 5.19 5.19 5.19 102 8.74 8.74 8.74 8.74

P-388D 1: Mouse lymphoma (ATCC CCL-46);AT549: Human lung carcinoma (ATCC CCL-185);HT-29: Human colon carcinoma (ATCC HTB-38);SK-MEL-28: Human melanoma (ATCC HTB-72).

Tabla 8. Cytotoxic activity in Cultured Cell Lines (IC 50 M) of (103-107).

P-388 A-549 HT-29 MEL-28 P-388 A-549 HT-29 MEL-28

95 0.54 0.54 1.07 1.07 105 9.58 9.58 9.58 9.58

103 0.20 0.20 0.42 0.42 106 5.06 5.06 5.06 5.06

104 0.18 0.22 0.22 0.22 107 1.08 1.08 2.17 1.08

P-388 : mouse lymphoma (ATCC:CCL 46);A-549: human lung carcinoma (ATCC:CL85);HT-28: human colon carcinoma (ATCC: HTB38);MEL-28: human melanoma (ATCC:HTB72).


17/27


2.4. Mechanism of ActionThe mechanism of action of these type of compounds

have not been elucidated yet. However, we advance aplausible mechanism of action for the quinone methide. Thismay interact with nucleophilic groups (:Nu) of the moleculartargetvia Michael addition, orvia nucleophilic addition toC-6 in 6-oxo-catechol triterpenes, orvia nucleophilicsubstitution with the 6-substituted-catechol triterpenes.

Our hypothesis has been recently reinforced by Setzeret al [181]. These researchers carry out molecular orbitalcalculations using semi-empirical PM3 and Hartee-Fock 3-21G ab initio techniques on the quinonemethide tingenone

(28), as well as on the nucleotides bases adenine, gucytosine, and thymine. The molecular mechanism calculatisuggested that a possible mode of action of quinone-minvolves quasi-intercalative interaction of the compwith DNA, followed by nucleophilic addition of thebase to the C-6 of the triterpenoid.

2.5. Summary of Structure-activity Relationships

Considering all the available information on struactivity relationships, we have distilled the followingthat may be accounted for to foresee or advanccititoxicity of a product:

Fig. (15). Modifications on (95 ).

Fig. (16). Plausible mechanism of action.

HO

HO

O

OH

NN

MeO

MeOCHO O

O

OH

HO

HO

CHO O

O

OH

AcO

AcOCHO O

O

OAcAcO

AcOCHO O

O

OAcH

O

OH

O

CHO

MeO

MeO

(106)

(105)

(107)CH2N2 /CHCl3

Mel/K2CO3

NH2NH2 /C7H8

(95)

NOE

(104)(103)

Ac2O/py

O

HO

HO

HO

O

HO

HOL2

L'Leaving group

:Nu-TargetNucleophilic Substitution

:Nu-TargetNucleophilic Addition

:Nu-TargtMichael Addition


18/27


For Quinone methide triterpenes: Presence of the extended conjugation of double bonds in

ring B. Presence of the carbonyl group on C-2. Small ester groups as substituent on C-3. The type and number of hydrogen-bond donors and

acceptors present in the E ring do not modify thecytotoxicity in a significant way.For Triterpene catechols: Presence on C-6 of a carbonyl group or other substituents

(e.g. OH, SCH2PH..). With the 6-oxo derivatives, the presence of the C-7-C-8

double bond, or the presence of an electron withdrawingsubstituent in ring A, or both.

With the 6-substituted catechol triterpenes, the schemistry enhances citotoxicity. No voluminsubstituents can be present in ring A.

Therefore, small structural modification may lesubstantial improvement in selectivity and several exahave been presented and discussed.

As a result of these studies several metabolite

forward above have been selected by the BiolEvaluation Committee for Cancer Drugs (BEC/C) NCI for preliminary testing in the hollow fiber-based These tests have been developed for the preliminaryin vivoassessment of cancer chemotherapeutic efficacy [182].

3. NATURAL NAPHTHOQUINONES POSSESSINGCANCER CHEMOPREVENTIVE ACTIVITY

Prevention is the most reliable strategy for remedydiseases, including cancer, and the prevention has

Fig. (17). Naturally occurring naphthoquinones.

O R

O

OOH

O

O

O

OCH3

H O

O O

OOCH3

O

O OHH

HOH

O

O

O

H

OH

O

O

O R'

R O

O

O

R

O

O

OH

O

O

O R

OH O

O

HO

OHO

OCH3

OH

OCH3

O

OCH3

OH

OCH3

R

O

O

O

O

OCH3

(108) R= =O(109) R= OH

(110) (111) (112)

(113) (117)(114) R=R'=OH(115) R=H; R'=OH(116) R=OMe; R'=H

(118) R=H(119) R=C(CH3)2OH

(120) R=H(121) R=OH

(122) (123) (124) R=H(125) R=OH

(126) (127)


19/27


widely anticipated as a foremost paradigm for cancer control.Cancer chemoprevention, coined by Spornet al . in 1976[183], is defined as a strategy for cancer control by theadministration of synthethic or natural compounds capable of halting or inhibiting the onset of cancer.

Most cancer prevention research is based on the conceptof multistage carcinogenesis: initiation promotion progression [184]. Among these stages, in contrast to bothinitiation and progression stages, animal studies indicate thatthe promotion stage takes a long time to occur and may bereversible, at least in its earlier stages. Therefore, theinhibition of tumor promotion is expected to be an efficientapproach to cancer control. Promotion involves clonalproliferation of the initiated cells, and convertion of theminto premalignant tumor cells. During this phase, a varietyof intracellular signalling pathways are activated. Tumorpromotion research was started with the identification of atumor-promoting constituent in croton oil, 12-O-

tetradecanoylphorbol-13-acetate [185]. TPA-type promoters such as phorbol esters, teleocidins, or aplysiatocan activate both phospholipid and Ca+2-dependent protkinase C (PKC). PKC is widely accepted as one of theintracellular targets of TPA-type tumor promoters.

Studies on TPA allowed to establish detection ausing its biological and physiological activities. Insense, a short-termin vitro assay using the activationEpsteinBarr virus (EBV) genome-carrying hlymphoblastoid cells has been used to detect tupromoters (e.g. 12-O-tetradecanoylphorbol-13-acetate) anti-tumour promoters. This assay system is compoEBV-non-producer cells as the indicator,n-butyrate as ttrigger, TPA as the EBV-activator and the test sub[186-187]. Epstein-Barr virus (EBV) is an ubiquitous herpes-virus that is associated with several malignand diseases, together with some types of lymphomimmunocompromised hosts (e.g., AIDS and post-tran

Table 9. Percentage of Epstein-barr Virus Early Antigen Induction.

Concentration (mol ratio/TPA) a

Compounds1000 500 100 10

108 0 (0) 0 (0) 0 (0) 9.2 (20)

109 0 (0) 0 (0) 0 (0) 53.7 (70)110 0 (60) 55.2 100 100

111 32.7 (70) 63.8 88.1 100

112 0 (70) 32.9 57.3 93.8

113 0 (30) 0 (60) 62.9 88.5

114 36.1 (60) 88.4 100 100

115 11.2 (60) 27.9 56.3 86.7

116 83.8 (70) 92.0 100 100

117 17.5 (70) 44.6 63.6 97.8

118 21.3 (70) 30.7 70.6 100.0119 19.2 (70) 36.6 79.5 100.0

120 1.9 (70) 24.2 65.2 94.6

121 1.4 (60) 23.5 63.5 93.1

122 9.9 (70) 28.7 70.1 100.0

123 5.9 (70) 27.7 72.6 95.0

124 13.4 (70) 29.2 73.2 100.0

125 2.7 (70) 24.3 66.2 92.7

126 17.7 (70) 33.9 76.4 100.0

127 12.1 (70) 30.2 73.6 100.0-carotene 8.6 (70) 34.2 82.1 100.0

aMol ratio/TPA (32 pmol=20 ng/mL), 1000 mol ratio=32 nmol, 500 mol ratio= 16 nmol, 100 mol ratio=3.2 nmol, and 10 mol ratio=0.32 nmol.bValues represepercentages of EBV-EA induction in the presence of the test compound relative to the positive control (100%). Values in parantheses represent viability pecells; unless otherwise stated, the viability percentages of Raji cells were more than 80%.


20/27


patients)[188]. Different kinds of natural products haveshown anti-tumor promoting activity. Some examples arecarotenoids [189], flavanoids [190], sesquiterpenes [191] andanthraquinones [192]. Also, several natural naphthoquinonesinhibit the EBV-EA activation. They have been isolatedfrom different plant species belonging to the Bignoniaceaefamily (Tabebuia rosae , T. avellanedae , Catalpa ovata )[193,194] and the Avicenniaceae family ( Avicenniarumphiana , A. alba) [195]. The structures of thesenaphthoquinones are depicted in Fig. (17) and the resultsreported in the literature are summarized in Table9.Compounds (108-110 ), (112-113 ), (115), (117), (120-123)and (125) were found to be more effective than-carotene, avitamin A precursor that has been intensively studied incancer prevention using animal models [196]. Structuralcomparison of (112), (113) and (115) with other naphtho-quinones such as (110), (111 ), (114) and (116) indicated thatthe absence of substituents on the A ring and the presence of a hydroxy group on the C ring may be important structure-activity information. Lapachol (117) was found to showinhibitory activities of 55.4 and 36.4% at 5x102 and 1x102mol ratio/TPA, respectively. Among furanonaphthoquinonesand their analogs (121) and (125), with an OH group at C-3,exhibited potent dose-dependent inhibitory activity (onset of inhibition of activation at 1x103 mol ratio/TPA; 75, 35 and7% inhibition of activation at 5x102, 1x102 and 1x10 molratio/TPA, respectively). Compounds (120) and (123)displayed about the same inhibitory activity as lapachol(117). Other furano-naphthoquinones (118-119 ) and theiranalogs (122-124) were found to be less active than lapachol(117). On the other hand, both the linear-type pyranon-aphtoquinones (126) and (127) displayed a weaker activitythan that of (117). The related naphthoquinones (108) and(109) showed significant dose-dependent inhibitory effects.In particular, the activity of (108) was 100-fold greater thanthat of (109), and greater also than that of (117).Based on the results obtainedin vitro , the inhibitory effectof (125) in an in vivo two-stage carcinogenesis test focusingon mouse skin papilomas induced by DMBA as an initiator,and TPA as a promoter, was studied. The control animalsshowed a 100% incidence of papillomas 10 weeks afterDMBA-TPA tumor promotion, while treatment with (125)along with the initiator and promoter remarkably reduced thepercentage of tumor-carrying mice to 33% after 10 weeks.The number of papillomas/mouse was reduced to about 57%after 20 weeks for (125) treated mice as compared withuntreated mice [195].From these studies, one concludes that naphthoquinonesrelated to lapachol have a notable cancer preventive potential.Consequently, efforts were made to obtain a series of derivatives resulting from modifications on C-2, on the sidechain, and on the C-1 carbonyl. We also tried to obtaintricyclic derivatives [176][197] (See Fig. (18)). The resultsare presented below.

3.1. Modifications of the C-2 Hydroxy Group:Introduction of COR

Derivatives (128), (129), (130) and (131) were obtainedby treating compound (117) with a variety of acylating

agents of different nature and lipophilic character chloride, p-bromo-benzoyl chloride, lauroyl chloridediazomalonyl chloride) using a small excess of acyagents, dry CH2Cl2 and lutidine as base. Derivatives (129)(130) and (131) showed activities similar to thoslapachol, while the acetyl derivative (128) turned out to more active than (117). It was even more effective thacarotene (see Table10). Compound (128) has strong an

tumor promoting activity, even at 10 mol ratio/TPA (inhibitory activity at 1000 mol ratio/TPA, and mor75% and 40% at 500 and 100 mol ratio/TPA , respectand preserved high viability of Raji cells (more than 710 to 1000 mol ratio/TPA). These data suggestreplacing a donating hydrogen-bond substituent on C-a hydrogen-bond acceptor substituent increases the awhen the acyl group does not have more than two carb

3.2. Modifications on the Side Chain

Compound (132) was obtained by Hookoxidation.The shortening of the side chain produstructure flatter than that of lapachol (117), and this effe

could explain the observed increase in inhibitory acWe also modified the double bond of the lateral chainderivative (128), which is already more potent than lapa(128) was treated with MCPBA to obtain the correspo(+ ) epoxy derivative (133) in 60% yield, which untreatment with HClO4, in catalytic amounts affordedcompound (134) in 96% yield. In addition, the reactio(128) with NBS yielded the hydroxy-halogenated com(135) in 94% yield.

All modifications of the side chain resulting iformation of (133), (134) and (135) increase the inhibitactivity with respect to lapachol (117), but not with respto the acetylated compound (128). This fact suggests thatpresence of the double bond on the isoprenyl side charequirement for high activity. Besides, the presence epoxy group or two hydroxyl groups on the carbons C12-Cproduced an effect on inhibitory activity similar tpresence of the double bond. Compound (136) was formby treating (117) with chloranil. This compound hnaphthol type-structure whose lateral chain has been pproduced by a hetero Diels-Alder reaction between odicarbonyl system of chloranil and the double bond C12-C13.

3.3. Modifications on the C-1 Carbonyl

The reaction of compound (117) with hydroxylamhydrochloride produced compound (137) in a regioselectform. This fact can be explained analyzing the diresonance structures where the carbonyl group on Cpull electrons from the oxygen atom located on C-2.

The structure of (137) was confirmed by analysis ofHMBC spectrum, which showed three bond coubetween the hydrogens H-11 and the carbonyl gro184.2. Compound (137), under acetylating conditioyielded the corresponding acetyl derivative (138). Thederivatives turned out to be less active than compound117)which indicates that the carbonyl group plays an improle for the activity.


21/27


3.4. Tricyclic Derivatives

Treatment of (117) with MCPBAvia an epoxideintermediate yielded several orthoquinones: the angular o-dihydrofuran derivative (139), the angular pyran derivative(140), and the linear dihydrofuran 1,4-naphthoquinone (119).Derivative-lapachone (141), and its -isomer derivative(142), were obtained under treatment with diluted H2SO4 viathe corresponding cationic intermediate.

Table 2 shows the results obtained when the deriva(139-142) were biologically evaluated. All triccompounds were more active than lapachol, witcompounds that present 5-member rings being more efthat the corresponding derivatives with 6-member With respect to the series of 6-member rings, the mostwas the one with an hydroxy group (140). compounds (139)(140) and (141) have a chromophoricortho -quinonic systestructurally different from that of lapachol. In

Fig. (18). Modifications on lapachol.

OOH

O

11

12

13

14

1

3

987

6

510

O

O

OH

O

O

OAc

O

HClO4

OO

O

KMnO4

H2SO4

OCOOR

O

RCOCl

OOAc

O HOOH

MCPBA

O

O

NBS

Br

NH2OH-HCl

MCPBA

O

OAc

OOH

Br

NOROR

O

O

O

OOH

OMeOH

O O

Cl

ClCl

Cl

OH

O

OO

OH

O

O

O

OH

(133)

(134)

(135)

(128) R=Me(129) R=

(130) R=(CH2)10CH3(131) R=CHN2COEt

(137) R=H(138) R=COCH3(132)

(117)

(136)

o-Chloranil

(119)(140)

(139)

(141) (42)

+


22/27


compounds the degrees of freedom of the side chaindisappear, being these molecules practically planar. Theseplanar molecules turned out to be slightly more active thanlapachol. The best result in the tricyclic series was obtainedwith the derivative (119), which presents the 1,4-naphthoquinone system.

ACKNOWLEDGEMENTS

This work has been realized within an ICIC program andit has been partly funded by the Spanish MCYT (PPQ 2000,1655, C02-01). We thank Pharma Mar S.A. and Prof.Tokuda for conducting the biological assays.

REFERENCES[1] Chang, H.M.; But, P.P.H.Pharmacology and Applications of

Chinese Materia Medica , World Scientific Publishing , Singapore1986, Vol 1,2.

[2] Dev, S.; Environ. Health Perspect . 1999 , 107 , 783.[3] Kapoor, L.D.CRC Handbook of Ayurvedic Medicinal Plants , CRC

Press, Boca Raton1990 .[4] Schultes, R.E.; Raffauf, R.F.The Healing Forest , Dioscorides

Press, Portland,1990.[5] Arvigo, R.; Balick, M. Rainforest Remedies , Lotus Press, Twin

Lakes,1993 .

[6] Farnsworth, N. R.; Akerele, O.; Bingel, A.S.; Soejarto, D.DZ. Bull. WHO , 1985 , 63 , 965.

[7] Cragg, G.M.; Newman, D. J.; Snader, K. M. J. Nat. Prod . 199760 , 52-60.

[8] Cordell, G. A.Phytochemistry , 1995 , 40 , 1585-1612.[9] Newman, D. J.; Cragg, G. M.; Snader, K.M. Nat. Prod. Rep . 2000

17 , 215-34.[10] Hinterding, K.; Alonso-Diaz, D.; Waldman, H. Angew. Chem. Int.

Ed. 1998 , 37 , 688-749.[11] Hung, D.T.; Jamison, T.F.; Schreiber, S.L.Chem. Biol. 1996, 3

623-639.[12.] Lenz, G.R.; Hash, H.M.; Jindal, S. Drug Discovery Today 2000 ,

145-156.[13] Razvi, E. S.; Leytes, L. J. Mod. Drug Discovery 2000, 41-42.[14] Rosamond, J.; Allsop, A.Science 2000, 287 , 1973-1976.[15] Garret, M. D.; Workman, P. Eur. J. Cancer . 1999 , 35 , 2010-2030[16] Barry, C.E.; Slayden, R.A.; Sampson, A.E.; Lee, R.E. Biochem

Pharmacol. 1999 , 59 , 221-231.[17] Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R

Schreiber, S. L.; Mitchison, T. J.Science , 1999, 286 , 971-974.[18] Silverman, L.; Campbell, R.; Broach, J. R.Curr.Opin.Chem.Biol

1998 , 2397-403.[19] Cordell, G.A.; Shin, Y.G.Pure Appl. Chem . 1999 , 71 , 1089-1094[20] Corley, D. G.; Durley, R. C. J. Nat. Prod. 1994 , 57 , 1484-1490.[21] Watson,C. Angew. Chem. Int. Ed . 1999, 38 , 1903-1908.[22] Bertels, S.; Frorman, S.; Jas, G.; Bindseil, K. U. Drug Discovery

Nat . 1999, 72-105.[23] Schreibers, S. L. Bioorg. Med. Chem . 1998 , 6 , 1127-1152.

Table 10. Percentage of Epstein-barr Virus Early Antigen Induction.

Concentration (mol ratio /TPA) aCompounds

1000 500 100 10

117 17.5b (70) 44.6 63.6 97.8

128 0 (70) 22.7 56.0 82.1129 21.3 (70) 48.0 71.9 100

130 17.3 (60) 49.5 76.9 100

131 16.9 (60) 46.7 72.5 100

132 2.5 (70) 33.8 61.3 89.3

133 4.7 (70) 35.2 62.4 90.2

134 5.2 (70) 38.5 63.7 91.7

135 14.7 (60) 45.9 71.6 100

136 23.6 (60) 50.3 73.9 100

137 19.9 (70) 47.8 68.5 100138 20.7 (70) 49.7 73.7 100

139 10.5 (60) 42.2 66.2 94.8

140 12.3 (60) 40.6 63.9 92.9

119 7.7 (60) 40.8 65.0 92.5

141 15.7 (60) 43.1 68.2 96.3

142 14.6 (60) 41.7 64.0 94.7

-carotene 8.6 (70) 34.2 82.1 100aMol ratio/TPA (32 pmol=20 ng/mL), 1000 mol ratio=32 nmol, 500 mol ratio= 16 nmol, 100 mol ratio=3.2 nmol, and 10 mol ratio=0.32 nmol.bValues represepercentages of EBV-EA induction in the presence of the test compound relative to the positive control (100%). Values in parantheses represent viability pe

cells; unless otherwise stated, the viability percentages of Raji cells were more than 80%.


23/27


[24] Xiao, X.-Y.; Parandoosh, Z.; Nova, M. P. J.Org. Chem. 1997 , 62 ,6029-6033.

[25] Nicolaou, K. C.; Winssinger, N.; Pastor, J.; Ninkovic, S.; Sarabia,F.; He, Y.; Vourloumis, D.; Yang, Z.; Li, T.; Giannakakon, P.;Hamel, E. Nature 1997 , 387 , 268-72.

[26] Nicolaou, K. C.; Vourloumis, D.; Li, T.; Pastor, J.; Winssinger, N.;He, Y.; Ninhovic, S.; Sarabia, F.; Vallberg, H., Roschangor, F.;King, N.P.; Finlay, M.R.V.; Giannakakon, P.; Verdierd-Pinard, P.;Hamel, E. Angew. Chem. Int. Ed. Engl. 1997 , 36 , 2097-2103.

[27] Nicolaou, K.C.; Pfefferkorn, J.A ; Roecker, A.J.; Cao, G.-Q.;Barluenga, S. ; Mitchell, H.J. J. Am. Chem. Soc . 2000 , 122 , 9939-9953.

[28] Nicolau, K.C.; Pfefferkorn, J.A.; Mitchell, H.J.; Roecker,A.J.;Barluenga, S.; Cao, G.-Q.; Affleck, R. L.; Lillig. J. E.

J.Am.Chem. Soc , 2000 ,122 , 9954-9967.[29] Nicolaou, K.C.; Pfefferkorn, J.A.; Barluenga, S.; Mitchell, H. J.;

Koecker, A.J. Cao, G.-Q. J. Am. Chem. Soc. 2000, 122 , 9968-9976.

[30] Evans. B.E.; Rittle, K. E.; Bock. M.G.; Di Pardo, R. M.;Freindinger, R.M.; Whitter, W.L.; Lundell. G.F.; Veber. D. F.;Anderson, P. S.; Chang, R. S. L.; Lotte, V. J.; Cerino, D. J., Chen,T. B.; Kling, P. J.; Kimkel, K. A.; Springer, J. P.; Hirshfield, J. J.

Med. Chem. 1988 , 31 , 2235-2246.[31] Mason, J.S.; Morize, I.; Menard, P.R.; Cheney D.L.; Hulme, C.;

Labandiniere, R. F. J. Med. Chem . 1999 , 42 , 3251-3264.[32] Brohm, D.; Metzger, S.; Bhargara, A.; Muller, O.; Lieb, F.;

Waldmann. H. Angew. Chem. Int. Ed. 2002, 41 , 307-311.[33] Stahl, P.; Kissan, L.; Mazitschek, R.; Giannis, A.; Waldmann, H. Angew. Chem. Int. Ed . 2002 , 41 , 1174-78.[34] Hartwell, J. L.Plants Used Against Cancer , Quarterman,

Lawrence.1982.[35] Cragg, G. M.; Boyd, M. R.; Cardellina J. H.; Newman, D. J. ;

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

[36] Mi, Q.; Lantvit, D. ; Reyes-Lim, E.; Chai, H.; Zhao, W. I.-S. Lee,Peraza-Sanchez, S.; Ngassapa, O.;. Kardono, L.B.S.; S. Riswan,M.G. Hollingshead, J. G. Mayo, N. R. Farnsworth, G.A. Cordell,A. D. Kinghorn, J. M. Pezzuto. J. Nat. Prod, 2002, 65, 842-850,and references cited therein.

[37] Monga, M.; Sausville, E.A. Leukemia 2002 , 16 , 520-526.[38] Da Rocha, A.B.; Lopes, R.; Schwartsmann, M.Curr. Opin.

Pharmacol. 2001 , 1(4) , 364-69.[39] Mukherjee, A. K.; Basu, S.; Sarkar, N.; Ghosh, A.C.Curr. Med.

Chem . 2001 , 8(12) , 1467-1486.[40] Gragg, G.M.; Newman D.J. Exper t Opinion on Investigational Drugs. 2000 , 9, 2783-2797.[41] Vishnu R.; Seema K. Drug News & Perspectives. 2001 , 14 , 465-

482.[42] Sausville, E. A.; Johnson, J. I.; Cragg, G. M.; Decker. S. ACS

Symposium Series 2001 , 796 (Anticancer agents), 1-15.[43] Croce, C. M.Proceeding of the National Academy of Science of

the United States of America . 2001 , 98 , 10986-10988.[44] Kuo, Y. H.; King, M. L. Bioactive Compounds from Natural

Sources. 2001, 189-281.[45] Alvi, K. A. J. Liq. Chromat. & Relates Technologies . 2001, 24 ,

1765-1773.[46] Taraphdar, A.K.; Roy, M.; Bhattacharya, R. K.Science. 2001 ,80,

1387-1396.[47] Konoshima, T.; Takasaki, M.Studies in Natural Products

Chemistry. 2000 , 24 , Part E 215-267.[48] Von Angerer, E.Curr. Op. in Drug Discov&Develop. 2000, 3575-

584.[49] Huang, Z.Curr. Op. in Drug Discov&Develop. 2000 , 3, 565-574.[50] Itokawa, H.; Takeda, K.; Hitotsuyanagi, Y.; Morita, H. J.

Biochem. Mol. Biol . and Biophys . 2000, 4, 213-222.[51] Crews, C. M.; Yeh, J.; Mohan, R.; Meng, L.; Kim, K.; Splittberger,

U.; Know, B. H. B.; E lofsson, M. Book of Abstract, 219 th AC S National Meeting San Francisco, Ca. March, 26-30,2000 .

[52] Hostettmann, K.; Terreaux, C.Chimia , 2000, 54 , 652-657.[53] Glen, V.G.; Hutson, P.R.; Boothman, D. A.,et al . Intern. Pharm.

Abstracts , 1996 , 33 , 2316.[54] Frydman, B.; Marton, L.J.; Sun, J. S. Cancer Res.,1995 , 57 , 620-

627.[55] Planchon, S. M.; Wuerzberger, S. M.; Frydman, B.Cancer Res.

1995, 55 , 3706-3711.

[56] Li, C. J.; Wang, C.; Pardee, A. B.Cancer Res ., 1995, 55 , 3713715.

[57] Wuerzberger, S. M.; Pink, J.J., Planchon, S.M.Cancer Res. , 199858 , 1876-1885.

[58] Li, C.J.; Li, Y.-Z.; Pinto, A. V. Proceedings of the NAcademy of Sciences of the USA,1999, 96 , 13369-13374.

[59] Alvi. K. A. ; Pu, H. J. of Antibiotics 1999 , 52 , 215-223.[60] Bunton, V.G.; Steele, G.; Lewis, A. D.et al . Cancer

Chemotherapy and Pharmacology , 1998 , 41 , 417-422.[61] Nimmapalli, R.; O`Bryan, E.; Bhalla, K.Cancer Res ., 2001 , 61

1799-1804.[62] Mauunster, P. N; Basso, A.; Solit, D.; Rosen, N.Clinical Cancer Res ., 2001, 7 , 2228-2236.

[63] Han, R.Stem. Cells , 1994, 12 , 53-63.[64] Wang, X.-W. Drugs of the Future , 1999, 24 , 613-617.[65] Elsenbrand, G.; Meiers, S.; Niederberger E.90 th Annual Meeting

of the American Association for Cancer Research. 1999 , 2.[66] Ikemoto, S.;Sugimura, K.;Nakatani, T. J. of Urology (Suppl)

1999 ,161 , 115.[67] Zi, X.;Zhang, J.; Agerwal, R.; Pollak, M.Cancer Res. 2000 , 60

5617-20.[68] Rong, Y.;Yang, E. B.;Zhang. K.;Mack, P. Anticancer Res . 2000

20 , 4339-46.[69] Chopra, M.; Fitzsimons, P. E. E.; Strain, J. J.; Thurnham

Howard, A. N.Clinical Chemistry . 2000, 46 , 1162-1170.[70] Ferry, D. R.; Smith, A.; Malkhandi, J.; Fyfe, D. W.; de Taka

Clinical Cancer Res. 1996, 2, 659-668.[71] Shoskes, D. A.;Zeitlin, S. I.;Shahed, A.; Rajfer, J.Urology , 199954 , 960-63.[72] Matsunaga, K.; Yoshimi, K.; Yamada, Y. Japan. J. of Cancer Res

1998 , 89 , 496-501.[73] Komaki, H.; Nemoto, A.; Tanaka, Y. J. of Antibiotics 1999, 52 , 13

19.[74] Komaki, H.; Tanaka, Y.; Yazawz, K.; Takagi, H.; Ando, . J. o

Antibiotics 2000 , 53 , 75-77.[75] Es-Saady, D.; Simon, A.; Jayat-Vignoles, C.; Chulia, A.J.;

C. Anticancer Res . 1996 , 16 , 481-486.[76] Cha, H.-J.; Baes, S.-K.; Lee, H.-Y; Cancer Res.,1996 , 56 , 228

2284.[77] Subbaramaiah, K.; Michalurat. P.; Sporn. M. B.; Dannenb

J. Cancer Res . 2000, 60 , 2399-2404.[78] Lee, J.S.; Yang, M.Y.; Yeo, H.; Kim, J.; Lee, H.S. Bioorg. Med.

Chem. Lett., 1999 , 9, 1429-1432.[79] Draetta, G.; Eckstein, J. Biochim. Biophys. Acta , 1997 , 1332.[80] Corey, E.J.; Roberts, B.E. J. Am. Chem. Soc. 1997, 119 , 124212431.[81] Magnuson, S. R.; Sepp-Lorenzino, L.; Rosen, N.; Danishs

S. J. J . Am, Chem. Soc. 1998, 120 , 1615-1616.[82] Boukouvalas, J.; Cheng, Y.-X.; Robichaud, J. J. Org. Chem. 1998

63 , 228-229.[83] Takahashi, M.; Dodo, K.; Yashimoto, Y.; Shirai, R.Tetrahedron

Lett. 2000 , 41 , 2111-2114.[84] Jung, M.; Nishimura, N.Org. Lett. 2001, 3, 2113-2115.[85] Piers, E.; Caill, S.; Chen, G.Org. Lett . 2000 , 2, 2483-2486.[86] Demeke, D. ; Forsyth, C. J.Org. Lett. 2000, 2, 3177-3179.[87] Miyaoka, H.; Kajiwara, Y.; Yamada, Y.Tetrahedron Lett. 2000

41 , 911-914.[88] Ecktein, J. W. Invest. New Drugs , 2000, 18 , 149-156.[89] Wang, L.; Woods K.W.; Li, Q.; Barr, K.J.; McCrosKey,

Hannick, S.M.; Gherke L.; Credo, R.B.; Hui, Y.H.; MarWagner R.; Lee, J.Y.; Zielinski-Mozng, N.; Frost, D. RoseS.H.; Sham, H.L. J. Med. Chem , 2002, 45 , 1697-1711.[90] Sackett, D.L.Pharmacol. Ther .1993, 59 , 163-228.

[91] Zhao, S.; Smith, K.S.; Deveau, A.M.; Dieckhaus C.M.; JM.A. Macdonald, T.L. and CooK, J.M. J. Med. Chem , 2002, 451559-1562.

[92] Folkman, J. Nat. Med. 1995, 1, 27-31.[93] Giannis, A.; Rbsam, F. Angew. Chem. Int. 1997 , 36 , 588-590.[94] Folkman, J. N. Engl. J. Med . 1971 , 285 , 1182-1186.[95] Carmeliet, P. Jain, R. K. Nature , 2000 , 407 , 249-257.[96] Yancopoulos, G. D.; Davis, S.; Gale, N.W.; Rudge, J.S.; W

S.J.; Holash, J. Nature , 2000, 407 , 242-248.[97] Maisonpierre. P. C.; Suri, C.; Jones, P. F.; Bartunkov

Wiegand, S.J.; .Radziejeewski, C.; Compton, D.; McCl


24/27


Aldrich, T. H.; Papadopoulos, N.; Daly, T. J.; Davis, S.; Sato, T.N.; Yancopoulos, G.D.Science 1997 , 277, 55-60.

[98] Hiratsuka, S; Maru, Y.; Okada, A.; Seiki, M.; Noda, T.; Shibuya,M.Cancer Res. 2001 , 61 , 1207-1213.

[99] Kubo, H.; Fujiwara, T.; Jussila, L.; Hashi, H.; Ogawa, M.; Shimizu,K.; Awanw, M.; Sakai, Y.; Takabayashi, A.; Alitalo, K.;Yamaokas, Y.; Nishikawa. S. I. Blood 2000 , 96 , 546-553.

[100] Stratmann, A.; Acker, T.; Burger, A. M.; Amann, K.; Risau, W.;Plate, K. H. Int. J. Cancer , 2001 , 91 , 273-282.

[101] Stacker, S. A.; Caesar, C.; Baldwin, M.E.; Thorton, G. E.; Willians,R. A.; Prevo, R.; Jacksons, D.G.; Nishikawa, S.; Kubo, H.; Achen,M.G. Nat. Med . 2001 , 7 , 186-191.

[102] Skobe, M.; Hawighorst, T.; Jackson, D.G.; Prevo, R.; Janes, L.;Velasco, P. ; Riccardi, L. ; Alitalo, K.; Claffey, K.; Detmar, M.

Nat. Med . 2001 , 7 , 192-198.[103] Lin, P.; Buxton, J. A.; Acheson, A.; Radziejewski, C.;

.Maisonpierre, P.C.; Yancopoulos, G.D.; Chanon, K. M.; Hale, L.P.; Dewhirst, M. W.; George, S.E. Peters, K.G.Proc. Nat. Acad,Sci. USA 1998 , 95 , 8829-8834.

[104.] Drevs, J.; Hoffmann, I.; Hugenschmidt, H.; Wittig, C.; Madjar, H.;Muller, M.; Wood, J.; Martiny-Baron, G.; Unger, C.; Marme, D.Cancer Res. 2000 , 60 , 4819-24.

[105] Boschelli, D.H. Drugs Future . 1999, 24 , 515-537.[106] G. Bold, K.-H.; Altmann, J.; Frei, Marc, L. P. W.; Manley, P.;

Traxler, B.; Wietfeld, J.; Brggen E.; Buchdunger, R.; Cozens, S.;Ferrari, P.; Furet, F.; Hofmann, G.; Martiny-Baron, J.; Mestan, J.;Lsser, E.; Masso, R.; Roth, C.; Schlatchter, W.; Vetterli, D.;Wyss, J.; Wood, M. J. Med. Chem . 2000 , 10 , 2310-2323.[107] L.D. Arnold, D.J. Calderwood, R.W. Dixon, D.N. Johnston, J.S.Kamens , R. Munschauer, P. Raffer ty, S. E. Rafnotsky. Biorg.

Med. Chem. Lett. 2000 , 10, 2167-70.[108] Burchadt, A.F.; Calderwood, D. J.; Hirst, G.C.; Holman, N. J.;

Johnston, D. N.; Munschauer, R.; Rafferty, P.; Tometzki. G. B. Biorg. Med. Chem. Lett. 2000 , 10 , 2171-74.

[109] Kirkin, V.; Mazitschek, R.; Krishnan, J.; Steffen,A.;Waltenberger, J.; Pepper, M.S.; Giannis, A.,Sleeman, J. P.

Eur.J.Biochem . 2001 , 268 , 5530-5540.[110] Stahl, P.; Kissau, L.; Mazitschek, R.; Huwe, A.; Furet, P.; Giannis,

A.; Waldmann, H. J. Am. Chem. Soc. 2001 , 123 , 11586-11593.[111] Syrigos, K.N.; Epenetos, A.A. Anticancer Res. 1999 , 19 , 605-614.[112] Duowchick, G.M.;Walker, M.A.Pharmacol, Ther. 1999 , 83 , 67-

123.[113] Bagshawe. K. D. Br. J. Cancer . 1987, 56 , 531-532.[114] Tietze, L.F.; Beller, M.; Fischer, R.; Lgers, M.; Jhde, E.;

Glsenkamp, K. H. ; Rajewsky, Angew. M. FChem. Int. De. Engl.1990, 29 , 782-783.[115] Tietze, L.F.; Herzig,T.; Fecher, A.; Haunert, F.; Schuberth, I.

Chem.BioChem 2001 , 2, 758-765.[116] Boger, D.L.; Garbaccio, R.M.; Acc. Chem. Res. 1999, 32 , 1043-

1052.[117] Boger, D.L.; Boyce, S.W.; Garbaccio, R.M.; Searcey, M.; Jin,Q.

Synthesis , 1999 , 1505-1509.[118] Coux, O.; Tanaka, K.; Goldberg, A. L. Annu, Rev. Biochem. 1996,

65 , 801-847.[119] Goettrup, M.; Soza, A.; Kuckelkorn, U. Kloetzel, P. M. Immunol.

Today, 1996 , 17 , 429-435.[120] Voges, D.; Zwickl, P.; Baumeister. W., Annu. Rev. Biochem. 1999,

8, 1015-68.[121] Bochtler, M.; Ditzel, L.; Groll, M.; Hartmann, C.; Huber, R.; Annu.

Rev. Biophys, Biomol. Atruct . 1999, 28 , 295-317.[122] Lwe, J.; Stock, D.; Jap, B.; Zwickl, P.; Baumeister, W.; Huber, R.

Science , 1995, 268 , 533-539.[123] Groll, M.; Ditzel, L.; Lwe, J.; Stock, D.; Bochtler, M.; Bartunik,H.D.; Huber, R. Nature 1997 , 386 , 463-471.

[124] Orlowski, M.; Wilk, S. Arch. Biochem. Biophys , 2000 , 383 ,1-16.[125] Dou, Q.P.; Nam, S. Expert Opin. Ther. Pat. 2000 , 10 ,1263-1272.[126] Adams, J.;Palombella, V.J.;Elliot, P. J. Invest. New Drugs , 2000 , 18 ,

109-121.[127] Wjcik, C. Drug Discovery Today , 1999 , 4,188.[128] Kisselev, A.F.; Goldberg, A.L.Chem. Biol. 2001 , 8, 739-758.[129] Koguchi, Y.; Kohno, J.; Nishio, M.; Takahashi, K.; Okuda, T.;

Ohnuki, T.; Komatsubara, S. J. Antibiot . 2000 , 53 , 105-109.[130] Lin,S.; Danishefsky, S.J. Angew. Chem. Int.Ed. 2001 , 40 , 1967-

1970.[131] Ma, D. ; Wu, Q. ;Tetrahedron Lett. 2001, 42 , 5279-5281.

[132] Ma, D.; Wu, Q.Tetrahedron Lett .2000 , 41 , 9089-9093.[133] Albrecht, B. K.; Williams, R.M.Tetrahedron Lett. 2001 , 42 , 275

2757.[134] Inoue, M.;Furuyama, H.;Sakazaki, H.;Hirama, M.Org. Lett . 2001

114 ,530.[135] Lin,S.; Danishefsky, S.J. Angew. Chem. Int.Ed . 2002 , 41 , 530-533[136] Kaiser, M.; Groll, M.; Renner, C.; Huber, R.; Moroder, L. Angew

Chem. Int.Ed. , 2002 , 41 ,780-783.[137] Wang, J. C. Annu. Rev. Biochem . 1996 , 65 , 635-692.[138] Vicker, N.; Hancox, C.; Burgess, L.; Chuckowree, I. S.; D

Folkes, A. J.; Hardick, D. J. T.; Miller, W.; Milton, J.; SohWan

recent studies on natural products as anticancer agents

Documents