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Cytotoxicity of some natural compounds

ARTCULO ORIGINAL

Cytotoxic effect of some natural compounds isolated

from Lauraceae plants and synthetic derivatives

Luis Enrique Cuca1, Ericsson David Coy1, Marln Andrea Alarcn2, Andrs Fernndez2,Fabio Anczar Aristizbal2

1 Laboratorio de Investigacin en Productos Naturales Vegetales, Departamento de Qumica, Facultad deCiencias, Universidad Nacional de Colombia, Bogot, D.C., Colombia

2 Laboratorio de Farmacogentica del Cncer, Departamento de Farmacia, Facultad de Ciencias, UniversidadNacional de Colombia, Bogot, D.C., Colombia

Introduction. The antiproliferative effect of eleven neolignans, two lignans and one diterpeneisolated from three Lauraceae plants, four benzofurans and two bicyclooctanes synthetic derivativeswas evaluated in vitroon a set of five human cancer cells from solid tumors with a high incidence inColombia.Objective. To evaluate the cytotoxic effect of twenty compounds on the tumor cell lines HeLa, A-549,Hep-2, PC-3, and MCF-7.Materials and methods. Fourteen natural compounds were isolated by chromatographic techniques

from three native colombian plants (Pleurothyrium cinereum, Ocotea macrophylla and Nectandraamazonum), whose structures were established by spectroscopic methods; six synthetic derivativeswere prepared by oxyarylation and diazomethane methylation. Antiproliferative effect and cell recoverywere performed by means of in vitrotreatment of tumor cell lines with test compounds, evaluating cellviability by resazurin staining.Results. Among test compounds, only neolignans ocophyllal A, cinerin D, kaurenoic acid, twobenzofuran-derivatives, and synthetic (-)-cinerin A were found to have antiproliferative effect at differentlevels. Bicyclooctanoids as well as kaurenoic acid exhibited activity against all human cancer cellswhile benzofuranoids showed selective activity against HeLa. Furthermore, compounds (-)-cinerin Aand kaurenoic acid exhibited total lethal effect against all-five cell lines and PC-3, Hep-2, and A549 celllines, respectively.Conclusion. Test compounds exhibiting antiproliferative activity showed interesting results, whichwould promote their use as lead compounds on further studies for anticancer agents development.

Key words: biological products, Lauraceae, straining, diterpenes, in vitro.

Efecto citotxico de algunos compuestos naturales aislados de plantas Laureaceae y derivadossintticos

Introduccin. El efecto contra la proliferacin celular de once neolignanos, dos lignanos y un diterpeno,aislados de tres plantas de la familia Lauraceae, y cuatro benzofuranos y dos biciclooctanos sintticos,fue evaluado in vitrosobre cinco lneas celulares derivadas de tumores slidos de alta incidencia enColombia.Objetivo. Evaluar el efecto citotxico de veinte compuestos sobre las lneas tumorales HeLa, A-549,Hep-2, PC-3 y MCF-7.Materiales y mtodos. Los 14 compuestos de origen natural fueron aislados de tres plantas nativascolombianas (Pleurothyrium cinereum, Ocotea macrophylla y Nectandra amazonum) por tcnicascromatogrficas y se establecieron sus estructuras por mtodos espectroscpicos, y los seis derivadossintticos fueron preparados mediante reaccin de oxiarilacin y metilacin con diazometano. El efectocontra la proliferacin y la recuperacin celular se hicieron mediante tratamiento in vitrode las lneastumorales con los compuestos , evaluando la viabilidad celular por tincin con resazurina.Resultados. Entre los compuestos evaluados, solamente ocofilal A, cinerina D, cido kaurenoico,dos benzofuranos y la (-)-cinerina A sinttica presentaron actividad contra la proliferacin celular endiferentes niveles. Los biciclooctanos, as como el cido kaurenoico, fueron activos contra todaslas lneas celulares, mientras que los benzofuranoides mostraron actividad selectiva contra HeLa.Adems, la (-)-cinerina A exhibi un efecto letal total contra todas las lneas celulares, mientras que elcido kaurenicopresent efecto letal total contra PC-3, Hep-2 y A549.Conclusin. Los compuestos evaluados que exhibieron actividad contra la proliferacin celularmostraron resultados interesantes, lo cual sugiere su potencial uso como cabezas de serie o molculasplantilla en el desarrollo de agentes anticancergenos.

Palabras clave: productos biolgicos, Lauraceae, cribado, diterpenos, in vitro.

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Cuca LE, Coy ED, Alarcn MA, et al.

Drug-seeds discovery from natural products has ledto novel medical advances, mostly including researchon anti-cancer agents through exploration for sourcesof novel cytotoxic compounds (1). Cytotoxicity studiescan be regarded when compounds exhibit a potentialbiological activity on cell damage, whose causes might

be related to either agent concentration or exposuretime for cells to these agents, which compromises cellviability (2).

Cancer is one of the leading causes of deathworldwide, being the main reason to searching fornew antineoplastic agents, mostly from natural origin,which has became an important research approach(3). Currently, the drugs used to treat cancer mayhave two limitations: reduced activity, on the onehand, or acquired resistance by tumor cells, on theother (4). In Colombia, recent statistics indicated thatthe highest incidences of cancer disease correspond

to breast, cervix, stomach, leukemia, colon, andprostate cancer (5). Therefore, performing in vitroscreening in order to discover cytotoxic agentsagainst tumor cell lines still remains as an importantobjective of anticancer research (6).

Lignans and neolignans (C6C3 dimers to the shikimatebiosynthetic pathway) (7), are well-recognized fortheir wide-range of important biological activities,because they have been used as lead compoundsfor the development of new drugs (8). An importantbioactive class of these type of natural productsis related to the benzofuran moiety, which has

exhibited antitumor (8), antiangiogenic (8), cytotoxic(9-11), antileishmanial (11),antitrypanosomal (12),antibacterial (13), PAF-antagonistic (14), amongothers, the cytotoxicity being a significant propertyof this type of compounds (8-11). Lauraceae plantsare well-known to contain benzofuran neolignansas well as bicyclo[3.2.1]octane neolignans (7),which are considered as benzofuran-derived(15),and they have exhibited excellent PAF-antagonisticactivity (16).

As part of our research on bioactive naturalcompounds, the phytochemical exploration carriedout on the leaves of three Lauraceae plants(Pleurothyrium cinereum, Ocotea macrophylla and

Corresponding author:Fabio Anczar Aristizbal, Laboratorio de Farmacogentica delCncer, Departamento de Farmacia, Universidad Nacional deColombia, Carrera 30 N 45-03, Edificio 450, Oficina 206, AA14490, Bogot, D.C., ColombiaTelfono: (571) 314 5000 extensin14643; fax: (571) 316 [email protected]

Recibido: 24/09/10; aceptado:28/04/11

Nectandra amazonum) yielded five benzofuranneolignans 1-5, six bicyclo[3.2.1]octane neolignans6-11, two diastereomeric dibenzylbutane lignans12-13 and a diterpene 14. Including a group ofsix synthetic neolignan derivatives 15-20, thenaturally-occurring compounds were screened in

order to determine their in vitro antiproliferativeeffect on a set of five human cancer cells consistingof cervical carcinoma HeLa, lung carcinoma A-549,larynx carcinoma Hep-2, prostate adenocarcinomaPC-3, and breast carcinoma MCF-7. Structuresof test compounds were determined by extensivespectroscopic analyses, and their absoluteconfiguration was defined by circular dichroism(CD) comparing with data reported in the literaturefor related compounds.

Materials and methods

General experimental procedures

CD spectra were obtained using a JASCO J-720spectropolarimeter. 1H and 13C Nuclear MagneticResonance (NMR) spectra were recordedon a Bruker Avance 400 spectrometer, usingtetramethylsilane (TMS) as internal shift reference.High Resolution Mass Spectrometry (HRMS) wasdetermined on a Shimadzu LCMS-IT-TOF (with anelectrospray (ESI) source and in the positive ionmode) mass spectrometer. Cell growth indicationswere performed by resazurin method using aTECAN GENios plate reader with excitationwavelength at 535nm and emission at 595nm.

Plant material

Whole plants of Pleurothyrium cinereum, Ocoteamacrophylla and Nectandra amazonum werecollected in different regions of Colombia: Tumacocounty, Department of Nario, on the NocaimaVergara road at Nocaima county, Department ofCundinamarca, and near the biological stationEl Zafire, 6 hours from Leticia city, Departmentof Amazonas, respectively. Reference specimens(numbers COL518334 for P. cinereum, COL517191for O. macrophylla, COL519818 for N. amazonum)are kept at Herbario Nacional Colombiano,Universidad Nacional de Colombia.

Extraction and isolation

Detailed extraction and isolation processes, andspectroscopic features for the test compoundswere previously reported (19-24) (figure 1) includingliliflol A 1 (17), 3-methoxy-3,4-methylendioxy-4,7-epoxy-nor-8.5-neolignan-7.8-diene 2 (12),(+)-licarin A 3 (18), ocophyllal A 4 (19), (+)-mirandin

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A 5 (20), cinerin C 6 (21), (-)-cinerin D 7 (21), 2-epi-guianin 8 (19), ocophyllol C 9 (19), kadsureninC 10 (22), nectamazin C 11 (23), meso- and threo-3,4,5,3,4,5-hexamethoxy-8.8-neolignan 12-13and kaurenoic acid 14 (24). Compounds 2, 5-7 wereisolated from P. cinereum, compounds 4, 8-9 fromO. macrophylla, and compounds 1, 3, 10-11 from N.amazonum, using conventional chromatographicmethods.

Chemical synthesis

Benzofurans 15-17 were synthesized via metal-catalyzed oxyarylation reaction (25). Benzofuran18 was obtained by direct O-methylation ofliliflol A using diazomethane prepared in situ bydecomposition of N-methyl-N-nitrosourea, followingthe reported methodology (26). Bicyclooctanes

O R5

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1 -OCH2O- H allyl OH H

3 OCH3 OH H (E)-p ro pen yl H O CH315 H OCH3 H H H Cl

16 -OCH2O- OCH3 H CO OCH3 H17 H OCH3 H H H H

18 -OCH2O- H allyl OCH3 H

O

R1O

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4 CH3 CHO

O O

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R1 R2 R3 R4 R5 R6

6 -OCH2O- OCH3 OCH3 =O OH7 -OCH2O- OCH3 OCH3 -OH =O

9OCH3 OCH3 OCH3 H =O OH

10OCH3 OCH3 H H =O OH

20 -OCH2O- OCH3 OCH3 =O =O

R5

O

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11 O CH3 OCH3 OCH3 OCH3 =O OH

19 -OCH2O- OCH3 OCH3 =O =O

R1

O

O

O

O

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H

HO

OH

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12 -CH 313 -CH3

Figure 1. Structures of compounds 1-20.

19-20 were obtained by HPLC kinetic resolutionon a racemic product [()-cineric A], whichwas synthesized following a reaction protocoldeveloped by us, starting from the correspondingbenzofuran precursor (25,35). Semi-preparativeKinetic resolution was performed in a HPLC systemconsisting of a Jasco PU-980 pump and Jascomultiwavelength detector MD-910 equipped with aChiralcel OD-H semi-preparative column (100x10mm) (Daicel Chemical Industries LTD) isocraticallyeluted with hexane: iPrOH 93:7 at a flow rate of 5ml/min. Levorotatory compound, 20 was eluted for11.3 min, and dextrorotatory 19, for 13.2 min.

Cytotoxicity assay

The cytotoxic effect of test compounds 1-20 wasevaluated in vitro against a panel of five human

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cancer cell-lines including cervical carcinomaHeLa, lung carcinoma A-549, larynx carcinomaHep-2, prostate adenocarcinoma PC-3 and breastcarcinoma MCF-7 (all adherent growth), derivedfrom human solid tumors, obtained from the CellBank of the Laboratorio de Inmunologa at Instituto

Nacional de Cancerologa de Colombia in Bogot.All tumor cell-lines were cultured in MinimumEssential Medium (MEM) supplemented with 5%fetal bovine serum (FBS), gentamicin (50 mg/ml)and sodium bicarbonate (2.2 g/L), in 75 cm2 boxesfor sterile cultures, and incubated under standardconditions (27). Medium was changed every twodays for maintaining the culture by washing cellswith phosphate buffer solution (PBS) 298 mOsm/L,pH= 7.3. Cultures with 90% confluence were treatedwith 0.025% trypsin solution and 0.03% EDTA for 5minutes at 37C, obtaining a cell suspension whichwas counted in a Neubauer chamber by trypan blueexclusion method. Cell growth was ensured withminimal essential medium (MEM) supplementedwith bovine fetal serum (FBS) and preserved withantibiotic to achieve the appropriate cell densityaccording to each cell line, using 96-well plates (100l/well). Incubation conditions were maintained in amoistened atmosphere at 37C and 5% CO2.

After adjusting the cell densities established by usin previous studies (28-32), cells were seeded into96-well plates, incubated for 24 hours in standardconditions to allow cell adhesion to the well surface,and after this time, test compounds 1-20 were

placed as treatments (at three concentrations:6, 18 and 54 g/ml, using three replicates perconcentration) and doxorrubicin HCl (EbewePharma) as positive control, by up to a volumeof 100 l per well, obtained by serial dilution inMEM supplemented with 5% FBS (28). After 48hours of treatment, the viable cell population on theplates was determined using 100 l of rezasurin(0.25 mg/ml in PBS), which was placed in eachwell with medium without supplement to avoidinterferences during test. Plates were incubatedfor 4 hours under standard conditions, and thenread using a TECAN GENios reader. Resorufinformed by rezasurin reduction contains suitablyboth chromophoric and fluorophoric moieties,allowing performing absorbance (at 570 nm) and/or fluorescence (excitation at 535 nm and emissionat 595 nm) measurements, in a non-destructivetesting. It shows almost no cytotoxic activity,allowing discrimination of the cytotoxic activity ofthe cytostatic. Supernatant was then carefullyremoved and 100 l of medium supplement was

added, whose plates were further incubated for 48hours, repeating again the procedure describedabove. Resulting values were taken as an indirectmeasure of viable cell mass.

Results

Figure 1 shows the chemical structures of isolatedcompounds 1-20, whose antiproliferative effectswere established using resazurin, which exhibitedslightly more sensitivity than MTT for proliferativeestimation. However, only compounds 4, 7, 14, 16,17, and 20 showeda considerable activity againstthe tumor cell-lines at the tested concentrations.Table shows the antiproliferative results as survivalpercentage at three concentrations (6, 18, and 54g/ml) for those compounds as a dose-dependentbehavior. The most important results are describedbelow.

Liliflol A, 1, a dihydrobenzofuran neolignan isolatedfrom N. amazonum, exhibited no significantantiproliferative activity against all the cell lines. AnO-methylation, using diazomethane prepared insitu by decomposition of N-methyl-N-nitrosourea,was performed starting from compound 1 toyield the synthetic derivative 18 (Figure 2a). Theantiproliferative effect of 18 remained identical tothat of 1, indicating no important effect when thestructure was slightly transformed.

Kaurenoic acid 14was found to be the most potentantiproliferative compound,showing activity against

the five evaluated human cancer cells lines at threeconcentrations, whose relative survival percentageswere estimated at 0%. Remarkably, kaurenoic acidpresented activity against the PC-3 and A-549 celllines, which exhibited resistance as compared withthe positive control (doxorubicin HCl), Similarly,(+)-cinerin D 7 and (-)-cinerin A 20 also showedactivity against all the tested human cancer cellsat different levels. Relative survival percentagesof 7 were 37.2% and 7.4% at 18 and 54 ug/ml,respectively, while compound 20 displayed 5.0and 0% survival percentages at 18 and 54 g/ml,respectively, against the HeLa cell line. Synthetic

()-cinerin A was separated into its enantiomersthrough semi-preparative HPLC kinetic resolution,obtaining (+)-cinerin A 19, and (-)-cinerin A 20 (figure2b). Interestingly, the dextrorotatory derivative 19,showed no significant activity against all cell lines,while the levorotatory enantiomer 20, was activeagainst all the tumor cell lines tested.

On the other hand, benzofurans 16, 17 and 4exhibited selective activity against the HeLa cell

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line. Compound 17 was found to be the most potentantiproliferative compound among the benzofuran-type compounds, whose survival percentagevalues were similar at the concentrations tested.Furthermore, ocophyllal A, 4, having a benzofuranaldehyde moiety, presented activity against A549cell line with survival values of 32.0% and 11.2 %at 18 and 54 ug/ml, respectively. However, allyl,propenyl and chlorobenzofuran 1-3, 15, 18 and

cyclohexadienone 5 derivatives, bicyclooctaneneolignans 6, 9, 10 and diarylbutane lignans 12-13 showed no significant antiproliferative activityagainst the cell lines evaluated with survival valuesof >50%.

After 48 h, medium was replaced by fresh medium, todetermine the cell recovery by using new resazurin.Thus,cell recovery of tested human tumor cell lineswas qualitatively determined after evaluation ofthe antiproliferative effect. Compounds 20 and 14exhibited total lethal effect against four cell linesand PC-3, Hep-2, and A549 cell lines, respectively.

However, it is suggested to carry out trials in shortertimes to establish the recovery effect, avoiding raisedadditional effects in the development of the test.

In addition, the effects of the most active compounds4, 7, 14, 16, 17 and 20, were also tested against theMCF-7 breast carcinoma cell line, which has beenextensively studied by the Pharmacogenetics ofCancer Group. This cell line showed survival valuesabove 50 percent, indicating that the cytotoxic effect

Figure 2. (a)Methylation of (+)-liliflol A; (b) kinetic resolution of ()-cinerina A.

O

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HPLC kineticresolution

()-cinerin A

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+

CH 2Cl2 ,MNU,

50% KOH,

2 h, 0 C

(a )

(b)

1 18

on this cell line is lower in comparison to the effectagainst other tumor cell lines. Susceptibility of theMCF-7 cell line was exhibited in the presence of14, 17 and 20 (table 1).

Discussion

At the Pharmacogenetics of Cancer laboratory themethods for indirect assessment of cell survivalusing MTT and resazurin (29) were previously

compared. Resazurin was found to be slightly moresensitive than MTT for proliferative estimation,leading to use the resazurin method with morereliable results, even during recovery time aftertreatment (29). Furthermore, different types ofcontrols, as medium, vehicle (DMSO), and growthwere previously optimized in order to ensure thetest quatlity (29-32).

Test compounds from natural sources, 1-14, andsynthetic benzofurans, 15-17, were obtained inprevious studies (19-25). Here the O-methylationof liliflol A, 1, affording 18, and the racemicresolution of synthetic ()-cinerin A into 19 and 20are decribed. In a previously reported cytotoxicevaluation against the tumor cell lines MCF-7 andHCT-8, compound 14 was found to exhibit a 45%survival rate at 78 M (33), and an effect against thePC-3 cell-line at 3.7 g/ml (34); compound 14 alsoshowed an IC50 of 1.6 g/ml in KB cells derived fromnasopharyngeal cancer (35), results which are inagreement with those of the present work. By meansof electrophoretic analyses, the mode of action of

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Table 1A. Concentrations used for each molecule of interest in the study (M). B. Survival percentage results from the citotoxicityassay for most potent compounds against five tumor cell lines

A.

Compounds mw I II III

4 310,30 19,3 58,0 174,0

7 402,44 14,9 44,7 134,214 302,45 19,8 59,5 178,516 318,75 18,8 56,5 169,417 240,30 25,0 74,9 224,720 400,42 15,0 45,0 134,9

Doxorubicin 543,52 11,0 33,1 99,4

mw: molecular weight

B HeLaa,b cell line

Compounds I M II M III M

4 Na 18.3 (18.2) 7.2 (2.1)7 Na 37.2 (3.3) 7.4 (2.1)14 0.0(0,4) 0.0 (0,4) 0.0 (0,1)16 Na Na 36.3 (4.3)17 34.2(3.9) 30.9( 3.5) 20.8 (2,9)20 Na 5.9(3.2) 0.0(0.3)

Doxorubicin c 110(1.2) 86.5(11.2) 15.3(1.1)

A549a,b cell line

4 Na 32.0 (3.7) 11.2 (2.3)7 Na 27.4 (16.0) 7.6 (6.2)14 9.2 (12.8) 0.0 (0.7) 0.0 (0.6)16 Na Na Na17 Na Na Na20 Na 0.1 (1.1) 0.0 (0.5)

Doxorubicin c 3.6(0.3) 0.3(0.1) 0.6(0.2)

Hep-2a,b cell line

4 Na Na Na7 25.5(23.6) 3.1 (1.4) 0.0 (0.1)

14 18.7(9.8) 0.0(0.2) 0.0 (0.2)16 Na Na Na17 Na Na Na20 Na 9.8(5.1) 0.2 (0.3)

Doxorubicin c 74.7(0.9) 51.5(1.8) 36.0(9.8)

PC-3,b cell line

4 Na Na Na7 Na 12.1(2.0) 0.0(0.2)14 3.1(1.4) 0.0(0.1) 0.0(0.3)16 Na Na Na17 Na Na Na20 Na 0.0 (0.2) 0.0(0.1)

Doxorubicin c 79.1(2.3) 18.6(2.3) 14.8(1.4)

MCF-7a,b cell line

4 Na Na Na7 Na Na Na14 Na Na 0.0(1.5)16 Na Na Na17 Na Na 13.1(3.6)20 Na Na 3.3(1.3)

Doxorubicin c 64.0(7.7) 13.0(2.0) 19.3(4.5)

a Values are means of three experiments, standard deviation is given in parentheses (Na = not active).b Negative values are approximated to zero survival percentage.c Doxorubicin as positive control.

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compound 14 was established as nucleosomalfragmentations, indicating apoptotic activity of 14involved on differentiation of protein expressionassociated with the regulation of apoptosis (34).

Our results also indicate that compound 14 exhibitssignificant activity against HeLa, A-549, Hep-2, and

PC-3 tumor cell lines, showing enhanced activityand behavior against HeLa as compared to thatof doxorubicin HCl (figure 3), which indicated anexcellent antitumor parameter. These resultspoint to the promising activity of compound 14for antitumor research purposes as a leadingcompound for structural optimization. Likewise,Perseal D, a compound isolated from Perseaobovatifolia (Lauraceae), having a benzofuranaldehyde moiety (like compounds 4, 16, 17), alsoexhibited antiproliferative activity against the A549cell line with ED50 of 0.59 g/ml (10), which indicatesthis chemical skeleton could be a good candidateto be used for further antitumor studies.

Conversely, compounds 7 and 20 are related tothe bicyclooctane core (21), and they share a verysimilar chemical structure (differenced solely by thefunctional group at C-2). Activity exhibited by thesetwo compounds on the HeLa cell line indicated that20 possesses a more forceful cytotoxic profile ascompared to that of 7 (figure 4). Both compoundsshowed similar effect against the PC-3 and A549cell lines, while the antiproliferation of compound7 was greater to that of 20 against the Hep-2 cellline. Interestingly, no significant antiproliferativeactivity was found for the neolignans having anOH group at C-4, as in compounds 6, 8-11 and19, with survival values of >50%. Additionally, inorder to determine the configuration-dependent

antiproliferative activity of bicyclo[3.2.1]octanenuclei, ()-cinerin A, prepared in a previous workby total synthesis from commercially availablematerials (36), were separated into compounds 19and 20, enantiomers with a significantly differentantiproliferative effect. Dextrorotatory enantiomer19 showed no relevant cytotoxic activity againstfour cell lines, while levorotatory enantiomer 20[which shares an identical absolute configurationto that of natural product (-)-cinerin A, which wasnot suitable to be included on this study due toa low isolated amount (21)], displayed activitytowards four cell lines at 54 g/ml, having the

greatest antiproliferative effect on cell lines PC-3,A549 and Hep-2 at 18 g/ml. This fact indicatedthe importance of the absolute configurationdetermination in rational drug design.

Although some compounds showed activity abovethe selection criteria, further biological studiesare suggested to complete their activity profile.Additionally, we found some compounds with noantiproliferative activity like 8, 17, 19, since theyexhibited no significant effect on the cell growth,indicating either no promising anticancer activity orthe lack of previous bioactivation.

In summary, the antiproliferative effect of twentycompounds, related to the bicycle[3.2.1]octaneand benzofuran neolignans, kaurane diterpene anddiarylbutane lignans classes, obtained by isolationfrom three lauraceae plants and synthesis, wasevaluated in vitro against six human cancer celllines. Several of them showed their anticancerpotential because they have a CL50 under 10mM, the reference value recommended by NCI

Figure 4. Antiproliferative activity of compounds7 and 20 on theHeLa cell line.

Figure 3. Comparison of survival rate against the logarithm of thetested concentration on the HeLa cell line between compound14(black-line) and doxorubicin HCl (gray-line).

18 /mLg

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for compounds (37). Three benzofuran-typecompounds exhibited selective activity againstHeLa cell lines, which might be used in furtheroptimization studies to improve their action on thetreatment of cervical carcinoma diseases. Kaurenoicacid and (-)-cinerin A were found to be the most

potent antiproliferative compounds at reasonablelevels, which would be good candidates to beused as lead compounds for further SAR studiesfor anticancer agent development. However, it isnecessary to perform many other studies both invitroand in vivoto determine their true potential forthe development of medicines

Acknowledgements

Authors thank the Chemistry and PharmacyDepartments and the Divisin de Investigacin,Sede Bogot (DIB) at Universidad Nacional deColombia, Bogot, for the financial support. Our

gratitude is extended to L. M. Escobar and C. P.Cordero for their support on the development andoptimization of the used methodology.

Conflict of interests

Authors declare no conflicts of interest.

Financial support

This work was carried out with financial support byColciencias and Divisin de Investigaciones deBogot (DIB) at Universidad Nacional de Colombia(Call 2009, Project Code No. 8003383).

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