Phytochemistry 72 (2011) 2237–2243

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Phytochemistry

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Absolute configuration of podophyllotoxin related lignans from Bursera fagaroidesusing vibrational circular dichroism

René Velázquez-Jiménez a, J. Martín Torres-Valencia a,⇑, Carlos M. Cerda-García-Rojas b,⇑,Juan D. Hernández-Hernández c, Luisa U. Román-Marín c, J. Jesús Manríquez-Torres a,Mario A. Gómez-Hurtado a, Alejandro Valdez-Calderón a, Virginia Motilva d, Sofía García-Mauriño e,Elena Talero d, Javier Ávila d, Pedro Joseph-Nathan b

a Área Académica de Química, Universidad Autónoma del Estado de Hidalgo, Km 4.5 Carretera Pachuca-Tulancingo, Mineral de la Reforma, Hidalgo 42184, Mexicob Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apartado 14-740, México D.F. 07000, Mexicoc Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Apartado 137, Morelia, Michoacán 58000, Mexicod Facultad de Farmacia, Universidad de Sevilla, Profesor García González No. 2, Sevilla 41012, Spaine Facultad de Biología, Universidad de Sevilla, Profesor García González No. 2, Sevilla 41012, Spain

a r t i c l e i n f o

Article history:Received 17 February 2011Received in revised form 25 June 2011Available online 15 August 2011

Keywords:Podophyllotoxin derivativesBurseraceaeBursera fagaroidesCytotoxicityAbsolute configurationVibrational circular dichroism

0031-9422/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.phytochem.2011.07.017

⇑ Corresponding authors. Tel.: +52 771 717 2002000x6502 (J.M. Torres-Valencia), tel.: +52 55 5747 4(C.M. Cerda-García-Rojas).

E-mail addresses: jmartin@uaeh.edu.mx (J.M. Torresmx (C.M. Cerda-García-Rojas).

a b s t r a c t

The ethanol extract from the dried exudate of Bursera fagaroides (Burseraceae) showed significantcytotoxic activity in the HT-29 (human colon adenocarcinoma) test system. The extract provided fourpodophyllotoxin related lignans, identified as (70R,8R,80R)-(�)-deoxypodophyllotoxin (3), (70R,8R,80R)-(�)-morelensin (4), (8R,80R)-(�)-yatein (5), and (8R,80R)-(�)-50-desmethoxyyatein (6), whose spectro-scopic and chiroptical properties were compared with those of (7R,70R,8R,80R)-(�)-podophyllotoxin (1)and its acetyl derivative (2). Their absolute configurations were assigned by comparison of the vibrationalcircular dichroism spectra of 1 and 3 with those obtained by density functional theory calculations.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Bursera species are of relevance in traditional medicine, andseveral of them have domestic uses in the central region of Mexico.Some species have ethnomedical applications against diarrhoea,fever, gingivitis, cough, and measles (Yasunaka et al., 2005;Hernández-Hernández et al., 2005). Chemical studies on this genusdemonstrated the presence of bioactive lignan derivatives thatwere isolated from Bursera ariensis (Hernández et al., 1983; Xiaet al., 2009), Bursera graveolens (Nakanishi et al., 2005), Burseramicrophylla (Cole et al., 1969), Bursera permollis (Wickramaratneet al., 1995), and Bursera simaruba (Peraza-Sánchez and Peña-Rodríguez, 1992), respectively. Particularly significant bioactivelignans are the podophyllotoxin derivatives which are also found

ll rights reserved.

0x2205; fax: +52 771 717035; fax: +52 55 5747 7137

-Valencia), ccerda@cinvestav.

in several Bursera species (Lautié et al., 2008). Podophyllotoxin(1) (Dewick, 2009; Cragg and Newman, 2009; Schrecker andHartwell, 1956; Petcher et al., 1973) was originally isolated fromthe rhizomes and roots of Podophyllum hexandrum and Podophyl-lum peltatum (Berberidaceae), whose extracts are used as tincturesfor genital warts. The clinically-active agents, etoposide, etoposidephosphate, and teniposide, are semisynthetic derivatives of naturaloccurring epipodophyllotoxin, the 7-epimer of 1, which areemployed as very effective drugs for the treatment of small-celllung cancer, testicular cancer, and lymphomas. These medicationsare frequently used in combination therapies with other anticancerdrugs (Dewick, 2009; Cragg and Newman, 2009).

In the present paper, the chemical constituents from the cyto-toxic resin of Bursera fagaroides (Burseraceae) was reported whichafforded (�)-deoxypodophyllotoxin (3), (�)-morelensin (50-des-methoxydeoxypodophyllotoxin) (4), (�)-yatein (5) and (�)-50-des-methoxyyatein (6), as verified by optical rotation and NMRspectroscopic measurements. Previous work on this species, direc-ted to a search for new anticancer agents, yielded b-peltatin Amethyl ether and 50-desmethoxy-b-peltatin A methyl ether(Bianchi et al., 1969).

Table 1Thermochemical parameters of podophyllotoxin (1).

Conformer DEMMFFa %MMFF

b DEDFTc %DFT

d DGOPTe %OPT

f

1a 0.00 27.21 0.00 26.10 0.00 35.131b 1.82 1.26 0.24 17.29 0.29 21.691c 0.05 24.88 0.97 5.85 0.41 17.681d 4.67 0.01 3.82 0.04 1.11 5.431e 1.57 1.92 0.59 9.61 1.14 5.101f 1.57 1.91 1.61 1.72 1.15 5.081g 0.47 12.27 3.55 0.07 1.48 2.901h 3.46 0.08 0.84 6.27 1.62 2.271i 1.87 1.16 2.35 0.49 1.71 1.971j 2.85 0.22 1.68 1.54 1.87 1.501k 3.18 0.13 1.40 2.46 1.99 1.23

a Molecular mechanics energies relative to 1a with EMMFF = 74.77 kcal/mol.b Population in % calculated from molecular mechanics energies according to

DEMMFF � �RT ln K.c Single point B3LYP/6-31G(d) energies relative to 1a with E6-

31G(d) = �911,896.72 kcal/mol.d Population in % calculated from B3LYP/6-31G(d) energies according to DE6-

2238 R. Velázquez-Jiménez et al. / Phytochemistry 72 (2011) 2237–2243

The absolute configuration of (�)-podophyllotoxin (1) has beendetermined by chemical correlation with D-phenylalanine(Schrecker and Hartwell, 1956) and by X-ray diffraction analysisof 20-bromopodophyllotoxin (Petcher et al., 1973). Now, the abso-lute configuration of (�)-podophyllotoxin (1) was studied by vibra-tional circular dichroism (VCD) which was essential to determine,by the same methodology, the absolute configuration of (�)-deoxypodophyllotoxin (3) isolated from B. fagaroides. This isrelevant since the literature reports gave erroneous absolute con-figuration assignments based on the application of either empiricalECD rules (Batista et al., 2010) or optical rotation values compari-son (Nakahashi et al., 2011) that VCD has now resolved. Thus, areliable way to determine absolute configuration is by comparisonof experimental ORD, ECD or in particular VCD data with calculatedvalues. Furthermore, VCD spectra comparison of conformationallyrestricted aryltetralin lactone lignans (1, 3 and 4) and conforma-tionally mobile dibenzyl butyrolactone lignans (5 and 6) provideevidence for the absolute configuration of these natural products.

31G(d) � �RT ln K.e Gibbs free energies relative to 1a with GDGDZVP = �911,862.03 kcal/mol.f Population in % calculated from DGDZVP Gibbs free energies according to

DG = �RT ln K.

2. Results and discussion

The cytotoxic activity of the ethanol extract from the dried exu-dates of B. fagaroides, showed a typical concentration-dependenteffect against HT-29 cells with IC50 values of 0.40 ± 0.001 and0.41 ± 0.01 lg/mL after 48 and 72 h, respectively. Systematiccolumn chromatography fractionation of the extract led to theisolation of (�)-deoxypodophyllotoxin (3) (Jolad et al., 1977),(�)-morelensin (4) (Jolad et al., 1977), (�)-yatein (5) (McDonieland Cole, 1972) and (�)-50-desmethoxyyatein (6) (McDoniel andCole, 1972) (Fig. 1). Identification of all compounds was supportedby spectroscopic analyses, including 1D and 2D NMR spectroscopy,in comparison with published data (Jolad et al., 1977; Hartwell andSchrecker, 1951; McDoniel and Cole, 1972; Brewer et al., 1979;Ikeda et al., 1998).

Some podophyllotoxin (1) related lignans found in Bursera spe-cies contain a 8,80-cis-lactone ring arrangement, as burseranin andpicropolygamain, isolated from B. graveolens (Nakanishi et al.,2005) and B. simaruba (Peraza-Sánchez and Peña-Rodríguez,1992), respectively, while other lignans, like morelensin (4), whichwas identified in B. morelensis (Jolad et al., 1977), or 40-demethyl-desoxypodophyllotoxin, isolated from B. tonkinensis (Jutiviboonsuket al., 2005), have a 8,80-trans-lactone ring. Observation of the 1HNMR coupling constant between H-8 and H-80 enables the distinc-tion between these two stereochemical variants, since for cis mol-ecules the J value is around 9 Hz, while in trans compounds it is

1

2

1

23

4

56

7 8 9

1'2'

3'

4'5'

6'

7' 8' 9'

1 2

1 2

1 2

1 2

Fig. 1. Structures of podophyllotoxin (1), acetyl podophyllotoxin (2), and lignans 3–6 isolated from Bursera fagaroides.

approximately 14 Hz. Our NMR spectroscopic data for lignans 3and 4 clearly showed they retained the trans stereochemistry ofpodophyllotoxin (1) (Brewer et al., 1979), given that the H-8/H-80coupling constant value was 14.5 Hz. Lignans 5 and 6 presentedsimilar coupling constant values also supporting a trans-lactonering, a biogenetic analogy since 3–6 were isolated from the samesource.

(�)-Podophyllotoxin (1) and (�)-deoxypodophyllotoxin (3)were selected as model compounds for the absolute configurationdetermination of lignans 3–6 by VCD. This technique has beenincreasingly employed to study the absolute configuration of natu-ral products (Nafie, 2008). The method is based on comparison ofthe experimental VCD spectrum and the theoretical curve obtainedby density functional theory (DFT) calculations. Molecular modelsof (7R,70R,8R,80R)-podophyllotoxin (1) and (70R,8R,80R)-deoxypodo-phyllotoxin (3) were built according to the absolute configurationrepresented in Fig. 1 and subjected to a conformational searchusing the Monte Carlo method and the MMFF94 molecularmechanics force field. The searching process was repeated severaltimes starting from different geometries to completely cover theconformational hypersurface affording 20 conformers for 1 and11 conformers for 3. DFT energy calculation at the B3LYP/6-31G(d) level of theory followed by geometry optimization usingthe B3LYP/DGDZVP level of theory yielded eleven relevant con-formers for podophyllotoxin (1) and nine for deoxypodophyllotox-in (3) within an energy range (DG) of 2 kcal/mol. In a previousconformational analysis of aryltetralin lactone lignans using DFTcalculations at the B3LYP/6-311G(d,p) level, morelensin (4) affor-ded a comparable number of minimum energy structures withinthe same 2 kcal/mol range (Casanovas et al., 2005).

Table 1 contains the calculated thermochemical parameters forthe calculation of the conformational equilibrium of podophyllo-toxin (1) which includes the three major contributing species1a–1c and several minor contributing conformers 1d–1k. TheBoltzmann distribution was calculated according to the DG = DH– TDS and DG = �RT ln K equations, considering the B3LYP/DGDZVP calculated energies. The eleven low-energy structures,accounting for the total conformational population, are repre-sented in Fig. 2. The conformers essentially preserve the same spa-tial arrangements with changes in the rotation of methoxyl groups.The VCD frequencies of the eleven conformers 1a–1k were calcu-lated and Boltzmann-averaged to generate the calculated spectrum

Fig. 2. DFT//B3LYP/DGDZVP minimum energy structures of podophyllotoxin (1).

R. Velázquez-Jiménez et al. / Phytochemistry 72 (2011) 2237–2243 2239

which was plotted using Lorentzian bandshapes and bandwidths of6 cm�1. Fig. 3 shows a comparison of the theoretical and experi-mental VCD spectra of podophyllotoxin (1), which showed verygood agreement. Quantitative evaluation of this concordance wasachieved by applying the CompareVOA algorithm (BioTools, Inc.,Jupiter, FL 33458, USA), which calculates the integrated overlapof the experimental and theoretical data as a function of a relativeshift (Debie et al., 2010). Application of this procedure allowed usto obtain the optimal anharmonicity factor (anH = 0.974), and theVCD spectral similarity for the correct (SE = 77.2%) and the incor-rect enantiomer (S�E = 15.2%) with a 100% confidence level forthe absolute configuration assignment.

The same procedure was applied for the determination of theabsolute configuration of deoxypodophyllotoxin (3). The calculatedthermochemical analysis for the conformational equilibrium of 3,composed by the four relevant species 3a–3d, and several less con-tributing conformers 3e–3i is given in Table 2. The nine structures,accounting for the total conformational population, are illustratedin Fig. 4. These conformers have the same spatial arrangement forthe molecular skeleton but showed minor variations in the rotationof the C03-OMe, C04-OMe and C05-OMe bonds. The VCD frequencies ofconformers 3a–3i were Boltzmann-averaged to generate the corre-sponding calculated spectrum which is shown in Fig. 5 togetherwith the experimental spectrum. Application of the confidence

Δε

103

Wavenumbers (cm -1)

-40

-20

0

20

40

-40

-20

0

20

40

9501050115012501350145015501650

(a)

(b)

Fig. 3. (a) Experimental and (b) B3LYP/DGDZVP DFT Boltzmann-weighted VCDspectra of podophyllotoxin (1).

Table 2Thermochemical parameters of deoxypodophyllotoxin (3).

Conformer DEMMFFa %MMFF

b DEDFTc %DFT

d DGOPTe %OPT

f

3a 1.47 2.23 2.64 0.51 0.00 24.333b 0.00 26.46 0.73 12.75 0.08 21.333c 1.40 2.49 0.00 43.81 0.09 20.763d 0.11 22.05 2.97 0.29 0.41 12.203e 1.32 2.83 0.29 26.67 0.83 6.023f 1.15 3.80 1.82 2.02 0.84 5.943g 0.48 11.76 4.18 0.04 0.89 5.443h 1.69 1.52 4.59 0.02 1.31 2.663i 1.13 3.90 0.82 10.99 1.73 1.31

a Molecular mechanics energies relative to 3b with EMMFF = 80.62 kcal/mol.b Population in % calculated from molecular mechanics energies according to

DEMMFF � �RT ln K.c Single point B3LYP/6-31G(d) energies relative to 3c with E6-31G(d)=�864,707.05 kcal/mol.

d Population in % calculated from B3LYP/6-31G(d) energies according toDE6-31G(d) � �RT ln K.

e Gibbs free energies relative to 3a with GDGDZVP = �864,656.66 kcal/mol.f Population in % calculated from DGDZVP Gibbs free energies according to

DG = �RT ln K.

2240 R. Velázquez-Jiménez et al. / Phytochemistry 72 (2011) 2237–2243

level algorithm afforded the optimal anharmonicity factor(anH = 0.972), and the VCD spectral similarity for the correct(SE = 86.5%) and the incorrect enantiomer (S�E = 11.8%) with a100% confidence level for the absolute configuration assignment.It is relevant to mention that comparison of the experimentalVCD spectra of (�)-podophyllotoxin (1) and (�)-deoxypodophyllo-toxin (3) established a high degree of similarity, except in the1170–1100 cm�1 region due to the presence of C–O vibration fre-quencies of the hydroxyl group of compound 1.

A conformational search employing the Monte Carlo methodusing molecular mechanics showed that aryltetralin lactone lignan4 exhibited 14 conformations within an energy range of 5 kcal/mol,while (�)-podophyllotoxin (1) afforded 20 conformers, and 3

yielded 11 conformers. In contrast, the more flexible dibenzylbutyrolactone lignans 5 and 6 displayed a high number of confor-mational variants when these structures were subjected to thesame searching protocol. (�)-Yatein (5) afforded 45 conformerswhile (�)-50-desmethoxyyatein (6) yielded 60 conformers in anenergy interval of 5 kcal/mol. The molecular flexibility differencesbecome evident when the experimental spectra of 1, 3 and 4 (Figs.3, 5 and 6), which display sharper peaks, were compared with thespectra of 5 and 6 (Fig. 6) which exhibit broader signals. The fiveVCD spectra display the same phase pattern indicating that(�)-podophyllotoxin (1) and compounds 3–6, isolated fromB. fagaroides, belong to the same enantiomeric series.

3. Conclusion

Our results indicate the experimental VCD curves of commer-cially available (�)-podophyllotoxin (1) and (�)-deoxypodophyllo-toxin (3) isolated from B. fagaroides show good agreement with theDFT//B3LYP/DGDZVP VCD calculated spectra, as evidenced in Figs.3 and 5, respectively, thus affording conclusive and direct evidencefor the absolute configuration of these and related compounds, asfollows: (7R,70R,8R,80R)-(�)-podophyllotoxin (1), (7R,70R,8R,80R)-(�)-acetyl podophyllotoxin (2), (70R,8R,80R)-(�)-deoxypodophyllo-toxin (3), (70R,8R,80R)-(�)-morelensin (4), (8R,80R)-(�)-yatein (5)and (8R,80R)-(�)-50-desmethoxyyatein (6).

4. Experimental

4.1. General experimental procedures

Melting points were determined on a Fisher–Johns meltingpoint apparatus and are uncorrected. Optical rotations were deter-mined in CHCl3 on a Perkin–Elmer 341 polarimeter, whereas IRspectra were recorded on a Perkin–Elmer 16F PC IR-FT spectropho-tometer using thin films of the compounds deposited on a CsIcrystal. VCD measurements were performed on a BioTools ChiralIRFT-VCD spectrophotometer equipped with dual photoelasticmodulation and a long-term detector. Samples of 1 (10 mg), 3(10 mg), 4 (4 mg), 5 (6 mg), and 6 (4 mg) were dissolved in 100%atom-D CDCl3 (150 lL), placed in a BaF2 cell with a pathlength of100 lm and data were acquired at a resolution of 4 cm�1 for 6–7 h. NMR spectroscopic measurements, including gCOSY, HETCOR,gHMQC, and gHMBC experiments, were performed at 400 MHz for1H and 100 MHz for 13C on a JEOL Eclipse 400 or a Varian VNMRS400 spectrometers from CDCl3 solutions using TMS as internalstandard. Low-resolution mass spectra were recorded at 70 eV ona Hewlett–Packard 5890 Series II spectrometer and at 70 eV on aHewlett–Packard 5989A spectrometer. Column chromatographywas carried out on Merck silica gel 60 (Aldrich, 230–400 meshASTM). Podophyllotoxin (1) was commercially available (Aldrich).

4.2. Plant material

Specimens of B. fagaroides (H.B.K.) Engler were collected at17�520180 0N, 99�340340 0W near the Iguala-Chilpancingo state roadNo. 95, State of Guerrero, Mexico, during July 2009. A voucherspecimen (Juan Diego Hernández 195) is deposited at theHerbarium of Instituto de Ecología, A.C., Centro Regional del Bajío,Pátzcuaro, Michoacán, México, where Prof. Jerzy Rzedowski kindlyidentified the plant material.

4.3. Extraction and isolation

Air-dried resins of B. fagaroides (64 g) were extracted withEtOH (250 mL) under condition of reflux for 4 h. Filtration and

Fig. 4. DFT//B3LYP/DGDZVP Minimum energy structures of deoxypodophyllotoxin (3).

R. Velázquez-Jiménez et al. / Phytochemistry 72 (2011) 2237–2243 2241

evaporation of the extract afforded a yellow viscous oil (46 g), aportion (1.2 g) of which was applied to a silica gel column(120 g) using hexanes–EtOAc (4:1, 7:3 and 1:1), EtOAc and MeOHas eluents. Fractions of 20 mL of each polarity were collected, mon-itored by TLC and analysed by 1H NMR spectroscopy. The resultingmaterial from each fraction was labelled as A (26 mg), B (52.0 mg),C (127.7 mg), D (149.0 mg), E (93.4 mg) and F (200.0 mg). FractionC was subjected to over silica gel (15 g) column chromatography(CC) using hexane–EtOAc (4:1 and 1:1) and EtOAc as eluents pro-viding 6 (10 mg) which showed ½a�20

D = �33.1 (c 1.1, CHCl3) lit.½a�20

D = �45.3 (c 3.8%, CHCl3) (McDoniel and Cole, 1972), respec-tively. Fraction D was purified over silica gel employing CHCl3–EtOAc (19:1) and MeOH as eluents affording 5 (12 mg) of½a�20

D = �33.2 (c 0.9, CHCl3) lit. ½a�20D = �29.8 (c 3.0%, CHCl3) (McDon-

iel and Cole, 1972), respectively. Separation of fraction E by meansof preparative TLC using CHCl3–EtOAc (4:1) as the mobile phaseallowed to obtain pure 3 (10 mg) which showed ½a�20

D = �95.2 (c1.0, CHCl3) lit. ½a�20

D = �105.0 (c 0.66, CHCl3) (Wickramaratneet al., 1995) and 4 (6.2 mg) of ½a�20

D = �127.0 (c 0.4, CHCl3) lit.½a�20

D = �125.0 (c 1.4, CHCl3) (Jolad et al., 1977).

4.4. Acetylation of 1

A solution of 1 (22 mg) in pyridine (3.0 mL) was treated withAc2O (1.0 mL). The reaction mixture was heated on a steam bath

for 3 h, poured over ice-H2O and extracted with EtOAc. The organiclayer was washed with 10% HCl, H2O, aqueous NaHCO3, and H2O,dried, filtered, and evaporated to give a yellow residue, whichwas subjected to column chromatographic purification usingCHCl3–EtOAc (5.5:1 and 4:1) as eluent. Fractions eluted withCHCl3–EtOAc (4:1), yielded pure 2 (17 mg, 70%) (Hartwell and Sch-recker, 1951). Compound 1 showed ½a�20

D = –134.3 (c 1.0, CHCl3) lit.½a�20

D = –132.0 (c 1.0, CHCl3) (Hartwell and Schrecker, 1951) andacetate 2 showed ½a�20

D = –130.3 (c 1.7, CHCl3) lit. ½a�20D = –143.0 (c

1.0, CHCl3) (Hartwell and Schrecker, 1951).

4.5. Cytotoxic assay

4.5.1. Media and cell lineHuman colon cell line HT-29 for the cytotoxic assay was incu-

bated in a standard Macoy’s medium supplemented with 10% foe-tal calf serum containing 50 U/mL of penicillin and 50 U/mL ofstreptomycin.

4.5.2. Cytotoxic assayCellular viability in the presence and absence of active mole-

cules was determined using the standard sulforhodamine B assay(SRB) (Skehan et al., 1990). Briefly, HT-29 cells in their log phaseof growth were harvested, counted and seeded (105 cell/well in100 lL volume) in a 96-well plate. After 24 h of incubation at

Δε

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Wavenumbers (cm-1)

(a)

(b)

-40

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0

20

40

9501050115012501350145015501650

-40

-20

0

20

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Fig. 5. (a) Experimental and (b) DFT//B3LYP/DGDZVP Boltzmann-weighted VCDspectra of deoxypodophyllotoxin (3).

-40

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0

20

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9501050115012501350145015501650

Wavenumbers (cm-1)

Δε10

3

(a)

(b)

(c)

Fig. 6. Experimental VCD spectra of (a) (70R,8R,80R)-(�)-morelensin (4), (b) (8R,80R)-(�)-yatein (5), and (c) (8R,80R)-(�)-50-desmethoxyyatein (6).

2242 R. Velázquez-Jiménez et al. / Phytochemistry 72 (2011) 2237–2243

37 �C with 5% CO2 to allow cell attachment, cultures were treatedwith varying concentrations (0.1–200 lg/mL) of test-extract. Tworeplicate wells were set up for each experimental condition.Extracts were left in contact with the cells for 48 and 72 h, respec-tively, SRB was then added to each culture and cell viability mea-sured as described previously (Skehan et al., 1990). Results wereexpressed as the concentration of compounds producing 50% inhi-bition (IC50). The IC50 values were calculated from at least 4 sig-nificant concentrations and the assays were performed intriplicate.

4.6. Molecular modelling

Geometry optimizations for 1, 3–6 were carried out using themolecular mechanics (MMFF94) force-field calculations as imple-mented in the Spartan’04 program (Wavefunction, Inc., Irvine CA92612, USA). A Monte Carlo search protocol was carried out con-sidering an energy cut-off of 10 kcal/mol. Single point energy ofthe conformers found within the EMMFF 10 kcal/mol range were cal-culated using the DFT B3LYP/6-31G(d) level of theory in the Spar-tan’04 program. This procedure provided a fast method forselecting low energy conformers within a quantum mechanicsframework and allows pre-optimization for further DFT calcula-tions. For compounds 1 and 3, DFT conformers found in the first5 kcal/mol were optimized at the B3LYP/DGDZVP level of theoryemploying the GAUSSIAN 03W program (Gaussian, Inc., WallingfordCT 06492, USA). The minimized structures in the first 3 kcal/molwere used to calculate the thermochemical parameters and theIR and VCD frequencies at 298 K and 1 atm. The VCD spectra wereBoltzmann-averaged using those conformers within 2 kcal/mol.Molecular visualization was carried out with the GaussianView3.0 program. Calculations required between 17 and 24 h computa-tional time per conformer when using a desktop computer operat-ing at 3 GHz with 8 Gb RAM.

Acknowledgments

Partial financial support from CONACYT, Mexico (Grant No. U2-80555) is acknowledged. R.V.J. thanks CONACYT for fellowship225823. We are grateful to Professor Jerzy Rzedowski (Institutode Ecología, A.C., Centro Regional del Bajío, Pátzcuaro, Michoacán,Mexico) for identifying the plant material.

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