Phytochemistry 80 (2012) 109–114

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Phytochemistry

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Determination of absolute configuration of salvic acid, an ent-labdanefrom Eupatorium salvia, by vibrational circular dichroism

Marcelo A. Muñoz a, Alejandro Urzúa b, Javier Echeverría b, Maria A. Bucio c,Angelina Hernández-Barragán c, Pedro Joseph-Nathan c,⇑a Instituto de Ciencias Químicas, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chileb Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chilec Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apartado 14-740, DF 07000, Mexico

a r t i c l e i n f o

Article history:Received 3 September 2011Received in revised form 16 March 2012Available online 31 May 2012

Keywords:Eupatorium salviaAsteraceaeVCDSalvic acident-LabdaneAbsolute configurationX-ray crystal structure

0031-9422/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.phytochem.2012.04.014

⇑ Corresponding author. Tel.: +52 55 5747 7112; faE-mail address: pjoseph@nathan.cinvestav.mx (P.

a b s t r a c t

The relative stereochemistry at C13 and the absolute configuration of salvic acid, a constituent of theleaves of Eupatorium salvia, were established as the 13-(R)-ent-labdane 1. The results follow from vibra-tional circular dichroism measurements of the derived O-methyl ether methyl ester 3 which were com-pared to DFT B3LYP/DGDZVP calculated spectra. The relative stereochemistry of salvic acid at C13 wasindependently verified by single crystal X-ray diffraction measurements of 1, and of its derived diol 4.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Labdane diterpenes are a well known class of compounds,widely distributed in nature, for which a long and continuousinterest has evolved (Chinou, 2005; Hanson, 2009; Peters, 2010;Reddy et al., 2009). This ongoing interest is mainly due to the rel-evant pharmacological activities many of these compound posses,as very recently reviewed (Frija et al., 2011). From the stereochem-ical point of view, labdanes occur in nature as two antipodalgroups of bicyclic molecules known as the ‘‘normal’’ and the entseries (Scheme 1), in which members of the same group are in gen-eral isolated together, although eventually the co-occurrence ofnormal and ent-labdanes has been surveyed (Carman and Duffield,1993). Assignment to either stereochemical series has been madeeither by chemical correlations with compounds of known abso-lute configuration, or indirectly through comparison with reportedoptical activity data. A recent related case established the coexis-tence of normal labdanes and ent-clerodanes in Chromolaenapulchella (Gómez-Hurtado et al., 2011).

Salvic acid (1), ([a]D + 21.5, c 0.98 CHCl3) (Scheme 2), originallyisolated from the leaves of the Chilean plant Eupatorium salvia, wascharacterized using 1H-NMR and MS. Its relative stereochemistry

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x: +52 55 5747 7137.Joseph-Nathan).

at C5, C9 and C10 was proposed based on comparison of 13C-NMR chemical shifts and lanthanide induced shifts with those of7a-hydroxymanool. Although no absolute configuration was pro-posed, the structure was depicted as a normal labdane with noconfiguration assignment at the C13 stereogenic center (Hoeneisenet al., 1979).

Afterwards, a study of the aerial parts of Cistus symphytifoliuslead (Calabuig et al., 1981) to the isolation of cistenolic acid([a]D-21.5, c 0.232 CHCl3), which turned out to be the enantiomerof salvic acid (1). Since chemical correlation of cistenolic acidmethyl ester with cativic acid methyl ester indicated the samenormal labdane absolute configuration, salvic acid (1) was assignedto the ent-labdane series, still with unknown configuration at C13(Calabuig et al., 1981).

Re-examination of the aerial parts of E. salvia led to re-isolate 1as the major component, with its identity confirmed through com-parison with reported spectroscopic data. However its absoluteconfiguration was again depicted as a normal labdane (Gonzálezet al., 1990) since the authors ignored the previous paper (Calabuiget al., 1981), generated in the same city. Furthermore, the 13-(S)absolute configuration for salvic acid (1) was drawn (Gonzálezet al., 1990) without any justification. In a fast auto-correction pro-cedure the authors postulate, after chemical correlation withmethyl dihydroepurate of known absolute configuration, that sal-vic acid (1), is an ent-labdane (González et al., 1991). This time,

12

3 57

9

10

12

13

16

20

17

1819

normal series

ent-series

H

H

15

Scheme 1. Stereochemistry and carbon atom numbering for the labdane ringsystem.

OR'

R

H

OH

COOH

1: R = COOH, R' = H2: R = COOMe, R' = H3: R = COOMe, R' = Me4: R = CH2OH, R' = H

5

6

OMe

COOMe

H

Scheme 2. Structures of labdanes 1–6.

110 M.A. Muñoz et al. / Phytochemistry 80 (2012) 109–114

thus was in agreement with the work they originally ignored (Cal-abuig et al., 1981), and where the 13-(R) absolute configurationwas depicted. In addition, and quite surprisingly, an authoritariandictionary of terpenoids (Connolly and Hill, 1992) shows stereo-structure 5 assigned to both salvic and cistenolic acid, as syn-onyms, levorotatory optical rotation, and as constituent of bothE. salvia and C. symphytifolius.

Recent studies accounting for the antibacterial (Urzúa et al.,1998, 2008) and antifungal (Mendoza et al., 2009) activities of salvic

acid (1) and related molecules again assumed the normal labdanestereochemistry and an unknown configuration at C13. Therefore,due to the inconsistencies found in the literature as far as the C13relative stereochemistry and the absolute configuration of salvicacid (1) are concerned, herein we definitively establish its chiralityas the 13-(R)-ent-labdane shown in 1.

The method of choice, vibrational circular dichroism (VCD), re-lies on comparison of the experimental spectrum of the moleculeunder study with a theoretical spectrum obtained by density func-tional theory (DFT) calculations. This method was used recently todetermine the relative stereochemistry and absolute configurationof a stereogenic center found on a side chain, one bond removedfrom the bicyclic ring system of the diterpene leubethanol(Molina-Salinas et al., 2011). Thus, it was of interest to use VCDto study a molecule like a labdane or ent-labdane diterpene, withan unknown stereogenic center three bonds away from the bicyclicring system.

2. Results and discussion

As a strategy to avoid condensed-phase intermolecular interac-tions between the carboxylic acid and hydroxyl groups in 1,O-methyl ether methyl ester 3 was selected for the VCD study. Thiscompound was prepared by treatment of salvic acid (1) (Hoeneisenet al., 1979) with acid in methanol. Compound 3 shows the ele-mental composition C22H38O3 as established by HR-EIMS, and theester carbonyl absorption at 1729 cm�1 is evident in the IR spec-trum. The 1H NMR spectrum of 3 showed the carbomethoxy groupsinglet at d 3.66, the C7 methoxy group singlet at d 3.14, threemethyl group singlets at d 0.87, 0.78 and 0.65, the secondarymethyl group as a doublet (J = 6.5 Hz) at d 0.94, the H7 signal, gem-inal to the methoxy group, as a broadened signal at d 3.72 and thetwo exocyclic double bond hydrogen atoms as two triplets at d 5.02(J = 1.3 Hz) and 4.72 (J = 1.6 Hz). These spectroscopic data are quitesimilar to those of 2 (Hoeneisen et al., 1979) and of its enantiomer(Calabuig et al., 1981), excepting the presence of the C7 methoxysignal, and the shift of the hydrogen geminal to that methoxygroup. The 13C NMR spectrum of 3 showed the carbomethoxy sig-nals at d 173.7 and 51.3, the exocyclic methylene group at d 147.1and 110.8, the methoxy group at d 55.0, the C7 signal at d 82.8, andfour methyl resonances at d 33.2, 21.4, 20.0, and 13.6. Again, thesedata are similar to those of salvic acid methyl ester (2) exceptingthe presence of the C7 methoxy group signal. The replacement ofthe hydroxyl group hydrogen atom by a methyl group occurs atthe oxygen atom and the C7 stereogenic center remains untouchedas further evidenced by the 1H NMR multiplicity of H7.

In order to obtain theoretical IR and VCD spectra, which can becompared to the experimental spectra, the conformational prefer-ences of O-methyl ether methyl ester 3 and of its C13 diastereoiso-mer 6 were addressed. The pertinent details are given in theSupplementary information. Only two conformations of the bicyclicskeleton were observed, the chair–chair disposition being 2.8 kcal/mol more stable than the boat–chair conformation. Single pointenergy calculations at the DFT//B3LYP/DGDZVP level of theoryshowed that for each diastereoisomer four conformations ac-counted for 98.5% and 97.5% of the entire conformational distribu-tions of 3 and 6, respectively, which were found in a 3.0 kcal/molgap. Further calculations of these eight optimized structures, per-formed at the same level of theory allowed prediction of the corre-sponding free energy values, which showed that only three and twoconformations accounted for more than 99.9% of the conforma-tional distributions of 3 and 6, respectively, as shown in Table S1of the Supplementary information. In both cases, the observedconformations showed chair–chair arrangements for the bicyclicring system, and the extended syn orientation of the methoxy group

Fig. 1. Low energy conformations (3a–3c) of the ent-13-(R) diastereoisomer ofmethyl O-methylsalvate.

Fig. 2. Low energy conformations (6a–6b) of the ent-13-(S) diastereoisomer ofmethyl O-methylsalvate.

Fig. 3. Experimental (center) and calculated Boltzmann weighted IR spectra of 3(top) and 6 (bottom). Frequencies of the calculated spectra are scaled usinganharmonicity factors of 0.975 and 0.968 for 3 and 6, respectively.

Fig. 4. Experimental (center) and calculated Boltzmann weighted VCD spectra of 3(top) and 6 (bottom). Frequencies of the calculated spectra are scaled usinganharmonicity factors of 0.975 and 0.968 for 3 and 6, respectively.

M.A. Muñoz et al. / Phytochemistry 80 (2012) 109–114 111

towards the side-chain, while the main conformational differencesarise from free rotations of the side-chain single bonds beyond C-12,as shown in Fig. 1 for 3 and in Fig. 2 for 6. Boltzmann-weightedvibrational spectra were synthesized using the fractional

populations of these conformational models and the harmonicvibrational frequencies and intensities calculated for these models.

Fig. 3 shows a comparison of the calculated IR spectra of 3 and 6with the experimental IR spectrum of the sample derived from thenatural product. In turn, Fig. 4 shows the comparison of the corre-sponding VCD spectra. Visual inspection of the later spectra sup-ports structure 3 as the correct stereoisomer. These observationswere further corroborated by a software-guided comparison which

Table 1Confidence level data for the IR and VCD spectra of ent-13R (3) and ent-13S (6)diastereoisomers.

Epimer anHa SIRb SE

c S�Ed ESIe Cf

3 0.975 88.2 72.9 20.6 52.3 1006 0.968 88.7 50.7 27.3 23.4 42

a Anharmonicity factor.b IR spectral similarity.c VCD spectral similarity for the correct enantiomer.d VCD spectral similarity for the incorrect enantiomer.e Enantiomer similarity index, calculated as the SE � S�E difference.f Confidence level for the stereochemical assignment.

Fig. 5. X-ray structure of salvic acid (1).

Fig. 6. X-ray structure of diol 4.

112 M.A. Muñoz et al. / Phytochemistry 80 (2012) 109–114

provided the same conclusion with a 100% confidence level, as de-tailed below.

A good band-to-band comparison of calculated and observedVCD spectra is very important for configuration assignment sincecalculated frequencies derive from a harmonic force field whilemeasured IR and VCD frequencies derive from anharmonic forcefield. Thus, the calculated data were corrected using the optimizedfrequency scaling factor from Compare VOA (BioTools Co, Jupiter,FL 33458, USA) that is based on neighborhood similarity (Kuppenset al., 2005, 2007; Debie et al., 2010). The algorithm takes advan-tage of a correlation function that describes the integrated overlapof the theoretical and experimental data as a function of a relativefrequency shift, being the optimal shift known as the anharmonic-ity (anH) factor. Thus, the calculated spectra of diastereomers 3 and6 were compared in silico to the experimental IR and VCD spectraof the O-methyl ether methyl ester derivative of salvic acid (1),the pertinent results being summarized in Table 1. The anH factor,a measure of band alignment of experimental and theoreticalabsorption signals, and the IR spectral similarity (SIR) are slightlydifferent for diastereoisomers 3 and 6. The VCD spectral similarity(SE) for 3 is 72.9 and that of its enantiomer (S�E) is 20.6, while forthe 13-(S) alternative 6 those values are 50.7 and 27.3, respec-tively, which provide enantiomer similarity indexes of 52.3 and23.4 for 3 and 6, respectively. Finally, the software generated con-fidence level for 3 is 100%, in contrast to 42%, for the 13-(S) epimer

6, from where the 13-(R) absolute configuration for salvic acid (1)follows.

In an independent effort to determine the relative stereo-chemistry of salvic acid (1), in particular at the C13 stereogeniccenter, a single crystal X-ray diffraction study of 1 was under-taken. The molecular structure is shown in Fig. 4, in which the13-(R) stereochemistry is evident for an ent-labdane structure.However, since after diffracting several crystals of salvic acid(1) the best data convergence obtained from the X-ray studywas a modest R-factor of 6.9%, it was decided to explore a re-lated derivative in search to obtain a better data convergence.For this purpose, diol 4, obtained by reduction of 1 (Hoeneisenet al., 1979) was studied, whose data nicely converged to pro-vide an R-factor of 3.9%, which again allowed to confirm the13-(R) stereochemistry for the ent-labdane structure shown inFig. 5. Furthermore, the solid state structures of 1 and 4 showmany conformational features also observed in the quantummechanical calculations used for the VCD study of 3. In particu-lar, the fused bicyclic skeleton of 1 and 4 Fig. 6 closely resemblethose calculated for the three low energy conformers of 3, show-ing the same chair–chair disposition with almost identical endo-cyclic dihedral angles. In addition, a very similar disposition ofthe side-chain atoms is observed when comparing the crystalstructures and the two more stable conformers calculated for3, in which differences are only observed beyond C-13.

3. Conclusion

The results obtained herein definitively establish the relativestereochemistry at C13 and the absolute configuration of salvicacid (1) as deduced from VCD measurements in comparison to cal-culated spectra. In addition, conformational information of 3 in thegas phase, and of 1 and 4 in the solid state, which could be useful togain insights into the mechanisms of action of these compounds,particularly in structure–activity relationship studies, is alsoprovided.

M.A. Muñoz et al. / Phytochemistry 80 (2012) 109–114 113

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, IR and VCDmeasurements were performed on a BioTools ChiralIR FT-VCDspectrophotometer equipped with dual photoelastic modulationand a long-term detector using a sample of 10 mg in 100% atom-D CDCl3 (150 lL) placed in a BaF2 cell with a pathlength of100 lm for which data were acquired at a resolution of 4 cm�1

during 7 h. NMR measurements, including gCOSY, HETCOR,gHMQC, and gHMBC experiments, were performed at 300 MHzfor 1H and 75 MHz for 13C on a Varian Merccury 300 spectrometerfrom CDCl3 solutions using TMS as internal standard, while thelow-resolution mass spectrum was recorded at 70 eV on a VarianSaturn 2000 spectrometer.

4.2. Plant material

Aerial parts of very resinous E. salvia were collected from LoPrado Pass (Región Metropolitana, Chile, 31� 280S, 71� 270W) atan altitude of 785 m over the average sea level during the flower-ing season, March 2008. Voucher specimens were deposited in theHerbarium of the National Museum of Natural History, Santiago,Chile (SGO 108833).

4.3. Compounds

4.3.1. Isolation of salvic acid (1)Aerial parts of E. salvia (1.8 kg) were extracted by dipping the

fresh plant material into of CH2Cl2 (5 L) at room temperature dur-ing 30 s. This procedure was repeated twice to assure the total andselective extraction of the epicuticular components. The combinedCH2Cl2 extracts were evaporated, and the residue (133 g) was frac-tionated by CC (silica gel) using pentane–CH2Cl2 andCH2Cl2–MeOH step gradients. The fractions eluted with CH2Cl2–MeOH (99:1) spontaneously crystallized to yield salvic acid (1)(40 g), which was identified by direct comparison with an authen-tic sample (Urzúa et al., 2008; Mendoza et al., 2009).

4.3.2. Diol 4The compound was obtained after LiAlH4 reduction of 1 follow-

ing the described procedure (Hoeneisen et al., 1979). The mp 106–108 �C and spectral data of 4 were in agreement with thosepublished.

4.3.3. Methyl O-methylsalvate (3)A solution of 200 mg (0.62 mmol) of salvic acid (1) in anhydrous

MeoH (5 mL) was treated with two drops of 85% H2SO4 and heatedunder reflux began, this being continued for 1 h. The solvent wasthen evaporated under reduced pressure, with the residue dis-solved in CH2Cl2 (10 mL) and the whole washed with an aqueous5% solution of NaHCO3 (2 � 10 mL) and H2O. After drying (anhy-drous Na2SO4), the solvent was evaporated to yield a yellowishoil which was purified by silica gel column chromatography (cc)with CH2Cl2 to yield 156 mg (72%) of the title molecule. Compound3 shows [a]589 + 5.3, [a]578 + 5.3, [a]546 + 6.6, [a]436 + 15.9 (c 0.98CHCl3); IR (CDCl3) mmax 1729, 1646 cm�1; 1H NMR (CDCl3,300 MHz) d: 5.02 (t, J = 1.3 Hz, H-17a), 4.72 (t, J = 1.6 Hz, H-17b),3.72 (br, H-7), 3.66 (s, COOMe), 3.14 (s, OMe), 2.32 (dd, J = 14.6,5.9 Hz, H-14a), 2.10 (dd, J = 14.6, 8.3 Hz, H-14b), 1.92 (m, H-13),1.91 (m, H-6a), 1.89 (m, H-9), 1.70 (d, H-1a), 1.55 (m, H-11a),1.52 (m, H-5), 1.51 (m, H-6b0), 1.49 (2H, m, 2H-2), 1.45 (m,

H-12a), 1.36 (m, H-3a), 1.26 (m, H-11b), 1.20 (m, H-3b), 1.07 (m,H-1b), 0.97 (m, H-12b), 0.94 (d, J = 6.5 Hz, H-16), 0.87 (s, H-18),0.78 (s, H-19), 0.65 (s, H-20); 13C NMR (CDCl3, 75 MHz) d: 173.7(C-15), 147.1 (C-8), 110.8 (C-17), 82.8 (C-7), 55.0 (OMe), 51.3 (2C,C-9 and COOMe), 48.2 (C5), 42.0 (C-3), 41.3 (C-14), 39.6 (C-10),38.6 (C-1), 35.3 (C-12), 33.2 (C-18), 33.0 (C-4), 31.1 (C-13), 29.8(C-6), 21.4 (C-19), 20.3 (C-11), 20.0 (C-16), 19.3 (C-2), 13.6 (C-20); EI-MS m/z (rel. int.): 350 [M]+ (16), 318 (49), 303 (50), 221(100), 189 (54), 133 (41), 123 (59), 121 (69), 119 (68), 111 (52),107 (72), 67 (58), 39 (56); HR-EI-MS m/z: 350.2833 [M]+ (calc.for C22H38O3, 350.2821).

4.4. Molecular modeling

Conformational distributions for 3 and 6 were obtained throughstochastic Monte Carlo guided conformational searches at themolecular mechanics (MMFF) level of theory using the Spartan’04software package (Wavefunction Inc, Irvine, CA 92612, USA).Geometry optimizations and calculations of harmonic vibrationalfrequencies, dipole strengths and rotational strengths were carriedout at the quantum mechanical level using the Gaussian 03 W soft-ware suite (Gaussian Inc, Wallingford, CT 06492, USA). Vibrationalspectra (IR/VCD) were calculated for each conformational modelusing these vibrational properties and fitting the subsequent linespectra with Lorentzian bandshapes (half-widths of 6 cm�1). Thesespectra were then weighted using Boltzmann statistics and com-bined to simulate VCD and IR spectra for 3 and 6. Quantitativeagreements between calculated and measured IR and VCD spectrawere assessed using CompareVOA (BioTools Co., Jupiter, FL 33458,USA). A data summary is given in Table S1 of the Supplementaryinformation.

The used functional and basis set have shown to provide a goodbalance between computational cost and spectral accuracy(Molina-Salinas et al., 2011; Gordillo-Román et al., 2012), speciallywhen compared with other hybrid functionals and basis sets(Burgueño-Tapia et al., 2010; Cerda-García-Rojas et al., 2008).

4.5. Single crystal X-ray diffraction analysis of 1 and 4

Studies for 1 were done on a Bruker Smart 6000 CCD diffrac-tometer using MoKa radiation (k = 0.7073 Å). A total of 1321frames were collected at a scan width of 0.3� and an exposuretime of 10 s/frame. These data were processed with the SAINTsoftware package, provided by the diffractometer manufacturer,by using a narrow-frame integration algorithm. An empiricalabsorption correction was applied. Crystal data were: C20H34O3,M = 322.47, monoclinic, space group C2, a = 29.905(6) Å,b = 6.060(1) Å, c = 11.089(2) Å, b = 106.06(3), V = 1931.1(7) Å3,Z = 4, q = 1.11 mg/mm3, l(MoKa) = 0.072 mm�1, total reflections16348, unique reflections 1682 (Rint 0.01%), observed reflections1682, final R indices [I > 2r (I)] R1 = 6.9%, wR2 = 17.8%. The datafor 4 were collected on a Bruker–Nonius CAD4 diffractometerequipped with CuKa radiation (k = 1.54184 Å) at 293(2) K in thex � 2h scan mode. Unit cell refinements using 25 machine cen-tered reflections were done using the CAD4 Express v2.0 software.Crystal data were: C20H36O2, M = 308.49, monoclinic, space groupC2, a = 29.746(6) Å, b = 6.113 (1) Å, c = 11.047(2) Å, b = 102.24(3)V = 1963.2(7) Å3, Z = 4, q = 1.04 mg/mm3, l(CuKa) = 0.495 mm�1,total reflections 3155, unique reflections 2756 (Rint 0.62%),observed reflections 2538, final R indices [I > 2 r (I)] R1 = 3.9%,wR2 = 10.6%. The structures were solved by direct methods usingthe SHELXS-97 program included in the WinGX v1.70.01 crystallo-graphic software package. For the structural refinement, thenon-hydrogen atoms were treated anisotropically, and the hydro-gen atoms, included in the structure factor calculation, were re-fined isotropically. Crystallographic data (excluding structure

114 M.A. Muñoz et al. / Phytochemistry 80 (2012) 109–114

factors) have been deposited at the Cambridge CrystallographicData Centre. Copies of the data can be obtained free of chargeon application to CCDC, 12 Union Road, Cambridge CB2 IEZ, UK.Fax: +44 (0) 1223 336033 or e-mail: deposit@ccdc.cam.ac.uk.The CCDC deposition number for 1 is 862753, and that for 4 is862754.

Acknowledgement

Partial financial support from Conacyt Mexico (Grant No.152994) is acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytochem.2012.04.014.

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