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Spectral Assignments and Reference DataReceived: 1 April 2009 Revised: 13 July 2009 Accepted: 14 July 2009 Published online in Wiley Interscience: 10 August 2009

(www.interscience.com) DOI 10.1002/mrc.2499

Structure determination of chamaedryosidesA – C, three novel nor-neo-clerodaneglucosides from Teucrium chamaedrys, by NMRspectroscopyAntonio Fiorentino, Brigida D’Abrosca,∗ Andreina Ricci, Severina Pacifico,Simona Piccolella and Pietro Monaco

Three new nor-neo-clerodane diterpenes, named chamaedryoside A (1), B (2) and C (3), have been isolated from the organicextracts of Teucrium chamaedrys (L.) and their structural characterization has been accomplished by 1H and 13C-NMR spectra, andDEPT, by COSY, TOCSY, HSQC, HSQC-TOSCY and HMBC experiments, as well as by ESI-MS/MS techniques. The stereostructureshave been elucidated by NOESY and computational calculations. Copyright c© 2009 John Wiley & Sons, Ltd.

Keywords: Teucrium chamaedrys; Lamiaceae; nor-neo-clerodane glucosides; chamaedryoside A – C; spectroscopic analysis

Introduction

The genus Teucrium (Labiatae) is a rich source of neo-clerodanediterpenes,[1] secondary metabolites with strong antifeedantactivity.[2,3] Teucrium chamaedrys (L.), also known as Germander, isa characteristic shrub of Mediterranean area, used in the past for itsmedicinal properties. Although T. chamaedrys is, still today, usedin the preparation of vermouth and other liquor beverages, withinspecified safety levels, teucrin A, a neo-clerodane metabolite wasfound to cause midzonal hepatic necrosis[4,5] and, as consequence,its use in pharmaceutical preparation was forbidden.

In our studies on chemical interactions among plants of Mediter-ranean ecosystems,[6 – 8] we undertook a systematic investigationon secondary metabolites from T. chamaedrys. Neo-clerodanediterpenes have been already isolated from this plant,[9,10]

and in the present paper we describe the structural charac-terization of three new nor-neo-clerodane glucosides, namedChamaedryosides A–C.

Results and Discussion

Compound 1, chamaedryoside A, showed 25 signals in its 13C NMRspectrum, and according to the molecular weight of 508 amu,deduced by MS experiments, a molecular formula C25H32O11 washypothesized. The UV-Vis spectrum, registered in MeOH showedpeaks at 208 and 223 nm. These data, together with the presence ofbands at 1705, 1506 and 875 cm−1 in the IR spectrum, suggestedthe presence of a furane ring and an α,β-unsaturated acidicmoieties in the molecule.[10 – 12]

The 1H NMR of new metabolite 1 showed three signal of thefuran ring as multiplets at δ 7.58, 7.52 and 6.47 (all δ values arein ppm); in the region of protons geminal to oxygen, a triplet atδ 5.48, a singlet at δ 5.00, one doublet at δ 4.45, a methylene at δ

3.81 and 3.65, a doublet of doublets at δ 3.13, a triplet at δ 2.97,and other overlapped protons ranging from 3.2 to 3.4 ppm were

evident. In the low-frequency region of the same spectrum, signalsof a doublet methyl at δ 0.95 as well as those of a diastereotopicmethylene at δ 2.41 and 2.63 and other overlapped protons,ranging from 1.4 to 2.3 ppm, were present.

The DQ-COSY and TOCSY experiments showed correlations ofthe following spin systems: the singlet at δ 5.00 correlated with amethylene at δ 1.88/2.14, showing cross peaks with a methine atδ 2.25 which was, in turn, correlated to the doublet methyl; themethine at δ 2.97 showed correlations with the methylene protonsat δ 1.46/2.15, which were both correlated to the methyleneprotons atδ 1.59/1.74, correlated with the methylene atδ 2.16/2.36;the doublet at δ 4.45 showed cross peaks with the doublet ofdoublet at δ 3.13, and correlations, in the TOCSY, with otherthree protons, in the range 3.33–3.37 ppm and a methylene at δ

3.65/3.81. These latter data suggested the presence of a glycosidicmoiety in the molecule. The 13C-NMR experiment showed 25carbons identified, on the basis of a DEPT experiment, as 1 methyl,6 methylenes, 12 methines and 6 tetrasubstituted carbons, two ofthem carboxylics.

The HSQC allowed the assignment of each proton of themolecule to the correlated carbons. HSQC-TOCSY experimentwas useful to resolve overlapped proton signals and allowedthe proton-carbon connectivities to be determined for each spinsystem. In particular, the anomeric proton at δ 4.45 correlated withthe C-2′, C-3′, C-4′ C-5′ and C-6′ carbons of the sugar moiety atδ 75.4, 78.3, 71.7, 77.6 and 62.7,[13] suggesting the presence ofglucose in the molecule. The GC-MS analysis of the glucitol acetatederivative, obtained by hydrolysis, reduction and acetylation of 1,

∗ Correspondence to: Brigida D’Abrosca, Dipartimento di Scienze della Vita,Laboratorio di Fitochimica, Seconda Universita degli Studi di Napoli, Via Vivaldi43, 81100 Caserta, Italy. E-mail: brigida.dabrosca@unina2.it

Dipartimento di Scienze della Vita, Laboratorio di Fitochimica, SecondaUniversita degli Studi di Napoli, Via Vivaldi 43, 81100 Caserta, Italy

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compared with pure standard, confirmed this hypothesis. In thesame HSQC – TOCSY experiment, the proton at δ 5.00 correlatedwith the carbons at δ 76.9, 35.8 and 33.9, and with the methyl atδ 17.5; the proton at δ 2.97 showed correlations with the carbonsat δ 41.6, 28.6, 26.4 and 21.9. The HMBC experiment allowedto assembling the partial structures as follows. The H-12 protonshowed heterocorrelations with the carbons at δ 180.0, 42.0 and55.2, assigned to the C-20, C-11 and C-9, respectively. The lattercarbon showed correlations with the methyl at δ 0.95, the methineat δ 2.25, the H-12 proton and the methine at δ 2.97. This protoncorrelated with the methylene carbon at δ 26.4 and with theolefin carbons at δ 134.5 and 160.1. The methine at δ 2.25 showedcorrelations with the methylene carbon at δ 35.8, which, in turn,correlated with the methyl and the methine at δ 5.00. This protonshowed, besides the correlations with the olefinic carbon a δ 134.5,a cross peak with the anomeric carbon at δ 103.3.

The positive and negative ESI mass spectra of compound 1showed the ions at m/z 507, 531 and 547 corresponding tothe [M−H]− ion and to the [M+Na]+, and [M+K]+ adducts,respectively. The CAD mass spectrum of the ion at m/z 507displayed the fragment at m/z 327 (−180 Da), suggesting thepresence of a hexose sugar bonded to an alcoholic oxygen. Theinduced collisional dissociation of the ion at m/z 327, obtained byin-source CAD, revealed the presence of fragments at m/z 283 and239 arising from two consecutive losses of 44 Da (CO2), of the ionsat m/z 255, 233, 203 and 189. The CAD [M+Na]+ mass spectrumshowed 351 (−180 Da) and 203.

These data indicated the presence of 15,16-epoxy-6-hydroxy-19-nor-neo-clerodane-20,12-olide-18-oic acid as aglycone with aglucose moiety bonded to the hydroxyl group at C-6 carbon bymeans the anomeric carbon. The β configuration for the sugar wasdetermined on the basis of the coupling constant value (7.8 Hz) ofanomeric proton, as detected in the 1H NMR spectrum.

The absolute configurations of the chiral carbons ofchamaedryoside A was assigned on the basis of NOESY exper-iments and computational calculations. The 9R and 12S config-urations for neo-clerodane diterpenes were previously reportedin the literature.[14 – 16] The observed NOE, in the NOESY exper-iment, between the H-12α and the H-11 at δ 2.63 allowed todefine the latter as proS. This proton showed NOE with the H-10proton at δ 2.97 permitting to evidence its β-orientation and,consequently, the S configuration for the C-10 carbon. The H-8β

methine gave NOE with the H-10 and the H-11 proR proton at δ

2.41, confirming the α-orientation for the H-17 methyl and an Rconfiguration for the C-7 carbon. Furthermore, the H-8β protongave NOE with the H-7 proton at δ 1.88 (β-oriented), while theH-17 methyl showed cross peak with the H-7 proton at δ 2.14(α-oriented), and both the H-7 protons showed NOE with the H-6proton. Computing the two possible C-6 epimers, using molecularmechanics method (MM+) as implemented in HyperChem 7.5,[17]

the observed NOEs can be justified only for the stereostructurewith the 6β-glucopyranosyloxy derivative: in this structure (Fig. 2),the calculated distance between the proS H-11 and H-10 was2.47 Å, while that between the H-10 and H-8 was 2.69 Å. Fur-thermore, the latter proton forms a dihedral angle of −56.9◦ and−173.3◦ with the H-7β and H-7α, respectively, and the calculateddistance between the H-8 and H-7β protons was 2.45 Å, justifyingthe observed NOEs. Finally, the anomeric proton gave NOE withthe H-6 methine and with the H-7β proton at δ 1.88 confirmingunequivocally the β-orientation for the glucopyranosyloxy groupand, consequently, an R configuration for the C-6 carbon.

The structural elucidations of the new metabolites 2 and 3 wereperformed in mixture. The 1H NMR spectrum displayed the signalsof the furane ring at δ 6.48, 7.53 and 7.59 (H-14, H-15 and H-16)and the triplet proton at δ 5.49 due to the H-12 proton, as well asa singlet methine at δ 5.11, two doublets at δ 5.08 and 4.48, twodouble doublets at δ 4.47 and 4.26, and other overlapped protonsranging from 3.3 to 3.6 ppm. In the low-frequency region of thesame spectrum, signals of a doublet methyl at δ 0.95 as well asthose of a diasterotopic methylene at δ 2.45 and 2.66 were present.The intensity of some 1H NMR signals indicated the presence of amixture of two different compounds, but all attempts to separatethis mixture by RP-HPLC failed because of a re-equilibration ofboth the purified metabolites in the initial mixture.

The positive and negative ESI mass spectra, obtained bydirect infusion of the mixture in the ionic source of themass spectrometer, showed the ions at m/z 507, 531 and 547corresponding to the [M−H]− ion, and to the [M+Na]+ and[M+K]+ adducts, respectively. The [M−H]− CAD mass spectrum,displayed the fragment at m/z 345 (−162 Da), suggesting thepresence of a hexose sugar, and the fragments at m/z 327, 233and 203. The induced collisional dissociation of the ion at m/z 345,obtained by in-source CAD, revealed the presence of a fragmentat m/z 327 (−18 Da) arising from the loss of a water molecule.By analogy with previously reported ESI/CAD mass spectra[18] itcould be indicative of the presence of a hydroxyl substituent inthe molecule. The CAD mass spectrum of the ion at m/z 345 alsodisplayed the ions at m/z 283 arising from the loss of 44 Da, likelyCO2, from the ions at m/z 327 and the fragments at m/z 239 and123. The CAD [M+Na]+ mass spectrum showed the ions at m/z471, 411, 351 and 203.

The 13C NMR revealed signals of a diterpene metabolite, besidestwelve carbons attributable to two hexoses. The values of the sugarmoiety were in accordance with a glucose having the anomericcarbon not involved in a glycoside bond. This hypothesis couldexplain the inability of HPLC technique to separate 2 and 3 dueto the mutarotation equilibrium established in aqueous solutions.The 13C NMR value at δ 64.8 and the H-6 values in the 1H NMR,suggested the presence of a mixture of 6-O-β-glucopyranosideand 6-O-α-glucopyranoside. On the basis of this evidence, the DEPTexperiment allowed the identification of 1 methyl, 6 methylenes, 12methines and 6 tetrasubstituted carbons for both the glycosides.

The comparison of the 13C NMR with the DEPT experimentindicated the presence in the aglycone of two further olefincarbons at δ 160.0 and 130.1, in addition to those of the furan ring,both tetrasubstituted, and of a carbinol methine at δ 66.7.

These data suggested the presence of a neo-clerodane asaglycone. In the DQ-COSY experiment, the H-12 proton at δ

5.49 showed cross peaks with the H-11 protons at δ 2.45 and2.66. The proton at δ 5.11 correlated with the methylene protonsat δ 1.71 and 2.12 which were both correlated with the methineat δ 2.20 that correlated with the H-17 methyl. This sequence,confirmed by a TOCSY experiment, allowed to locating a hydroxylgroup at the C-6 carbon. Finally, the methine at δ 2.97, attributedto the H-10 proton, correlated with the remaining signals of theCH/CH2/CH2/CH2 spin system bonded to the C-10/C-1/C-2/C-3carbons. The HMBC experiment confirmed the proton sequencesand allowed to identify the glucosilation site. In fact the C-18carboxyl carbon at δ 170.4 showed heterocorrelations with boththe glucidic H-6 protons, while the olefin carbon at δ 130.1and 160.0 showed cross peaks with the protons at δ 5.11 and2.23. Unfortunately, the H-6 protons were overlapped with theanomeric proton of the β-glucose. To confirm our hypothesis, an

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Structure determination of chamaedryosides A – C

O

O

O

OO

1

O

OH

OH

OHHO

OH

23

α glcβ glc

O

O

O

OHOO

OH

O

HO OH

OH

H H1

3 610

1112

14

181′

17

20

13

Figure 1. Chemical structures of chamaedryosides 1–3.

aliquot of the mixture was acetylated with Ac2O in anhydrouspyridine. The 1H NMR of the product showed a downfield shiftfor all the geminal to hydroxyl protons. In particular the α andβ anomeric protons resonated at δ 6.35 and 5.75, while the H-6glucosidic protons remained at δ 4.30 and 4.21. Heteronuclear2D NMR experiments allowed to assign all the carbons of themolecule and to associate each proton to the correspondingcarbon. These data, together with the other data reported inTable 2, confirmed the proposed structures for chamaedryosidesB and C (Fig. 1). The NOESY experiment (Table 2) allowed todefining the stereochemistry of the C-10 carbon. In fact the H-10proton gave NOEs with the H-11 proS proton at δ 2.66 and withthe H-8β proton, indicating its β-orientation. The configurationat the C-6 carbon has been established by comparison withchamaedryoside A.

Experimental

General procedures

The preparative HPLC apparatus consisted of a pump LC-10AD(Shimadzu, Japan), a refractive index detector (Shimadzu RID-

10A) and a Shimadzu Chromatopac C-R6A recorder. PreparativeHPLC was performed using a 250 × 10 mm i.d., 10 µm, Luna RP-8(Phenomenex, Torrance, Canada, CA) column. Analytical TLC wasperformed on Merck Kieselgel (Darmstadt, Germany) 60 F254 orRP-8 F254 plates with 0.2 mm layer thickness. Spots were visualizedby UV light or by spraying with H2SO4−AcOH−H2O (1 : 20 : 4).The plates were then heated for 5 min at 110 ◦C. Preparative TLCwas performed on Merck Kieselgel 60 F254 plates, with 0.5 or1 mm film thickness. Flash column chromatography (FCC) wasperformed on Merck Kieselgel 60 (230–400 mesh) at mediumpressure. Column chromatography (CC) was performed on MerckKieselgel 60 (70–240 mesh), Reversed Phase silica gel 100 C8(230–400 mesh) (Fluka, Buchs, Switzerland), Fluka Amberlite XAD-4, or Sephadex LH-20 (Pharmacia, Piscataway, USA) media.

IR spectra were determined in the KBr pellet using a FT-IR Perkin-Elmer ‘Spectrum GX’ spectrometer. UV spectra were performed onUV-1700 Shimadzu spectrophotometer in MeOH solution. Opticalrotations were measured on a Perkin-Elmer 141 in MeOH solution.

Electrospray mass spectra (ESI-MS) were acquired using aQuattro Micro (Micromass, Manchester, UK) triple quadrupoleinstrument operating in the negative and positive ion mode.Nitrogen was used as nebulization and desolvation gas at flowrates of 50 and 500 l/h, respectively. Source and desolvationtemperatures were set at 120 and 400 ◦C. Potential applied onthe electrospray capillary and on the cone ranged from 2.7 to3.0 kV and from 30 to 50 V respectively, and were optimized foreach molecule and for the ionization mode. CAD/MS experimentswere performed using argon as collision gas at a pressure of6.5 × 10−3 mbar with collision energy varying from 20 to 30 eV.All mass spectra were performed by direct introduction of purifiedcompounds as 10−4 M solutions in MeOH/H2O (1 : 1, v/v) with a10 µl/min flow rate. Full scan mass spectra were acquired fromm/z = 50–1500 in MS and from 20 to the m/z ratio of the precursorion in MS/MS experiments, with a scan time of 2s.

NMR spectroscopy

The NMR spectra were recorded on a Varian Mercury Plus300 spectrometer equipped with 5-mm probes. Samples weredissolved in 650 µl of CD3OD. The 1H and 13C-NMR spectra (at 300and 75 MHz respectively) were measured at 298 K. Chemical shiftswere reported in parts per million (ppm) and are referenced to the

H-8β

H-7β

H-7α

H-10H-1’

H-17

H-6α

H-11pro-R

H-11 pro-S

Figure 2. Geometry-optimized structure of MM+ low-energy conformations of chamaedryoside A (1). Selected NOEs are reported as dashed curves.

Magn. Reson. Chem. 2009, 47, 1007–1012 Copyright c© 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/mrc

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Table 1. 1H-, 13C- and 2D-NMR Data of chamaedryoside A (1) at 300/75 MHz, in CD3OD; δ in ppm

Position δH COSY H → H δC HMBC C → H NOESY H → H

1 α 1.46 m 1β ,2α,2β ,10 26.4 t 2,3 1β ,12

β 2.15 ov 1α,2α,2β ,10 1α,10,11 proS

2 α 1.74 m 1α,1β ,2β ,3α,3β 21.9 t 1,3 2β ,3α

β 1.59 m 1α,1β ,2α,3α,3β 2α,3β ,10

3 α 2.16 ov 2α,2β ,3β 28.6 t 1,2 2α,3β

β 2.36 m 2α,2β ,3α 2β ,3α

4 – – 134.5 s 3 –

5 – – 160.1 s 1,3 –

6 5.00 s 7α,7β 76.9 d 8 7α,7β , 1′

7 α 2.14 ov 7β ,6, 8 35.8 t 8,17 6,17

β 1.88 d (13.2) 7α,6, 8 8,17 6,8, 1′

8 2.25 ov 7α,7β , 17 33.9 d 7,11,17 7β ,10,11proR,17

9 – – 55.2 s 10,11,17

10 2.97 t (7.8) 1α, 1β 41.6 d 1,8, 11 1β , 2β , 8, 11proS

11 ProR 2.41 dd (13.8, 8.4) 11proS,12 42.0 t 8,10,12 8

Pros 2.63 dd (13.8, 9.0) 11proR,12 8,10,12 1β ,10,12

12 5.48 t (8.7) 11proS, 11proR 73.7 d 11 1α,11proS

13 – – 127.1 s 11,12,14,15 –

14 6.47 m 15 109.3 d 12,15,16 15

15 7.52 m 14 145.4 d 14,16 14

16 7.58 s 141.3 d 14,15

17 0.95 d (6.0) 8 17.5 q 7,8 7α,8,

18 – – 170.4 s 6′, 8,11 –

20 – – 180.0 s 8,10,11 –

1′ 4.45 d (7.8) 2′ 103.3 d 2′ , 3′ 6,7β , 2′

2′ 3.13 dd (8.7, 7.8) 1′,3′ 75.4 d 1′ , 3′ 1′ , 3′

3′ 3.35 ov 2′,4′ 78.3 d 2′ , 4′ 2′ , 4′

4′ 3.37 ov 3′,5′ 71.7 d 3′ , 5′ 3′ , 5′

5′ 3.33 ov 4′,6′ 77.6 d 4′ , 6′ 6a′,6b′

6 a′ 3.81 dd (12.1, 2.0) 5′ 62.7 t 5′ 4′,6b′

b′ 3.65 dd (12.1, 4.5) 5′ 5′ 4′ ,6a′

d = doublet; dd = doublet of doublet; m = multiplet; ov = overlapped; q = quartet; s = singlet; t = triplet. The J value in Hz are reported in thebrackets.

residual solvent peak of CD3OD at δ 3.31 and 49.0 ppm. Couplingconstants (J) were expressed in hertz.

The pulse conditions in CD3OD-d4 were as follows: for the 1HNMR spectrum, observation frequency = 300.03 MHz, acquisitiontime (AQ) = 1.998 s, number of scans (NS) = 256, relaxation delay(RD) = 1.0 s, 90◦ pulse width = 9.2 µs, spectral width (SW) =4506 Hz, number of data points (NP) = 18 000 and line broadening= 1.00 Hz. For the 13C NMR spectrum, observation frequency =75.450 MHz, AQ = 0.5 s, NS = 12 800, RD = 2.0 s, 90◦ pulse width= 10.2 µs, SW = 18 762 Hz, NP = 18 800 and line broadening= 1.0 Hz. For the DEPT-135 spectrum, observation frequency =75.450 MHz, AQ = 0.50 s, NS = 12 800, 90◦ pulse width = 10.2 µs,SW = 18 762Hz, NP = 18 800 and line broadening = 1.00 Hz. Forthe gradient 1H–1H COSY spectrum, AQ = 0.222 s, RD = 1.0 s, SW= 2306.8 Hz, number of points (NP) = 1024, number of increments(NI) = 256; for the NOESY spectrum, AQ = 0.461 s, RD = 1.00 s,SW = 2222.2 Hz, NP = 2048, NI = 256 and mixing time = 0.5 s; forthe TOCSY spectrum, AQ = 0.222 s, RD = 1.00 s, SW = 2306.8 Hz,NP = 2048, NI = 200 and mixing time = 0.09 s. The TOCSYand NOESY experiments were performed in the phase-sensitivemode. Proton-detected heteronuclear correlations were measuredusing a gradient heteronuclear single-quantum coherence (HSQC),

optimized for 1JHC = 140 Hz and a gradient HMBC, optimized fornJHC = 8 Hz. For the HSQC spectrum, AQ = 0.22 s, RD = 1.50 s, SW= 2306.8 (1H) and 12 820.5 (13C)Hz, NP = 1024, NI = 128 and FTsize = 1024 × 2048, and for the HMBC spectrum, the parameterswere very similar to those used in the HSQC experiments exceptthe NI = 400. For the HSQCTOCSY spectrum, AQ = 0.467 s, RD =1.00 s, SW = 2306.8 (1H) and 12 820.5 (13C)Hz, NP = 1024, NI =224, FT size = 1024 × 2048 and mixing time = 0.09 s.

The HSQC and HSQCTOCSY experiments were performed inthe phase sensitive mode with field gradient, while the HMBCexperiment was performed in the absolute value mode with fieldgradient.

Plant material

Plant materials of Teucrium chamaedrys (L.) were collected inthe vegetative state, in ‘Castel Volturno’ Nature Reserve, a flatcoastal area in the north of Naples (Southern Italy). The sampleswere carried to the laboratory, separated as roots and leaves,dried in a ventilated thermostat at 40 ◦C, and stored at −20 ◦Cuntil extraction. A voucher specimen (T. chamaedrys CE0037) wasdeposited in the Herbarium of the Dipartimento di Scienze dellaVita of Second University of Naples.

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Structure determination of chamaedryosides A – C

Table 2. 1H-, 13C- and 2D NMR Data of Chamaedryosides B and C mixture at 300/75 MHz, in CD3OD; δ in ppm

Position δH COSY (H → H) δC HMBC (C → H) NOESY (H → H)

1 α 1.49 ov 1β ,2α,2β ,10 26.4 t 2,3 1β ,12

β 2.17 ov 1α,2α,2β ,10 1α,11proS

2 α 1.79 m 1α,1β ,2β ,3α,3β 21.9 t 1,3 2β ,3α

β 1.51 ov 1α,1β ,2α,3α,3β 2α,3β ,10

3 α 2.23 m 2α,2β ,3β 28.1 t 1,2 2α,3β

β 2.32 ov 2α,2β ,3α 2β ,3α

4 – – 130.1 s 3 –

5 – – 160.0 s 1,3 –

6 5.11 s 7α,7β 66.7 d 8 7α,7β

7 α 2.12 ov 7β ,6, 8 37.7 t 8,17 6,17

β 1.71 d (11.4) 7α,6, 8 8,17 6,8, 1′

8 2.20 ov 7α,7β , 17 33.6 d 7,11,17 7β ,10,11proR,17

9 – – 55.8 s 10,11,17

10 2.97 t (7.8) 1α, 1β 41.6 d 1,8, 11 1β , 2β , 8, 11proS

11 ProR 2.45 dd (13.8, 8.4) 11proS,12 41.7 t 8,10,12 8

Pros 2.66 dd (13.8, 9.0) 11proR,12 8,10,12 1β ,12

12 5.49 t (8.7) 11proS, 11proR 73.8 d 11 1α,11proS

13 – – 126.9 s 11,12,14,15 –

14 6.48 m 15 109.3 d 12,15,16 15

15 7.53 m 14 145.4 d 14,16 14

16 7.59 s – 141.3 d 14,15 –

17 0.95 d (6.0) 8 17.5 q 7,8 7α,7β ,8,

18 – – 170.4 s 6′ , 8,11 –

20 – – 179.6 s 8,10,11 –

1′ 5.08 d (3.6) 4.48 d (7.8) 2′ 94.0 d 98.3 d 2′, 3′ 6,7β , 2′

2′ 3.36 dd (8.7, 7.8) 3.13 dd (8.7, 7.8) 1′,3′ 73.9 d 75.4 d 1′, 3′ 1′, 3′

3′ 3.36 ov 3.36 ov 2′,4′ 74.8 d 78.0 d 2′, 4′ 2′, 4′

4′ 3.99 ov 3.38 ov 3′,5′ 71.7 d 70.7 d 3′, 5′ 3′, 5′

5′ 3.35 ov 3.33 ov 4′,6′ 72.0 d 76.2 d 4′, 6′ 6a′ ,6b′

6 a′ 4.26 dd (12.3, 2.0) 5′ 64.8 t 5′ 4′ ,6b′

b′ 4.47 dd (12.3, 4.5) 5′ 5′ 4′ ,6a′

Extraction and isolation of compounds

Dried leaves of T. chamaedrys (761.3 g) were powdered andextracted in methanol at 4 ◦C in a dark refrigerated room. After5 days, the solution was filtered on Whatman paper, dried withNa2SO4 and concentrated under vacuum, yielding 108.0 g of cruderesidue, which was stored at −80 ◦C until purification. The crudemethanol extract was fractionated by liquid-liquid extraction. Theobtained aqueous fraction was chromatographed on AmberliteXAD-4 eluting first with H2O and then with MeOH. The leaf organicfraction (27.0 g) from Amberlite XAD-4 was chromatographedby flash chromatography on SiO2 eluting with the lower phaseof a biphasic solution CHCl3/MeOH/H2O (13 : 7 : 2) to obtain afraction which was chromatographed by RP-18 CC eluting withH2O/MeOH/MeCN (3 : 1 : 1). The eluate was purified by RP-18 HPLCusing as mobile phase H2O/MeOH/MeCN (4 : 5 : 1) to obtain thepure compound 1 (87.0 mg).

Dried T. chamaedrys roots (300.2 g) were powdered andextracted in MeOH for 5 days at 4 ◦C in a dark refrigerated room.After solvent removal by Rotavapor, the methanol crude extract(20.0 g) was dissolved in water and then shaken by liquid–liquidextraction using EtOAc as extracting solvent, obtaining an organicand an aqueous fraction. The last one was chromatographed onAmberlite XAD-4 eluting first with H2O and then with MeOH.Fractions, eluted with MeOH, were chromatographed by FCC

on SiO2 eluting with the organic phase of a CHCl3/MeOH/H2O(13 : 9 : 5) to obtain a fraction that, on purification by RP-18 HPLCusing as mobile phase H2O/MeOH/MeCN (4 : 5 : 1), furnished aninseparable mixture of metabolites 2 and 3 (12 mg).

Chamaedryoside A (1)

Colorless oil; C25H32O11; [α]D25 = +30.3◦ (c = 0.56, MeOH). IR

νmax cm−1 3392, 2935, 1704, 1704, 1515, 874. UV λmax nm: 208.1(3.91), 223.2 (3.27). 1H- and 13C-NMR (300 and 75 MHz respectively,CD3OD): Table 1. ESI/MS: m/z 531 [M+Na]+ , m/z 547 [M+K]+ andm/z 507 [M−H]− .

Chamaedryosides B and C (2–3)

Colorless oil; C25H32O11; [α]D25 = +61.6◦ (c = 0.38, MeOH). IR

νmax cm−1 3392, 2929, 1751, 875. UV λmax nm: 203 (3.95), 230(3.24); 1H- and 13C-NMR (300 and 75 MHz respectively, CD3OD):Table 2. ESI/MS: m/z 531 [M+Na]+ , m/z 547 [M+K]+ and m/z 507[M−H]− .

Acetylation of chamaedryosides B and C mixture

Aliquot of chamaedryosides B and C mixture (4 mg) was stirred in a1 : 1 mixture (1 ml) of acetic anhydride and dry pyridine overnight.

Magn. Reson. Chem. 2009, 47, 1007–1012 Copyright c© 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/mrc

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A. Fiorentino et al.

Successively, the mixture was treated with toluene and methanoland dried under nitrogen stream. The residue was dissolved inCDCl3 and analyzed by NMR. 1H-NMR (CDCl3, 300 MHz): δ 7.46(1H, brs, H-16) 7.44 (1H, m, H-15), 6.41 (1H, brs, H-14), 6.35 (1H, d,J = 3.3 Hz, H-1′ α-Glc), 6.23 (1H, m, H-6), 5.75 (1H, d, J = 7.8 Hz,H-1′ β-Glc), 5.47 (1H, t, J = 9.3 Hz, H-12), 4.27 (2H, ov, H-6′), 2.11-2.00 (15H, ov, CH3CO), 0.98 (3H, d, J = 6.6 Hz, H-17). 13C-NMR(CDCl3, 75 MHz): δ 171-169 (C O), 160.4 (C-5), 144.3 (C-15), 139.8(C-14), 131.1 (C-4), 129.0 (C-13), 108.3 (C-14), 91.9 (C-1′ β-Glc), 89.2(C-1′ α-Glc), 73.1 (C-12), 73.0-68.4 (glucidic carbons), 62.6 (C-6′),54.1 (C-9), 41.7 (C-11), 41.1 (C-10), 34.2 (C-8), 26.8 (C-3), 25.4 (C-1),24.0 (C-2), 21-5-20.6 (acetyl carbons), 17.1 (C-17).

Acknowledgement

The authors wish to thank Dr Agostino Russo of Department ofEnvironmental Sciences (SUN) for IR measurements.

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www.interscience.wiley.com/journal/mrc Copyright c© 2009 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2009, 47, 1007–1012