Fitoterapia 80 (2009) 404–407

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Fitoterapia

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New clerodane diterpenoids from Casearia sylvestris

Wei Wang a, Zulfiqar Ali a, Xing-Cong Li a, Troy A. Smillie a, De-An Guo c, Ikhlas A. Khan a,b,⁎a National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, Oxford, MS 38677, USAb Department of Pharmacognosy, School of Pharmacy, University of Mississippi, Oxford, MS 38677, USAc Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, CAS, Shanghai 201203, PR China

a r t i c l e i n f o

⁎ Corresponding author. Tel.: +1 662 915 7821; faxE-mail address: ikhan@olemiss.edu (I.A. Khan).

0367-326X/$ – see front matter © 2009 Elsevier B.V.doi:10.1016/j.fitote.2009.05.013

a b s t r a c t

Article history:Received 4 March 2009Accepted in revised form 14 May 2009Available online 22 May 2009

Two new clerodane diterpenoids 2β-methoxy-cleroda-3,13-dien-18-carboxy-15,16-olide (1)and 15ξ-methoxy-cleroda-3,12-dien-18-carboxy-15,16-olide (2) and one new nitrogen-containing clerodane diterpenoid 15-oxo-echinophyllin A (3), along with six knowncompounds, namely, echinophyllin A, (−)-patagonic acid, tyrosol, oplopanone, 4-hydroxy-4-methyl-2-[(1R)-1-(1-methylethyl)-4-oxopentyl]-2-cyclohexen-1-one and 1β,6α-dihydroxy-eudesman-4(15)-ene were isolated from the leaves of Casearia sylvestris. Their structureswere determined by extensive NMR techniques.

© 2009 Elsevier B.V. All rights reserved.

Keywords:FlacourtiaceaeCasearia sylvestrisClerodane diterpenoid

1. Introduction

Casearia sylvestris (Flacourtiaceae) is a Brazilian andParaguayan folk medicinal plant called as “Guaçatonga” or“Chá de Bugre” and used to treat snakebite, trauma, ulcera-tion, obesity, and cough [1–5]. A number of clerodanediterpenes were reported from the leaves of C. sylvestris,some of which possess antitumoral, trypanocidal and DNA-modifying bioactivities [6–12]. In continuation to search fornew chemical markers of the dietary supplement “Chá deBugre” has led to the identification of two new clerodanediterpenoids 2β-methoxy-cleroda-3,13-dien-18-carboxy-15,16-olide (1) and 15ξ-methoxy-cleroda-3,12-dien-18-car-boxy-15,16-olide (2) and one new nitrogen-containingclerodane diterpenoid 15-oxo-echinophyllin A (3) (Fig. 1),along with six known compounds, namely, echinophyllin A(4) [13], (−)-patagonic acid (5) [14], tyrosol (6) [15],oplopanone [16,17], 4-hydroxy-4-methyl-2-[(1R)-1-(1-methylethyl)-4-oxopentyl]-2-cyclohexen-1-one (1,10-seco-4ξ-hydroxy-muurol-5-ene-1,10-diketone) [18] and 1β,6α-dihydroxy-eudesman-4(15)-ene [19,20]. Herein, we reportthe isolation and structure elucidation of these three newclerodane diterpenoids. Their structures were identified onthe basis of extensive NMR experiments.

: +1 662 915 7989.

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2. Experimental

2.1. General

Optical rotation: Rudolph Research AutoPol IV. UV:Hewlett-Packard 8453. IR: Bruker Tensor 27 FT-IR andMIRacle ATRFT-IR. HRESIMS: Agilent Series 1100 SL. NMRspectra: American Varian Mercury plus 400 (1H 400 MHz, 13C100 MHz). HPLC: Waters LC Module I. HPLC column:Phenomenex Gemini C18 5 µm ODS column (10×250 mm).

2.2. Plant material

The leaves of C. sylvestris (Chá de Bugre) were purchasedfrom Raintree Nutrition Inc (Carson City, NV 89701, USA), andwere identified by TLC and HPLC analyses with the authenti-cated sample offered by Dr. Rainer W. Bussmann (MissouriBotanical garden). Voucher specimens (#3247 and 3812)were deposited at the National Center for Natural ProductsResearch, Research Institute of Pharmaceutical Sciences,University of Mississippi, USA.

2.3. Extraction and isolation

The air-dried and powdered leaves of C. sylvestris (3 kg) wereextracted by percolationwithMeOH (4×4 L). The pooledMeOHsolutionwas evaporated in vacuo to give a residue (342 g; 11.3%).

Fig. 1. Structures of 1–6.

405W. Wang et al. / Fitoterapia 80 (2009) 404–407

The MeOH extract was partitioned between H2O and petroleumether and then betweenH2O andEtOAc. The EtOAc layer affordeda waxy residue (207 g), which was further separated into 9fractions by column chromatography on silica gel (2500 g,120×8 cm)with gradient elution of petroleum ether–EtOAc andCHCl3–MeOH. Fraction 1 (118 mg) was chromatographed over asilica gel column (10 g, 60×2.5 cm), which was eluted withpetroleum CHCl3–Me2O (15:1, 0.75 L) to afford oplopanone(35.1 mg). 4-hydroxy-4-methyl-2-[(1R)-1-(1-methylethyl)-4-oxopentyl]-2-cyclohexen-1-one (6.0 mg) and 6 (15.6 mg) wereobtained from rechromatography of fraction 3 by silica gelcolumn(80g, 60×6cm)withpetroleumether–EtOAc (1:4,1.5 L).The residue (2.0 g) of fraction 4 was subjected to silica gelchromatography (80 g, 60×6 cm) using CHCl3–(CH3)2CO (20:1,15:1 and 10:1 1.5 L each eluent) to yield 6 subfractions.Subfraction 5 (31.4 mg) was chromatographed over a Phenom-enex Gemini C18 5 µm ODS column (10×250 mm, flow rate5.0 mL/min) by preparative HPLC with MeOH–H2O (60:40) asmobile phase to obtain compounds 1 (2.8 mg, tR=6.6 min), 4(5.5 mg, tR=7.8 min) and 3 (2.0 mg, tR=12.1 min) respectively.The subfraction6 (29.8mg)was chromatographedover the sameODS column (flow rate 6.0mL/min)withMeOH–H2O (58:42) asmobile phase to yield compounds 5 (7.9 mg, tR=9.1 min) and 2(2.0 mg, tR=10.4 min) respectively. 1β,6α-dihydroxy-eudes-man-4(15)-ene (10.2 mg) was obtained from rechromatographyof fraction 6 by silica gel column (80 g, 60×6 cm) with EtOAc–MeOH (4:1, 1.5 L).

2β-methoxy-cleroda-3,13-dien-18-carboxy-15,16-olide (1):Colorless gum; [α]: −23.2 (c 0.14, MeOH); UV, λmax

(MeOH) nm (logε): 251 (3.44); IR (KBr), νmax cm−1: 2932,1754, 1711, 1551, 1452, 1382, 1227, 1081, 829; HR-ESI-MS m/z385.1996 [M+Na]+ (calcd for C21H30O5Na, 385.1991); For 1Hand 13C NMR data: see Table 1.

15ξ-methoxy-cleroda-3,12-dien-18-carboxy-15,16-olide (2):Colorless gum; [α]: −56.0 (c 0.10, MeOH); UV, λmax (MeOH)nm (logε): 224 (3.87), 251 (3.47); IR (KBr), νmax cm−1: 2958,1762, 1682, 1458, 1385, 1190, 1115, 1015, 1030, 941 cm−1; HR-ESI-MS m/z 385.1988 [M+Na]+ (calcd for C21H30O5Na,385.1991); For 1H and 13C NMR data: see Table 1.

15-oxo-echinophyllin A (3): Yellow gum; [α]:−10.0 (c 0.10,MeOH); UV, λmax (MeOH) nm (logε): 232 (4.03), 251 (3.62),

278 (3.22); IR (KBr), νmax cm−1: 2930, 2870, 1706, 1679, 1631,1551, 1537, 1451, 1383, 1308, 1228, 1171, 994 cm−1; HR-ESI-MSm/z 488.2419 [M+Na]+ (calcd for C28H35NO5Na, 488.2413);For 1H and 13C NMR data: see Table 1.

3. Results and discussion

Compound 1was isolated as colorless gum and HR-ESI-MSestablished the molecular formula as C21H30O5 (positive m/z=385.1996 [M+Na]+ calcd. for C21H30O5Na: 385.1991). The1H NMR spectrum (Table 1) showed two quaternary methyl(δH 0.76 and 1.48), one secondary methyl (δH 0.79, d,J=6.0 Hz) and one methoxy group (δH 3.41, s). The 13CNMR (Table 1) and DEPT spectra indicated the presence of 21carbons, consisting of two carboxyl (δC 175.0 and 170.5), fourolefinic (δC 147.0, 145.4, 134.8 and 132.5), one oxygenatedmethine (δC 74.1), one oxygenated methylene (δC 71.0) andonemethoxy group (δC 56.9) in the low-field region and threemethyl, five methylene, two methine and two quaternarycarbons in the high-field region. These data are consistentwith the HR-MS empirical formula and similar to those of(−)-patagonic acid (5) [14], the major difference is anadditional resonance of methoxy group (δH 3.41, δC 56.9)that appeared in 1. The methoxy group was assigned at C-2based on the HMBC correlations (Fig. 2) of H-2 (δH 3.80, t,J=4.4 Hz) to the carbon of methoxy group, C-3 (δC 132.5), C-4(δC 147.0) and C-10 (δC 42.6) respectively. 1H–1H COSYcorrelations (Fig. 2) of H-2 to H-3 (δH 6.99, d, J=4.0 Hz)and H-1b (δH 1.97, br d, J=14.0 Hz) also supported methoxygroup at C-2. In the ROESY experiment, H-2 (δH 3.80, t,J=4.4 Hz) correlated with H-3 (δH 6.99) but had nocorrelation with H-10 (δH 1.75, d, J=12.8 Hz), indicatingthat H-2 should have α-orientation. The trans-AB ringjunction was confirmed by the chemical shift of C-19 angularmethyl (δC 19.5) which is 10 ppm lower than that of cis-clerodanes [14,21,22]. Thus, 1 was characterized as 2β-methoxy-cleroda-3,13-dien-18-carboxy-15,16-olide.

Compound 2 was isolated as colorless gum and has thesame molecular formula C21H30O5 as 1 calculated from HR-ESI-MS (positive m/z=385.1988 [M+Na]+ calcd. forC21H30O5Na: 385.1991). Similarities in the 1H and 13C

Table 11H (400 MHz) and 13C (100 MHz) NMR data for 1–4.

No. 1 2 3 4

δH (mult) δC (mult) δH (mult) δC (mult) δH (mult) δC (mult) δH (mult) δC (mult)

1 1.59 (1H, m) 23.9 (t) 1.54 (1H, m) 18.5 (t) 1.48 (1H, m) 18.1 (t) 1.46 (1H, m) 18.2 (t)1.97 (1H, br d, J 14.0) 1.64 (1H, m) 1.70 (1H, m) 1.72 (1H, m)

2 3.80 (1H, t, 4.4) 74.1 (d) 1.29 (1H, m) 27.5 (t) 1.32 (1H, m) 27.7 (t) 1.36 (1H, m) 27.8 (t)1.32 (1H, m) 1.35 (1H, m) 1.38 (1H, m)

3 6.99 (1H, d, J 4.0) 132.5 (d) 6.91 (1H, br s) 137.0 (d) 6.96 (1H, t, J 4.0) 137.1 (d) 6.98 (1H, t, J 4.0) 137.2 (d)4 147.0 (s) 143.4 (s) 144.0 (s) 143.8 (s)5 38.9 (d) 38.3 (d) 38.4 (d) 38.4 (d)6 1.33 (1H, m) 36.3 (t) 1.31 (1H, m) 36.0 (t) 1.30 (1H, m) 36.7 (t) 1.30 (1H, m) 37.0 (t)

2.67 (1H, br d, J 12.0) 2.77 (1H, br d, J 13.6) 2.83 (1H, br d, J 12.8) 2.85 (1H, br d, J 12.0)7 1.33 (1H, m) 27.8 (t) 1.30 (1H, m) 27.7 (t) 1.33 (1H, m) 27.9 (t) 1.35 (1H, m) 28.1 (t)

2.16 (1H, m) 2.21 (1H, m) 2.20 (1H, m) 2.19 (1H, m)8 1.51 (1H, m) 36.8 (d) 1.32 (1H, m) 38.1 (d) 1.53 (1H, m) 36.9 (d) 1.54 (1H, m) 36.9 (d)9 39.0 (s) 41.4 (s) 40.1 (s) 39.3 (s)10 1.75 (1H, d, J 12.8) 42.6 (d) 1.29 (1H, m) 49.1 (d) 1.46 (1H, m) 47.4 (d) 1.46 (1H, m) 47.5 (d)11 1.65 (1H, m) 36.4 (t) 2.13 (1H, m) 39.4 (t) 1.52 (1H, m) 36.4 (t) 1.59 (1H, m) 36.8 (t)

1.67 (1H, m) 2.22 (1H, m) 1.68 (1H, m) 1.70 (1H, m)12 2.18 (1H, m) 19.2 (t) 6.96 (1H, br s) 138.2 (d) 2.20 (1H, m) 19.8 (t) 2.18 (1H, m) 20.3 (t)

2.21 (1H, t, J 13.6) 2.31 (1H, t, J 14.0) 2.29 (1H, t, J 14.0)13 134.8 (s) 127.3 (s) 141.0 (s) 141.0 (s)14 7.17 (1H, s) 145.4 (d) 2.84 (1H, d, J 17.2) 33.3 (t) 6.54 (1H, s) 126.8 (d) 6.63 (1H, br s) 134.9 (d)

3.10 (1H, dd, J 17.2, 6.4)15 4.74 (2H, br s) 71.0 (t) 5.64 (1H, d, J 6.4) 103.2 (d) 171.6 (s) 3.75 (2H, br s) 51.7 (t)16 175.0 (s) 170.0 (s) 172.5 (s) 171.8 (s)17 0.79 (3H, d, J 6.0) 16.3 (q) 0.75 (3H, d, J 5.6) 16.6 (q) 0.79 (3H, d, J 6.0) 16.4 (q) 0.81 (3H, d, J 6.4) 16.5 (q)18 170.5 (s) 170.0 (s) 170.1 (s) 170.2 (s)19 1.48 (3H, s) 19.5 (q) 1.45 (3H, s) 20.9 (q) 1.48 (3H, s) 21.3 (q) 1.51 (3H, s) 21.4 (q)20 0.76 (3H, s) 18.6 (q) 0.73 (3H, s) 17.9 (q) 0.74 (3H, s) 18.7 (q) 0.75 (3H, s) 18.9 (q)MeO– 3.41 (3H, s) 56.9 (q) 3.46 (3H, s) 56.7 (q)1′ 3.82 (1H, t, J 7.6) 39.4 (t) 3.79 (1H, t, J 7.6) 44.8 (t)2′ 2.93 (1H, t, J 7.6) 34.4 (t) 2.89 (1H, t, J 7.6) 34.9 (t)3′ 129.3 (s) 130.2 (s)4′, 8′ 7.18 (2H, d, J 8.4) 130.7 (d) 7.22 (2H, d, J 8) 130.6 (d)5′, 7′ 7.12 (2H, d, J 8.4) 116.7 (d) 7.15 (2H, d, J 8) 116.7 (d)6′ 157.9 (s) 157.9 (s)

In pyridine-d5; δ in ppm, J in Hz.

406 W. Wang et al. / Fitoterapia 80 (2009) 404–407

spectroscopic data (Table 1) indicated that 2was correlated to1. The major difference between 1 and 2 was the positions ofone double bond and one methoxy group. The double bondwas assigned between C-12 and C-13 due to the HMBCcorrelations (Fig. 2) of H2-11 (δH 2.13, 2.22) to C-8 (δC 38.1),C-10 (δC 49.1), C-12 (δC 138.2) and C-13 (δC 127.3) and of H-12(δH 6.96) to C-13 (δC 127.3), C-14 (δC 33.3) and C-16 (δC

Fig. 2. Selected 1H–1H COSY and HM

170.0). The methoxy group was located at C-15 based onHMBC correlations of H-15 (δH 5.64, d, J=6.4 Hz) to MeO (δC56.7), C-14 (δC 33.3) and C-16 (δC 170.0). 1H–1H COSYcorrelations (Fig. 2) of H-15/H2-14 and H-12/H2-11 con-firmed the existence of a rare 3,12-diene structure. A transA/B ring junction was also evident from the chemical shift ofC-19 (δC 20.9). Although a ROESY correlation can be observed

BC correlations for 1 and 2.

Scheme 1. Possible biosynthetic pathway for 3.

407W. Wang et al. / Fitoterapia 80 (2009) 404–407

between MeO and H-14b (δH 3.10, dd, J=17.2, 6.4 Hz), theorientation of the methoxy group remains to be determined.Therefore 2 was assigned as 15ξ-methoxy-cleroda-3,12-dien-18-carboxy-15,16-olide.

Compound 3 was obtained as yellow gum and has themolecular formula C28H35NO5 as calculated from HR-ESI-MS(positive m/z=488.2419 [M+Na]+ calcd. for C28H35NO5Na:488.2413). The 1H NMR spectroscopic data (Table 1) showedresonances for a typical tricyclic clerodane diterpenoidskeleton as 1 and 2: two tertiary methyl (δH 0.74 and 1.48),a secondary methyl (δH 0.79, d, J=6.0 Hz), and two low-fieldolefinic protons at δH 6.54 and 6.96. The NMR spectroscopicdata (Table 1) were very similar to those of a nitrogen-containing clerodane diterpenoid echinophyllin A (4) [13],which was reported from Echinodorus macrophyllus but theprevious assignments for the C-4′, C-5′, C-7′ and C-8′ seemincorrect and need to be revised. This revision (Table 1) issupported by the corresponding data of echinophyllin D [23].When the 13C NMR spectroscopic data (Table 1) of 3 werecompared with those of 4, the resonance for an oxygenatedmethylene group (δC-15 51.7) was missing in 3 showinginstead of an oxo group (δC 171.6). HMBC correlations of H-1′(δH 3.82, t, J=7.6 Hz) to C-15 (δC 171.6) and C-16 (δC 172.5)confirmed that the oxo groupwas located at C-15. Thus, 3wascharacterized as 15-oxo-echinophyllin A. Possible biosyn-thetic pathway for 3 was considered (Scheme 1). 3 might bederived from 5 and tyramine, because 5 and 6 were found inC. sylvestris and 6 was derived from tyramine.

Acknowledgements

This work was supported by the United States Departmentof Agriculture Specific Cooperative Research AgreementNumber 58-6408-6-067 and the FDA/CFSAN grant entitled“Science Based Authentication of Dietary Supplements”

Number 2 U01 FD 002071-07. We thank Dr. Bharathi Avulafor conducting the HR-ESI-MS spectra and Mr. Frank Wiggersfor NMR assistance.

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