Antimicrobial Diterpenoids of Wedelia trilobata (L.) Hitchc

Continued interest in the metabolites of Wedelia trilobata (L.) Hitchc, a notoriously invasive weed in South China, led to the isolation of twenty-six ent-kaurane diterpenoids, including seven new ones 1–7. Their structures and relative configuration were elucidated on the basis of extensive spectroscopic analysis, including 1D- and 2D-NMR experiments. The antimicrobial activities of all isolated diterpenoids were evaluated against a panel of bacteria and fungi.


Introduction
Wedelia trilobata is a notoriously invasive weed in a wide range of tropical and subtropical areas [1]. In southern China, this creeping, matforming perennial herb has caused significant damage to farmlands, forests, and orchards [2,3]. Studies have shown that W. trilobata has a strong allelopathic potential on neighboring native plants [4,5]. The major chemical constituents of W. trilobata are ent-kaurane diterpenes, sesquiterpene lactones, and triterpenes with a variety of biological activities, such as antibacterial, antitumor, hepatoprotective, and central nervous system depressant properties [6]. We previously reported ten eudesmanolides isolated from this plant as potential inducers of plant systemic acquired resistance [7]. As continuation of that work, twenty-six ent-kaurane diterpenoids including seven new ones 1-7 were obtained from the whole plant W. trilobata ( Figure 1). All diterpenoids were evaluated against a panel of bacteria and fungi, and compounds 2, 4, 7, 10, 12, and 13 showed weak inhibitory activities against Monilia albicans with MICs of ca. 125 µg/mL. Herein, we report the isolation and structural elucidation of these compounds, as well as their antimicrobial properties. Apart from five carbon signals assigned to the angeloyloxy group (δC 167.9, 128.1, 138.6, 20.9, and 16.0), the 13 C-NMR (DEPT) spectrum of 1 (Table 1) also exhibited 20 carbons composed of three methyls, eight methylenes, four methines (one oxygenated), and five quaternary carbons, which were consistent with a skeleton of an ent-kauranoid [9]. In particular, the NMR spectroscopic features of 1 are similar to those of 8 (16α-hydroxy-ent-kauran-19-oic acid), and only differed in the appearance of an angeloyloxy group at C-3 in 1. It was also confirmed by the chemical shift value of C-3 (δC 78.9, CH), C-9 (δC 56.1, CH) and the HMBC correlations ( Figure 2) from H-3 (δH 4.50, dd, J = 12.2, 4.7 Hz) to C-1′ (δC 167.9, C), C-1 (δC 38.9, CH2), and C-18 (δC 24.7, CH3) as well as the correlations from Me-20, H-12, and H-15 to C-9, and from the methyl at C-4 (Me-18) to a downfield quaternary carbon (C-19) at δC 178.1. The ROESY correlations of H-3 with H-5 and H3-18 suggested that the angeloyloxy was α-orientated, and the hydroxy at C-16 was also assigned as α-orientated by the ROESY correlations of H3-17 with H2-11 and H-14β along with the ROESY correlations of H3-20 with H2-15. Consequently, the structure of 1 was finally determined as 3α-angeloyloxy-16α-hydroxy-ent-kauran-19-oic acid. Compound 2 had the molecular formula C25H38O6 as determined by the HREIMS, with 16 mass units more than 1. The 1 H-and 13 C-NMR data similarities between 2 and 1 (Tables 1 and 2) suggested that they were structural analogues. As compared with compound 1, the main differences were due to the presence of a hydroxymethyl group (δC 66.8) and the absence of a methyl group in 2. The hydroxymethyl group was assigned to C-17 by the HMBC correlations of H2-17 to C-14, C-15, and C-16. Therefore, the structure of 2 was established as shown.

Structure Elucidation of Compounds
Compound 1 was obtained as a white amorphous powder, with a molecular formula determined as C 25 H 38 O 5 on the basis of HREIMS which indicated a molecular ion peak at m/z 418.2722 M + (calcd. for C 25 H 38 O 5 , 418,2719). The IR spectrum revealed absorption bands of hydroxyl (3431 cm´1) and carbonyl (1711 cm´1) groups. In the 1 H-NMR spectrum (Table 1), the downfield olefinic proton at δ H 6.02 (1H, q, J = 7.0 Hz) and two methyl signals at δ H 1.82 (3H, s) and 1.92 (3H, d, J = 7.0 Hz), indicated the presence of an angeloyloxy group in 1 [8].
C-1′ (δC 167.9, C), C-1 (δC 38.9, CH2), and C-18 (δC 24.7, CH3) as well as the correlations from Me-20, H-12, and H-15 to C-9, and from the methyl at C-4 (Me-18) to a downfield quaternary carbon (C-19) at δC 178.1. The ROESY correlations of H-3 with H-5 and H3-18 suggested that the angeloyloxy was α-orientated, and the hydroxy at C-16 was also assigned as α-orientated by the ROESY correlations of H3-17 with H2-11 and H-14β along with the ROESY correlations of H3-20 with H2-15. Consequently, the structure of 1 was finally determined as 3α-angeloyloxy-16α-hydroxy-ent-kauran-19-oic acid. Compound 2 had the molecular formula C25H38O6 as determined by the HREIMS, with 16 mass units more than 1. The 1 H-and 13 C-NMR data similarities between 2 and 1 (Tables 1 and 2) suggested that they were structural analogues. As compared with compound 1, the main differences were due to the presence of a hydroxymethyl group (δC 66.8) and the absence of a methyl group in 2. The hydroxymethyl group was assigned to C-17 by the HMBC correlations of H2-17 to C-14, C-15, and C-16. Therefore, the structure of 2 was established as shown. Compound 2 had the molecular formula C 25 H 38 O 6 as determined by the HREIMS, with 16 mass units more than 1. The 1 H-and 13 C-NMR data similarities between 2 and 1 (Tables 1 and 2) suggested that they were structural analogues. As compared with compound 1, the main differences were due to the presence of a hydroxymethyl group (δ C 66.8) and the absence of a methyl group in 2. The hydroxymethyl group was assigned to C-17 by the HMBC correlations of H 2 -17 to C-14, C-15, and C-16. Therefore, the structure of 2 was established as shown.   4). The NMR data suggested that compounds 3 and 4 possessed the same relative configuration as those of 1 and 2, respectively. Thus, compounds 3 and 4 were determined as 3α-tigloyloxy-16α-hydroxy-ent-kauran-19-oic acid and 3α-tigloyloxy-16α, 17-dihydroxy-ent-kauran-19-oic acid, respectively.
Compound 5, a white powder, possessed the molecular formula C 29 H 38 O 4 , as determined by the HREIMS, 13 C-NMR (Table 2) and DEPT data. Comparison of the 1D-and 2D-NMR spectroscopic data of 5 with those of 3α-cinnamoyloxy-ent-kaur-16-en-19-oic acid (15) revealed that their structures were closely similar to each other. The only difference between them was that the double bond of the cinnamoyloxy group at C-3 in 15 was reduced in 5, which was supported by the molecular weights of 5, showing two mass units more than those of 15. This was further confirmed by the HMBC cross-peaks of H-2 1 and H-3 1 with C-1 1 and C-4 1 . The α-orientation of the 3-dihydrocinnamoyloxy group was apparent from the ROESY correlations of H-3β with H-5β and H 3 -18β. Thus, compound 5 was determined as 3α-dihydrocinnamoyloxy-ent-kaur-16-en-19-oic acid.
Compound 7 was isolated as a white powder, and its molecular formula was determined as Further analyses demonstrated that compound 7 showed a closely similar NMR pattern to that of 6, indicating that compound 7 was a structural analogue of ent-kaurane-19-oic acid. The double bond was located between C-15 and C-16 by the HMBC cross-peaks of H-15 with C-8, C-14, C-16 and C-17. Meanwhile, the O-bearing methylene group was only connected to C-17 by the HMBC correlations from H 2 -17 to C-14, C-15, and C-16. At last, the O-bearing quaternary carbon could be attributed to C-9 due to the HMBC correlations of H 2 -7, H 2 -11, and H 3 -20 to C-9. The relative configuration of 7 was shown to be identical with that of 6 by NMR analysis. Thus, compound 7 was determined as 3α-cinnamoyloxy-9β, 17-dihydroxy-ent-kaur-15-en-19-oic acid.

Evaluation of Anti-Micobial Activity
In summary, seven new and nineteen known ent-kaurane diterpenoid metabolites were obtained from whole plant W. trilobata, and some compounds exhibited weak antimicrobial activities. Moreover, we previously reported ten eudesmanolides as potential inducers of plant systemic acquired resistance isolated from this species [7]. Above all, a conclusion that can be drawn is that diterpenes and sesquiterpenes are the main metabolites of W. trilobata and they may be significant as chemical defenses allowing this notoriously invasive weed to adapt to varying surroundings rapidly and effectively ( Figure 3).

Evaluation of Anti-Micobial Activity
In summary, seven new and nineteen known ent-kaurane diterpenoid metabolites were obtained from whole plant W. trilobata, and some compounds exhibited weak antimicrobial activities. Moreover, we previously reported ten eudesmanolides as potential inducers of plant systemic acquired resistance isolated from this species [7]. Above all, a conclusion that can be drawn is that diterpenes and sesquiterpenes are the main metabolites of W. trilobata and they may be significant as chemical defenses allowing this notoriously invasive weed to adapt to varying surroundings rapidly and effectively ( Figure 3).

General Procedures
1D-and 2D-NMR spectra were recorder on either an AM-400 or a DRX-500 or an Avance III-600 spectrometer (Bruker, Karlsruhe, Germany) with TMS as an internal standard. Unless otherwise specified, chemical shifts (δ) were expressed in ppm. MS were measured on a HPLC-Thermo Finnigan LCQ Advantage ion trap mass spectrometer (Waters, Milford, PA, USA). Optical rotation was determined on a SEPA-300 polarimeter (Horiba, Tokyo, Japan). UV spectroscopic data were measured on a 210A double-beam spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra of samples in KBr discs were recorded on a Tensor-27 spectrometer with KBr pellets (Bruker, Rheinstetten, Germany). Column chromatography (CC) was carried out on silica gel G (

Plant Material
The whole plant of Wedelia trilobata (L.) Hitchc was collected in Simao, Yunnan Province, China, in August 2011. The specimen was identified by Yu Chen of Kunming Institute of Botany (KIB), Chinese Academy of Sciences (CAS). A voucher specimen (H20110805) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany.