Novel Diterpenoids from the Twigs of Podocarpus nagi

Phytochemical investigation of the twigs of Podocarpus nagi (Podocarpaceae) led to the isolation of two new abietane-type diterpenoids, named 1β,16-dihydroxylambertic acid (1) and 3β,16-dihydroxylambertic acid (2), along with two new ent-pimarane-type diterpenoids, named ent-2β,15,16,18-tetrahydroxypimar-8(14)-ene (3) and ent-15-oxo-2β,16,18-trihydroxypimar-8(14)-ene (4). Their respective structures were elucidated on the basis of spectroscopic analyses, including 1D- and 2D-NMR, IR, CD, and HR-ESI-MS. This is the first time ent-pimarane-type diterpenoids from the genus Podocarpus has been reported. All four new compounds were tested for cytotoxic activity. The MTT assay results showed that compounds 3 and 4 significantly inhibited the proliferation of human cervical cancer Hela cells, human lung cancer A549 cells, and human breast cancer MCF-7 cells at a concentration of 10 μM. Furthermore, using the lipopolysaccharide (LPS)-stimulated RAW264.7 cells, compounds 2 and 4 were found to significantly inhibit nitrogen oxide (NO) production with IC50 values of 26.5 ± 6.1 and 17.1 ± 1.5 μM, respectively.


Introduction
Podocarpus nagi (Thunb) Zoll. et Mor ex Zoll (Podocarpaceae) is one of the most ancient Gymnosperms widely distributed in East Asia and the Southern Hemisphere [1]. As a traditional herbal medicine, the leaves and roots of P. nagi have been used for the treatment of rheumatism and arthritis, as well as venereal diseases [2]. A number of diterpenoids [3][4][5][6] and flavonoids [7] have been identified from this plant in previous investigations, and some of them exhibited cytotoxic [4,[7][8][9], insecticidal [10], and antifungal [3] properties. Previously, we identified two cyclopeptides from the stem barks of P. nagi [11], and found that nagilactone B, a major diterpenoid from this plant, suppressed atherosclerosis in apoE deficient mice [12]. In our ongoing research, two new abietane-type diterpenoids and two new ent-pimarane-type diterpenoids were identified from the twigs of P. nagi. and arthritis, as well as venereal diseases [2]. A number of diterpenoids [3][4][5][6] and flavonoids [7] have been identified from this plant in previous investigations, and some of them exhibited cytotoxic [4,[7][8][9], insecticidal [10], and antifungal [3] properties. Previously, we identified two cyclopeptides from the stem barks of P. nagi [11], and found that nagilactone B, a major diterpenoid from this plant, suppressed atherosclerosis in apoE deficient mice [12]. In our ongoing research, two new abietane-type diterpenoids and two new ent-pimarane-type diterpenoids were identified from the twigs of P. nagi. (Figure 1). Herein, details of the isolation and structure elucidation of these compounds, as well as their cytotoxic and anti-inflammatory activities are described.

Results
Compound 1 was obtained as a white amorphous powder. The molecular composition of 1, C20H28O5, was deduced from the positive ion peak at m/z 349. 2001 Figure S3). The 1 H-and 13 C-NMR data of 1 were quite similar to those of the aglycone of 19-O-Dglucopyranoside of 16-hydroxylambertic acid except that a methylene group in ring A of the aglycone of 19-O-D-glucopyranoside of 16-hydroxylambertic acid was replaced by an oxymethine group in compound 1, as well as the down-field shift of an aromatic acid (8.05 in 1; 6.54 in 19-O-D-glucopyranoside of 16-hydroxylambertic acid) [13].
The structure of 1 was constructed by the detailed analysis of the HSQC and HMBC spectra ( Figure 2 and Supplementary Figure S4 and S5). The correlations from H-11 to C-10, from H-14 to C-7, from H-15 to C-12, C-13, and from H2-16 to C-13 manifested a hydroxyl group on C-12, as well as a hydroxyl-substituted isopropyl substituent on C-13. Furthermore, the HMBC crosspeaks from H-1 to C-9 and C-20 suggested a hydroxyl group was substituted on C-1. The downfield shift of H-11 also supported a hydroxyl group was substituted on C-1 [14,15]. In addition, the correlated signals of H3-18 and H-5 to C-19 confirmed the carboxyl group on C-19. The relative configuration of 1 was subsequently deduced from the NOESY experiment. The hydroxyl group on C-1 was assigned as β-oriented based on the correlations from H3-18 to H-1 and H-5, as well as H-5 to H-1 ( Figure 3 and Supplementary Figure S6). The absolute configuration of

Results
Compound 1 was obtained as a white amorphous powder. The molecular composition of 1,   [13].
The structure of 1 was constructed by the detailed analysis of the HSQC and HMBC spectra ( Figure 2 and Supplementary Figure S4 and S5). The correlations from H-11 to C-10, from H-14 to C-7, from H-15 to C-12, C-13, and from H 2 -16 to C-13 manifested a hydroxyl group on C-12, as well as a hydroxyl-substituted isopropyl substituent on C-13. Furthermore, the HMBC cross-peaks from H-1 to C-9 and C-20 suggested a hydroxyl group was substituted on C-1. The down-field shift of H-11 also supported a hydroxyl group was substituted on C-1 [14,15]. In addition, the correlated signals of H 3 -18 and H-5 to C-19 confirmed the carboxyl group on C-19. The relative configuration of 1 was subsequently deduced from the NOESY experiment. The hydroxyl group on C-1 was assigned as β-oriented based on the correlations from H 3 -18 to H-1 and H-5, as well as H-5 to H-1 ( Figure 3 and Supplementary Figure S6). The absolute configuration of compound 1 was determined by CD spectrum. The Cotton effect of compound 1 (∆ε 211 4.14, ∆ε 227 3.91, ∆ε 265 −2.99) was consistent with that of ferruginol (∆ε 211 4.11, ∆ε 227 3.97, and ∆ε 265 −2.95), indicating a 10S configuration [16]. Thus, the structure of 1 was established as shown in Figure 1, and it was named 1β,16-dihydroxylambertic acid.
Compound 2 was isolated as a white amorphous powder. The molecular formula was determined to be C 20 Figure 2). The NOE cross-peaks from H 3 -18 to H-3 and H-5, and H-5 to H-3 indicated the hydroxyl group was β-oriented ( Figure 3). Moreover, the identical Cotton effect of compounds 2 and 1 suggested they shared the same absolute configuration at C-10. Thus, the structure of 2 was determined as shown in Figure 1, and it was named 3β,16-dihydroxylambertic acid.  [16]. Thus, the structure of 1 was established as shown in Figure 1, and it was named 1β,16-dihydroxylambertic acid. Compound 2 was isolated as a white amorphous powder. The molecular formula was determined to be C20H28O5 from the ion peak at m/z 349.2004 [M + H] + (calcd. 349.2010) in its HR-ESI-MS. The IR spectrum of 2 displayed the absorptions for hydroxyl groups (3530 and 3385 cm −1 ), an aromatic ring (1616 and 1383 cm −1 ), and a carboxyl group (1688 cm −1 ). The 1 H-and 13 C-NMR spectra of 2 were quite similar to those of the aglycone of the 19-O-D-glucopyranoside of 16-hydroxylambertic acid except that a methylene group in ring A of the aglycone of the 19-O-Dglucopyranoside of 16-hydroxylambertic acid was replaced by an oxymethine group in compound 2, which inferred a hydroxyl group might substitute at ring A [13]. The hydroxyl group was assigned on C-3 based on the HMBC correlations from H-3 to C-5 and C-18 ( Figure 2). The NOE cross-peaks from H3-18 to H-3 and H-5, and H-5 to H-3 indicated the hydroxyl group was β-oriented ( Figure 3). Moreover, the identical Cotton effect of compounds 2 and 1 suggested they shared the same absolute configuration at C-10. Thus, the structure of 2 was determined as shown in Figure 1, and it was named 3β,16-dihydroxylambertic acid.    [16]. Thus, the structure of 1 was established as shown in Figure 1, and it was named 1β,16-dihydroxylambertic acid.  [13]. The hydroxyl group was assigned on C-3 based on the HMBC correlations from H-3 to C-5 and C-18 ( Figure 2). The NOE cross-peaks from H3-18 to H-3 and H-5, and H-5 to H-3 indicated the hydroxyl group was β-oriented ( Figure 3). Moreover, the identical Cotton effect of compounds 2 and 1 suggested they shared the same absolute configuration at C-10. Thus, the structure of 2 was determined as shown in Figure 1, and it was named 3β,16-dihydroxylambertic acid.      Table 2). The 13 C-NMR spectrum revealed 20 carbon signals attributed to three methyl carbons, eight methylene carbons, four sp 3 methine carbons, three sp 3 quaternary carbons, one sp 2 methine carbon, and one sp 2 quaternary carbon ( Table 2). The 1 H-and 13 C-NMR data of 3 quite resembled to those of kirenol except for the chemical shifts of H 3 -18, C-18, and C-19 [17,18]. Detailed analysis of the 1 H-1 H COSY, HSQC, and HMBC spectra resulted in the construction of planar structure of 3 ( Figure 4). Subsequently, 2-hydroxyl group and H 3 -18 were assigned as β-oriented and α-oriented, respectively, by the correlations from H 3 -20 to H-2, and H-2 to H 3 -18 in the NOESY spectrum ( Figure 5). The correlations from H-5 to H-9, together with H-9 to H 3 -17 indeed confirmed the two protons and H 3 -17 were β-oriented. Thus, the structure of 3 was elucidated as ent-2β,15,16,18-tetrahydroxypimar-8(14)-ene (Figure 1).
Compound 4 was obtained as a white amorphous powder. The molecular formula was determined to be C20H28O5 from the ion peak at m/z 337.2374 [M + H] + (calcd. 337.2373) in its HR-ESI-MS, suggesting the presence of five degrees of unsaturation. The IR spectrum showed absorptions for a double bond at 1618 cm −1 , a ketone carbonyl group at 1710 cm −1 and hydroxyl groups at 3426 cm −1 . The 1 H-and 13 C-NMR spectra of 4 were quite similar to those of compound 3 ( Table 2) Figure 4). The NOESY experiment further confirmed the relative configuration of 4 ( Figure 5). Thus, the structure of 4 was established as ent-15-oxo-2β, 16,18-trihydroxypimar-8(14)-ene as shown in Figure 1.     Compound 4 was obtained as a white amorphous powder. The molecular formula was determined to be C 20 H 28 O 5 from the ion peak at m/z 337.2374 [M + H] + (calcd. 337.2373) in its HR-ESI-MS, suggesting the presence of five degrees of unsaturation. The IR spectrum showed absorptions for a double bond at 1618 cm −1 , a ketone carbonyl group at 1710 cm −1 and hydroxyl groups at 3426 cm −1 . The 1 H-and 13 C-NMR spectra of 4 were quite similar to those of compound 3 (Table 2). After careful comparison, the chemical shifts of C-15 (80.8 ppm in 3; 215.3 ppm in 4) and H 2 -16 (3.38 and 3.74 ppm in 3; 4.36 and 4.32 ppm in 4) were obviously downfield shifted, implying that a ketone group in 4 might replace the hydroxyl group on C-15 in 3. The HMBC correlations from H-14, H-16, and H 3 -17 to the carbonyl carbon assigned it as C-15 ( Figure 4). The NOESY experiment further confirmed the relative configuration of 4 ( Figure 5). Thus, the structure of 4 was established as ent-15-oxo-2β,16,18-trihydroxypimar-8(14)-ene as shown in Figure 1.
Additionally, the new compounds were evaluated for their inhibitory effect on NO production in LPS-stimulated RAW264.7 cells. The results showed compounds 2 and 4 significantly reduced NO production, with IC 50 values of 26.5 ± 6.1 and 17.1 ± 1.5 µM, respectively, which were comparable with that of the positive control indomethacin (IC 50 4.5 ± 0.2 µM). The other two compounds did not show any inhibitory effect up to 100 µM. On the other hand, compounds 1-4 did not show obvious cytotoxicity at 100 µM against RAW264.7 cells. . NMR spectra were recorded on a Bruker Avance III (Bruker, Zurich, Switzerland) for 500M and 600M NMR spectrometer and a Varian MR-400 (Varian, Palo Alto, CA, USA) for 400M NMR spectrometer with TMS as the internal standard. CD spectra were measured on a Jasco J-180 spectrophotometer (Mitsubishi Chemical Industries, Tokyo, Japan). HR-ESI-MS were measured on a Waters Xevo Q-Tof mass detector and an Agilent G6520 Q-TOF mass detector (Santa Clara, CA, USA). All solvents used for CC and HPLC were of analytical grade (Shanghai Chemical Reagents Co. Ltd., Shanghai, China) and gradient grade (Merck KGaA, Darmstadt, Germany), respectively.

Plant Material
The twigs of P. nagi were collected in Ledong County, Hainan Province, China, and identified by Professor Chang-Qiang Ke, Shanghai Institute of Materia Medica. A voucher specimen (No. 20140611) was deposited at the herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Extraction, Isolation, and Characterization
Air-dried twigs of P. nagi (39.3 kg) were grounded and extracted with 95% EtOH (

Cell Culture
The human cervical cancer Hela cells were acquired from American Type Culture Collection (Rockville, MD, USA) and cultured in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin. The human non-small cell lung cancer A549 cells were obtained from American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI 1640 medium containing 10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin. The human breast cancer MCF-7 cells were acquired from KeyGEN Biotech (Nanjing, Jiangsu, China) and cultured in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin. The murine macrophage RAW264.7 cells were obtained from American Type Culture Collection (Rockville, MD, USA) and maintained in DMEM supplemented with 10% FBS. Cells were grown in a standard humidified incubator with 5% CO 2 at 37 • C.