Norditerpenoids and Dinorditerpenoids from the Seeds of Podocarpus

Jul 18, 2017 - (9, 10) Plants of the genus Podocarpus (Podocarpaceae) are widely ... and dinorditerpenoids were identified from the seeds of Podocarpu...
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Norditerpenoids and Dinorditerpenoids from the Seeds of Podocarpus nagi as Cytotoxic Agents and Autophagy Inducers Zhe-Ling Feng,†,# Le-Le Zhang,†,# Yuan-Dong Zheng,‡,§,⊥,# Qian-Yu Liu,† Jing-Xin Liu,† Lu Feng,‡ Li Huang,† Qing-Wen Zhang,† Jin-Jian Lu,*,† and Li-Gen Lin*,† †

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao 999078, People’s Republic of China ‡ State Key Laboratory of Drug Research & Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China ⊥ School of Life Science and Technology, Shanghai Tech University, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new norditerpenoids and dinorditerpenoids, 2-oxonagilactone A (1), 7β-hydroxynagilactone D (2), nagilactones K and L (3 and 4), 3β-hydroxynagilactone L (5), 2β-hydroxynagilactone L (6), 3-epi-15-hydroxynagilactone D (7), 1α-chloro-2β,3β,15-trihydroxynagilactone L (8), and 15hydroxynagilactone L (9), were isolated from the seeds of Podocarpus nagi, along with eight known analogues. The structures of the new compounds were established based on detailed NMR and HRESIMS analysis, as well as from their ECD spectra. The absolute configuration of the known compound 1-deoxy-2α-hydroxynagilactone A (16) was confirmed by single-crystal X-ray diffraction. All of the isolates were tested for their cytotoxic activities against cancer cells. The results indicated that compounds 4 and 6, as well as several known compounds, displayed cytotoxicity against A2780 and HEY cancer cells. Among the new compounds, 2β-hydroxynagilactone L (6) showed IC50 values of less than 2.5 μM against the two cell lines used. Furthermore, compound 6 induced autophagic flux in A2780 cells, as evidenced by an enhanced expression level of the autophagy marker phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II) and increased mRFP-GFP-LC3 puncta. Also, compound 6 activated the c-Jun N-terminal kinase (JNK) pathway, while pretreatment with the JNK inhibitor SP600125 decreased compound 6-induced autophagy.

C

ancer is one of the leading causes of mortality worldwide.1 Although targeted therapy and immunotherapy have resulted in breakthroughs in the treatment of cancer in the last few decades, chemotherapy is still recommended as first-line therapy for the treatment of many types of cancer.2−4 There is an urgent need for developing new anticancer agents that have higher efficacy and lesser side effects than the presently available agents and also can be acquired at an affordable cost. Autophagy is a homeostatic cellular degradation process that mediates degradation and recycling of damaged or unnecessary cellular organelles and proteins to maintain basic energy levels. To better understand autophagy in diseases such as cancer, neurological disease, diabetes, infection, and ischemic injury, the screening for novel autophagy regulators has attracted substantial research interest in recent years.5,6 Autophagy inducers have been reported to provide therapeutic benefits for cancer via inducing autophagic cancer cell death, and they may also help in clearing aggregated proteins that abnormally accumulate in neurodegenerative diseases.7,8 Owing to their structural diversity, natural products have attracted considerable attention in the discovery and develop© 2017 American Chemical Society and American Society of Pharmacognosy

ment of new anticancer compounds and autophagy regulators.9,10 Plants of the genus Podocarpus (Podocarpaceae) are widely distributed in southern mainland China, and the roots, stems, leaves, and flowers of plants from this genus are used as traditional medicines for the treatment of fevers, asthma, coughs, cholera, distemper, chest complaints, and venereal diseases.11 Extensive phytochemical studies resulted in the identification of many totarane-type diterpenoids, norditerpenoids, and dinorditerpenoids from plants of this genus, which showed cytotoxic activities against several cancer cell lines.12−14 In a search for new anticancer compounds, nine new norditerpenoids and dinorditerpenoids were identified from the seeds of Podocarpus nagi (Thunb.) Pilg. Herein, the isolation and structure elucidation of these new compounds, as well as their cytotoxic and autophagy-inducing activities on cancer cells, are reported. Received: April 21, 2017 Published: July 18, 2017 2110

DOI: 10.1021/acs.jnatprod.7b00347 J. Nat. Prod. 2017, 80, 2110−2117

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RESULTS AND DISCUSSION The EtOAc- and n-butanol-soluble fractions of a 95% ethanol extract of P. nagi were purified by column chromatography over silica gel, MCI gel, and preparative HPLC to afford 17 norditerpenoids and dinorditerpenoids, including nine new compounds (1−9, Figure 1) and eight known analogues (10− 17).

8.7, 4.4 Hz), 5.16 (dd, J = 8.7, 5.8 Hz), and 4.45 (d, J = 4.4 Hz), an olefinic proton at δH 6.80 (s), and two other protons at δH 5.94 (d, J = 4.4 Hz) and 5.58 (d, J = 4.4 Hz). The septet proton and two doublet methyl signals indicated the presence of a typical isopropyl group in the structure of 1. The 13C NMR spectrum (Table 2) exhibited 19 resonances, including four methyls at δC 20.7 (C-16), 20.5 (C-17), 25.2 (C-18), and 15.4 (C-20), one methylene at δC 42.6 (C-3), six methines at δC 78.7 (C-1), 49.5 (C-5), 74.0 (C-6), 59.2 (C-7), 108.0 (C-11), and 28.9 (C-15), and eight quaternary carbons at δC 207.7 (C-2), 43.3 (C-4), 111.6 (C-8), 162.9 (C-9), 42.5 (C-10), 161.7 (C12), 169.3 (C-14), and 180.0 (C-19). The 1H and 13C NMR data 1 closely resembled those of nagilactone A,15,16 except for the presence of a ketone carbonyl carbon at δC 207.7 in 1 rather than a methylene in nagilactone A. The HMBC correlations between the low-field proton at δH 4.45 and C-5, C-9, C-10, and C-20 supported its assignment as H-1, and the HMBC correlations from the geminal protons to C-4, C-5, C-18, and C-19 suggested them to the H2-3. The ketone carbonyl carbon was assigned as C-2 based on the HMBC cross-peaks from H-1, OH-1 at δH 5.58, H2-3, and H3-18 at δH 1.36 to this carbon. The relative configuration of 1 was inferred on the basis of the analysis of the ROESY spectrum. The NOE correlations of H1/H-5, H-1/H3-18, H-5/H-6, and H-6/H-7 suggested the hydroxy groups at C-1 and C-7 as being β-oriented. Accordingly, the structure of 2-oxonagilactone A (1) was established as shown (Figure 1). Compound 2 was obtained as colorless needles and exhibited an elemental formula of C18H20O7 on the basis of the ion peak at m/z 347.1114 [M − H]− in its HRESIMS. The IR absorption at 3461 cm−1, along with the signals of two protons at δH 5.80 (d, J = 4.2 Hz) and 5.31 (d, J = 4.9 Hz) in the 1H NMR spectrum, suggested the occurrence of two hydroxy groups in 2. Comparison of the NMR data of 2 (Tables 1 and 2) with those of nagilactone D15,16 revealed close similarities, except for the presence of an oxygenated methine at δH 5.16 (dd, J = 8.5, 4.1 Hz)/δC 59.5 in 2 instead of the methylene (C7) in nagilactone D. The HMBC correlations from this proton to C-5 (δC 50.4), C-6 (δC 72.6), C-8 (δC 112.7), C-9 (δC 165.3), and C-14 (δC 124.7) supported its assignment as H-7.

Figure 1. Compounds isolated from the seeds of Podocarpus nagi.

Compound 1 was isolated as a white, amorphous powder, and its molecular formula was designated as C19H22O7 according to the ion peak at m/z 385.1260 [M + Na]+ (calcd for C19H22O7Na, 385.1263) in its HRESIMS. The IR spectrum exhibited absorptions for hydroxy (3492 cm−1) and carbonyl (1777 and 1704 cm−1) groups. The 1H NMR spectrum of 1 (Table 1) displayed signals ascribed to two singlet methyls at δH 1.36 and 1.14, two doublet methyls at δH 1.20 (J = 7.1 Hz) and 1.18 (J = 7.1 Hz), a pair of geminal protons at δH 2.95 (d, J = 18.6 Hz) and 2.61 (d, J = 18.6 Hz), a septet proton at δH 3.26 (q, J = 6.8 Hz), three oxygenated methines at δH 5.24 (dd, J =

Table 1. 1H NMR Spectroscopic Data (600 MHz, DMSO-d6) for Compounds 1−6 (δH in ppm, J in Hz) position 1α 1β 2α 2β 3α 3β 5 6 7α 7β 11 15 16/17 18 20 OH-1 OH-2 OH-3 OH-7

1 4.45, d (4.4)

2.61, 2.95, 2.55, 5.16, 5.24,

d (18.6) d (18.6) d (5.8) dd (8.7, 5.8) dd (8.7, 4.4)

6.80, 3.26, 1.20, 1.36, 1.14, 5.58,

s q (6.8) d (7.1); 1.18, d (7.1) s s d (4.4)

5.94, d (4.4)

2

3

4

3.57, d (4.3) 3.38, m

3.88, dd (10.8, 4.8) 3.51, m

4.23, dd (6.0, 4.9)

4.00, dd (7.9, 3.4)

2.04, d (5.2) 4.88, dd (8.5, 5.2) 5.16, dd (8.5, 4.1)

1.98, 5.08, 3.41, 2.64, 6.26, 2.56, 1.12, 1.19, 1.03, 5.15, 5.32, 4.94,

6.29, 2.65, 1.19, 1.31, 1.34,

s q (7.5) t (7.5) s s

5.31, d (4.9) 5.80, d (4.2)

d (6.5) dt (9.8, 6.4) m dd (16.6, 6.2) s q (7.5) t (7.5) s s d (4.8) d (6.3) d (3.4)

2111

5

6

3.84, m

3.78, dt (11.2, 4.4)

3.95, dd (7.0, 4.2)

1.75, 1.48, 2.05, 1.47, 1.78, 5.07, 3.38, 2.74, 6.54, 2.55, 1.13, 1.34, 1.03, 5.21,

1.93, ddd (11.2, 5,9, 4.1) 1.68, m 3.56, m

3.80, m

m m m m d (5.5) m m dd (17.0, 4.8) s q (7.5) t (7.5) s s d (4.2)

1.71, 5.06, 3.38, 2.79, 6.54, 2.54, 1.12, 1.32, 1.06, 5.25,

d (5.5) dt (9.9, 5.5) m dd (17.0, 4.8) s q (7.6) t (7.6) s s d (4.9)

1.52, 1.95, 1.88, 5.09, 3.43, 2.66, 6.27, 2.56, 1.12, 1.26, 1.07, 4.95, 5.06,

dd (13.2, dd (13.2, d (6.4) m dd (16.7, dd (16.7, s q (7.6) t (7.6) s s d (4.2) d (6.8)

4.6) 7.1)

10.0) 4.9)

4.73, d (7.8)

DOI: 10.1021/acs.jnatprod.7b00347 J. Nat. Prod. 2017, 80, 2110−2117

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Table 2. 13C NMR Spectroscopic Data (150 MHz) for Compounds 1−9 (δC in ppm) 1b

position 1 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17 18 19 20 a

78.7 207.7 42.6 43.3 49.5 74.0 59.2 111.6 162.9 42.5 108.0 161.7 169.3 28.9 20.7 20.5 25.2 180.0 15.4

CH C CH2 C CH CH CH C C C CH C C CH CH3a CH3a CH3 C CH3

2b 57.3 51.1 66.8 49.4 50.4 72.6 59.5 112.7 165.3 37.3 106.3 161.7 167.4 24.0 12.4

3b

CH CH CH C CH CH CH C C C CH C C CH2 CH3

26.0 CH3 177.2 C 18.7 CH3

69.0 67.9 66.8 45.4 45.0 73.2 24.6 107.0 165.2 40.7 105.7 161.8 161.8 23.4 11.4

CH CH CH C CH CH CH2 C C C CH C C CH2 CH3

16.6 CH3 180.4 C 16.6 CH3

4b 69.9 28.7 27.9 41.7 48.6 73.9 25.3 108.0 165.2 41.4 106.6 162.3 162.3 24.1 12.1

CH CH2 CH2 C CH CH CH2 C C C CH C C CH2 CH3

24.5 CH3 180.9 C 14.0 CH3

5b 73.3 25.2 71.1 45.4 49.5 69.0 38.5 108.6 164.8 42.1 106.6 162.4 162.4 24.2 12.1

6b

CH CH2 CH C CH CH CH2 C C C CH C C CH2 CH3

69.7 64.4 34.6 41.5 44.6 73.6 25.3 107.5 165.9 42.2 106.1 162.3 162.4 24.0 12.0

22.1 CH3 177.8 C 13.5 CH3

CH CH CH2 C CH CH CH2 C C C CH C C CH2 CH3

23.8 CH3 181.5 C 16.5 CH3

7c 57.9 68.8 64.5 49.4 52.4 74.1 25.6 107.6 163.8 38.6 108.4 162.7 163.0 56.6 18.6

CH CH CH C CH CH CH2 C C C CH C C CH2 CH3

21.1 CH3 181.2 C 18.7 CH3

8b 64.2 72.2 70.4 44.1 44.9 72.9 24.1 108.0 161.5 41.6 108.0 161.6 162.2 63.3 20.6

9b

CH CH CH C CH CH CH2 C C C CH C C CH CH3

22.8 CH3 178.4 C 23.3 CH3

69.4 28.1 27.2 41.2 47.8 73.1 24.2 107.6 164.6 40.9 107.2 160.9 161.3 62.6 20.1

CH CH2 CH2 C CH CH CH2 C C C CH C C CH CH3

23.9 CH3 180.4 C 13.5 CH3

Exchangeable. bRecorded in DMSO-d6. cRecorded in acetone-d6.

Table 3. 1H NMR Spectroscopic Data (600 MHz) for Compounds 7−9 (δH in ppm, J in Hz) 7b

position 1α 1β 2α 2β 3α 3β 5 6 7α 7β 11 15 16 18 20 OH-1 OH-2 OH-3 OH-15 a

8a

4.64, d (5.3)

3.84, dd (7.7, 4.9) 4.87, d (3.0) 4.12, dd (5.0, 3.0)

4.08, dd (5.3, 3.3)

3.89, dd (10.8, 5.0) 3.18, 2.27, 4.99, 3.77, 2.70, 6.33, 5.10, 1.43, 1.39, 0.94,

9a

dd (4.7, 3.3) d (6.5) m dd (16.7, 9.8) dd (16.7, 6.6) s dq (9.7, 6.5) d (6.5) s s

2.27, 5.17, 3.63, 2.76, 6.17, 4.77, 1.31, 1.45, 1.40,

d (4.6) td (10.3, 4.6) dd (17.7, 10.4) dd (17.7, 4.1) s m d (6.5) s s

6.11, d (5.0) 4.08, d (10.8) 5.48, d (5.0)

3.63, d (4.7) 4.98, m

1.77, 1.51, 2.05, 1.47, 1.77, 5.04, 3.63, 2.70, 6.60, 4.79, 1.31, 1.17, 1.05, 5.17,

m m m m m m m dd (16.8, 5.8) s m d (6.5) s s d (4.9)

5.43, d (5.1)

Recorded in DMSO-d6. bRecorded in acetone-d6.

of H-2/H-3, H-2/H-5, H-2/H3-18, H-3/H-5, H-5/H-6, H-5/ H3-18, and H-6/H3-18 suggested the α-orientation of H-2, H-3, H-5, H-6, and H3-18. Furthermore, the NOE correlations of H1/H3-20 and H-1/OH-2 supported the β-orientation of H-1 and H3-20. Thus, the structure of nagilactone K (3) was determined as shown (Figure 1). Compound 4 was obtained as colorless needles, and its molecular formula was assigned as C18H22O5 on the basis of the ion peak at m/z 319.1543 ([M + H]+) in the HRESIMS. The NMR data for this compound (Tables 1 and 2) were similar to those of 3, except that three oxygenated methines in ring A of 3 were evident instead of one oxygenated methine at δH 3.84 (m)/δC 69.9 and two methylenes at δH 1.75 (m) and 1.48 (m)/ δC 28.7, and δH 2.05 (m) and 1.47 (m)/δC 27.9 in 4. The correlations between the protons at δH 3.84 and C-9 (δC

Furthermore, the NOE correlations of H-7/H-5 and H-7/H-6 in the ROESY spectrum indicated H-7 to be α-oriented. Therefore, the structure of 7β-hydroxynagilactone D (2) was elucidated as shown (Figure 1). Compound 3 was isolated as colorless crystals. The HRESIMS deprotonated ion peak at m/z 349.1274 [M − H]− was consistent with a molecular formula of C18H22O7. The NMR data for 3 (Tables 1 and 2) were almost identical to those of 3-epi-nagilactone D,17 except for the presence of two additional proton resonances. Considering the molecular weight of 3 was 18 amu more than that of 3-epi-nagilactone D, it was inferred that the epoxide ring at C-1 and C-2 in 3-epinagilactone D might be opened to form two hydroxy groups in 3. Detailed analysis of the HMBC spectrum confirmed the planar structure of 3. In the ROESY spectrum, the correlations 2112

DOI: 10.1021/acs.jnatprod.7b00347 J. Nat. Prod. 2017, 80, 2110−2117

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Figure 2. Compound 6 induces autophagic flux in cancer cells. (A, B) A2780 and HEY cells were treated with the indicated concentrations of compound 6 for 24 h, and expression levels of the proteins were detected by Western blot analysis. (C) A2780 cells were transiently transfected with the mRFP-GFP-LC3 plasmid and then treated with compound 6 (10 μM), HBSS, or BAF (50 nM) for 24 h. mRFP-GFP-LC3 punta were examined using a confocal microscope.

165.2), C-10 (δC 41.4), and C-20 (δC 14.0) in the HMBC spectrum supported its assignment as H-1. The methylenes were assigned as C-2 and C-3 based on the HMBC correlations from the protons at δH 1.75 and 1.48 to C-1 (δC 69.9) and C-10 and from the protons at δH 2.05 and 1.47 to C-1, C-4 (δC 41.7), C-5 (δC 48.6), C-18 (δC 24.5), and C-19 (δC 180.9), respectively. Next, the relative configuration of 4 was established by analysis of the ROESY spectrum. The NOE cross-peaks from H-1 to H-5 (δH 1.78, d, J = 5.5 Hz) and H3-18 (δH 1.34, s) indicated the hydroxy group at C-1 to be βoriented. Accordingly, the structure of 4 was determined as shown in Figure 1, and this compound was named nagilactone L. Compounds 5 and 6 were isolated as white, amorphous powders. Their molecular formula, C18H22O6, was deduced from the deprotonated molecular ion peak at m/z 333.1324 (5) and the molecular adduct molecular ion peak at m/z 357.1309 (6) in the HRESIMS, respectively. Comparison of the NMR data of 5 and 6 (Tables 1 and 2) with those of 4 revealed high similarities, except for the occurrence of an additional oxygenated methine and the absence of a methylene in 5 and 6, respectively. The molecular weights of 5 and 6 were 16 amu more than that of 4, which suggested one more hydroxy group in the structure of both 5 and 6. NMR spectroscopic data interpretation revealed a β-hydroxy group at C-3 in 5 and C-2 in 6, respectively. The structures of 3β-hydroxynagilactone L (5) and 2β-hydroxynagilactone L (6) were thus elucidated as shown (Figure 1). Compound 7, a white, amorphous powder, was assigned the molecular formula C18H20O7 based on the HRESIMS ion peak at m/z 347.1111 [M − H]−. A comparison of the NMR data of 7 (Tables 2 and 3) and those of 15-hydroxynagilactone D16,18 indicated their close similarity in structure. The NOE correlations of H-1/H-5 and H-3/H 3 -20 enabled the determination of α-oriented hydroxy group at C-3, as well as the β-oriented epoxide ring at C-1 and C-2. Thus, the structure of 7 was elucidated, and this compound was assigned as 3-epi15-hydroxynagilactone D (Figure 1). Compound 8, colorless needles, showed a cluster of deprotonated ion peaks at m/z 383.1/385.1 with a ratio of

3:1 in the ESIMS, indicative of a monochlorinated compound. A molecular formula of C18H21O7Cl was assigned to 8 as evidenced by the HRESIMS ion peak at m/z 383.0883 [M − H]−. The IR absorption at 3413 cm−1, together with the downfield proton signals at δH 6.11 (d, J = 5.0 Hz), 5.48 (d, J = 5.0 Hz), and 4.08 (d, J = 10.8 Hz) in the 1H NMR spectrum, indicated the presence of three hydroxy groups in the structure of 8. The NMR data of 8 (Tables 2 and 3) resembled those of 1-chloro-2β-hydroxynagilactone D.19 Detailed analysis revealed the occurrence of an oxygenated methine at δH 4.77 (m)/δC 63.3 and a doublet methyl at δH 1.31 (d, J = 6.5 Hz)/δC 20.6 in the structure 8 instead of the ethyl group in 1-chloro-2βhydroxynagilactone D. Analysis of the HMBC spectrum supported the occurrence of a hydroxy group at C-15 in the structure of 8. A ROESY experiment was carried out to determine the relative configuration of 8, and its structure was established as 1α-chloro-2β,3β,15-trihydroxynagilactone L (8) (Figure 1). Compound 9 was obtained as a white, amorphous powder. The molecular formula of 9, C18H22O6, was inferred from the ion peak at m/z 335.1495 [M + H]+ in its HRESIMS. The 1H and 13C NMR spectra of 9 (Tables 2 and 3) were very similar to those of 4, except that a doublet methyl at δH 1.31 (d, J = 6.5 Hz)/δC 20.1 and an oxygenated methine at δH 4.79 (m)/δC 62.6 rather that an ethyl group were observed in 9. The HMBC cross-peaks from the doublet methyl protons to C-14 (δC 161.3) and from the oxygenated proton to C-8 (δC 107.6) and C-14 supported the presence of a 1-hydroxyethyl group at C14. Furthermore, the ROESY spectrum indicated the relative configuration of 9 to be the same as that of 4. Thus, the structure of 15-hydroxynagilactone L (9) was assigned as shown (Figure 1). Eight known compounds, nagilactone A (10),12,15 nagilactone B (11),12 nagilactone C (12),12 nagilactone D (13),12 nagilactone G (14),13,20 1-chloro-2β-hydroxynagilactone D (15),19 1-deoxy-2β-hydroxynagilactone A (16),17 and 3-epinagilactone D (17),17 were isolated. Their structures were identified by comparison of the NMR and MS spectra with the reported data. 2113

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Figure 3. Compound 6 induces autophagy by JNK/c-Jun pathway activation. (A) A2780 cells were treated with indicated concentrations of compound 6 for 24 h, and expression levels of the proteins were detected by Western blot analysis. (B) A2780 cells were treated with compound 6 (10 μM) for 24 h with or without SP600125 pretreatment (10 μM, 1 h), and expression levels of the proteins were detected by Western blot analysis.

istics of autophagy after treatment with compound 6, A2780 cells were transfected with mRFP-GFP-LC3 plasmid, and autophagic flux was evaluated by assessment of mRFP-LC3 and GFP-LC3 puncta colocalization. GFP fluorescence is stable only in autophagosomes and can be quenched easily in acidic environments such as autolysosomes, whereas mRFP is more stable under acidic conditions and can be detected in both autophagosomes and autolysosomes.24 As shown in Figure 2C, exposure to bafilomycin A1 (BAF), which suppresses autophagy by inhibiting fusion between autophagosomes and lysosomes,25 caused pronounced formation of LC3 puncta displaying both red and green fluorescence, and the puncta appeared yellow in merged images. Conversely, starvation with Hanks’ Balanced Salt Solution (HBSS), as an autophagy inducer, led to the production of large amounts of red-only puncta. Upon 24 h of treatment of compound 6, large amounts of red-only puncta were observed, indicating that compound 6 induced autophagic flux without disrupting the lysosomal function and/or autophagosome−lysosome fusion (Figure 2C). c-Jun NH2-terminal kinase (JNK) is representative of a subgroup of mitogen-activated protein kinases that can be activated primarily by cytokines and exposure to some chemicals or environmental stress.26 JNK is activated through phosphorylation on Thr183 and Tyr185 residues, and, once activated, JNK phosphorylates a number of substrates including the transcription factor c-Jun. JNK participates in multiple cellular events via activating c-Jun through phosphorylation on the N-terminal Ser63 and Ser73 residues.27 Previous studies have also indicated that JNK/c-Jun pathway activation can induce autophagy under certain conditions.28,29 Thus, the role of the JNK/c-Jun pathway in autophagy induced by compound 6 was evaluated. After 24 h of treatment with the indicated concentrations of compound 6, the expression of phosphorylated JNK was upregulated, along with the downstream signals c-Jun and phosphorylated c-Jun, suggesting that the JNK/c-Jun pathway was activated by compound 6 (Figure 3A). Thereafter, the JNK/c-Jun pathway inhibitor SP600125 was used to further confirm whether compound 6 induced autophagy via JNK/cJun activation. As shown in Figure 3B, compound 6-induced activation of phosphorylated JNK and c-Jun was decreased after pretreatment with SP600125 (10 μM, 1 h). Also, the upregulation of the autophagy marker LC3-II induced by

Colorless needle crystals of 1-deoxy-2β-hydroxynagilactone A (16) were obtained by recrystallization from MeOH. The structure of 16 and its absolute configuration (2S,4S,5R,6S,7R,10S) were confirmed by X-ray crystallographic analysis using graphite-monochromated Cu Kα radiation (Table S1). Similar Cotton effects in the ECD spectra confirmed that the absolute configurations of the core skeleton of compounds 1−9 were identical with that of 16. Previous studies have identified a number of totarane-type diterpenoids, norditerpenoids, and dinorditerpenoids from the genus Podocarpus, and some of them demonstrated cytotoxic activity against cancer cells.21 Herein, two human cancer cell lines, A2780 and HEY, were used for cytotoxicity screening of compounds 1−17. The results showed compounds 1−3, 5, and 7−9 did not show growth inhibitory effects on the two cell lines at concentrations up to 10 μM. Compound 6 showed the highest potential, with IC50 values of less than 2.5 μM on the two cell lines. Compound 4 showed IC50 values of 3.8 ± 0.9 μM against A2780 cells and 6.4 ± 1.4 μM against HEY cells, respectively. In addition, the known compounds 10−14, 16, and 17 also showed cytotoxic effects on both cell lines (Table S2). Herein, paclitaxel was used as the positive control, with IC50 values of 164.2 ± 35.7 and 104.1 ± 27.7 nM against A2780 and HEY cells, respectively. In the process of autophagy, microtubule-associated protein 1 light chain 3 (LC3) is cleaved by ATG4 to generate the cytoplasmic form LC3-I, which is subsequently modified and converted into the active form LC3-II through conjugating with phosphatidylethanolamine.22 The conversion of LC3-I to LC3II, as detected by Western blotting, is commonly used as a marker to evaluate autophagy activity. The results of primary screening indicated compounds 6 and 17 to be potential autophagy regulators, as evidenced by induction of accumulation of LC3-II (data not shown). Herein, compound 6, exhibiting the most potent cytotoxic activity in both A2780 and HEY cells, was chosen for further study. Western blot analysis showed that compound 6 induced accumulation of LC3-II in both cell lines (Figure 2A and B), indicating that compound 6 might affect autophagy. Increased expression level of LC3-II responses to the agent may result from enhanced autophagic flux and/or autolysosome degradation inhibition.23 To further determine the character2114

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Fraction B2A was purified by preparative HPLC, eluting with H2O/ CH3CN (7:13, v/v), to obtain compound 1 (7.3 mg). Subfraction B2B was further separated over silica gel, eluted with CHCl3/CH3OH (60:1 to 5:1, v/v), to yield six subfractions (B2B1 to B2B6). Subfraction B2B5 was further separated by preparative HPLC, eluting with H2O/ CH3CN (13:7, v/v), to obtain compounds 5 (6.4 mg), 8 (6.5 mg), and 15 (3.2 mg). Fractions B3 was subjected to CC over ODS gel eluted with H2O/CH3OH (19:1 to 1:19, v/v) to yield four subfractions (B3A to B3D). Subfraction B3A was further separated over silica gel, eluted with CHCl3/CH3OH (40:1 to 5:1, v/v), to yield two subfractions (B3A1 and B3A2). Subfraction B3A1 was further separated by preparative HPLC, eluting with H2O/CH3CN (19:1, v/v), to obtain compounds 2 (19.3 mg), 3 (45.5 mg), 4 (14.7 mg), and 6 (13.8 mg). 2-Oxonagilactone A (1): white, amorphous powder; [α]20D +13.0 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 300 (0.45) nm; ECD (MeOH, nm) λmax (Δε) 190.7 (−5.2), 207.7 (+5.2); IR (KBr) νmax 3492 (strong, broad), 2970, 1777, 1704, 1447, 1547, 1400, 1267, 1097, 913, 875 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 385.1260 [M + Na]+ (calcd for C19H22O7Na, 385.1263). 7β-Hydroxynagilactone D (2): colorless needles; mp 262−263 °C; [α]20D +7.1 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 300 (0.53) nm; ECD (MeOH, nm) λmax (Δε) 190.7 (−5.2), 207.7 (+5.2); IR (KBr) νmax 3436 (strong, broad), 2920, 2344, 1772, 1685,1400, 1093, 662, 480 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 347.1114 [M − H]− (calcd for C18H19O7, 347.1130). Nagilactone K (3): colorless crystals; mp 264−266 °C; [α]20D +48.6 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 303 (0.33) nm; ECD (MeOH, nm) λmax (Δε) 200 (−19.7), 212 (+51.1); IR (KBr) νmax 1766, 1712, 1633, 1549, 1461, 1384, 1205, 1025, 998, 762 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 349.1274 [M − H]− (calcd for C18H21O7, 349.1287). Nagilactone L (4): colorless needles; mp 280−282 °C; [α]20D +25.8 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 302 (0.49) nm; ECD (MeOH, nm) λmax (Δε) 198.0 (−31.3), 212.0 (+54.3); IR (KBr) νmax 2981, 1769, 1717, 1636, 1548, 1461, 1400, 1203, 1142, 1061, 1028, 996, 927, 858, 838, 794, 765 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 319.1543 [M + H]+ (calcd for C18H23O5, 319.1545). 3β-Hydroxynagilactone L (5): white, amorphous powder; [α]20D −1.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 301 (0.18) nm; ECD (MeOH, nm) λmax (Δε) 211 (+14.3), 239 (−1.3); IR (KBr) νmax 1777, 1682, 1537, 1384, 1009, 668 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 333.1324 [M − H]− (calcd for C18H21O6, 333.1338). 2β-Hydroxynagilactone L (6): white, amorphous powder; [α]20D +31.5 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 303 (0.34) nm; ECD (MeOH, nm) λmax (Δε) 197.0 (−30.8), 212.0 (+34.2); IR (KBr) νmax 3429, 2977, 1759, 1633, 1546, 1455, 1382, 1356, 1323, 1296, 1275, 1079, 957, 894 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 333.1326 [M − H]− (calcd for C18H21O6, 333.1338). 3-epi-15-Hydroxynagilactone D (7): white, amorphous powder; [α]20D −7.0 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 235 (0.08) nm; ECD (MeOH, nm) λmax (Δε) 195.7 (−0.2), 196.5 (+0.1); IR (KBr) νmax 3400 (strong, broad), 2922, 2851, 2349, 1647, 1469, 1121, 667, 534 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 347.1111 [M − H]− (calcd for C18H19O7, 347.1130). 1α-Chloro-2β,3β,15-trihydroxynagilactone L (8): colorless needles; mp 267−268 °C; [α]20D +91.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 300 (0.23) nm; ECD (MeOH, nm) λmax (Δε) 293 (−14.5), 195 (+5.3); IR (KBr) νmax 3413, 1764, 1709, 1384, 1107 cm−1; 1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 383.0883 [M − H]− (calcd for C18H20O7Cl, 383.0898). 15-Hydroxynagilactone L (9): white, amorphous powder; [α]20D +9.0 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 300 (0.59) nm; ECD (MeOH, nm) λmax (Δε) 198.2 (−8.5), 215.5 (+7.6); IR (KBr) νmax 3431, 1705, 1633, 1384, 1121, 668, 504, 476 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 335.1495 [M + H]+ (calcd for C18H23O6, 335.1495). X-ray Analysis of Compound 16. Colorless needle crystals of 16 were obtained by recrystallization from MeOH. The crystal data were

compound 6 was also reversed by SP600125, indicating that the JNK/c-Jun pathway activation seems to be pivotal for compound 6-induced autophagy in A2780 cells.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained on a TA DSC Q2000 apparatus. Optical rotation data were obtained using an Autopol VI polarimeter. UV data were recorded with a Varian Cary 50 spectrophotometer. Electronic circular dichroism (ECD) spectra were recorded with a JASCO J-810 circular dichroism spectrophotometer. IR spectra were recorded on a PerkinElmer spectrum-100 FTIR spectrometer using KBr disks. NMR spectra were recorded on an ASCEND 600 MHz/54 mm NMR spectrometer. The chemical shift (δ) values are given in ppm with tetramethylsilane as internal standard, and coupling constants (J) are in Hz. ESIMS and HRESIMS spectra were recorded on an LTQOrbitrap XL spectrometer. All solvents were analytical grade (Tianjing Chemical Plant, Tianjing, People’s Republic of China). Silica gel used for flash chromatography and precoated silica gel GF254 plates used for TLC were produced by Qingdao Haiyang Chemical Co., Ltd. TLC spots were viewed at 254 nm and visualized by spraying with 10% sulfuric acid in alcohol. MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.) was used for column chromatography (CC). Preparative HPLC was performed on a Shimadzu LC-20AP instrument with an SPD-M20A PDA detector. Chromatographic separation was carried out on a C18 column (19 × 250 mm, 5 μm, Waters, SunFire), using a gradient solvent system composed of H2O (A) and CH3CN (B) at a flow rate of 10 mL/min. Plant Material. The seeds of P. nagi were collected from Aihua, Lincang, Yunnan Province, People’s Republic of China, in October 2013, and identified by Professor Jingui Shen from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. A voucher was deposited at the herbarium of the Institute of Chinese Medical Sciences, University of Macau (LL-20150901). Extraction and Isolation. The air-dried seeds of P. nagi (14.97 kg) were ground into a powder and extracted with 95% ethanol at room temperature (40 L × 3 times, each 2 days). After evaporation of the collected percolate, the crude extract (1.37 kg) was suspended in 4 L of H2O and extracted with petroleum ether (2 L × 3), EtOAc (3 L × 3), and n-butanol (2 L × 3), successively. The EtOAc fraction (138.7 g) was subjected to CC over MCI gel eluted with H2O/CH3OH (1:0 to 0:1) to yield eight major fractions (E1 to E8). Fractions E1 and E2 were combined and subjected to CC over silica gel eluted with petroleum ether/acetone (10:1 to1:1) to yield six subfractions (E1A to E1F). Compound 11 (376.7 mg) was crystallized from subfraction E1B. Fraction E1E was purified with Sephadex LH-20, eluting with CHCl3/CH3OH (1:1), to yield three fractions (E1E1 to E1E4). Compound 10 (37.2 mg) was crystallized from subfraction E1E2. E1E3 was subjected to a C18 gel column eluted with H2O/CH3OH (1:1 to 0:1) to obtain compound 12 (23.9 mg). Fractions E3 and E4 were combined and subjected to CC over silica gel, eluted with petroleum ether/acetone (14:1 to 1:1, v/v), to yield nine subfractions (E3A to E3I). Fraction E3D was separated by ODS gel eluted with H2O/CH3OH (1:1 to 0:1, v/v) to yield four subfractions (E3D1 to E3D7). Compound 13 (17.4 mg) was crystallized from subfraction E3D4. Fraction E3D5 was further purified by preparative HPLC, eluting with H2O/CH3CN (7:13, v/v), to obtain compounds 1 (18.6 mg), 9 (8.4 mg), and 16 (27.8 mg). Fraction E3G was purified with Sephadex LH-20, eluting with CHCl3/CH3OH (1:1, v/v), to yield three fractions (E3G1 to E3G2). 3G2 was further separated by preparative HPLC, eluting with H2O/CH3CN (4:1, v/v), to obtain compounds 7 (3.7 mg) and 17 (7.6 mg). Fraction E6 was subjected to CC over silica gel, eluted with petroleum ether/acetone (16:1 to1:1), to yield six subfractions (E6A to E6F). Compound 14 (8.7 mg) was crystallized from fraction E6B. The n-butanol fraction was subjected to CC over MCI gel, eluted with H2O/CH3OH (19:1 to 1:19, v/v), to yield five fractions (B1 to B5). Fraction B2 was subjected to CC over ODS gel eluted with H2O/ CH3OH (19:1 to 1:19, v/v) to yield six subfractions (B2A to B2E). 2115

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collected on a Bruker D8 Venture diffractometer using graphitemonochromated Cu Kα radiation (λ = 1.541 78 Å). A total of 9951 frames were detected with the detector set at different positions of 2θ. Each frame covered 0.20° in ω. Of the 9951 reflections accumulated with 3.8° ≤ 2θ ≤ 68.2°, 3427 were independent (Rint = 0.049) and representing 99.8% of the unique data to the maximum value of 2θ. The structure of 16 was solved by direct methods using SHELXS-97. Refinements were performed with SHELXL-2013 using full-matrix least-squares calculations on F2, with anisotropic displacement parameters for all the non-hydrogen atoms. The full-matrix leastsquares of the 243 variables produced values of the conventional crystallographic residuals R1 = 0.049 (wR2 = 0.1034) for the 3427 observed data points with I > 2σ(I) and R1 = 0.0428 (wR2 = 0.1094) for all data. The goodness-of-fit was 1.153. A final difference Fourier map indicated a residual density between −0.26 and 0.33 e/Å3. The absolute structure parameter with a refined value of 0.18(6) was used to assign the absolute configuration. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. Crystal data of 16: C19H24O6·H2O; fw 366.40; space group P43212; tetragonal; colorless block; 0.2 × 0.2 × 0.2 mm; unit cell dimensions a = 16.3895(3) Å, b = 16.3895(3) Å, c = 13.9454(3) Å, V = 3745.95(16) Å3; Z = 8; dcalc = 1.299 mg/m3. Crystallographic data of 16 have been deposited in the Cambridge Crystallographic Data Center (deposition no. CCDC 1545211), which can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk or from the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB21EZ, UK [fax: (+44) 1223-336-033 or e-mail: [email protected]]. Reagents. The compounds were dissolved in dimethyl sulfoxide (DMSO) as the stock solutions of 20 mM and stored at −20 °C. Paclitaxel, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenysltetrazolium bromide (MTT), phenylmethanesulfonyl fluoride (PMSF), and DMSO were purchased from Sigma (St. Louis, MO, USA). The protease and phosphatase inhibitor cocktail were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). SP600125 and radio immunoprecipitation assay buffer (RIPA) were obtained from Beyotime Biotechnology (Shanghai, People’s Republic of China). Bafilomycin A1 was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin−streptomycin, phosphatebuffered saline (PBS), 0.25% trypsin-EDTA, and HBSS were purchased from Gibco (Carlsbad, CA, USA). Primary antibodies against LC3, p-JNK (Thr183/Tyr185), JNK, p-c-Jun (Ser63), c-Jun, and GAPDH and the secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Cell Culture. Human ovarian cancer A2780 cells were purchased from KeyGEN Biotech Co. Ltd. (Nanjing, Jiangsu, People’s Republic of China), and HEY cells were obtained from Dr. Wen-An Qiang (Feinberg School of Medicine, Northwestern University, Chicago, IL, USA). The two cell lines were routinely cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics (100 units/mL of penicillin and 100 μg/mL of streptomycin) at 37 °C in a standard humidified incubator with 5% CO2. MTT Assay. Exponentially growing A2780 and HEY cells were seeded in 96-well plates at a density of 4 × 103 cells/well and then treated with the indicated concentrations of the test compounds for 48 h upon reaching approximately 70−80% confluence. Paclitaxel was used as the positive control. Thereafter, 1 mg/mL MTT solution was added to each well, and the plates were incubated for 4 h at 37 °C. A 100 μL aliquot of DMSO was added to each well after removal of the MTT solution to dissolve the needle-like formazan crystals formed by viable cells. Then, the optical density at 570 nm was determined using a microplate reader (1420 Multilabel Counter Victor 3, PerkinElmer, Wellesley, MA, USA). Western Blot Analysis. Cells were seeded in six-well plates for overnight incubation. After 24 h of treatment, whole cell extracts were lysed in ice-cold RIPA lysis buffer supplemented with 1% PMSF and 1% protease and phosphatase inhibitor cocktail. Lysates were then centrifuged at 14000 g for 20 min to remove the insoluble fraction. Protein concentrations were then quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Then, 20 μg of total proteins

was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and blocked with 5% nonfat milk in TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween-20) for 2 h at room temperature. The membranes were then incubated with relative primary antibodies overnight at 4 °C, washed with TBST, and probed with anti-rabbit IgG with HRPlinked secondary antibody for 1 h at room temperature. Thereafter, protein bands were visualized with an ECL advanced Western blot detection kit (GE Healthcare, Uppsala, Sweden) using the CheniDoc MP Imaging System. Transfection and Immunofluorescence. The mRFP-GFP-LC3 plasmid was obtained from Addgene (supplied by Tamotsu Yoshimori, Addgene plasmid #21074).24 A2780 cells were seeded in six-well plates for overnight incubation and then transiently transfected with mRFPGFP-LC3 plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. After 24 h of treatment with compound 6, BAF, and HBSS, cells were fixed with 4% formaldehyde for 30 min and then washed with PBS. Immunofluorescence images were obtained using a confocal laser scanning microscope with a 60× oil immersion objective (Leica TCS SP8, Solms, Germany). Statistical Analysis. Data are presented as mean values and standard errors of the mean. Statistical analyses were performed using one-way analysis of variance (SPSS 17 software, Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL, USA). *p < 0.05 and **p < 0.01 were considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00347. 1D and 2D NMR, IR, and HRESIMS spectra of compounds 1−9 and the cytotoxic activity of eight known compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +853-88224674. Fax: +853-28841358. E-mail: jinjianlu@ umac.mo. (J.-J. Lu). *Tel: +853-88228041. Fax: +853-28841358. E-mail: ligenl@ umac.mo. (L.-G. Lin). ORCID

Li-Gen Lin: 0000-0002-6799-5327 Author Contributions #

Z.-L. Feng, L.-L. Zhang, and Y.-D. Zheng contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the Science and Technology Development Fund, Macao S.A.R (FDCT 120/2013/A3), and the Research Fund of University of Macau (MYRG2015-00153-ICMSQRCM, MYRG2015-00091-ICMS-QRCM, MYRG201500101-ICMS-QRCM, and MYRG2017-00109-ICMS) is gratefully acknowledged. The authors thank Prof. J.-G. Shen for identification of plant material and Dr. W.-A. Qiang for the HEY cell line.

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