Nitrobenzoyl Sesquiterpenoids with Cytotoxic Activities from a Marine

16 Aug 2017 - Marine-Derived Aspergillus ochraceus Fungus ... Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical ...
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Nitrobenzoyl Sesquiterpenoids with Cytotoxic Activities from a Marine-Derived Aspergillus ochraceus Fungus Yanhui Tan,†,‡,§ Bin Yang,†,§ Xiuping Lin,† Xiaowei Luo,† Xiaoyan Pang,† Lan Tang,‡ Yonghong Liu,† Xiaojuan Li,*,‡ and Xuefeng Zhou*,† †

Chinese Academy of Sciences (CAS) Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, CAS, Guangzhou 510301, China ‡ Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China S Supporting Information *

ABSTRACT: Nitrobenzoyl sesquiterpenoids are rare from natural sources. Two new nitrobenzoyl sesquiterpenoids, insulicolide B (1) and insulicolide C (3), and the new natural product 14-O-acetylinsulicolide A (2) were isolated from culture extracts of the marine-derived fungus Aspergillus ochraceus Jcma1F17, together with three known nitrobenzoyl sesquiterpenoids (4−6) and a derivative sesquiterpenoid (7). The structures of the new compounds, including their absolute configurations, were determined by NMR and MS spectroscopic data analyses and comparison between the calculated and experimental ECD spectra. The nitrobenzoyl sesquiterpenoids (1−6) were evaluated for their cytotoxicities against three renal carcinoma cell lines, ACHN, OS-RC-2, and 786-O cells, and compounds 2, 4, and 5 displayed activities with IC50 values of 0.89 to 8.2 μM. Further studies indicated that 2 arrested the cell cycle at the G0/G1 phase at a concentration of 1 μM and induced late apoptosis at a concentration of 2 μM after a 72 h treatment of 786-O cells.

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aturally occurring nitro compounds display great structural diversity and a wide range of biological activities.1 Nitrobenzoyl sesquiterpenoids are rare from natural sources, and only five such compounds have been isolated from marine fungi.2−6 In our previous work, 6β,9α-dihydroxy-14-pnitrobenzoylcinnamolide (4) and its known analogue insulicolide A (5) were obtained from the marine-derived fungus Aspergillus ochraceus Jcma1F17 and displayed significant cytotoxicities against 10 cancer cell lines with IC50 values of 2.0 to 6.4 μM.2 Insulicolide A was also reported to exhibit moderately selective cytotoxicity toward a panel of renal tumor cell lines, suggestive of its greater pharmacological potential.3 The inhibitory activities of other nitrobenzoyl sesquiterpenoids against renal tumor cell lines and their structure−activity relationships have been unexplored. Limited amounts of 4 and 5 were obtained in our first 15 L fermentation of the marine Aspergillus strain.2 Additional insulicolide A and its congeners were needed to study their pharmacological effects, especially for the selective inhibition of renal tumor cells. So, the A. ochraceus Jcma1F17 strain was fermented again on a larger scale (50 L). Herein, we describe the isolation, structure elucidation, and biological evaluations of the two new (1, 3) and four known (2, 4−6) nitrobenzoyl sesquiterpenoids. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The EtOAc extract (52 g) of the 50 L culture of A. ochraceus Jcma1F17 was obtained and subjected to successive silica gel and Sephadex LH-20 column chromatography. Compounds 1− 7 were isolated and purified by semipreparative HPLC. Insulicolide A (5), the main secondary metabolite of this strain, was obtained as a pure compound (320 mg), followed by 6β,9α-dihydroxy-14-p-nitrobenzoylcinnamolide (4; 32 mg). Received: August 16, 2017

A

DOI: 10.1021/acs.jnatprod.7b00698 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. NMR Spectroscopic Data (700 MHz, CD3OD) for 1, 2, and 3 1

2

δC, type

δH (J in Hz)

δC, type

1

41.0, CH2

32.1, CH2

2

18.3, CH2

3

37.1, CH2

a: 1.37, ddd (13.7, 13.7, 3.9) b: 1.72, brd (13.7) a: 1.50, m b: 1.72, m a: 1.19, ddd (13.4, 13.4, 2.9) b: 2.09, brd (13.4)

4 5 6 7 8 9 10 11

38.8, C 57.0, CH 64.4, CH 136.8, CH 127.8, C 52.0, CH 34.6, C 68.6, CH2

12 13 14

171.8, C 26.3, CH3 69.2, CH2

15 1′ 2′ 3′, 7′ 4′, 6′ 5′ 1″ 2″

15.6, CH3 165.5, C 136.4, C 130.9, CH 124.0, CH 151.4, C

pos.

1.56, d (4.8) 4.76, dd (4.8, 3.6) 6.77, dd (3.6, 3.6) 2.87, m a: 4.51, dd (9.2, 9.2) b: 4.18, dd (9.2, 9.2) 1.33, s a: 5.13, d (11.4) b: 4.98, d (11.4) 1.09, s

8.24, brd (8.9) 8.36, brd (8.9)

3 δH (J in Hz)

a: 1.38, m b: 2.22, m a: 1.58, m b: 1.68, m a: 1.14, m b: 2.01, m

17.7, CH2 37.1, CH2 37.9, C 47.1, CH 68.1, CH 132.3, CH 134.1, C 76.9, C 39.6, C 75.5, CH2

2.51, d (4.2) 6.11, dd (4.2, 4.2) 6.81, d (4.2)

a: 4.57, dd (9.6, 9.6) b: 4.29, dd (9.6, 9.6)

170.1, C 26.5, CH3 67.4, CH2

1.11, s a: 4.64, d (11.3) b: 4.25, d (11.3) 1.35, s

19.7, CH3 164.3, C 135.3, C 131.4, CH 124.1, CH 151.5, C 172.2, C 20.7, CH3

8.25, brd (8.7) 8.36, brd (8.7)

1.90, s

Compounds 1−3, 6, and 7 were obtained in amounts less than 10 mg. Compound 1 was obtained as a white, amorphous powder; HRESIMS revealed a negative adduct ion peak of m/z 450.1324 for C22H25ClNO7 [M + Cl]− and suggested the molecular formula C22H25NO7. The 1H NMR spectrum of 1 (Table 1) displayed signals attributable to two methyl groups at δH 1.33 (s, H3-13) and 1.09 (s, H3-15); two oxygenated methylenes at δH 4.18 (1H, t, J = 9.2 Hz, H-11b), 4.51 (1H, d, J = 9.2 Hz, H-11a), 4.98 (1H, d, J = 11.4 Hz, H-14b), and 5.13 (1H, d, J = 11.4 Hz, H-14a); an oxygenated methine at δH 4.76 (1H, m, J = 2.6, 4.1 Hz, H-6); and five olefinic or aromatic protons at δH 6.77 (1H, t, J = 3.6 Hz, H-7), 8.24 (2H, brd, J = 8.9 Hz, H-3′, 7′), and 8.36 (2H, brd, J = 8.9 Hz, H-4′, 6′). The 1 H and 13C NMR spectra of 1 were similar to those of the known compound 4.2 Compound 1 was suggested to have a pnitrobenzoate moiety connected to C-14, which is supported by HMBC correlations from H2-14 (δH 4.98, 5.13) to C-1′ (δC 165.5) and from H-3′ (δH 8.24) to C-1′, C-2′ (δC 136.4), and C-5′ (δC 151.4) (Figure 1). The only difference in the 1H and 13 C NMR spectra of 1, compared to 4, is the absence of the C-9 hydroxy signal. The chemical shifts and coupling constants of H-7 (δH 6.77, 1H, t, J = 3.6 Hz), and H2-11 (δH 4.18, 4.51) of 1 suggest that the C-9 hydroxy in 4 was replaced by the proton H-9 (δH 2.87, m, J = 9.2, 3.0 Hz) in 1. This was further confirmed by HMBC correlations from H-9 to C-7 (δC 136.8), C-8 (δC 127.8), C-10 (δC 34.6), C-11 (δC 68.6), and C-15 (δC 15.6) and COSY correlations of H-9/H2-11. Thus, the planar structure of compound 1 was suggested to be 6-hydroxy-14-pnitrobenzoylcinnamolide.

δC, type

δH (J in Hz)

40.9, CH2

a: 1.48, ddd (13.5, 13.5, 3.9) b: 2.05, brd (13.5) a: 1.53, m b: 1.68, m a: 1.21, m b: 1.81, brd (13.3)

18.1, CH2 37.3, CH2 38.1, C 54.9, CH 68.5, CH 129.8, CH 132.2, C 52.0, CH 35.1, C 68.4, CH2 170.7, C 26.1, CH3 67.4, CH2 16.1, CH3 164.3, C 135.5, C 131.4, CH 124.1, CH 151.6, C 172.1, C 19.8, CH3

1.95, d (4.3) 6.15, m 6.75, dd (3.8, 3.8) 3.04, m a: 4.57, dd (9.2, 9.2) b: 4.24, dd (9.2, 9.2) 1.12, s a: 4.62, d (11.3) b: 4.25, d (11.3) 1.26, s

8.25, br.d (9.0) 8.36, br.d (9.0)

1.91, s

Figure 1. Key HMBC, COSY, and NOESY correlations of 1−3.

The 1H−1H coupling constant values of H-5 (δH 1.56, d, J = 4.8 Hz) and H-6 (δH 4.76, dd, J = 4.8, 3.6 Hz) and the NOESY correlations (Figure 1) of H2-14/H3-15 (δH 1.09), H3-15/H11b (δH 4.18), H-11a (δH 4.51)/H-9, H-9/H-1a (δH 1.37), H9/H-5, H-5/H-6, H-6/H-7, H-6/H3-13 (δH 1.33), and H-5/H313 help to establish the orientation of H-5, H-6, H-9, and Me13 on the bottom face of the molecule. Compound 1 was elucidated to have the same relative configuration as the reported compounds 4 and 5. The absolute configuration was determined by experimental and calculated electronic circular dichroism (ECD) data.7 Time-dependent density functional theory (TD-DFT) calcuB

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of 1 and 5.2 This compound, named 14-O-acetylinsulicolide A (2), was isolated in this study as a new natural product. Compound 2 was previously reported as a synthetic compound, obtained by treatment of insulicolide A (5) with acetic anhydride in the presence of pyridine.6 The molecular formula C24H27NO8 and NMR data (Table 1) of compound 3 suggested that it also is a nitrobenzoyl sesquiterpenoid. Comparison of the 1H and 13C NMR data of 3 with those of 14-O-acetylinsulicolide A (2) and known 9-deoxy insulicolide A (6) indicated the structure of 3 as 9-deoxy-14-Oacetylinsulicolide A, named insulicolide C here. The relative and absolute configuration of 3 were also determined by the NOESY correlations (Figure 1), specific rotation data, and the ECD spectrum (Figure 2). The structures of the other three known nitrobenzoyl sesquiterpenoids were characterized by comparison of their NMR and MS data with literature data and were determined to be 6β,9α-dihydroxy-14-p-nitrobenzoylcinnamolide (4),2 insulicolide A (5),2−4 and 9-deoxyinsulicolide A (6).3 Moreover, a derivative sesquiterpenoid was also obtained and identified as 6β,14-dihydroxy-7α-methoxyconfertifolin (7), which had been reported as a hydrolysis product of insulicolide A.3 It is likely to be an artifact, rather than a natural product. All of the nitrobenzoyl sesquiterpenoids (1−6) were evaluated for their cytotoxicities against three human renal cell carcinoma cell lines, ACHN, OS-RC-2, and 786-O cells (Figure 3). Insulicolide A (5) displayed the strongest activities, with IC50 values of 1.5, 1.5, and 0.89 μM, against ACHN, OSRC-2, and 786-O cells, respectively (Table 2). 14-O-

lations performed at the B3LYP/6-311G(d,p) level were used to generate ECD spectra for the lowest-energy conformers of 1 (Tables S2, S3, Figure S1, Supporting Information). The resulting ECD spectra were combined by Boltzmann weighting to give a composite spectrum for each enantiomer. Comparison of the experimental and calculated ECD spectra for 1 showed good agreement with the 4S,5R,6R,9R,10R-1 enantiomer (Figure 2). So, the absolute configuration of 1 was assigned

Figure 2. Experimental and calculated ECD spectra of 1−3.

as 4S,5R,6R,9R,10R. This absolute configuration is in agreement with the configuration established for insulicolide A by Xray.4 Taken together, the complete structure of 1 was resolved and named insulicolide B. Compound 2 had the molecular formula C24H27NO9, as established by HRESIMS. The 1H and 13C NMR spectra (Table 1) suggested 2 is a nitrobenzoyl sesquiterpenoid, with a p-nitrobenzoate moiety connected to C-6, as supported by HMBC correlations from H-6 (δH 6.11, t, J = 4.2 Hz) to C-1′ (δC 164.3). The NMR data of 2 were nearly identical to those of the known compound insulicolide A (5), except for the presence of an additional acetyl group, which is supported by HMBC correlations from H2-14 (δH 4.25, 4.64) to C-1″ of the acetyl moiety. Furthermore, the key HMBC and COSY correlations (Figure 1) supported 2 as C-14 hydroxy acetylated insulicolide A. The 1H−1H coupling constant and NOESY correlations (Figure 1) suggested that 2 has the same relative configuration as 1. The specific rotations (1: [α]20 D −140; 2: 20 [α]20 −270; 5: [α] −210) and similar Cotton effects in the D D ECD spectra of 1, 2 (Figure 2), and insulicolide A (5)2 indicated that 2 shared the same absolute configuration as those

Table 2. Cytotoxicities of Compounds 1−6 IC50 (μM) compound

ACHN

OS-RC-2

786-O

sorafenib 1 2 3 4 5 6

3.4 30 4.1 13 11 1.5 25

7.0 23 5.3 11 8.2 1.5 30

4.9 24 2.3 14 4.3 0.89 20

Acetylinsulicolide A (2) showed potent inhibitory activities at low μM levels, comparable to the positive control, sorafenib, a drug (Nexavar) approved for the treatment of primary kidney cancer (advanced renal cell carcinoma). Additionally, 2 and 4

Figure 3. Inhibitory activities of the nitrobenzoyl sesquiterpenoids (1−6) against the proliferation of three renal carcinoma cells. Data represent mean ± SD (n = 3). C

DOI: 10.1021/acs.jnatprod.7b00698 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 4. Effects of 2 on cell cycle and apoptosis in 786-O cells. (A) Cell cycle in 786-O cells treated with 2 (1 and 2 μM) for 24 and 72 h. (B) Apoptosis in 786-O cells induced by 2 (1 and 2 μM) for 24 and 72 h.

good resource to produce such compounds. Although more investigations are needed, the significant inhibitory activities against renal carcinoma cells described here indicate the antitumor potential of these unusual natural products.

exhibited stronger cytotoxicity to 786-O cells (IC50 2.3 and 4.3 μM, respectively) than to OS-RC-2 (IC50 5.3 and 8.2 μM, respectively) and ACHN (IC50 4.1 and 11 μM, respectively). Our results revealed that the C-9 hydroxy group might contribute more to the cytotoxic activities against renal carcinoma cells. Based on its selective cytotoxicity against 786-O cells, the ability to arrest the cell cycle and cell apoptosis effects induced by 14-O-acetylinsulicolide A (2) were further investigated in 786-O cells (Figure S2). To determine if an alteration of the cell cycle occurred after treatment with 2, the DNA content of treated cells was measured by flow cytometry. The 786-O cells were treated with 2 (1 and 2 μM) for 24 and 72 h. The percentages of each phase in the cell cycle are shown in Figure 4A. The results showed that the cell population at the G0/G1 phase was enhanced from 45% to 54% after 72 h of exposure to 2 at a concentration of 1 μM, although the effect on the cell cycle arrest after 24 h was not obvious. Moreover, the apoptotic cells induced by 2 were quantified by flow cytometry using annexin V (AV)-FITC/propidium iodide (PI) double staining. The 786-O cells were treated with 2 (1 and 2 μM) for 24 and 72 h, stained with AV-FITC and PI, and then analyzed by flow cytometry. The results (Figure 4B) showed that 14-Oacetylinsulicolide A (2) did not induce apoptosis in 786-O cells at 24 h, but slightly induced late apoptosis (AV+/PI+) at 72 h. NF-κB is considered to be an important transcription factor in the tumorigenic process, due to its strong antiapoptotic functions in cancer cells.8 Many NF-κB inhibitors from natural products are suggested to be useful for the inhibition of cancer cell growth.9 In this study, the three new nitrobenzoyl sesquiterpenoids (1−3) and the derivative sesquiterpenoid (7) were evaluated for their inhibitory activities of lipopolysaccharide (LPS)-induced NF-κB activation in RAW264.7 cells, and only 1 displayed weak activity with 16% inhibition at 4 μM (Figure S3). Moreover, 1−3 and 7 exhibited weak toxic effects on RAW264.7 cells (Figure S4). Nitrobenzoyl sesquiterpenoids are relatively rare in nature, and the new natural nitrobenzoyl sesquiterpenoids identified in this research expand this class of natural products. The yield of insulicolide A (5), 320 mg from a 50 L fermentation, and the diversity of the isolated nitrobenzoyl sesquiterpenoids suggest that the marine-derived fungus A. ochraceus Jcma1F17 may be a

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were performed on a PerkinElmer 341 polarimeter. UV and ECD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics); IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu). The NMR spectra including 1D and 2D NMR were recorded on a Bruker AC 700 MHz spectrometer using tetramethylsilane as standard. HR-ESIMS were determined with a Bruker maXis Q-TOF in positive/negative ion mode. Column chromatography (CC) was performed on silica gel (200−300 mesh, 300−400 mesh) and Sephadex LH-20 (Amersham Biosciences), respectively. Thin-layer chromatography (TLC) was carried out on silica gel GF254 (10−40 μm) plates (Qingdao Marine Chemical Factory). All solvents used were of analytical grade (Tianjin Fuyu Chemical and Industry Factory). Semipreparative HPLC (Agilent Technologies, 1260 Infinity series) was performed using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 1.5 mL/min). Fungal Material and Fermentation. The fungal strain Jcma1F17 was isolated from a marine alga Coelarthrum sp. collected in the South China Sea and deposited at the China General Microbiological Culture Collection (Beijing) as CGMCC 8180.2 Strain Jcma1F17 was cultured on MB agar plates and then incubated for 7 days. Aspergillus ochraceus Jcma1F17 was fermented in 500 mL flasks and incubated at 28 °C under static stations and daylight. The seed medium was the same as reported in our previous study, while the production medium was adjusted, adding 0.625 g of MgSO4 in 1000 mL of production medium.2 After 7 days, cultures from 170 flasks (about 300 mL broth/ flask) were harvested for chemical study. Isolation and Purification of the Compounds. The broth (about 50 L) was extracted with 50 L of EtOAc stirring three times for 30 min. The EtOAc was concentrated in vacuo to yield an organic extract (about 52 g). The extract was subjected to a silica gel column chromatograph and was separated by a linear gradient of petroleum ether/EtOAc (50:0, 50:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:1) to yield nine fractions (fr.I−fr.IX). Fractions fr.V−fr.VII were further purified after HPLC analysis showed the characteristic UV (250−260 nm) peaks of the potential nitrobenzoyl sesquiterpenoids. Most of insulicolide A (5) was obtained from fr.VII by recrystallization. Fr.V, fr.VI, and the rest of fr.VII were further subjected to Sephadex LH-20 CC (CHCl3/MeOH, 1:1), followed by semipreparative HPLC (ODS column, 10 × 250 mm, 5 μm, 2 mL/min) with a gradient solvent system from 10% to 50% MeCN/H2O over 30 min, to yield D

DOI: 10.1021/acs.jnatprod.7b00698 J. Nat. Prod. XXXX, XXX, XXX−XXX

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NF-κB Inhibition Assay. To examine NF-κB inhibition activities, RAW264.7 cells stably transfected with a luciferase reporter gene were used.12 Cells were plated in 96-well plates and pretreated with the compounds (4, 2, 1, and 0.5 μM) for 30 min, followed by 1 μg/mL LPS stimulation for 8 h. BAY11-7082 (Sigma-Aldrich) was used as positive control (5 μM). Cells were harvested, and luciferase activities of the triplicate tests were measured using the luciferase assay system (Promega, Madison, WI, USA). The MTT method was also used to evaluate cell viability of RAW264.7 cells treated with the compounds at the same concentrations.

compounds 1 (5.6 mg), 2 (8.5 mg), 3 (4.4 mg), 4 (32.0 mg), 6 (6.2 mg), 7 (3.5 mg), and the remainder of 5. The total amount of pure insulicolide A (5) obtained was 320 mg. Insulicolide B (1): white, amorphous powder; [α]20 D −140 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 258 (4.12) nm; ECD (c 0.1, MeOH) λmax (Δε) 217 (−2.59), 262 (−0.50); IR νmax 3420, 2924, 2853, 1717, 1526, 719 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 450.1324 [M + Cl]− (calcd for C22H25ClNO7, 450.1325). 14-O-Acetylinsulicolide A (2): white, amorphous powder; [α]20 D −270 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 258 (4.21) nm; ECD (c 0.08, MeOH) λmax (Δε) 229 (−2.03); IR νmax 3420, 2920, 2851, 1722, 1531, 719 cm−1; 1H and 13C NMR data, Table 1; HRESIMS (m/z 508.1383 [M + Cl]− (calcd for C24H27ClNO9, 508.1380). Insulicolide C (3): white, amorphous powder; [α]20 D −61 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 254 (4.27) nm; ECD (c 0.08, MeOH) λmax (Δε) 220 (−1.50), 260 (−0.35); IR νmax 3354, 2920, 2851, 1747, 1635 cm−1; 1H and 13C NMR data, Table 1; HR-ESIMS m/z 492.1428 [M + Cl]− (calcd for C24H27ClNO8, 492.1431). 6β,9α-Dihydroxy-14-p-nitrobenzoylcinnamolide (4): white, amor20 phous powder; [α]20 D −99 (c 0.3, EtOH), literature [α]D −99 (c 0.3, 2 EtOH); ECD (c 0.03, EtOH) λmax (Δε) 225 (−7.86), 262 (−1.97); 1 H and 13C NMR data, Table S1; HR-ESIMS m/z 454.1480 [M + Na]+ (calcd for C22H25NO8Na, 454.1472). Insulicolide A (5): white, amorphous powder; [α]20 D −210 (c 0.3, EtOH), literature [α]D −204 (c 0.4, MeOH);3 ECD (c 0.03, EtOH) λmax (Δε) 213 (−12.12), 263 (−2.32); 1H and 13C NMR data, Table S1; ESIMS m/z 454.1 [M + Na]+. 9-Deoxyinsulicolide A (6): white, amorphous powder; [α]20 D −81 (c 0.3, EtOH), literature [α]D −76 (c 0.5, MeOH);3 1H and 13C NMR data, Table S1; HR-ESIMS m/z 450.1318 [M + Cl]− (calcd for C22H25ClNO7, 450.1325). 6β,14-Dihydroxy-7α-methoxyconfertifolin (7): clear oil; 1H and 13 C NMR data, Table S1; HR-ESIMS m/z 295.1551 [M − H]− (calcd for C16H23O5, 295.1551). Energy Minimization and ECD Calculations. Conformational analysis of 1 was initially performed using the MMFF94 method (Table S2). The chosen conformers based on Boltzmann distribution were optimized at the B3LYP/6-311G(d,p) level in MeOH (Table S3, Figure S1), and TD-DFT ECD calculations were performed with Gaussian 09. The ECD curves were extracted by SpecDis and weighted by Boltzmann distribution after UV correction. Cytotoxic Bioassay. Three renal carcinoma cell lines, ACHN, OSRC-2, and 786-O cells, were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. ACHN cells were grown and maintained in MEM medium with 10% fetal bovine serum (FBS), while OS-RC-2 and 786-O cells were grown in RPMI1640 medium with 10% FBS. Cell viability was determined by the CCK-8 (Dojindo) assay.10 The cells were seeded at a density of 400 to 800 cells/well in 384-well plates and then treated with various concentrations (100, 25.0, 6.25, 1.56, 0.391, 0.0977, 0.0244, 0.00610, and 0.00153 μM) of compounds or solvent control. Sorafenib was used as positive control. After 72 h of incubation, CCK-8 reagent was added, and absorbance of the triplicate tests was measured at 450 nm by an Envision 2104 multilabel reader (PerkinElmer). Dose−response curves were plotted to determine IC50 using Prism 5.0 (GraphPad Software Inc.). Cell Cycle and Apoptosis Assay. Cell cycle arrest by 14-Oacetylinsulicolide A (2) in 786-O cells was analyzed by PI DNA staining using flow cytometry.11 Briefly, 786-O cells were treated with 2 (1 and 2 μM) for 24, 48, and 72 h, respectively. After treatment, cells were harvested, prepared, and then fixed overnight. The fixed cells were harvested, washed, resuspended, and finally stained with PI (Sigma-Aldrich). Cell cycle distribution was studied using an Accuri C6 (BD) flow cytometer. Cell apoptosis was analyzed using a FITC annexin V apoptosis detection kit (BD), according to the manufacturer’s protocol.9 Briefly, 786-O cells were treated with 2 (1 and 2 μM) for 24, 48, and 72 h, stained with annexin V-FITC and PI solution, examined, and analyzed quantitatively using an Accuri C6 (BD) flow cytometer.

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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.7b00698. NMR spectroscopic data of 4−7; conformational analysis of 1; NF-κB inhibition data of 1−3 and 7; and HRMS, IR, 1H, 13C, and 2D NMR spectra of 1−3 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(X. Li) E-mail: [email protected]. *(X. Zhou) Tel/Fax: +86-20-89023174. E-mail: xfzhou@scsio. ac.cn. ORCID

Lan Tang: 0000-0002-2345-0886 Yonghong Liu: 0000-0001-8327-3108 Xuefeng Zhou: 0000-0001-9601-4869 Author Contributions §

Y. Tan and B. Yang contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (41376162, 81073119, 81611130078, 81673677), Pearl River S&T Nova Program of Guangzhou Scientific Research Project (201610010017), Natural Science Foundation of Guangdong Province (2015A030313287, 2014A030313765), Science and Technology Project of Guangdong Province (2016A020222009), and the Open Project of Guangdong Provincial Key Laboratory of New Drug Screening, Southern Medical University.

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REFERENCES

(1) Parry, R.; Nishinob, S.; Spain, J. Nat. Prod. Rep. 2011, 28, 152− 167. (2) Fang, W.; Lin, X.; Zhou, X.; Wan, J.; Lu, X.; Yang, B.; Ai, W.; Lin, J.; Zhang, T.; Tu, Z.; Liu, Y. MedChemComm 2014, 5, 701−705. (3) Belofsky, G. N.; Jensen, P. R.; Renner, M. K.; Fenical, W. Tetrahedron 1998, 54, 1715−1724. (4) Rahbaek, L.; Christophersen, C.; Frisvad, J.; Bengaard, H. S.; Larsen, S.; Rassing, B. R. J. Nat. Prod. 1997, 60, 811−813. (5) Wu, Q. X.; Jin, X. J.; Draskovic, M.; Crews, M. S.; Tenney, K.; Valeriote, F. A.; Yao, X. J.; Crews, P. Phytochem. Lett. 2012, 5, 114− 117. (6) Zhao, H. Y.; Anbuchezhian, R.; Sun, W.; Shao, C. L.; Zhang, F. L.; Yin, Y.; Yu, Z. S.; Li, Z. Y.; Wang, C. Y. Curr. Pharm. Biotechnol. 2016, 17, 271−274. (7) Wang, J.; Wei, X.; Qin, X.; Tian, X.; Liao, L.; Li, K.; Zhou, X.; Yang, X.; Wang, F.; Zhang, T.; Tu, Z.; Chen, B.; Liu, Y. J. Nat. Prod. 2016, 79, 59−65.

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(8) Olivier, S.; Robe, P.; Bours, V. Biochem. Pharmacol. 2006, 72, 1054−1068. (9) Ban, J. O.; Oh, J. H.; Hwang, B. Y.; Moon, D. C.; Jeong, H. S.; Lee, S.; Kim, S.; Lee, H.; Kim, K. B.; Han, S. B.; Hong, J. T. Mol. Cancer Ther. 2009, 8, 1613−1624. (10) Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.; Ohkura, Y.; Ueno, K. Biol. Pharm. Bull. 1996, 19, 1518−1520. (11) Chen, L.; Chai, W.; Wang, W.; Song, T.; Lian, X. Y.; Zhang, Z. J. Nat. Prod. 2017, 80, 1450−1456. (12) Hong, S. S.; Lee, S. A.; Han, X. H.; Jin, H. Z.; Lee, J. H.; Lee, D.; Lee, J. J.; Hong, J. T.; Kim, Y.; Ro, J. S.; Hwang, B. Y. J. Nat. Prod. 2007, 70, 632−636.

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DOI: 10.1021/acs.jnatprod.7b00698 J. Nat. Prod. XXXX, XXX, XXX−XXX