LC-MS-Guided Isolation of Penicilfuranone A: A New Antifibrotic

Dec 18, 2015 - Penicilfuranone A (1), a novel furancarboxylic acid, and its proposed biosynthetic precursor, gregatin A (2), were isolated from the cu...
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LC-MS-Guided Isolation of Penicilfuranone A: A New Antifibrotic Furancarboxylic Acid from the Plant Endophytic Fungus Penicillium sp. sh18 Wei-Guang Wang,†,⊥ Ao Li,‡,⊥ Bing-Chao Yan,†,§,⊥ Shu-Bin Niu,∥ Jian-Wei Tang,†,§ Xiao-Nian Li,† Xue Du,† Gregory L. Challis,# Yongsheng Che,∥ Han-Dong Sun,† and Jian-Xin Pu*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China ‡ College of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 400054, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China ∥ State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, People’s Republic of China # Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom S Supporting Information *

ABSTRACT: Penicilfuranone A (1), a novel furancarboxylic acid, and its proposed biosynthetic precursor, gregatin A (2), were isolated from the cultures of the fungus Penicillium sp. sh18 endophytic to the stems of Isodon eriocalyx var. laxif lora guided by HPLC-MS. X-ray crystallography was applied to the structure determination of furancarboxylic acid for the first time, allowing unambiguous assignment of 1. Penicilfuranone A displays a significant antifibrotic effect in activated hepatic stellate cells via negative regulation of transforming growth factor-β (TGF-β)/Smad signaling. epatic fibrosis, characterized by excessive synthesis and deposition of extracellular matrix (ECM) components in the liver, is a wound-healing response that occurs in most types of chronic liver diseases.1 Aggravated hepatic fibrosis may progress to cirrhosis, ultimately leading to liver failure and even hepatocellular carcinoma. Few effective therapies for hepatic fibrosis are available. Thus, research identifying antifibrotic agents that are innocuous is of high priority and is urgently needed. Natural products are one of the most important sources of medicinal products to treat diseases,2 including organ fibrosis.3 However, during the past 20 years, the chances of discovering new natural products are decreasing.4 LC-MS-guided isolation is an effective method, both in finding new natural products and in characterizing trace components in a complex mixture of natural products.5 The furancarboxylic acids, a class of naturally occurring phytotoxins generally bearing a C13-, C15-, or C17-methyl ester,6 were named aspertetronins A and B,7 gregatins A−E,8

H

© XXXX American Chemical Society and American Society of Pharmacognosy

graminins A9 and B,10 and penicilliols A and B.11 Up to now, less than 20 of these furancarboxylic acids (Figure 1) have been reported from fungi, including Aspergillus rugulosus, Cephalosporium gregatum, and Penicillium daleae,6b showing phytotoxic8 and antibiotic10 activity and selective inhibition of eukaryotic Y-family DNA polymerases.11 Some furancarboxylic acids such as gregatins A−D6,12 and aspertetronin A6,12,13 have been synthesized. In efforts to identify novel pharmaceutical lead structures, especially for the prevention and treatment of hepatic fibrosis, from endophytic fungi, we first focused on the proprietary collection of endophytes that were isolated from healthy plants in the genus Isodon, one of the most important medicinal plant taxa distributed widely in Sichuan, Guizhou, and Yunnan Provinces. An endophytic strain of Penicillium sp. sh18 (GenBank Accession No. KP404098) was isolated from the Received: September 10, 2015

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structure of this secondary metabolite was elucidated using 2D NMR techniques, and the compound was named penicilfuranone A (1; Figure 2D). Penicilfuranone A is a unique furancarboxylic acid featuring a 4-(methoxycarbonyl)furan3(2H)-one core fused with an oxygenated C9 unit. The C24 skeleton of 1 was quite different from the C13-, C15-, or C17basic skeletons of the previously reported furancarboxylic acids.6b,10 The structure for 1 was finally confirmed by singlecrystal X-ray diffraction analysis using anomalous scattering of Cu Kα radiation (CCDC 1042018; Figure 3). As far as we are

Figure 1. Structures and MS data of previously reported furancarboxylic acids.

stems of I. eriocalyx var. laxif lora and was found to be a source of furancarboxylic acids based on HPLC-MS analysis of the crude extract (Figures 1, 2, and S25).

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Figure 3. X-ray crystallographic structure of 1.

RESULTS AND DISCUSSION HPLC-MS analysis determined the ion peak at m/z 299 (M + Na) as a furancarboxylic acid and further guided the isolation of gregatin A (2) ([α]18D −122, c 1.8, CHCl3) (Figures 2B and D). The structure was determined on the basis of NMR spectroscopic data and chiral HPLC analysis (Figure S23).3 In addition, analysis of the total ion chromatograms (Figure 2A and C) revealed the presence of an ion peak at m/z 509 (M + Na), corresponding to the molecular formula C26H30O9 as determined by HRESIMS. This compound showed a similar UV profile6b,10,11,14 (Figures 2A and S25) to furancarboxylic acid, but has a much larger molecular weight than those previously reported, possibly indicating the presence of a dimeric furancarboxylic acid in the extract. Subsequently, the

aware, this is the first crystal structure of a furancarboxylic acid to be reported. Here, we report the isolation, structure elucidation, and antifibrotic activity for 1. Penicilfuranone A (1) was obtained as gold needles. The molecular formula C26H30O9 (12 degrees of unsaturation) was established by HRESIMS analysis and NMR spectroscopic data (Table 1). The 1H NMR spectrum of 1 showed resonances attributed to one primary (δH 0.91, t, 7.4 Hz), one secondary (δH 1.11, d, 6.8 Hz), and two tertiary (δH 1.18 and 1.84, s) methyl groups and two methoxyls (δH 3.72 and 3.80, s). Analysis of the 13C NMR and DEPT data (Table 1) showed the presence of 26 carbons, which were assigned as six methyls

Figure 2. Total ion chromatograms of the crude extract of Penicillium sp. sh18 (A) and the extracted ions of gregatin A (2; B) and penicilfuranone A (1; C), and the structures of 1 and 2 (D). B

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Table 1. 13C and 1H NMR Spectroscopic Data of Compound 1 (δ in ppm, J in Hz) no.

δ Ca

2 3 4 5 6

195.4 109.6 197.0 90.2 124.9

s s s s d

7

130.0 d

8

127.6 d

9

138.5 d

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 12-OCH3 22-OCH3

25.2 t 13.2 q 162.7 s 22.4 q 53.5 d 40.0 d 195.0 s 122.2 s 125.8 s 73.4 s 142. Eight s 140.1 s 147.3 s 100.6 d 13.1 q 29.5 q 51.3 q 55.8 q

δHa

5.27 br t (15.3) 5.22 dd (15.3, 10.1) 5.79 dd (15.1, 10.1) 5.43 dt (15.1, 6.6) 1.97 m 0.91 t (7.4) 1.18 s 4.73 d (5.4) 3.30 (overlap)

7.07 1.11 1.84 3.72 3.80

s d (6.8) s s s

δ Cb 195.8 111.1 197.3 91.6 125.8

δHb s s s s d

131.1 d 128.5 d 139.4 d 26.2 13.5 164.8 22.8 54.8 41.4 195.2 123.9 126.0 75.1 143.5 140.8 148.3 101.9 13.5 30.2 51.8 56.3

t q s q d d s s s s s s s d q q q q

5.26 t (15.1) 5.25 dd (15.1, 8.2) 5.78 ddd (15.1, 8.2, 1.3) 5.46 dt (15.1, 6.6) 1.93 m 0.92 t (7.5)

Figure 4. 1H−1H COSY (bold), selected HMBC (green arrow), and key ROESY correlations for 1 (blue arrow).

the hydroxy proton signals for OH-20 and OH-21 were not observed in the 1H NMR spectra acquired with different deuterated solvents. This information, together with the proton spin-system deduced from the 1H−1H COSY correlations between H-14 and H-15, suggested the presence of the partial structure part B in 1 (Figure 4). Key HMBC correlations of H14 with C-2 and C-3 and of H-15 with C-2, C-14, C-16, C-17, C-19, and C-24 permitted connection of the partial structures part A and part B via C-2/C-14 linkage. In the ROESY spectrum of 1, NOE correlations of H3-25/H-15, H3-25/H-14, and H-15/H-14 implied that H-14, H-15, and H3-25 might adopt the same orientation (Figure 4). However, if the internuclear distance was less than 3 Å, the NOE correlations could also be observed even for the two spinsystems with opposite orientations.15 It is not possible to assign the relative configuration at C-14, C-15, and C-19 using the NOE correlations observed between vicinal protons (H-14 and H-15; H-14 and H3-25). In addition, for either α or β orientation, NOE correlations could be observed between H-14 and H-15 and between H-14 and H3-24, suggesting that the NOE experiment failed to predict the orientation for H-14 and H-15. Therefore, the relative configuration of 1 is difficult to propose based on the existing evidence. Single-crystal X-ray diffraction analysis of 1 using the anomalous scattering of Cu Kα radiation yielded a Flack parameter of −0.1(2) and a Hooft parameter of 0.04 (5) for 1888 Bijvoet pairs (CCDC 1042018),16 which confirmed the above conjecture and established the 5R, 14S, 15R, and 19S absolute configuration for 1 (Figure 3). Penicilfuranone A (1) is presumably biosynthesized via conjugate addition of the anion derived from the polyketide 3 to gregatin A (2), followed by an intramolecular vinylogous aldol reaction17 (Figure 5). Hepatic stellate cells are identified as the major extracellular matrix-producing cells in the liver. Hepatic stellate cell activation and trans-differentiation into myofibroblasts are believed to be a key process in liver injury and fibrogenesis. Thus, inhibition of activation and function of hepatic stellate cells is regarded as the primary therapeutic target for hepatic fibrosis.18 The activation of hepatic stellate cells is mediated by different inflammatory cytokines and growth factors. Among them, transforming growth factor-β1 (TGF-β1) is recognized as a pivotal profibrogenic mediator. TGF-β1 accelerates hepatic stellate cells’ progressive activation to become myofibroblastlike cells by increasing the expression of cellular markers such as α-smooth muscle actin (α-SMA) and vimentin and up-regulates extracellular matrix component expression, especially type I collagen.19 Western blot analysis revealed that TGF-β1 induced a considerable increase in α-SMA and vimentin protein

1.26 s 4.85 d (5.2) 3.26 dq (6.8, 5.2)

7.21 1.23 2.00 3.75 3.87

s d (6.8) s s s

a

Recorded at 600 MHz in DMSO-d6. bRecorded at 600 MHz in acetone-d6.

including two O-methyls, one methylene, seven methines (four olefinic and one aromatic carbon), and 12 other carbons including two oxygenated, two olefinic, five aromatic, and three carbonyl carbons. These data suggested that 1 was a highly oxygenated furancarboxylic acid with a C24 skeleton different from those previously reported with C 13 , C 15 , or C 17 skeletons.6b,7 The HMBC spectrum of 1 showed correlations from one of the tertiary methyl signals, H3-13 (δH 1.18), to C-4, C-5, C-6, and C-7, from the primary methyl signal, H3-11 (δH 0.91), to C-9 and C-10, and from 12-OCH3 (δH 3.72) to C-3 and C-12. Furthermore, the methylene protons H2-10 (δH 1.97, m) showed HMBC correlations to C-8, C-9, and C-11. Other correlations were H-6 (δH 5.27, br t, 15.3) with C-4, C-5, C-7, C-8, and C-13, H-7 (δH 5.22, dd, 15.3, 10.1) with C-5, C-6, C-8, and C-9, H-8 (δH 5.79, dd, 15.1, 10.1) with C-6, C-7, C-9, and C-10, and H-9 (δH 5.43, dt, 15.1, 6.6) with C-7, C-8, C-10, and C-11. These observed HMBC correlations, coupled with a spinsystem (E,E-CH3CH2CHCHCHCH, H3-11/H2-10/H-9/ H-8/H-7/H-6) determined by 1H−1H COSY and HSQC data, established the partial structure part A (Figure 4). In the HMBC spectrum of 1, H-14 (δH 4.73, d, 5.4 Hz) showed correlations to C-15, C-16, C-18, C-19, C-24, and C25, H3-24 (δH 1.11) correlated with C-14, C-15, and C-16, H325 (δH 1.84) correlated with C-14, C-18, and C-19, H-23 (δH 7.07) correlated with C-17, C-18, C-21, and C-22, and 22OCH3 (δH 3.80) correlated with C-22 and C-23. It is noted that C

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Figure 5. Proposed biosynthetic pathway to 1.

Figure 6. Compound 1 inhibits TGF-β1-induced fibrosis-related protein expression in hepatic stellate cells. Serum-starved (A) rat hepatic stellate cells T6 and (B) human hepatic stellate cells LX-2 were treated with TGF-β1 (10 ng/mL) for 48 h in the absence or presence of DMSO (vehicle), the indicated concentrations of 1 (1, 2, or 4 μM), or 10 μM SB431542 (positive control). Whole cell lysates were collected and subjected to Western blot analysis using antibodies specific for α-SMA, vimentin, and type I collagen. β-Actin level served as the loading control.

Figure 7. Compound 1 blocks TGF-β1-induced Smad2/3 phosphorylation and down-regulation of Smad7 expression in hepatic stellate cells. Serumstarved (A) rat hepatic stellate cells T6 and (B) human hepatic stellate cells LX-2 were treated with TGF-β1 (10 ng/mL) for 1 h in the absence or presence of DMSO (vehicle), the indicated concentrations of 1 (1, 2, or 4 μM), or 10 μM SB431542 (positive control). Whole cell lysates were probed with antibodies against phospho-Smad2/3 and Smad7. Membranes were reprobed for β-actin and Smad2/3 to confirm equal protein loading.

In addition, connective tissue growth factor is considered an important downstream modulator protein of TGF-β1 in chronic liver injury. Connective tissue growth factor synergizes with the action of TGF-β1 to promote extracellular matrix accumulation and fibrogenesis.20 As expected, TGF-β1 significantly induced increased connective tissue growth factor expression in hepatic stellate cells, while 1 markedly attenuated TGF-β1-induced protein expression of connective tissue growth factor in a concentration-dependent manner. Together, these results demonstrate that 1 inhibits hepatic stellate cell activation and extracellular matrix deposition in TGF-β1activated hepatic stellate cells. Extracellular TGF-β signals are transduced by membranebound serine/threonine kinase receptors type II and type I,

expression in both rat hepatic stellate cells T6 and human hepatic stellate cells LX-2 (Figure 6). Pretreatment of hepatic stellate cells with 1 substantially reduced TGF-β1-enhanced expression of these markers of hepatic stellate cell activation. In particular, the results suggested that 1 suppressed the expression of vimentin far more effectively than the positive control, SB431542, a potent and specific inhibitor of TGF-β type I receptor kinase. Since activated hepatic stellate cells are responsible for increased collagen synthesis and deposition in the liver, we also monitored the expression of type I collagen. Treatment with TGF-β1 stimulated a persistent increase in the expression of type I collagen in hepatic stellate cells. Remarkably, this pro-fibrogenic effect of TGF-β1 was concentration-dependently inhibited by 1. D

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which phosphorylate intracellular substrates Smad2 and Smad3 (R-Smads) proteins, the canonical downstream effectors of TGF-β signaling. Phosphorylated Smad2/3 form a heteromeric complex with Smad4 (Co-Smad), and then the complex translocates into the nucleus, where it binds to and transactivates the CAGA consensus sequence in the promoters of TGF-β-responsive fibrogenic genes, including those encoding α-SMA and type I collagen.21 In contrast, Smad7 is a negative regulator of TGF-β1 signaling. Smad7 can specifically decrease Smad2/3 phosphorylation and activation by blocking their access to TGF-β type I receptor.22 Here, we observed robust Smad2/3 phosphorylation and loss of Smad7 during hepatic stellate cell activation induced by TGF-β1 (Figure 7). Similar to SB431542, 1 remarkably reduced TGF-β1-induced Smad2/3 phosphorylation and up-regulated expression of Smad7 in a concentration-dependent manner, whereas Smad2/3 remained unchanged. These data suggest that up-regulation of Smad7 expression preventing Smad2/3 from phosphorylation may be an underlying molecular mechanism through which 1 exerts its inhibitory effect on hepatic stellate cell activation and hepatic fibrosis. To exclude the possibility that effects of 1 mentioned above are related to its cytotoxicity, an MMT assay was used to measure the viability of human normal liver L-02 and hepatic stellate cells-T6 and LX-2 cell lines after treatment with 1. The preferential activity of 1 against hepatic stellate cells under concentrations that do not perturb normal hepatocytes was confirmed (Figure 8). Meanwhile, 1 showed no effects on cell

Article

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. ECD spectra were measured on an Applied Photophysics Chirascan spectrophotometer. Scanning IR spectroscopy was performed using a Bruker Tensor-27 spectrophotometer with KBr pellets. NMR spectra were recorded on Bruker AM-600 spectrometers. ESIMS, HRESIMS, and HREIMS experiments were performed on a Bruker HCT/Esquire spectrometer and a Waters AutoSpec Premier P776 spectrometer. Column chromatography was performed with silica gel (200−300 mesh), Lichroprep RP-18 gel (40−63 μm), MCI gel (75−150 μm), and Sephadex LH-20 gel (40−70 μm). Preparative HPLC was performed on a liquid chromatograph with a PRC−ODS column. Semipreparative HPLC was performed on a liquid chromatograph with a 9.4 mm × 25 cm column. Fractions were monitored by TLC, and compounds were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. All solvents including petroleum ether (60−90 °C) were distilled prior to use. Fungal Material. The culture of Penicillium sp. sh18 was isolated from the stems of Isodon eriocalyx var. laxif lora collected from Kunming Botanical Garden, Kunming, People’s Republic of China, in December 2012. The isolate was identified based on sequence (GenBank Accession No. KP404098) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 25 °C for 7 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions, and 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract); the final pH of the media was adjusted to 6.5, and the flasks were sterilized by autoclave. Three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 20 Fernbach flasks (500 mL), each containing 80 g of rice. Spore inoculum was prepared in sterile, distilled H2O to give a final spore/cell suspension of 1 × 106/mL. Distilled H2O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 28 °C for 40 days. Extraction, Isolation, and Purification. The fermented material was extracted with EtOAc (4 × 4.0 L), and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (20 g). The crude extract (20 g) was purified by CC (column chromatography on SiO2 with a CHCl3−Me2CO gradient system 1:0, 9:1, 8:2, 7:3, 6:4, and 1:1) to yield six main fractions, Fr. A−F. Compound 2 was mainly detected in fraction B (CHCl3−Me2CO, 9:1; 1.5 g); then this fraction was subjected to repeated chromatography over silica gel (petroleum ether−Me2CO, 60:1, 30:1, 10:1) to yield fractions B1−B3. Fraction B2 (petroleum ether−Me2CO, 30:1; 120 mg) was fractionated by RP-18 CC (petroleum ether−Me2CO, from 50:50 to 100:0) to afford fractions B2/1-B2/3. Fraction B2/3 (40 mg) was purified by preparative HPLC (10 mL/min, detector UV λmax 230 nm, MeCN− H2O, 60:40) to yield 2 (25 mg, 18.0 min). Compound 1 was mainly observed in fraction C (CHCl3−Me2CO, 8:2, 2 g). Fraction C was purified by silica gel CC with petroleum ether−EtOAc (20:1, 10:1, and 5:1) to yield subfractions C1−C3. Subfraction C3 (100 mg, petroleum ether−EtOAc, 5:1) was purified by preparative HPLC (12 mL/min, detector UV λmax 230 nm, MeCN−H2O, 45:55) to yield 1 (15 mg, 15.0 min). Penicilfuranone A (1): gold needle crystals; [α]22D −6 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 196 (4.5), 222 (4.6), 359 (4.0), 495 (2.6) nm; IR (KBr) Vmax 3444, 3026, 2999, 2933, 2872, 1715, 1704, 1679, 1658, 1504, 1462, 1440, 1381, 1320, 1278, 1257, 1205, 1175, 1144, 1108, 1084, 1061, 1022, 992, 969, 938, 860, 797, 670 cm−1; positive ESIMS m/z 487 [M + H]+, 509 [M + Na]+; positive HRESIMS [M + Na]+ m/z 509.1792 (calcd for C26H30O9Na, 509.1782). X-ray Crystallographic Analysis of Penicilfuranone A (1): C26H30O9, M = 486.50, orthorhombic, a = 8.4640(3) Å, b =

Figure 8. Inhibitory effect of 1 on L-02, hepatic stellate cells-T6, and hepatic stellate cells-LX-2 cell viability measured by the MTT assay. Cells were incubated for 48 h with 1 at different concentrations (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, or 128 μM) in serum-free medium, respectively. Results are expressed as % of control cell viability at the corresponding concentration. Data are presented as mean ± SEM of three independent experiments.

viability of all treated cell lines at concentrations less than 4 μM, suggesting that 1 exerts its antifibrotic effects independently of cytotoxicity on hepatic stellate cells. In conclusion, to the best of our knowledge, this is the first report of the furancarboxylic acids with antifibrotic potential and also the first report of natural products isolated from the endophytes of the plants in the genus Isodon. E

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15.4214(5) Å, c = 19.4594(7) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2539.97(15) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.802 mm−1, 20 530 reflections measured, 4551 independent reflections (Rint = 0.0444). The final R1 values were 0.0521 (I > 2σ(I)). The final wR(F2) values were 0.1648 (I > 2σ(I)). The final R1 values were 0.0540 (all data). The final wR(F2) values were 0.1741 (all data). The goodness of fit on F2 was 1.046. Flack parameter = −0.1(2). The Hooft parameter is 0.04(5) for 1888 Bijvoet pairs. The intensity data for penicilfuranone A (1) were collected on a Bruker APEX DUO diffractometer using graphite-monochromated Cu Kα radiation. Its structure was solved by direct methods (SHELXS97), expanded using difference Founier techniques, and refined by the program and full-matrix least-squares calculations. The non-hydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions. Crystallographic data for the structure of penicilfuranone A (1) has been deposited in the Cambridge Crystallographic Data Centre (deposition number CCDC 1042018). Copies of the data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk. Cell Culture. Immortalized rat hepatic stellate cell line HSC-T6 with characteristics of an activated HSC phenotype and human embryonic hepatocyte cell line L-02 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium (Hyclone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and antibiotics (100 U/mL penicillin G and 100 μg/ mL streptomycin) at 37 °C with 5% CO2. Immortalized human hepatic stellate cell line HSC-LX-2 was purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone). The culture conditions were the same as the conditions described above. The T6, LX-2, and L-02 cells were prepared with passages between 18 and 25. When cells were 70% confluent, they were cultured in medium without FBS for 24 h to synchronize, and then the cells were preincubated for 2 h with DMSO (vehicle control), compound 1, or SB431542 (a potent and specific inhibitor of TGF-β type I receptor kinase, positive control, Cayman Chemicals, Ann Arbor, MI, USA). After preincubation, human recombinant TGF-β1 (Peprotech, Rocky Hill, NJ, USA) at a final 10 ng/mL concentration was added to the culture medium without wash for different periods until further assays. Western Blot Analysis. Lysate preparations were performed and used for Western blot analysis as described previously.23 Briefly, equal amounts of protein samples (30 μg) were separated on 6−12% SDS− polyacrylamide gels and electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) using the Trans-Blot SD semidry transfer cell system (Bio-Rad, Hercules, CA, USA). Membranes were incubated overnight at 4 °C with the primary antibodies as follows: mouse monoclonal anti-α-SMA (Beyotime, Jiangsu, China), rabbit monoclonal anti-vimentin (Cell Signaling, Beverly, MA, USA), rabbit polyclonal antitype I collagen (Boster, Wuhan, China), rabbit polyclonal anti-CTGF (GeneTex, Irvine, CA, USA), rabbit monoclonal anti-phospho-Smad2/3 (Cell Signaling), rabbit monoclonal anti-total-Smad2/3 (Cell Signaling), rabbit monoclonal anti-Smad7 (Epitomics, Burlingame, CA, USA), and mouse monoclonal anti-β-actin (Bioworld Technology, Minneapolis, MN, USA). Subsequently, membranes were incubated for 1 h at 25 °C with corresponding horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse antibodies (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China). Visualization was performed using the Tanon-5200 system (Biotanon, Shanghai, China). Measurement of Cell Viability. Cell viability was determined by use of an MTT assay. Cells were seeded in 96-well plates at a density of 1 × 104 cells/well and grown to 70% confluence in culture medium. The medium was replaced by serum-free medium containing different concentrations of compound 1 (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, or 128 μM) for 48 h. After treatment, the medium was removed, and 10 μL of MTT reagent (5 mg/mL in phosphate-buffered saline; Sigma, St. Louis, MO, USA) in 90 μL of medium was added into each

well. After incubation at 37 °C for an additional 4 h, 100 μL of DMSO (Sigma) was added to each well to dissolve formazan crystals. The optical density was measured at 570 nm wavelength using a universal microplate autoreader (Varioskan, Thermo Electron Co., Waltham, MA, USA). The assays were performed in three independent experiments.

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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.5b00814. 1D and 2D NMR, MS, IR, UV, and ECD spectra for compound 1 (PDF) X-ray crystal structure for compound 1 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: (86) 871-65223616. E-mail: [email protected]. Author Contributions ⊥

W.-G. Wang, A. Li, and B.-C. Yan contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the NSFC-Joint Foundation of Yunnan Province (U1302223), the National Natural Science Foundation of China (Nos. 21322204, 21402213, 81172939), the reservation-talent project of Yunnan Province (2011CI043), the Youth Innovation Promotion Association, and the West Light Foundation of the Chinese Academy of Science (W.-G. Wang).

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DOI: 10.1021/acs.jnatprod.5b00814 J. Nat. Prod. XXXX, XXX, XXX−XXX