Hypoxia-Protective Azaphilone Adducts from Peyronellaea glomerata

May 8, 2018 - A1 is the absorbance of the control and A2 is the absorbance of the test sample. The EC50 value was determined as the concentration that...
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Cite This: J. Nat. Prod. 2018, 81, 1148−1153

Hypoxia-Protective Azaphilone Adducts from Peyronellaea glomerata Tian-Xiao Li, Rui-Huan Liu, Xiao-Bing Wang, Jun Luo, Jian-Guang Luo, Ling-Yi Kong,* and Ming-Hua Yang* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Peyronellones A and B (1 and 2), a pair of rare tetracyclic caged adducts of azaphilone with pyruvic acid, along with four new analogues (3−6), were isolated from solid cultures of the endophytic fungus Peyronellaea glomerata. Their structures were elucidated through spectroscopic analysis, and their absolute configurations were unambiguously determined by a combination of single-crystal X-ray crystallography, Rh2(OCOCF3)4-induced ECD experiments, ECD calculations, and modified Mosher methods. Compound 2 (5 μM) was found to have a significant hypoxia-protective effect that improved the survival rate of hypoxia/reoxygenation-treated human umbilical vein endothelial cells from 35% to 70%, which was equal to the potency of the positive control, verapamil. Flow cytometry analysis suggested 2 could inhibit H/R-induced late-stage apoptosis of this cell line.

V

suggested to be present by UV characteristics during HPLC analysis. Further chemical studies were carried out, and six new azaphilone analogues, peyronellones A−F (1−6), were isolated. Compounds 1 and 2 were the rare tetracyclic caged adducts of azaphilone with pyruvic acid through the similar oxa-[4+2]cycloaddition that generated chaetophenol C.18 Compound 1 possesses an unprecedented enantiomeric core differing from that of chaetophenol C and 2, indicating that during biosynthesis the pyruvic acid was added to the opposite side of the azaphilone plane. Compounds 1 and 2 exhibited potent antioxidant effects, and 2 could significantly protect hypoxia/ reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs) with a potency equal to that of the positive control.6 Herein, the isolation, structural elucidation, and bioactive studies are described.

ascular endothelium cells, the primary barrier between blood and tissues, play vital roles in the cardiovascular system including influencing fluid and nutrition exchange, immune response coagulation, and extracellular matrix production.1 Endothelial cell injury can cause multiple cardiovascular diseases such as thrombosis, atherosclerosis, hypertension, and cardiac failure.1,2 It has been known that hypoxia/oxidative stress is a critical factor in the endothelial cell injury.3,4 Such injury can induce endothelium cell apoptosis and further lead to endothelium desquamation and inflammatory reactions as well as cardiovascular disorders. 1,5 Thus, antihypoxic and antioxidative protections are key and fundamental procedures in the treatment of cardiovascular diseases.6 Natural products with free radical scavenging effects have exhibited clinical value in treating cardiovascular diseases.7 For decades, the application of antioxidant agents such as flavonoids, phenolic acids, and carotenoids has been known to prevent hypoxia/reoxygenation induced injury.8,9 Azaphilones, pyranoquinone bicyclic polyketides produced by multiple fungal species, could represent a new class of cardiovascularprotective natural products, since they were reported to be potential antioxidants with similar effects to those of salicylic acid and trolox.10−13 In our ongoing search for new antioxidant and antihypoxic compounds,14−16 the endophytic strain Peyronellaea glomerata attracted our attention; a crude extract showed a half-maximal effective concentration (EC50) of 187.5 μM in the 2,2-diphenyl1-picrylhydrazyl (DPPH) assay. Moreover, azaphilone analogues are known to be produced by P. glomerata,17 which were © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION P. glomerata was isolated from Ilex cornuta and was identified by 18S rDNA sequence analysis. The EtOAc extract of the solidstate fermentation was fractionated by silica gel column chromatography (CC) and preparative HPLC to yield compounds 1−6. The molecular formula (C18H22O6) of peyronellone A (1) was determined by HRESIMS and was indicative of eight indices of unsaturation. The IR absorption bands at 3445 and 1772 cm−1 suggested the presence of hydroxy and ester Received: July 31, 2017 Published: May 8, 2018 1148

DOI: 10.1021/acs.jnatprod.7b00663 J. Nat. Prod. 2018, 81, 1148−1153

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experiment as an ester carbonyl, six aromatic, a ketal, a tertiary alcohol, a quaternary, two methine, two methylene, and four methyl carbons. One benzene ring and a carbonyl accounted for five degrees of unsaturation. Thus, the remaining three indices suggested the presence of three other rings, which combined with the aforementioned benzene confirmed the tetracyclic skeleton of 1. HMBC correlations (Figure 1) unambiguously established the bicyclic azaphilone core. The correlations from H-5 to C-7/

carbonyl groups. The 1H NMR data (Table 1) were indicative of four methyls (δH 0.32, 3H, d, J = 6.7 Hz; 0.83, 3H, t, J = 6.6 Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for 1−3 1a position 1

67.0

3 4 4a 5 6 7 8 8a 9 10

112.2 51.9 135.4 105.7 157.4 111.7 150.2 120.3 41.5 24.1

11

12.6

12

13.8

13 14 1′ 2′ 3′

12.4 9.4 179.0 75.1 42.8

6-OH 8-OH 2′-OH a

δC

δH (mult, J in Hz) 5.50, d (4.3)

6.58, s

1.76, m 1.79, m 1.11, m 0.83, t (6.6)

0.32, d (6.7) 1.48, s 2.15, s

2.66, dd (13.9, 4.4) 1.50, d (13.9) 7.77, br s 8.22, br s 4.86, br s

2a δC 66.9 112.1 51.7 135.5 105.6 157.4 111.7 150.1 120.4 41.2 23.9 11.8

13.0 12.5 9.4 178.9 75.2 42.8

3b

δH (mult, J in Hz) 5.50, d (4.4)

6.56, s

1.80, m 0.99, m 0.38, m 0.64, t (7.5)

0.96, d (6.7) 1.45, s 2.15, s

δC

δH (mult, J in Hz)

168.3 156.1 110.0 139.5 101.1 165.3 111.8 162.6 99.6 32.1 38.3 60.7

18.8 12.1 8.0

6.48, s

Figure 1. Key HMBC and ROESY correlations of 1 and 2.

C-8a and from H-14 to C-6/C-7/C-8 confirmed the presence of the 6,8-dihydroxy-7-methyl benzene ring A; the correlations from H-1 and CH3-13 to C-3/C-4a denoted a tetrahydropyran ring B with a methyl substituent at C-4; and the correlations from H-10 and CH3-12 to C-3 placed the sec-butyl side chain at C-3. More importantly, the correlations from H-1 to C-2′/C3′, from H-3′ to C-1′/C-4/C-8a, and from CH3-13 to C-2′ established the six-membered carbo cyclic ring C bearing both one hydroxy moiety and an ester carbonyl at C-2′ (δC 75.1). The remaining one index of unsaturation and the 3-ketal carbon (δC 112.2) suggested the presence of γ-lactone ring D, which gave 1 a caged skeleton. To analyze the absolute configuration, a Rh2(OCOCF3)4induced electronic circular dichrosim (ECD) experiment was conducted to determine the 2′-chiral tertiary alcohol. According to the bulkiness rule,19 the induced positive Cotton effect at approximately 350 nm (Figure 2) confirmed the 2′S configuration, thus establishing the core of 1 as being in the 1S, 3R, 4S, 2′S configuration. These results were further verified by ECD calculations (Figure 2). Peyronellone B (2) had an identical molecular formula and very similar NMR data to those of 1 (Table 1). The same planar structure was easily confirmed by their identical HMBC correlations (Figure 1). The approximate mirror image Cotton effects (Figure 2) indicated that 2 might be an enantiomer of 1. Their relationship was confirmed by the Rh2(OCOCF3)4induced ECD experiments, which suggested opposite configurations for their caged skeletons. However, obviously different NOE correlations were observed for the side chains of 1 and 2 (Figure 1), indicating they had different relative configurations of CH3-12. This assignment was more directly verified by the differences in the proton chemical shifts of their side chains,

3.33, m 1.93, m 1.77, m 3.57, dt (10.8, 5.9) 3.47, dt (10.8, 6.6) 1.26, d (6.9) 2.13, s 2.09, s

2.65, dd (14.0, 4.6) 1.49, d (14.0) 7.76, br s 8.21, br s 4.85, br s

Measured in acetone-d6. bMeasured in methanol-d4.

Hz; and 1.48 and 2.15, each 3H, s), two methylenes (δH 1.11 and 1.79, each 1H, m; 1.50, 1H, d, J = 13.9 Hz and 2.66, 1H, dd, J = 13.9, 4.4 Hz), and three methines (δH 1.76, 1H, m; 5.50, 1H, d, J = 4.3 Hz; 6.58, 1H, s). The downfield singlet at δH 6.58 indicated the existence of a pentasubstituted benzene, while the triplet and doublet methyls together with the methylene multiplet and methane multiplet demonstrated the presence of a sec-butyl side chain. The 13C NMR spectrum (Table 1) showed 18 signals, which were further classified by an HSQC 1149

DOI: 10.1021/acs.jnatprod.7b00663 J. Nat. Prod. 2018, 81, 1148−1153

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Figure 2. Rh2(OCOCF3)4-induced and calculated ECD spectra of 1 and 2.

indicated the 9R configuration based on comparison to a series of (S)-(+)- and (R)-(−)-α-methylbutyric acid derivatives.27,28 Peyronellones D and E (4 and 5), two isomers of 3, had the same molecular formula (C15H18O5) based on their HRESIMS data. A comparison of their NMR data (Table 2) with those of 3 revealed the side chains of 4 and 5 were hydroxylated at a different position; both were found to be at C-10 by the downfield shifts of H-10 and C-10 as well as by the HMBC correlations from CH3-12 to C-10 and from H-10 to C-9. Moreover, the 9R configurations of 4 and 5 were revealed by comparison of their negative optical rotations with those of several methyl nilate derivatives.29 The absolute configurations of C-10 were determined by modified Mosher methods using (R)- and (S)-MTPA-Cl.14 In conjunction with the 1H-1H COSY data, the 1H NMR signals of their MTPA esters (Supporting Information Figure S1) were assigned, and the calculated ΔδH(S−R) values suggested 10S and 10R configurations for 4 and 5, respectively. The molecular formula (C14H20O4) of peyronellone F (6) was determined from its HRESIMS data. The UV absorption peak at 340 nm suggested the presence of extended conjugation. Its 1H NMR data combined with its 1H-1H COSY data disclosed two spin systems, namely, a −CH2− CHOH− group and a sec-butyl group. Further analysis of its NMR data (Table 2) suggested that 6 was a hydrogenated azaphilone, which made it structurally similar to berkazaphilone A.30 In particular, the HMBC correlations from H-5 to C-4 and from CH3-13 to C-3/C-4/C-4a revealed the presence of a 4methyldienone, which connected the −CH2−CHOH− fragment based on the correlations from H-5 to C-7 and from H-7 and H-8 to C-6 (Figure 3). The bicyclic azaphilone core was confirmed by the HMBC correlations from H-1 and H-8 to C4a and from H-1 to C-8. As with the aforementioned compounds 1−5, its sec-butyl side chain was also located at C-3. Thus, the planar structure of 6 was confirmed.

which were unequally affected by the shielding of the aromatic rings.20 For compound 1, CH3-12 (δH 0.32, d, J = 6.7 Hz) was shifted upfield due to shielding, while for 2, H-10 (δH 0.99, 1H, m, H-10a; δH 0.38, 1H, m, H-10b) and CH3-11 (δH 0.64, 3H, t, J = 7.5 Hz) were both downfield. Thus, in combination with their well-determined caged skeletons, the configurations of the side chains were both assigned as 9R. The interesting configurations of 1 and 2 could be explained by their biosynthetic origin (Supporting Information Scheme S1). Generally, oxonium intermediates (such as 1a and 2a) are thermodynamically unstable and would rapidly rearrange to give ring cleavage products, thus preventing subsequent reactions.21−23 However, in this case, the crucial hemiketal intermediates 1b and 2b are proposed to trap these, allowing them to react with carboxyl moieties to form γ-lactone rings.24−26 The simultaneous isolation of 1 and 2 suggests that the [4+2]-cycloaddition is a pivotal step in the formation of both of their skeletons. This is the first direct evidence that oxa-[4+2]-cycloadditions can generate enantiomeric skeletons. The molecular formula (C15H18O5) of peyronellone C (3) was determined by HRESIMS. Overall analysis of its NMR data (Table 1) revealed that 3 was an isocoumarin, which are azaphilone analogues and have the same biosynthetic origin (Supporting Information Scheme S1).10,11 The isocoumarin core of 3 was confirmed by the HMBC correlations from H-5 to C-4/C-7/C-8a and from CH3-13 to C-3/C-4/C-4a. In addition, its 6,8-dihydroxy-7-methyl substitution was suggested by the HMBC correlations from H-14 to C-6/C-7/C-8. The absence of a triplet methyl signal and the presence of an additional hydroxy methylene signal (δH 3.57, 1H, dt, J = 10.8, 5.9 Hz and 3.47, 1H, dt, J = 10.8, 6.6 Hz; δC 60.7) as well as the HMBC correlation from H-11 to C-9 suggested the 11hydroxylation of its sec-butyl side chain, which was further corroborated to be at C-3 by the HMBC signals from H-9 and H-10 to C-3. The negative optical rotation ([α]25 D −74.1) 1150

DOI: 10.1021/acs.jnatprod.7b00663 J. Nat. Prod. 2018, 81, 1148−1153

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Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for 4−6 4a position

a

δC

δH (mult, J in Hz)

5a δC

δH (mult, J in Hz)

6b δC

1

167.6

167.7

72.3

3 4 4a 5 6 7

155.9 110.3 139.5 101.6 164.9 111.6

155.8 110.2 139.7 101.3 164.6 111.2

165.1 104.4 149.7 115.7 196.0 42.1

8 8a 9

162.9 100.2 44.0

10

71.1

11

22.6

1.11, d (6.0)

22.1

12

15.9

15.4

13 14 8-OH

12.9 8.5

1.31, d (6.8) 2.13, s 2.12, s 11.83, s

6.60, s

3.01, dq (7.1, 6.8) 3.91, dq (7.1, 6.0)

162.6 100.4 44.2 70.4

12.9 8.5

6.60, s

3.04, dq (6.8, 6.6) 3.96, dq (6.8, 7.0)

its hypoxia-protective mechanism against H/R-induced cell death, HUVECs were pretreated with 2 and were analyzed for their apoptotic signals through flow cytometry using annexin V−FlTC/PI staining. Compared to the apoptotic percentages of the control group, hypoxia/reoxygenation successfully induced cell apoptosis in the model group. However, after pretreatment with 1 μM or 5 μM of 2, the apoptotic cells were obviously inhibited, especially for the late-stage cells (from 22.8% to 9.8% and 9.4%). Thus, compound 2 could protect H/ R-treated HUVECs by inhibiting their late-stage apoptosis (Supporting Information Figure S3).

71.7 66.8 37.2

δH (mult, J in Hz) 4.23, d (11.7) 4.16, d (11.7)

■

5.79, s

General Experimental Procedures. Optical rotations were collected on a JASCO P-1020 polarimeter, and UV spectra were recorded on a UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan). A JASCO J-810 spectrometer and a Bruker Tensor 27 spectrometer were used to acquire ECD and IR data, respectively. NMR data were measured on a Bruker AVIII-500 NMR instrument (1H NMR, 500 MHz; 13C NMR, 125 MHz) with tetramethylsilane as internal standard, and HRESIMS data were obtained using an Agilent 1100 series LC/MSD-Trap-SL mass analyzer and Agilent 6520B QTOF mass instrument. A Shimadzu LC-8A system equipped with a binary channel UV detector at 210 and 265 nm and Shim-pack RP-C18 column (10 μm, 200 mm × 20 mm i.d., Shimadzu, Tokyo, Japan) was used for preparative HPLC. CCs were carried out with silica gel (Qingdao Marine Chemical Co. Ltd., Qingdao, China), Sephadex LH20 (Pharmacia, Sweden), and MCI (Mitsubishi, Japan) as packing materials. After fluorescence observation, silica gel GF254 plates were sprayed with vanillin−sulfuric acid to visualize the spots. Fungal Material. The fresh bark of Ilex cornuta was obtained from the suburb of Nanjing, Jiangsu, People’s Republic of China, in May 2012. After being disinfected by 75% ethanol and 3% NaClO, the bark was incubated on potato dextrose agar (PDA) at 28 °C for four days, and the title strain was isolated. The fungus was preliminarily identified as P. glomerata from morphological and microscopic characteristics, and the result was further reinforced by its internal transcribed spacer and 18S rDNA sequences with 100% identity to those reported (GenBank accession no. KF293971.1). P. glomerata was cultivated on PDA for five days, and 32 pieces of the agar were added into eight Erlenmeyer flasks (500 mL) each containing 100 mL of potato dextrose liquid medium. The cultures were incubated using a rotatory shaker at 28 °C and 120 rpm for seven days to prepare the seed culture. Solid fermentation was carried out in 50 Erlenmeyer flasks (500 mL) each containing 100 g of rice and 150 mL of water, which were sterilized at 115 °C for 30 min. After being incubated with 20 mL of seed culture, the flasks were cultivated at 28 °C for 30 days. Extraction and Isolation. The fermented rice cultures were extracted with EtOAc three times, and the crude extract (27.2 g) was obtained by removing EtOAc under reduced pressure. With a gradient elution (petroleum ether−EtOAc, 40:1 to 1:2), the crude extract was fractionated through silica gel CC, giving fractions A−G. Fractions D− F (9.5 g) were combined and further submitted to MCI CC with

3.12, dd (17.2, 3.0) 2.57, dd (17.2, 2.4) 4.22, m 2.80, m

27.5

1.61, m

1.25, d (6.6)

12.1

1.47, m 0.87, t (7.4)

1.18, d (7.0) 2.12, s 2.11, s 11.83, s

17.7

1.14, d (6.9)

12.1

1.82, s

EXPERIMENTAL SECTION

Measured in acetone-d6. bMeasured in CDCl3.

Subsequently, slow evaporation of its n-hexane−EtOAc solution afforded colorless needle-like crystals that were suitable for the single-crystal X-ray analysis. Based on the final Flack parameter of x = 0.06(9), the 8S, 8aR, 9R configuration of 6 was unambiguously established (Figure 3). Azaphilones have been reported to be antioxidants, and compounds 1−6 were evaluated for their free radical scavenging effects through the ABTS (2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) and DPPH assays using trolox as the positive control.12,13 Peyronellones A and B (1 and 2) showed potent effects with EC50 values from 83.5 to 126.3 μM (Supporting Information Table S1). Compound 2 was further revealed to have significant antihypoxic effects on H/R-treated HUVECs. At a concentration of 5 μM, it increased the cell viability from 35% to 70%. This effect was therefore equal to that of the positive control, verapamil (Supporting Information Figure S2).6,16 To elucidate

Figure 3. Selected HMBC and 1H−1H COSY correlations and the ORTEP drawing of 6. 1151

DOI: 10.1021/acs.jnatprod.7b00663 J. Nat. Prod. 2018, 81, 1148−1153

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MeOH−H2O as eluent to obtain seven subfractions, M1−M7. Fraction M5 (65% MeOH) was purified by Sephadex LH-20 CC using CH2Cl2−MeOH (1:1) as eluent to afford five subfractions, and the third subfraction was further separated by a Shimadzu LC-8A preparative HPLC system with MeOH−H2O (60:40) as the mobile phase, giving compounds 3 (1.8 mg, tR 27.0 min), 5 (2.3 mg, tR 28.8 min), and 4 (10.9 mg, tR 30.6 min). In the same manner using Sephadex LH-20 CC and preparative HPLC (50% MeOH), 6 (110.0 mg, tR 25.6 min), 1 (4.5 mg, tR 52.9 min), and 2 (6.2 mg, tR 56.0 min) were isolated from fraction M4. Peyronellone A (1): orange oil; [α]25 D +6.6 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 210 (4.70), 260 (3.99), 281 (3.96) nm; ECD (0.2 mg/ml, MeOH) λ (Δε) 204 (−19.96), 228 (+12.21) nm; IR (KBr) νmax 3445, 2966, 2927, 2851, 1868, 1634, 1339 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 335.13 [M + H]+; HRESIMS m/z 357.1308 [M + Na] + (calcd for 357.1309, C18H22NaO6). Peyronellone B (2): orange oil; [α]25 D −13.6 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 211 (4.83), 260 (3.96), 279 (3.95) nm; ECD (0.2 mg/ml, MeOH) λ (Δε) 206 (+21.70), 261 (−12.15) nm; IR (KBr) νmax 3445, 2965, 2924, 2851, 1771, 1631, 1339 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 335.09 [M + H]+; HRESIMS m/z 357.1306 [M + Na] + (calcd for 357.1309, C18H22NaO6). Peyronellone C (3): white powder; [α]25 D −74.1 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 198 (3.86), 247 (4.78), 330 (4.17) nm; IR (KBr) νmax 3450, 2921, 1661, 1623 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 277.11 [M − H]−; HRESIMS m/z 301.1049 [M + Na]+ (calcd for 301.1046, C15H18NaO5). Peyronellone D (4): white powder; [α]25 D −89.5 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 200 (3.67), 248 (4.14), 329 (3.32) nm; IR (KBr) νmax 3450, 2375, 2347, 1659, 1630 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 277.08 [M − H]−; HRESIMS m/ z 301.1048 [M + Na]+ (calcd for 301.1046, C15H18NaO5). Peyronellone E (5): white powder; [α]25 D −53.6 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 198 (3.75), 247 (4.34), 331 (3.42) nm; IR (KBr) νmax 3422, 2925, 1665, 1625, 1121 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 277.07 [M − H]−; HRESIMS m/ z 301.1047 [M + Na]+ (calcd for 301.1046, C15H18NaO5). Peyronellone F (6): colorless crystal; mp 168−169 °C; [α]25 D +165.6 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 233 (3.31), 338 (4.59) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 211 (+6.06), 248 (+11.31), 320 (−9.90), 357 (+15.69) nm; IR (KBr) νmax 3445, 2353, 2318, 1643 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 353.20 [M + H]+; HRESIMS m/z 275.1253 [M + Na]+ (calcd for 275.1254, C14H20NaO4). Rh2(OCOCF3)4-Induced ECD Experiments of 1 and 2. The samples of 1 and 2 (each 0.4 mg) were dissolved in CH2Cl2 (800 μL). Then 0.8 mg of Rh2(OCOCF3)4 salts was added respectively and the first induced ECD spectra were recorded at once. The following spectra were measured every 5 min three times. The absolute configurations of the 2′-tertiary alcohols in 1 and 2 were determined by the induced Cotton effects at around 350 nm. Theoretical Calculated ECD Experiments of 1 and 2. The ECD calculations were carried out using the Gaussian 09 program package. The conformations with a 1.5 kcal/mol energy window from the global minimum were generated, which were further geometrically optimized applying DFT with B3LYP using the 6-31G* basis set. At the b3lyp/6-31+G(d,p) level of theory, the final ECD calculations were performed with DFT calculations with sigma = 0.30 eV and 2.0 nm UV correction for 1 and sigma = 0.27 eV and 5.0 nm UV correction for 2, respectively. Preparation of (S)- and (R)-MTPA Esters of 4 and 5. Two samples of 4 (each 1.0 mg) together with several pieces of dimethylaminopyridine (DMAP) were dried under vacuum overnight. After being dissolved in 120 μL of anhydrous pyridine, 5 μL of (R)and (S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPACl) was added, respectively. The mixtures were kept at room temperature for 8 h, and 200 μL of methanol was added respectively to quench the reactions. Using a Shimadzu LC-8A preparative HPLC

system with MeOH−H2O (88:12) as the mobile phase, (S)- and (R)MTPA esters (4a and 4b) were obtained at 32.7 and 33.5 min, respectively. In the same manner, (S)- and (R)-MTPA esters of 5 were prepared at room temperature overnight and were also purified through preparative HPLC with 88% MeOH [(S)-MTPA ester 5a, tR 33.3 min; (R)-MTPA ester 5b, tR 32.4 min]. X-ray Crystallographic Analysis of Peyronellone F (6). From n-hexane−EtOAc (2:1) solution, colorless crystals were obtained, and a needlelike crystal (0.42 × 0.35 × 0.32 mm3) was supplied for the Xray crystallographic analysis. Through direct methods with the ShelXS structure solution program, the structure was demonstrated, which was refined with the ShelXL refinement package by least squares minimization. The H atoms were placed in calculated positions and refined by a riding model. The molecular graphic was drawn using Ortep-3. A total of 16 050 measurements (8.654° ≤ 2Θ ≤ 139.24°) yielded 2134 unique reflections (Rint = 0.0207, Rsigma = 0.0128), and the final R1 was determined as 0.0562, and wR2 was 0.1869 (I > 2σ(I)). The absolute configuration was determined correctly with the Flack parameter value x = 0.06(9). Crystal data for peyronellone F (6): C14H20O4, M = 252.30 g/mol, hexagonal, space group P65, a = 15.77120(10) Å, b = 15.77120(10) Å, c = 10.46120(10) Å; α = β = 90°, γ = 120°, V = 2253.42(4) Å3, Z = 6, T = 290(2) K, μ(Cu Kα) = 0.662 mm−1, F = 816.0, Dcalc = 1.116 g/cm3. Crystallographic data of 6 have been deposited at the Cambridge Crystallographic Data Centre with the deposition number CCDC 1427120, and they can be obtained from the Cambridge Crystallographic Data Center {12 Union Road, Cambridge CB2 1EZ, UK [fax: + 44-(0)1223-336033; email: [email protected]]} or through www.ccdc.cam.ac.uk/ products/csd/request, free of charge. ABTS and DPPH Radical Cation Scavenging Activity. The ABTS radical cation scavenging assay was performed to determine the antioxidant activities of 1−6 with trolox as the positive control.12,13 After mixing the ABTS and oxidant solutions equally and reacting at room temperature in the darkness for 16 h, the mixture was diluted with 80% ethanol to the final absorbance of 0.7 ± 0.05 at 734 nm. Compound solutions were prepared in the range of 0.05−3.00 mM. Then 10 μL of prepared solution together with 200 μL of ABTS solution was added to the 96-well microtiter plate (Thermo, USA), which was kept at room temperature in the dark for 6 min before the absorbance measurement at 734 nm. The ABTS radical scavenging activity was measured as follows: scavenging activity (%) = [(A1 − A2)/A1 × 100]. A1 is the absorbance of the control and A2 is the absorbance of the test sample. The EC50 value was determined as the concentration that caused 50% reduction of the absorbance. In the DPPH assay, 100 μL of sample solution and 100 μL of 0.15 mM DPPH solution were added and reacted at 25 °C in the dark for 30 min. Finally, the absorbance was measured at 517 nm and the EC50 values of the isolated compounds were calculated in the same manner as above, using trolox as the positive control. In Vitro Protection on H/R-Induced HUVECs. HUVECs (ATCC, USA) were cultivated in RPMI-1640 medium (Sigma, USA) at 37 °C and 5% CO2. H/R wells were pretreated with peyronellone B RPMI-1640 solutions (1, 5, 10, and 50 μM) for 1 h. Then the solutions were changed to low-glucose Dulbecco minimum essential medium (DMEM), and the 96-well plates were transferred into a hypoxic incubator (95% N2 + 5% CO2, SANYO, Japan) to hypoxia at 37 °C for 12 h. Subsequently, peyronellone B RPMI-1640 solutions were added, and the reoxygenation was carried out in an incubator under normal oxygen conditions for 3 h. The model groups were pretreated with an equal volume of DMSO, and the positive control groups were treated with verapamil RPMI-1640 solution. The control groups were incubated with RPMI-1640 medium under the normal oxygen conditions. Cell viability was evaluated by MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. In particular, 20 μL of MTT PBS solution was transferred into each well to incubate for 4 h. Then the supernatant was removed and the crystals were dissolved using 100 μL of DMSO. After shaking for 5 min, the absorbance measurements were performed through a microplate reader (Tecan, Switzerland) at 570 nm. 1152

DOI: 10.1021/acs.jnatprod.7b00663 J. Nat. Prod. 2018, 81, 1148−1153

Journal of Natural Products

Article

Cell Apoptotic Assay. For the cell apoptotic assay, HUVECs were pretreated with peyronellone B solutions at concentrations of 1, 5, and 10 μM for 1 h. Afterward, the solutions were replaced by DMEM and the 96-well plates were put in a hypoxic incubator at 37 °C for 12 h. Then, peyronellone B solutions were added again and the reoxygenation was performed in a normal oxygen incubator for another 3 h. The control groups were kept in RPMI-1640 medium under the normal oxygen conditions, and the model groups were pretreated with an equal amount of DMSO in RPMI-1640 medium. After being trypsinized and washed twice by cold phosphate-buffered saline, the cells were centrifuged at 2000 rpm for 5 min and were suspended into 500 mL of binding buffer containing 5 mL of APC annexin V−FITC and 5 mL of propidium iodide (BD Biosciences, USA). After staining at room temperature in the dark for 20 min, the apoptotic signals were analyzed by flow cytometry using 488 nm excitation and 600 nm emission filters.

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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.7b00663. HRESIMS, 1H NMR, 13C NMR, HSQC, HMBC, and ROESY spectra of 1−6, ECD spectra of 1 and 2, and data for MTPA esters 4a, 4b, 5a, and 5b (PDF) Crystallographic data for 6 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.-Y. Kong). Tel/Fax: +86-258327-1405. *E-mail: [email protected] (M.-H. Yang). Tel/Fax: +86-258618-5039. ORCID

Xiao-Bing Wang: 0000-0001-5044-3721 Ling-Yi Kong: 0000-0001-9712-2618 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research work was funded by the National Natural Science Foundation of China (81503218), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Youth Fund Project of Basic Research Program of Jiangsu Province (Natural Science Foundation, BK20130651), and the Fundamental Research Funds for the Central Universities (2016ZZD010).

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DOI: 10.1021/acs.jnatprod.7b00663 J. Nat. Prod. 2018, 81, 1148−1153