p-Terphenyl Derivatives from the Endolichenic Fungus Floricola striata

Article pubs.acs.org/jnp

p‑Terphenyl Derivatives from the Endolichenic Fungus Floricola striata Wei Li,†,⊥ Wei Gao,†,⊥ Ming Zhang,‡ Yue-Lan Li,† Lin Li,† Xiao-Bin Li,† Wen-Qiang Chang,† Zun-Tian Zhao,*,‡ and Hong-Xiang Lou*,† †

Department of Natural Products Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, No. 44 Wast Wenhua Road, Jinan 250012, China ‡ College of Life Sciences, Shandong Normal University, No. 88 East Wenhua Road, Jinan 250014, China S Supporting Information *

ABSTRACT: Ten new p-terphenyl derivatives, floricolins A−J (1−10), together with six known compounds (11−16), were isolated from the extract of the endolichenic fungus Floricola striata. Chemical structures of these compounds were elucidated using spectroscopic data (HRESIMS and NMR). Among them, 9 and 10 were enantiomeric mixtures, and their configurations were established by single-crystal X-ray diffraction analysis using Cu Kα radiation. Evaluation of the isolated compounds against Candida albicans revealed that the most active compound, 3 (MIC 8 μg/mL), exerted fungicidal action by destruction of the cell membrane. methines (δ 6.75−7.66) and a methoxy group (3H, δ 3.34, OCH3-3). One monosubstituted phenyl unit was established on the basis of the coupling patterns of aromatic protons at δ 7.66 (2H, dd, J = 7.2, 1.1 Hz, H-2″/6″), 7.40 (2H, td, J = 7.4, 1.2 Hz, H-3″/5″), and 7.29 (1H, tt, J = 7.4, 1.2 Hz, H-4″) (Table 1). A second p-substituted phenyl unit was inferred according to two pairs of ortho-coupled aromatic protons (2H, δ 6.92, dd, J = 8.6, 2.1 Hz, H-3′/5′; 2H, δ 7.35, dd, J = 8.6, 2.1 Hz, H-2′/6′), while a third pentasubstituted phenyl unit was identified on the basis of an isolated aromatic proton at δ 6.75 (1H, s, H-6), which was in accord with the number of aromatic carbons as shown by the 13 C NMR data. The key HMBC correlations from H-2′/H-6′ to C-2 (δC 122.8) and from H-2″/H-6″ to C-5 (δC 128.1) (Figure 1) disclosed the connection of C-1′−C-2 and C-1″−C5, respectively, which suggested the presence of a typical pterphenyl-type skeleton. The methoxy was placed at C-3 according to the HMBC correlation from OCH3-3 to C-3 (δC 147.0) (Figure 1). By comparison of 1H and 13C NMR data of 1 with those of BTH-II0204-207 (A),23 1 was inferred to have an additional hydroxy group at C-4′. This conclusion was certified by the chemical shift (δC 157.6) at C-4′ as well as the molecular formula of 1. Floricolin B (2), giving the molecular formula C19H14O4 by HRESIMS, was obtained as a yellow, amorphous powder. By the same strategy as that used for 1, a p-substituted phenyl unit and a monosubstituted one were also identified in compound 2. The phenolic hydroxy groups at the middle ring of 1 were oxidized to a para-quinone moiety with two quinone carbonyl carbons (δC 187.6, C-1; δC 183.3, C-4) in 2, which were in accord with their molecular formulas and the HMBC

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ndolichenic fungi are a valuable source of bioactive products, and only a limited number of strains have been chemically investigated.1 A variety of bioactive metabolites, including chromones,2 heptaketides,3 quinones,4 isocoumarins,5 terpenoids,6 alkaloids,7 and p-terphenyls,8 have been reported from endolichenic fungi. So far, most natural pterphenyl derivatives were isolated from macrofungi such as Thelephora aurantiotincta,9 Thelephora ganbajun,10 Boletopsis grisea,10 Lenzites betulina,11 and Polyozellus multiplex,12 and few of them have been reported from endolichenic fungi.8 The chemical investigation of p-terphenyl pigments can be traced back to 1877,13 and these compounds exhibit a broad spectrum of biological effects including immunosuppressive,14 antioxidative,15 neuroprotective,16 antimicrobial,17 and cytotoxic activities.18 As DNA topoisomerase inhibitors,19 they were also found to have antitumor activity. During our ongoing search for antifungal natural metabolites from endolichenic fungi,20−22 a chemical investigation of the fungus Floricola striata, inhabiting the lichen Umbilicaria sp. collected from Mount Jiaozi of Yunnan Province in China, was carried out. Ten new p-terphenyl derivatives, floricolins A−J (1−10), together with six known compounds (11−16) were obtained. Floricolin C (3) exhibited significant fungicidal action against Candida albicans. A mechanism of action analysis showed that 3 caused the destruction of the cell membrane, resulting in the death of C. albicans.

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RESULTS AND DISCUSSION Structure Elucidation. The molecular formula of floricolin A (1) was determined as C19H16O4 by HRESIMS and 13C NMR data. The IR spectrum indicated absorption bands of hydroxy (3472 cm−1) and aromatic rings (1657, 1605 cm−1). The 1H NMR data showed the presence of 10 aromatic © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 7, 2016

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DOI: 10.1021/acs.jnatprod.6b00197 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data for Compounds 1−4 1a

a

position

δC, type

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″/6″ 3″/5″ 4″ OMe-3

148.4, C 122.8, C 147.0, C 141.4, C 128.1, C 112.9, CH 125.8, C 132.8, CH 115.9, CH 157.6, C 115.9, CH 132.8, CH 139.6, C 130.1, CH 128.9, CH 127.6, CH 60.6, CH3

2b

δH, mult. (J in Hz)

6.75, s 7.35, dd (8.6, 2.1) 6.92, dd (8.6, 2.1) 6.92, dd (8.6, 2.1) 7.35, dd (8.6, 2.1) 7.66, 7.40, 7.29, 3.34,

dd (7.2, 1.2) td (7.4, 1.2) tt (7.4, 1.2) s

δC, type 187.6, C 128.1, C 155.2, C 183.3, C 144.5, C 132.9, CH 122.3, C 132.4, CH 115.2, CH 156.3, C 115.2, CH 132.4, CH 128.8, C 129.3, CH 128.7, CH 130.2, CH 61.4, CH3

3b


6.89, s 7.29, d (8.7) 6.91, dd (8.7, 1.9) 6.91, dd (8.7, 1.9) 7.29, d (8.7) 7.54, 7.47, 7.47, 3.84,

m m m s

δC, type 187.7, C 125.5, C 155.8, C 182.6, C 144.7, C 133.3, CH 118.4, C 154.1, C 120.8, CH 131.0, CH 117.9, CH 132.3, CH 132.4, C 129.3, CH 128.7, CH 130.3, CH 61.7, CH3

4c


6.92, s

7.02, 7.33, 7.02, 7.18,

m m m dd (1.4, 7.6)

7.54, 7.48, 7.48, 3.83,

m m m s

δC, type 188.4, C 126.1, C 157.3, C 184.1, C 145.9, C 134.0, CH 120.2, C 149.4, C 117.8, CH 117.2, CH 150.9, C 119.0, CH 134.3, C 130.4, CH 129.4, CH 130.8, CH 61.0, CH3


6.86, s

6.72, m 6.72, m 6.56, m 7.56, 7.46, 7.46, 3.78,

m m m s

Measured in acetone-d6. bMeasured in CDCl3. cMeasured in CD3OD.

correlations of 2 (Figure 1). The conversion of compound 1 to 2 at room temperature in air confirmed the above conclusion. Floricolin C (3) displayed the same molecular formula as and similar NMR resonances to those of 2. The difference between them was the presence of an ortho-substituted phenyl unit (ring A) in 3 instead of the para-substituted unit found in 2, as confirmed by the HMBC correlations from H-6′ (δH 7.18) to C-2 (δC 125.5), C-2′ (δC 154.1), and C-4′ (δC 131.0) and from H-4′ (δH 7.33) to C-6′ (δC 132.3). Floricolin D (4) was a hydroxylated derivative of 3, as determined by its formula C19H14O5 as well as their almost identical 1H and 13C resonances (Table 1) except for an additional hydroxy group at C-5′ (δC 150.9) in 4. HMBC correlations from H-3′ (δH 6.72) to C-5′ (δC 150.9) and from H-6′ (δH 6.56) to C-4′ (δC

117.2) and C-2′ (δC 149.4) confirmed the location of the additional hydroxy group. Floricolin E (5) gave the molecular formula C18H10O5 by HRESIMS analysis, corresponding to 14 indices of hydrogen deficiency. Comparison of the NMR data of 5 with those of 4, the absence of the methyl group on the methoxy group at C-3, and the similar chemical shift for C-3 and C-2′ (Table 2) suggested the presence of a furan ring with an oxygen bridge connecting C-3 and C-2′. One more index of hydrogen deficiency in 5 further supported the above conclusion. In addition, the H-6 proton in 5 was replaced by a hydroxy group as determined by the noticeable downfield chemical shift for C6 (δC 161.3), which determined the structure of 5 as depicted. Floricolins F (6) and G (7) shared the same structural skeleton B



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The enantiomer mixture, floricolin I (9), was obtained as yellow crystals from MeOH with [α]20 D 0 (c 0.10, MeOH). The molecular formula was determined as C19H14O6 by HRESIMS. Compound 9 had the same structural skeleton as 5, the difference between them being a double bond between C-2 and C-3, which was oxidized to OH-2 and OCH3-3, confirmed by the upfield chemical shift for C-2 (δC 86.0) and C-3 (δC 106.7). The HMBC correlations from H-6 and H-6′ to C-2 and from OMe-3 to C-3 (Figure 1) supported this deduction. In order to determine the absolute configuration, a single-crystal X-ray diffraction experiment was employed using Cu Kα radiation. The P21 space group of the crystallographic data is shown in Figure 2. The optically inactive property suggests that 9 may be enantiomeric mixtures. Further analysis using chiral HPLC-CD spectroscopy showed two peaks with approximately a 1:1 integration ratio and almost mirror-image CD curves (S64, Supporting Information). The n−π* and π−π* transitions in the CD curves are clearly related to the helicity rule of the α,βunsaturated ketone.25,26 Therefore, the absolute configuration of the faster eluting enantiomer (tR = 14.3 min) was assigned as 2S and 3R (9a) according to the observed negative Cotton effect at 320 nm and positive Cotton effect at 250 nm, while the absolute configuration of the slower eluting enantiomer (tR = 16.0 min) was assigned as 2R and 3S (9b) according to the opposite Cotton effect. Floricolin J (10), giving the molecular formula C20H18O6 by HRESIMS and 13C NMR, was obtained as yellow crystals from MeOH. The 13C NMR of ring B displayed resonances that were assigned to one carbonyl carbon (δC 194.1, C-1), two olefinic carbons (δC 129.8, C-5; δC 128.8, C-6), two oxygenated

Figure 1. Selected 1H−1H COSY (bold lines) and HMBC (H → C) correlations of compounds 1, 2, 5, 9, and 10.

with 5, while the hydroxy group at C-6 in 5 was methylated in 6 and 7 (Table 2), and 7 was a C-5′ dehydroxy derivative of 6. Floricolin H (8), an orange powder, had the molecular formula C19H14O3, which was determined by HRESIMS and 13 C NMR data. The 1H and 13C NMR data (Table 2) of rings A and C were nearly identical to those of the known compound 2-phenyl-[1H-2-benzopyran][4,3-e][p]benzoquinone (15).24 Ring B in 8 was a pentasubstituted benzene ring instead of the para-quinone moiety in 15, which was confirmed by the HMBC correlations from H-6′ to C-2 (δC 112.2) and from H2″/H-6″ to C-5 (δC 129.7). At room temperature 8 could be easily oxidized to compound 15. Table 2. 1H and 13C NMR Data for Compounds 5−10 5a position

δC, type

1 2 3 4 5 6 1′ 2′ 3′

174.5, 117.4, 151.2, 174.6, 117.1, 161.3, 125.1, 151.2, 113.8,

C C C C C C C C CH

4′

116.6, CH

5′

156.9, C

6′

107.2, CH

1″ 2″/6″

136.7, C 132.6, CH

3″/5″

128.4, CH

4″

126.4, CH


7.49, d (8.7) 6.92, dd (2.5, 8.7)

6b δC, type 177.7, 114.3, 153.7, 180.4, 120.9, 157.4, 124.0, 151.5, 114.3,

7c δH, mult. (J in Hz)

C C C C C C C C CH

7.46, m

177.5, 120.4, 152.2, 179.6, 128.3, 156.5, 122.4, 156.6, 113.1,

119.2, CH

7,16, m

129.1, CH

157.0, C 7.36, d (2.5) 7.42, dd (8.0, 1.2) 7.31, dd (7.6, 8.0) 7.16, tt (7.6, 1.2)

δC, type C C C C C C C C CH

126.2, CH

8a δH, mult. (J in Hz)

7.69, d (8.4) 7.58, dt (1.2, 7.4) 7.51, m 8.19, d (7.7)

δC, type 149.3, 112.2, 145.3, 137.2, 129.7, 110.8, 131.3, 133.0, 125.2,

C C C C C CH C C CH


7.46, m

127.8, CH

7.24, m

119.4, CH

7.16, m

117.6, CH

128.9, CH

7.32, dt (7.2, 1.5) 8.50, d (7.8)

151.1, C

123.4, CH

131.7, C 131.5, CH

7.39, m

130.0, C 130.8, CH

7.37, m

139.7, C 130.2, CH

128.5, CH

7.45, m

128.2, CH

7.51, m

128.9, CH

129.1, CH

7.41,m

128.9, CH

7.51, m

127.9, CH

a

62.0, CH3

δC, type

7.24, m

7.52, m

3.94, s


194.1, C 56.0, CH 95.3, C 98.4, C 129.8, C 128.8, CH 119.2, C 146.1, C 118.2, CH

107.2, CH

62.1, CH3

δC, type

10b

192.6, C 86.0, C 106.7, C 191.6, C 152.8, C 134.2, C 128.9, C 154.5, C 112.1, CH

127.8, CH

6.54, s

7.61, dd (1.3, 7.9) 7.38, dd (7.5, 7.8) 7.28, tt (1.3, 7.5)

OMe-3 OMe-6 CH2-2′

9d

δH, mult. (J in Hz) 4.00, s

6.14, s

6.80, d (8.8) 6.76, dd (2.8, 8.8)

152.8, C

112.2, CH

7.52, m

115.1, CH

6.68, d (2.8)

133.7, C 129.5, CH

7.39, m

136.6, C 129.5, CH

7.70, m

129.8, CH

7.45, m

128.7, CH

7.40, m

131.8, CH

7.41, m

129.1, CH

7.40, m

53.7, CH3

3.65, s

52.0, CH3 48.9, CH3

3.40, s 3.25, s

3.89, s 70.2, CH2

5.07, s

Measured in CD3OD at 150 MHz. bMeasured in acetone-d6 at 150 MHz. cMeasured in CDCl3 at 100 MHz. dMeasured in acetone-d6 at 100 MHz. C



Article

Figure 2. X-ray crystallographic structures of compounds 9 and 10 (the relative configuration).

Table 3. In Vitro Susceptibilities of Isolated Compounds against Candida albicans by Broth Microdilution Method MIC80 (μg/mL)

1

2

3

4

7

8

9

10

11

12

14

15

16

16

8

8

64

>128

>128

>128

>128

>128

>128

64

>128

>128

carbons (δC 95.3, C-3; δC 98.4, C-4), and one methine (δC 56.0, C-2) (Table 2). The positions of the two methoxy moieties were both assigned at C-3 by HMBC correlations from methoxy groups (δH 3.40 and 3.25) to C-3 (δC 95.3) (Figure 1). The HMBC correlations from H-6 to C-1″, H-2″/H-6″ to C-5, and H-2 to C-1′, C-2′, and C-6′ suggested the connections of C-5−C-1″ and C-2−C-1′. The structure of rings A and C was determined by the same strategy used for 5. The absolute structure of 10 was further determined by X-ray crystallography. An X-ray crystal structure analysis was conducted using Cu Kα radiation, and the crystallographic data displayed a P21 space group, suggesting 10 consisted of a pair of enantiomers. Further analysis by HPLC using a chiral column showed the ratio of 10a:10b to be in 83:16 (S73, Supporting Information). The known compounds were identified as betulinan C (11),17 BTH-II0204-207 (A) (12),23 betulinan A (13),11 terphenyl 2 (14),27 2-phenyl-[1H-2-benzopyran][4,3-e][p]benzoquinone (15)24 and betulinan B (16).11 by comparion of their spectroscopic data with those reported. Antifungal Activity and Determination of the Minimum Inhibitory Concentration (MIC). The isolated compounds were assayed for their activity against C. albicans using a broth microdilution checkerboard assay. The MICs of all tested compounds, based on a reduction of 80% of C. albicans growth, are summarized in Table 3. Floricolin A (1) displayed an MIC of 16 μg/mL, and floricolin B (2) and floricolin C (3) exhibited MICs of 8 μg/mL. Taking into account the yield and activity, 3 was chosen for further antifungal investigations. Fungicidal Action of Floricolin C (3) against C. albicans. The antifungal activity of floricolin C (3) was further assessed by plotting its time−killing curve against C. albicans SC5314 strain; 16 μg/mL of 3 caused 99.8% cell death within 12 h, while the same fungicidal efficacy was achieved within 2 h using 32 μg/mL of 3 (Figure 3). Floricolin C (3) Disrupted the Integrity of Plasma Membrane. The cell membrane probes of DPH (1,6diphenyl-1,3,5-hexatriene) and propidium iodide were applied to investigate changes in the cell membrane caused by 3. DPH, as a membrane probe interacting with an acyl group of the plasma membrane, was used to monitor the changes in membrane dynamics. As shown in Figure 4, DPH fluorescence intensity of the plasma membrane was significantly decreased after treatment with 3 in a dose-dependent manner. A decrease in fluorescence intensity indicates disruption of the plasma membrane by 3 as well as by a positive control, amphotericin B.

Figure 3. Time−killing curves of floricolin C (3) against C. albicans. Overnight cultured C. albicans SC5314 cells were incubated with different concentrations of 3 in RPMI 1640 medium (1 × 106 cells/ mL) at 30 °C. The number of viable cells was determined by the colony counting method at specific times (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 12 h). Bars represent means ± SD.

The influx of propidium iodide was monitored by flow cytometry to indicate the permeability of the cell membrane when treated with floricolin C (3). 3 induced an increase in the fluorescence of propidium iodide in a dose-dependent manner, demonstrating that the fungal membrane was injured by 3. In conclusion, this data suggested that floricolin C (3) exerted fungicidal action through inducing high fluidity and permeabilization of the plasma membrane.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting point (uncorrected) was measured on an X-6 melting-point apparatus (Beijing TECHInstrument Co. Ltd.). Optical rotations were determined using a PerkinElmer 241MC polarimeter. UV data were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan). IR spectra were recorded using a Thermo Nicolet NEXUS 470 FT-IR spectrometer in KBr discs. NMR spectra were recorded on a Bruker AV spectrometer operating at 400 (1H) and 100 (13C) MHz or a Bruker Avance DRX600 spectrometer operating at 600 (1H) and 150 (13C) MHz with tetramethylsilane as an internal standard. HPLC was performed on an Agilent 1100 G1310A isopump equipped with a G1322A degasser, a G1314A VWD detector (210 nm), and a ZORBAX SB-C18 5 μm column (9.4 × 250 mm). Enantiomeric excesses were determined by HPLC using a Daicel Chiralpak AD-H column (5 μm, 250 × 10 mm) with hexane−i-PrOH as the eluent. Column chromatographies (CCs) were taken using silica gel (200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, China) and Sephadex LH-20 (25−100 D



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Figure 4. Effect of floricolin C (3) on cell membrane integrity. (A) Effect of 3 on cell membrane dynamics. Changes in DPH fluorescence intensity are expressed as relative fluorescence after treatment with various doses of 3 (0, 4, 8, 16, and 32 μg/mL) or 8 μg/mL of AMB (positive control) for 3 h followed by staining of DPH for spectrofluorophotometer detection. Bars represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Change of membrane permeabilization after cells were treated with 3 or 8 μg/mL of AMB (positive control). Treated cells were stained with PI and analyzed by flow cytometry. mL/min; 13.8 mg, tR = 13.8 min), 4 (HPLC, 50% MeOH−H2O, 1.8 mL/min; 14.3 mg, tR = 22.4 min), and 6 (HPLC, 50% MeOH−H2O, 1.8 mL/min; 1.2 mg, tR = 23.0 min). Floricolin A (1): yellow, amorphous powder; UV (MeOH) λmax (log ε) 316 (4.01) nm; IR (KBr) νmax 3472, 2942, 1657, 1605, 1322, 1261, 1168, 889 cm−1; 1H (acetone-d6, 400 MHz) and 13C NMR (acetoned6, 100 MHz), see Table 1; HRESIMS m/z 309.1120 [M + H]+ (calcd for C19H17O4, 309.1121) and 331.0938 [M + Na]+ (calcd for C19H16O4Na, 331.0941). Floricolin B (2): yellow, amorphous powder; UV (MeOH) λmax (log ε) 248 (4.27), 351 (3.64) nm; IR (KBr) νmax 3442, 2924, 1643, 1608, 1384, 1263, 1093 cm−1; 1H (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Table 1; HRESIMS m/z 307.0967 [M + H]+ (calcd for C19H15O4, 307.0965) and 329.0785 [M + Na]+ (calcd for C19H14O4Na, 329.0783). Floricolin C (3): yellow, amorphous powder; UV (MeOH) λmax (log ε) 320 (3.80) nm; IR (KBr) νmax 3434, 2936, 1655, 1603, 1497, 1450, 1320, 1289, 1171, 1080, 895, 756 cm−1; 1H (CDCl3, 400 MHz) and 13 C NMR (CDCl3, 100 MHz), see Table 1; HRESIMS m/z 307.0967 [M + H]+ (calcd for C19H15O4, 307.0965). Floricolin D (4): yellow, amorphous powder; UV (MeOH) λmax (log ε) 306 (4.06) nm; IR (KBr) νmax 3396, 2075, 1641, 1590, 1496, 1446, 1325, 1213, 1083, 787, 702 cm−1; 1H (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz), see Table 1; HRESIMS m/z 323.0917 [M + H]+ (calcd for C19H15O5, 323.0915) and 345.0736 [M + Na]+ (calcd for C19H14O5Na, 345.0734). Floricolin E (5): brown, amorphous powder; UV (MeOH) λmax (log ε) 210 (4.28), 301 (4.05) nm; IR (KBr) νmax 3609, 3387, 1669, 1490, 1410, 1241, 1029 cm−1; 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 2; HRESIMS m/z 307.0602 [M + H]+ (calcd for C18H11O5, 307.0601) and 329.0422 [M + Na]+ (calcd for C18H10O5Na, 329.0420). Floricolin F (6): red, amorphous powder; UV (MeOH) λmax (log ε) 209 (4.44), 253 (4.38), 296 (3.77) nm; IR (KBr) νmax 3630, 3330, 3106, 1657, 1566, 1492, 1444, 1309, 1220, 1118, 1089, 1049, 870, 829, 738 cm−1; 1H (acetone-d6, 600 MHz) and 13C NMR (acetone-d6, 150 MHz), see Table 2; HRESIMS m/z 321.0760 [M + H]+ (calcd for C 19 H 13 O5 , 321.0758) and 343.0578 [M + Na]+ (calcd for C19H12O5Na, 343.0577). Floricolin G (7): red crystal (MeOH); mp 144−146 °C; UV (MeOH) λmax (log ε) 207 (4.51), 248 (4.47), 336 (3.88) nm; IR (KBr) νmax 3321, 3298, 3073, 3056, 3020, 2958, 2858, 1659, 1555, 1444, 1303, 1121, 1049, 947, 871, 758 cm−1; 1H (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Table 2; HRESIMS m/z 305.0811 [M + H]+ (calcd for C19H13O4, 305.0808) and 322.1075 [M + NH4]+ (calcd for C19H16O4N, 322.1074). Floricolin H (8): red, amorphous powder; UV (MeOH) λmax (log ε) 292 (4.32), 336 (3.88) nm; IR (KBr) νmax 3459, 3057, 2851, 1633, 1593, 1469, 1414, 1225, 1092, 1037, 974, 910, 770 cm−1; 1H (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), see Table 2;

mm; Pharmacia Biotek, Denmark). TLC for monitoring was carried out with precoated silica gel GF-254 glass plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). The compounds were visualized under UV (254 nm) light and by spraying with H2SO4−EtOH (1:9, v/ v) followed by heating. Fungal Material. The fungus Floricola striata was isolated from the lichen Umbilicaria sp., collected from Mount Jiaozi of Yunnan Province in China. The strain, assigned no. 20128462B, was identified based on the nuclear 18S rDNA sequences (GenBank: GU479751) and deposited in the Key Lab of Chemical Biology of Ministry of Education, Shandong University, Jinan. It was cultured in 200 mL of potato dextrose broth at 30 °C on a rotary shaker (120 rpm) for 7 days to prepare the seed culture. Large-scale fermentation was carried out in 20 Erlenmeyer flasks (500 mL), each containing rice (80 g) and H2O (120 mL), and the contents were soaked overnight before autoclaving at 120 °C for 30 min. After cooling to room temperature, seed broth (10 mL) was added to each flask, and these flasks were maintained at room temperature for 30 days in stationary phase. Extraction and Isolation. The culture medium containing the mycelium was cut into small pieces and extracted using EtOAc. After removal of the EtOAc, the crude extract (67.0 g) was applied to a silica gel column with a gradient of cyclohexane−EtOAc from 1:0 to 0:1 (v/ v) to give six fractions, A−F. Compound 11 (616.3 mg) was crystallized from fraction A as a yellow crystal. Fraction C (1.0 g) was separated into three subfractions (C1−C3) by silica gel CC, eluting with a step gradient of cyclohexane−EtOAc from 1:0 to 0:1 (v/v). Fraction C2 (105.1 mg) was chromatographed over Sephadex LH-20 CC (MeOH) to afford two subfractions, C2A and C2B. Further separation of C2A and C2B by HPLC gave compound 13 (72% MeOH−H2O, 1.8 mL/min, 2.1 mg, tR = 20.4 min) and 12 (70% MeOH−H2O, 1.8 mL/min, 4.9 mg, tR = 11.3 min), respectively. Fraction C3 (53.4 mg) was further purified using HPLC (50% CH3CN−H2O, 1.8 mL/min) to yield 8 (4.7 mg, tR = 29.1 min) and 16 (1.6 mg, tR = 53.1 min). Fraction E (3.6 g) was fractionated using silica gel CC, eluting with a step gradient of petroleum ether−EtOAc from 1:0 to 0:1 (v/v) to yield four subfractions (E1−E4). Fraction E2 (72.8 mg) was further purified by HPLC (62% CH3CN−H2O, 1.8 mL/min) to yield 15 (6.6 mg, tR = 26.3 min). Fraction E3 (188.6 mg) was further separated by CC over silica gel using a gradient of petroleum ether−EtOAc from 1:0 to 0:1 (v/v) to give three subfractions (E3A− E3C). Further separation of E3A and E3C by HPLC gave compounds 3 (70% MeOH−H2O, 1.5 mL/min, 19.9 mg, tR = 17.0 min) and 10 (55% MeOH−H2O, 1.5 mL/min, 1.0 mg, tR = 17.1 min), respectively. Fraction E3B (57.8 mg) was fractionated using MPLC (ODS, MeOH−H2O from 30:70 to 100:0) to yield compounds 1 (7.3 mg), 2 (6.5 mg), and 7 (13.4 mg). Compounds 5 and 9 were separated from fraction E4 by HPLC (5: 30% MeOH−H2O, 1.8 mL/min, 1.0 mg, tR = 9.6 min; 9: 55% MeOH−H2O, 1.8 mL/min, 39.0 mg, tR = 20.7 min). Separation of fraction F following a procedure similar to that used for fraction E afforded 14 (HPLC, 50% MeOH−H2O, 1.8 E



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intensity of cells was analyzed using flow cytometry (FACS Calibur; BD Biosciences, San Jose, CA, USA). DPH was used to monitor the changes in the membrane dynamic challenged floricolin C (3). Fungal cells (1 × 106/mL YPD) were fixed with 0.37% formaldehyde for 30 min after treatment with 3 (concentrations ranging from 0 to 32 μg/mL) for 3 h and washed with PBS. The cells were then frozen with liquid nitrogen, thawed, and resuspended in PBS. The suspension was incubated with 1 μM DPH for 45 min at 30 °C and washed with PBS buffer. The fluorescence intensity of DPH was measured by a spectrofluorophotometer (Berthold Biotechnologies, Bad Wildbad, Germany) at 350 nm excitation and 450 nm emission wavelengths.

HRESIMS m/z 291.1018 [M + H]+ (calcd for C19H15O3, 291.1016) and 313.0838 [M + Na]+ (calcd for C19H14O3Na, 313.0835). Floricolin I (9): yellow crystal (MeOH); mp 184−186 °C; [α]20 D 0.1 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 300 (4.05) nm; IR (KBr) νmax 3411, 3324, 3065, 1728, 1691, 1586, 1494, 1375, 1027, 1167, 1087, 1051, 988, 825, 771 cm−1; 1H (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz), see Table 2; HRESIMS m/z 356.1131 [M + NH4]+ (calcd for C19H18O6N, 356.1129) and 361.0679 [M + Na]+ (calcd for C19H14O6Na, 361.0683). Floricolin J (10): yellow crystal (MeOH); mp 206−209 °C; [α]20 D −20.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 255 (3.81), 282 (3.86) nm; IR (KBr) νmax 3547, 3498, 3159, 2831, 1672, 1635, 1457, 1308, 1196, 1156, 1077, 1013, 951, 865 cm−1; 1H (acetone-d6, 600 MHz) and 13C NMR (acetone-d6, 150 MHz), see Table 2; HRESIMS m/z 355.1179 [M + H]+ (calcd for C20H19O6, 355.1176) and 377.0996 [M + Na]+ (calcd for C20H18O6Na, 377.0996). X-ray Crystallographic Analysis of Compounds 9 and 10. Upon crystallization from MeOH, yellow crystals were obtained for compounds 9 and 10. X-ray data were collected on an Oxford Diffraction Xcalibur Eos Gemini diffractometer with a graphite monochromator at 291.15 K, Cu Kα radiation. The structure was solved with the olex2.solve28 structure solution program using charge flipping and refined with the SHELXL29 refinement package using least squares minimization. Crystallographic data for the structure of this racemic mixture have been deposited in the Cambridge Crystallographic Data Center with the deposition numbers 1446394 and1446396, respectively. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: + 44-(0)1223-336033 or e-mail: deposit@ccdc. cam.ac.uk). Crystal data of 9: C19H14O6, M = 338.30, space group triclinic, P21; unit cell dimensions were determined to be a = 17.1992(14) Å, b = 11.1810(10) Å, c = 8.2334(7) Å, β = 98.336(4)°, V = 1566.6 (2) Å3, Z = 4, T = 291.15 K, Dcalc = 1.434 mg/mm3, F(000) = 704, μ(Cu Kα) = 0.904 mm−1, 7045 reflections measured (2.60 ≤ 2θ ≤ 50.42), 1487 unique (Rint = 0.0551), which were used in all calculations. The final R1 was 0.0351 (I > 2σ(I)) and wR2 was 0.0939 (all data). Crystal data of 10: C20H18O6·H2O, M = 372.36, space group triclinic, P21; unit cell dimensions were determined to be a = 8.0545(2) Å, b = 20.6132(6) Å, c = 11.6495(4) Å, β = 99.0520(10)°, V = 1910.07(10) Å3, Z = 4, T = 291.15 K, Dcalc = 1.295 mg/mm3, F(000) = 784, μ(Cu Kα) = 0.825 mm−1, 10 406 reflections measured (10.07 ≤ 2θ ≤ 65.36), 2246 unique (Rint = 0.0519), which were used in all calculations. The final R1 was 0.0552 (I > 2σ(I)) and wR2 was 0.1331 (all data). In Vitro Susceptibility Test of the Compounds against C. albicans. The MICs of all tested compounds against C. albicans were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (M27A3) as previously described.30 Overnight cultured C. albicans SC5314 were adjusted to an initial density of (2−5) × 103 cells/mL in RPMI 1640 medium; then compounds were added to suspensions at final concentrations ranging from 2 to 128 μg/mL and incubated at 35 °C for 48 h. The OD600 values for each well were determined with a plate reader, and the MICs were defined as the concentrations of drugs that reduced growth by 80%. Time−Killing Curve Assay.31 SC5314 cells from overnight cultures were washed and resuspended in RPMI 1640 medium at a cell density of 1 × 106 cells/mL buffered with MOPS. Floricolin C (3) was added to the cultures at final concentrations of 8, 16, and 32 μg/ mL, respectively, and incubated at 30 °C in a reciprocating shaker. At specific times (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 12 h), 100 μL aliquots were taken, washed, diluted, and placed on YPD agar plates for survival colony counting. Integrity of the Plasma Membrane Assay.32 The membrane perturbation is detected by a propidium iodide influx assay. C. albicans in the log phase was harvested by centrifugation, suspended in RPMI 1640 medium, and treated with floricolin C (3) for 3 h at 30 °C. Subsequently, the cells were stained with 5 μM propidium iodide and incubated for another 30 min at room temperature. The fluorescence

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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.6b00197. 1D and 2D NMR spectra of the new compounds 1−10 and HPLC analysis of 9 and 10 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Fax (Z.-T. Zhao): +86-531-8618-0749. E-mail: ztzhao@sohu. com. *Fax (H.-X. Lou): +86-531-8838-2019. E-mail: louhongxiang@ sdu.edu.cn. Author Contributions ⊥

W. Li and W. Gao contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 81273383 and 81473107). We thank Mr. H.-B. Zheng and Mr. B. Ma for the NMR measurements and Mrs. Y.-H. Gao for the HRESIMS determination.

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REFERENCES

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p-Terphenyl Derivatives from the Endolichenic Fungus Floricola striata

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