Cytotoxic pâTerphenyls from the Endolichenic ... - ACS Publications

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Cite This: J. Nat. Prod. 2018, 81, 2041−2049

Cytotoxic p‑Terphenyls from the Endolichenic Fungus Floricola striata Ke Xu,† Yun Gao,† Yue-Lan Li,† Fei Xie,† Zun-Tian Zhao,*,‡ and Hong-Xiang Lou*,† †

Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan 250012, People’s Republic of China ‡ College of Life Sciences, Shandong Normal University, No. 88 East Wenhua Road, Jinan 250014, People’s Republic of China

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S Supporting Information *

ABSTRACT: Eleven new p-terphenyls, floricolins K−U (1−11), together with 13 biosynthetically related known compounds (12−24) were isolated from an endolichenic fungus, Floricola striata. Their structures were elucidated by extensive spectroscopic analyses and single-crystal X-ray diffraction measurements. The newly isolated p-terphenyls inhibited the growth of A2780, MCF-7, and A549 cell lines. Further evaluation for the multidrug resistance (MDR) reversal activity of compound 5 revealed it enhanced the sensitivity of MCF-7/ADR cells toward adriamycin 39-fold at 10 μM through modulating P-glycoprotein-mediated drug exclusion.

T

Multidrug resistance (MDR), defined as a phenotype with cells resistant to multiple drugs with different structures and molecular targets, is a major obstacle in cancer chemotherapy.15 MDR is largely attributed to the overexpression of P-glycoprotein (P-gp), serving as a transmembrane efflux pump extruding anticancer drugs from cells.16,17 Inhibiting the overexpression of P-gp is an effective strategy to reverse drug resistance and thus restore cell sensitivity to drugs. Herein, all the new compounds were tested for cytotoxicity against A2780, MCF-7, and A549 cell lines using the MTT assay. Compound 5 was selected for evaluation in MDR-reversal activity against MCF-7/ADR cells.

erphenyls are aromatic hydrocarbons composed of a chain of three benzene rings and include p-terphenyls, m-terphenyls, and o-terphenyls. The most common natural terphenyls are p-terphenyl derivatives, in which two terminal benzene rings connect to the middle ring at the p-position.1 Over 230 p-terphenyl derivatives have been isolated mainly from basidiomycete macrofungi, with some from endolichenic fungi, actinomycetes, and mosses.2 In recent years, p-terphenyls have been reported to exhibit diverse biological activities, such as cytotoxic,3 antimicrobial,4 antioxidative,5 phosphodiesterase inhibitory,6 neuraminidase inhibitory,7 topoisomerase inhibitory8 and α-glucosidase inhibitory properties.5 In addition, terphenyls are easy to synthesize since they contain fewer chiral centers,1 generating a wide range of total synthesis products. For example, cytotoxic p-terphenyls kehokorins A−E9 and p-terphenyl glycosides terfestatins B and C exhibiting neuroprotective activity10 have been recently synthesized. During our ongoing study on endolichenic fungi,11−13 we have reported 10 p-terphenyl derivatives, floricolins A−J, with fungicidal action from an endolichenic fungus, Floricola striata.14 A chemical investigation on the same species F. striata found in a different lichen, Pseudosyphellaria spp. collected from Wangqing County of Jilin Province in China, led to the discovery of 11 new p-terphenyls, floricolins K−U (1−11), together with 13 known compounds (12−24). © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Structure Elucidation. Floricolin K (1) was obtained as a white, amorphous powder. Its molecular formula was determined as C19H14O4 with 13 degrees of unsaturation according to the HRESIMS and NMR data. It showed UV maxima at 211, 233, 278, and 337 nm, and the IR spectrum indicated absorption bands of the hydroxy group (3331 cm−1) and aromatic rings (1639, 1598 cm−1). The 1H NMR spectrum showed signals for one methoxy group (δH 3.98), nine aromatic Received: May 7, 2018 Published: August 23, 2018 2041

DOI: 10.1021/acs.jnatprod.8b00362 J. Nat. Prod. 2018, 81, 2041−2049

Journal of Natural Products

Article

Chart 1

Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 1, 5, 7, and 8.

C-5 and from OH-5′ to C-4′, C-5′, and C-6′ (Figure 1). Thus, the structure for compound 1 was unambiguously established as depicted and was given the serial nomenclature floricolin K after the congeners from the same species.14 Floricolin L (2), a white, amorphous powder, had the molecular formula C19H14O5 as determined by HRESIMS data. Comparison of the NMR data for compounds 1 and 2 suggested they shared the same substructures of rings A and B but differences in ring C. An additional phenolic hydroxy group (δH 9.12) revealed by the 1H NMR data and the obvious downfield chemical shift of C-4″ (δC 156.4) indicated the H-4″ in 1 was replaced by a hydroxy group in 2. Hence, compound 2 was elucidated as the 4″-hydroxyated analogue of 1 as shown. Floricolin M (3) was obtained as a white, amorphous powder, and HRESIMS data established the molecular formula C19H14O4. Its mass was 16 Da lower than that of 2, implying 3 was a dehydroxyated derivative of 2. Further comparison of the NMR data revealed their similarities in rings A and C, the presence of one more aromatic proton, and the absence of a hydroxy group at C-5′ of ring B in 3, which also supported the assignment of the structure for 3. Floricolin N (4), obtained as a brown, amorphous solid, had the same molecular formula as that of compound 2 on the

methines (δH 6.77−7.66), and two exchangeable protons (δH 9.32 and 9.40). 13C NMR spectrum showed 18 sp2 carbons. The 1H−1H COSY correlations of five aromatic protons [δH 7.45 (2H, t, J = 7.2 Hz, H-3″/5″), 7.66 (2H, d, J = 7.2 Hz, H-2″/6″), 7.35 (1H, t, J = 7.2 Hz, H-4″)] led to the identification of a monosubstituted benzene ring (Figure 1). A trisubstituted phenyl unit was established according to the coupling patterns of three aromatic protons [δH 6.91 (1H, dd, J = 8.8, 2.4 Hz, H-4′), δH 7.49 (1H, d, J = 8.8 Hz, H-3′), and δH 7.43 (1H, d, J = 2.4 Hz, H-6′)]. The remaining six aromatic carbons and an isolated aromatic proton at δH 6.77 (1H, s, H-6) indicated the presence of a pentasubstituted phenyl ring, which was also supported by the key HMBC correlations as shown in Figure 1. The key HMBC correlations from H-6 to C-1″ (δC 138.5) and from H-6′ to C-2 (δC 113.3) disclosed the connection of C-1″−C-5 and C-2−C-1′ (Figure 1), respectively, suggesting a typical p-terphenyl type skeleton in 1. An oxygen bridge connecting C-3 and C-2′ was deduced based on the requirement of degrees of unsaturation and the chemical shifts of C-3 (δC 147.0) and C-2′ (δC 148.9). The HMBC correlation of the methoxy signal at δH 3.98 with C-1 placed this group on C-1. Two hydroxy groups were located at C-4 and C-5′, confirmed by the HMBC correlations from OH-4 to 2042



Article

Figure 2. X-ray crystallographic structure of compounds 5 and 7 (relative configuration).

basis of HRESIMS and NMR data. Its 1H and 13C NMR data suggested similar structural features to those of 2, apart from the upfield shift of C-3′ (δC 98.0) and C-1′ (δC 115.1) and the splitting pattern of H-3′ (δH 7.01) in ring B. The above analysis indicated that the hydroxy group at C-5′ in 2 was connected to C-4′ in 4. Therefore, the structure of 4 was determined as depicted. Floricolin O (5) was isolated as red acerose crystals. A molecular formula of C20H16O4 was assigned for 5 from the HRESIMS data. The NMR data of 5 were very similar to those of the known compound floricolin B (15),14 except for an extra methoxy group instead of a phenolic hydroxy group, suggesting 5 was a methylated analogue of floricolin B. Two carbonyl signals resonating at δC 183.0 and 186.3 supported the presence of the para-quinone moiety. The HMBC correlation from OCH3-4′ (δH 3.80) to C-4′ (δC 159.2) (Figure 1) unambiguously verified the position of the new methoxy group was at C-4′. Finally, a single-crystal X-ray diffraction analysis further confirmed the structure of 5 (Figure 2). Floricolin P (6) was isolated as a rufous, amorphous solid and had a molecular formula of C19H14O5 as determined from HRESIMS data. Compound 6 was identified as a terphenyl derivative with the same ring A as 5 by analysis of the NMR spectra. As shown by the 1H NMR and 1H−1H COSY spectra, the aromatic protons at δH 6.86 (2H, d, J = 8.8 Hz, H-3″/5″) and 7.47 (2H, d, J = 8.8 Hz, H-2″/6″) revealed the presence of a para-substituted phenyl ring, and four interrelated aromatic methines (δH 6.85, 6.87, 7.02, and 7.22) constructed an orthosubstituted phenyl unit. The molecular formula and low-field chemical shifts of C-2′ (δC 155.1) and C-4″ (δC 159.2) indicated the two carbons should be connected to hydroxy groups. Thus, the structure of 6 was elucidated as shown. Floricolin Q (7) was obtained as colorless acerose crystals. The molecular formula was determined to be C18H14O5 by HRESIMS. Comparing its NMR data with those of 1, three of the same moietiesa monosubstituted phenyl, a trisubstituted phenyl, and a furan ringwere identified. In contrast to 5 and 6, the 1D NMR and HSQC data for 7 showed the presence of one methylene, one oxygenated methine, and one oxygenated carbon (δC 79.0) and the absence of the two aromatic/olefinic carbons and the one carbonyl signals found in 5 and 6. Furthermore, the HMBC correlations from OH-4 (δH 6.07) to C-4 (δC 70.0) and from OH-5 (δH 5.50) to C-5 (δC 79.0) indicated the two hydroxy groups were located at C-4 and C-5, respectively. The above evidence supported ring A of 7 being a 4,5-hydroxyated cyclohexenone. The relative configuration of

7 was assigned on the basis of the NOESY data. Key NOESY correlations of OH-5 (δH 5.50) with Hβ-6 (δH 2.63) and of Hα-6 (δH 3.37) with H-4 (δH 5.44) (Figure 3) indicated a

Figure 3. Key NOESY correlations of compound 7.

cofacial relationship between OH-5 and OH-4, which was also supported by the single-crystal X-ray diffraction analysis using Mo Kα radiation (Figure 2). The absolute configuration was determined by analysis of the electronic circular dichroism (ECD) spectrum and the helicity rule of α,β-unsaturated ketones (S90 and S91, Supporting Information).18,19 It should be emphasized that the sign of the n → π* transition in the CD curves depends on the helicity of the enone moiety. The enone helicity of 7 was defined as negative ring chirality by the conformation of the cyclohexenone ring (S91, Supporting Information). Therefore, the negative Cotton effect at 311 nm (Δε311 −0.2973) suggested the absolute configuration of 7 should be 4S, 5R. Floricolin R (8) was obtained as a yellow, amorphous solid. HRESIMS data gave the molecular formula C21H20O4. Compound 8 shared the same structural skeleton with the known compound floricolin A (23),14 as shown by similar NMR data. However, the presence of two more methoxy groups and the absence of two signals for hydroxy groups in the 1H NMR data suggested that two hydroxy groups in floricolin A were methylated in 8. In addition, HMBC correlations from OMe-4 (δH 3.59) to C-4 (δC 60.8) and from OMe-4′ (δH 3.88) to C-4′ (δC 55.3) further confirmed the connection of these two methoxy groups as depicted. Floricolin S (9), a white, amorphous powder, had the molecular formula C22H22O4 based on HRESIMS data. Interpretation of the MS and 1D NMR spectra suggested that 9 was an analogue of 8, with the hydroxy group at C-1 in 8 being methylated in 9. Further observation of the HMBC correlation from OMe-1 (δH 3.74) to C-1(δC 153.2) also provided evidence for the assignment of the structure for 9. 2043



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Figure 4. Effect of compound 5 on intracellular accumulation of ADM. (a) The ADM accumulation (%) was measured in the presence or absence of 5 or Ver in MCF-7 and MCF-7/ADR cells by flow cytometry. The fluorescence intensity in cells without treatment of inhibitory agents was set as the negative control. Ver (10 μg/mL) was the positive control. Data were expressed as the mean ± SD from three independent experiments. **P < 0.01. (b) The fluorescence intensity of ADM was used to display its accumulation in MCF-7/ADR cells with or without 5 (10 μM) or Ver (10 μg/mL). Bar = 20 and 10 μm.

Figure 5. Effect of compound 5 on intracellular accumulation of Rh123. (a) Rh123 accumulation (%) was evaluated by flow cytometry to monitor the accumulation level of Rh123 in cells treated with different concentrations of 5. The fluorescence intensity in cells without inhibitors was set as the negative control. Ver (10 μg/mL) was the positive control. The results are presented as the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001. (b) The fluorescence intensity of Rh123 represents the accumulation in MCF-7/ADR cells in the presence or absence of 5 (10 μM) or Ver (10 μg/mL). Bar = 20 and 10 μm.

floricolin B (15),14 betulinan A (16),23 floricolin E (17),14 2,4dimethoxy-3,6-di(p-methoxyphenyl)phenol (18),20 2′,3′,5′trimethoxy-p-terphenyl (19),24 pentamethoxy-p-terphenyl (20),25 terphenyl 2 (21),26 terphenyl 3 (22),26 floricolin A (23)14 and BTH-II0204-207 (A) (24)27 by comparison of the spectroscopic data with those reported. Cytotoxicity and the Ability to Reverse Adriamycin (ADM) Resistance. All new p-terphenyls (1−11) were tested for their cytotoxicity against the A2780, MCF-7, and A549 cell lines using the MTT assay with ADM as a positive control. The results (Table 4) showed that compounds 5 and 6, containing a para-quinone moiety, exhibited significant cytotoxicity (IC50 < 10 μM) against the A2780 cell line but weak cytotoxicity (IC50 > 10 μM) against the MCF-7 cell line, whereas compounds 1, 2, 3, 4, and 10 displayed weak cytotoxicity. Based on the preliminary results, the para-quinone moiety in 5 and 6 played a vital role for the cytotoxicity, while the hydroxy group at C-4 in the structures as for 1, 2, 3, 4, and 10 would enhance the cytotoxicity. Furthermore, compound 4 exhibited higher potency than 1, 2, and 3 against

Floricolin T (10) was isolated as a white, amorphous solid that analyzed for the molecular formula C21H20O4 by HRESIMS data. The NMR data of rings A and C were similar to those of the known compound 2,4-dimethoxy-3,6-di(p-methoxyphenyl)phenol (18).20 Considering the absence of a methoxy group signal as well as the upfield chemical shift of C-4′ (δC 127.3), 10 was determined to be a 4′-demethoxylated derivative of 18 as depicted. Floricolin U (11) was obtained as a yellow, amorphous solid, with a molecular formula of C22H22O5 as derived from HRESIMS data. Similarities in the NMR data between 11 and the known compound pentamethoxy-p-terphenyl (20)25 indicated they had the same substructures of rings A and C. Ring B of 11 was identified as an ortho-substituted benzene ring based on the 1 H−1H COSY correlations of four remaining contiguous aromatic methines (δH 7.04−7.33). The OH-2′ (δH 5.72) were proved to be located at C-2′ through analyzing the corresponding HMBC correlations. Thus, the structure of 11 was established. The known compounds were determined as kehokorins D (12),21 floricolin D (13),14 3,5-diarylbenzoquinones (14),22 2044



Article

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data in DMSO-d6 for Compounds 1−4 1 position

δC, type

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

147.8, C 113.3, C 147.0, C 133.2, C 127.8, C 105.7, CH 123.9, C 148.9, C 111.5 CH 114.6, CH 153.5, C 107.5, CH 138.5, C 129.5, CH 128.0, CH 126.8, CH 55.9, CH3

2

δH, mult. (J in Hz)

6.77, s

7.49, d (8.8) 6.91, dd (8.8, 2.4) 7.43, d (2.4) 7.66, 7.45, 7.29, 3.98, 9.32, 9.40,

d (7.2) t (7.2) t (7.2) s s s

δC, type 147.7, C 112.6, C 147.1, C 132.9, C 128.0, C 105.5, CH 124.0, C 148.8, C 111.4, CH 114.3, CH 153.5, C 107.4, CH 129.1, C 130.6, CH 114.8, CH 156.4, C 55.8, CH3

3


6.72, s

7.47, d (8.8) 6.88, dd (8.8, 2.8) 7.40, d (2.8) 7.49, d (8.8) 6.84, d (8.8) 3.97, s

several tumor cell lines, suggesting the OH-4′ increased cytotoxicity. Among the tested compounds in the cytotoxicity assay, compound 5 showed the most potent activity, and enough material was available to do further experiments. Herein, compound 5 was selected for evaluation in MDR reversal activity in ADM-selected P-gp-overexpressing MCF-7/ADR cells. As shown in Table 5, compound 5 could enhance the cytotoxicity of ADM 39-fold against MCF-7/ADR cells at a nontoxic concentration of 10.0 μM. Effect of Compound 5 on Adriamycin Accumulation. To investigate whether the reversal activity of 5 is associated with a concomitant increase in intracellular ADM accumulation, an accumulation assay using flow cytometry and fluorescence microscopy was carried out. Figure 4(a) shows that 5 can increase ADM accumulation in a dose-dependent manner in MCF-7/ADR cells. In contrast, the accumulation of ADM in MCF-7 cells remained nearly invariant in the presence of 5 or verapamil (Ver). Figure 4(b) further visually displays that the fluorescence intensity of ADM was very weak in the MCF-7/ ADR cells without treatment of inhibitory agents, whereas in those cells treated with 10 μM 5 or 10 μg/mL Ver, the fluorescence intensity was remarkably intense. These results suggested that 5 enhanced the sensitivity of MCF-7/ADR cells toward ADM through increasing intracellular ADM accumulation. Inhibitory Effect of Compound 5 on P-gp-Mediated Rhodamine 123 (Rh123) Efflux. The decrease in intracellular ADM accumulation in MCF-7/ADR cells is probably due to P-gp-mediated drug efflux. Rh123 is a P-gp-specific fluorescent dye that has been widely applied to assess the potency of P-gp inhibitors. Hence, we detected the intracellular fluorescence intensity of Rh123 by flow cytometry and fluorescence microscopy to further prove compound 5 could interfere with the P-gp function. As depicted in Figure 5(a) and (b), the accumulation of Rh123 in MCF-7/ADR cells treated with 5 or Ver was obviously higher than that in the negative control and was significantly increased in a dose-dependent

δC, type 147.8, C 112.2, C 146.4, C 132.9, C 128.5, C 106.0, CH 123.4, C 154.9, C 111.2, CH 126.3, CH 123.2, CH 122.3, CH 129.0, C 130.6, CH 114.9, CH 156.5, C 55.8, CH3

4


6.78, s

7.69, 7.48, 7.40, 8.04,

d (8.0) m t (7.6) d (7.6)

7.51, d (8.8) 6.85, d (8.8) 3.99, s 9.24, s 9.53, s

δC, type 147.0, C 112.7, C 146.2, C 132.8, C 129.2, C 106.0, CH 115.1, C 156.5, C 98.0, CH 157.1, C 111.8, CH 122.6, CH 126.8, C 130.6, CH 114.8, CH 156.3, C 55.8, CH3


6.71, s

7.01, d (2.0) 6.84, dd (8.0, 2.0) 7.78, d (8.0) 7.47, d (8.8) 6.83, d (8.8) 3.95, s

pattern. All the experimental data indicated that compound 5 could reverse MDR by modulating P-gp-mediated drug efflux effectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-6 melting-point apparatus (Beijing TECH Instrument Co. Ltd.). Optical rotations were acquired by a PerkinElmer 241MC polarimeter at 20 °C. HRESIMS data were determined on a Finnigan LC-QDECA mass spectrometer. ECD spectra were performed on a Chirascan spectropolarimeter. UV data were recorded using a UV-2550 spectrophotometer (Shimadzu, Japan). IR spectra were obtained on a Nicolet iN 10 Micro FTIR spectrometer. NMR spectra were recorded on a Bruker Avance spectrometer operating at 400 (1H) and 100 (13C) MHz or at 600 (1H) and 150 (13C) MHz with tetramethylsilane as an internal standard. HPLC were performed on an Agilent 1200 G1311A pump equipped with a G1322A degasser, a G1315D DAD detector, and an Eclipse XDB-C18 5 μm column (9.4 × 250 mm). Column chromatography (CC) was carried out using silica gel (200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, China) and Sephadex LH-20 (25−100 mm; Pharmacia Biotek, Denmark). Precoated silica gel GF-254 glass plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, China) were used for TLC. 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 Pseudosyphellaria spp. collected from Wangqing County of Jilin Province in China. The strain, assigned the accession no. 8938a, was identified using nuclear 18S rDNA sequences (GenBank: GU479751.1) and was deposited in the Key Lab of Chemical Biology of Ministry of Education, Shandong University, Jinan, China. The fungus was cultured in three 300 mL Erlenmeyer flasks containing 100 mL of potato dextrose broth at 25 °C on a rotary shaker (110 rpm) for 7 days to obtain the seed culture. Large-scale fermentation proceeded in 20 Erlenmeyer flasks (500 mL) containing 80 g of rice and 120 mL of distilled water, which were autoclaved at 120 °C. After cooling to room temperature, each flask was inoculated with 10 mL of seed culture and incubated at room temperature for 50 days. Extraction and Isolation. The culture medium including the mycelium was extracted three times with EtOAc. After removal of the organic solvent, the crude extract (20.0 g) was separated into eight 2045



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Table 2. 1H (400 MHz) and 13C (100 MHz) NMR Data in DMSO-d6 for Compounds 5−7 5 position

δC, type

1 2 3 4 5 6

187.0, 128.2, 155.2, 182.6, 144.2, 131.8,

C C C C C CH

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

122.3, C 131.9, CH 113.2, CH 159.2, C 113.2, CH 131.9, CH 132.7, C 129.4, CH 128.3, CH 129.7, CH 60.8, CH3 55.1, CH3

6 δH, mult. (J in Hz)

6.94, s

7.27, d (8.8) 7.01, d (8.8) 7.01, d (8.8) 7.27, d (8.8) 7.59, 7.49, 7.49, 3.84, 3.80,

m m m s s

δC, type 186.3, 125.6, 155.9, 183.0, 143.6, 129.9,

C C C C C CH

118.4, C 155.1, C 115.3, CH 129.5, CH 118.3, CH 131.3, CH 123.2, C 131.0, CH 115.2, CH 159.2, C 60.1, CH3

7 δH, mult. (J in Hz)

6.83, s

6.87, 7.22, 6.85, 7.02,

m td (7.6, 1.6) m dd (7.6, 1.6)

7.47, d (8.8) 6.86, d (8.8)

δC, type 191.6, C 114.6, C 169.8, C 70.0, CH 79.0, C 51.8, CHβ CHα 124.0, C 148.6, C 112.1, CH 113.5, CH 154.8, C 105.6, CH 144.7, C 125.9, CH 127.8, CH 126.9, CH

δH,mult. (J in Hz)

5.44, d (7.6) 2.63, d (16) 3.37, m

7.51, d (8.8) 6.81, dd (8.8, 2.4) 7.29, m 7.64, d (7.6) 7.39, t (7.6) 7.27, m

3.73, s 6.07, d (7.6) 5.50, d (1.2) 9.45, s

fractions (A−H) by a silica gel column with a step gradient of CH2Cl2−MeOH from 100:0 to 0:100 (v/v). Fraction A (4.5 g) was chromatographed over Sephadex LH-20 CC eluted with CH2Cl2−MeOH (1:1) twice to yield five subfractions, A1−A5. Fractions A3 (1.6 g) and A4 (249.0 mg) were both separated into six parts (A3A−A3F and A4A−A4F) using MPLC (ODS, MeOH− H2O from 40:60 to 100:0). Fraction A3A (345.6 mg) was further fractionated by Sephadex LH-20 CC with CH2Cl2−MeOH (1:1) and afforded three subfractions, A3A1−A3A3. Further purification of A3A1, A3A2, and A4A by HPLC gave compounds 16 (74% MeOH−H2O, 1.5 mL/min, 1.5 mg, tR = 20.0 min), 10 (74% MeOH−H2O, 1.5 mL/min, 6.9 mg, tR = 21.6 min), and 24 (70% MeOH−H2O, 1.5 mL/min, 51.3 mg, tR = 14.4 min), respectively. Fraction A3B was purified with HPLC (84% MeOH−H2O, 1.5 mL/min) to yield compounds 9 (3.3 mg, tR = 22.7 min) and 19 (6.0 mg, tR = 24.1 min). Fraction B (1.8 g) was subjected to Sephadex LH-20 CC by elution with CH2Cl2−MeOH (1:1) to afford six subfractions (B1−B6). Compound 5 (16.0 mg) was crystallized out from fraction B as red needle crystals. Fraction B4 (1.5 g) was separated into eight subfractions (B4A−B4H) with MPLC (ODS, MeOH−H2O from 30:70 to 100:0). Fraction B4B (30.5 mg) was prepared using HPLC (75% MeOH−H2O, 1.5 mL/min) to afford compounds 8 (1.2 mg, tR = 22.7 min), 14 (2.2 mg, tR = 20.7 min), and 18 (6.9 mg, tR = 27.4 min). Compound 20 (1.2 mg, tR = 30.5 min) was obtained from fraction B4C (27.9 mg) by HPLC (80% MeOH−H2O, 1.5 mL/min). Separation of fraction C (3.5 g) was performed by Sephadex LH-20 CC eluted with CH2Cl2−MeOH (1:1), producing seven subfractions, C1−C7. Fractions C3, C5, and C6 were fractionated using MPLC (ODS, MeOH−H2O from 10:90 to 100:0). Preparation of fractions C5E and C5F by HPLC yielded compounds 22 (65% MeOH−H2O, 1.5 mL/min, 8.2 mg, tR = 13.2 min) and 23 (64% MeOH−H2O, 1.5 mL/min, 4.0 mg, tR = 14.3 min) and compounds 15 (70% MeOH−H2O, 1.5 mL/min, 1.8 mg, tR = 18.9 min) and 17 (64% MeOH−H2O, 1.5 mL/min, 1.9 mg, tR = 17.9 min), respectively. Fractions C6C and C3C were subjected to HPLC to obtain compounds 1 (73% MeOH−H2O, 1.5 mL/min, 4.1 mg, tR = 16.2 min) and 11 (74% MeOH−H2O, 1.5 mL/min, 2.8 mg, tR = 23.7 min), respectively. Compounds 3 (1.6 mg, tR = 17.6 min) and 12 (1.8 mg, tR = 23.2 min) were purified from fraction C6E using HPLC (76% MeOH−H2O, 1.5 mL/min).

Isolation of fraction E (1.8 g) following a similar procedure to that used for fraction C afforded 2 (HPLC, 60% MeOH−H2O, 1.5 mL/min; 1.8 mg, tR = 17.8 min), 4 (HPLC, 60% MeOH−H2O, 1.5 mL/min; 2.0 mg, tR = 29.2 min), 6 (HPLC, 42% MeCN−H2O, 1.5 mL/min; 1.6 mg, tR = 22.7 min), 7 (HPLC, 50% MeOH−H2O, 1.5 mL/min; 0.6 mg, tR = 20.7 min), 13 (HPLC, 43% MeCN−H2O, 1.5 mL/min; 3.3 mg, tR = 17.7 min), and 21 (HPLC, 43% MeCN− H2O, 1.5 mL/min; 40.6 mg, tR = 16.0 min). Floricolin K (1): white, amorphous powder; UV (MeOH) λmax (log ε) 211 (4.35), 233 (4.24), 278 (4.13), 337 (3.94) nm; IR νmax 3331, 2936, 1639, 1598, 1447, 1223, 1181, 820 cm−1; 1H (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 1; HRESIMS m/z 305.0818 [M − H]− (calcd for C19H13O4, 305.0814). Floricolin L (2): white, amorphous powder; UV (MeOH) λmax (log ε) 214 (5.17), 285 (4.94), 338 (4.82) nm; IR νmax 3345, 2958, 1607, 1503, 1449, 1224, 1180, 1099, 826 cm−1; 1H (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 1; HRESIMS m/z 321.0769 [M − H]− (calcd for C19H13O5, 321.0763). Floricolin M (3): white, amorphous powder; UV (MeOH) λmax (log ε) 213 (4.51), 286 (4.21), 325 (4.04) nm; IR νmax 3361, 2933, 1607, 1502, 1448, 1221, 1095, 829 cm−1; 1H (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 1; HRESIMS m/z 305.0818 [M − H]− (calcd for C19H13O4, 305.0814). Floricolin N (4): brown, amorphous powder; UV (MeOH) λmax (log ε) 211 (4.37), 278 (4.14), 324 (4.11) nm; IR νmax 3360, 2936, 1611, 1504, 1444, 1223, 1096, 829 cm−1; 1H (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 1; HRESIMS m/z 321.0766 [M − H]− (calcd for C19H13O5, 321.0763). Floricolin O (5): red, acerose crystals; mp 174−175 °C; UV (MeOH) λmax (log ε) 245 (4.29) nm; IR νmax 3263, 2923, 1642, 1606, 1511, 1443, 1253, 1094, 808 cm−1; 1H (DMSO-d6, 400 MHz) and 13 C NMR (DMSO-d6, 100 MHz), see Table 2; HRESIMS m/z 321.1124 [M + H]+ (calcd for C20H17O4, 321.1127) and 343.0942 [M + Na] + (calcd for C20H16O4Na, 343.0946). Floricolin P (6): rufous, amorphous powder; UV (MeOH) λmax (log ε) 246 (4.63) nm; IR νmax 3370, 2920, 1667, 1603, 1511, 1447, 1222, 1084, 837 cm−1; 1H (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz), see Table 2; HRESIMS m/z 323.0917 [M + H]+ (calcd for C19H15O5, 323.0919). 2046



Article

Table 3. 1H (400 Mz) and 13C NMR Data in CDCl3 for Compounds 8−11 8a position

δC, type

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

149.1, C 121.8, C 151.4, C 144.3, C 135.9, C 111.6, CH 124.2, C 131.5, CH 114.7 CH 159.5, C 114.7, CH 131.5, CH 137.9, C 129.1, CH 128.2, CH 127.3, CH

9a


δC, type

7.37, d (8.8) 7.07, d (8.8) 7.07, d (8.8) 7.37, d (8.8) 7.59, d (7.2) 7.45, t (7.2) 7.36, m

60.9, CH3 60.8, CH3 55.3, CH3


153.2, C 124.5, C 151.9, C 144.8, C 134.8, C 108.0, CH 125.9, C 131.6, CH 113.3, CH 158.6, C 113.3, CH 131.6, CH 138.4, C 129.2, CH 128.2, CH 127.3, CH 56.1, CH3 60.8, CH3 60.9, CH3 55.2, CH3

6.77, s

3.68, s 3.59, s 3.88, s

10b 150.2, C 122.7, C 145.3, C 140.3, C 126.4, C 108.4, CH 133.5, C 130.6, CH 128.0, CH 127.3, CH 128.0, CH 130.6, CH 130.1, C 130.2, CH 113.8, CH 158.9, C 56.4, CH3 60.6, CH3

6.70, s 7.37, d (8.8) 7.00, d (8.8) 7.00, d (8.8) 7.37, d (8.8) 7.60, 7.47, 7.39, 3.74, 3.64, 3.62, 3.86,

δC, type

d (7.2) m m s s s s

55.3, CH3

11b


6.73, s 7.51, 7.47, 7.38, 7.47, 7.51,

d (7.2) t (7.2) t (7.2) t (7.2) d (7.2)

7.63, d (8.8) 7.02, d (8.8) 3.72, s 3.37, s

3.87, s

δC, type 153.0, C 119.1, C 152.1, C 145.1, C 130.3, C 108.5, CH 121.0, C 153.5, C 117.1, CH 129.3, CH 120.4, CH 132.2, CH 135.8, C 130.2, CH 113.8, CH 159.1, C 56.4, CH3 61.2, CH3 60.9, CH3 55.3, CH3


6.74, s

7.08, 7.33, 7.04, 7.31,

dd (8.4, 0.8) m m m

7.55, d (8.8) 7.01, d (8.8) 3.78, s 3.64, s 3.61, s 3.88, s

4.87, s 5.74, s 5.72, s

a13

C NMR was recorded at 150 MHz.

b13

C NMR was recorded at 100 MHz. 276 (+2.33), 311 (−0.30); UV (MeOH) λmax (log ε) 214 (3.41), 238 (3.32), 273 (2.92) nm; IR νmax 3363, 1658, 1586, 1491, 1458, 1201, 1030, 802 cm−1; 1H (DMSO-d6, 400 MHz) and 13C NMR (DMSOd6, 100 MHz), see Table 2; HRESIMS m/z 311.0916 [M + H]+ (calcd for C18H15O5, 311.0919). Floricolin R (8): yellow, amorphous solid; UV (MeOH) λmax (log ε) 206 (4.43), 262 (3.97) nm; IR νmax 3387, 2931, 1604, 1517, 1460, 1245, 1060, 821 cm−1; 1H (CDCl3, 400 MHz) and 13 C NMR (CDCl3, 150 MHz), see Table 3; HRESIMS m/z 337.1436 [M + H]+ (calcd for C21H21O4, 337.1440). Floricolin S (9): white, amorphous powder; UV (MeOH) λmax (log ε) 264 (4.24) nm; IR νmax 3050, 2940, 1607, 1518, 1461, 1225, 1107, 835 cm−1; 1H (CDCl3, 400 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 3; HRESIMS m/z 351.1589 [M + H]+ (calcd for C22H23O4, 351.1596) and m/z 373.1409 [M + Na]+ (calcd for C22H22O4Na, 373.1416). Floricolin T (10): white, amorphous solid; UV (MeOH) λmax (log ε) 264 (4.10), 310 (3.80) nm; IR νmax 3494, 2936, 1609, 1518, 1462, 1247, 1100, 830 cm−1; 1H (CDCl3, 400 MHz) and 13 C NMR (CDCl3, 100 MHz), see Table 3; HRESIMS m/z 337.1434 [M + H]+ (calcd for C21H21O4, 337.1440). Floricolin U (11): yellow, amorphous solid; UV (MeOH) λmax (log ε) 206 (4.45), 261 (4.05) nm; IR νmax 3411, 2934, 1607, 1516, 1462, 1249, 1106, 832 cm−1; 1H (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Table 3; HRESIMS m/z 367.1542 [M + H]+ (calcd for C22H23O5, 367.1545). X-ray Crystallography of Compounds 5 and 7: Crystal data of 5: C20H16O4, M = 320.34, monoclinic, space group P21/c, a = 16.2197(8) Å, b = 7.3971(4) Å, c = 14.6244(8) Å, β = 113.672(3)°, V = 1606.98(15) Å3, Z = 4, Dcalcd = 1.312 g/cm3, μ(Cu Kα) = 1.542 mm−1, F(000) = 668, 6297 reflections measured (5.957° ≤ 2θ ≤ 68.573°), 2169 unique, which were used in all calculations. The final R1 was 0.1081 (>2σ(I)) and wR2 was 0.2434 (all data). Crystal data of 7: C18H14O5, M = 310.08, monoclinic, space group P21/c, a = 7.1391(9) Å, b = 13.972(2) Å, c = 15.793(2) Å, β = 101.538(4)°, V = 1543.5(4) Å3, Z = 2, Dcalcd = 1.443 g/cm3,

Table 4. Cytotoxicity of Compounds 1−11 against A2780, MCF-7, and A549 Cell Lines IC50 (μM) compound

A2780

MCF-7

A549

1 2 3 4 5 6 7 8 9 10 11 adriamycin

25.1 ± 2.3 21.4 ± 1.9 40.1 ± 3.8 14.9 ± 1.4 3.4 ± 0.6 8.6 ± 1.0 >40 >40 >40 >40 >40 0.5 ± 0.1

13.4 ± 1.4 17.9 ± 2.4 17.5 ± 1.7 11.7 ± 1.2 19.9 ± 2.1 16.7 ± 0.8 >40 >40 >40 38.5 ± 4.0 >40 1.3 ± 0.2

>40 >40 >40 27.8 ± 2.0 40.1 ± 3.8 >40 >40 >40 >40 12.5 ± 2.5 >40 1.3 ± 0.1

Table 5. Multidrug Resistance Reversal Activity of Compound 5 against the MCF-7/ADR Cell Line IC50 (μM) treatment 5 adriamycin adriamycin + 5 (5 μM) adriamycin + 5 (10 μM) adriamycin + verapamil (10 μg/mL)

MCF-7/ADR

MCF-7

RFa (MCF-7/ADR)

23.5 ± 2.2 39.1 ± 2.4 29.8 ± 1.9 1.0 ± 0.3 4.3 ± 1.1

19.9 ± 2.1 1.3 ± 0.2 1.1 ± 0.4 0.1 ± 0.03 1.5 ± 0.4

1.3 39.1 9.1

a

Reversal fold (RF) = IC50(adriamycin)/IC50(adriamycin + 5).

Floricolin Q (7): colorless, acerose crystals; mp 216−217 °C; [α]20D 24.2 (c 0.05, MeOH); ECD (MeOH) λmax (Δε) 232 (−3.73), 2047



Article

μ(Mo Kα) = 0.711 mm−1, F(000) = 704, 4076 reflections measured (2.633° ≤ 2θ ≤ 26.014°), 3916 unique, which were used in all calculations. The final R1 was 0.0551 (>2σ(I)) and wR2 was 0.0885 (all data). Crystallographic data of 5 and 7 have been deposited with the Cambridge Crystallographic Data Centre with the deposition numbers CCDC 1816352 and CCDC 1833857, respectively. The data can be obtained free of charge via www.ccdc.cam.ac.uk/products/csd/ request. Cytotoxicity and MDR Reversal Activity Assays. The cytotoxicity of compounds 1−11 against human ovarian cancer cell line A2780, human non-small-cell lung cancer cell line A549, and human breast carcinoma cell line MCF-7 was tested using the MTT assay. Cells were seeded into 96-well plates at 5000−10 000 cells/well and incubated for 12 h (37 °C, 5% CO2). Different concentrations of the positive control ADM and the test compounds 1−11 were subsequently added and incubated for 48 h. Then 10 μL of MTT was added into each well. After 4 h, the absorbance at 570 nm was measured and the percentage of cell growth inhibition was calculated to get the IC50 values. The MDR reversal activity of compound 5 was tested by the same method. Various concentrations of ADM were added into wells in the presence or absence of compound 5 (5, 10 μM) or Ver (10 μg/mL), which were then seeded with MCF-7 cells and its ADM-selected P-gpoverexpressing cell line MCF-7/ADR, respectively, for 48 h for the MTT assay. Finally, the IC50 values of ADM as well as ADM in combination with compound 5 or Ver were calculated to obtain the reversal fold. Intracellular Adriamycin Accumulation Assay. The ADM accumulation assay was carried out by reference to the reported procedure.28 Briefly, 4 × 105 MCF-7 and MCF-7/ADR cells were incubated with 10.0 μM ADM alone or in combination with different concentrations of 5 (5, 10, 20 μM) or Ver (10 μg/mL) for 1 h at 37 °C. The cells were collected and washed twice with ice-cold phosphate-buffered saline. The ADM accumulation (%) was determined by flow cytometry to monitor the accumulation level of ADM. The pictures of ADM accumulation in MCF-7/ADR cells were taken using a fluorescence microscope after incubation for 24 h in the presence or absence of 5 (10 μM) or Ver (10 μg/mL). Intracellular Rh123 Accumulation Assay. The Rh123 accumulation assay was performed according to the reported procedure28 with slight modifications. In brief, MCF-7 and MCF-7/ADR cells at a density of 3 × 105/mL in exponential growth were incubated with Rh123 (5 μM) in the presence or absence of various concentrations of 5 (5, 10, 20 μM) or Ver (10 μg/mL) at 37 °C for 1 h. The intracellular Rh123 accumulation (%) was measured by flow cytometry. The argon ion laser was operated at 488 nm, and the emitted fluorescence was collected by a 530 nm pass filter. After 24 h, pictures of MCF/ADR cells in negative control or treated with 5 (10 μM) or Ver (10 μg/mL) were taken by a fluorescence microscope.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the National Natural Science Foundation of China (Nos. 81473107 and 81630093) for financial support and Mrs. Y.-N. Qiao for the analysis of single-crystal X-ray diffraction.

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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.8b00362. 1D and 2D NMR spectra and HRESIMS data of compounds 1−11 and the ECD spectrum of compound 7 (PDF)

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REFERENCES

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. ORCID

Hong-Xiang Lou: 0000-0003-3300-1811 2048



Article

(26) Zhang, C. W.; Ondeyka, J. G.; Herath, K. B.; Guan, Z. Q.; Collado, J.; Pelaez, F.; Leavitt, P. S.; Gurnett, A.; Nare, B.; Liberator, P.; et al. J. Nat. Prod. 2006, 69, 710−712. (27) Biggins, J. B.; Liu, X.; Feng, Z.; Brady, S. F. J. Am. Chem. Soc. 2011, 133, 1638−1641. (28) Li, X.; Sun, B.; Zhu, C. J.; Yuan, H. Q.; Shi, Y. Q.; Gao, J.; Li, S. J.; Lou, H. X. Toxicol. In Vitro 2009, 23, 29−36.

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