Usnic Acid Derivatives with Cytotoxic and Antifungal Activities from the

Article pubs.acs.org/jnp

Usnic Acid Derivatives with Cytotoxic and Antifungal Activities from the Lichen Usnea longissima Xuelong Yu,†,‡,# Qiang Guo,†,‡,# Guozhu Su,†,‡ Ailin Yang,†,‡ Zhongdong Hu,*,† Changhai Qu,† Zhe Wan,§ Ruoyu Li,§ Pengfei Tu,*,† and Xingyun Chai*,† †

Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡ School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic of China § Department of Dermatology and Venereology, Peking University First Hospital, Research Center for Medical Mycology, Peking University, Beijing 100034, People’s Republic of China S Supporting Information *

ABSTRACT: Eight usnic acid derivatives, that is, usenamines A−F (1−6), usone (7), and isousone (8), together with the known (+)-usnic acid (9), were isolated from the lichen Usnea longissima. Their structures were elucidated using 1D and 2D NMR and MS data, and the absolute configurations of compounds 1 and 2 were defined by single-crystal X-ray diffraction analyses. Compounds 1, 2, and 8 showed inhibitory effects on the growth of human hepatoma HepG2 cells with IC50 values of 6.0−53.3 μM compared with methotrexate as the positive control, which had an IC50 value of 15.8 μM. Furthermore, 1 induced apoptosis of HepG2 cells in a dosedependent manner at concentrations of 0−15.0 μM. The isolated compounds were also evaluated for their antifungal and antibacterial activities, with 7 and 8 exhibiting weak inhibitory effects on fungal Trichophyton rubrum spp. with an MIC value of 41.0 μM.

U

of monosubstituted phenyls, depsides, anthraquinones, dibenzofurans, and terpenoids,19 which have been shown to exhibit insecticidal20 and antioxidant properties.21−23 As part of a comprehensive exploration of UA and its derivatives with significant bioactivities and fewer side effects, phytochemical investigations using 1H NMR-guided fractionation of U. longissima led to the isolation of eight UA derivatives: usenamines A−F (1−6), usone (7), and isousone (8), along with the known (+)-UA (9). Their structures were elucidated using MS, 1D, and 2D NMR, electronic circular dichroic (ECD), as well as single-crystal X-ray diffraction data. Herein, the isolation, structural elucidation, inhibitory effects against the growth of human hepatoma HepG2 cells, and evaluation of the antifungal and antibacterial activities of these constituents are discussed.

snic acid (UA) has been of interest to chemists and pharmacologists since it was first isolated in 1844.1−3 As one of the most characteristic metabolites isolated from lichen, UA is widely distributed in Usnea (Usneaceae), Cladonia (Cladoniaceae), Lecanora (Lecanoraceae), and other lichen genera, with a highest isolated yield of 26%.4 UA exhibits diverse biological activities such as antitumor, antiviral, antimicrobial, anti-inflammatory, and insecticidal effects. It is one of the few commercially available lichen metabolites and has been the most extensively investigated. Nevertheless, its adverse effects, particularly severe liver damage, greatly limit its use in medical applications.3,5,6 Over the past few decades, significant efforts have been made to reduce the side effects of UA by encapsulating it in microspheres or nanocapsules7,8 and by searching for derivatives through synthesis9−13 or phytochemical isolation from natural resources.14−16 These efforts have ultimately led to the discovery of lead compounds with better pharmacological and toxicity profiles. Usnea longissima is widely distributed throughout the northern temperate zones and are especially prominent in the subarctic and coastal rainforests of Europe, Asia, and North America. It has been used to treat a wide range of ailments in developing countries, such as phlegm-heat, malaria, asthma, carbuncles, headache, blood in stool, and ascariasis.17 Its crude extract has been used to treat leg and loin injuries, fractured bones, and gynecological diseases.18 Previous studies revealed the presence © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was obtained as yellow crystals (MeOH-H2O, 90:10). Its molecular formula, C18H17NO6, was deduced from HRESIMS (m/z 344.1120 [M + H]+, calcd for C18H18NO6, 344.1129) and 13C NMR data, which indicated it had 11 indices of hydrogen deficiency. The IR spectrum displayed absorption bands at 3440, 1697, 1625, 1215, and 1187 cm−1, indicating the Received: February 5, 2016

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DOI: 10.1021/acs.jnatprod.6b00109 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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presence of hydroxy and carbonyl functionalities. The 1H NMR spectrum displayed signals for four methyl groups (δH 1.63, 1.96, 2.52, and 2.63), two hydroxy-group protons (δH 13.40, 12.30), two amino protons (δH 11.53, 9.82), and an olefinic proton at δH 5.85 (H-4). The 13C NMR spectrum showed 18 carbon resonances that consisted of three ketocarbonyls, four oxygenated olefinic, six olefinic, four methyl, and one sp3 quaternary carbon at δC 56.1 (C-9b). The HMBCs from H3-10 to C-1, C-4a, and C-9a indicated that the angular methyl group was located at C-9b, while those from H3-15 to C-14 and C-6 suggested that C-14 was connected to C-6. Comparison of the NMR data (Tables 1 and 2) with those of 9 revealed their structural similarities, especially of the A and B rings. This was demonstrated by the presence of cross-peaks in the HMBC spectrum, from H3-13 to C-9 and C-7, from H-4 to C-2, C-4a, and C-9b, from HO-7 to C-6 and C-8, and from HO-9 to C-8 (Figure 1). The above assignment combined with analysis of its molecular formula C18H17NO6 suggested the presence of an enamine group (−CC-NH2) in 1, which was supported by the observation of NOESY correlations of NH2b (δ 9.82) with H3-12 (δ 2.52) and NH2a (δ 11.53). HMBCs from H3-12 to C-11 and C-2 established the connection of C-11 with C-2. The (9bR) configuration and the E-geometry of the enamine functionality were confirmed by single-crystal X-ray diffraction analysis (Figure 2). Thus, the structure of 1, usenamine A, was established as shown.9 Compound 2 was obtained as pale yellow needles (MeOHH2O, 90:10). Its molecular formula was assigned as C20H23NO7 according to 13C NMR spectroscopic data and the observed protonated molecule at m/z 390.1546 [M + H]+ (calcd for C20H24NO7, 390.1547) in the positive-ion HRESIMS spectrum. Its 1H and 13C NMR data (Tables 1 and 2) were similar to those of 1, suggesting their structural similarities. Comparing the NMR data of 2 with those of 1 revealed the absence of a double bond and the presence of a methylene carbon (C-4), a dioxygenated secondary carbon (C-4a), and O-ethyl signals at δH 3.86 (2H, q, J = 7.0 Hz) and 1.21 (3H, t, J = 7.0, Hz). The HMBCs from H2-16 to C-4a suggested the connection of an ethoxy group at C-4a. The 2D structure of 2 was supported by analyses of the HMBC data, especially correlations from H3-10 to C-1, C-4a, C-9a, and C-9b and from H2-4 to C-2, C-3, C-4a, and C-9b (Figure 1). A NOESY correlation between H2-16/H3-10 suggested the angular C-10 methyl and C-4a ethoxy groups were cis-orientated.

The absolute configuration of the compound was defined by single-crystal X-ray diffraction analysis, revealing a (4aR, 9bR) absolute configuration. Notably, in the X-ray data was the presence of two conformers; one had the ethoxy group rotated toward the A/B-ring portion (2a) and the other was rotated in the opposite direction (2b) (Figure 2). Therefore, the structure of 2, usenamine B, was elucidated as shown. The molecular formula of compound 3 was determined to be C20H23NO7 based on the 13C NMR and positive-ion HRESIMS data (m/z 390.1548 [M + H]+, calcd for C20H24NO7, 390.1547). Interpretation of the NMR data revealed that the structure of 3 was similar to that of 2, but showed major chemical shift differences at C-5a, C-9, C-14, and C-15, presumably due to exchange of the C-13 methyl and C-14 acetyl groups in 3 compared with 2. The HMBC correlations from H3-13 to C-6, C-7, and C-5a and from H3-15 to C-8 supported this deduction. These differences may be explained by deshielding of the resonances due to the location of the acetyl group in the anisotropic deshielding zone of ring A. This phenomenon was also evident for the shielded C-6 resonance (δ 100.5) compared with 2. R configurations for C-4a and C-9b of 3 were presumed through the NOESY correlation of H2-16/H3-10 and comparison of the specific rotation and NMR data with those of 2, as well as those of the normal usnic acid and isousnic acid series,24 which were identical to the reported compounds in this series.14 The absolute configurations at C-4a and C-9b were both determined to be R, due to the sequential negative and positive Cotton effects at 298 (Δε − 0.83) and 274 nm (Δε + 0.63) observed in ECD spectrum, which was similar sign to that of 2 {300 nm (Δε − 1.49), 274 nm (Δε + 1.36) nm}. Thus, the structure of 3, usenamine C, was defined as shown. Compound 4 had the molecular formula of C23H25NO8 on the basis of its 13C NMR and HRESIMS data (m/z 444.1649 [M + H]+, calcd for C23H26NO8, 444.1653). Comparison of the 1 H and 13C NMR data with those of 1 revealed that most of the signals were similar, except for the presence of resonances at δH 3.62 (H-1′)/δC 44.0, δH 2.02 (H-2′)/δC 25.4, and δH 2.51 (H-3′)/δC 31.7, which were assigned as a −CH2CH2CH2− unit, and a methoxy signal at δ 3.69 (H3-5′). The HMBCs from H3-5′ to a carbonyl (C-4′), from both H2-3′ and H2-2′ to C-4′, and from H2-1′ to C-11, in combination with the presence of a singlet at δ 13.48 (NH) in the 1H NMR spectrum, revealed the presence of a C-2 enamine moiety in 4. A previous report demonstrated B


18.82, s (OH-3)

the phenomenon of imine-enamine tautomerism for this class of usnic acid derivatives.9 The presence of an enamine group was determined by comparing the NMR data of H-4 and C-12 in 4 with those of corresponding imine analogues. The NMR (500/125 MHz, in CDCl3) chemical shift of H-4 (δH 5.79) and C-12 (δC 18.4) in 4 differed from those of usimine C (δH 6.07, H-4; δC 19.3, C-12),14 but were in good agreement with those of the enamine analogues.9 The NOESY correlation between H 3-12 and H 2-1′ suggested an E-configuration for the enamine functionality. The similar sign of the specific rotation {([α]21 D + 167 (c 0.1, MeOH)} and the positive Cotton effects observed in the ECD spectrum {328 nm (Δε + 1.01), 274 nm (Δε + 0.67), 224 nm (Δε + 0.43)} of 4 compared to those of 1 demonstrated that the compound had a 9bR absolute configuration. Therefore, the structure of 4, usenamine D, was elucidated as shown. Compound 5 was obtained as pale yellow oil. Its molecular formula was defined as C24H27NO8 based on 13C NMR and positive-ion HRESIMS data (m/z 458.1825 [M + H]+, calcd for C24H28NO8, 458.1809), indicating that the molecular mass of 5 was 14 mass units more than that of 4. Comparison of the 1 H and 13C NMR data suggested that their structures were similar, except for the methoxy group in 4 being replaced with an ethoxy group in 5. The structural identification of 5 was supported by analyses of the HMBC data. The enamine moiety was presumed to be in an E-configuration based on the NOESY correlation between H3-12 and H2-1′. The absolute configuration of 5 was identical to that of 1 as determined by comparison of their specific rotations and ECD data. Therefore, the structure of 5, usenamine E, was elucidated as shown. Compound 6 was isolated as a colorless oil. Its positive-ion HRESIMS data showed a pseudomolecular ion at m/z 504.2232 [M + H]+ (calcd for C26H34NO9, 504.2228) which, in conjunction with the 13C NMR data, assigned a molecular formula of C26H33NO9. A comparison of the 1H and 13C NMR data of 6 with those of 5 revealed their structural similarities, except for the presence of ethoxy signals at δH 1.22 (CH3) and δH 3.86 (OCH2), a methylene signal at δH 3.13/3.09 (H2-4), and the absence of the H-4 olefinic proton in 6 (Table 1). The HMBCs from H2-16 to C-17 and C-4a suggested that the ethoxy group was linked to C-4a. The (4aR, 9bR) absolute configuration of the compound was defined by comparing their NMR, specific rotation, and ECD data with those of 2 and 3. The enamine moiety adopted an E-configuration based on the NOESY correlation between H3-12 and H2-1′. Thus, the structure of 6, usenamine F, was elucidated as shown. Compound 7 had the molecular formula of C20H22O8 based on its 13C NMR and HRESIMS data. Comparison of its NMR data with those of 2 showed that most of the signals were similar, except that an amino unit (−NH2) in 2 was replaced by a ketocarbonyl group. Compound 7 had an additional ethoxy group compared with compound 9, which was supported by the HMBC data. The NOESY correlation between H2-16 and H3-10 revealed that they were in a cis orientation, and the (4aR, 9bR) absolute configuration was presumed to be analogous to 2. Thus, the structure of 7, usone, was elucidated as shown. Compound 8 represented an isomer of 7, as shown by analyses of its HRESIMS and 13C NMR data. The IR and UV spectra were similar to those of 7. Comparison of their NMR data revealed that the major chemical shift differences involved C-5a, C-9, C-14, and C-15, due to the alternating substitution of methyl and acetyl groups on ring A, as was also observed for 3 and 2. This difference was also supported by their HMBCs. The (4aR, 9bR)

9.82, s (b) 11.53, s (a) NH2

1′ 2′ 3′ 5′ 6′

13.40, s 12.30, s 1.63 (3H, s) 2.52 (3H, s) 1.96 (3H, s) 2.63 (3H, s) 7 9 10 12 13 15 16 17

Measured in DMSO-d6; bmeasured in methanol-d4; cmeasured in CDCl3. a

1.57 (3H, s) 2.48 (3H, s) 1.90 (3H, s) 2.65 (3H, s) 3.82 (2H, q, 7.0) 1.19, (3H, t, 7.0)

1.63 (3H, s) 2.63 (3H, s) 1.99 (3H, s) 2.61 (3H, s)

3.62 (2H, td, 7.0; 3.0) 2.02 (2H, m) 2.51 (2H, t, 7.0) 3.69 (3H, s)

1.59 (3H, s) 2.62 (3H, s) 1.96 (3H, s) 2.57 (3H, s)

3.61(2H, td, 7.0; 4.0) 2.01 (2H, m) 2.49 (2H, t, 7.0) 4.15 (2H, q, 7.0) 1.26 (3H, t, 7.0)

3.58 (2H, t, 7.0) 1.98 (2H, m) 2.45 (2H, t, 7.0) 4.11 (2H, q, 7.0) 1.23 (3H, t, 7.5)

1.54 (3H, s) 2.51 (3H, s) 1.95 (3H, s) 2.54 (3H, s) 3.86 (2H, q, 7.0) 1.22 (3H, t, 7.0)

1.63 (3H, s) 2.47 (3H, s) 1.89 (3H, s) 2.50 (3H, s) 3.84 (2H, q, 7.0) 1.20 (3H, t, 7.0)

1.64 (3H, s) 2.49 (3H, s) 1.86 (3H, s) 2.61 (3H, s) 3.80 (2H, q, 7.0) 1.17 (3H, t, 7.0)

13.29, s 11.00, s 1.75 (3H, s) 2.66 (3H, s) 2.09 (3H, s) 2.67 (3H, s)

5.85, s

1.51 (3H, s) 2.47 (3H, s) 1.92 (3H, s) 2.52 (3H, s) 3.86 (2H, q, 7.0) 1.21 (3H, t, 7.0)

3.34 (2H, overlapped) 3.34 (2H, overlapped) 3.13 (d, 15.5) 3.09 (d, 15.5)

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4

3.08 (d,15.5) 3.10 (d, 15.5)

3.06 (d, 15.5) 3.08 (d, 15.5)

5.72, s

4b 3b 2b 1a no.

Table 1. 1H NMR Data of Compounds 1−9 (δ in ppm, J in Hz, 500 MHz)

5.67, s

5b

6b

7b

8b

5.97, s

9c


C



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Table 2. 13C NMR Data of Compounds 1−9 (δ in ppm, 125 MHz)

a

no.

1a

2b

3b

4b

5b

6b

7b

8b

9c

1 2 3 4 4a 5a 6 7 8 9 9a 9b 10 11 12 13 14 15 16 17 1′ 2′ 3′ 4′ 5′ 6′

197.6, C 101.2, C 188.5, C 102.6, CH 173.0, C 155.7, C 100.9, C 162.5, C 106.3, C 157.7, C 105.1, C 56.1, C 31.6, CH3 175.8, C 24.5, CH3 7.5, CH3 200.9, C 31.0, CH3

200.5, C 106.3, C 194.3, C 44.0, CH2 111.6, C 158.4, C 102.2, C 163.8, C 106.5, C 161.1, C 107.5, C 61.1, C 19.3, CH3 177.1, C 24.3, CH3 7.4, CH3 202.7, C 31.4, CH3 60.7, OCH2 15.7, CH3







44.0, CH2 25.4, CH2 31.7, CH2 174.7, C 52.3, OCH3

44.0, CH2 25.4, CH2 31.9, CH2 174.2, C 61.7, OCH2 14.5, CH3

200.0, C 106.8, C 194.1, C 43.8, CH2 111.5, C 158.5, C 102.3, C 163.8, C 106.5, C 161.1, C 107.5, C 61.2, C 19.2, CH3 176.1, C 18.0, CH3 7.4, CH3 202.8, C 31.4, CH3 60.7, OCH2 15.7, CH3 44.1, CH2 25.3, CH2 31.9, CH2 174.2, C 61.7, OCH2 14.5, CH3

Measured in DMSO-d6; bmeasured in methanol-d4; cmeasured in CDCl3.

experimental data. In an effort to prove their origin from a natural resource, (+)-UA (9) was refluxed with 95% EtOH at 75 °C for 12 h. However, the assumed product (7) was not detected by TLC and HPLC. LC-MS analysis was further performed on the MeOH and MeCN extracts of U. longissima, respectively, but constituents without an ethoxy substituent at C-4a were not detected, demonstrating that 2, 3, 7, and 8 were not artifacts. Moreover, the MeOH extract of U. longissima was subjected to LC-MS analysis. The presence of 2 was observed in both the HPLC and LC-MS systems (Figure 3), suggesting that 2 occurs naturally. Therefore, the C-4a ethoxy-substituted isolates were natural secondary metabolites of U. longissima rather than artifacts. However, compounds 4−6 with ester groups were probably artifacts generated during ethanol extraction because no ion chromatogram peaks were observed at the corresponding times in the negative and positive mode analyses of the MeCN extract of U. longissima. Notably, the free acid of esters, 4 and 5, has been previously synthesized.9 It was reported that tautomerism may have been involved in the imine-enamine and enol groups of the UA derivatives.14,15 The presence of enamine rather than imine functionalities in 1 and 2 was revealed by X-ray diffraction analyses. Similar functionalities were presumed for 3−6 by comparing their observed and reported NMR data. Compound 2 is the first UA derivative that has been shown to have two conformers due to the presence of the ethoxy group. Similarly, it may be presumed that 3 and 6−8 also possessed similar conformers, as was also reported for (−)-placodiolic acid and (±)-9-O-methyl placodiolic acid possessing C-4a methoxy groups.15 UA enamines or derivatives with substituents at C-4a not only possessed improved solubility, which contributed to an increase in their bioavailability, but also demonstrated the potential for generating new leads with better bioactivities or fewer severe side effects.

Figure 1. Selected HMBC and NOESY correlations of 1−4.

absolute configuration was based on the observed NOESY correlations between H2-16 and H3-10, and comparison of the specific rotation and ECD data with 2. Therefore, the structure of 8, isousone, was elucidated as shown. Also isolated was the major product (+)-usnic acid (9) 14 25 ([α]21 D + 422 (c 0.1, CH2Cl2) [lit. [α]D + 300 (c 0.54, CH2Cl2) ], which was identified by comparing its NMR data with reported data.25 It should be noted that although 1 had been previously synthesized, this paper reports its first isolation as a natural product and its absolute configuration, including the E configuration of the enamine group as resolved by X-ray diffraction analysis. Among these compounds, 2, 3, and 6−8 possess unique ethoxy substituents at C-4a. Although U. longissima was extracted with EtOH and these ethoxy-substituted isolates were possible artifacts derived from 1, 4-Michael addition reaction of EtOH to the enone moiety, this observation was not supported by further D



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Figure 2. ORTEP drawing of compounds 1 and 2.

exhibited cytotoxicities against HepG2 cells with IC50 values of 6.0, 50.2, and 53.3 μM, respectively, compared with the positive control methotrexate, which had an IC50 value of 15.8 μM. A variety of bacteria and fungi, including Escherichia coli, Candida albicans, Bacillus subtilis, Mycobacterium smegmatis, Trichophyton, and Aspergillus, were used to examine the in vitro susceptibilities of these constituents, but only 7 and 8 displayed weak inhibitory activities against T. rubrum spp. fungi; both had MIC values of 41.0 μM, whereas others that exhibited MIC values above 200 μM were inactive. The above data suggest the potential of UA enamines toward the development of drugs to treat hepatic cancer. Apoptosis is physiological and orderly cell death regulated by genes.26 As such, drug-induced apoptosis in cancer cells is important for cancer therapy. Thus, the effects of 1 on the apoptosis of HepG2 cells by staining them with Annexin V-FITC/propidium iodide (PI) and analysis by flow cytometry were investigated. The results showed that the rate of apoptosis increased in a dosedependent manner in HepG2 cells after treatment with 0−15 μM of 1 (Figure 4). The cytotoxicity and induction of apoptosis, in combination with its high yield from 9 through a one-step synthesis,9 lay a solid foundation for further toxicological assessments and pharmacological evaluations against hepatic cancer in vivo.

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EXPERIMENTAL SECTION General Experimental Procedures. Melting points were measured on an XT4 digital micromelting point apparatus and is uncorrected. Optical rotations were measured on a Rudolph Autopol IV automatic polarimeter at room temperature. The UV and IR spectra were measured on a Shimadzu UV-2450 spectrophotometer and Thermo Nicolet Nexus 470 FT-IR spectrometer with KBr pellets, respectively. The HRESIMS data were obtained with an LCMS-IT-TOF system (Shimadzu, Kyoto, Japan). The NMR spectra were recorded on Varian INOVA-500 spectrometer. ECD spectra were measured using a Jasco J-815 spectropolarimeter. The X-ray data were collected on an Agilent Gemini E single-crystal X-ray diffractometer with Cu Kα radiation (Agilent Technologies, Yarnton, Oxfordshire, U.K.). Column chromatography was performed with silica gel (200−300 mesh, Qingdao Haiyang Chemical Co., Qingdao, People’s Republic of China), Sephadex LH-20 (Pharmacia), MCI gel (CHP20/P120, Mitsubishi Chemical Corp., Japan), and LiChroprep RP-C18 gel

Figure 3. HPLC-PDA (254 nm) profiles of a fraction by preparative TLC of the MeOH extract of U. longissima (A) and 2 (B), and total ion chromatograms in negative mode of the MeOH extract of U. longissima (C) and 2 (D).

Inspired by the remarkable antibacterial, antifungal, and cytotoxic activities against various cancer cell lines of UA and its derivatives,1,2 all the isolates were screened for their cytotoxic, antifungal, and antibacterial activities. Compounds 1, 2, and 8 E



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Figure 4. Apoptosis increased after treatment with 1 in HepG2 cells. Cells in the Q2 and Q4 quadrants were considered to be late apoptotic and early apoptotic stages, respectively. Cells in the Q2 + Q4 quadrants were considered to be apoptotic. Data are presented as the mean ± SEM, n = 3, **P < 0.01.

(40−63 μm, Merck, Germany). HPLC separation was conducted using a Waters 2535 pump system equipped with a Shim-pack PREP-ODS (H) (250 mm × 20 mm, 5 μm). TLC was performed using GF254 plates sprayed with 1% vanillin-H2SO4 reagent. Plant Material. Whole U. longissima plants were collected in Chengdu, Sichuan Province, People’s Republic of China, in September 2013 and were authenticated by Prof. Pengfei Tu. A voucher specimen (UL-201309) was deposited in the Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine. Extraction and Isolation. A whole U. longissima plant (30 kg) was refluxed twice in 70% EtOH (500 L). After removal of the solvent under reduced pressure, the residue (2.5 kg) was suspended in H2O (20 L) and sequentially extracted with petroleum ether (PE) (40 L), EtOAc (60 L), and n-BuOH (40 L). The EtOAc-soluble extract (1.0 kg) was subjected to silica gel column chromatography (CC) and was eluted with a stepwise gradient of CH2Cl2-MeOH (30:1−0:1) to afford fractions A−I. Compound 9 (2 g) was obtained via recrystallization in acetoneH2O from fraction E. Because UA and its derivatives feature isolated phenolic protons and isolated methyl signals in their 1 H NMR spectra, 1H NMR-guided fractionation was applied for these fractions. Fraction D (125 g) exhibited the characteristic signals of UA derivatives in its 1H NMR spectrum and was subjected to silica gel CC; eluting with PE-EtOAc (6:1−1:1) to give subfractions D1−D5. Subfraction D3 (20 g) was further separated on a Sephadex LH-20 column (CH2Cl2-MeOH, 1:1) to yield three portions (D3a−D3c). D3b (8 g) that were purified by silica gel CC using PE-EtOAc (4:1) to afford 7 (500 mg) and 8 (800 mg). Subfraction D5 (10 g) was further fractionated on an MCI column with MeOH-H2O (50:50−100:0) to yield six portions (D5a−D5f). Portion D5d (200 mg) was subjected to semipreparative HPLC using MeOH-H2O (77:23) to afford 1 (10.2 mg, retention time (tR) = 48.08 min), 2 (5.5 mg, tR = 43.67 min), and 3 (4.7 mg, tR = 52.45 min). Compounds 4 (5.6 mg, tR = 57.60 min), 5 (1.8 mg, tR = 77.49 min), and 6 (1.8 mg, tR = 64.29 min) were purified from D5f (173 mg) by semipreparative HPLC using MeOH-H2O (70:30). Usenamine A (1). Yellow crystals (MeOH-H2O, 90:10); mp 300.5−301.0 °C; [α]21 D + 351 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 291 (4.51), 225 (4.40) nm; ECD (c 9.72 × 10−4 M, MeOH), λmax (Δε) 320 (+1.06), 274 (+0.80), 224 (+0.42) nm; IR (KBr) νmax 3440, 2850, 1697, 1625, 1548, 1445, 1374, 1215, 1187, 1058, 966 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 344.1120 [M + H]+ (calcd for C18H18NO6, 344.1129); 1H NMR spectroscopic data (methanol-d4, 500 MHz) δ 5.80 (1H, s, H-4), 2.66 (3H, s, H-12),

2.60 (3H, s, H-15), 2.03 (3H, s, H-13), 1.68 (3H, s, H-10); C NMR (methanol-d4, 125 MHz) δ 202.3 (C-14), 200.4 (C-1), 191.3 (C-3), 177.9 (C-11), 175.8 (C-4a), 164.5 (C-7), 159.4 (C-9), 157.4 (C-5a), 108.6 (C-8), 106.4 (C-9a), 103.3 (C-4), 102.9 (C-2), 102.3 (C-6), 58.2 (C-9b), 32.2 (C-10), 31.4 (C-15), 25.1 (C-12), 7.6 (C-13). Usenamine B (2). Pale yellow needles (MeOH-H2O, 90:10); mp 191.0−191.5 °C; [α]21 D − 240 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 292 (4.44), 258 (4.15), 226 (4.18) nm; ECD (c 5.14 × 10−4 M, MeOH), λmax (Δε) 300 (−1.49), 274 (+1.36) nm; IR (KBr) νmax 3423, 2918, 2850, 1630,1573,1455, 1371 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 390.1546 [M + H]+ (calcd for C20H24NO7, 390.1547); 1H NMR (DMSO-d6, 500 MHz) δ 13.41 (OH-7, s), 11.30 (s, NHa), 10.94 (OH-9), 9.83 (s, NHb), 3.79 (2H, m, H-16), 3.16 (d, H-4a), 3.12 (d, H-4b), 2.52 (3H, s, H-15), 2.40 (3H, s, H-12), 1.90 (3H, s, H-13), 1.50 (3H, s, H-10), 1.14 (3H, t, H-17). Usenamine C (3). White powder, [α]21 D − 59 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 290 (4.49), 258 (4.21), 227 (4.25), 203 (4.29) nm; ECD (c 4.30 × 10−4 M, MeOH), λmax (Δε) 298 (−0.83), 274 (+0.63) nm; IR (KBr) νmax 3426, 2919, 2850, 1631, 1573, 1421, 1363 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 390.1548 [M + H]+ (calcd for 390.1547, C20H24NO7); 1H NMR (DMSO-d6, 500 MHz) δ 13.41 (OH-7, s), 11.30 (s, NHa), 10.94 (OH-9), 9.84 (s, NHb), 3.79 (2H, m, H-16), 3.16 (d, H-4a), 3.12 (d, H-4b), 2.62 (3H, s, H-15), 2.40 (3H, s, H-12), 1.90 (3H, s, H-13), 1.50 (3H, s, H-10), 1.14 (3H, t, H-17). Usenamine D (4). White oil, [α]21 D + 167 (c 0.1, MeOH); UV (MeOH) λmax (logε) 294 (4.44), 225 (4.35) nm; ECD (c 7.52 × 10−4 M, MeOH), λmax (Δε) 328 (+1.01), 274 (+0.67), 224 (+0.43) nm; IR (KBr) νmax 3446, 2918, 2850, 1738, 1699, 1627, 1558, 1471, 1384, 1188, 1139, 1056 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; positive-ion HRESIMS m/z 444.1649 [M + H]+ (calcd for C23H26NO8, 444.1653); 1H NMR (CDCl3, 500 MHz) δ 13.48 (NH, br s), 13.35 (OH-7), 11.92 (OH-9), 5.79 (1H, s, H-4), 3.71 (3H, s, H-5′), 3.55 (2H, t, H-1′), 2.67 (3H, s, H-15), 2.64 (3H, s, H-12), 2.49 (2H, t, H-3′), 2.10 (3H, s, H-13), 2.05 (2H, m, H-2′), 1.71(3H, s, H-10); 13C NMR (CDCl3, 125 MHz) δ 200.8 (C-14), 198.5 (C-1), 175.3 (C-11), 174.3 (C-4a), 172.8 (C-4′), 163.7 (C-7), 158.4 (C-9), 156.0 (C-5a), 108.2 (C-8), 105.2 (C-9a), 102.6 (C-2), 101.5 (C-6), 57.3 (C-9b), 52.1 (C-5′), 43.1 (C-1′), 32.1 (C-10), 31.4 (C-15), 30.9 (C-3′), 24.5 (C-2′), 18.4 (C-12), 7.6 (C-13). Usenamine E (5). Pale yellow oil, [α]21 D + 191 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 295 (4.46), 225 (4.36) nm; 13

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ECD (c 7.29 × 10−4 M, MeOH), λmax (Δε) 328 (+1.26), 276 (+0.85), 224 (+0.58) nm; IR (KBr) νmax 3442, 2918, 2850, 1734, 1627, 1577, 1542, 1472, 1420, 1384, 1188, 1058 cm−1; 1 H and 13C NMR spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 458.1825 [M + H]+ (calcd for 458.1809, C24H28NO8). Usenamine F (6). Colorless oil, [α]21 D − 190 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 296 (4.34), 225 (4.13), 202 (4.14) nm; ECD (c 6.62 × 10−4 M, MeOH), λmax (Δε) 306 (−2.52), 276 (+2.35) nm; IR (KBr) ν max 3445, 2957, 2918, 2850, 1733, 1628, 1576, 1541, 1471, 1384, 1033 cm−1; 1H and 13 C NMR spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 504.2232 [M + H]+ (calcd for C26H34NO9, 504.2228). Usone (7). Dark yellow powder, [α]21 D − 83 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 282 (4.26), 226 (4.18) nm; ECD (c 8.54 × 10−4 M, MeOH), λmax (Δε) 294 (−0.42), 274 (+0.64) nm; IR (KBr) νmax 3448, 2957, 2918, 2850, 1630, 1578, 1400, 1027 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; positive-ion HRESIMS m/z 391.1382 [M + H]+ (calcd for C20H23O8, 391.1387). Isousone (8). Yellow powder, [α]21 D − 17 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 282 (4.32), 226 (4.23) nm; ECD (c 8.54 × 10−4 M, MeOH), λmax (Δε) 296 (−0.81), 272 (+1.40) nm; IR (KBr) ν max 3442, 2984, 2918, 2850, 1633, 1455, 1400, 1367, 1318, 1291, 1211, 1030 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; negative-ion HRESIMS m/z 389.1256 [M − H]+ (calcd for C20H21O8, 389.1242). HPLC-MS Analyses. Dried U. longissima (1.0 g) was pulverized and extracted for 60 min at 25 °C using ultrasonic action with MeOH and MeCN (each 50 mL), respectively. The filtered extracts were evaporated in vacuo to dryness. After separation of the MeOH extracts using preparative TLC (200 × 200 × 0.5 mm, 4 plates, PE-EtOAc, 4:1), the silica gel band with the same Rf value as compound 2 was scratched and extracted with MeOH. The MeCN extract was prepared in the same manner, and the silica gel band with the corresponding Rf values of compounds 4, 5, and 6 were cut and extracted with MeOH. Both the samples were subjected to LC-MS analysis using an Agilent Zorbax SB-C18 (4.6 × 250 mm, 5 μm) column with UV (254 nm) detection. An MeCN (A)/water mixture was used as the mobile phase for HPLC analysis. The elution condition was applied with a linear gradient program of 5−95% of A from 0−95 min. X-ray Crystal Data for Usenamines A (1) and B (2). The crystal structures and absolute configurations were determined using data collected with Cu Kα radiation (λ = 1.54178 Å) on an Agilent Gemini E X-ray single crystal diffractometer, equipped with an Oxford Cryostream cooler at T = 100.8 K, and 101.8 K, respectively. Structures were solved by direct methods using SHELXS-97 and refined anisotropically by fullmatrix least-squares on F2 using SHELXL-97.27 The proton atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with ORTEP-3.28 The absolute configurations were determined by refinement of the Flack parameter based on resonant scattering of the light atoms.29 Crystal data: Usenamine A (1): yellow needles, C18H17NO6, M = 343.33, monoclinic, crystal size 0.24 × 0.18 × 0.09 mm3, space group C2, a = 17.138 (2) Å, b = 9.7525 (5) Å, c = 13.831 (2) Å, α = 90.00°, β = 134.45 (3)°, γ = 90.00°, V = 1650.1 (4) Å3, Z = 4, μ(Cu Kα) = 0.879 mm−1, Dcalcd = 1.382 mg/m3, F(000) = 720, reflections collected 5546, independent reflections

2981 (Rint = 0.0240), final R indices for I > 2σ (I), R1 = 0.0325, wR 2 = 0.0843, R indices for all data R 1 = 0.0335, wR2 = 0.0852, Flack parameter =0.04 (15). Usenamine B (2): pale yellow needles, C20H23NO7, M = 389.39, monoclinic, crystal size 0.50 × 0.25 × 0.25 mm3, space group P21, a = 11.62935 (18) Å, b = 9.99614 (17) Å, c = 16.9428 (2) Å, α = 90.00°, β = 93.8401 (13)°, γ = 90.00°, V = 1965.15 (5) Å3, Z = 4, μ(Cu Kα) = 0.837 mm−1, Dcalcd = 1.316 mg/m3, F(000) = 824, reflections collected 13261, independent reflections 7421 (Rint = 0.0220), final R indices for I > 2σ (I), R1 = 0.0329, wR2 = 0.0848, R indices for all data R1 = 0.0340, wR2 = 0.0857, Flack parameter = −0.10 (10). Crystallographic data for 1 and 2 has been deposited in Cambridge Crystallographic Data Centre (deposition numbers: CCDC 1054502 and 1054505, respectively). Copies of these data can be obtained free of charge on application to Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: + 44(0)1223−336033 or e-mail: [email protected]). Cytotoxicity Assay. HepG2 cells were seeded in 96-well plates at a density of 3 × 103 cells/well in 100 μL of DMEM medium that was supplemented with 10% fetal bovine serum and incubated for 24 h. The drugs were added, and the samples were incubated for 48 h. Ten microliters of MTT (5 mg/mL in phosphate-buffered saline) was added to the medium and incubated for 3 h. The supernatant was removed, and 150 μL of DMSO was added. The plate was placed on a shaker for dissolution. After 10 min, the optical density was measured with a microplate reader at a wavelength of 490 nm. Methotrexate was used as a positive control.30 Flow Cytometry Analysis. HepG2 cells were seeded in a six-well plate at a density of 1 × 105 cells/well and were treated with 1. The cells were collected 48 h later and analyzed for apoptosis using an Annexin V-FITC apoptosis detection kit (BD Pharmingen), according to the manufacturer’s instructions. Annexin V-FITC positive/PI negative cells were regarded as early apoptotic, while Annexin V-FITC positive/PI positive cells were regarded as late apoptotic. In Vitro Antifungal Activity. The microtiter plate-based antifungal activity assay was tested as described in the literature.31,32 The fungi strains used were two Candida isolates, including one C. albicans, one C. parapsilosis isolate, and 15 Trichophyton isolates consisting of nine T. mentagrophytes and six T. rubrum isolates, and two Aspergillus isolates, A. fumigatus and A. flavus. All of the isolates were collected from patients and kept at the Peking University Research Center for Medical Mycology. The detailed protocol can be found in the Supporting Information.

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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.6b00109. The in vitro antifungal and antibacterial assays, HRESIMS, ECD, and 1D and 2D NMR spectra of compounds 1−8, and LC-MS analyses of extract of U. longissima and 2 (PDF) X-ray crystallographic data of 1 (CIF) X-ray crystallographic data of 2 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for Z.H.: [email protected]. Tel/Fax: 86-1064286180. G



Article

*E-mail for P.T.: [email protected]. Tel/Fax: 86-1082802750. *E-mail for X.C.: [email protected]. Tel/Fax: 86-1064286350.

(21) Odabasoglu, F.; Yildirim, O. S.; Aygun, H.; Halici, Z.; Halici, M.; Erdogan, F.; Cadirci, E.; Cakir, A.; Okumus, Z.; Aksakal, B.; Aslan, A.; Unal, D.; Bayir, Y. Eur. J. Pharmacol. 2012, 674, 171−178. (22) Atalay, F.; Halici, M. B.; Mavi, A.; Cakir, A.; Odabasoglu, F.; Kazaz, C.; Aslan, A.; Kufrevioglu, O. I. Turk. J. Chem. 2011, 35, 647−661. (23) Halici, M.; Odabasoglu, F.; Suleyman, H.; Cakir, A.; Aslan, A.; Bayir, Y. Phytomedicine 2005, 12, 656−662. (24) Kutney, J. P.; Sanchez, I. H.; Yee, T. Can. J. Chem. 1976, 54, 3721−3731. (25) Rashid, M. A.; Majid, M. A.; Quader, M. A. Fitoterapia 1999, 70, 113−115. (26) Wong, R. S. J. Exp. Clin. Cancer Res. 2011, 30, 87−100. (27) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112−122. (28) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565−565. (29) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, A39, 876−881. (30) Li, N.; Zhang, J. Y.; Zeng, K. W.; Zhang, L.; Che, Y. Y.; Tu, P. F. Fitoterapia 2012, 83, 1042−1045. (31) Li, Y. L.; Wan, Z.; Liu, W.; Li, R. Y. Antimicrob. Agents Chemother. 2015, 59, 1365−1369. (32) Wang, X. J.; Wan, Z.; Li, R. Y.; Liu, W. Chin. J. Mycol. 2014, 9, 75− 78.

Author Contributions #

X.Y. and Q.G. contributed equally to this research.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was financially supported by the National Natural Science Foundation of China (Nos. 81473426 and 81403147) and a grant from Beijing University of Chinese Medicine (2015JYB-XS088).

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