Article pubs.acs.org/jnp
Metabolites from the Plant Endophytic Fungus Aspergillus sp. CPCC 400735 and Their Anti-HIV Activities Xu Pang, Jian-Yuan Zhao, Xiao-Mei Fang, Tao Zhang, De-Wu Zhang, Hong-Yu Liu, Jing Su, Shan Cen, and Li-Yan Yu* Institute of Medicinal Biotechnology, Academy of Medical Science & Peking Union Medical College, Beijing 100050, China S Supporting Information *
ABSTRACT: Thirty-three metabolites including five phenalenone derivatives (1−5), seven cytochalasins (6−12), thirteen butenolides (13−25), and eight phenyl derivatives (26−33) were isolated from Aspergillus sp. CPCC 400735 cultured on rice. The structures of all compounds were elucidated by NMR, MS, and CD experiments, of which 1−5 (asperphenalenones A−E), 6 (aspochalasin R), and 13 (aspulvinone R) were identified as new compounds. Specifically, asperphenalenones A−E (1−5) represent an unusual structure composed of a linear diterpene derivative linked to a phenalenone derivative via a C−C bond. Compounds 1, 4, 10, and 26 exhibited antiHIV activity with IC50 values of 4.5, 2.4, 9.2, and 6.6 μM, respectively (lamivudine 0.1 μM; efavirenz, 0.4 × 10−3 μM).
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spectrometry (HRESIMS). In the 1H NMR spectrum, the characteristic signals at δ 14.30 (1H, s, 13-OH), 13.08 (1H, s, 7OH), 6.79 (1H, s, H-8), 5.02 (2H, t, H-29), 4.84 (1H, t, H-21), 4.77 (1H, t, H-17), 3.86 (2H, s, H-33), 2.81 (3H, s, H-15), 2.52 (1H, d, H-16), 2.11 (3H, s, H-14), 1.58 (3H, s, H-31), 1.50 (3H, s, H-15), 1.36 (3H, s, H-34), 1.24 (3H, s, H-35) were clearly observed. Starting from these characteristic proton signals, the following key heteronuclear multiple bond correlations (HMBCs) of 13-OH/C-13, C-3, C-2, H-14/C12, C-11, C-13, 7-OH/C-7, C-8, C-5, C-2, H-8/C-5, C-10, C15, C-7, C-6, H-15/C-8, C-9, C-10, H-16/C-1, C-2, C-6, C-17, C-18, H-17/C-16, C-19, C-35, H-35/C-19, C-17, C-18, H-21/ C-34, C-20, C-19, H-34/C-23, C-21, C-22, H-25/C-23, C-24, C-27, C-33, H-33/C-25, C-26, C-27, H-29/C-27, C-32, H-31/ C-29, C-30, and H-32/C-29, C-30 were found, together with the 1H−1H correlation spectroscopy (COSY) correlations of H-17/H-16, H-25/H-24, H-24/H-23, H-29/H-28, and H-28/ H-27, which allowed the planar structure of 1 to be established (Figure 1). Based on analysis of nuclear Overhauser effect spectroscopy (NOESY) data, the correlations of H-17/H-19, H-21/H-23, and H-24/H-33 demonstrated the configurations for 17-ene, 21-ene, and 25-ene to be E-, E-, and Z-, respectively. The absolute configuration of C-1 was assigned by the Rh2(OCOCF3) induced CD experiment and electronic circular dichroism (ECD) calculation. The Rh2(OCOCF3) induced CD
cquired immunodeficiency syndrome (AIDS), an immunosuppressive disease that can result in life-threatening opportunistic infections and malignancies, is caused by human immunodeficiency virus (HIV). Substantial research efforts have been dedicated to find compounds from synthetic products and natural products that can be developed as therapeutic agents against HIV. Microbial secondary metabolites are one potential resource for finding anti-HIV agents. Aspergillus (Moniliaceae) is a genus consisting of many filamentous fungal species, several of which have substantial medical value. The Aspergillus genus is well-known for its ability to produce diverse secondary metabolites, so it is regarded as an important source for discovering new bioactive natural products. CPCC 400735, an Aspergillus sp., was isolated from a medicinal plant Kadsura longipedunculata. While searching for HIV-1 inhibitors from plant endophytes, it was found that the EtOAc-soluble fraction of an extract from rice cultures of CPCC 400735 displayed significant HIV-1 inhibitory effect. Investigation of the chemical constituents of the bioactive fraction led us to obtain 33 metabolites, including five new phenalenone derivatives, asperphenalenones A−E (1−5), one new aspochalasin, aspochalasin R (6), one new aspulvinone, aspulvinone R (13) (Chart 1), and 26 known compounds (7− 12 and 14−33, Chart 2); all were tested for anti-HIV-1 activity.
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RESULTS AND DISCUSSION Compound 1 had a molecular formula of C35H44O7 as determined by high-resolution electrospray ionization mass © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 27, 2016
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DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Chart 1. Structures of the New Compounds
the planar structure of 3 to be established. In the NOESY spectrum, the correlations of H-17/H-19 and H-21/H-23 determined the configurations of 17-ene and 21-ene to be E and E, respectively, and the Z-configuration of 25-ene was determined by NOESY correlation of H-24/H-27. The CD spectrum of 3 showed Cotton effects identical to 1 (Figure S6), so the absolute configuration of C-1 was assigned as S. The Mo(Ac)4 induced CD spectrum of 3, which presented a negative Cotton effect at 317 nm and a positive Cotton effect at 380 nm (Figure S7), allowed the R-configuration to be assigned for C-29. Consequently, the structure of 3 was elucidated as drawn and named asperphenalenone C. Compound 4 had a molecular formula of C37H50O10 as determined by HRESIMS. Comparison of spectroscopic data of 4 with those of 1 revealed that the planar structure of 4 was the same as 1 except for the structure fragment of C-24 to C-33. The 13 degrees of unsaturation corresponding to the molecular formula C37H50O10 suggested a cyclic structure in the fragment of C-24 to C-33. Based on the HMBC correlations of H-31/C29, C-30, H-32/C-29, C-30, H-29/C-27, H-33/C-25, C-26, C27, and H-25/C-23, and the 1H−1H COSY correlations of H24/H-23, H-25, H-26/H-25, H-27, H-33, and H-28/H-29, the C-24 to C-32 structure was established. The correlations of H17/H-19 and H-21/H-23b observed in the NOESY spectrum revealed the E-configurations for 17-ene and 21-ene. The Cotton effects in the CD spectrum (Figure S8) of 4, which was identical to 1, suggested the S configuration of C-1. The absolute configurations of chiral carbons C-25, C-26, and C-29 were not identified in this work, whereas their relative configurations were determined by establishing the relative structure of the C-25 to C-29 cyclic ring based on the key NOESY correlations of H-25/H-26 and H-25/H-29. Consequently, the structure of 4 was elucidated as drawn and named asperphenalenone D. Compound 5 with a molecular formula of C35H46O9 determined by HRESIMS had two hydrogen atoms more
spectrum of 1 presented a negative Cotton effect at 350 nm (Figure S1); thus the configuration of C-1 was considered to be S based on the empirical rule.1 Moreover, the result of ECD calculations of 1 (details of ECD calculations are available in the Supporting Information) showed that the theoretically calculated ECD spectrum of (1S)-1 was in good agreement with the experimental ECD spectrum of 1 in the 200−400 nm region (Figure 2), which allowed the assignment of the S configuration for C-1. Consequently, the structure of 1 was elucidated as drawn and named asperphenalenone A; its 1H and 13 C NMR data were fully assigned (Tables 1 and 2). Compound 2 had a molecular formula of C35H44O10 as determined by HRESIMS. The NMR data suggested that 2, as an analogue of 1, had the same structural fragment of C-1 to C22 as 1 but differed in the structural fragment of C-23 to C-33. By combined analyses of 1H−1H COSY and HMBC spectra, the structural fragment of C-23 to C-33 was finally established. In NOESY spectrum, the correlations of H-17/H-19, H-21/H23, and H-24/H-27 determined the configurations for 17-ene, 21-ene, and 25-ene as E, E, and E. The CD spectrum of 2 was identical to that of 1 (Figures S2 and S3), and like 1, the Rh2(OCOCF3) induced CD spectrum of 2 presented a negative Cotton effect at 347 nm (Figure S4); thus the 1Sconfiguration was assigned for 2. The absolute configuration of C-29 was determined by Mo(Ac)4 induced CD experiment. The CD spectrum of 2 in solution of Mo(Ac)4 in CH3Cl presented a negative Cotton effect at 317 nm and a positive Cotton effect at 375 nm (Figure S5), thus the R-configuration of C-29 was determined according to the empirical rule.2 Consequently, the structure of 2 was elucidated as drawn and named asperphenalenone B. Compound 3 with a molecular formula of C35H44O10 determined by HRESIMS was an isomer of 2. By comparing the NMR data of 3 with those of 2, it was found that they had identical structure except for the difference in C-23 to C-27. Combined use of 1H−1H COSY and HMBC spectra allowed B
DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Chart 2. Structures of the Known Compounds
Figure 1. Key HMBC (H → C), COSY (H → H), and NOESY (H → H) correlations of compounds 1−6.
(C-28 to C-32). Compariaon of the NMR data of 5 with those of 3 revealed that they had the same structure of C-28 to C-32. Based on the analyses of HSQC, COSY, and HMBC spectra,
than 1 and two oxygen atoms more than 1. Comparison of spectroscopic data of 5 with those of 1 revealed that 5 had the same planar structure as 1 except for terminal isopentyl group C
DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
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and fully assigned its NMR data, as well as established its absolute configuration for the first time. Compound 6 was obtained as a white solid. HRESIMS gave a molecular formula of C28H33NO6. From the 1H NMR data, the proton signals at δ 7.25 (2H, t, J = 7.3 Hz), 7.18 (1H, t, J = 7.3 Hz), and 7.16 (2H, d, J = 7.3 Hz) supported a single substituted benzene in the molecule. In addition, the 1H NMR data also clearly showed an amide NH (δ 8.52), three CH3 doublets (δ 0.58, 1.04, and 1.11), a CH3 singlet (δ 1.13), and four olefinic protons (δ 5.21, 5.53, 5.70, and 6.17). The 13C NMR spectrum presented 28 carbon signals including a ketone CO (δ 212.1), an amide CO (δ 169.3), a carbonate CO (δ 148.7), and four olefinic protons (δ 117.1, 127.9, 131.5 and 139.5), as well as other 15 sp3 carbons. These data suggested that 6 shared several structural similarities with 10-phenyl-cytochalasin. Combined analysis of the key HMBC correlations and COSY correlations (Figure 1) allowed us to establish the planar structure of 6, and its 1H and 13C NMR data were fully assigned. The NOESY correlations of H-11/H-3, H-11/H-12 suggested the β-configurations for H-4 and H-5, as well as the α-configurations for CH3-6 and H-7, and the correlations of H13/H-7, H-13/H-24, H-18/H-24, H-19/H-25 revealed the βconfigurations of H-8 and H-16 and the α-configuration of H18 (Figure 1). The big coupling constant J13,14 = 15.3 Hz and J19,20 = 12.5 Hz suggested trans-configurations for 13-ene and 19-ene. Consequently, the structure of 6 was elucidated as drawn and named aspochalasin R. Compound 13 was obtained as a yellow solid. HRESIMS gave a molecular formula of C18H14O7 (12 degrees of unsaturation). From the 1H NMR data, the obvious signals at δ 7.78 (2H, dt, J = 10.6, 2.5 Hz), 6.68 (2H, dt, J = 10.6, 2.5 Hz), 7.00 (1H, d, J = 2.0 Hz), and 6.78 (1H, d, J = 2.0 Hz), indicated a 1,4-disubstitued benzene and a 1,3,4,5-tetrasubstitued
Figure 2. Experimental ECD spectra of compound 1 and the calculated ECD spectra of (1S)-1 and (1R)-1.
the planar structure of 5 was established. The NOESY correlations of H-17/H-19, H-21/H-23, and H-23/H-24 indicated that the configurations of 17-ene, 21-ene, and 25ene were E, E, and Z, respectively. The Cotton effects (Figure S9) of 5 were identical to those of 1, which suggested its S configuration at C-1. The Mo(Ac)4 induced CD spectrum of 5 presented a negative Cotton effect at 317 nm and positive Cotton effect at 380 nm (Figure.S10) resulted in the R configuration of C-29 being assigned. Consequently, the structure of 5 was elucidated as drawn and named asperphenalenone E. To the best of our knowledge, the planar structure of 5 was already registered for commercial sources in CAS (CAS 1083197-82-3), but no data and references are available for it. In this work, we regard it as a new compound Table 1. 1H NMR Data of 1−5a position 8 14 15 16 17 19 20 21 23 24 25 26 27 28 29 31 32 33 34 35 35-OCH3 35-OCH3 7-OH 13-OH a
1
2
3
6.79 2.11 2.81 2.52 4.77 1.59 1.48 4.84 1.82 2.00 5.02
s s s d (7.9) t (7.7) overlap m t (6.4) t (7.4) dd (14.7, 7.3) t (6.8)
6.79 2.11 2.80 2.52 4.79 1.60 1.51 4.90 1.93 2.18 6.51
s s s d (8.0) t (8.0) t (7.9) m t (7.1) t (7.6) dd (15.0, 7.5) t (7.4)
6.80 s 2.13 s 2.82 s 2.54 d (8.0) 4. 80 t (7.5) 1.61 m 1.51 m 4.89 t (6.6) 1.91 t (7.4) 2.37 m 5.71 t (7.3)
1.96 1.98 5.02 1.58 1.50 3.86 1.36 1.24
m m t (6.8) s s s s s
2.38 1.53 3.00 0.98 0.92
m, 2.09 m m, 1.10 m d (10.1) s s
2.40 1.58 3.00 1.00 0.95
13.08 br s 14.30 br s
m, 2.05 m m, 1.17 m dd (10.4, 1.9) s s
1.39 s 1.24 s
1.38 s 1.26 s
13.07 br s 14.29 br s
13.09 br s 14.31 br s
4 6.80 s 2.11 s 2.81 s 2.53 d (8.1) 4. 77 t (9.1) 1.61 m 1.49 m 4.86 t (7.7) 1.98 m, 1.88 m 1.70 m, 1.21 m 3.02 dt (9.6, 2.5) 1.37 m 1.75 m, 1.22 m 1.61 m, 1.12 m 2.76 dt (10.7, 2.0) 1.01 s 0.95 s 4.12 d (3.6) 1.36 s 1.24 s 3.24 s 3.23 s 13.07 br s 14.29 br s
5 6.80 2.11 2.80 2.52 4.78 1.59 1.47 4.85 1.83 2.00 5.06
s s s d (7.9) t (8.0) t (7.9) m t (7.1) t (7.6) dd (15.0, 7.3) t (7.2)
2.24 1.57 3.00 0.99 0.94 3.85 1.39 1.24
m, 1.89 m m, 1.12 m d (10.1) s s dd (25.0, 12.1) s s
13.07 br s 14.30 br s
Conditions: 600 MHz, DMSO-d6, J in Hz. D
DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. 13C NMR Data of Compounds 1−5a
a
position
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 35-OCH3 35-OCH3
81.2 200.6 101.9 135.2 106.1 202.9 163.3 118.0 149.1 112.9 163.2 107.2 165.8 8.1 26.2 41.2 115.3 139.7 39.2 25.8 123.8 134.1 39.4 25.3 125.2 139.0 34.5 26.4 124.4 134.0 25.5 17.5 58.0 15.4 15.6
81.2 200.6 102.0 135.1 106.1 202.8 163.3 118.0 149.0 112.8 162.9 107.2 165.8 8.1 26.2 41.2 115.4 139.6 39.2 25.9 124.2 133.7 38.0 26.4 141.2 132.8 23.9 30.8 77.3 71.5 26.2 24.6 168.6 15.3 15.6
81.2 200.6 102.0 135.2 106.2 202.8 163.4 118.0 149.1 112.9 163.2 107.2 165.9 8.2 26.2 41.3 115.3 139.8 39.2 25.9 124.0 134.0 38.7 27.4 138.7 133.0 31.7 30.6 76.9 71.6 26.2 24.6 169.2 15.3 15.6
81.3 200.7 102.0 135.1 106.1 202.9 163.3 118.0 149.0 112.8 163.2 107.2 165.8 8.1 26.2 41.3 115.3 139.7 39.3 25.9 123.5 134.5 34.6 30.9 77.2 43.0 23.0 24.4 83.6 70.4 27.5 24.3 106.0 15.4 15.5 55.3 55.2
81.2 200.7 102.0 135.2 106.1 202.9 163.3 118.0 149.0 112.8 162.9 107.2 165.8 8.1 26.2 41.3 115.3 139.7 39.2 25.9 123.7 134.3 39.4 25.5 124.9 139.8 31.9 29.6 77.2 71.6 26.1 24.6 58.2 15.3 15.6
corresponding to R-configuration for C-4 was deduced. Compound 23, a 2-O-methyl product of 22, was a known butyrolactone derivative (CAS 1246066-84-1) with no NMR data and ambiguous stereochemistry of C-4. By extensive spectroscopic analysis, its 1H and 13C NMR data were fully assigned. The biogenetic relationship suggested it should share the 4R-configuration with 22. The remaining compounds were identified as rosellichalasin (7),6 cytochalasin Z17 (8),7 cytochalasin Z9 (9),8 cytochalasin Z8 (10),8 cytochalasin Z7 (11),8 cytochalasin Z 13 (12), 9 aspulvinone Q (14), 10 aspulvinone P (15),10 aspulvinone E (16),3 3′,4′,4-trihydroxypulvinone (17),11 3,4,4′-trihydroxypulvinone (18),11 aspernolide H (19),12 aspernolide I (20),12 aspernolide K (21),12 butyrolactone II (24),13 2-O-methyl butyrolactone II (25),14 epicocconigrone A (26),15 epicoccolide B (27),16 1,3-dihydro4,6-dihydroxy-7-methylisobenzofuran (28),17 monomethylosoic acid (29),10,18 asterric acid (30),19,20 methyl-dichloroasterrate (31),20 sulochrin (32),20 and dihydrogeodin (33)21 by comparing their NMR data with those reported. The anti-HIV-1 activities of compounds 1−33 were evaluated (Table 3). A total of 33 compounds were subjected to preliminary screening by testing their HIV-1 inhibition rates at 100 μM. The IC50 and CC50 values of compounds with inhibition rates greater than 60% were then determined. Remarkably, 1, 4, 10, and 26 exhibited anti-HIV activities with IC50 values of 4.5, 2.4, 9.2, and 6.6 μM, respectively, and their CC50 values all were greater than 100 μM. The isolation of several different kinds of secondary metabolites from rice cultures of Aspergillus sp. CPCC 400735, and especially the discovery of new bioactive secondary metabolites, emphasizes that Aspergillus is an important source for discovering bioactive natural products. Asperphenalenones A−E (1−5) are composed of a linear diterpene linked to a phenalenone via a C−C bond. Although phenalenones have been reported frequently both from higher plants and microbial sources,22 this is the first report of the isolation of the phenalenone derivatives of this type and their potential bioactivities.
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Conditions: 150 MHz, DMSO-d6.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P2000 automatic digital polarimeter. UV spectra were measured on a JASCO V-650 spectrophotometer. CD spectra were measured on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 spectrometer. High resolution electrospray ionization mass spectra (HRESIMS) were recorded on a Synapt MS (Waters Corporation). Nuclear magnetic resonance (NMR) spectra were performed on Agilent-NMR-vnmrs 600 (600 MHz for 1H NMR and 150 MHz for 13C NMR) and Bruker spectrometer (500 MHz for 1H NMR and 125 MHz for 13C NMR), and the chemical shifts were given in δ (ppm). High performance liquid chromatography (HPLC) analyses were performed on an Agilent 1100 system equipped with an Agilent Eclipse C18 column (4.6 mm × 250 mm, 5 μm) and an Alltech 2000ES evaporative light scattering detector (115 °C, Gas 2.5 L/min). Semipreparative HPLC separations were performed on a NP7000 pump (Hanbon Sci. & Tech. Jiangsu, China) equipped with a Shodex RID 101 detector as well as an Agilent SB-C18 column (8.0 mm × 250 mm, 5 μm). Thin layer chromatography was performed on silica gel GF254 plates (Qingdao Marine Chemical, Qingdao, China). Silica gel H (Qingdao Marine Chemical, Qingdao, China), Lichroprep RP-C18 (Merck), and Sephadex LH-20 (Amersham Pharmacia) were used for column chromatography. Fungal Material. The endophytic fungus Aspergillus sp. CPCC 400735 was isolated from the plant Kadsura longipedunculata collected
benzene, and signal at δ 3.80 (3H, s) suggested a methoxyl group in 13. The 13C NMR data showed 18 carbon signals including a lactone group at δ 168.2, 16 olefinic carbons at δ 162.0, 156.4, 148.3, 145.9, 140.3, 136.0, 128.5 (overlapping), 123.1, 120.8, 115.1 (overlapping), 108.1, 106.3, and 99.7 (six of which were oxygenated), and a methoxyl group δ 55.8. The one-dimensional NMR data for 13 was similar to those of pulvinone analogues. By detailed analysis of 1H NMR, 13C NMR, and HMBC data, the relative structure of 13 was established, and its 1H and 13C NMR data were fully assigned. The Z-configuration of the 4-ene was deduced from the carbon signals at δ 99.6 (C-2) and 107.6 (C-5), which were much lower than those (about 105 and 115 ppm) of E-configuration.3 Therefore, the structure of 13 was elucidated as drawn and named aspulvinone R. Comparison of NMR data with reported values allowed 22 to be identified as a synthetic product 3-hydroxy-5-(4-hydroxybenzyl)-4-(4-hydroxyphenyl)-furan-2(5H)-one with ambiguous stereochemistry of C-4.4 Due to the biogenetic relationship and its optical rotation value [α]25 D −1.9 (c 0.052, MeOH) similar to that reported in literature,5 the β-configuration of H-4 E
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Table 3. Anti-HIV Activities of Compounds 1−33, Lamivudine, and Efavirenz compd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a
inhibition rate (% at 100 μM)
IC50 (μM)
CC50 (μM)
compd
± ± ± ± ± ± ± ± ± ± ± ± ±
4.5 32.6 b 2.4 22.1 b 41.6 28.9 14.4 9.2 25.8 b 78.3 b 43.7 b 40.0 72.4
>100 78.6 b >100 >100 b 98.6 70.8 >100 >100 95.1 b >100 b >100 b >100 >100
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 lamivudine efavirenz
99.99 96.82 48.08 99.84 98.60 54.26 81.48 79.45 98.72 99.98 99.99 44.88 69.83 a 52.30 a 72.27 74.76
0.01 0.11 1.69 0.12 0.53 4.25 0.60 0.03 1.01 0.02 0.01 2.73 0.07
± 2.48 ± 2.10 ± 2.21
inhibition rate (% at 100 μM)
IC50 (μM)
CC50 (μM)
± ± ± ± ± ± ± ± ± ± ± ±
82.3 67.2 48.0 54.2 68.7 84.5 74.8 6.6 20.2 49.3 b b b b 56.3 0.1 0.4 × 10−3
94.3 >100 93.0 >100 96.5 >100 >100 >100 >100 91.4 b b b b >100 b b
64.35 73.37 84.77 62.21 72.77 60.80 68.69 99.91 94.65 90.78 25.46 8.53 a 53.81 91.07 b b
0.13 2.83 1.91 1.46 0.03 1.36 1.94 0.02 3.10 2.33 4.05 0.07
± 5.55 ± 2.07
No Activity. bNot tested. 347.4 (3.9); IR (KBr) νmax 3383, 2926, 1603, 1459, 1336, and 1187 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2. HRESIMS m/z 575.3018 [M − H]− (calcd for C35H43O7, 575.3009). Asperphenalenone B (2). Brown gum; C35H44O10; [α]25 D = +1.9 (c 0.0792, CH3OH); λmax (log ε): 216.8 (4.7), 256.8 (4.5), 342.2 (4.2); IR (KBr) νmax 3381, 2933, 1602, 1460, 1336, and 1188 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2; HRESIMS m/z 623.2900 [M − H]− (calcd for C35H43O10, 623.2856). Asperphenalenone C (3). Brown gum; C35H44O10; [α]25 D = −12.5 (c 0.1075, CH3OH); λmax (logε): 215.4 (4.3), 256.6 (4.1), 342.8 (3.8); IR (KBr) νmax 3375, 2933, 1604, 1459, 1337, and 1188 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2; HRESIMS m/z 623.2893 [M − H]− (calcd for C35H43O10, 623.2856). Asperphenalenone D (4). Brown gum; C37H50O10; [α]25 D = +3.3 (c 0.0918, CH3OH); UV (MeOH) λmax (logε): 216.0 (4.4), 257.0 (4.3), 349.2 (4.0); IR (KBr) νmax 3402, 2932, 1604, 1480, 1337, and 1187 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2; HRESIMS m/z 653.3368 [M − H]− (calcd for C37H49O10, 653.3326). Asperphenalenone E (5). Brown solid; C35H46O9; [α]25 D = +13.3 (c 0.0975, CH3OH); UV (MeOH) λmax (logε): 209.0 (4.5), 257.2 (4.4), 346.8 (4.1); IR (KBr) νmax 3373, 2933, 1604, 1460, 1337, 1234, and 1150 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2; HRESIMS m/z 609.3094 [M − H]− (calcd for C35H45O9, 609.3064). Aspochalasin R (6). White amorphous powder; C28H33NO6; 1H NMR (DMSO-d6, 600 MHz) δ 8.52 (1H, s, 2-NH), 7.25 (2H, dd, J = 7.4, 6.9 Hz, H-3′/5′), 7.18 (1H, t, J = 7.3 Hz, H-4′), 7.16 (2H, d, J = 7.3 Hz, H-2′/6′), 6.17 (1H, d, J = 12.5 Hz, H-20), 5.70 (1H, dd, J = 15.3, 9.9 Hz, H-13), 5.53 (1H, t, J = 10.5 Hz, H-19), 5.27 (1H, m, H14), 3.64 (1H, brs, H-3), 3.15 (1H, m, H-18), 2.84 (1H, m, H-10a), 2.80 (1H, m, H-10b), 2.79 (1H, m, H-16), 2.69 (1H, brs, H-4), 2.62 (1H, d, J = 4.7 Hz, H-7), 2.31 (1H, d, J = 9.8 Hz, H-8), 2.27 (1H, m, H-15a), 2.11 (1H, m, H-15b), 1.83 (1H, m, H-5), 1.13 (3H, s, H-12), 1.11 (3H, d, J = 7.1 Hz, H-23), 1.04 (3H, d, J = 6.9 Hz, H-23), 0.58 (3H, d, J = 7.2 Hz, H-11); 13C NMR (DMSO-d6, 150 MHz) δ 212.1 (C-17), 169.3 (C-1), 148.7 (C-22), 139.5 (C-20), 136.9 (C-1′), 131.5 (C-14), 130.0 (C-2′/6′), 128.2 (C-3′/5′), 127.9 (C-13), 126.5 (C-4′), 117.1 (C-19), 86.7 (C-9), 59.2 (C-7), 57.1 (C-6), 53.0 (C-3), 47.9 (C8), 46.3 (C-4), 43.0 (C-18), 42.3 (C-16), 42.2 (C-10), 36.4 (C-15), 35.2 (C-5), 19.5 (C-12), 18.1 (C-24), 16.6 (C-25), 12.5 (C-11);
in Leishan of Guizhou province, China, and was identified on the basis of its 18S rRNA gene sequence (Genbank accession no. MF576131). The fungus was maintained in China Pharmaceutical Culture Collection (CPCC), Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. Fermentation and Extraction. The strain was cultured on PDA (0.3% potato extract, 2% glucose, 1.5% agar power) at 28 °C for 5 days. Under aseptic conditions, agar plugs were cut into small pieces and inoculated in 500 mL Erlenmeyer flasks, each containing 100 mL of seed medium [glucose 2%, sucrose 1%, soybean powder 0.2%, peptone 1%, K2HPO4 0.03%, poly(ethylene glycol) 0.25%, NaNO3 0.3%, and (NH4)2SO4 0.3%; the final pH of the medium was adjusted to 6.0 before sterilization with an autoclave at 121 °C for 20 min]. Cultures were incubated in flasks at 28 °C on a rotary shaker at 190 rpm for 2 days to prepare the seed culture. We filled each of 32 500 mL Erlenmeyer flasks with 80 g of rice and 120 mL of distilled H2O. After soaking for 5 h, the contents were sterilized with an autoclave at 121 °C for 20 min. After cooling to room temperature, each flask was inoculated with 10 mL of seed culture and incubated at 28 °C for 40 days. The fermented rice was supersonically extracted with CH3OH (3 × 2.5 L), and after recovering the organic solvent, the crude extract was further partitioned between EtOAc and H2O. Isolation and Purification. The EtOAc-soluble fraction (50 g) was subjected to silica gel column chromatography (CH3Cl−CH3OH, v/v, 100:0→20:1→7:1→0:100) to finally yield 12 combined fractions (Fr. A to M). By separation on silica gel column, ODS column, Sephadex LH-20 column and repeated purification by semipreparative HPLC, compounds 1 (14.7 mg), 4 (5.0 mg), 6 (4.5 mg), 7 (32.5 mg), 30 (22.0 mg), and 33 (13.8 mg) were obtained from Fr. B; compounds 8 (5.0 mg), 9 (13.0 mg), 10 (30.0 mg), 11 (26.2 mg), 12 (21.5 mg), 21 (200.2 mg), 23 (13.1 mg), 25 (215.7 mg), 31 (5.0 mg), 32 (30.0 mg), and 33 (18.0 mg) were obtained from Fr. C; compounds 19 (40.0 mg) and 20 (64.5 mg) were obtained from Fr. D; compounds 2 (52.0 mg), 3 (5.0 mg), 5 (197.6 mg), 24 (604.0 mg), and 28 (167.4 mg) were obtained from Fr. E; compounds 5 (125.0 mg), 14 (30.9 mg), 15 (32.0 mg), 22 (34.0 mg), 26 (15.8 mg), 27 (52.8 mg), and 29 (2.0 mg) were obtained from Fr. F; compounds 13 (110.0 mg), 17 (17.0 mg), and 18 (6.7 mg) were obtained from Fr. J; and compounds 14 (25.6 mg) and 16 (52.8 mg) were obtained from Fr. L. The details of the extraction and isolation are available in the Supporting Information. Asperphenalenone A (1). Brown gum; C35H44O7; [α]25 D = −5.5 (c 0.1050, CH3OH); UV (MeOH) λmax (logε): 204.8 (4.4), 256.8 (4.2), F
DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
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HRESIMS m/z 478.2157 [M − H]− (calcd for C28H32NO6, 478.2230). Aspulvinone R (13). Yellow amorphous powder; C18H14O7; 1H NMR (DMSO-d6, 500 MHz) δ: 9.58 (1H, s, 4′−OH), 9.29 (1H, s, 5″−OH), 8.83 (1H, s, 4″−OH), 7.78 (2H, dt, J = 10.6, 3.5, 2.5 Hz, H2′/6′), 7.00 (1H, d, J = 2.0 Hz, H-6″), 6.82 (2H, dt, J = 10.6, 3.5, 2.5 Hz, H-3′/5′), 6.78 (H, d, J = 2.0 Hz, H-2″), 6.50 (1H, s, H-5); 13C NMR (DMSO-d6, 125 MHz) δ 168.3 (C-1), 162.0 (C-3), 156.4 (C4′), 148.3 (C-3″), 145.9 (C-5″), 140.3 (C-4), 136.0 (C-4″), 128.5 (C2′/6′), 123.1 (C-1″), 120.8 (C-1′), 115.1 (C-3′/5′), 111.1 (C-6″), 108.1 (C-5), 106.3 (C-2″), 99.7 (C-2), 55.8 (3″-OCH3); HRESIMS m/z 341.0694 [M − H]−(calcd for C18H13O7, 341.0661). Anti-HIV Assay. SupT1 cells (2 × 105) were cotransfected with 0.6 mg of pNL-Luc-E and 0.4 mg of pHIT/G. Then the VSV-G pseudo typed viral supernatant (HIV-1) was harvested by filtration through a 0.45 μm filter after 48 h and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux, Lyon, France). SupT1 cells were exposed to VSV-G pseudotyped HIV-1 (MOI = 1) at 37.8 °C for 48 h in the absence or presence of test compounds. A Luciferase Assay System (Promega, Madison, USA) was used to determine the inhibition rate.23 The cytotoxicity was measured by the CCK-8 method. SupT1 cells (5 × 105) were seeded into a 96-well microtiter plate in the absence or presence of test compounds (negative control, DMSO) in triplicate and incubated at 37 °C. After 48 h incubation, cell viability was measured by the CCK-8 method. The dilutions of the tested compounds were independently performed three times.
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(3) Gao, H. Q.; Guo, W. Q.; Wang, Q.; Zhang, L.; Zhu, M.; Zhu, T.; Gu, Q.; Wang, W.; Li, D. Bioorg. Med. Chem. Lett. 2013, 23, 1776− 1778. (4) Cotelle, P.; Cotelle, N.; Teissier, E.; Vezin, H. Bioorg. Med. Chem. 2003, 11, 1087−1093. (5) Nong, X.; Wang, Y.; Zhang, X.; Zhou, M.; Xu, X.; Qi, S. Mar. Drugs 2014, 12, 6113−6124. (6) Zhang, H. Q.; Deng, Z. S.; Guo, Z. Y.; Peng, Y.; Huang, N. Y.; He, H. B.; Tu, X.; Zou, K. Molecules 2015, 20, 7940−7950. (7) Lin, Z. J.; Zhang, G. J.; Zhu, T. J.; Liu, R.; Wei, H. J.; Gu, Q. Q. Helv. Chim. Acta 2009, 92, 1538−1544. (8) Liu, R.; Gu, Q. Q.; Zhu, W. M.; Cui, C. B.; Fan, G. T.; Fang, Y. C.; Zhu, T. J.; Liu, H. B. J. Nat. Prod. 2006, 69, 871−875. (9) Liu, R.; Lin, Z. J.; Zhu, T. J.; Fang, Y. C.; Gu, Q. Q.; Zhu, W. M. J. Nat. Prod. 2008, 71, 1127−1132. (10) Zhang, L. H.; Feng, B. M.; Zhao, Y. Q.; Sun, Y.; Liu, B.; Liu, F.; Chen, G.; Bai, J.; Hua, H. M.; Wang, H. F.; Pei, Y. H. Bioorg. Med. Chem. Lett. 2016, 26, 346−350. (11) Gill, M.; Kiefel, M. J.; Lally, D. A.; Ten, A. Aust. J. Chem. 1990, 43, 1497−1518. (12) Li, L. J.; Li, T. X.; Kong, L. Y.; Yang, M. H. Phytochem. Lett. 2016, 16, 134−140. (13) Rao, K. V.; Sadhukhan, A. K.; Veerender, M.; Ravikumar, V.; Mohan, E. V.; Dhanvantri, S. D.; Sitaramkumar, M.; Babu, J. M.; Vyas, K.; Reddy, G. O. Chem. Pharm. Bull. 2000, 48, 559−562. (14) Chen, M.; Wang, K. L.; Liu, M.; She, Z. G.; Wang, C. Y. Chem. Biodiversity 2015, 12, 1398−1406. (15) El Amrani, M.; Lai, D.; Debbab, A.; Aly, A. H.; Siems, K.; Seidel, C.; Schnekenburger, M.; Gaigneaux, A.; Diederich, M.; Feger, D.; Lin, W. H.; Proksch, P. J. Nat. Prod. 2014, 77, 49−56. (16) Talontsi, F. M.; Dittrich, B.; Schüffler, A.; Sun, H.; Laatsch, H. Eur. J. Org. Chem. 2013, 2013, 3174−3180. (17) Ishikawa, Y.; Ito, T.; Lee, K. H. Nihon Yukagakkaishi 1996, 45, 1321−1325. (18) Natori, S.; Nishikawa, H. Chem. Pharm. Bull. 1962, 10, 987− 989. (19) Ohashi, O.; et al. J. Antibiot. 1992, 45, 1684−1685. (20) Liu, D. S.; Yan, L.; Ma, L. Y.; Huang, Y. L.; Pan, X. H.; Liu, W. Z.; Lv, Z. H. Arch. Pharmacal Res. 2015, 38, 1038−1043. (21) Inamori, Y.; Kato, Y.; Kubo, M.; Kamiki, T.; Takemoto, T.; Nomoto, K. Chem. Pharm. Bull. 1983, 31, 4543−4548. (22) Elsebai, M. F.; et al. Nat. Prod. Rep. 2014, 31, 628−645. (23) Zhang, Q.; Liu, Z. L.; Mi, Z. Y.; Li, X. Y.; Jia, P. P.; Zhou, J. M.; Yin, X.; You, X. F.; Yu, L. Y.; Guo, F.; Ma, J.; Liang, C.; Cen, S. Antiviral Res. 2011, 91, 321−329.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00878. NMR spectra of 1−33, UV, IR, and CD spectra of 1−5, and NMR data of known compounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
*L. Y. Yu. E-mail: [email protected]. Tel: +86-10-63187118. ORCID
De-Wu Zhang: 0000-0001-6289-0617 Li-Yan Yu: 0000-0002-8861-9806 Notes
Compounds 1, 2, 4, and 5 presented in this work have been reported previously in a Chinese patent (Application number 201610424586; Publication Number 106085868A) applied by our group. The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge financial support from the National Natural Science Foundation of China (NSFC) (Nos. 31170041 and 81373452), the National Infrastructure of Microbial Resources (No. NIMR-2016-3), National S & T Major Special Project on Major New Drug Innovation (No. 2017ZX09101003-007-001), and CAMS Initiative for Innovative Medicine (No. 2016-I2M2-002). Liyan Yu is supported as a Xiehe Scholar.
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REFERENCES
(1) Frelek, J.; Szczepek, W. J. Tetrahedron: Asymmetry 1999, 10, 1507−1520. (2) Di Bari, L.; Pescitelli, G.; Pratelli, C.; Pini, D.; Salvadori, P. J. Org. Chem. 2001, 66, 4819−4825. G
DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
Metabolites from the Plant Endophytic Fungus Aspergillus sp. CPCC 400735 and Their Anti-HIV Activities Xu Pang, Jian-Yuan Zhao, Xiao-Mei Fang, Tao Zhang, De-Wu Zhang, Hong-Yu Liu, Jing Su, Shan Cen, and Li-Yan Yu* Institute of Medicinal Biotechnology, Academy of Medical Science & Peking Union Medical College, Beijing 100050, China S Supporting Information *
ABSTRACT: Thirty-three metabolites including five phenalenone derivatives (1−5), seven cytochalasins (6−12), thirteen butenolides (13−25), and eight phenyl derivatives (26−33) were isolated from Aspergillus sp. CPCC 400735 cultured on rice. The structures of all compounds were elucidated by NMR, MS, and CD experiments, of which 1−5 (asperphenalenones A−E), 6 (aspochalasin R), and 13 (aspulvinone R) were identified as new compounds. Specifically, asperphenalenones A−E (1−5) represent an unusual structure composed of a linear diterpene derivative linked to a phenalenone derivative via a C−C bond. Compounds 1, 4, 10, and 26 exhibited antiHIV activity with IC50 values of 4.5, 2.4, 9.2, and 6.6 μM, respectively (lamivudine 0.1 μM; efavirenz, 0.4 × 10−3 μM).
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spectrometry (HRESIMS). In the 1H NMR spectrum, the characteristic signals at δ 14.30 (1H, s, 13-OH), 13.08 (1H, s, 7OH), 6.79 (1H, s, H-8), 5.02 (2H, t, H-29), 4.84 (1H, t, H-21), 4.77 (1H, t, H-17), 3.86 (2H, s, H-33), 2.81 (3H, s, H-15), 2.52 (1H, d, H-16), 2.11 (3H, s, H-14), 1.58 (3H, s, H-31), 1.50 (3H, s, H-15), 1.36 (3H, s, H-34), 1.24 (3H, s, H-35) were clearly observed. Starting from these characteristic proton signals, the following key heteronuclear multiple bond correlations (HMBCs) of 13-OH/C-13, C-3, C-2, H-14/C12, C-11, C-13, 7-OH/C-7, C-8, C-5, C-2, H-8/C-5, C-10, C15, C-7, C-6, H-15/C-8, C-9, C-10, H-16/C-1, C-2, C-6, C-17, C-18, H-17/C-16, C-19, C-35, H-35/C-19, C-17, C-18, H-21/ C-34, C-20, C-19, H-34/C-23, C-21, C-22, H-25/C-23, C-24, C-27, C-33, H-33/C-25, C-26, C-27, H-29/C-27, C-32, H-31/ C-29, C-30, and H-32/C-29, C-30 were found, together with the 1H−1H correlation spectroscopy (COSY) correlations of H-17/H-16, H-25/H-24, H-24/H-23, H-29/H-28, and H-28/ H-27, which allowed the planar structure of 1 to be established (Figure 1). Based on analysis of nuclear Overhauser effect spectroscopy (NOESY) data, the correlations of H-17/H-19, H-21/H-23, and H-24/H-33 demonstrated the configurations for 17-ene, 21-ene, and 25-ene to be E-, E-, and Z-, respectively. The absolute configuration of C-1 was assigned by the Rh2(OCOCF3) induced CD experiment and electronic circular dichroism (ECD) calculation. The Rh2(OCOCF3) induced CD
cquired immunodeficiency syndrome (AIDS), an immunosuppressive disease that can result in life-threatening opportunistic infections and malignancies, is caused by human immunodeficiency virus (HIV). Substantial research efforts have been dedicated to find compounds from synthetic products and natural products that can be developed as therapeutic agents against HIV. Microbial secondary metabolites are one potential resource for finding anti-HIV agents. Aspergillus (Moniliaceae) is a genus consisting of many filamentous fungal species, several of which have substantial medical value. The Aspergillus genus is well-known for its ability to produce diverse secondary metabolites, so it is regarded as an important source for discovering new bioactive natural products. CPCC 400735, an Aspergillus sp., was isolated from a medicinal plant Kadsura longipedunculata. While searching for HIV-1 inhibitors from plant endophytes, it was found that the EtOAc-soluble fraction of an extract from rice cultures of CPCC 400735 displayed significant HIV-1 inhibitory effect. Investigation of the chemical constituents of the bioactive fraction led us to obtain 33 metabolites, including five new phenalenone derivatives, asperphenalenones A−E (1−5), one new aspochalasin, aspochalasin R (6), one new aspulvinone, aspulvinone R (13) (Chart 1), and 26 known compounds (7− 12 and 14−33, Chart 2); all were tested for anti-HIV-1 activity.
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RESULTS AND DISCUSSION Compound 1 had a molecular formula of C35H44O7 as determined by high-resolution electrospray ionization mass © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 27, 2016
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Chart 1. Structures of the New Compounds
the planar structure of 3 to be established. In the NOESY spectrum, the correlations of H-17/H-19 and H-21/H-23 determined the configurations of 17-ene and 21-ene to be E and E, respectively, and the Z-configuration of 25-ene was determined by NOESY correlation of H-24/H-27. The CD spectrum of 3 showed Cotton effects identical to 1 (Figure S6), so the absolute configuration of C-1 was assigned as S. The Mo(Ac)4 induced CD spectrum of 3, which presented a negative Cotton effect at 317 nm and a positive Cotton effect at 380 nm (Figure S7), allowed the R-configuration to be assigned for C-29. Consequently, the structure of 3 was elucidated as drawn and named asperphenalenone C. Compound 4 had a molecular formula of C37H50O10 as determined by HRESIMS. Comparison of spectroscopic data of 4 with those of 1 revealed that the planar structure of 4 was the same as 1 except for the structure fragment of C-24 to C-33. The 13 degrees of unsaturation corresponding to the molecular formula C37H50O10 suggested a cyclic structure in the fragment of C-24 to C-33. Based on the HMBC correlations of H-31/C29, C-30, H-32/C-29, C-30, H-29/C-27, H-33/C-25, C-26, C27, and H-25/C-23, and the 1H−1H COSY correlations of H24/H-23, H-25, H-26/H-25, H-27, H-33, and H-28/H-29, the C-24 to C-32 structure was established. The correlations of H17/H-19 and H-21/H-23b observed in the NOESY spectrum revealed the E-configurations for 17-ene and 21-ene. The Cotton effects in the CD spectrum (Figure S8) of 4, which was identical to 1, suggested the S configuration of C-1. The absolute configurations of chiral carbons C-25, C-26, and C-29 were not identified in this work, whereas their relative configurations were determined by establishing the relative structure of the C-25 to C-29 cyclic ring based on the key NOESY correlations of H-25/H-26 and H-25/H-29. Consequently, the structure of 4 was elucidated as drawn and named asperphenalenone D. Compound 5 with a molecular formula of C35H46O9 determined by HRESIMS had two hydrogen atoms more
spectrum of 1 presented a negative Cotton effect at 350 nm (Figure S1); thus the configuration of C-1 was considered to be S based on the empirical rule.1 Moreover, the result of ECD calculations of 1 (details of ECD calculations are available in the Supporting Information) showed that the theoretically calculated ECD spectrum of (1S)-1 was in good agreement with the experimental ECD spectrum of 1 in the 200−400 nm region (Figure 2), which allowed the assignment of the S configuration for C-1. Consequently, the structure of 1 was elucidated as drawn and named asperphenalenone A; its 1H and 13 C NMR data were fully assigned (Tables 1 and 2). Compound 2 had a molecular formula of C35H44O10 as determined by HRESIMS. The NMR data suggested that 2, as an analogue of 1, had the same structural fragment of C-1 to C22 as 1 but differed in the structural fragment of C-23 to C-33. By combined analyses of 1H−1H COSY and HMBC spectra, the structural fragment of C-23 to C-33 was finally established. In NOESY spectrum, the correlations of H-17/H-19, H-21/H23, and H-24/H-27 determined the configurations for 17-ene, 21-ene, and 25-ene as E, E, and E. The CD spectrum of 2 was identical to that of 1 (Figures S2 and S3), and like 1, the Rh2(OCOCF3) induced CD spectrum of 2 presented a negative Cotton effect at 347 nm (Figure S4); thus the 1Sconfiguration was assigned for 2. The absolute configuration of C-29 was determined by Mo(Ac)4 induced CD experiment. The CD spectrum of 2 in solution of Mo(Ac)4 in CH3Cl presented a negative Cotton effect at 317 nm and a positive Cotton effect at 375 nm (Figure S5), thus the R-configuration of C-29 was determined according to the empirical rule.2 Consequently, the structure of 2 was elucidated as drawn and named asperphenalenone B. Compound 3 with a molecular formula of C35H44O10 determined by HRESIMS was an isomer of 2. By comparing the NMR data of 3 with those of 2, it was found that they had identical structure except for the difference in C-23 to C-27. Combined use of 1H−1H COSY and HMBC spectra allowed B
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Chart 2. Structures of the Known Compounds
Figure 1. Key HMBC (H → C), COSY (H → H), and NOESY (H → H) correlations of compounds 1−6.
(C-28 to C-32). Compariaon of the NMR data of 5 with those of 3 revealed that they had the same structure of C-28 to C-32. Based on the analyses of HSQC, COSY, and HMBC spectra,
than 1 and two oxygen atoms more than 1. Comparison of spectroscopic data of 5 with those of 1 revealed that 5 had the same planar structure as 1 except for terminal isopentyl group C
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and fully assigned its NMR data, as well as established its absolute configuration for the first time. Compound 6 was obtained as a white solid. HRESIMS gave a molecular formula of C28H33NO6. From the 1H NMR data, the proton signals at δ 7.25 (2H, t, J = 7.3 Hz), 7.18 (1H, t, J = 7.3 Hz), and 7.16 (2H, d, J = 7.3 Hz) supported a single substituted benzene in the molecule. In addition, the 1H NMR data also clearly showed an amide NH (δ 8.52), three CH3 doublets (δ 0.58, 1.04, and 1.11), a CH3 singlet (δ 1.13), and four olefinic protons (δ 5.21, 5.53, 5.70, and 6.17). The 13C NMR spectrum presented 28 carbon signals including a ketone CO (δ 212.1), an amide CO (δ 169.3), a carbonate CO (δ 148.7), and four olefinic protons (δ 117.1, 127.9, 131.5 and 139.5), as well as other 15 sp3 carbons. These data suggested that 6 shared several structural similarities with 10-phenyl-cytochalasin. Combined analysis of the key HMBC correlations and COSY correlations (Figure 1) allowed us to establish the planar structure of 6, and its 1H and 13C NMR data were fully assigned. The NOESY correlations of H-11/H-3, H-11/H-12 suggested the β-configurations for H-4 and H-5, as well as the α-configurations for CH3-6 and H-7, and the correlations of H13/H-7, H-13/H-24, H-18/H-24, H-19/H-25 revealed the βconfigurations of H-8 and H-16 and the α-configuration of H18 (Figure 1). The big coupling constant J13,14 = 15.3 Hz and J19,20 = 12.5 Hz suggested trans-configurations for 13-ene and 19-ene. Consequently, the structure of 6 was elucidated as drawn and named aspochalasin R. Compound 13 was obtained as a yellow solid. HRESIMS gave a molecular formula of C18H14O7 (12 degrees of unsaturation). From the 1H NMR data, the obvious signals at δ 7.78 (2H, dt, J = 10.6, 2.5 Hz), 6.68 (2H, dt, J = 10.6, 2.5 Hz), 7.00 (1H, d, J = 2.0 Hz), and 6.78 (1H, d, J = 2.0 Hz), indicated a 1,4-disubstitued benzene and a 1,3,4,5-tetrasubstitued
Figure 2. Experimental ECD spectra of compound 1 and the calculated ECD spectra of (1S)-1 and (1R)-1.
the planar structure of 5 was established. The NOESY correlations of H-17/H-19, H-21/H-23, and H-23/H-24 indicated that the configurations of 17-ene, 21-ene, and 25ene were E, E, and Z, respectively. The Cotton effects (Figure S9) of 5 were identical to those of 1, which suggested its S configuration at C-1. The Mo(Ac)4 induced CD spectrum of 5 presented a negative Cotton effect at 317 nm and positive Cotton effect at 380 nm (Figure.S10) resulted in the R configuration of C-29 being assigned. Consequently, the structure of 5 was elucidated as drawn and named asperphenalenone E. To the best of our knowledge, the planar structure of 5 was already registered for commercial sources in CAS (CAS 1083197-82-3), but no data and references are available for it. In this work, we regard it as a new compound Table 1. 1H NMR Data of 1−5a position 8 14 15 16 17 19 20 21 23 24 25 26 27 28 29 31 32 33 34 35 35-OCH3 35-OCH3 7-OH 13-OH a
1
2
3
6.79 2.11 2.81 2.52 4.77 1.59 1.48 4.84 1.82 2.00 5.02
s s s d (7.9) t (7.7) overlap m t (6.4) t (7.4) dd (14.7, 7.3) t (6.8)
6.79 2.11 2.80 2.52 4.79 1.60 1.51 4.90 1.93 2.18 6.51
s s s d (8.0) t (8.0) t (7.9) m t (7.1) t (7.6) dd (15.0, 7.5) t (7.4)
6.80 s 2.13 s 2.82 s 2.54 d (8.0) 4. 80 t (7.5) 1.61 m 1.51 m 4.89 t (6.6) 1.91 t (7.4) 2.37 m 5.71 t (7.3)
1.96 1.98 5.02 1.58 1.50 3.86 1.36 1.24
m m t (6.8) s s s s s
2.38 1.53 3.00 0.98 0.92
m, 2.09 m m, 1.10 m d (10.1) s s
2.40 1.58 3.00 1.00 0.95
13.08 br s 14.30 br s
m, 2.05 m m, 1.17 m dd (10.4, 1.9) s s
1.39 s 1.24 s
1.38 s 1.26 s
13.07 br s 14.29 br s
13.09 br s 14.31 br s
4 6.80 s 2.11 s 2.81 s 2.53 d (8.1) 4. 77 t (9.1) 1.61 m 1.49 m 4.86 t (7.7) 1.98 m, 1.88 m 1.70 m, 1.21 m 3.02 dt (9.6, 2.5) 1.37 m 1.75 m, 1.22 m 1.61 m, 1.12 m 2.76 dt (10.7, 2.0) 1.01 s 0.95 s 4.12 d (3.6) 1.36 s 1.24 s 3.24 s 3.23 s 13.07 br s 14.29 br s
5 6.80 2.11 2.80 2.52 4.78 1.59 1.47 4.85 1.83 2.00 5.06
s s s d (7.9) t (8.0) t (7.9) m t (7.1) t (7.6) dd (15.0, 7.3) t (7.2)
2.24 1.57 3.00 0.99 0.94 3.85 1.39 1.24
m, 1.89 m m, 1.12 m d (10.1) s s dd (25.0, 12.1) s s
13.07 br s 14.30 br s
Conditions: 600 MHz, DMSO-d6, J in Hz. D
DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. 13C NMR Data of Compounds 1−5a
a
position
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 35-OCH3 35-OCH3
81.2 200.6 101.9 135.2 106.1 202.9 163.3 118.0 149.1 112.9 163.2 107.2 165.8 8.1 26.2 41.2 115.3 139.7 39.2 25.8 123.8 134.1 39.4 25.3 125.2 139.0 34.5 26.4 124.4 134.0 25.5 17.5 58.0 15.4 15.6
81.2 200.6 102.0 135.1 106.1 202.8 163.3 118.0 149.0 112.8 162.9 107.2 165.8 8.1 26.2 41.2 115.4 139.6 39.2 25.9 124.2 133.7 38.0 26.4 141.2 132.8 23.9 30.8 77.3 71.5 26.2 24.6 168.6 15.3 15.6
81.2 200.6 102.0 135.2 106.2 202.8 163.4 118.0 149.1 112.9 163.2 107.2 165.9 8.2 26.2 41.3 115.3 139.8 39.2 25.9 124.0 134.0 38.7 27.4 138.7 133.0 31.7 30.6 76.9 71.6 26.2 24.6 169.2 15.3 15.6
81.3 200.7 102.0 135.1 106.1 202.9 163.3 118.0 149.0 112.8 163.2 107.2 165.8 8.1 26.2 41.3 115.3 139.7 39.3 25.9 123.5 134.5 34.6 30.9 77.2 43.0 23.0 24.4 83.6 70.4 27.5 24.3 106.0 15.4 15.5 55.3 55.2
81.2 200.7 102.0 135.2 106.1 202.9 163.3 118.0 149.0 112.8 162.9 107.2 165.8 8.1 26.2 41.3 115.3 139.7 39.2 25.9 123.7 134.3 39.4 25.5 124.9 139.8 31.9 29.6 77.2 71.6 26.1 24.6 58.2 15.3 15.6
corresponding to R-configuration for C-4 was deduced. Compound 23, a 2-O-methyl product of 22, was a known butyrolactone derivative (CAS 1246066-84-1) with no NMR data and ambiguous stereochemistry of C-4. By extensive spectroscopic analysis, its 1H and 13C NMR data were fully assigned. The biogenetic relationship suggested it should share the 4R-configuration with 22. The remaining compounds were identified as rosellichalasin (7),6 cytochalasin Z17 (8),7 cytochalasin Z9 (9),8 cytochalasin Z8 (10),8 cytochalasin Z7 (11),8 cytochalasin Z 13 (12), 9 aspulvinone Q (14), 10 aspulvinone P (15),10 aspulvinone E (16),3 3′,4′,4-trihydroxypulvinone (17),11 3,4,4′-trihydroxypulvinone (18),11 aspernolide H (19),12 aspernolide I (20),12 aspernolide K (21),12 butyrolactone II (24),13 2-O-methyl butyrolactone II (25),14 epicocconigrone A (26),15 epicoccolide B (27),16 1,3-dihydro4,6-dihydroxy-7-methylisobenzofuran (28),17 monomethylosoic acid (29),10,18 asterric acid (30),19,20 methyl-dichloroasterrate (31),20 sulochrin (32),20 and dihydrogeodin (33)21 by comparing their NMR data with those reported. The anti-HIV-1 activities of compounds 1−33 were evaluated (Table 3). A total of 33 compounds were subjected to preliminary screening by testing their HIV-1 inhibition rates at 100 μM. The IC50 and CC50 values of compounds with inhibition rates greater than 60% were then determined. Remarkably, 1, 4, 10, and 26 exhibited anti-HIV activities with IC50 values of 4.5, 2.4, 9.2, and 6.6 μM, respectively, and their CC50 values all were greater than 100 μM. The isolation of several different kinds of secondary metabolites from rice cultures of Aspergillus sp. CPCC 400735, and especially the discovery of new bioactive secondary metabolites, emphasizes that Aspergillus is an important source for discovering bioactive natural products. Asperphenalenones A−E (1−5) are composed of a linear diterpene linked to a phenalenone via a C−C bond. Although phenalenones have been reported frequently both from higher plants and microbial sources,22 this is the first report of the isolation of the phenalenone derivatives of this type and their potential bioactivities.
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Conditions: 150 MHz, DMSO-d6.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO P2000 automatic digital polarimeter. UV spectra were measured on a JASCO V-650 spectrophotometer. CD spectra were measured on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 spectrometer. High resolution electrospray ionization mass spectra (HRESIMS) were recorded on a Synapt MS (Waters Corporation). Nuclear magnetic resonance (NMR) spectra were performed on Agilent-NMR-vnmrs 600 (600 MHz for 1H NMR and 150 MHz for 13C NMR) and Bruker spectrometer (500 MHz for 1H NMR and 125 MHz for 13C NMR), and the chemical shifts were given in δ (ppm). High performance liquid chromatography (HPLC) analyses were performed on an Agilent 1100 system equipped with an Agilent Eclipse C18 column (4.6 mm × 250 mm, 5 μm) and an Alltech 2000ES evaporative light scattering detector (115 °C, Gas 2.5 L/min). Semipreparative HPLC separations were performed on a NP7000 pump (Hanbon Sci. & Tech. Jiangsu, China) equipped with a Shodex RID 101 detector as well as an Agilent SB-C18 column (8.0 mm × 250 mm, 5 μm). Thin layer chromatography was performed on silica gel GF254 plates (Qingdao Marine Chemical, Qingdao, China). Silica gel H (Qingdao Marine Chemical, Qingdao, China), Lichroprep RP-C18 (Merck), and Sephadex LH-20 (Amersham Pharmacia) were used for column chromatography. Fungal Material. The endophytic fungus Aspergillus sp. CPCC 400735 was isolated from the plant Kadsura longipedunculata collected
benzene, and signal at δ 3.80 (3H, s) suggested a methoxyl group in 13. The 13C NMR data showed 18 carbon signals including a lactone group at δ 168.2, 16 olefinic carbons at δ 162.0, 156.4, 148.3, 145.9, 140.3, 136.0, 128.5 (overlapping), 123.1, 120.8, 115.1 (overlapping), 108.1, 106.3, and 99.7 (six of which were oxygenated), and a methoxyl group δ 55.8. The one-dimensional NMR data for 13 was similar to those of pulvinone analogues. By detailed analysis of 1H NMR, 13C NMR, and HMBC data, the relative structure of 13 was established, and its 1H and 13C NMR data were fully assigned. The Z-configuration of the 4-ene was deduced from the carbon signals at δ 99.6 (C-2) and 107.6 (C-5), which were much lower than those (about 105 and 115 ppm) of E-configuration.3 Therefore, the structure of 13 was elucidated as drawn and named aspulvinone R. Comparison of NMR data with reported values allowed 22 to be identified as a synthetic product 3-hydroxy-5-(4-hydroxybenzyl)-4-(4-hydroxyphenyl)-furan-2(5H)-one with ambiguous stereochemistry of C-4.4 Due to the biogenetic relationship and its optical rotation value [α]25 D −1.9 (c 0.052, MeOH) similar to that reported in literature,5 the β-configuration of H-4 E
DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 3. Anti-HIV Activities of Compounds 1−33, Lamivudine, and Efavirenz compd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a
inhibition rate (% at 100 μM)
IC50 (μM)
CC50 (μM)
compd
± ± ± ± ± ± ± ± ± ± ± ± ±
4.5 32.6 b 2.4 22.1 b 41.6 28.9 14.4 9.2 25.8 b 78.3 b 43.7 b 40.0 72.4
>100 78.6 b >100 >100 b 98.6 70.8 >100 >100 95.1 b >100 b >100 b >100 >100
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 lamivudine efavirenz
99.99 96.82 48.08 99.84 98.60 54.26 81.48 79.45 98.72 99.98 99.99 44.88 69.83 a 52.30 a 72.27 74.76
0.01 0.11 1.69 0.12 0.53 4.25 0.60 0.03 1.01 0.02 0.01 2.73 0.07
± 2.48 ± 2.10 ± 2.21
inhibition rate (% at 100 μM)
IC50 (μM)
CC50 (μM)
± ± ± ± ± ± ± ± ± ± ± ±
82.3 67.2 48.0 54.2 68.7 84.5 74.8 6.6 20.2 49.3 b b b b 56.3 0.1 0.4 × 10−3
94.3 >100 93.0 >100 96.5 >100 >100 >100 >100 91.4 b b b b >100 b b
64.35 73.37 84.77 62.21 72.77 60.80 68.69 99.91 94.65 90.78 25.46 8.53 a 53.81 91.07 b b
0.13 2.83 1.91 1.46 0.03 1.36 1.94 0.02 3.10 2.33 4.05 0.07
± 5.55 ± 2.07
No Activity. bNot tested. 347.4 (3.9); IR (KBr) νmax 3383, 2926, 1603, 1459, 1336, and 1187 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2. HRESIMS m/z 575.3018 [M − H]− (calcd for C35H43O7, 575.3009). Asperphenalenone B (2). Brown gum; C35H44O10; [α]25 D = +1.9 (c 0.0792, CH3OH); λmax (log ε): 216.8 (4.7), 256.8 (4.5), 342.2 (4.2); IR (KBr) νmax 3381, 2933, 1602, 1460, 1336, and 1188 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2; HRESIMS m/z 623.2900 [M − H]− (calcd for C35H43O10, 623.2856). Asperphenalenone C (3). Brown gum; C35H44O10; [α]25 D = −12.5 (c 0.1075, CH3OH); λmax (logε): 215.4 (4.3), 256.6 (4.1), 342.8 (3.8); IR (KBr) νmax 3375, 2933, 1604, 1459, 1337, and 1188 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2; HRESIMS m/z 623.2893 [M − H]− (calcd for C35H43O10, 623.2856). Asperphenalenone D (4). Brown gum; C37H50O10; [α]25 D = +3.3 (c 0.0918, CH3OH); UV (MeOH) λmax (logε): 216.0 (4.4), 257.0 (4.3), 349.2 (4.0); IR (KBr) νmax 3402, 2932, 1604, 1480, 1337, and 1187 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2; HRESIMS m/z 653.3368 [M − H]− (calcd for C37H49O10, 653.3326). Asperphenalenone E (5). Brown solid; C35H46O9; [α]25 D = +13.3 (c 0.0975, CH3OH); UV (MeOH) λmax (logε): 209.0 (4.5), 257.2 (4.4), 346.8 (4.1); IR (KBr) νmax 3373, 2933, 1604, 1460, 1337, 1234, and 1150 cm−1; 1H NMR data (DMSO-d6, 600 MHz) see Table 1; 13C NMR data (DMSO-d6, 150 MHz) see Table 2; HRESIMS m/z 609.3094 [M − H]− (calcd for C35H45O9, 609.3064). Aspochalasin R (6). White amorphous powder; C28H33NO6; 1H NMR (DMSO-d6, 600 MHz) δ 8.52 (1H, s, 2-NH), 7.25 (2H, dd, J = 7.4, 6.9 Hz, H-3′/5′), 7.18 (1H, t, J = 7.3 Hz, H-4′), 7.16 (2H, d, J = 7.3 Hz, H-2′/6′), 6.17 (1H, d, J = 12.5 Hz, H-20), 5.70 (1H, dd, J = 15.3, 9.9 Hz, H-13), 5.53 (1H, t, J = 10.5 Hz, H-19), 5.27 (1H, m, H14), 3.64 (1H, brs, H-3), 3.15 (1H, m, H-18), 2.84 (1H, m, H-10a), 2.80 (1H, m, H-10b), 2.79 (1H, m, H-16), 2.69 (1H, brs, H-4), 2.62 (1H, d, J = 4.7 Hz, H-7), 2.31 (1H, d, J = 9.8 Hz, H-8), 2.27 (1H, m, H-15a), 2.11 (1H, m, H-15b), 1.83 (1H, m, H-5), 1.13 (3H, s, H-12), 1.11 (3H, d, J = 7.1 Hz, H-23), 1.04 (3H, d, J = 6.9 Hz, H-23), 0.58 (3H, d, J = 7.2 Hz, H-11); 13C NMR (DMSO-d6, 150 MHz) δ 212.1 (C-17), 169.3 (C-1), 148.7 (C-22), 139.5 (C-20), 136.9 (C-1′), 131.5 (C-14), 130.0 (C-2′/6′), 128.2 (C-3′/5′), 127.9 (C-13), 126.5 (C-4′), 117.1 (C-19), 86.7 (C-9), 59.2 (C-7), 57.1 (C-6), 53.0 (C-3), 47.9 (C8), 46.3 (C-4), 43.0 (C-18), 42.3 (C-16), 42.2 (C-10), 36.4 (C-15), 35.2 (C-5), 19.5 (C-12), 18.1 (C-24), 16.6 (C-25), 12.5 (C-11);
in Leishan of Guizhou province, China, and was identified on the basis of its 18S rRNA gene sequence (Genbank accession no. MF576131). The fungus was maintained in China Pharmaceutical Culture Collection (CPCC), Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. Fermentation and Extraction. The strain was cultured on PDA (0.3% potato extract, 2% glucose, 1.5% agar power) at 28 °C for 5 days. Under aseptic conditions, agar plugs were cut into small pieces and inoculated in 500 mL Erlenmeyer flasks, each containing 100 mL of seed medium [glucose 2%, sucrose 1%, soybean powder 0.2%, peptone 1%, K2HPO4 0.03%, poly(ethylene glycol) 0.25%, NaNO3 0.3%, and (NH4)2SO4 0.3%; the final pH of the medium was adjusted to 6.0 before sterilization with an autoclave at 121 °C for 20 min]. Cultures were incubated in flasks at 28 °C on a rotary shaker at 190 rpm for 2 days to prepare the seed culture. We filled each of 32 500 mL Erlenmeyer flasks with 80 g of rice and 120 mL of distilled H2O. After soaking for 5 h, the contents were sterilized with an autoclave at 121 °C for 20 min. After cooling to room temperature, each flask was inoculated with 10 mL of seed culture and incubated at 28 °C for 40 days. The fermented rice was supersonically extracted with CH3OH (3 × 2.5 L), and after recovering the organic solvent, the crude extract was further partitioned between EtOAc and H2O. Isolation and Purification. The EtOAc-soluble fraction (50 g) was subjected to silica gel column chromatography (CH3Cl−CH3OH, v/v, 100:0→20:1→7:1→0:100) to finally yield 12 combined fractions (Fr. A to M). By separation on silica gel column, ODS column, Sephadex LH-20 column and repeated purification by semipreparative HPLC, compounds 1 (14.7 mg), 4 (5.0 mg), 6 (4.5 mg), 7 (32.5 mg), 30 (22.0 mg), and 33 (13.8 mg) were obtained from Fr. B; compounds 8 (5.0 mg), 9 (13.0 mg), 10 (30.0 mg), 11 (26.2 mg), 12 (21.5 mg), 21 (200.2 mg), 23 (13.1 mg), 25 (215.7 mg), 31 (5.0 mg), 32 (30.0 mg), and 33 (18.0 mg) were obtained from Fr. C; compounds 19 (40.0 mg) and 20 (64.5 mg) were obtained from Fr. D; compounds 2 (52.0 mg), 3 (5.0 mg), 5 (197.6 mg), 24 (604.0 mg), and 28 (167.4 mg) were obtained from Fr. E; compounds 5 (125.0 mg), 14 (30.9 mg), 15 (32.0 mg), 22 (34.0 mg), 26 (15.8 mg), 27 (52.8 mg), and 29 (2.0 mg) were obtained from Fr. F; compounds 13 (110.0 mg), 17 (17.0 mg), and 18 (6.7 mg) were obtained from Fr. J; and compounds 14 (25.6 mg) and 16 (52.8 mg) were obtained from Fr. L. The details of the extraction and isolation are available in the Supporting Information. Asperphenalenone A (1). Brown gum; C35H44O7; [α]25 D = −5.5 (c 0.1050, CH3OH); UV (MeOH) λmax (logε): 204.8 (4.4), 256.8 (4.2), F
DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
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HRESIMS m/z 478.2157 [M − H]− (calcd for C28H32NO6, 478.2230). Aspulvinone R (13). Yellow amorphous powder; C18H14O7; 1H NMR (DMSO-d6, 500 MHz) δ: 9.58 (1H, s, 4′−OH), 9.29 (1H, s, 5″−OH), 8.83 (1H, s, 4″−OH), 7.78 (2H, dt, J = 10.6, 3.5, 2.5 Hz, H2′/6′), 7.00 (1H, d, J = 2.0 Hz, H-6″), 6.82 (2H, dt, J = 10.6, 3.5, 2.5 Hz, H-3′/5′), 6.78 (H, d, J = 2.0 Hz, H-2″), 6.50 (1H, s, H-5); 13C NMR (DMSO-d6, 125 MHz) δ 168.3 (C-1), 162.0 (C-3), 156.4 (C4′), 148.3 (C-3″), 145.9 (C-5″), 140.3 (C-4), 136.0 (C-4″), 128.5 (C2′/6′), 123.1 (C-1″), 120.8 (C-1′), 115.1 (C-3′/5′), 111.1 (C-6″), 108.1 (C-5), 106.3 (C-2″), 99.7 (C-2), 55.8 (3″-OCH3); HRESIMS m/z 341.0694 [M − H]−(calcd for C18H13O7, 341.0661). Anti-HIV Assay. SupT1 cells (2 × 105) were cotransfected with 0.6 mg of pNL-Luc-E and 0.4 mg of pHIT/G. Then the VSV-G pseudo typed viral supernatant (HIV-1) was harvested by filtration through a 0.45 μm filter after 48 h and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux, Lyon, France). SupT1 cells were exposed to VSV-G pseudotyped HIV-1 (MOI = 1) at 37.8 °C for 48 h in the absence or presence of test compounds. A Luciferase Assay System (Promega, Madison, USA) was used to determine the inhibition rate.23 The cytotoxicity was measured by the CCK-8 method. SupT1 cells (5 × 105) were seeded into a 96-well microtiter plate in the absence or presence of test compounds (negative control, DMSO) in triplicate and incubated at 37 °C. After 48 h incubation, cell viability was measured by the CCK-8 method. The dilutions of the tested compounds were independently performed three times.
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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.6b00878. NMR spectra of 1−33, UV, IR, and CD spectra of 1−5, and NMR data of known compounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
*L. Y. Yu. E-mail: [email protected]. Tel: +86-10-63187118. ORCID
De-Wu Zhang: 0000-0001-6289-0617 Li-Yan Yu: 0000-0002-8861-9806 Notes
Compounds 1, 2, 4, and 5 presented in this work have been reported previously in a Chinese patent (Application number 201610424586; Publication Number 106085868A) applied by our group. The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge financial support from the National Natural Science Foundation of China (NSFC) (Nos. 31170041 and 81373452), the National Infrastructure of Microbial Resources (No. NIMR-2016-3), National S & T Major Special Project on Major New Drug Innovation (No. 2017ZX09101003-007-001), and CAMS Initiative for Innovative Medicine (No. 2016-I2M2-002). Liyan Yu is supported as a Xiehe Scholar.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.6b00878 J. Nat. Prod. XXXX, XXX, XXX−XXX
