Eremophilane Sesquiterpenes from an Endophytic Fungus Periconia

Jul 19, 2016 - After our paper was published on the Web on July 19, 2016, we were made aware that one of the compounds we reported, compound 6, had al...
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Eremophilane Sesquiterpenes from an Endophytic Fungus Periconia Species Jimei Liu,† Dewu Zhang,† Min Zhang, Jinlian Zhao, Ridao Chen, Nan Wang, Dan Zhang, and Jungui Dai* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new polyoxygenated eremophilane sesquiterpenes, periconianones C−K (1−9), including one unusual isoeremophilane sesquiterpene, periconianone C (1), and four trinor-eremophilane sesquiterpenes, periconianones H−K (6−9), were isolated from the endophytic fungus Periconia sp. F-31. Compound 1 is the first furan-type isoeremophilane reported containing a linkage of C-8/C-11 and a 7,12-epoxy moiety. These compound structures, including absolute configurations, were elucidated through extensive spectroscopic data analysis, electronic circular dichroism, Mo2(AcO)4-induced circular dichroism, and single-crystal X-ray diffraction (Cu Kα). Compounds 2, 5, and 9 showed inhibition effects on lipopolysaccharide-induced NO production in BV2 cells by 10.2%, 18.3%, and 16.1% at a concentration of 1.0 μM, respectively, which is comparable to the positive control curcumin (12.9% at 1.0 μM).

T

he family of eremophilane sesquiterpenes is widely distributed in terrestrial and marine organisms and possesses a wide range of biological activities including antiinflammatory, antimicrobial, and antitumor properties.1 In recent years, new bioactive eremophilane sesquiterpenes have been discovered from various natural sources.2 Endophytic fungi, a distinct group of microorganisms that asymptomatically colonize living tissues of healthy plants, are an important source of biologically active eremophilane sesquiterpenes.3 We previously isolated three eremophilane sesquiterpenes (periconianones A and B, dihydronaphthalene-2,6-dione) from the endophytic fungus Periconia sp. F-31, which was originally isolated from the medicinal plant Annona muricata.4 Further biological studies revealed that periconianones A and B exhibited strong neural anti-inflammatory activities.4b Toward our goal of finding structurally novel eremophilane sesquiterpenes with interesting biological activities, we have carried out a further investigation of the metabolites of this strain. As a result, we have discovered one unusual furan-type isoeremophilane sesquiterpene (periconianone C, 1), four new eremophilanetype sesquiterpenes (periconianones D−G, 2−5), and four stereoisomeric trinor-eremophilane sesquiterpenes (periconianones H−K, 6−9) (Figure 1). Herein, we report their isolation, structural elucidation, and biological activities.

Figure 1. Structures of compounds 1−9.

presence of hydroxy (3475 and 3307 cm−1) and conjugated carbonyl (1675 cm−1) groups. The 1H NMR spectroscopic data (Table 1) contained signals for three olefinic protons [δH 7.06 (1H, d, J = 9.6 Hz), 6.14 (1H, s), and 5.77 (1H, d, J = 9.6 Hz)], two methylenes [δH 3.73 (1H, dd, J = 7.8, 7.8 Hz) and 3.53 (1H, dd, J = 11.4, 7.8 Hz), oxygen-bearing methylene; δH 1.95 (1H, d, J = 13.8 Hz) and 1.65 (1H, d, J = 13.8 Hz)], two methines [δH 2.16 (1H, m) and 2.07 (1H, q, J = 6.6 Hz)], and three methyls [δH 1.19 (3H, s), 0.95 (3H, d, J = 6.6 Hz), and 0.87 (3H, d, J = 6.6 Hz)]. The 13C NMR and DEPT spectra (Table 1, Figures S6 and S7) displayed signals for one carbonyl carbon (δC 203.0), one dioxygenated secondary carbon (δC 103.0), one oxygenated tertiary carbon (δC 76.1), two quaternary carbons (δC 136.7 and 40.0), five methines (δC 144.6, 136.8, 124.2, 52.4, and 44.6, including three olefinic), two methylenes (δC 70.0 and 41.7, including one oxygenated), and three methyls (δC 26.1, 14.2, and 9.1). Among the 15

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RESULTS AND DISCUSSION Periconianone C (1) was obtained as colorless, columnar crystals (cyclohexane−acetone), and its molecular formula was determined as C15H20O4 according to the HRESIMS peak at m/z 265.1439 [M + H]+ (calcd for C15H21O4, 265.1434) with six degrees of unsaturation. The IR spectrum indicated the © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 5, 2016

A

DOI: 10.1021/acs.jnatprod.6b00299 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR and 13C NMR Data of Compounds 1−5 1a position

δC type

2b δH (J in Hz)

δC type

3b δH (J in Hz)

δC type

4b δH (J in Hz)

5b

δC type

δH (J in Hz)

δC type

1

144.6, CH

7.06, d (9.6)

145.9, CH

7.07, d (9.6)

145.8, CH

7.05, d (9.6)

82.3, CH

4.32, dd (1.2, 5.4)

128.8, CH

2

124.2, CH

5.77, d (9.6)

127.5, CH

5.86, d (9.6)

125.5, CH

5.74, d (9.6)

42.1, CH2

2.58, ddd (1.2, 5.4, 16.8, Ha); 2.41, dt (1.2, 16.8, Hb)

70.2, CH

3 4

203.0, C 52.4, CH

2.07, q (6.6)

200.8, C 54.4, CH

2.42, q (7.2)

203.7, C 54.8, CH

2.19, q (7.2)

5 6

40.0, C 41.7, CH2

7 8

103.0, C 76.1, C

9

136.8, CH

10 11 12

136.7, C 44.6, CH 70.0, CH2

1.95, d (13.8, Ha) 1.65, d (13.8, Hb)

40.6, C 40.1, CH2

74.9, C 69.2, CH 6.14, s

13 14

9.1, CH3 14.2, CH3

2.16, m 3.73, dd (7.8, 7.8, Ha) 3.53, dd (7.8, 11.4, Hb) 0.95, d (6.6) 0.87, d (6.6)

15 2-OH

26.1, CH3

1.19, s

132.4, CH 142.4, C 151.2, C 111.3, CH2

2.10, d (13.2, Ha) 1.91, dd (1.2, 13.2, Hb) 4.02, m 6.13, d (5.4)

19.4, CH3 7.7, CH3

5.02, q (1.2, Ha) 4.88, quin (1.2, Hb) 1.89, s 1.08, d (7.2)

20.8, CH3

1.19, s

40.4, C 35.9, CH2

75.2, C 69.1, CH 135.4, CH 139.5, C 151.1, C 111.4, CH2

2.35, d (13.8, Ha) 1.47, dd (1.2, 13.8, Hb) 4.06, dd (5.4, 5.4) 6.28, d (5.4)

5.01, s, Ha

19.3, CH3 14.4, CH3

4.88, t (1.2, Hb) 1.90, s 0.96, d (7.2)

27.9, CH3

1.37, s

210.7, C 49.0, CH 44.6, C 39.7, CH2

1.66, dd (1.2, 14.4, Ha) 1.28, d (14.4, Hb)

80.1, C 115.7, C 37.7, CH2 78.3, C 39.8, CH 74.7, CH2

9.1, CH3 8.0, CH3 22.1, CH3

2.80, Hac ; 2.06, Hbc 2.21, m 3.95, dd (7.8, 7.8, Ha) 3.48, dd (7.8, 10.2, Hb) 0.91, d, (7.2) 0.92, d (7.2) 1.32, s

3-OH 7-OH 8-OH 9-OH

3.51, d (1.2) 3.71, d (7.8)

3.58, s 3.77, d (5.4)

77.1, CH 40.6, CH

2.94, q (7.2)

3.59, s 4.74, brs

δH (J in Hz) 5.65, d (3.6) 3.87, m

3.69, brs 1.73, qd (2.4, 7.2)

38.2, C 38.6, CH2

1.55, m

39.5, CH 73.2, CH

2.80c 3.87, m

79.8, CH

4.06, m

145.0, C 148.5, C 110.9, CH2

4.80, s 4.75, s

22.7, CH3 12.3, CH3 21.8, CH3

1.79, s 1.07, d (7.2) 1.35, s 2.94, d (6.0) 3.62, d (4.2) 3.75, brs 3.97, d (3.0)

a

Data were recorded at 600 MHz for proton and at 150 MHz for carbon in DMSO-d6. bData were recorded at 600 MHz for proton and at 150 MHz for carbon in acetone-d6. cOverlapped with other signals.

and C-13; and H3-13/C-8, C-11, and C-12 along with the spin system of H3-13/H-11/H2-12 based on the 1H−1H COSY correlations confirmed the presence of a furan ring, indicating a novel eremophilane sesquiterpenoid bearing the linkage of C-8 and C-11 and a 7,12-epoxy moiety. In addition, the chemical shift of C-7 (δC 103.0) indicated the existence of a furan hemiacetal moiety. Thus, the planar structure of 1 was elucidated as shown in Figure 1. To the best of our knowledge, this finding is the first example with the linkage of C-11 to C-8 and a furan hemiacetal moiety in the eremophilane-type sesquiterpenoid family. The relative configuration of 1 was determined by NOESY spectroscopic data analysis (Figure 2). The NOESY correlations of H3-15/H-4 and H-6b and of H-6a/H-11 and H3-14 indicated that H3-15, H-4, and H3-13 were on the same face of the ring system, whereas H3-14 and H-11 were on the opposite face of the molecule. However, due to the lack of key definitive correlations at 7-OH and 8-OH in the NOESY experiments, the relative stereochemistry of C-7 and C-8 could not be determined. To support the above deduction and determine the absolute configuration of 1, a single-crystal X-ray diffraction pattern was obtained using the anomalous scattering of Cu Kα radiation. Figure 3 shows an ORTEP drawing and unambig-

carbons, the existence of one carbonyl and two double bonds accounted for three degrees of unsaturation, illustrating the presence of the tricyclic ring system in 1. The HMBC correlations (Figure 2) of H-1/C-3, C-5, C-9, and C-10; H-2/C-4 and C-10; H-4/C-3, C-5, C-10, C-14, and C-15; H2-6/C-4, C-5, C-7, C-8, C-10, and C-15; and H-9/C-1, C-5, C-7, and C-11 were observed, which indicated the presence of a naphthalenone moiety. Furthermore, the HMBC cross-peaks for H-11/C-12 and C-13; H-12/C-7, C-8, C-11,

Figure 2. 1H−1H COSY (blue lines), key HMBC (red →), and NOESY (↔) correlations of compounds 1, 2, and 6. B

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Periconianone F (4) was obtained as a colorless powder. The molecular formula was C15H22O5, corresponding to five degrees of unsaturation, on the basis of the HRESIMS peak at m/z 283.1533 [M + H]+. The IR spectrum showed absorption bands at 3393 and 1701 cm−1 for hydroxy and carbonyl groups, respectively. The general features of its 1H NMR spectroscopic data (Table 1) were similar to those of 1 except for the absence of three olefinic protons in 4 and the presence of two sets of nonequivalent methylenes [δH 2.06 (1H, overlapped with solvent peak)/2.80 (1H, overlapped with H2O peak); δH 2.41 (1H, dt, J = 1.2, 16.8 Hz)/2.58 (1H, ddd, J = 1.2, 5.4, 16.8 Hz)] and one oxygenated methine [δH 4.32 (1H, dd, J = 1.2, 5.4 Hz)]. The 13C NMR and DEPT spectra displayed 15 carbon resonances (Table 1), which consisted of one carbonyl carbon (δC 210.7), one dioxygenated secondary carbon (δC 115.7), two oxygenated tertiary carbons (δC 80.1 and 78.3), one quaternary carbon (δC 44.6), three methine carbons (δC 82.3, 49.0, and 39.8, including one oxygenated carbon), four methylene carbons (δC 74.7, 42.1, 39.7, and 37.7, including one oxygenated carbon), and three methyl carbons (δC 22.1, 9.1, and 8.0). By analyzing the HSQC, 1H−1H COSY, and HMBC spectra, the naphthalenone moiety and the furan ring were established (Figure 1). However, the HMBC correlations of H6b/C-11; H2-12/C-7 and C-8; and H3-13/C-7 and C-12 indicated the O-linkage of C-8 and C-12, which differs from compound 1. In addition, the chemical shift values of C-1 (δC 82.3) and C-10 (δC 78.3) together with the molecular formula indicated the presence of a 1,10-epoxide. The HMBC correlations of 7-OH (δH 3.59)/C-6, C-7, C-8, and C-11 along with the corresponding shifts (C-8 at δC 115.7; C-7 at δC 80.1) demonstrated the presence of a 7,8-diol moiety (Figure S43). Therefore, the planar structure of 4 was elucidated. The relative configuration of 4 was established by NOESY experiments. The enhancement of H3-15 when H-4 was irradiated suggested the anti-orientation of H3-15 and H3-14. The NOEs of H3-15/H-6a, H3-13, and H-9a; H-6a/H3-13 and H3-15; 7-OH/H3-13, H3-15, and H-6a; and 8-OH/H3-13, H315, H-9a, and 7-OH indicated the syn-orientation of H-6a, H315, H3-13, 8-OH, and 7-OH. The NOEs of H-11/H-6b and H1/H-2a and H-9b established the syn-orientation of H-6b, H-1, and H-11. The absolute configurations of the 7,8-diol moieties were determined by Snatzke’s method (the dimolybdenum method).6 In the Mo2(AcO)4-induced CD spectrum, positive Cotton effects at 303 and 408 nm (Figure S50) supported the 7S and 8R configurations. Thus, the absolute configuration of 4 was assigned as 1S, 4R, 5S, 7S, 8R, 10R, 11S. Periconianone G (5) was isolated as a colorless gum, and it gave an HRESIMS ion peak at a m/z of 291.1559 [M + Na]+, which corresponded to a molecular formula of C15H24O4. The IR spectrum showed an absorption band at 3330 cm−1 corresponding to hydroxy group(s). The 1H and 13C NMR spectroscopic data of 5 were similar to those of 7βH-eremophil1(10),11(12)-diene-2β,8β-diol.7 The obvious difference was the presence of two additional hydroxy groups in 5 that were located at C-3 and C-9 according to the 1H−1H COSY correlations of 3-OH (δH 3.62)/H-3 (δH 3.69) and 9-OH (δH 3.97)/H-9 (δH 4.06). The relative configuration of 5 was determined by NOESY experiments. The enhancement of H-3 was observed with irradiation of H-4, which indicated a synorientation of H-4 and H-3. The NOEs of H3-15/H3-14, and H-7; 9-OH/H3-15; and H-2/H3-14 and H3-15 suggested the syn-orientation of H3-14, H3-15, 9-OH, H-2, and H-7. The NOESY correlations of H 3 -13/H-8 indicated the syn-

uously demonstrates the absolute configuration of 4S, 5R, 7R, 8S, 11S for 1.

Figure 3. Structure of 1 resulting from single-crystal X-ray diffraction.

The asymmetric diene in 1 is a chromophore that can cause the Cotton effect. Therefore, the absolute configuration of the chiral center (C-5) in the nonplanar skeleton can also be determined using the helicity rule.5 A positive Cotton effect at 274.5 nm for the π−π* transition (Figure S15) is indicative of the 5R configuration, which is in accord with X-ray diffraction data. Periconianone D (2) was obtained as colorless needle crystals (MeOH). The molecular formula of C15H20O3 was determined from the HRESIMS peak at m/z 249.1479 [M + H]+ (calcd for C15H21O3, 249.1485). The IR spectrum showed the presence of hydroxy (3399 cm−1) and conjugated carbonyl (1648 cm−1) groups. The 1H and 13C NMR spectroscopic data of 2 were similar to those of periconianone B.4b The differences include the presence of an oxymethine carbon (δC 69.2, C-8) and a vinylidene carbon (δC 111.3, C-12 and δC 151.2, C-11) in 2 instead of a carbonyl carbon (δC 198.6, C-8) and a carboxyl carbon (δC 176.4, C-12) in periconianone B. In addition, the chemical shift of C-7 (δC 74.9) together with the molecular formula of 2 indicated the attachment of a hydroxy at C-7. These assignments were confirmed by the HMBC correlations of C-8/H-9, 8-OH, and H2-6; C-7/H-8, H-9, H2-6, H2-12, and H3-13; C-11/H2-6, H3-13; and C-12/H3-13. On the basis of the above observations, the planar structure of 2 was elucidated (Figure 1). The NOESY correlations of H3-15/H3-14, H-6b, and 7-OH indicated their syn-orientation, whereas the correlations of H-8/H3-13 and H-6a/H-4, H-12a, and H3-13 indicated that these protons were on the opposite face of the ring system. In the CD spectrum, a positive Cotton effect at 278.5 nm was observed, which is similar to that of 1 (Figure S26), indicating a 5R configuration for 2. Accordingly, the absolute configuration of 2 was determined as 4R, 5R, 7R, 8R. Periconianone E (3) was obtained as a colorless gum and had the same molecular formula as 2 as determined by the HRESIMS peak at m/z 249.1478 [M + H]+ (calcd for C15H21O3, 249.1485). The general features of its 1H and 13C NMR spectroscopic data closely resembled those of 2 except those for the different chemical shifts for H-4, H2-6, H3-15, C-6, C-14, and C-15 (Table 1). Analysis of DEPT, 1H−1H COSY, and HMBC correlations suggested that 3 was the stereoisomer of 2. The NOESY correlations of H3-15/H-4 and H-6b and of H-8/H3-13 and H3-15 indicated that these protons were on the same face of the ring system, whereas the correlations of H314/H-6a implied that these protons were on the opposite face. The absolute configuration of 3 was assigned as 4S, 5R, 7S, 8S according to the helicity rule with a positive Cotton effect at 279.5 nm in the CD spectrum (Figure S37). C

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(Figures S82, S83, and S84). The large coupling constant of JH‑6b,H‑7 (7.2 Hz) indicated their axial orientation, while the small coupling constants of JH‑7,H‑8 (3.6 Hz) and JH‑8,H‑9 (3.6 Hz) implied an equatorial arrangement of H-8. The NOEs of H-6a/H-4, H-7, and H-8 together with H-4/H-7 established the syn-orientation of H-4, H-7, and H-8, whereas the NOEs of H-6b/H3-11 and H3-12 indicated these protons were on the opposite face of the ring system. Therefore, 7 was identified as a 7-epimer of 6, which was further supported by the CD spectrum with a positive Cotton effect at 278 nm (Figure S88). Periconianone J (8) and periconianone K (9) had the same molecular formula as 6 as deduced from HRESIMS. The 1H NMR spectroscopic data were similar to those of 6 except that the methyl and olefinic protons in 8 and 9 resonated at a much lower field [(δH 6.12, d, J = 2.4 Hz, H-9 and δH 1.18, s, H3-12 in 8) and (δH 6.01, d, J = 2.4 Hz, H-9 and δH 1.21, s, H3-12 in 9) vs (δH 5.86, d, J = 3.0 Hz, H-9 and δH 1.03, s, H3-12 in 6)]. The large coupling constants of JH-6b,H-7 (10.8 Hz) and JH-7,H-8 (7.2 Hz) indicated that both H-7 and H-8 were disposed in an axial orientation and were on opposite sides in 8. In addition, the NOEs of H3-11/H-6a and H-7 established the syn-orientation of H3-11 and H-7; the NOEs of H3-12/H-6b and H-4 indicated the syn-orientation of H3-12 and H-4, which further indicated that 8 was a stereoisomer of 6. In the NOESY spectrum of 9, the NOEs of H-6b/H3-12, H-4, and H-7 established the synorientation of H3-12, H-4, and H-7; the NOEs of H3-11/H-6a indicated the anti-orientation of H3-11 and H3-12. Furthermore, the large coupling constants of JH-6a,H-7 (12.6 Hz) and JH-7,H-8 (8.4 Hz) proved a trans-axial orientation of H-7 and H8. Thus, 9 was identified as the 11-epimer of compound 6. On the basis of CD spectral analysis, the absolute configurations of 8 and 9 were proposed to be 4S, 5R, 7S, 8S and 4S, 5R, 7R, 8R, respectively (Figures S98 and S115). We isolated and fully characterized nine eremophilane-type sesquiterpenoids bearing the vic-diols moiety (1−9) from the endophytic fungus Periconia sp. Interestingly, three different configurations appeared at C-4 and C-5 (4S, 5R; 4R, 5R; and 4R, 5S), which might arise from the skeleton formation process by nonstereospecific cyclization of sesquiterpene synthases. Biogenetically, 1 might be formed from 3 via a pinacol rearrangement, which would lead to the migration of the isopropenyl group from C-7 to C-8, followed by hydroxylation and intramolecular acetalization (Supporting Information, Figure S4). In addition, the elimination of the isopropenyl group in four trinor-eremophilanes (periconianones H−K, 6− 9) might be biosynthesized from 2 and 3 via oxidation and decarboxylation. In summary, 1−9 were proposed to originate from different biosynthetic intermediates 1a, 1b, and 1c. Different types of tailoring reactions including hydroxylation, oxidation, isomerization, acetalization, decarboxylation, and rearrangements might create the structural diversity (Figure S4). All compounds were evaluated for their neural antiinflammatory, cytotoxic, and anti-HIV activities. Compounds 2, 5, and 9 exhibited anti-inflammatory activity indirectly by suppressing LPS-induced NO production in BV2 cells with inhibition rates of 10.2%, 18.3%, and 16.1% at a concentration of 1.0 μM, respectively, which is comparable to that of curcumin, a positive control with an inhibition rate of 12.9% (at 1.0 μM). Compound 3 exhibited weak cytotoxic activity against the human MCF-7 with an IC50 value of 17.9 μM (paclitaxel as the positive control, IC50 = 0.2 nM), and 6 displayed low cytotoxic activity against the HeLa cancer cell line with an IC50

orientation of H-8 and the isopropenyl group. The absolute configuration of 5 was calculated using the TD-DFT method at the B3LYP/6-31G(d) level. After analyzing the relative configuration of 5, only two stereoisomers existed, (2R,3R,4R,5R,7S,8S,9R)-5a and (2S,3S,4S,5S,7R,8R,9S)-5b (Figure S3). On the basis of a comparison of the theoretically calculated and experimental electronic circular dichroism (ECD) curves (Figure 4), the best match of the absolute configuration of 5 was 2R, 3R, 4R, 5R, 7S, 8S, 9R.

Figure 4. Calculated ECD spectra of 5a and 5b and the experimental ECD spectrum of 5.

Periconianone H (6) was obtained as a pale yellow gum with the molecular formula C12H16O3 as determined from the HRESIMS peak at m/z 209.1168 [M + H]+. The IR spectrum showed characteristic absorption bands of hydroxy and conjugated carbonyl groups at 3393 and 1678 cm −1 , respectively. The 13C NMR spectrum showed 12 carbon resonances, and its 1H NMR spectrum was similar to that of 2 except for the absence of the isopropenyl group, suggesting it was a trinor-eremophilane sesquiterpene. This deduction was confirmed by the HMBC correlations of H-1/C-3, C-5, C-9, and C-10; H-9/C-1, C-5, and C-7; H-2/C-3 and C-4; H-8/C-7, C-9, and C-10; H-7/C-5, C-6, and C-8; H-4/C-11, C-12, C-5, C-6, C-10, C-2, and C-3; H2-6/C-12, C-5, C-4, C-7, C-8, and C-10; H3-11/C-4, C-5, and C-3; and H3-12/C-4, C-6, and C10. The relative configuration of 6 was established by NOE experiments (Figures S67 and S68). The NOEs of H-6a/H3-11, H3-12, and H-7 and H-7/H-6a and H3-12 indicated the synorientation of H3-11, H3-12, and H-7, whereas the NOEs of H6b/H-4 and H-8 implied these protons were on the opposite face of the ring system. The absolute configuration of C-5 in 6 was determined to be R by analyzing the helicity rule in the CD spectrum with a positive Cotton effect at 280 nm (Figure S77). Thus, the absolute configuration of 6 was assigned as 4R, 5R, 7R, 8R. Periconianone I (7) was given as a pale yellow gum with the molecular formula C12H16O3 , as determined from the HRESIMS peak at m/z 209.1168 [M + H]+. The IR and 1H and 13C NMR spectroscopic data were very similar to those of 6 except that the oxymethine proton appeared at δH 4.07 (dd, J = 3.6, 3.6 Hz, H-8) in 7 instead of at δH 4.11 (dd, J = 3.0, 8.4 Hz, H-8) in 6, indicating that the compounds might be a pair of stereoisomers. The relative configuration of 7 was determined by the coupling constants and the NOESY experiments D

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Table 2. 1H NMR and 13C NMR Data of Compounds 6−9a 6 position 1 2 3 4 5 6

δC type 144.7, 127.1, 200.1, 52.9, 41.5, 43.3,

7

CH CH C CH C CH2

70.0, CH

8 9 10 11 12 7-OH 8-OH

74.9, 135.0, 142.0, 7.3, 20.1,

CH CH C CH3 CH3

7 δH (J in Hz)

7.01, dd (1.2, 9.6) 5.83, d (9.6) 2.46, q (6.6) 1.92, dd (4.2, 12.6, Ha) 1.63, dd (12.6, 12.6, Hb) 3.77, ddd (4.2, 8.4, 12.6) 4.11, dd (3.0, 8.4) 5.86, d (3.0) 1.01, d (6.6) 1.03, s 4.07b and 4.30,b brs

δC type 145.7, 127.3, 200.6, 52.8, 40.3, 38.7,

CH CH C CH C CH2

71.1, CH 70.6, 133.2, 143.0, 7.9, 22.0,

CH CH C CH3 CH3

8 δH (J in Hz)

7.06, d (10.2) 5.84, d (10.2) 2.44, q (6.6) 1.97, dd (3.6, 14.4, Ha) 1.74, dd (7.2, 14.4, Hb) 3.87, ddd (3.6, 3.6, 7.2) 4.07, dd (3.6, 3.6) 6.02, d (3.6) 1.06, 1.09, 3.98, 4.15,

d (6.6) s brs brs

δC type 145.5, 124.8, 202.8, 55.1, 41.0, 40.4,

CH CH C CH C CH2

72.6, CH 72.6, 137.5, 139.0, 15.0, 30.5,

CH CH C CH3 CH3

9 δH (J in Hz)

7.06, d (10.2) 5.70, d (10.2) 2.14, q (7.2) 1.97, dd (4.8, 14.4, Ha) 1.47, dd (10.8, 14.4, Hb) 3.63, ddd (4.8, 7.2, 10.8) 4.06, dd (2.4, 7.2) 6.12, d (2.4) 0.99, d (7.2) 1.18, s

δC type 144.7, 125.1, 203.0, 53.6, 41.2, 39.5,

CH CH C CH C CH2

69.6, CH 75.3, 138.1, 138.9, 14.5, 27.3,

CH CH C CH3 CH3

δH (J in Hz) 6.99, d (9.6) 5.71, d (9.6) 2.14, q (7.2) 1.92, dd (12.6, 12.6, Ha) 1.48, dd (4.2, 12.6, Hb) 3.86, ddd (4.2, 8.4, 12.6) 4.12, dd (2.4, 8.4) 6.01, d (2.4) 0.97, 1.21, 4.04, 4.29,

d (7.2) s brs brs

a

Data were recorded at 600 MHz for proton and at 150 MHz for carbon in acetone-d6. bNot assigned in acetone-d6 and assigned in DMSO- d6 (Supporting Information, Table S3 and Figures S69−73).

value of 16.5 μM (paclitaxel as the positive control, IC50 = 6.4 nM). In addition, compound 6 displayed anti-HIV activity with an IC50 value of 11.0 μM, whereas efavirenz (positive control) gave an IC50 of 1.4 nM.

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(3.9 mg, tR 32.8 min). Purification of Fr3.4.3 (105.7 mg) by reversedphase semipreparative HPLC (CH3OH−H2O, 25:75, v/v) yielded 2 (4.4 mg, tR 27.4 min) and 3 (1.8 mg, tR 21.9 min). Fr4 (3.4 g) was fractionated with Sephadex LH-20 CC by eluting with CH3OH, yielding five fractions (Fr4.1−Fr4.5). Fr4.5 (950 mg) was further separated via normal-phase semipreparative HPLC (nhexane−EtOAC, 5:1, v/v) to furnish seven fractions (Fr4.5.1− Fr4.5.7). Purification of Fr4.5.2 (80.0 mg) through normal-phase semipreparative HPLC (n-hexane−isopropyl alcohol, 15:1, v/v) followed by reversed-phase semipreparative HPLC (CH3OH−H2O, 20:80, v/v) afforded 4 (1.5 mg, tR 25.8 min). Fr4.5.3 (74.8 mg) was separated by normal-phase semipreparative HPLC (n-hexane− isopropyl alcohol, 13:1, v/v) followed by reversed-phase semipreparative HPLC (CH3OH−H2O, 25:75, v/v) to yield 7 (8.5 mg, tR 28.3 min). Purification of Fr4.5.4 (99.1 mg) by normal-phase semipreparative HPLC (n-hexane−isopropyl alcohol, 13:1, v/v) followed by reversed-phase semipreparative HPLC afforded 6 (21.4 mg, tR 30.3 min, CH3OH−H2O, 20:80, v/v), 5 (1.0 mg, tR 18.5 min, CH3OH−H2O, 15:85, v/v), and 8 (6.3 mg, tR 40.5 min, CH3OH− H2O, 15:85, v/v). Fr4.5.5 (106.0 mg) was separated by normal-phase semipreparative HPLC (n-hexane−isopropyl alcohol, 20:1, v/v) to give 9 (24.5 mg, tR 54.2 min). Periconianone C (1): colorless columnar crystals (cyclohexane− acetone); [α]20546 −45.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 203 (3.61), 282 (3.87) nm; CD (MeOH) λmax (Δε) 274 (8.03), 338 (−3.95) nm; IR (νmax) 3307, 2974, 1675 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 265.1439 [M + H]+ (C15H21O4, calcd [M + H]+ 265.1434). Periconianone D (2): colorless needle crystals (MeOH); [α]20546 +672.0 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 279 (3.99) nm; CD (MeOH) λmax (Δε) 278 (22.49), 338 (−3.75) nm; IR (νmax) 3399, 2924, 1649 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 249.1479 [M + H]+ (C15H21O3, calcd [M + H]+ 249.1485). Periconianone E (3): colorless gum; [α]20546 +342.8 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 282 (4.49) nm; CD (MeOH) λmax (Δε) 279 (9.49), 338 (−1.59) nm; IR (νmax) 3433, 2968, 1658 cm−1; 1 H and 13C NMR data, see Table 1; HRESIMS m/z 249.1478 [M + H]+ (C15H21O3, calcd [M + H]+ 249.1485). Periconianone F (4): colorless powder; [α]20546 −33.4 (c 0.06, MeOH); Mo2(AcO)4-induced CD (DMSO) λmax (Δε) 303 (0.28), 408 (0.09) nm; IR (νmax) 3393, 2921, 1701 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 283.1533 [M + H]+ (C15H23O5, calcd [M + H]+ 283.1540).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer model-343 digital polarimeter. The CD spectra were recorded on a JASCO J-815 spectropolarimeter. The UV absorption spectra were measured in MeOH on a Thermo SpectronicVision32 Software V1.25. IR spectra were acquired on a Nicolet 5700 FT-IR microscope spectrometer (FTIR Microscope Transmission). 1D and 2D NMR spectra were obtained at 600 MHz for 1H NMR and 150 MHz for 13C NMR on VNOVA SYSTEM-600 and Bruker AVIIID 600 spectrometers. Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in hertz (Hz). HRESIMS data were measured using an Agilent Technologies 6520 Accurate Mass Q-TOF LC/MS spectrometer. Silica gel (200−300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, PR China) and Sephadex LH-20 gel (Amersham Biosciences, Sweden) were used for column chromatography (CC). Semipreparative reversed-phase and normal-phase HPLC were performed on a Shimadzu HPLC instrument equipped with a Shimadzu RID-10A detector and a Grace Adsorbosphere C18 column (250 mm × 10 mm, i.d., 5 μm) by eluting with mixtures of methanol and H2O at 3 mL/min or a Grace Allsphere silica column (250 mm × 10 mm, i.d., 5 μm) by eluting with mixtures of n-hexane and isopropyl alcohol at 4 mL/min, respectively. Analytical TLC was carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Industry, Qingdao, China), and spots were visualized under UV light or by spraying with 10% H2SO4 in EtOH followed by heating at 120 °C. Fungal Material, Fermentation, Extraction, and Isolation. The fermentation, extraction, and isolation of the fungal strain Periconia sp. F-31 were performed as described previously.4,8 The EtOAc extract (25.0 g) of the culture filtrate was subjected to silica gel CC eluting with a CH2Cl2−CH3OH gradient (100:0−0:100) to produce eight fractions (Fr1−Fr8) on the basis of TLC analysis. Fr3 (4.5 g) was initially subjected to Sephadex LH-20 CC by eluting with CH3OH to give four fractions (Fr3.1−Fr3.4). Fr3.4 (1.0 g) was then fractionated by reversed-phase semipreparative HPLC eluting with CH3OH−H2O (65:35, v/v) to afford four fractions (Fr3.4.1− Fr3.4.4). Purification of Fr3.4.2 (61.9 mg) by reversed-phase semipreparative HPLC (CH3OH−H2O, 25:75, v/v) resulted in 1 E

DOI: 10.1021/acs.jnatprod.6b00299 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Periconianone G (5): colorless gum; [α]20546 −50.0 (c 0.10, MeOH); IR (νmax) 3331, 2940, 1644 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 291.1559 [M + Na]+ (C15H24O4Na, calcd [M + Na]+ 291.1567). Periconianone H (6): pale yellow gum; [α]20546 +80.0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 283 (5.93) nm; CD (MeOH) λmax (Δε) 280 (7.81), 337 (−2.86) nm; IR (νmax) 3394, 2921, 1676 cm−1; 1 H and 13C NMR data, see Table 2; HRESIMS m/z 209.1168 [M + H]+ (C12H17O3, calcd [M + H]+ 209.1172). Periconianone I (7): pale yellow gum; [α]20546 +366.7 (c 0.21, MeOH); UV (MeOH) λmax (log ε) 279 (4.05) nm; CD (MeOH) λmax (Δε) 278 (17.03), 336 (−3.07) nm; IR (νmax) 3393, 2921, 1663 cm−1; 1 H and 13C NMR data, see Table 2; HRESIMS m/z 209.1168 [M + H]+ (C12H17O3, calcd [M + H]+ 209.1172). Periconianone J (8): pale yellow gum; [α]20546 +390.9 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 282 (5.94) nm; CD (MeOH) λmax (Δε) 282 (21.74), 334 (−3.36) nm; IR (νmax) 3391, 2921, 1661 cm−1; 1 H and 13C NMR data, see Table 2; HRESIMS m/z 209.1168 [M + H]+ (C12H17O3, calcd [M + H]+ 209.1172). Periconianone K (9): pale yellow gum; [α]20546 −110.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 285 (3.97) nm; CD (MeOH) λmax (Δε) 242 (3.81), 279 (3.89), 337 (−2.49) nm; IR (νmax) 3402, 2929, 1672 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 209.1170 [M + H]+ (C12H17O3, calcd [M + H]+ 209.1172). Anti-inflammatory Activity Assay.9 The BV2 microglia cell line was obtained from the Cell Culture Center at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. LPS (from Escherichia coli 055:B5) was obtained from Sigma-Aldrich. After preincubation for 24 h in a 96-well plate (at 37 °C with 5% CO2), the cells were treated with various concentrations of the test compounds (10−5, 10−6, 10−7, 10−8 M), followed by stimulation with LPS for 24 h. The production of NO was determined by measuring the concentration of nitrite in the culture supernatant. NaNO2 was utilized to generate a standard curve. The absorbance at 550 nm was measured. Curcumin was used as a positive control. Cytotoxicity Bioassay.10 The cytotoxicity of the compounds against the human cancer cell lines (HCT-8, Bel-7402, HeLa, and MCF-7) was measured using the MTT assay. The cells were maintained in an RRMI S7 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/ mL streptomycin. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2. Tumor cells were seeded in 96-well microtiter plates at 1200 cells/well. After 24 h, compounds were added to the wells. After incubation for 96 h, cell viability was determined by measuring the metabolic conversion of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) into purple formazan crystals by viable cells. The MTT assay results were read using an MK3 Wellscan (Labsystem Dragon, Helsinki, Finland) plate reader at 570 nm. All compounds were tested at five concentrations (10−5, 10−6, 10−7, 10−8, and 10−9 M) in 100% DMSO with a final concentration of DMSO of 0.1% (v/v) in each well. Paclitaxel was used as a positive control. Each concentration of the compounds was tested in three parallel experiments. IC50 values were calculated using Microsoft Excel software. HIV-Inhibitory Bioassay. 11 293T cells (2 × 105 ) were cotransfected with 0.6 μg of pNL-Luv-E−-Vpu− and 0.4 μg of pHIT/G. After 48 h, the VSV-G pseudotyped viral supernatant (HIV1) was harvested by filtration through a 0.45 μm filter and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux). SupT1 cells were exposed to VSV-G pseudotyped HIV-1 (MOI = 1) at 37 °C for 48 h in the absence or presence of test compounds (efavirenz was used as positive control). The inhibition rate was determined by using a firefly luciferase assay system (Promega).

Article

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00299. UV, IR, MS, 1D and 2D NMR, and CD spectra for 1−9, ECD calculation of 5, X-ray crystallographic data for 1, and proposed biosynthesis of 1−9. (PDF) Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (J. Dai): 86-10-63165195. Fax: 86-10-63017757. E-mail: [email protected]. Author Contributions †

J. Liu and D. Zhang contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National High Technology Research and Development Program of China (2012AA091606).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00299 J. Nat. Prod. XXXX, XXX, XXX−XXX