Nitric Oxide Inhibitory Meroterpenoids from the Fungus Penicillium

Article pubs.acs.org/jnp

Nitric Oxide Inhibitory Meroterpenoids from the Fungus Penicillium purpurogenum MHZ 111 Jing Sun,†,‡ Zhi-Xiang Zhu,† Yue-Lin Song,† Dan Dong,§ Jiao Zheng,† Ting Liu,§ Yun-Fang Zhao,† Daneel Ferreira,⊥ Jordan K. Zjawiony,⊥ Peng-Fei Tu,*,† and Jun Li*,† †

Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡ School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People’s Republic of China § Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, People’s Republic of China ⊥ Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States S Supporting Information *

ABSTRACT: Five new meroterpenoids, purpurogenolides A−E (1−5), and four known metabolites (6−9) were isolated from the solid substrate fermentation cultures of the fungus Penicillium purpurogenum MHz 111. The structures of the new meroterpenoids were elucidated by analysis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The absolute configurations of 1 and 5 were determined by single-crystal X-ray crystallographic analysis, and those of 2−4 were elucidated on the basis of experimental and calculated electronic circular dichroism spectra. Compounds 2−4 and 6 showed inhibition of nitric oxide production in lipopolysaccharideactivated BV-2 microglial cells with IC50 values of 0.8−30.0 μM.

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lovastatin, echinocandin B, and cyclosporin A serve to demonstrate the importance of the fungal secondary metabolites in drug discovery.9 Different strains of Penicillium purpurogenum were reported to represent productive sources of a variety of bioactive meroterpenoids.13−15 In the course of an ongoing search for antineuroinflammatory agents from fungal sources, an EtOAc extract of the solid substrate fermentation cultures of the fungus P. purpurogenum MHZ 111 was found to inhibit NO production in lipopolysaccharide (LPS)-activated BV-2 microglial cells (95% inhibition at 40 μg/mL). Subsequent isolation of the bioactive fraction afforded five new meroterpenoids, purpurogenolides A−E (1−5) and four known metabolites (6−9). Herein, the isolation and structural elucidation of the isolates as well as an evaluation of their inhibitory effects on NO production in LPS-activated BV-2 microglial cells are described.

euroinflammation is characterized by the excessive production of inflammatory mediators in the brain, and it plays an important role in the initiation and progression of neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington’s disease, among others.1−3 Microglia are the resident immune cells in the central nervous system (CNS), which physiologically play a key role in protecting neurons against exogenous injuries. Upon overactivation, microglia release various inflammatory mediators, including nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and reactive oxygen and nitrogen species. These neurotoxic mediators contribute to neuronal injuries and progression of inflammation in the CNS.4−8 Therefore, inhibition of microglial activation and subsequent neuroinflammation may represent an attractive and effective therapeutic strategy for the treatment of neurodegenerative diseases. Fungi continue to be a rich source of new metabolites belonging to highly diverse structural classes, including alkaloids, anthracenones, aromatic compounds, butenolides, coumarins, cytochalasans, macrolides, naphthalenones, mero- and other terpenoids, peptides, polyketides, and pyrones.9−12 These fungal secondary metabolites have been reported to exhibit a wide array of biological and pharmacological properties including antibacterial, anti-inflammatory, antitumor, antifungal, cholesterollowering, and immunosuppressive activities.9−12 Several wellknown fungi-derived pharmaceuticals such as the penicillins, © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The EtOAc extract of P. purpurogenum MHZ 111 was subjected to repeated silica gel, Sephadex LH-20, and RP-C18 gel column chromatography, followed by semipreparative RP-HPLC, to afford five new (1−5) and four known meroterpenoids (6−9). Purpurogenolide A (1) was obtained as colorless plates via crystallization from MeOH, [α]21 D +131 (c 0.2, MeOH). Its molecular formula was determined to be C25H28O7 by the Received: February 23, 2016

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DOI: 10.1021/acs.jnatprod.6b00160 J. Nat. Prod. XXXX, XXX, XXX−XXX

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deficiency required by the molecular formula. Such an array of five-membered rings is readily explicable via the acid-catalyzed cascade of cyclizations involving the C-9 hydroxy group in 7, effecting nucleophilic attack at C-22 of the oxirane moiety, and subsequent trans-esterification of the C-20 methyl ester functionality. The molecular structure of 1 was confirmed by the HMBC correlations between H2-6 and C-4/C-5/C-7/C-8/ C-22/C-24; H2-13 and C-5/C-11/C-12/C-14/C-15/C-19; H-14 and C-3/C-16; H3-17 and C-15/C-16/C-18; H3-18 and C-15/C-16/C-17; H3-19 and C-5/C-11/C-12/C-13; H3-21 and C-8/C-9/C-10; H2-23 and C-7/C-11/C-20/C-22; H3-24 and C-6/C-7/C-8/C-22; and H3-25 and C-3/C-4/C-5 (Figure 1). The relative configuration of 1 was established as shown in Figure 1 on the basis of the NOESY spectrum, which showed NOE correlations of H-5/H-6β, H-5/H-14, H-6α/H3-19, H-6α/ H-23α, H-6α/H3-24, H-6α/H3-25, H-6β/H3-24, H-13α/H3-19, H-13β/H-14, H3-19/H-23α, H-23α/H3-24, and H-23β/H3-24 (Figure 1). The absolute configuration of 1 was unequivocally defined as (5S,7R,9R,11R,12S,22R) on the basis of single-crystal X-ray diffraction analysis (Figure 2) using Cu Kα radiation with Flack and Hooft parameters of 0.02(16) and −0.02(11), respectively. The molecular formula of compound 2 (purpurogenolide B) was deduced as C26H32O9 from the 13C NMR and positive-ion HRESIMS data (m/z 489.2125 [M + H]+, calcd for C26H33O9, 489.2119). The IR spectrum showed the presence of hydroxy (3439 cm−1), carbonyl (1730, 1711 cm−1), and olefinic (1634 cm−1) functionalities. The 1H NMR data (Table 1) of 2 displayed resonances of six methyls (δH 1.21, 1.32, 1.41, 1.45, 1.50, 1.59, each 3H, s), one methoxy [δH 3.71 (3H, s)], two methylenes [δH 2.26 (1H, d, J = 13.5 Hz), 1.82 (1H, t, J = 13.5 Hz); 2.92 (1H, d, J = 14.5 Hz), 2.72 (1H, dd, J = 17.0, 9.0 Hz)], one oxygenated methylene [δH 3.25 (1H, d, J = 2.0 Hz), 3.05 (1H, d, J = 3.0 Hz)], one methine [δH 1.47 (1H, m)], and two olefinic protons [δH 6.14 (1H, m); 6.03 (1H, s)]. The 13C NMR data (Table 1) of 1 revealed 26 carbon resonances comprising six methyl (δC 16.4, 16.7, 18.2, 26.7, 27.1, 27.3), one methoxy (δC 53.1), three sp3 methylene (δC 36.1, 38.4, 51.5), one sp3 methine (δC 53.3), two sp2 methine (δC 116.3, 130.5), three sp3 quaternary (δC 49.7, 50.6, 69.2), four oxygenated sp3 tertiary (δC 59.9, 75.4, 78.7, 83.4), two sp2 tertiary (δC 135.8, 159.9), two ester carbonyl (δC 165.4, 168.1), and two ketocarbonyl (δC 203.1, 208.1) carbons. The aforementioned data suggested that 2 also possesses a carbon skeleton reminiscent of a polyketide−terpenoid hybrid meroterpenoid similar to 22,23-epoxyberkleydione (7).17 The main difference was the replacement of the Δ3,4 double bond in 7 by a Δ2,3 double bond in 2, as well as the presence of an additional hydroxy group at C-4 (δC 75.4) in 2. This deduction was supported by the significantly red-shifted UV absorption at λmax 273 nm (conjugated dienone, Δλ +62 nm) as well as the HMBC correlations from H-2 to C-15; from H-14 to C-3; from H3-17 and H3-18 to C-15; and from H3-25 to C-3, C-4, and C-5 (Figure 3). The relative configurations at C-5, -7, -9, -11, -12, and -22 were the same as those of 7 on the basis of the NOESY data (Figure 4). The NOE correlation of H3-25 and H-5 indicated that CH3-25 is β-oriented. The absolute configuration of 2 was established by the experimental and quantum chemical calculated electronic circular dichroism (ECD) spectra.19,20 The ECD spectra of (4R,5R,7R,9S,11R,12S,22R)-2 and its enantiomer (4S,5S,7S,9R,11S,12R,22S)-2 were calculated using TDDFT at the B3LYP/6-31G level with the CPCM model in MeOH. As shown in Figure 5, the calculated spectrum of

positive-ion HRESIMS (m/z 441.1909 [M + H]+, calcd for C25H29O7, 441.1908) and 13C NMR spectroscopic data, indicating 12 indices of hydrogen deficiency. The IR spectrum showed absorption bands at 1799, 1770, 1727, 1641, and 1631 cm−1, reminiscent of the presence of carbonyl and olefinic functionalities. The 1H NMR data (Table 1) displayed characteristic resonances for six methyls (δH 1.10, 1.27, 1.28, 1.33, 1.53, and 1.78, each 3H, s), three methylenes [δH 3.52 (1H, d, J = 21.0 Hz), 3.35 (1H, d, J = 21.0 Hz); 2.13 (1H, d, J = 13.5 Hz), 1.90 (1H, dd, J = 13.5, 3.0 Hz); 3.26 (1H, dd, J = 14.5, 8.5 Hz), 1.66 (1H, dd, J = 14.5, 5.0 Hz)], one oxygenated methylene [δH 4.84 (1H, m)], 4.67 (1H, d, J = 11.5 Hz)], one methine [δH 2.03 (1H, d, J = 12.5 Hz)], and one olefinic proton [δH 6.09 (1H, dd, J = 6.5, 5.0 Hz)]. The 13C NMR data of 1 (Table 1) exhibited 25 carbon resonances comprising six methyl (δC 9.8, 15.3, 17.7, 21.0, 26.7, 29.4), four sp3 methylene (δC 33.0, 34.7, 36.2, 69.8), one sp3 methine (δC 44.6), one sp2 methine (δC 129.8), three sp3 quaternary (δC 49.3, 62.1, 66.3), three oxygenated sp3 tertiary (δC 83.9, 92.5, 93.6), three sp2 quaternary (δC 127.7, 136.4, 141.7), two ester carbonyl (δC 168.4, 170.1), and two ketocarbonyl (δC 196.9, 212.1) carbons. The aforementioned data revealed that compound 1 is a polyketide−terpenoid hybrid meroterpenoid.16−18 The 1H and 13 C NMR spectroscopic data of 1 were comparable to those of 22,23-epoxyberkleydione (7), which was previously isolated from Penicillium minioluteum 03HE3-1,17 except for the significantly deshielded C-9 (δC 93.6; ΔδC +14.9), C-22 (δC 92.5; ΔδC +32.7), and C-23 (δC 69.8; ΔδC +19.5) resonances, as well as the absence of resonances for the methoxy group in 1. The significantly deshielded carbon chemical shifts of C-22 (ΔδC +32.7) and C-23 (ΔδC +19.5) indicated that the 22,23-epoxy group was absent in 1. This deduction was verified by the HMBC correlations from H2-6, H2-23, and H3-24 to C-22 and from H2-23 to C-7 and C-11 (Figure 1). The deshielded H2-23 (δH 4.84, 4.67) resonances in the 1H NMR spectrum as well as the HMBC correlations between H2-23 and the C-20 carbonyl carbon (δC 170.1) indicated that the C-23 hydroxy group is acylated and culminated in the formation of a γ-lactone ring between C-11 and C-23 (Figure 1). In view of the significantly deshielded carbon chemical shifts of the oxygenated sp3 tertiary carbons C-9 (δC 93.6; ΔδC +14.9) and C-22 (δC 92.5; ΔδC +32.7), an ether bridge between C-9 and C-22, forming two five-membered rings from the C-7,8,9,10,11,22 sixmembered ring, was proposed to satisfy the 12 indices of hydrogen B

DOI: 10.1021/acs.jnatprod.6b00160 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for Compounds 1−5a (δ in ppm, J in Hz) 1 position

δC

1 2a

168.4, C 34.7, CH2

2b 3 4 5 6a

127.7, C 136.4, C 44.6, CH 33.0, CH2

6b

2 δH 3.52, d (21.0) 3.35, d (21.0)

2.03, d (12.5) 2.13, d (13.5) 1.90, dd (13.5, 3.0)

δC 165.4, C 116.3, CH

159.9, C 75.4, C 53.3, CH 38.4, CH2

3 δH

δC

6.03, s

167.6, C 112.7, CH

1.47, m

147.6, C 156.9, C 39.4, CH

2.26, d (13.5) 1.82, t (13.5)

31.8, CH2

49.3, C 212.1, C 93.6, C

49.7, C 208.1, C 78.7, C

44.8, C 178.5, C 74.8, CH

10 11

196.9, C 66.3, C

203.1, C 69.2, C

209.0, C 63.7, C

62.1, C 36.2, CH2

13b 14a

129.8, CH

14b 15 16 17 18 19 20 21

141.7, 83.9, 29.4, 26.7, 21.0, 170.1, 9.8,

C C CH3 CH3 CH3 C CH3

3.26, dd (14.5, 8.5) 1.66, dd (14.5, 5.0) 6.09, dd (6.5, 5.0)

1.28, s 1.53, s 1.10, s 1.33, s

22

92.5, C

23a

69.8, CH2

4.84, m

17.7, CH3 15.3, CH3

4.67, d (11.5) 1.27, s 1.78, s

23b 24 25a

50.6, C 36.1, CH2

130.5, CH

135.8, 83.4, 27.3, 26.7, 16.7, 168.1, 16.4,

C C CH3 CH3 CH3 C CH3

2.92, d (14.5) 2.72, dd (17.0, 9.0) 6.14, m

1.50, s 1.59, s 1.41, s 1.45, s

59.9, C 51.5, CH2

3.25, d (2.0)

5.94, s

165.9, C 115.9, CH

2.55, m

156.6, C 59.6, C 40.5, CH

2.02, m

4.27, q (7.5)

29.0, CH2

43.9, C 35.7, CH2

2.52, m 1.94, dd (15.0, 5.0) 6.23, m

135.7, CH

133.3, 85.1, 28.2, 27.3, 19.0, 169.5, 19.1,

44.4, C 181.5, C 70.6, CH

5 δH

δC

6.07, d (1.0)

171.5, C 32.1, CH2

2.45, dd (13.5, 3.0) 1.98, dd (15.0, 3.0) 1.49, dd (15.0, 13.5)

3.35, q (6.0)

99.8, C 67.4, C

C C CH3 CH3 CH3 C CH3

1.64, s 1.69, s 0.85, s 1.34, d (6.5)

51.8, C 83.3, CH

127.4, CH

135.9, 84.4, 26.8, 26.5, 16.5, 173.6, 13.3,

C C CH3 CH3 CH3 C CH3

35.1, CH2

44.1, C 181.4, C 73.3, CH 214.4, C 54.1, CH

5.45, d (5.5)

6.18, dd (5.5, 1.5)

1.47, s 1.66, s 1.32, s

δH 3.67, d (21.0) 3.25, d (21.0)

129.3, C 139.5, C 141.7, C 3.00, d (16.0) 2.05, d (16.0)

4.27, q (7.0) 3.55, d (8.5)

53.7, C 213.2, C

45.4, CH2

129.1, 86.8, 27.8, 26.2, 27.7,

C C CH3 CH3 CH3

1.22, d (6.5)

16.6, CH3

51.0, CH

4.06, d (5.5)

42.6, CH

3.75, d (8.0)

42.2, CH

100.0, CH

6.31, d (6.0)

100.6, CH

6.23, d (8.5)

100.5, CH

28.1, CH3 118.1, CH2

1.45, s 5.82, s

3.88, d (12.0) 3.27, m

1.58, s 1.49, s 1.27, s 1.31, d (7.0) 3.41, t (8.0) 6.31, d (8.0)

3.05, d (3.0) 18.2, CH3 27.1, CH3

1.21, s 1.32, s

25b 26

δC

1.85, m

7 8 9

12 13a

4 δH

32.1, CH3 56.3, CH2

5.32, s 53.1, CH3

3.71, s

53.7, CH3

3.87, s

1.37, s 2.90, d (5.5) 2.63, d (5.0)

52.9, CH3

26.1, CH3 59.5, CH2

1.37, s 4.32, d (13.0) 4.29, d (13.0)

3.73, s

a

All assignments are based on HSQC, HMBC, and NOE experiments. The data for 1 and 3−5 were measured in methanol-d4, and 2 was measured in CDCl3.

at 263 nm. Therefore, the structure of purpurogenolide B (2) was elucidated as shown. Purpurogenolide C (3) was assigned a molecular formula of C26H30O8 on the basis of the 13C NMR and positive-ion HRESIMS data (m/z 471.2003, [M + H]+, calcd for C26H31O8, 471.2013), indicating 12 indices of hydrogen deficiency. The IR spectrum indicated the presence of carbonyl and olefinic functionalities (1788, 1725, 1632 cm−1). The 1H and 13C NMR data (Table 1) displayed resonances attributable to five methyls (δH 0.85, 1.34, 1.45, 1.64, 1.69; δC 19.0, 19.1, 28.1, 28.2, 27.3, respectively), one methoxy group (δH 3.87; δC 53.7), two methylenes [δH 1.85 (1H, m), 2.02 (1H, m); 1.94 (1H, dd, J = 15.0, 5.0 Hz), 2.52 (1H, m); δC 31.8, 35.7, respectively], two methines [δH 2.55 (1H, m), 4.06 (1H, d, J = 5.5 Hz);

Figure 1. Selected HMBC (arrows point from protons to carbons) and NOE correlations of compound 1.

(4R,5R,7R,9S,11R,12S,22R)-2 agreed well with the experimental spectrum, which showed a negative Cotton effect (Δε = −39.92) C

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spectroscopic data of 3 (Table 1) showed a close structural resemblance to berkeleyacetal B, which was previously isolated from Penicillium rubrum Stoll.18 The major difference was that the C-4 oxirane moiety (see the structures of 4 and 6 for a similar functionality) [δH 2.58 (1H, d, J = 5.4 Hz), 2.92 (1H, d, J = 5.4 Hz); δC 56.8, 59.5] in berkeleyacetal B was replaced by an exocyclic olefinic functionality [δH 5.32 (1H, s), 5.82 (1H, s); δC 118.1, 156.9] in 3. This deduction was supported by the HMBC correlations from H2-25 to C-3, C-4, and C-5 (Figure 3). The 2D structure of 3 was verified by the HMBC correlations between H-2 and C-1/C-3/C-15; H2-6 and C-5/C-7/C-8/C-12/C-24; H2-13 and C-5/C-11/C-12/C-14/C-15/C-19; H3-17 and C-15/ C-16/C-18; H3-18 and C-15/C-16/C-17; H3-19 and C-5/C-11/ C-12/C-13; H3-21 and C-9/C-10; H-22 and C-7/C-8/C-10/ C-11/C-12/C-20/C-24; H-23 and C-9/C-11/C-22; H3-24 and C-6/C-7/C-8/C-22; and H3-26 and C-20 (Figure 3). The relative configuration of 3 was established on the basis of the NOESY data (Figure 4). NOE correlations of H3-19/H-22, H-22/H-23, H-22/H3-24, and H-23/H3-24 indicated their cofacial orientations. In turn, NOEs of H-5/H3-26 and H3-21/ H3-26 placed these groups on the opposite face (Figure 4). The absolute configuration of 3 was defined by comparison of the experimental and calculated ECD spectra using the same procedures, conformational searches, geometry optimizations, and calculated ECD spectra applied as in the case of 2. The experimental and calculated ECD spectra of (4R,5R,7R,9S,11R,12S,22R)-3 showed good agreement (Figure 5). Thus, the structure and absolute configuration of purpurogenolide C (3) were defined as shown. Purpurogenolide D (4) was isolated as a white powder, [α]21 D +200 (c 0.1 MeOH). Its molecular formula was determined as C26H30O10 by 13C NMR and positive-ion HRESIMS data (m/z 503.1918, [M + H]+, calcd for C26H31O10, 503.1912), indicating 12 indices of hydrogen deficiency. The IR spectrum showed absorption bands (3427, 1772, 1713, 1636 cm−1) for hydroxy, carbonyl, and olefinic functionalities. The 1H NMR data (Table 1) of 4 showed resonances of five methyls [δH 1.32, 1.37, 1.47, 1.66, each 3H, s; δH 1.22 (3H, d, J = 6.5 Hz)], one methoxy group (δH 3.73), one methylene [δH 1.98 (1H, dd, J = 15.0, 3.0 Hz), 1.49 (1H, dd, J = 15.0, 13.5 Hz)], one oxygenated methylene [δH 2.90 (1H, d, J = 5.5 Hz), 2.63 (1H, d, J = 5.0 Hz)], two methines [δH 2.45 (1H, dd, J = 13.5, 3.0 Hz); 3.75 (1H, d, J = 8.0 Hz)], three oxygenated methines [δH 6.23 (1H, d, J = 8.0 Hz); 5.45 (1H, d, J = 5.5 Hz); 3.35 (1H, q, J = 6.0 Hz)], and two olefinic protons [δH 6.07 (1H, d, J = 1.0 Hz); 6.18 (1H, dd, J = 5.5, 1.5 Hz)]. The 13C NMR data (Table 1) revealed 26 carbon resonances corresponding to three ester/lactone carbonyl, one hemiketal,

Figure 2. ORTEP drawing of compound 1.

Figure 3. Selected HMBC correlations of compounds 2−5.

δC 39.4, 51.0, respectively], one oxygenated methine [δH 4.27 (1H, q, J = 7.5 Hz); δC 74.8], one acetal methine [δH 6.31 (1H, d, J = 6.0 Hz); δC 100.0], three quaternary carbons (δC 43.9, 44.8, 63.7), one oxygenated tertiary carbon (δC 85.1), one exocyclic double bond [δH 5.32 (1H, s), 5.82 (1H, s); δC 118.1, 156.9], two trisubstituted double bonds [δH 5.94 (1H, s), 6.23 (1H, m); δC 112.7, 133.3, 135.7, 147.6], three ester carbonyl carbons (δC 167.6, 169.5, 178.5), and one ketocarbonyl carbon (δC 209.0). These spectroscopic data revealed that compound 3 also belonged to the polyketide−terpenoid hybrid meroterpenoid class of compounds.17,18,21 Analysis of the 1D and 2D NMR

Figure 4. Selected NOESY correlations of 2−4. D

DOI: 10.1021/acs.jnatprod.6b00160 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

of 4 (Figure 3). The relative configuration of 4 was established on the basis of NOESY data (Figure 4). NOE correlations between H-2/H-25b, H-5/H-6a, H-5/H-25a, H-5/H-9, H-6b/ H3-19, H-6a/H-25a, H-13/H3-19, H3-19/H3-26, H-22/H-23, H-23/ H3-24, and H-22/H3-24 indicated the relative configuration of 4 to be as shown in Figure 4. A comparison of the experimental and calculated ECD spectra (Figure 5) facilitated assignment of the absolute configuration of 4 as (4S,5S,7R,9S,10R,11R,12R,13R,22S,23S). Therefore, the structure of purpurogenolide D (4) was established as shown. Purpurogenolide E (5) was isolated as colorless plates via crystallization from MeOH−H2O (95:5), [α]21 D −226 (c 0.1, MeOH). Its 13C NMR and positive-ion HRESIMS data, exhibiting a protonated molecule at m/z 445.1866 [M + H]+, corresponded to a molecular formula of C24H28O8 (calcd for C24H29O8, 445.1857). The IR spectrum showed absorption bands at 3442, 1777, 1717, and 1636 cm−1, suggesting the presence of hydroxy, carbonyl, and olefinic functionalities. The 1H NMR data (Table 1) of 5 displayed characteristic resonances of five methyls [δH 1,27, 1.37, 1.49, 1.58, each 3H, s; δH 1.31 (1H, d, J = 7.0 Hz)], one oxygenated methylene [δH 4.32 (1H, d, J = 13.0 Hz), 4.29 (1H, d, J = 13.0 Hz)], one oxygenated methine [δH 4.27 (1H, q, J = 7.0 Hz)], and one acetal [6.31 (1H, d, J = 8.0 Hz)] protons. The 13C NMR data of 5 exhibited 24 carbon resonances, including two ketocarbonyls (δC 214.4; 213.2), two ester carbonyls (δC 181.4; 171.5), two tetrasubstituted double bonds (δC 129.1, 129.3, 139.5, 141.7), five methyls, four methylenes (one oxygenated), four methines (one oxygenated, one acetal), one oxygenated tertiary carbon, and two quaternary carbons (Table 1). Comparison of the NMR spectroscopic data revealed that the 2D structure of 5 was closely related to berkeleyacetal C (6), which was previously isolated from Penicillium rubrum Stoll.18 The main differences were that the conjugated Δ2,3 and Δ14,15 double bonds in 6 were replaced by the conjugated Δ4,5 and Δ3,15 double bonds in 5. This deduction was supported by the HMBC correlations from H2-2 to C-1, C-3, and C-15; from H2-6 to C-4, C-5, C-7, C-8, C-12, C-22, and C-24; from H-11 to C-5, C-9, C-10, C-12, C-13, C-19, and C-22; from H2-14 to C-3, C-12, C-13, C-15, and C-16; from H3-17 to C-15, C-16, and C-18; from H3-18 to C-15, C-16, and C-17; from H3-19 to C-5, C-11, C-12, and C-13; and from H2-25 to C-3, C-4, and C-5 (Figure 3). In the NOESY spectrum, NOE correlations of H-11/H3-19, H-11/H3-21, H-11/H-22, H-11/H-23, H3-19/H-22, H-22/H-23, H-22/ H3-24, and H-23/H3-24 indicated that these groups are cofacially orientated (Figures S32 and S33, Supporting Information). The absolute configuration of 5 was defined as (7R,9R,11R,12R,22S,23R) by single-crystal X-ray diffraction analysis (Figure 6) using Cu Kα radiation with Flack and Hooft parameters of 0.01(9) and 0.00(9), respectively. By comparing spectroscopic and specific rotation data with literature values, the remaining four known compounds were identified as berkeleyacetal C (6),18 22,23-epoxyberkeleydione (7),17 chrodrimanin B (8),22 and chrodrimanin E (9).23 Compounds 1−9 were evaluated for their inhibitory activities against LPS-activated NO production in BV-2 microglial cells using the Griess assay.24−27 Indomethacin was used as a positive control (IC50 = 34.5 ± 1.2 μM). Compounds 2−4 and 6 showed inhibitory activity against NO production with IC50 values of 30.0 ± 1.5, 15.5 ± 0.5, 8.8 ± 0.1, and 0.8 ± 0.1 μM, respectively. Compounds 1, 5, and 7−9 were inactive (