Azaphilone Alkaloids with Anti-inflammatory Activity from Fungus

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Bioactive Constituents, Metabolites, and Functions

Azaphilone Alkaloids with Anti-inflammatory Activity from Fungus Penicillium sclerotiorum cib-411 Jialin Tang, Zongyuan Zhou, tao yang, Can Yao, linwei wu, and Guo-You Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05628 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 2, 2019

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Journal of Agricultural and Food Chemistry

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Azaphilone Alkaloids with Anti-inflammatory Activity from Fungus

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Penicillium sclerotiorum cib-411

3

Jia-Lin Tang,†,‡,§ Zong-Yuan Zhou,†,‡,§ Tao Yang,†,§ Can Yao,† Lin-Wei Wu,† and Guo-You Li†,*

4

†Key

5

Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences,

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Chengdu 610041, China.

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‡University

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ABSTRACT: Nine new azaphilone alkaloids, penazaphilones A–I (1–9), were isolated from the

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solid fermented rice culture of Penicillium sclerotiorum cib-411. The structures of compounds 1–9

10

were elucidated based on HRESIMS, NMR, and CD spectroscopic data. The structures of 5 and 8

11

were confirmed by X-ray crystallographic analyses. Biological evaluation showed that compounds

12

1, 5, 6 and 8 inhibited the production of nitric oxide (NO) on RAW 264.7 cells stimulated by

13

lipopolysaccharide with IC50 values of 15.29, 9.34, 9.50 and 7.05 μM, respectively. Meanwhile they

14

did not exhibit obvious cytotoxicity at the concentration of 50.0 μM.

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KEYWORDS: Penicillium sclerotiorum, azaphilones, anti-inflammatory activity, cytotoxicity

16

Laboratory of Environmental and Applied Microbiology, Environmental Microbiology Key

of Chinese Academy of Sciences, Beijing 100049, China.

 INTRODUCTION

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Azaphilones are a class of typical fungal polyketide pigments characteristic of a highly oxygenated

18

pyranoquinone or isoquinoline bicyclic core and a large conjugated system in their structures,

19

presenting different colors such as orange, purple, red and yellow.1 They are widely discovered as

20

secondary metabolites from ascomyceteous2 and basidiomyceteous fungi, such as Monascus,3-8

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Penicillium,9-12 Aspergillus,13,14 Chaetomium15-17 and Emericella.18,19 Many azaphilones exhibit

ACS Paragon Plus Environment


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diverse

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antioxidation,11 lipoxygenase and Hsp90 inhibition,13,20 nematicida15 and antimalaria.16 In Asian

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countries, such as China, Korea and Japan, Monascus-fermented rice has been used to produce

25

azaphilone food pigments for centuries.3 Azaphilone pigments are commercially available and

26

consumed as food colorants.21 Till now, there are no reports on the toxicity of azaphilone pigments

27

from the genus Monascus, which suggests azaphilone pigments from Monascus are safe for human

28

being.

biological

activities

including

anti-inflammation,4-7

cytotoxicity,5,8-10,12,14,16,17

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Recently, more and more consumers have been aware of the link between diet and health, which

30

leads to the increasing demand for natural pigments. Fungal pigments have aroused strong interest

31

of food colorant industries because they are suitable for large-scale industrial production and have

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no drawbacks of synthetic pigments.22 Because of the color diversity and multiple beneficial

33

bioactivities, azaphilones may be a class of potential food colorants.22 In search of fungal pigments,

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nine new azaphilone alkaloids, named penazaphilones A–I (1–9), were isolated and identified from

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the ethyl acetate extract of the solid-fermented rice culture of P. sclerotiorum cib-411. Here the

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isolation, structure elucidation, and anti-inflammatory activity of azaphilone alkaloids from P.

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sclerotiorum cib-411 were described.

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 MATERIALS AND METHODS

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General Experimental Procedures. Melting points were measured on an X-6 precise melting

40

point apparatus (Beijing Fukai Science and Technology Development Limited Company, Beijing,

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China). Infrared (IR) spectra, UV spectra and optical rotations were respectively obtained on a

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Perkin-Elmer Spectrum One FT-IR spectrometer (Perkin-Elmer, Inc., Waltham, MA, USA), Perkin-


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Elmer S2 Lambda 35 UV/VIS spectrometer and a Perkin-Elmer 341 polarimeter. ECD data were

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obtained using a JASCO J-815 ECD instrument (Jasco Analytical Instruments, Easton, MD, USA).

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Nuclear magnetic resonance (NMR) spectra were generated on Bruker Avance instruments (400

46

MHz, 600 MHz) (Bruker Biospin Gmbh, Rheistetten, Germany). A BioTOF-Q mass spectrometer

47

(Bruker Daltonics, Billerica, MA, USA) was used to obtain high-resolution electrospray ionization

48

mass (HRESIMS) data. HPLC separations and analyses were performed on a LC100 series

49

apparatus with an UV detector (Shanghai Wufeng Scientific Instruments Co., Ltd. Shanghai, China),

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equipped a Kromasil-100-10-C18 column (250*20 mm, (AkzoNobel, Bohus, Sweden). X-ray

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crystallographic data of 5 and 8 were respectively acquired on a Bruker SMART-1000 CCD and a

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Bruker APEX-II CCD diffractometers (Bruker AXS, Madison, WI, USA).

53

Reagents. Methanol and acetonitrile (HPLC grade), acetone, methanol, ethyl acetate, chloroform,

54

and petroleum ether (analytical grade) were purchased from KeLong (ChengDu, China).

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Chloroform-d and methanol-d4 were purchased from CIL CO. (YRTC, China). Dulbeccoʼs modified

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Eagleʼs medium (DMEM) and streptomycin/penicillin were purchased from Hyclone (Thermo

57

Scientific, Waltham, MA, USA). Fetal bovine serum (FBS) was bought from Tianhang Biological

58

Technology Stock Co., Ltd. (Huzhou, China). Alamar-Blue was provided by Sun-bio Medical

59

Biotechnology Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) prepared from Escherichia

60

coli

61

dimethylaminopropyl)carbodiimide

62

trifluoromethylphenylacetic acid (MTPA) were from J&K Chemical Ltd. (Beijing, China).

055:

B5,

BAY

11-7082,

dimethylaminopyridine hydrochloride

(EDCl),


(DMAP),

1-ethyl-3-(3-

and

α-methoxy-α-


63

Fungal Material. P. sclerotiorum cib-411 was isolated from a brick of Daqu from Wenjunjing

64

Distillery Co. Ltd., Chengdu, Sichuan, China. Daqu brick is a fermentation starter for the production

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of Chinese Baijiu.23 The fungus was identified to be P. sclerotiorum based on DNA amplification

66

and sequencing of the ITS and microscopic and macroscopic features. The fungus (No. CIB-411)

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was cultured on potato dextrose agar slant (PDA) at 4 °C and deposited at the fungus storage center

68

of Chengdu Institute of Biology.

69

Fermentation and Isolation. The procedures of fermentation were the same as those reported

70

previously.24 The rice culture (10.0 kg) was soaked with EtOAc (60 L) at 40 ºC for three times (1

71

day each time). The solvents were combined and evaporated under reduced pressure to give a crude

72

extract, which was fractionated over a silica gel column eluted successively with petroleum

73

ether/ethyl acetate (20:1, 10:1, 5:1, 2:1, 1:1, 0:1, v/v) and CHCl3/MeOH (15:1, 5:1, v/v) to give

74

seven fractions (Frs. A–G). Chromatographic separation of fraction D by a silica gel column with

75

petroleum ether/acetone afforded compound 2 (17.0 mg). Fraction E was purified over RP-18 silica

76

gel column eluted with MeOH-H2O (20: 80, 40: 60, 80: 20, 100: 0, v/v) to give subfractions E1–

77

E12. Compound 5 (15.0 mg) was crystallized from subfraction E1. Purification of subfraction E6

78

by HPLC using CH3CN-H2O (2.0 mL/min, 70: 30, v/v) as eluent yielded compounds 1 (65.0 mg, tR

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12.7 min) and 6 (11.0 mg, tR 23.1 min). Fraction F was separated over reverse-phase silica gel eluted

80

with MeOH-H2O (20: 80, 40: 60, 80: 20, 100: 0, v/v) to afford twenty subfractions F1–20. The

81

subfraction F15 was subjected to semipreparative HPLC using CH3CN-H2O (2.0 mL/min, 70: 30,

82

v/v) to yield compound 7 (42.0 mg, tR 27.5 min). Fraction F16 was subjected to Sephadex LH-20

83

column chromatography eluted with MeOH to afford 8 (63.0 mg). HPLC separation of fraction F19


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using CH3CN-H2O (2.0 mL/min, 75: 25, v/v) as the eluent yielded compound 9 (35.0 mg, tR 4.8

85

min). Separation of fraction G over RP-18 silica gel with MeOH-H2O (20: 80, 40: 60, 80: 20, 100:

86

0, v/v) as eluent gave ten subfractions (G1–10). Subfraction G2 was separated by HPLC using

87

CH3CN-H2O (2.0 mL/min, 33: 67, v/v) as eluent to yield compound 3 (3.0 mg, tR 7.5 min).

88

Preparative HPLC purification of fraction G7 using CH3CN-H2O (2.0 mL/min, 40: 60, v/v) afforded

89

compound 4 (8.0 mg, tR 18.5 min).

90

Penazaphilone A (1): red amorphous powder;  20D ‒ 430° (0.01, MeOH); UV (MeOH) λmax (log

91

ε) 471 (3.60), 369 (4.34), 235 (4.24); IR νmax (KBr) 3436, 2957, 1743, 1705, 1598, 1491, 1369, 1243,

92

1187, 1140, 1084, 1005 cm-1; HRESIMS m/z 504.2148 [M + H]+, (calcd for C27H35NO6Cl, 504.2153,

93

1H

and 13C NMR data were shown in Tables 1 and 2.

94

Penazaphilone B (2): red amorphous powder;  20 ‒ 350° (0.01, MeOH); UV (MeOH) λmax (log D

95

ε) 466 (3.68), 378 (4.43), 232 (4.35); IR νmax (KBr) 2957, 2920, 1743, 1705, 1603, 1505, 1365, 1243,

96

1187, 1136 cm-1; HRESIMS m/z 518.2309 [M + H]+ (calcd for C28H37NO6Cl, 518.2304); 1H and

97

13C

NMR data were shown in Tables 1 and 2.

98

Penazaphilone C (3): red amorphous powder;  20D ‒ 300° (0.01, MeOH); UV (MeOH) λmax (log

99

ε) 480 (3.20), 370 (4.00), 232 (3.87); IR νmax (KBr) 3436, 1724, 1701, 1579, 1491, 1329, 1229, 1145,

100

1080 cm-1; HRESIMS m/z 450.1668 [M + H]+ (calcd for C23H29NO6Cl, 450.1678); 1H and 13C NMR

101

data were shown in Tables 1 and 2.

102

Penazaphilone D (4): red amorphous powder;  20D ‒ 400° (0.01, MeOH); UV (MeOH) λmax (log

103

ε) 485 (3.60), 372 (4.39), 235 (4.24); IR νmax (KBr) 3427, 2960, 1731, 1590, 1496, 1400, 1229, 1085,



104

963, 775 cm-1; HRESIMS m/z 475.1991 [M + H]+ (calcd for C25H32N2O5Cl, 475.1994); 1H and 13C

105

NMR data were shown in Tables 1 and 2.

106

Penazaphilone E (5): red cubic crystal, mp 231‒232 ºC;  20D ‒ 380° (0.01, MeOH); UV (MeOH)

107

λmax (log ε) 488 (4.00), 336 (4.86), 235 (4.30); IR (KBr) 3427, 2957, 1733, 1701, 1584, 1551, 1495,

108

1467, 1225, 1136, 1080 cm-1; HRESIMS m/z 390.1468 [M + H]+ (calcd for C21H25NO4Cl, 390.1467);

109

1H

and 13C NMR data were shown in Tables 1 and 2.

110

Penazaphilone F (6): red amorphous powder;  20D ‒ 700° (0.01, MeOH); UV (MeOH) λmax (log

111

ε) 480 (3.28), 368 (4.09), 237 (3.91); IR (KBr) 2957, 1729, 1701, 1593, 1500, 1369, 1234, 1145,

112

1080 cm-1; HRESIMS m/z 490.1996 [M + H]+ (calcd for C26H33NO6Cl, 490.1991); 1H and 13C NMR

113

data were shown in Tables 1 and 2.

114

Penazaphilone G (7): red amorphous powder;  20D ‒ 220° (0.01, MeOH); UV (MeOH) λmax (log

115

ε) 485 (3.45), 367 (4.01), 235 (3.88); IR (KBr) 2957, 1733, 1705, 1593, 1500, 1369, 1248 cm-1;

116

HRESIMS m/z 833.3357 [M + H]+ (calcd for C46H55N2O8Cl2, 833.3335); 1H NMR (400 MHz,

117

CDCl3): δH 8.00 (s, 1H, H-1), 6.93 (s, 1H, H-4), 6.87 (1H, d, J = 15.3 Hz, H-10), 6.10 (1H, d, J =

118

15.3 Hz, H-9), 5.67 (1H, d, J = 9.6 Hz, H-12), 3.81 (2H, m, H-1′), 2.45 (1H, m, H-13), 2.13 (3H, s,

119

H-20), 1.75 (3H, s, H-17), 1.70 (2H, m, H-2′), 1.51 (3H, s, H-18), 1.26~1.42 (2H, m, H-14), 1.02

120

(3H, d, J = 6.6 Hz, H-16), 0.84 (3H, t, J = 7.4 Hz, H-15); 13C NMR (100 MHz, CDCl3): δC 194.3

121

(C-8), 184.0 (C-6), 170.3 (C-19), 148.5 (C-4a), 148.4 (C-12), 146.1 (C-3), 144.4 (C-10), 141.5 (C-

122

1), 132.1 (C-11), 114.9 (C-9), 114.2 (C-8a), 111.9 (C-4), 102.3 (C-5), 85.7 (C-7), 53.4 (C-1′), 35.2

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(C-13), 30.2 (C-14), 27.2 (C-2′), 23.7 (C-18), 20.6 (C-20), 20.4 (C-16), 12.7 (C-17), 12.2 (C-15).


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Penazaphilone H (8): red cubic crystal; mp 210‒211 ºC;  20D ‒ 420° (0.01, MeOH); UV (MeOH)

125

λmax (log ε) 475 (3.30), 367 (4.03), 235 (3.85); IR (KBr) 3408, 2920, 1705, 1579, 1485, 1330, 1225,

126

1078 cm-1; HRESIMS m/z 435.1761 [M + H]+ (calcd for C23H29NO5Cl, 435.1734); 1H NMR (400

127

MHz, CDCl3): δH 7.88 (1H, s, H-1), 7.00 (1H, s, H-4), 6.25 (1H, d, J = 15.3Hz, H-9), 6.90 (1H, d,

128

J = 15.3 Hz, H-10), 5.67 (1H, d, J = 9.2 Hz, H-12), 2.45 (1H, m, H-13), 1.40 (1H, m, H-14), 1.31

129

(1H, m, H-14), 0.84 (3H, t, J = 7.2 Hz, H-15), 0.99 (3H, d, J = 6.4 Hz, H-16), 1.81 (3H, s, H-17) ,

130

1.49 (3H, s, H-18), 2.11 (3H, s, H-20), 4.00 (2H, brs, H-1′), 3.86 (2H, brs, H-2′); 13C NMR (100

131

MHz, CDCl3): δC 142.6 (C-1), 145.6 (C-3), 111.9 (C-4), 149.1 (C-4a), 101.7 (C-5), 184.4 (C-6),

132

85.2 (C-7), 194.2 (C-8), 114.8 (C-8a), 115.3 (C-9), 145.5 (C-10), 132.0 (C-11), 148.3 (C-12), 35.2

133

(C-13), 30.2 (C-14), 12.2 (C-15), 20.4 (C-16), 12.8 (C-17), 23.5 (C-18), 170.5 (C-19), 20.5 (C-20),

134

56.0 (C-1′), 60.8 (C-2′).

135

Penazaphilone I (9): red amorphous powder;  20D ‒ 320° (0.01, MeOH); UV (MeOH) λmax (log

136

ε) 478 (3.22), 372 (4.05), 235 (3.85); IR (KBr) 2957, 2920, 17330, 1701, 1593, 1591, 1369, 1234,

137

cm-1; HRESIMS m/z 498.1639 [M + H]+ (calcd for C25H30NO6ClNa, 498.1654); 1H NMR (400 MHz,

138

CDCl3): δH 7.82 (1H, s, H-1), 7.03 (1H, s, H-4), 6.30 (1H, d, J = 15.3 Hz, H-9), 6.96 (1H, d, J =

139

15.3 Hz, H-10), 5.67 (1H, d, J = 9.7 Hz, H-12), 2.46 (1H, m, H-13), 1.39 (1H, m, H-14), 1.29 (1H,

140

m, H-14), 0.84 (3H, t, J = 7.4 Hz, H-15), 0.99 (3H, d, J = 6.4 Hz, H-16), 1.85 (3H, s, H-17), 1.51

141

(3H, s, H-18), 2.12 (3H, s, H-20), 3.95 (2H, m, H-1′), 2.00 (2H, m, H-2′), 2.46 (2H, m, H-3′); 13C

142

NMR (100 MHz, CDCl3): δC 141.7 (C-1), 148.7 (C-3), 111.6 (C-4), 145.5 (C-4a), 101.8 (C-5), 184.4

143

(C-6), 85.0 (C-7), 193.9 (C-8), 115.2 (C-8a), 114.5 (C-9), 145.9 (C-10), 132.3 (C-11), 148.7 (C-12),



144

35.2 (C-13), 30.3 (C-14), 12.2 (C-15), 20.4 (C-16), 12.7 (C-17), 23.4 (C-18), 170.5 (C-19), 20.5 (C-

145

20), 53.8 (C-1′), 25.3 (C-2′), 30.2 (C-3′) , 175.6 (C-4′).

146

Single Crystal X-ray Diffraction Data for Compound 5: C21H24NO4Cl; Mr = 389.86;

147

orthorhombic, space group P212121, a = 7.3616 (5) Å, b = 8.6802 (4) Å, c = 31.9635 (16) Å,  =

148

90°,  = 92.700°,  =90°, V = 2040.2 (2) Å3, Z = 4, Dcalc = 1.269 g/cm3,  = 0.71073 Å,  (Mo K)

149

= 0.213 mm-1, F (000) = 824.0, T = 293 K. Of the 3139 reflections that were collected, 2663 were

150

unique (Rint = 0.0412). The structure was solved by direct methods with SHELXL-97 and refined

151

by full-matrix least-squares on F2. Final refinement: data/restraints/parameters = 3139 / 1 / 249; R1

152

= 0.0517 (all data), wR2 = 0.0899 (all data). GOF = 1.052. Crystallographic data of compound 5

153

(CCDC1886552) has been deposited at the Cambridge Crystallographic Data Center. Copies of the

154

data are free of charge to be obtained by application to CCDC, 12 Union Road, CB2 1EZ, UK [fax:

155

+44-0-1223-336033 or e-mail: [email protected]].

156

Preparation of (S)- and (R)-MTPA Esters of Penazaphilone C (3a and 3b). A

157

dichloromethane solution of penazaphilone C (1.0 mg, 0.0022 mmol) was treated with DMAP

158

(catalytic amount), EDCl (1.0 mg, 0.0052 mmol), and (S)-MTPA or (R)-MTPA (2.0 mg, 0.0085

159

mmol), and the mixture was stirred at room temperature for 4 h. Then the solution was washed with

160

1 M aq. HCl for three times and concentrated under reduced pressure to yield a residue. The residue

161

was respectively dissolved in CD3OD for 1H NMR analysis without purification.

162

Cell Lines and Culture. Murine macrophage RAW264.7 cells were from the Cell Bank of

163

Chinese Academy of Sciences (Shanghai, China). DMEM with 10% FBS and 1%


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penicillin/streptomycin was used as a culture for cells in a humidified incubator with a 5% CO2

165

atmosphere at 37 ºC.

166

Cell viability assay. Alamar-Blue assay was used to determine cell viability. In brief, RAW264.7

167

cells at the concentration of 1 × 104 cells/ml were inoculated into 96-well plates, then treated with

168

compounds at 50.0 μM or DMSO for 24 h. The purities of compounds 1, 2, and 4‒9 were > 98%

169

checked by HPLC with UV detector, and the purity of 3 was > 90%. Each well was added with

170

Alamar-Blue reagent and measured by a Thermo Scientific Varioskan Flash Multimode Reader with

171

excitation at 544 nm and emission at 590 nm, respectively. Each sample was performed in triplicate.

172

Measurement of Nitric Oxide in RAW 264.7 Macrophage Cells. Nitric oxide (NO) was

173

determined according to the method described previously.25,26 The RAW264.7 cells at 5 × 105

174

cells/ml were plated in 24-well plates. After incubated overnight, cells were treated with different

175

concentrations of test compounds for 1 h, then treated with LPS (1.0 μg/ml) for an additional 24 h

176

(BAY as positive control, 10.0 μM). A commercially available kit based on the Griess reaction

177

(Beyotime) was used to measure NO directly in the cell cultue medium. Data reported are the mean

178

values from triplicate analyses.

179

Data Analysis. All experiments were repeated in triplicate and the results are presented as mean

180

± S.D. Significant differences were evaluated with one-way analysis of variance using GraphPad

181

Prism 5.0 (Graphpad Software, San Diego, CA). When p < 0.05, the difference was considered to

182

be significant.

183

 RESULTS AND DISSCUSION



184

Structural Elucidation. Compound 1 was obtained as a red amorphous powder and assigned a

185

molecular formula of C27H34ClNO6 based on the HRESIMS peak at m/z 504.2148 [M + H]+ and 13C

186

NMR spectrum, indicating 11 unsaturation degrees. The relative intensity radio of isotope peaks of

187

about 3:1 demonstrated that 1 contained a chlorine atom. The IR spectrum of 1 featured typical

188

absorption bands for hydroxyl (3436 cm-1), lactone (1743 cm-1), and conjugated ketone (1705 cm-

189

1).

190

spectrum of 1, along with the HSQC experiment, informed the presence of seven methyls [δH 2.11

191

(3H, s), 1.82 (3H, s), 1.54 (3H, s), 1.00 (3H, d, J = 6.6 Hz), 0.92 (3H, d, J = 6.6 Hz), 0.88 (3H, d, J

192

= 6.6 Hz), 0.84 (3H, t, J = 7.4 Hz)], and five olefinic protons [7.92 (1H, s), 6.98 (1H, s), 6.87 (1H,

193

d, J = 15.2 Hz), 6.15 (1H, d, J = 15.2 Hz), 5.66 (1H, d, J = 9.7 Hz)] (Table 1). By the same way, 27

194

carbon signals in the

195

ketonic carbonyl (δC 193.6, 184.9), one ester carbonyl (δC 170.7), one carboxyl (δC170.1)], ten

196

oleﬁnic C-atoms [139.1, 145.6, 112.9, 150.1, 102.8, 115.2, 115.7, 146.2, 132.0 and 148.4], one

197

oxygenated quaternary C-atom (δC 84.8), three methines (δC 61.9, 35.2, 24.9), two methylenes (δC

198

40.7, 30.2), and seven methyls (δC 23.4, 22.8, 21.7, 20.4, 20.4, 12.8, 12.1) (Table 2). In the HMBC

199

spectrum, the correlations of H-1 with C-2′, C-3, C-4a, C-8 and C-8a, H-4 with C-4a, C-5 and C-9,

200

and H-18 with C-6 and C-8 indicated an isoquinoline-6,8 (2H,7H)-dione moiety, a typical structural

201

core in azaphilone skeleton27. Meanwhile the HMBC correlations of H-9 with C-3, C-4, and C-10,

202

H-17 with C-10, C-11, and C-12, H-16 with C-12, C-13, and C-14, and H-15 with C-14 and C-13

203

confirmed the presence of the side chain [C-9/C-10/C-11(C-17)/C-12/C-13(C-16)/C-14/C-15]

204

connected with C-3, which confirmed 1 to be a sclerotioramine derivative. Comparing the NMR

The UV spectrum displayed absorption maximums at 471, 369, and 235 nm. The 1H NMR

13C

NMR spectrum can be recognized as four carbonyls [two conjugated


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205

spectra of compound 1 and sclerotioramine, we concluded that 1 had one more intact leucine residue

206

located at N-2,27 which was deduced from the HMBC correlations of H-5′ (δH 0.92 ) and H-6′ (δH

207

0.88) with C-4′ (24.9) and C-3′ (40.7), H-3′ (δH 1.91, 2.10) with C-1′ (δC 170.1) and C- 2′ (δC 61.9),

208

and H-1 with C-2′ (δC 61.9) (Figure 2).

209

The stereochemistry of C-13 in 1 was biogenetically established as (S), since the stereochemistry

210

at C-13 in compounds 5 and 8 from this same fungus were determined as (S) by X-ray

211

crystallographic analyses. It is known that 7-epi-sclerotiorin azaphilones afford (‒) ∆ε at ca. 390 nm

212

in their circular dichroism (CD) spectra.28, 29 In the CD spectrum of 1, the negative cotton effect at

213

385 nm revealed an S configuration at C-7 (see supporting information). In order to determine the

214

absolute configuration of leucine residue, compound 1 was hydrolyzed in 6 M aq. HCl at 100 ºC for

215

24 hours but failed to yield a leucine. Furthermore we failed to obtain crystals for X-ray

216

crystallographic analysis. Therefore the absolute configuration of C-2′ were not established here.

217

Finally, the structure of compound 1 was defined as in figure 1 and named penazaphilone A.

218

Compound 2 was a red solid and given a molecular formula C28H36ClNO6 from the HRESIMS

219

peak at m/z 518.2309 [M + H]+ and 13C NMR data. Compound 2 shared similar NMR spectra to 1,

220

except for one more OCH3 group (δH 3.78, δC 53.8) in 2, which formed an ester with C-1′ confirmed

221

by the HMBC correlation of CH3 at δH 3.78 with C-1′ (δC 169.2). The CD spectrum of 2 was similar

222

to that of 1 and had (‒) ∆ε at ca. 385 nm, indicative of (S) configuration at C-7. The configurations

223

of 2 at C-13 and C-2′ were biogenetically expected to be the same as 1. With the aid of HSQC and

224

HMBC correlations, the structure of 2 was finally determined and named penazaphilone B (Figure

225

2).



226

Compound 3, a red amorphous solid, had a molecular formula of C23H28ClNO6 inferred from the

227

m/z 450.1668 [M + H]+ peak in HRESIMS, indicative of 10 unsaturation degrees. The UV spectrum

228

of 3 showed maximal absorptions at 480, 370 and 232 nm, which were similar to those of

229

compounds 1 and 2. Compound 3 contained one more oxygen atom than isochromophilone VI.30 In

230

the 1H and 13C NMR spectra of compound 3, there were one more doublet methyl (δH 1.15, d, J =

231

6.3 Hz) and one more oxygenated methine at δc 72.5 with the disappearance of a triplet methyl at

232

δH 0.84 and a methylene at δc 30.2 in 9. In the HMBC spectrum, correlations from H-15 to C-13

233

and C-14, and H-16 to C-12, C-13, and C-14, demonstrated that a hydroxyl was located at C-14

234

(Figure 2). In order to determine the absolute configuration of C-14, (S)- and (R)-MTPA esters (3a

235

and 3b) were prepared by esterification of 3 with (S)- and (R)-MTPA acids, respectively.31 The

236

comparison of the 1H NMR of 3a and 3b showed that H-12 and H-16 had negative ΔδSR (δS – δR)

237

values (–0.017 and –0.016, respectively) and H-15 had a positive ΔδSR value (+0.018), suggesting

238

the configuration at C-14 to be R (Figure 3). The absolute stereochemistry at C-7 and C-13 were

239

determined in the same way as for 1 and 2. However after hydroxylation at C-14, the second largest

240

substitution of C-13 (C-14/C-15) became the largest one, which defined the absolute configuration

241

of C-13 to be R. Finally, the structure of 3 was confirmed with the aids of HSQC and HMBC

242

correlations and named penazaphilone C.

243

Compound 4 was purified as a red amorphous powder. The HRESIMS peak at m/z 475.1991 [M

244

+ H]+ and 25

245

unsaturation degrees. The UV spectrum [λmax (log ε) 485 (3.60), 372 (4.39), 235 (4.24)]

246

demonstrated it to be an azaphilone derivative. 1H and 13C NMR spectra of compound 4 were nearly

13C

NMR signals suggested a molecular formula of C25H31ClN2O5 for 4 with 11


Page 12 of 31

Page 13 of 31


247

identical to those of isochromophilone IX.32 However 4 had one more N atom than

248

isochromophilone IX, suggesting that carboxyl (C-4′) in isochromophilone IX presented as amide

249

in 4. The absolute stereochemistry at C-7 and C-13 were both biogenetically expected to be S in the

250

same way as for 1 and 2. All the correlations of HSQC and HMBC confirmed the above speculation

251

(Figure 2). The structure of compound 4 was determined and named penazaphilone D.

252

Compound 5 was isolated as red cubic crystals, and its HRESIMS ion peak at m/z 390.1468 [M

253

+ H]+ supported the molecular formula of C21H24ClNO4, indicating 10 degrees of unsaturation. The

254

molecular formula of 5 was identical to that of sclerotioramine.27 The 1H NMR and 13C NMR spectra

255

of 5 were similar to those of sclerotioramine. Detailed analyses of the HSQC and HMBC

256

correlations led to a conclusion that compound 5 shared the same planar structure as sclerotioramine,

257

suggesting they were diastereoisomers. The relative configuration of compound 5 could not be

258

determined using NOESY experiment. In the CD spectrum, the negative cotton effect at 385 nm

259

suggested that the absolute configuration at C-7 was S as in compounds 1‒4. Finally, the structure

260

of 5 was determined on the basis of X-ray single crystallographic analysis (Figure 4). Because of

261

the presence of one chlorine atom, the absolute configurations of C-7 and 13 were both determined

262

to S from the Flack parameter of 0.05 (4).

263

Compound 6 was also purified as a red amorphous powder, and a molecular formula

264

C26H32ClNO6 was assigned for 6 from the HRESIMS peak at m/z 490.19 96 [M + H]+, indicative of

265

14 mass unit larger than that of isochromophilone IX. The 1H and 13C NMR spectra of 6 were very

266

similar to those of isochromophilone IX,32 except for one more OCH3 group (δH 3.68, δC 53.4) in 6.

267

In the HMBC spectrum, the 1H NMR signal for OCH3 group at δH 3.68 showed correlation with the



268

carbonyl signal at δC 172.6, suggesting that compound 6 was the product of methyl esterification of

269

isochromophilone IX. The absolute configurations at C-7 and C-13 were both biogenetically

270

expected to be S for the same reason as in 1 and 2. Detailed analyses of HSQC and HMBC

271

correlations confirmed the structure of compound 6 (Figure 2).

272

Compounds 7‒9 were respectively identified to have the same planar structures as the

273

sclerotioramine dimer,33,34 isochromophilone VI,30, 35 and isochromophilone IX,32 by comparing

274

their mass, 1D and 2D NMR data with those reported. However compounds 7‒9 were all

275

levorotatory and exhibited negative cotton effects at ca. 385 nm in the CD spectra, suggesting that

276

the absolute configurations of C-7 (and C-7′ in 7) were S.35 Furthermore X-ray single

277

crystallographic analysis of compound 8 confirmed the absolute stereochemistry of C-7 and C-13

278

to be S [Flack parameter -0.04 (9)](Figure 4). The absolute configurations of C-13 (and C-13′ in 7)

279

of compounds 7 and 9 were assigned as S for the same reasons as in 1‒6.

280

Inhibition on NO Production in LPS-stimulated RAW 264.7 Cells. Murine macrophage

281

RAW264.7 cells were pretreated with compounds 1–9 at the concentration of 10.0 μM for the

282

measurement of NO production. As shown in Figure 5A, compounds 1, 2, 5, 6, and 8 significantly

283

inhibited the production of NO (Figure 5A). In the cytotoxicity assay, compound 2 was cytotoxic to

284

murine macrophage RAW264.7 cells at the concentration of 50.0 μM, which may partly contribute

285

to the production inhibition of NO. Therefore the half maximal inhibitory concentrations (IC50) of

286

compounds 1, 5, 6, and 8 were determined to be 15.29, 9.34, 9.50, and 7.05 μM, respectively (Figure

287

5C-F).


Page 14 of 31

Page 15 of 31


288

Cell viability assay. In order to determine whether the inhibition of NO production in LPS-

289

stimulated RAW 264.7 cells was resulted from the cytotoxicity of tested compounds, cell viabilities

290

of RAW264.7 cells treated with 1–9 at the concentration of 50.0 μM were measured (Figure 5B).

291

The results showed that compounds 1 and 3–9 did not exhibit any cytotoxicity to RAW264.7 cells,

292

indicating that the inhibition of NO production of 1, 5, 6, and 8 did not result from their cytotoxicity

293

to RAW 264.7 cells.

294

Till now, there are only dozens of azaphilone alkaloids discovered from natural resource.1 Our

295

chemical investigation on the secondary metabolites of P. sclerotiorum cib-411 led to the

296

identification of nine new azaphilone alkaloids, among of which 1 and 2 have an intact leucine

297

residue in their structures. Previous studies showed that azaphilones, especially from Monascus,

298

possess significant anti-inflammatory activity. In this study, all isolated compounds (1–9) were

299

evaluated for their anti-inflammatory activity, among which compounds 1, 5, 6, and 8 significantly

300

inhibited NO production on lipopolysaccharide-stimulated RAW 264.7 cells. Furthermore, in the

301

cytotoxicity assay, these compounds did not show any cytotoxicity at the concentration of 50.0 μM.

302

This study suggested that compounds 1, 5, 6, and 8 may be potential functional food colorants and

303

deserve further systematic investigation in the future.

304

 ASSOCIATED CONTENT

305

Supporting Information

306

The Supporting Information is available free of charge on the ACS Publications website.

307 308

HRESIMS, NMR, CD spectra of compounds 1–9, Single crystal X-ray diffraction Data of compound 8, and Crystallographic data (CIF) of compounds 5 and 8.



309

 AUTHOR INFORMATION

310

Corresponding Author

311

* Telephone/Fax: +86-28-82890829. E-mail: [email protected] (G.-Y. L.).

312

Funding

313

This research was financially supported by The National Key Research and Development Program

314

of China (2016YFD0400500).

315

Notes

316

The authors declare no competing financial interest.

317

§These

318 319 320

authors contributed equally to this work.

 ABBREVIATIONS USED HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond correlation.  REFERENCES

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415



Cl 5

O O

6 18

O

17 4a

B

A 7

8

O

4

8a

1

9 3

10

N

2' 3' 1'

2

O 1R=H 2 R = Me

OR

4'

Cl

16 13

11

12

S

O

15 14

1'

O

2'

R OH

OH

3 Cl S

O

N

O

R

O

S

O

N

O O

O

417

N

O

5'

Cl O

416

R

O

6'

4 5 6 8 9

R = CH2CH2CH2CONH2 R=H R = CH2CH2CH2COOCH3 R = CH2CH2OH R = CH2CH2CH2COOH

O O

N

O

S

O Cl 7

Figure 1. Compounds 1–9 isolated from P. sclerotiorum cib-411.


Page 22 of 31

Page 23 of 31


Cl

Cl O

O O

O

N O

O

N O

HO

O

O

1

Cl

Cl O

O

N

OH

OH

O O

O

Cl

N

COOCH3

Cl O

O

419

CONH2

4

O

418

N O

3

NH O

O

2

O O

O

O O

O 5

O 6

Figure 2. Key HMBC correlations of compounds 1–6.



-0.016

Cl

CH3

O

+0.018

R CH3

O

N

O

-0.017

H OH

OR

O

420 421

3a R = (S)-MTPA 3b R = (R)-MTPA

Figure 3. Selected ΔδS−R values around C-14 of the (S)- and (R)-MTPA esters of 3.


Page 24 of 31

Page 25 of 31


422 423

5

424 425 426

8 Figure 4. X-ray structures of compounds 5 and 8.



427 428

Figure 5. Effect of compounds 1–9 on NO production in RAW264.7 cells. (A) RAW264.7 cells

429

seeded in 24-well plates overnight were pretreated with the compounds respectively at the indicated

430

concentration for 1 h, and then stimulated with LPS (1.0 μg/ml) for 24 h. Levels of NO were

431

determined by Griess assay in culture medium. (B) RAW264.7 cells were seeded onto a 96-well

432

plate and treated with 1–9 at the indicated concentrations for 24 h. Cell proliferation was estimated

433

by Alamar Blue assay and expressed relative to the DMSO control. (C–F) IC50 of compounds 1, 5,

434

6, and 8 on NO production in RAW264.7 cells. **p

Azaphilone Alkaloids with Anti-inflammatory Activity from Fungus

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