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Bioactive Constituents, Metabolites, and Functions

Antiproliferative and Anti-inflammatory Lanostane Triterpenoids from the Polish Edible Mushroom Macrolepiota procera He-Ping Chen, Zhen-zhu Zhao, Zheng-Hui Li, Ying Huang, Shuai-Bing Zhang, Yang Tang, Jian-Neng Yao, lin Chen, Masahiko Isaka, Tao Feng, and Ji-Kai Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00287 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Journal of Agricultural and Food Chemistry

1

Antiproliferative

and

2

Triterpenoids

3

Macrolepiota procera

4

He-Ping Chen,† Zhen-Zhu Zhao,†,‡ Zheng-Hui Li,† Ying Huang,† Shuai-Bing Zhang,†

5

Yang Tang,†,‡ Jian-Neng Yao,†,‡ Lin Chen,† Masahiko Isaka,§ Tao Feng,†,* Ji-Kai

6

Liu†,*

7

†

8

Wuhan 430074, China

9

‡

from

Anti-inflammatory the

Polish

Edible

Lanostane Mushroom

College of Pharmaceutical Sciences, South-Central University for Nationalities,

State Key Laboratory of Phytochemistry and Plant Resources in West China,

10

Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

11

§

12

Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120,

13

Thailand

National Center for Genetic Engineering and Biotechnology (BIOTEC), 113

1

ACS Paragon Plus Environment


Page 2 of 42

14

ABSTRACT

15

This study features the isolation and identification of twelve lanostane-type

16

triterpenoids, namely lepiotaprocerins A–L, 1–12, from the fruiting bodies of the

17

Poland-collected edible mushroom Macrolepiota procera. The structures as well as

18

absolute configurations of the new compounds were ambiguously established by

19

extensive spectroscopic analyses, ECD calculation, and single crystal X-ray

20

diffraction analyses. Structurally, lepiotaprocerins A–F, 1–6, are distinguished by the

21

presence of a rare “1-en-1,11-epoxy” moiety which has not been previously described

22

in the lanostane class. Biologically, lepiotaprocerins A–F, 1–6, displayed more

23

significant inhibitions of nitric oxide (NO) production than the positive control

24

L-N

25

12, showed various cytotoxicity potencies against a panel of human cancer cell lines.

26

Compound 9 also displayed antitubercular activity against Mycobacterium

27

tuberculosis H37Ra with a minimal inhibitory concentration (MIC) 50 µg/mL.

28

Keywords:

29

anti-inflammatory activity; cytotoxicity

G

-monomethyl arginine (L-NMMA) (IC50 47.1 µM), and lepiotaprocerins G–L, 7–

Mushroom;

Macrolepiota

procera;

2


lanostane

triterpenoid;

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30

INTRODUCTION

31

According to the World Health Organization, cancer is the second leading cause of

32

death worldwide, and was responsible for 8.8 million deaths in 2015.1 Many cancers

33

arise from infection, chronic irritation, and inflammation. Recent researches expanded

34

the concept that inflammation is a critical component of tumor progression.2,3

35

Therefore, anti-inflammatory therapy is efficacious towards early neoplastic

36

progression and malignant conversion. A large number of scientific publications have

37

shown that natural products from medicinal/edible mushroom plays a dominant role

38

in the discovery of leads for the development of drugs for prevention and treatment of

39

this disease.4-6 Moreover, a meta-analysis results of observational studies suggested

40

that consumption of more mushrooms may be associated with decreased risk of breast

41

cancer.7

42

The mushroom Macrolepiota procera, also called “parasol mushroom” due to its

43

large fruiting body resembling a parasol, is widespread in temperate regions. In

44

Europe, M. procera is a highly sought-after and popular item due to its large size

45

fruiting bodies, frequent seasonal accessibility and versatility in the kitchen. However,

46

no reports have addressed the secondary metabolites of this kind of famous edible

47

mushroom so far. As our continuous research aiming at discovery drug leads from

48

edible mushroom, A chemical investigation on the constituents of the Poland-origin

49

parasol mushroom M. procera was carried out. Herein, we report the isolation,

50

structure elucidation, biological evaluation of twelve lanostane triterpenoids, namely

3



51

lepiotaprocerins A–L, 1–12, from the fruiting bodies of M. procera (Figure 1).

52

MATERIALS AND METHODS

53

General Experimental Procedures

54

Optical rotations were measured by a JASCO P-1020 digital polarimeter (Horiba,

55

Kyoto, Japan). A UV-2401PC UV-visible recording spectrophotometer (Shimadzu,

56

Kyoto, Japan) was used to recorded the Ultraviolet (UV) spectra. A Chirascan circular

57

dichroism spectrometer (Applied Photophysics Limited, Leatherhead, Surrey, UK)

58

was used to recorded the CD spectra. 1D and 2D NMR spectra were obtained on

59

Bruker Avance III 600 MHz or Ascend 800 MHz spectrometers (Bruker Corporation,

60

Karlsruhe, Germany). An Agilent 6200 Q-TOF MS system (Agilent Technologies,

61

Santa Clara, CA) was used to acquire the HRESIMS data. A Waters AutoSpec Premier

62

P776 MS system (Waters Corporation, Milford, MA) was used to acquire the

63

HREIMS datum. An APEX II DUO spectrophotometer (Bruker AXS GmbH,

64

Karlsruhe, Germany) was applied for performing the single crystal X-ray diffraction

65

experiment. Column chromatography (CC) were run on Sephadex LH-20 (Amersham

66

Biosciences, Uppsala, Sweden) and silica gel (Qingdao Haiyang Chemical Co., Ltd,

67

Qingdao, China). A Büchi Sepacore System (pump manager C-615, pump modules

68

C-605, and fraction collector C-660) (Büchi Labortechnik AG, Flawil, Switzerland)

69

was used to perform medium pressure liquid chromatography (MPLC), which

70

equipped with a column (400 mm × 7.4 mm i.d., 40–75 µm, flow rate 40 mL/min)

4


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filled with Chromatorex C-18 (Fuji Silysia Chemical Ltd., Kasugai, Japan) RP-C18

72

silica gel. An Agilent 1260 liquid chromatography system (Agilent) which equipped

73

with an ODS column (Zorbax SB-C18, 150 mm × 9.4 mm i.d., 5 µm, flow rate 10

74

mL/min) was used for preparative high performance liquid chromatography

75

(prep-HPLC).

76

Fungal Material

77

The mushroom M. procera was collected at a meadow near Wroclaw, Poland in

78

October 2014, and identified by Prof. Yu-Cheng Dai, who is a mushroom specialist of

79

Beijing Forestry University. A specimen (No. 20141005PL) was kept by Herbarium of

80

Ethnic Medicinal Plants of South-Central University for Nationalities (SCUEC). The

81

dried collection was permitted by the local authorities.

82

Extraction and Isolation

83

The dried fruiting bodies of M. procera (2.0 kg) were powdered and then extracted

84

with 10 L of 90% ethanol for three times (24 h each). The extract was evaporated and

85

re-dissolved in water followed by partition between H2O/EtOAc for three times (3 h

86

each). The EtOAc layers were combined and concentrated in vacuum to afford a

87

crude extract (85 g). This residue was separated by MPLC with MeOH/H2O (from

88

20:80→100:0, v/v, totally 4 L) to give eleven main fractions (A–K). Fraction F was

89

separated by Sephadex LH–20 CC (acetone) to give four subfractions F1–F4.

90

Subfraction F1 was subjected to normal phase silica gel CC (petroleum ether/acetone: 5



91

5:1→2:1, v/v, totally 1 L) to afford three subfractions F1a-F1c. Subfraction F1a was

92

purified by prep-HPLC (MeCN/H2O: 55/45→75:25, v/v, 10 mL/min) to obtain

93

compounds 8 (tR = 16.5 min, 1.2 mg), 9 (tR = 18.4 min, 3.5 mg), 10 (tR = 20.1 min, 1.3

94

mg). Subfraction F2 was purified by normal phase silica gel CC (petroleum

95

ether/acetone: 5:1→1:1, v/v, totally 1 L) to give compound 1 (1.8 mg) and F2b.

96

Subfraction F2b was further separated by prep-HPLC (MeCN/H2O: 50/50→75:25, v/v,

97

10 mL/min) to yield compounds 11 (tR = 17.4 min, 1.5 mg) and 12 (tR = 18.4 min, 1.4

98

mg). Subfraction F3 was subjected to normal phase silica gel CC (petroleum

99

ether/acetone: 5:1→1:1, v/v, totally 1.2 L) to give three subfractions F3a-F3c.

100

Subfraction F3a was further separated by Sephadex LH-20 CC (acetone) to give three

101

subfractions F3aa-F3ac. Subfraction F3aa was purified by HPLC (MeCN/H2O:

102

45/55→80:20, v/v, 10 mL/min) to give compound 2 (tR = 14.6 min, 0.8 mg).

103

Subfraction F3ab was purified by HPLC (MeCN/H2O: 45/55→75:25, v/v, 10 mL/min)

104

to give compounds 3 (tR = 18.2 min, 2.2 mg) and 7 (tR = 14.8 min, 2.2 mg).

105

Subfraction F3ac was purified by HPLC (MeCN/H2O: 35/65→65:35, v/v, 10 mL/min)

106

to give compounds 5 (tR = 21.4 min, 0.6 mg), 6 (tR = 22.0 min, 0.6 mg). Subfraction

107

F4 was purified repeatedly by HPLC to yield compound 4 (MeCN/H2O:

108

45/55→75:25, v/v, 10 mL/min, tR = 15.6 min, 1.0 mg).

109

Lepiotaprocerin A, 1: Colorless prisms (MeOH); C32H44O6; (+)-HRESIMS m/z

110

525.3211 [M + H]+, calcd for C32H45O6, 525.3211; [α]24D + 114.2; UV (MeOH) λmax

111

(log ε) 222 (3.01), 261 (3.30) nm; 1H NMR data (Table 1); 13C NMR (Table 3).

6


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112

Lepiotaprocerin B, 2: White powder; C33H46O6; (+)-HRESIMS m/z 539.3375

113

[M + H]+, calcd for C33H47O6, 539.3367; [α]24D + 81.7; UV (MeOH) λmax (log ε) 203

114

(3.87), 261 (3.99) nm; 1H NMR data (Table 1); 13C NMR (Table 3).

115

Lepiotaprocerin C, 3: White powder; C30H38O5; (+)-HRESIMS m/z 479.2794

116


117

(3.03), 214 (2.96), 260 (3.12) nm; CD (MeOH) λmax (∆ε) 208 (− 1.79), 223 (+ 5.67),

118

261 (+ 35.37), 306 (− 10.64) nm; 1H NMR data (Table 1); 13C NMR (Table 3).

119

Lepiotaprocerin D, 4: White powder; C30H40O5; (+)-HRESIMS m/z 481.2950

120


121


122

Lepiotaprocerin E, 5: White powder; C32H42O6; (+)-HRESIMS m/z 523.3054

123

[M + H]+, calcd for C32H43O6, 523.3054; [α]25D + 282.3; UV (MeOH) λmax (log ε) 193.4

124

(4.03), 206 (4.32), 261 (4.34) nm; CD (MeOH) λmax (∆ε) 196 (+ 7.56), 215 (+ 7.26),

125

261 (+40.98), 308 (−10.98) nm; 1H NMR data (Table 1); 13C NMR (Table 3).

126

Lepiotaprocerin F, 6: White powder; C32H42O6; (+)-HRESIMS m/z 523.3055 [M

127

+ H]+, calcd for C32H43O6, 523.3054; [α]25D + 106.3; UV (MeOH) λmax (log ε) 206

128

(4.01), 261 (4.03) nm; CD (MeOH) λmax (∆ε) 208 (− 15.96), 215 (− 12.06), 261 (+

129

50.53), 307 (− 14.11) nm; 1H NMR data (Table 1); 13C NMR (Table 3).

130

Lepiotaprocerin G, 7: Colorless crystals (MeOH); C30H38O5; (+)-HRESIMS m/z

131

479.2792 [M + H]+, calcd for C30H39O5, 479.2792; [α]21D + 179.8; UV (MeOH) λmax

132

(log ε) 204 (3.96), 254 (3.76) nm; 1H NMR data (Table 2); 13C NMR (Table 3).

7



133

Lepiotaprocerin H, 8: White powder; C31H44O5; (+)-HRESIMS m/z 497.3261

134


135


136

Lepiotaprocerin I, 9: White powder; C32H44O6; (−)-HRESIMS m/z 523.3064 [M

137

− H]−, calcd for C32H43O6, 523.3065; [α]25D + 199.7; UV (MeOH) λmax (log ε) 221

138


139

Lepiotaprocerin J, 10: White powder; C33H46O6; (+)-HREIMS m/z 538.3033

140

[M]+, calcd for C33H46O6, 538.3294; [α]25D + 85.7; UV (MeOH) λmax (log ε) 219 (3.85),

141

251 (3.49) nm; 1H NMR data (Table 2); 13C NMR (Table 3).

142

Lepiotaprocerin K, 11: White powder; C32H42O6; (+)-HRESIMS m/z 523.3055

143


144

(4.16), 246 (3.88) nm; CD (MeOH) λmax (∆ε) 201 (− 3.50), 215 (+ 4.28), 258 (+

145


146

Lepiotaprocerin L, 12: White powder; C32H42O6; (+)-HRESIMS m/z 523.3060

147


148

(4.23), 246 (3.97) nm; CD (MeOH) λmax (∆ε) 207 (− 26.91), 215 (− 22.93), 257 (+

149


150

Single Crystal X-ray Diffraction Data for 1: Crystal data for Cu_1_0m:

151

C32H44O6, M = 524.67, a = 5.9650(5) Å, b = 16.1958(11) Å, c = 14.5295(11) Å, α =

152

90°, β = 91.695(5)°, γ = 90°, V = 1403.05(18) Å3, T = 100(2) K, space group P21, Z =

153

2, µ(CuKα) = 0.674 mm−1, 13050 reflections measured, 4533 independent reflections

8


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154

(Rint = 0.0798). The final R1 values were 0.0595 (I > 2σ(I)). The final wR(F2) values

155

were 0.1450 (I > 2σ(I)). The final R1 values were 0.0981 (all data). The final wR(F2)

156

values were 0.1601 (all data). The goodness of fit on F2 was 1.065. Flack parameter =

157

−0.2(2). The crystallographic data were deposited to the Cambridge Crystallographic

158

Data Centre (CCDC) with the No. CCDC 1559796. Copies of the data were available

159

for free from Cambridge Crystallographic Data Centre.8

160

Single Crystal X-ray Diffraction Data for 7: Crystal data for Cu_7_0m:

161

C30H38O5, M = 478.60, orthorhombic, a = 6.2081(3) Å, b = 19.8715(11) Å, c =

162

20.2149(11) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2493.8(2) Å3, T = 100(2) K,

163

space group P212121, Z = 4, µ(CuKα) = 0.680 mm−1, 13924 reflections measured,

164

4386 independent reflections (Rint = 0.0502). The final R1 values were 0.0493 (I >

165

2σ(I)). The final wR(F2) values were 0.1274 (I > 2σ(I)). The final R1 values were

166

0.0547 (all data). The final wR(F2) values were 0.1319 (all data). The goodness of fit

167

on F2 was 1.061. Flack parameter = 0.4(3). The Hooft parameter is 0.27(13) for 1786

168

Bijvoet pairs. The crystallographic data were submitted to the Cambridge

169

Crystallographic Data Centre (CCDC) with the No. CCDC 1559976. These data can

170

be accessed free of charge from Cambridge Crystallographic Data Centre.9

171

Nitric Oxide Production in RAW 264.7 Macrophages

172

The RPMI 1640 medium (Hyclone, Logan, UT) containing 10% FBS was used to

173

culture the murine monocytic RAW 264.7 macrophages. The compounds were

174

dissolved in DMSO and further diluted in medium to produce different concentrations. 9



Page 10 of 42

175

The culture medium and cell mixture were dispensed into 96-well plates (2 × 105

176

cells/well) and maintained at 37 °C under 5% CO2 in a humidified atmosphere. After

177

preincubated for 24 h, serial dilutions of the test compounds were added into the cells,

178

up to the maximum concentration 25 µM, then added with LPS to a concentration 1

179

µg/mL and continued to incubation for 18 h. NO production in each well was assessed

180

After added 100 µL of Griess reagent (reagent A and reagent B, Sigma, St. Louis, Mo)

181

to 100 µL of each supernatant from the LPS-treated or LPS- and compound-treated

182

cells in triplicates and incubated for 5 min, NO production of each cell was assessed.

183

The sample absorbance was measured at 570 nm by a 2104 Envision Multilabel Plate

184

Reader. L-NG-monomethyl arginine (L-NMMA) were used as positive controls.

185

Cytotoxicities Against Five Human Cancer Cell Lines

186

The following five human cancer cell lines were used: the HL-60 (ATCC

187

CCL-240) human myeloid leukemia; SMMC-7721 human hepatocellular carcinoma;

188

A-549 (ATCC CCL-185) lung cancer; MCF-7 (ATCC HTB-22) breast cancer;

189

SW-480 (ATCC CCL-228) human colon cancer. The cell line SMMC-7721 was

190

bought from China Infrastructure of Cell Line Resources (Beijing, China), and others

191

were bought from American Type Culture Collection (ATCC, Manassas, VA). All

192

cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS)

193

(Hyclone) and maintained at 37 oC under 5% CO2 in a humidified atmosphere.

194

Colorimetric measurements of the amount of insoluble formazan which produced in

195

living

cells

based

on

the

10


reduction

of

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196

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St.

197

Louis, MO) was used to assess cell viability. In brief, each well of a 96-well cell

198

culture plate was seeded with 100 µL of adherent cells and kept for 12 h for adherence,

199

and then added with test compounds, however, suspended cells were seeded before

200

added with test compounds with both the same density of 1 ×105 cells/mL every 100

201

µL of culture medium. After different concentrations of test compounds addition, each

202

cancer cell line was incubated for 48 h in triplicates. Cisplatin was used as positive

203

control. After the incubation, each well was added with MTT (100 µg) and continued

204

to incubate for 4 h at 37 oC. After removed the 100-µL culture medium, the cells were

205

lysed with 20% SDS-50% DMF (100 µL). The remained lysates were subjected to

206

measure the optical density at 595 nm with a 96-well microtiter plate reader. The IC50

207

value for each compound was calculated by a published method.10

208

Antimycobacterial Assay

209

Antimycobacterial activity against Mycobacterium tuberculosis H37Ra was

210

determined using the green fluorescent protein microplate assay.11

211

RESULTS AND DISCUSSION

212

Structural Elucidations of Lepiotaprocerins A–L, 1–12

213

Lepiotaprocerin A, 1, was isolated as colorless needles. It was determined to have

214

the molecular formula C32H44O6 based on the protonated molecule ([M+H]+) on

215

HRESIMS analysis, corresponding to eleven degrees of unsaturation. The 1D NMR

11



216

data (Tables 1 and 2) displayed resonances assignable to eight methyls (one doublet),

217

six methylenes, seven methines, and eleven quaternary carbons. All these data are

218

reminiscent of those for (24Z)-3,11-dioxo-lanosta-8,24-dien-26-oic acid, a lanostane

219

triterpenoid from the mushroom Jahnoporus hirtus,12 indicating the same lanostane

220

skeleton of compound 1. A thorough analysis using a combination of 1H-1H COSY

221

and HMBC spectra allowed the completion of the planar structure of 1. The 1H-1H

222

COSY correlations between H-15 (δH 2.22, 1.49)/H-16 (δH 5.36), and H-16/H-17 (δH

223

1.96), and the HMBC correlations from H-16 and a methyl (δH 2.04) to a carbonyl at

224

δC 170.8 revealed the presence of an acetoxy group attached to C-16. In addition, the

225

HMBC correlations from H-11 (δH 5.14) to C-8 (δC 137.6)/C-9 (δC 132.6), from

226

Me-19 (δH 1.25) to a low field quaternary carbon at δC 187.6 (C-1), from H-2 (δH 5.19)

227

to C-4 (δC 44.1) and C-10 (δC 41.7), and C-1 were observed (Figure 2). All these data

228

indicated that the existence of a β-oxygenated-α,β-unsaturated functionality located at

229

C-1 and C-2, leading to the assignment of ten degrees of unsaturation. Although the

230

HMBC correlation from H-11 to C-1 was absent, the remaining one degree of

231

unsaturation was ascribed to a ring constructed by an epoxy group between C-1 and

232

C-11, which was confirmed by the downfield shifted of C-1, C-11 (δC 81.7), and the

233

HRESIMS result. Thus, the planar structure of compound 1 was established as shown

234

in Figure 1. Structurally speaking, the highly constrained 1-en-1,11-epoxy group is

235

unprecedented in the lanostane class. The relative configuration of 1 was determined

236

by a ROESY experiment. In the ROESY spectrum, the cross peaks between

12


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237

Me-18/H-11/Me-19, Me-30/H-16/H-17, and H-24/Me-27 suggested that an α

238

orientation of 1,11-epoxy group, β orientation of the 16-acetoxy group, and Z

239

configuration of C-24–C-25 double bond (Figure 2). A single crystal X-ray diffraction

240

analysis of 1 further confirmed the above assignments, and provided solid evidence

241

for the existence of the highly rigid 1,11-epoxy group (Figure 3). Therefore, the

242

structure

243

(24Z)-16β-acetoxy-1,11α-epoxy-3-oxo-lanosta-1,8,24-trien-26-oic acid.

of

lepiotaprocerin

A,

1,

was

determined

as

244

Lepiotaprocerin B, 2, was acquired as white powder. The molecular formula

245

C33H46O6 was established based on the protonated molecule ([M+H]+) on HRESIMS

246

analysis. Both the 1H and

247

of 1 (Tables 1 and 2). The only difference was the presence of a methoxy group,

248

which was substantiated by the 1H and 13C resonances at δH 3.73, δC 51.3, respectively.

249

Elucidating the HMBC spectrum of 2 revealed that the 26-carboxylic group in 1 was

250

transformed into the corresponding methyl ester in 2, which was supported by the

251

HMBC correlation from δH 3.73 to C-26 (δH 168.3). Therefore, lepiotaprocerin B was

252

established

253

(24Z)-16β-acetoxy-1,11α-epoxy-3-oxo-lanosta-1,8,24-trien-26-oate, 2. It may be an

254

artefact produced during the isolation processes.

13

C NMR spectra of 2 showed high similarities with those

as

methyl

255

Lepiotaprocerin C, 3, was purified as white powder. Its molecular formula was

256

established as C30H38O5 by HRESIMS on the basis of sodium adduct ion peak,

257

indicating twelve degrees of hydrogen deficiency. The 13C and DEPT data (Tables 1

13



Page 14 of 42

258

and 2) exhibited resonances assignable to seven methyls, five methylenes, seven

259

methines, and eleven quaternary carbons. The overall 1D NMR spectra displayed

260

significant similarities to those of 1, except for the absence of an acetyl group and a

261

methylene, while with the presence of dioxygen-bearing non-protonated carbon at δC

262

106.9. In the HMBC spectrum, H-16, H-22, and H-24 were correlated to the

263

aforementioned carbon (δC 106.9) (Figure 2), suggesting that the methylene C-23 in 1

264

was oxygenated into a ketal carbon in 3 and forms an epoxy bond between C-16 and

265

C-23. Additionally, all above assignments accounted for eleven degrees of

266

unsaturation, the residuary one degree of unsaturation was assigned as a γ-lactone

267

group connected by C-23 and C-26 carbonyl, which showed typical 1H and

268

resonance patterns same as those of spirochensilide A [3: δC 145.8 (C-24), 131.1

269

(C-25), 172.1 (C-26), 10.4 (C-27); spirochensilide A: δC 147.2 (C-24), 132.0 (C-25),

270

171.9 (C-26), 10.5 (C-27)].13 Thus, the planar structure of 3 was established as

271

depicted in Figure 1.

13

C

272

The absolute configurations of the chiral centers in the lanostane nucleus of 3

273

were established to be same with those of compound 1 by ROESY spectrum. The

274

relative configuration of C-23 was assigned as R* based upon the key ROESY

275

correlation between H-20 (δH 1.81) and H-24 (δH 6.86). The absolute configuration of

276

compound 3 was determined by comparison of experimental and theoretical electronic

277

circular dichroism (ECD) spectra.14-17 A conformation search by MMFF94s force

278

field of 3 only gave two conformers. Both the conformers were optimized at

14


Page 15 of 42


279

B3LYP/6-31G** level of theory. The theoretical calculations of ECD were performed

280

using time-dependent density functional theory (TDDFT) at B3LYP/6-31G** level in

281

MeOH with the IEFPCM model. As shown in Figure 4, the calculated ECD for 23R

282

matched with the experimental curve and thus determine the absolute configurations.

283

Based on the foregoing evidence, lepiotaprocerin C was established as

284

(23R,24Z)-1,11α-16β,23-diepoxy-3-oxo-lanosta-1,8,24-trien-26,23-olide, 3.

285

The white powder, lepiotaprocerin D, 4, was determined to have the molecular

286

formula C30H40O5 based upon the protonated molecule ([M+H]+) on HRESIMS

287

analysis, indicating eleven degrees of hydrogen deficiency. Comparison of its 1D

288

NMR data with those of 3 indicated that 4 is an analogue of 3 (Tables 1 and 2). In the

289

13

290

the presence of an oxygenated methine at δC 71.3 suggested that the difference

291

between them was that the α,β-unsaturated-γ-lactone group in 3 was opened to give 4

292

which presented an α,β-unsaturated carboxylic acid. These changes were further

293

confirmed by key HMBC correlation from H-16 (δH 4.10) to C-23 (δC 71.3), and one

294

degree of unsaturation less than compound 3. The configuration of C-23 was

295

determined as S by the ROESY correlation between H-16 and H-23 (δH 4.86) (Figure

296

2).

297

(23S,24Z)-1,11α-16β,23-diepoxy-3-oxo-lanosta-1,8,24-trien-26-oic acid, 4.

C NMR and DEPT spectra of 4, the absence of the ketal carbon (δC 106.9 in 3) and

Thus,

lepiotaprocerin

D

was

identified

as

298

Lepiotaprocerins E, 5, and F, 6, were two compounds with close retention time on

299

preparative HPLC, nearly the same NMR spectroscopic patterns, and the same

15



Page 16 of 42

300

molecular formula C32H42O6 established by HRESIMS, indicating that they were

301

isomeric with each other. Both the 1D NMR spectra exhibited resemblance with those

302

of compound 1 (Tables 1 and 2). The HMBC correlations from H-16 (δH 5.33) and a

303

methyl singlet at δH 2.05 to a carbonyl at δC 170.4, and from H-23 (δH 4.95) to C-26

304

(δC 174.1) observed for 5 suggested the absence of the 16,23-epoxy bond in the

305

structure of 5, but an acetoxy group attached to C-16 (Figure 2). The ROESY

306

correlation between H-16 and H-17 (δH 2.08) enabled the determination of 16-acetoxy

307

group as β-oriented. The remarkable discrepancies between the 1H and

308

chemical shifts of the position 23 revealed that compounds 5 and 6 differed from each

309

other by virtue of the absolute configurations of C-23. In many cases, S or R

310

configuration of C-23 were responsible for the respective positive or negative signs of

311

Cotton effects at approximately 215 nm, involving π→π* transitions due to the

312

5-substituted 2(5H)-furanone moiety.18-20 In accordance with these empirical rules,

313

the C-23 configurations of compounds 5 and 6 were determined as S and R,

314

respectively (Figure 5). The structures of compounds 5 and 6 were designated as

315

(23S,24Z)-

316

(23R,24Z)-16β-acetoxy-1,11α-epoxy-3-oxo-lanosta-1,8,24-trien-26,23-olide,

317

respectively.

13

C NMR

or

318

The colorless crystals compound 7 gave a protonated molecular ([M+H]+) on

319

HRESIMS analysis, indicating a molecular formula of C30H38O5 (calcd for C30H39O5,

320

479.2792) with twelve degrees of unsaturation. The NMR data of compound 7 were

16


Page 17 of 42


321

highly similar to those of compound 3 (Tables 2 and 3), where the discrepancies

322

involved the absence of the sp2 quaternary carbon at δC 187.3 (C-1) and the

323

oxygenated carbon at δC 81.2 (C-11), and the presence of an sp2 methine at δC 158.6

324

(C-1) and a carbonyl at δC 197.7 (C-11). These changes were proved to be the

325

removal of 1,11-ether bond and assignment of a carbonyl at C-11 of 3 to give the

326

resultant 7, which could be corroborated by the 1H-1H COSY correlation between H-1

327

(δH 8.04, d, J = 10.5 Hz) and H-2 (δH 5.91, d, J = 10.5 Hz), and HMBC correlations

328

from H-12 (δH 2.76, d, J = 16.0 Hz; δH 2.49, d, J = 16.0 Hz) to C-11. Other

329

assignments were same to those of compound 3. The relative configuration of 7 were

330

established by X-ray single crystal diffraction due to the poor Flack parameter 0.4(3),

331

which led to the assignment of C-23 as R* configuration which was the same as that

332

of 3 (Figure 6). Although no natural lanostane enantiomers existed to the best of our

333

knowledge, a definite conclusion of the absolute configuration of C-23 was R could

334

not be drawn. Finally, the absolute configuration of 7 was unequivocally determined

335

as 23R by ab initio calculation of its circular dichroism spectrum. As shown in Figure

336

7, the calculated ECD for 23R configuration showed similarity to those of the

337

experimental CD spectra.

338

Compounds 8, 9, and 10 were three lanostane congeners with similar NMR

339

spectroscopic characteristics. Their molecular formulas were established by

340

HRESIMS/HREIMS analyses as C31H44O5, C32H44O6, and C33H46O6, respectively.

341

Their spectroscopic data shared similarities with those of 7 (Tables 2 and 3), revealing

17



342

the presence of three α,β-unsaturated carbonyl groups and oxygenated pattern for

343

C-16 in the structures of 8–10. However, the absence of the spiroketal carbon at δC

344

106.8 (C-23) and the presence of methylenes (C-23, δC 28.0 for 8, 27.0 for 9, and 26.8

345

for 10) suggested that the C-17 side chain remained uncyclized. The 1H-1H COSY

346

correlation between the hydroxy proton 16-OH (δH 2.95, d, J = 4.0 Hz) and H-16 (δH

347

4.56) as well as the HMBC correlations from methoxy at δH 3.73 to the carbonyl at δC

348

168.4 (C-24), and from H3-27 to C-24, C-25, C-26 suggested that compound 8

349

possessed a hydroxy group at C-16 and an α,β-unsaturated carboxylic group with

350

methyl esterification. As for compounds 9 and 10, C-16 were substituted by an

351

acetoxy group deduced by the HMBC correlation from H-16 (δH 5.34, ddd, J = 8.0,

352

8.0, 5.0 Hz) and a methyl at δH 2.05/2.07 to the carbonyl at δC 170.7. Furthermore, the

353

HMBC correlation from a methoxy at δH 3.73 to the carbonyl (δC 168.2, C-26)

354

demonstrated that the C-26 carboxylic group of 10 was methyl esterified compared to

355

that of 9. The ROESY spectra of 8–10 which demonstrated cross peaks between

356

H-16/H-17 implied that the hydroxy/acetoxy of C-16 were β-oriented. Therefore,

357

compounds 8–10 were established as shown in Figure 1, and trivially named as

358

lepiotaprocerins H–J, respectively.

359

Lepiotaprocerins K, 11, and L, 12, were isolated as white powders. HRESIMS

360

results showed that these two compounds had the identical molecular formula of

361

C32H42O6. Comparison of their 1D NMR spectroscopic data which bore high

362

resemblance to those of compounds 5 and 6 revealed that these two compounds had

18


Page 18 of 42

Page 19 of 42


363

the same planar structure while differentiated at the configuration of C-23 (Tables 2

364

and 3). The HMBC correlations from H-12 (δH 2.75, 2.62 for 11, δH 2.74, 2.65 for 12)

365

to C-11 (δC 197.6/197.7), C-8, and C-9 as well as the 1H-1H COSY correlations

366

between H-1 (δH 8.07/8.09) and H-2 (δH 5.90/5.91) enabled the assignments of two

367

α,β-unsaturated carbonyl groups at rings A and C. The configuration of C-23 was

368

determined via the rules used in the structure elucidation of compounds 5 and 6. As

369

shown in Figure 8, the respective positive (∆ε +4.28) and negative (∆ε −22.93) Cotton

370

effects around 215 nm for compounds 11 and 12 indicated the S and R configuration

371

of C-23, respectively. Thus, lepiotaprocerins K, 11, and L, 12, were determined as

372

(23S)-16β-acetoxy-3,11-dioxo-lanosta-1,8,24-trien-26,23-olide

373

(23S)-16β-acetoxy-3,11-dioxo-lanosta-1,8,24-trien-26,23-olide, respectively.

374

Biological Activities of Lepiotaprocerins A–L, 1–12

375

Anti-inflammatory Activity

376

All the compounds were evaluated for their inhibitory activity against NO production.

377

However, compounds 7–12 were toxic to the subject murine monocytic RAW 264.7

378

macrophages at the concentration of 25 µM. As shown in Table 4, compounds 1–6

379

showed notable inhibitory activity on NO production in RAW 264.7 macrophages in

380

vitro, which were more significant than the positive control L-NMMA. The most

381

pronounced was compound 4, possessing an IC50 value of 17.9 µM. Comparison of

382

compounds 3 and 4 suggested that the structures with 26,23-lactone functionality

19


and


383

were less potent than those with the free carboxylic acid group. Comparison of 5 and

384

6 revealed that the 23R or 23S configuration of γ-lactone group showed no

385

differentiations for the inhibitory activities of NO production.

386

Antiproliferative Activity Against Five Human Cancer Cell Lines

387

All the compounds were screened for their cytotoxicity against five human cancer cell

388

lines (HL-60, A-549, SMMC-7721, MCF-7, SW480). Compounds 1–6 were inactive

389

in the cytotoxicity assay (IC50 > 40 µM). Compounds 7–12 displayed inhibitory

390

activity against the five human cancer cell lines. Among them, compounds 7, 8, and

391

12 exhibited significant cytotoxicity while compounds 9–11 display moderated

392

cytotoxicity (Table 5).

393

Antitubercular Activity

394

The scarcity of the compounds at our disposal precluded the screen for other

395

promising activity except for 9. Compound 9 was evaluated for its antitubercular

396

activity against the strain Mycobacterium tuberculosis H37Ra. The results indicated

397

that this compound showed weak antitubercular activity with an MIC value of 50

398

µg/mL. MIC values of the positive controls were isoniazid 0.0469 µg/mL, ethambutol

399

0.938 µg/mL.

400

In summary, chemical investigation on the Polish edible mushroom Macrolepiota

401

procera acquired six minor lanostane triterpenoids harboring a rare and rigid

402

1-en-1,11-epoxy moiety. The absolute configurations of all isolates were 20


Page 20 of 42

Page 21 of 42


403

unambiguously determined via single crystal X-ray diffraction analysis, Cotton effects,

404

and ECD calculation. Among the structures, lepiotaprocerin C, 3, is a lanostane

405

triterpenoid with inflexible polycyclic structure. All these lanostanoids displayed

406

remarkable

407

antiproliferative activity against five human cancer cell lines in vitro. This work

408

represents the first report of triterpenoids or even secondary metabolites from the

409

well-known edible mushroom M. procera, and also discloses its medicinal value from

410

the chemical aspects. From these point of view, efforts to promote the artificial

411

cultivation of this kind of mushroom may put into practice which could bring

412

economic values to the food industry.

413

ABBREVIATIONS USED

anti-inflammatory

activities

in

RAW

264.7

macrophages

or

414

HRESIMS, high resolution electrospray ionization mass spectroscopy; MPLC,

415

medium pressure liquid chromatography; DAD, diode array detector; HSQC,

416

heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond

417

correlation; 1H-1H COSY, 1H-1H correlation spectroscopy; ROESY, rotating frame

418

nuclear overhauser effect spectroscopy; ORTEP, Oak Ridge Thermal Ellipsoid Plot

419

Program; RMPI, Roswell Park Memorial Institute medium; LPS, lipopolysaccharide;

420

MIC, minimal inhibitory concentration; SCUEC, South-Central University for

421

Nationalities.

21



422

ACKNOWLEDGMENT

423

The authors thank Analytical & Measuring Center, School of Pharmaceutical

424

Sciences, South-Central University for Nationalities for MS and NMR spectra tests.

425

The computational work was supported by HPC Center, Kunming Institute of Botany,

426

CAS, China.

427

ASSOCIATED CONTENT

428

Supporting Information

429

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

430

website at DOI: .

431

Spectroscopic data including 1D & 2D NMR, HRMS, CD, X-ray crystallographic

432

data, and calculation details of compounds 1–12 (PDF).

433

Funding

434

This work was financially supported by National Natural Science Foundation of

435

China (31560010, 81561148013), the Key Projects of Technological Innovation of

436

Hubei Province (No. 2016ACA138), and the Fundamental Research Funds for the

437

Central Universities, South-Central University for Nationalities (CZW17094,

438

CZY16010, CSP17061, CZQ17008).

22


Page 22 of 42

Page 23 of 42


439

AUTHOR INFORMATION

440

Corresponding Authors

441

*E-mail: [email protected] (T. Feng)

442

*E-mail: [email protected] (J.-K. Liu)

443

23



444

REFERENCES

445

(1) World

Health

Organization,

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Cancer,

2017;

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http://www.who.int/mediacentre/factsheets/fs297/en/; Accessed January 15, 2018.

447

(2) Mantovani, A.; Allavena P.; Sica A.; Balkwill F. Cancer-related inflammation.

448

Nature 2008, 454, 436–444.

449

(3) Coussens, L. M.; Werb, Z., Inflammation and cancer, Nature 2002, 420, 860–867.

450

(4) Friedman, M. Chemistry, nutrition, and health-promoting properties of Hericium

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erinaceus (lion’s mane) mushroom fruiting bodies and mycelia and their

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bioactive compounds. J. Agric. Food Chem. 2015, 63, 7108–7123.

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(5) Guillamón, E.; García-Lafuente, A.; Lozano, M.; DArrigo, M.; Rostagno, M. A.;

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Villares, A.; Martínez, J. A. Edible mushrooms: role in the prevention of

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cardiovascular diseases. Fitoterapia 2010, 81, 715–723.

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(6) Chen, H. P.; Liu, J. K. Secondary metabolites from higher fungi. Prog. Chem. Org. Nat. Prod.. 2017, 106, 1–201.

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(7) Li, J.; Zou, L.; Chen, W.; Zhu, B.; Shen, N.; Ke, J.; Lou, J.; Song, R.; Zhong, R.;

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Miao, X. Dietary mushroom intake may reduce the risk of breast cancer: evidence

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from a meta-analysis of observational studies. PLoS One 2014, 9, e93437.

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1559796.

462

https://www.ccdc.cam.ac.uk/structures/Search?Ccdcid=1559796. (Accessed: 28

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(9) CCDC

Number:

1559976.

465

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February 2018).

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(10) Reed, L.J.; Muench H. A simple method of estimating fifty percent endpoint. Am. J. Hyg. 1938, 27, 493–497.

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(11) Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Improved green fluorescent

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protein reporter gene-based microplate screening for antituberculosis compounds

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by utilizing an acetamidase promoter. Antimicrob. Agents Chemother. 2003, 47,

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3682−3687.

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(12) Liu, X. T.; Winkler, A. L.; Schwan, W. R.; Volk, T. J.; Rott, M. A.; Monte, A.

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Antibacterial compounds from mushrooms I: a lanostane-type triterpene and

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prenylphenol derivatives from Jahnoporus hirtus and Albatrellus flettii and their

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activities against Bacillus cereus and Enterococcus faecalis. Planta Med. 2010,

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76, 182–185.

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(13) Zhao, Q.Q.; Song, Q. Y.; Jiang, K.; Li, D. G.; Wei, W. J.; Li Y.; Gao, K.

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Spirochensilides A and B, two new rearranged triterpenoids from Abies

480

chensiensis. Org. Lett. 2015, 17, 2760–2763.

481 482

(14) Gotō, H.; Ösawa, E. Corner flapping: a simple and fast algorithm for exhaustive generation of ring conformations. J. Am. Chem. Soc. 1989, 111, 8950–8951.

483

(15) Gotō, H.; Ösawa, E. An efficient algorithm for searching low-energy conformers

484

of cyclic and acyclic molecules. J. Chem. Soc. Perkin Trans. 2 1993, 187–198.

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(16) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;

486

Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.;

487

Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.;

488

Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;

489

Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven,

490

T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.;

491

Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.;

492

Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.;

493

Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo,

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C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;

495

Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.;

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Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.;

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Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.

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Gaussian 09, Revision D.01; Gaussian, Inc.: Wallingford, CT, 2013.

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(17) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis: Quantifying

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the comparison of calculated and experimental electronic circular dichroism

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spectra. Chirality 2013, 25, 243–249.

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(18) Hu, Z. X.; Hu, K.; Shi, Y. M.; Wang, W. G.; Du, X.; Li, Y.; Zhang, Y. H.; Pu, J. X.;

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Sun, H. D. Rearranged 6/6/5/6-fused triterpenoid acids from the stems of

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Kadsura coccinea. J. Nat. Prod. 2016, 79, 2590–2598.

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(19) Tanaka, R.; Wada, S. I.; Aoki, H.; Matsunaga, S.; Yamori, T. Spiromarienonols A

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and B: Two new 7(8→9)abeo-lanostane-type triterpene lactones from the stem

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bark of Abies mariesii. Helv. Chim. Acta 2004, 87, 240–249.

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(20) Wang, G. W.; Lv, C.; Yuan, X.; Ye, J.; Jin, H. Z.; Shan, L.; Xu, X. K.; Shen, Y. H.;

509

Zhang, W. D. Lanostane-type triterpenoids from Abies faxoniana and their DNA

510

topoisomerase inhibitory activities. Phytochemistry 2015, 116, 221–229.

511 512

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Page 28 of 42

Table 1. 1H NMR Data of Compounds 1–6 (CDCl3). No. 2 5 6 7

11 12 15 16

17 18 19 20 21 22 23 24 27 28 29 30 CH3COCH3Oa

1a

2b

3a

4b

5b

6b

5.19, s 1.36, dd (13.2, 2.0) 1.65, m 1.78, m 2.18, m 2.18, m 5.14, br. dd (9.6, 8.0) 2.50, dd (12.5, 8.0) 1.83, dd (12.5, 9.6) 2.22, dd (13.0, 8.0) 1.49, dd (13.0, 5.5) 5.36, ddd (8.0, 8.0, 5.5)

5.18, s 1.36, dd (13.2, 2.0) 1.79, m 1.65, m 2.18, m 2.19, m 5.14, br. dd (9.6, 8.5) 2.51, dd (12.5, 8.0) 1.83, dd (12.5, 9.6) 2.22, dd (13.0, 9.0) 1.50, dd (13.0, 5.5) 5.35, ddd (9.0, 8.0, 5.5)

5.19, s 1.38, dd (13.0, 2.0) 1.68, m 1.78, m 2.21, m 2.23, m 5.15, dd (8.0, 8.0) 2.28, dd (13.0, 8.0) 1.85, dd (13.0, 8.0) 1.96, overlapped 1.65, overlapped 4.10, dd (13.0, 8.0)

1.96, dd (10.7, 8.0) 1.14, s, 3H 1.25, s, 3H 1.92, m 1.06, d (6.5), 3H 1.38, m 1.06, m 2.44, m; 2.55, m 5.87, t (8.0) 1.88, s, 3H 1.16, s, 3H 1.10, s, 3H 1.02, s, 3H 2.05, s, 3H 3.73, s, 3H

5.19, s 1.36, br. d (13.0, 2.0) 1.79, m 1.65, m 2.18, m 2.19, m 5.14, dd (9.5, 8.0) 1.86, dd (12.0, 9.5) 2.50, dd (12.0, 8.0) 1.48, dd (13.5, 6.5) 2.25, dd (13.5, 8.0) 5.33, ddd (8.0, 8.0, 6.5) 2.08, dd (11.0, 8.0) 1.15, s, 3H 1.25, s, 3H 2.12, m 1.17, d (6.5), 3H 1.61, m 1.50, m 4.95, br. t (6.5) 7.06, br. s 1.92, s, 3H 1.16, s, 3H 1.10, s, 3H 1.04, s, 3H 2.05, s, 3H

5.19, s 1.36, br. d (13.0, 2.0) 1.79, m 1.66, m 2.17, m 2.19, m 5.16, dd (9.5, 8.0) 1.86, dd (12.0, 9.5) 2.53, dd (12.0, 8.0) 1.52, dd (13.5, 6.5) 2.20, dd (13.5, 8.0) 5.32, ddd (8.0, 8.0, 6.5)

1.96, dd (10.7, 8.0) 1.14, s, 3H 1.25, s, 3H 1.92, m 1.06, d (6.4), 3H 1.40, m 1.08, m 2.49, m; 2.60, m 6.02, t (7.0) 1.91, s, 3H 1.15, s, 3H 1.10, s, 3H 1.02, s, 3H 2.04, s, 3H

5.19, s 1.38, dd (13.0, 2.3) 1.79, m 1.67, m 2.22, m 2.22, m 5.14, br. t (7.5) 2.29, dd (12.0, 7.8) 1.90, overlapped 1.99, dd (12.5, 8.0) 1.66, dd (12.5, 7.5) 4.54, ddd (8.0, 7.5, 6.5) 1.82, m 1.10, s, 3H 1.26, s, 3H 1.81, overlapped 1.12, d (6.0), 3H 1.87, overlapped 1.87, overlapped 6.86, s 1.91, s, 3H 1.16, s, 3H 1.10, s, 3H 1.02, s, 3H

1.62, dd (16.0, 8.0) 1.15, s, 3H 1.26, s, 3H 1.90, m 1.06, d (6.5), 3H 1.55, m 1.68, m 4.86, m 6.16, m 1.95, s, 3H 1.16, s, 3H 1.11, s, 3H 1.00, s, 3H

Recorded at 600 MHz; b Recorded at 800 MHz.

28


1.92, dd (11.0, 8.0) 1.18, s, 3H 1.25, s 3H 2.39, m 1.16, d (6.5), 3H 1.34, m 1.31, m 4.99, br. t (6.5) 6.96, br. s 1.90, s, 3H 1.16, s, 3H 1.10, s, 3H 1.02, s, 3H 2.09, s, 3H

Page 29 of 42


Table 2. 1H NMR Data of Compounds 7–12 (CDCl3). No. 1 2 5 6 7 12 15 16

17 18 19 20 21 22 23 24 27 28 29 30 CH3COCH3O15-OH a

7a 8.04, d (10.5) 5.91, d (10.5) 1.80, overlapped 1.80, overlapped 1.65, m 2.40, m 2.40, m 2.76, d (16.0) 2.49, d (16.0) 2.07, dd (13.0, 8.0) 1.86, overlapped 4.55, ddd (8.0, 8.0, 6.5) 1.88, dd (10.5, 5.5) 1.00, s, 3H 1.35, s, 3H 1.76, m 1.05, d (6.5), 3H 1.88, m 1.88, m

6.86, br. s 1.92, s, 3H 1.15, s, 3H 1.16, s, 3H 1.18, s, 3H

8b 8.11, d (10.5) 5.89, d (10.5) 1.79, overlapped 1.67, m 1.78, m 2.38, dd (11.0, 6.0) 2.48, overlapped 2.57, d (17.0) 2.67, d (17.0) 2.09, dd (13.0, 8.0) 1.87, dd (13.0, 5.5) 4.56, m

9a 8.08, d (10.3) 5.89, d (10.3) 1.78, overlapped 1.62, m 1.78, overlapped 2.40, dd (20.0, 6.0) 2.35, dd (10.7, 7.0) 2.72, d (17.0) 2.63, d (17.0) 2.28, dd (13.0, 8.0) 1.72, dd (13.0, 5.0) 5.34, ddd, (8.0, 8.0, 5.0)

1.76, overlapped 1.04, s, 3H 1.34, s, 3H 1.90, overlapped 0.98, d (6.5), 3H 1.17, overlapped 1.58, overlapped 2.31, m; 2.48, m 6.11, t (7.5) 1.89, s, 3H 1.15, s, 3H 1.16, s, 3H 1.11, s, 3H 3.73, s, 3H

2.01, dd (10.6, 8.0) 1.02, s, 3H 1.32, s, 3H 1.88, m 0.96, d (6.6), 3H 1.41, m 1.07, m 2.58, m; 2.50, m 6.02, t (7.4) 1.91, s, 3H 1.14, s, 3H 1.15, s, 3H 1.15, s, 3H 2.05, s, 3H

10b 8.09, d (10.5) 5.90, d (10.5) 1.79, overlapped 1.63, overlapped 1.79, overlapped 2.38, overlapped 2.34, overlapped 2.63, d (17.0) 2.72, d (17.0) 2.28, dd (13.0, 8.0) 1.72, dd (13.0, 5.0) 5.34, ddd (8.0, 8.0, 5.0) 2.01, dd (11.0, 8.0) 1.03, s, 3H 1.34, s, 3H 1.88, overlapped 0.97, d (6.5), 3H 1.39, m 1.06, m 2.53, m; 2.43, m 5.87, t (7.5) 1.89, s, 3H 1.15, s, 3H 1.16, s, 3H 1.16, s, 3H 2.07, s, 3H 3.73, s, 3H

2.95, d (4.0) b

Recorded at 600 MHz; Recorded at 800 MHz.

29


11b 8.07, d (10.5) 5.91, d (10.5) 1.80, overlapped 1.80, overlapped 1.62, overlapped 2.37, dd (11.0, 6.8) 2.40, ddd (20.0, 7.0, 1.6) 2.75, d (17.0) 2.62, d (17.0) 2.31, dd (13.0, 7.8) 1.72, dd (13.0, 5.0) 5.31, ddd (7.8, 7.8, 5.0)

2.15, dd (11.0, 7.8) 1.04, s, 3H 1.34, s, 3H 2.08, m 1.08, d (6.5), 3H 1.62, overlapped 1.50, m 4.95, br. d (9.5) 7.06, s 1.92, s, 3H 1.15, s, 3H 1.16, s, 3H 1.18, s, 3H 2.06, s, 3H

12b 8.09, d (10.5) 5.90, d (10.5) 1.80, overlapped 1.81, overlapped 1.65, overlapped 2.41, dd (20.0, 5.0) 2.36, overlapped 2.74, d (17.0) 2.65, d (17.0) 2.25, dd (13.0, 8.0) 1.75, dd (13.0, 5.0) 5.32, ddd (8.0, 8.0, 5.0) 1.97, dd (11.0, 8.0) 1.08, s, 3H 1.34, s, 3H 2.35, overlapped 1.07, d (6.5), 3H 1.35, overlapped 1.32, overlapped 4.98, br. d (9.5) 6.96, s 1.91, s, 3H 1.15, s, 3H 1.16, s, 3H 1.16, s, 3H 2.11, s, 3H


Page 30 of 42

Table 3. 13C NMR Data of Compounds 1–6 (CDCl3). No. 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 CH3COCH3CO-OMe a

1a 187.6, C 97.1, CH 206.8, C 44.1, C 48.8, CH 18.3, CH2 25.8, CH2 137.6, C 132.6, C 41.7, C 81.7, CH 33.6, CH2 48.4, C 49.2, C 39.3, CH2 74.9, CH 52.6, CH 16.7, CH3 19.4, CH3 30.5, CH 18.7, CH3 35.8, CH2 27.3, CH2 146.1, CH 126.2, C 171.8, C 20.8, CH3 21.0, CH3 29.3, CH3 26.4, CH3 170.8, C 21.4, CH3

2b 187.5, C 97.0, CH 206.7, C 44.1, C 48.8, CH 18.3, CH2 25.8, CH2 137.6, C 132.5, C 41.6, C 81.6, CH 33.5, CH2 48.3, C 49.2, C 39.3, CH2 74.8, CH 52.6, CH 16.6, CH3 19.4, CH3 30.5, CH 18.6, CH3 35.8, CH2 27.0, CH2 143.4, CH 126.9, C 168.3, C 20.8, CH3 21.0, CH3 29.3, CH3 26.4, CH3 170.7, C 21.4, CH3 51.3, CH3

3a 187.3, C 97.0, CH 206.5, C 44.0, C 48.7, CH 18.1, CH2 25.8, CH2 137.9, C 132.4, C 41.5, C 81.2, CH 34.3, CH2 47.3, C 48.6, C 37.3, CH2 75.0, CH 52.7, CH 19.0, CH3 19.4, CH3 25.3, CH 21.1, CH3 37.7, CH2 106.9, C 145.8, CH 131.1, C 172.1, C 10.4, CH3 20.9, CH3 29.2, CH3 26.1, CH3

4b 187.7, C 97.0, CH 206.7, C 44.1, C 48.8, CH 18.3, CH2 25.9, CH2 138.2, C 132.4, C 41.6, C 81.6, CH 34.2, CH2 47.8, C 48.4, C 37.9, CH2 76.2, CH 52.1, CH 18.8, CH3 19.4, CH3 24.3, CH 21.8, CH3 37.4, CH2 71.3, CH 145.5, CH 128.2, C 169.6, C 20.6, CH3 21.0, CH3 29.3, CH3 26.4, CH3

5b 187.4, C 97.2, CH 206.7, C 44.1, C 48.8, CH 18.3, CH2 25.8, CH2 137.5, C 132.7, C 41.6, C 81.4, CH 33.6, CH2 48.4, C 49.2, C 39.4, CH2 74.8, CH 52.6, CH 16.6, CH3 19.4, CH3 28.3, CH 19.9, CH3 39.9, CH2 80.1, CH 148.8, CH 130.0, C 174.1, C 10.9, CH3 21.0, CH3 29.3, CH3 26.5, CH3 170.4, C 21.5, CH3

6b 187.5, C 97.1, CH 206.7, C 44.1, C 48.9, CH 18.3, CH2 25.8, CH2 137.5, C 132.7, C 41.7, C 81.5, CH 33.7, CH2 48.5, C 49.2, C 39.3, CH2 73.8, CH 52.9, CH 16.7, CH3 19.4, CH3 28.0, CH 18.4, CH3 40.7, CH2 78.3, CH 149.4, CH 129.9, C 174.2, C 10.8, CH3 21.0, CH3 29.3, CH3 26.4, CH3 171.4, C 21.4, CH3

7 158.6, CH 126.1, CH 204.6, C 44.5, C 48.8, CH 17.9, CH2 29.3, CH2 165.8, C 134.7, C 40.0, C 197.7, C 51.1, CH2 46.4, C 49.6, C 39.4, CH2 74.3, CH 53.0, CH 19.3, CH3 24.4, CH3 25.5, CH 20.4, CH3 37.4, CH2 106.8, C 145.7, CH 131.2, C 172.0, C 10.4, CH3 21.3, CH3 28.2, CH3 26.7, CH3

8 159.1, CH 125.9, CH 204.8, C 44.5, C 48.8, CH 17.9, CH2 29.2, CH2 166.6, C 134.8, C 40.0, C 198.5, C 51.4, CH2 46.9, C 49.7, C 42.5, CH2 71.4, CH 54.7, CH 17.3, CH3 24.5, CH3 30.8, CH 18.4, CH3 36.3, CH2 28.0, CH2 143.9, CH 127.4, C 168.4, C 20.5, CH3 21.3, CH3 28.2, CH3 26.9, CH3

51.6, CH3

Recorded at 150 MHz; b Recorded at 200 MHz.

30


9 158.8, CH 126.0, CH 204.7, C 44.5, C 48.7, CH 17.9, CH2 29.1, CH2 165.7, C 135.0, C 39.9, C 198.1, C 51.1, CH2 47.2, C 50.1, C 41.4, CH2 73.9, CH 52.9, CH 17.2, CH3 24.5, CH3 30.1, CH 17.9, CH3 35.3. CH2 27.0, CH2 146.0, CH 126.2, C 172.5, C 20.6, CH3 21.3, CH3 28.2, CH3 27.0, CH3 170.7, C 21.2, CH3

10 158.8, CH 126.0, CH 204.7, C 44.5, C 48.7, CH 17.9, CH2 29.1, CH2 165.7, C 135.0, C 39.9, C 198.0, C 51.1, CH2 47.2, C 50.1, C 41.5, CH2 73.9, CH 52.9, CH 17.2, CH3 24.5, CH3 30.1, CH 17.8, CH3 35.3, CH2 26.8, CH2 143.2, CH 126.9, C 168.2, C 20.7, CH3 21.3, CH3 28.2, CH3 27.0, CH3 170.7, C 21.2, CH3 51.3, CH3

11 158.6, CH 126.1, CH 204.6, C 44.5, C 48.6, CH 17.9, CH2 29.1, CH2 165.5, C 135.1, C 39.9, C 197.6, C 51.1, CH2 47.2, C 50.0, C 41.5, CH2 73.8, CH 52.7, CH 17.1, CH3 24.5, CH3 27.8, CH 19.1, CH3 39.3, CH2 79.6, CH 148.6, CH 129.9, C 173.8, C 10.7, CH3 21.3, CH3 28.2, CH3 27.0, CH3 170.4, C 21.3, CH3

12 158.6, CH 126.1, CH 204.6, C 44.5, C 48.7, CH 17.8, CH2 29.1, CH2 165.5, C 135.1, C 40.0, C 197.7, C 51.1, CH2 47.2, C 50.0, C 41.3, CH2 72.8, CH 53.2, CH 17.3, CH3 24.5, CH3 27.7, CH 17.7, CH3 40.3, CH2 78.2, CH 149.2, CH 129.8, C 174.0, C 10.7, CH3 21.3, CH3 28.2, CH3 27.0, CH3 171.2, C 21.2, CH3

Page 31 of 42


Table 4. Inhibitory Activity of NO Production of Compounds 1–6. Samples L-NMMA 1 2 3 4 5 6

IC50 (µM) 47.1 33.8 24.3 34.9 17.9 29.9 26.7

31



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Table 5. Cytotoxicity of 7–12 Against Five Human Cancer Cell Line. Samples

HL-60

A-549

SMMC-7721

MCF-7

SW480

IC50 (µM ± SD)

7

3.52 ± 0.79

6.99 ± 0.29

5.00 ± 0.11

12.95 ± 0.73

5.21 ± 0.39

8

2.88 ± 0.08

4.96 ± 0.10

3.17 ± 0.04

8.27 ± 0.12

3.57 ± 0.22

9

14.59 ± 0.39

18.86 ± 0.36

10.77 ± 0.85

25.89 ± 0.87

16.77 ± 0.84

10

12.70 ± 0.06

17.68 ± 0.52

12.61 ± 0.16

15.50 ± 0.80

17.38 ± 0.71

11

14.09 ± 0.86

21.03 ± 0.74

15.28 ± 0.61

23.23 ± 0.88

19.73 ± 0.60

12

3.09 ± 0.03

5.70 ± 0.18

3.14 ± 0.06

12.48 ± 0.50

4.38 ± 0.39

Cisplatin

2.95 ± 0.13

15.97 ± 0.69

10.17 ± 0.41

23.64 ± 1.6

9.26 ± 0.98

32


Page 33 of 42


FIGURE CAPTIONS Figure 1. Structures of lepiotaprocerins A–L (1–12). Figure 2. Key 2D NMR correlations of compounds 1, 3–6. Figure 3. Oak Ridge Thermal Ellipsoid Plot Program (ORTEP) drawing of compound 1. Figure 4. Experimental CD and calculated ECD spectra for compound 3, and calculated ECD for 3a (enantiomer of 3). Figure 5. CD spectra of compounds 5 and 6. Figure 6. ORTEP drawing of compound 7. Figure 7. Experimental CD and calculated ECD spectra for compound 7. Figure 8. CD spectra of compounds 11 and 12.

33



Figure 1

34


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Page 35 of 42


H

COOH O

O

O

O

H

O

O H

O

COOH O

H

1

1 H

O

O O

O

O

H

O

O

O O

H 3

3 H O

O

O

O H

H

COOH

O O

O

O O

O

H 4 1

5/6 1

H- H COSY

HMBC

ROESY

Figure 2

35



Figure 3

36


Page 36 of 42

Page 37 of 42


40

Calc ECD for 3

30

Calc ECD for 3a

20

Exptl CD for 3

∆ε

10 0

-10 -20 -30 -40 195

220

245

270

295

320

345

370

395

Wavelength (nm)

Figure 4

37



60 Compound 5

50

Compound 6

40

∆ε

30 20 10 0 -10 -20 195 215 235 255 275 295 315 335 355 375 395 Wavelength (nm)

Figure 5

38


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Figure 6

39



Calcd ECD for 7

35

Exptl CD for 7

25

∆ε

15 5 -5 -15 -25 195

245

295

345

395

445

495

545

595

Wavelength (nm)

Figure 7

40


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45

Compound 11

35

Compound 12

25

∆ε

15 5 -5 -15 -25 -35 195 215 235 255 275 295 315 335 355 375 395

Wavelength (nm)

Figure 8

41



Table of Contents Graphic

42


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Antiproliferative and Anti-inflammatory Lanostane ... - ACS Publications

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