Polyoxygenated Cyclohexenoids with Promising Î±-Glycosidase

Subscriber access provided by UNIVERSITY OF LEEDS

Article

Polyoxygenated Cyclohexenoids with Promising #-Glycosidase Inhibitory Activity Produced by Phomopsis sp. YE3250, an Endophytic Fungus Derived from Paeonia delavayi Rong Huang, Bo-Guang Jiang, Xiao-Nian Li, Ya-Ting Wang, Si-Si Liu, Kai-Xuan Zheng, Jian He, and Shao-Hua Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04998 • Publication Date (Web): 15 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

Journal of Agricultural and Food Chemistry

Polyoxygenated Cyclohexenoids with Promising α-Glycosidase Inhibitory Activity Produced by Phomopsis sp. YE3250, an Endophytic Fungus Derived from Paeonia delavayi Rong Huang,‡ Bo-Guang Jiang,† Xiao-Nian Li,§ Ya-Ting Wang,† Si-Si Liu,† Kai-Xuan Zheng,† Jian He,*,⊥ and Shao-Hua Wu*,†

†

Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, China

‡ §

School of Chemical Science and Technology, Yunnan University, Kunming 650091, China State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

⊥

Group of Peptides and Natural Products Research, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China

Corresponding Authors *(S.-H. Wu) E-mail: [email protected]. Phone/Fax: +86-871-65034264. *(J. He) E-mail: [email protected]. Phone/Fax: +86-20-61648717.

ACS Paragon Plus Environment

1


Page 2 of 26

1

ABSTRACT: Seven new polyoxygenated cyclohexenoids, namely, phomopoxides A–G (1–7),

2

were isolated from the fermentation broth extract of an endophytic fungal strain Phomopsis sp.

3

YE3250 from the medicinal plant Paeonia delavayi Franch. The structures of these compounds

4

were established by spectroscopic interpretation. The absolute configurations of compounds 1

5

and 4 were confirmed by X-ray crystallographic analysis and chemical derivative approach. All

6

isolated compounds showed weak cytotoxic activities towards three human tumor cell lines (Hela,

7

MCF-7, and NCI-H460), and weak antifungal activities against five pathogenic fungi (Candida

8

albicans, Aspergillus niger, Pyricularia oryzae, Fusarium avenaceum, and Hormodendrum

9

compactum). In addition, compounds 1−7 showed a promising α-glycosidase inhibitory activity

10

with IC50 values of 1.47, 1.55, 1.83, 2.76, 2.88, 3.16, and 2.94 mM, respectively, as compared

11

with a positive control of acarbose (IC50 = 1.22 mM).

12 13

KEYWORDS: Phomopsis, Paeonia delavayi, polyoxygenated cyclohexenoids, α-glucosidase

14

inhibitor

15 16 17 18 19 20 21 22 23 24


2

Page 3 of 26

25


INTRODUCTION

26

Endophytic fungi have gained much more attention from natural product chemists because of

27

their extraordinary abilities in producing diverse and structurally unprecedented secondary

28

metabolites.1–6 Most of these metabolites are potential candidates for drug discovery. Among

29

them, Phomopsis is a creative genus known for making a large number of exclusive and

30

structurally significant bioactive compounds.7 Interestingly, many Phomopsis species are

31

symbiotic prevalently inside their host plant growing in temperate and tropical regions,7 which

32

intrigued us to search for novel metabolites from these materials. As a consequence, in our

33

previous work, several new compounds including ten-membered lactones and steroids have been

34

isolated and identified from endophytic species of Phomopsis. 8,9

35

The medicinal plant Paeonia delavayi Franch. is an important source of traditional Chinese

36

medicine “mudanpi”. It is not only a major herbal material used as an anti-inflammatory and

37

sedative agent, but also curing cardiovascular and female diseases in oriental traditional

38

medicine.10–12 To our surprise, up to now, few work has been performed on the endophytic fungi

39

from this plant except for our previous report on new sesquiterpenes from an endophytic

40

Trichoderma sp.13

41

As a result of our continuous bioactive screening of the endophytic fungi isolated from P.

42

delavayi, the EtOAc extract of the fermentation broth from the strain Phomopsis sp. YE3250

43

exhibited potent α-glucosidase inhibitory activity with an IC50 value of 1.08 mg/mL, meanwhile

44

the IC50 value of positive control (acarbose) was 0.79 mg/mL. In this paper, we report the

45

isolation and structure elucidation of bioactive secondary metabolites from this strain, as well as

46

their α-glycosidase inhibitory activity. Besides that, the in vitro cytotoxic and antifungal

47

activities of the metabolites are also addressed.


3


48

Page 4 of 26

MATERIALS AND METHODS

49

General. An XRC-1 apparatus was used for measuring melting points (mp), and the reported

50

data were uncorrected. A HORIBA SEPA-300 polarimeter was used to obtain optical rotations.

51

A Bio-Rad FTS-135 infrared spectrophotometer was used for recording infrared (IR) spectra with

52

KBr pellets. Ultraviolet (UV) spectra were obtained on Shimadzu double-beam 210A

53

spectrophotometer. Circular dichroism (CD) spectra were measured with an Applied

54

Photophysics Chirascan spectrometer. Nuclear magnetic resonance (NMR) spectra were acquired

55

on a Bruker DRX-500 spectrometer with tetramethylsilane (TMS) as internal standard. High-

56

resolution electrospray ionization mass spectrometry (HR-ESIMS) were determined on an

57

Agilent G3250AA LC/MSD TOF spectrometer. Silica gel (200−300 mesh, Qingdao Marine

58

Chemical Inc., China), reversed-phase silica gel C18 (40-63 µm, Merck), and Sephadex LH-20

59

(Pharmacia) were employed for column chromatography (CC).

60

Fungal Material. The endophytic strain YE3250 was isolated from the fresh stems of Paeonia

61

delavayi in Songming County, Yunnan, China, following an isolation protocol described

62

previously.13 The strain was deposited in our institute. It was identified to be Phomopsis sp. on

63

the basis of morphological characteristic and analysis of its internal transcribed spacer (ITS)

64

sequences (GenBank accession No. KF733183) according to the reported method previously.8

65

Fermentation, Extraction, and Isolation. The fresh mycelium of the strain was grown on

66

potato dextrose agar (PDA) medium at 28 °C for 7 days. Then, it was inoculated into 500 mL

67

Erlenmeyer flasks (×50) containing 120 mL of potato dextrose broth (PDB) medium (200 g

68

potato and 20 g dextrose in 1 L H2O). After incubation at 28 °C with shaking (200 rpm) for 4

69

days, each 30 mL of the seed culture solution was added into 1 L Erlenmeyer flasks (×150)

70

containing 300 mL of PDB medium. The following cultivation was carried out at 28 °C with


4

Page 5 of 26


71

shaking (200 rpm) for 7 days. The culture broth was filtered and concentrated to 10 L, and

72

then sufficiently partitioned with EtOAc (10 L) for three times. After evaporating of the organic

73

solvent under vacuum, the final brown gum (32.6 g) was obtained and chromatographed on silica

74

gel column, eluting gradiently with a mixture solvents of CHCl3/MeOH from 1:0 to 0:1 (v/v) to

75

obtain seven fractions (Frs. 1–7). The α-glucosidase inhibitory activity test showed that fractions

76

3–6 were active. Fr. 3 (2.1 g) was purified by CC over reversed-phase silica gel C18, eluting with

77

MeOH/H2O (3:7, 4:6, 1:1, 6:4), and then on silica gel, eluting with petroleum ether (PE)/acetone

78

(7:3, 6:4) afforded compound 6 (3.9 mg) and compound 7 (6.3 mg). Further separation of Fr. 4

79

(2.9 g) by CC over reversed-phase silica gel C18 (MeOH/H2O, 1:1, 6:4, 7:3) and Sephadex LH-

80

20 (MeOH) obtained compound 4 (50.6 mg) and compound 5 (8.7 mg). Purification of Fr. 5 (1.8

81

g) by CC over Sephadex LH-20 (MeOH) and silica gel (CHCl3/MeOH, 9:1) yielded compound 3

82

(5.7 mg). Fr. 6 (2.6 mg) was separated by CC over silica gel (CHCl3/MeOH, 9:1, 8:2) and

83

reversed-phase silica gel C18 (MeOH/H2O, gradiently from 2:8 to 4:6) to give compound 1 (15.5

84

mg) and compopund 2 (7.4 mg).

85

Phomopoxide A (1): colorless crystals; TLC Rf 0.36 (CHCl3/MeOH, 85:15); mp 186−187 °C;

86

[α]20 D −14.8 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 238 (4.28) nm; CD (c 0.29, MeOH) λmax

87

(∆ε) 233 (−3.26) nm; IR (KBr) νmax 3377 (br), 3331, 2955, 2922, 2852, 1643, 1463, 1406, 1384,

88

1217, 1040, 996 cm−1; HRESIMS m/z 367.2095 [M + Na]+ (calcd for C18H32O6Na, 367.2097);

89

1

H and 13C NMR spectral data, see Tables 1 and 2.

90

Phomopoxide B (2): white powder; TLC Rf 0.39 (CHCl3/MeOH, 85:15); [α]20 D +25.7 (c 0.21,

91

MeOH); UV (MeOH) λmax (log ε) 197 (4.02) nm; CD (c 0.64, MeOH) λmax (∆ε) 196 (+7.49), 222

92

(+2.48) nm; IR (KBr) νmax 3457 (br), 3211, 2958, 2928, 2857, 1666, 1462, 1266, 1106, 996 cm−1;


5


93

HRESIMS m/z 367.2094 [M + Na]+ (calcd for C18H32O6Na, 367.2097); 1H and

94

spectral data, see Tables 1 and 2.

Page 6 of 26

13

C NMR

95

Phomopoxide C (3): white powder; TLC Rf 0.48 (CHCl3/MeOH, 85:15); [α]20 D −35.1 (c 0.23,

96

MeOH); IR (KBr) νmax 3405 (br), 3377, 2955, 2926, 2856, 1642, 1403, 1251, 1090, 994 cm−1;

97


98


13

C NMR

99

Phomopoxide D (4): white powder; TLC Rf 0.23 (PE/acetone, 6:4); [α] 20 D −68.3 (c 0.38,

100

MeOH); UV (MeOH) λmax (log ε) 237 (4.25) nm; CD (c 0.27, MeOH) λmax (∆ε) 234 (−9.87) nm;

101

IR (KBr) νmax 3366 (br), 2957, 2925, 2855, 1638, 1465, 1257, 1087, 1030, 966 cm−1; HRESIMS

102

m/z 349.1985 [M + Na]+ (calcd for C18H30O5Na, 349.1991); 1H and 13C NMR spectral data, see

103

Tables 1 and 2.

104

Phomopoxide E (5): white powder; TLC Rf 0.37 (PE/acetone, 6:4); [α]20 D +9.1 (c 0.24, MeOH);

105

UV (MeOH) λmax (log ε) 196 (4.02) nm; CD (c 0.64, MeOH) λmax (∆ε) 204 (−4.65), 233 (−1.08)

106

nm; IR (KBr) νmax 3346 (br), 3194, 2962, 2932, 2858, 1633, 1495, 1252, 1098, 1012, 960 cm−1;

107


108


13

C NMR

109

Phomopoxide F (6): colorless gum; TLC Rf 0.51 (PE/acetone, 6:4); [α] 20 D −28.7 (c 0.17,

110

MeOH); IR (KBr) νmax 3446, 3367 (br), 2950, 2924, 2851, 1464, 1412, 1347, 1258, 1119, 1090,

111

971 cm−1; HRESIMS m/z 331.1887 [M + Na]+ (calcd for C18H28O4Na, 331.1885); 1H and

112

NMR spectral data, see Tables 1 and 2.

13

C

113

Phomopoxide G (7): colorless gum; TLC Rf 0.47 (PE/acetone, 6:4); [α] 20 D −15.1 (c 0.21,

114

MeOH); IR (KBr) νmax 3451 (br), 3387, 2956, 2927, 2856, 1711, 1466, 1408, 1351, 1271, 1120,


6

Page 7 of 26


115

1038, 943 cm−1; HRESIMS m/z 347.1837 [M + Na]+ (calcd for C18H28O5Na, 347.1834); 1H and

116

13

C NMR spectral data, see Tables 1 and 2.

117

Chemical Transformation of 4 to 4a. 50 µL concentrated HCl (37%) was added into the

118

MeOH solution (5 mL) of compound 4 (10 mg). The transformation was performed by stirring

119

for 1 h at 38 °C. The reaction mixture was concentrated and then added to 10 mL with CHCl3.

120

The obtained solution was washed sufficiently with 10 mL saturated NaHCO3 for three times and

121

neutralized to pH 7. The CHCl3 extract was concentrated and purified by CC over silica gel

122

eluting with PE/acetone (8:2), and then crystallized in MeOH/H2O (1:1) to afford 4a (8.3 mg).

123

Compound 4a: colorless crystals; TLC Rf 0.56 (PE/acetone, 6:4); mp 173−174 °C; [α]20 D −54.7

124

(c 0.24, MeOH); IR (KBr) νmax 3358, 2953, 2916, 2852, 1629, 1469, 1217, 1128, 1100, 963 cm−1;

125

HRESIMS m/z 367.1652 [M + Na]+ (calcd for C18H29O4ClNa, 367.1652); 1H and

126


13

C NMR

127

X-ray Crystallography of Compounds 1 and 4a. The crystallographic data of 1 and 4a were

128

collected with Cu Kα radiation (λ = 1.54178 Å) and graphite-monochromated Mo Kα radiation

129

(λ = 0.71073 Å), respectively, at T = 100 (2) K on the Bruker APEX DUO diffractometer,

130

equipped with an Oxford Cryostream 700+ cooler. The structures were solved using direct

131

method with SHELXS-97, and then refined using full-matrix least-squares on F2 with SHELXL

132

package software14. The absolute configurations were determined by refinement of the Flack

133

parameter15 on the basis of resonant scattering of the light atoms.

134

Crystallographic data for 1: C18H32O6·H2O, Mr = 362.45, orthorhombic, crystal dimensions

135

0.09×0.12×0.90 mm, space group P212121, a = 4.668 Å, b = 9.78500(10) Å, c = 41.7773(5) Å, V

136

= 1908.24(3) Å3, α = 90.00°, β = 90.00°, γ = 90.00°, Z = 4, Dcalcd = 1.262 mg/m3, F(000) = 792,

137

µ(CuKα) = 0.790 mm-1, Tmin/Tmax = 0.54/0.93, 2.11° ≤ θ ≤ 69.62°, 11914 reflections measured,


7


Page 8 of 26

138

3359 unique reflections (Rint = 0.0229), final R1 = 0.0278 and wR(F2) = 0.0742 (I > 2σ(I)), final

139

R1 = 0.0280 and wR(F2) = 0.0744 (all data), goodness of fit on F2 = 1.096. The Flack parameter

140

is 0.06(13).

141

Crystallographic data for 4a: C18H29ClO4· H2O, Mr = 362.88, monoclinic, crystal dimensions

142

1.14 × 0.45 × 0.09 mm, space group P2(1), a = 4.6368(6) Å, b = 7.8718(9) Å, c = 25.885(3) Å, α

143

= 90°, β = 93.657(2)°, γ = 90°, V = 942.9(2) Å3, Z = 2, Dcalcd=1.278 mg/m3, F(000)= 392,

144

µ(MoKα) = 0.226 mm-1, Tmin/Tmax = 0.78/0.98, 10463 reflections measured, 5362 unique

145

reflections (Rint = 0.0217), final R1 = 0.0275 and wR(F2) = 0.0737 (I > 2σ(I)), final R1 = 0.0307

146

and wR(F2) = 0.0811 (all data), goodness of fit on F2 = 1.133. The Flack parameter is 0.016(17).

147

The above crystallographic data have been deposited with the Cambridge Crystallographic

148

Data Centre as supplementary publication numbers CCDC 966947 for compound 1 and CCDC

149

1577168

150

www.ccdc.cam.ac.uk/data_request/cif.

for

compound

4a.

These

data

can

be

acquired

free

of

charge

via

151

α-Glucosidase Inhibitory Effect Assay. Evaluations of all isolated compounds for enzyme

152

inhibitory activity against α-glycosidase were conducted following the method reported

153

previously,16,17 by using acarbose as a positive control.

154

Cytotoxicity Assay. Three cancer cell lines, human cervical carcinoma (Hela), human breast

155

carcinoma (MCF-7), and human non-small cell lung carcinoma (NCI-H460) were employed in

156

this experiment. The in vitro cytotoxic activities of compounds 1−7 and 4a were assessed by

157

using

158

tetrazolium bromide) assay, as described in the literature.18 Each cell line was tested in triplicates

159

with paclitaxel (Sigma, USA) as a positive control. Cytotoxic effects were estimated by

160

comparing the cell survival rate of compound-treated with that of solvent-treated. The IC50

tetrazolium-based

colorimetric

MTT

(3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl


8

Page 9 of 26

161


values were calculated by using Reed and Muench’s method.19

162

Antifungal Assays. Five pathogenic fungal strains, Candida albicans (YM 2005), Aspergillus

163

niger (YM 3029), Pyricularia oryzae (YM 3051), Fusarium avenaceum (YM 3065), and

164

Hormodendrum compactum (YM 3077) were used as indicators in this experiment. The minimal

165

inhibitory concentrations (MICs) for antifungal activity were measured by the broth

166

microdilution method described previously9 with nystatin as positive control.

167

RESULTS AND DISCUSSION

168

Structural Elucidation. The current chemical investigations on fermentation product of

169

Phomopsis sp. YE3250 resulted in the isolation of seven new polyoxygenated cyclohexenoids,

170

named as phomopoxides A–G (1–7, Figure 1). Their structures were respectively determined by

171

one-dimensional (1D) and two-dimensional (2D) NMR experiments, combined with X-ray

172

crystallographic analysis.

173

The molecular formula of phomopoxide A (1) was determined as C18H32O6 according to its

174

positive-ion HRESIMS at m/z 367.2095 [M + Na]+ (calcd 367.2097) and NMR spectroscopic

175

data. The 1H NMR spectrum (Table 1) exhibited a primary methyl signal at δH 0.92 (t, J = 5.3

176

Hz), five oxymethine protons (δH 3.48, 3.55, 4.13, 4.22, 4.26), an oxymethylene singlet at δH 4.37,

177

and two olefinic protons at δH 6.45 (d, J = 15.9 Hz) and 5.94 (dd, J = 15.9, 6.5 Hz), which

178

indicated a trans-double bond. The

179

signals including four olefinic carbons, five oxymethines, one oxymethylene, seven aliphatic

180

methylenes, and a methyl group. A monocyclic structure was proposed for 1 by three degrees of

181

unsaturation deduced from the molecular formula except for two double bonds. Furthermore, a

182

broad IR absorption band at 3377 cm–1, together with the signals of five oxygenated carbons in

183

the 13C NMR spectrum, indicated the presence of multiple hydroxy groups. Interpretation of the

13

C NMR spectrum of compound 1 (Table 2) displayed 18


9


Page 10 of 26

184

1

H–1H correlation spectroscopy (COSY) spectrum (Figure 2) revealed that four adjacent

185

oxymethine groups were linked together. The heteronuclear multiple bond correlations (HMBCs)

186

for both oxymethine protons at δH 4.22 (H-3) and 4.26 (H-6) to C-1 (δC 136.8), C-2 (δC 135.9), C-

187

4 (δC 76.7), and C-5 (δC 76.3) established construction of a cyclohexene moiety (Figure 2). The

188

tracking cross-peaks between H-2' (δH 5.94) and H-1' (δH 6.45) and H-3' (δH 4.13) in the 1H–1H

189

COSY spectrum deduced that the trans-double bond was connected with an aliphatic linear chain

190

through an oxymethine group. The double bond was linked to C-2 in the monocyclic system

191

based on the HMBC correlations from H-2' to C-2 and from H-1' to C-1 and C-2. Similarly, the

192

hydroxy methylene group at δC 59.7 (C-7) was attached to C-1 in the cyclohexene ring by the

193

HMBC correlations from δH 4.37 (H-7) to C-1, C-2 and C-6. Thus, the planar structure of 1 was

194

assigned. The 6.1–9.3 Hz coupling constants observed for the contiguous protons of H-3/H-4, H-

195

4/H-5, and H-5/H-6 suggested that these four hydrogens are trans relationship to each other,

196

which was also supported by selective 1D nuclear Overhauser effect spectroscopy (NOESY)

197

experiment. The selective irradiation of H-4 (δH 3.55) caused an NOE enhancement only for the

198

signal of H-6 (δH 4.26). In a similar way, the selective irradiation of H-5 (δH 3.48) led to NOE

199

intensification only for the signal of H-3 (δH 4.22).

200

A single colorless crystal of compound 1 was obtained from a mixed solvent of methanol and

201

acetone (1:5) after several crystallization attempts. Its absolute configuration was ultimately

202

established by a low-temperature single crystal X-ray diffraction experiment performed with Cu

203

Kα. The last Flack parameter 0.06(13) and Hooft parameter 0.03(3) unambiguously confirmed

204

the stereochemistry of 3R, 4S, 5S, 6R in the cyclohexene moiety, and C-3' as R in the side chain

205

(Figure 3). Consequently, the structure of compound 1 was formulated as in Fig. 1 and given its

206

name of phomopoxide A.


10

Page 11 of 26


207

Phomopoxide B (2) was isolated as a white powder and determined to have the same

208

molecular formula of C18H32O6 as 1 due to its HRESIMS at m/z 367.2094 [M + Na]+. The NMR

209

spectroscopic data of 2 showed only slight differences in chemical shifts with those of 1,

210

suggesting that they were stereoisomers. On the basis of the similar coupling constants for each

211

of H-3/H-4, H-4/H-5, and H-5/H-6 with those of 1, together with the view of their biogenetic

212

consideration, the chiral centers of cyclohexene moiety in 2 could be assigned as the same 3R, 4S,

213

5S, 6R-configuration. A careful comparison of their NMR data indicated that the most obvious

214

difference is the chemical shift of C-3' at δC 69.2 in 2 and δC 74.4 in 1. This was indicated that

215

compound 2 was an epimer of 1 at C-3'. In the CD spectra of 1 and 2 (Figures S10 and S20 of the

216

Supporting Information), a negative Cotton effect appeared at 233 (∆ε −3.26) nm in 1, while a

217

positive Cotton effect was observed at 196 (∆ε +7.49) and 222 (∆ε +2.48) nm in 2. Furthermore,

218

20 the opposite optical rotation value of 2 ([α]20 D +25.7) to that of 1 ([α] D −14.8) indicated that

219

compound 2 possessed the different configuration of C-3' with that in 1. As the absolute

220

configuration of C-3' in 1 was R, 3'S configuration was accordingly established in 2. The

221

relatively small coupling constant (J = 11.3 Hz) for H-1' with H-2' clearly confirmed Z-geometry

222

of the ∆1'-double bond in the aliphatic side chain. Thus, the structure of compound 2 was

223

formulated as in Fig. 1 and given its name of phomopoxide B.

224

Phomopoxide C (3) was isolated as a white powder, and possessed the molecular formula of

225

C18H30O5 due to its HRESIMS at m/z 349.1993 [M + Na]+ (calcd 349.1991), which was 18 mass

226

units less than that of 1. The NMR spectrometry of 3 showed resemblance to those of 1.

227

Comprehensive analysis of its 2D NMR spectra led to the determination of the 3,4,5,6-

228

tetrahydroxycyclohexene moiety. The main difference in

229

was the downfield chemical shift of the oxymethylene group at C-7 to δ 75.5 and one of the

13

C NMR spectrum between 3 and 1


11


Page 12 of 26

230

oxymethine group to δ 88.4 in 3. These two carbons were directly correlated to the downfield

231

shifted signals of two nonequivalent protons at δH 4.61 (dd, J = 12.2, 4.5 Hz) and 4.78 (dd, J =

232

12.2, 3.9 Hz), and the oxymethine proton at δH 5.27 (br s), respectively in the 1H NMR spectrum,

233

according to heteronuclear multiple-quantum coherence (HMQC) experiment. Its 1H−1H COSY

234

spectrum showed the weak couplings from two nonequivalent oxymethylene protons at δH 4.61

235

and 4.78 to H-6 (δH 4.06) and the oxymethine proton at δH 5.27. In addition, the weak correlation

236

between this oxymethine proton and H-3 (δH 4.18) could also be observed. The presence of an

237

additional ring in 3 was supported by four degrees of unsaturation deduced from the molecular

238

formula except for a cyclohexane ring and two double bonds. Based on the above data, it could

239

be easily deduced that the oxymethine group at δC 88.4 (C-1') was linked not only to C-7 through

240

an oxygen atom but also to the olefinic quaternary carbon C-2 (δC 137.5). Thus, a five-membered

241

ring was formed, though it could not be sufficiently elucidated from the HMBC spectrum. The

242

correlation between the oxymethine proton (δH 5.27) and the olefinic proton (δH 5.46) in 1H−1H

243

COSY spectrum established that the decyl terminated double bond was connected to C-1'. The

244

large coupling constant of J = 15.3 Hz between two olefinic protons at δH 5.46 and 5.82

245

confirmed the trans-double bond between C-2' and C-3'. The absolute configuration of

246

cyclohexene moiety in 3 could be also determined as 3R, 4S, 5S, 6R according to its homologues

247

1 and 2. The stereochemistry of the remaining chiral center C-1' in compound 3 was assigned to

248

be R on the basis of its negative optical rotation ([α]20 D −35.1). Thus, the structure of compound 3

249

was formulated as in Fig. 1 and given its name of phomopoxide C.

250

Phomopoxide D (4) was also isolated as a white powder. The quasi-molecular ion peak at m/z

251

349.1985 [M + Na]+ in HRESIMS suggested a molecular formula of C18H30O5. Its NMR signals

252

displayed resemblance with those of 1, indicating the similar oxygenated cyclohexene ring and


12

Page 13 of 26


253

the same side chain with 1. The chemical shifts of two oxymethine groups (δH 3.45, 3.52; δC 55.9,

254

56.6) clearly demonstrated the presence of a 1,2-disubstituted epoxide20 in compound 4. The 1H−

255

1

256

oxymethine protons at δH 4.66 (H-3) and 4.58 (H-6) for a small coupling constant. Thus, the

257

substructure of C3–C6 unit could be confirmed. Further analysis of 2D NMR spectra indicated

258

that the aliphatic side chain was linked to C-2 in a same manner with 1. The planar structure of 4

259

was accordingly determined, containing an epoxy unit between C-4 and C-5.

H COSY spectrum showed the couplings of each epoxide proton with one of the other two

260

Chlorination of compound 4 was carried out in the hope of introducing an extra heavy atom in

261

4 to confirm its absolute configuration by single crystal X-ray analysis. Compound 4 was

262

transformed into 4a as the major product after adding hydrochloric acid to its methanol solution.

263

The structure of 4a could be determined by the elucidation of NMR spectra. A chlorine atom and

264

a hydroxyl group were connected with C-4 and C-5, respectively in 4a, instead of an epoxy

265

moiety. With the similarity of compound 3, the existence of a 2, 5-dihydrofuran unit in 4a could

266

be deduced by the weak couplings between the nonequivalent oxymethylene protons H2-7 (δH

267

4.62 and 4.79) and the oxymethine proton H-1' (δH 5.27) in the 1H−1H COSY spectrum. A

268

colorless crystal of 4a was obtained in the MeOH-H2O (1:1, v/v) mixture solution. It was

269

unambiguously feasible to use single crystal X-ray diffraction analysis for assignment of the

270

absolute configurations of all chiral centers as 3R, 4S, 5S, 6R, 1'R (Figure 4) with the Flack

271

parameter 0.016(17). The X-ray crystallographic analysis of 4a also established that 3-OH and 5-

272

OH were β-oriented, whereas 6-OH was in α-orientation, which were identical with those of 1−3.

273

The chlorine atom was added to C-4 from α-orientation due to its less steric hindrance. The

274

relative configuration of epoxy unit in 4 could be easily confirmed as β-orientation based on the

275

structure and configuration of its derivative 4a. The configurations at the chiral centers of C-3


13


Page 14 of 26

276

and C-6 were identical to those in 4a. The chemical shift of C-3' at δC 74.4 in compound 4 was

277

identical to that in compound 1. The CD spectrum of 4 (Figure S38 in Supporting Information)

278

resembled that of 1 with the negative Cotton effect at 234 (∆ε −9.87) nm. These data, combined

279

20 with the similar negative optical rotation value of 4 ([α] 20 D −68.3) as that of 1 ([α] D −14.8),

280

implied that both compounds possessed the same 3'R configuration. The absolute configuration

281

of 4 was accordingly established as 3R, 4R, 5S, 6R, 3'R. Consequently, the structure of compound

282

4 was formulated as in Fig. 1 and given its name of phomopoxide D.

283

Phomopoxide (5), isolated as a white powder, possessed the same molecular formula of

284

C18H30O5 as 4 based on its HRESIMS at m/z 349.1997 [M + Na]+. The NMR spectral data of 5

285

were closely resembled with those of 4. The major difference between these two compounds was

286

the chemical shift of C-3'. In a similar manner with 1 and 2, compound 5 was also the C-3'

287

epimer of 4. In its 13C NMR spectrum, the chemical shift of C-3' at δC 68.4 in 5 was close to the

288

signal of C-3' at δC 69.2 in 2. The CD spectrum of 5 (Figure S56 of the Supporting Information)

289

showed the negative Cotton effect at 204 (∆ε −4.65) and 233 (∆ε −1.08) nm. In addition, the

290

20 similar positive optical rotation value of 5 ([α] 20 D +9.1) to that of 2 ([α] D +25.7), while the

291

negative optical rotation value of 4 ([α]20 D −68.3), indicated an absolute configuration of 3'S in

292

compound 5. The stereochemistry of the other chiral centers in 5 was the same as those of 4 to be

293

3R, 4R, 5S, 6R from biogenetic consideration. Z-geometry of the ∆1'-double bond in side chain

294

could be deduced by the relatively small coupling constant (J = 11.5 Hz) for H-1' with H-2'.

295

Therefore, the structure of compound 5 was formulated as in Fig. 1 and given its name of

296

phomopoxide E.

297

Phomopoxide F (6) was isolated as a colorless gum. The quasi-molecular ion peak at m/z

298

331.1887 [M + Na]+ in its HRESIMS confirmed a molecular formula of C18H28O4, suggesting


14

Page 15 of 26


299

the presence of five degrees of unsaturation. Comparison of its 1H and

13

300

those of 5 indicated the existence of the same 4,5-epoxy cyclohexenoid moiety in 6. Due to five

301

degrees of unsaturation calculated for compound 6, one more ring should present in the structure

302

except for two double bonds, a cyclohexene system, and an epoxy unit. With the similar

303

structural elucidation of 3, a five-membered dihydrofuran ring was formed by the linkage of C-7

304

with C-1' through an oxygen atom. This was deduced by the correlations between two

305

nonequivalent oxymethylene protons H2-7 (δH 4.53 and 4.86) and the oxymethine proton H-1' (δH

306

5.28) in the 1H−1H COSY spectrum, whereas H2-7 were only showed HMBC correlations with

307

C-1 and C-2. A double bond was located between C-2' and C-3' based on the coupling sequence

308

from H-1' to H-4' by tracking correlations in the 1H−1H COSY spectrum. A large coupling

309

constant of J = 15.1 Hz between two olefinic protons at δH 5.39 and 5.84 indicated E-geometry of

310

the double bond between C-2' and C-3'. Interestingly, compound 6 could be also transformed into

311

4a by adding hydrochloric acid to its methanol solution. Based on the determination of 4a, the

312

absolute configuration of 6 was definitely confirmed to be 3R, 4R, 5S, 6R, 1'R. Thus, the

313

structure of compound 6 was formulated as in Fig. 1 and given its name of phomopoxide F.

C NMR spectra with

314

Phomopoxide G (7) was also isolated as a colorless gum. A molecular formula of C18H28O5

315

was confirmed by the quasi-molecular ion peak at m/z 347.1837 [M + Na]+ in the HRESIMS. Its

316

NMR spectroscopic data were resemble with those of 6, except for an additional carbonyl group

317

(δC 211.5) and one more aliphatic methylene group instead of a double bond in 7. The cross-

318

peaks between H-1' (δH 5.27) and H-7 (δH 4.51 and 4.76) in its 1H−1H COSY spectrum indicated

319

the same five-membered dihydrofuran ring formed through the linkage of C1'-O-C7 as that in 6.

320

The carbonyl group was positioned at C-3' due to HMBC correlations from H-1' (δH 5.27) and

321

H2-2' (δH 2.73 and 2.93) to the quaternary carbon at δC 211.5. The close optical rotation value of


15


Page 16 of 26

322

7 (−15.1) to that of 6 (−28.7) implied the same 3R, 4R, 5S, 6R, 1'R configuration, which could be

323

also supported by a shared biogenesis for both compounds. Consequently, the structure of

324

compound 7 was formulated as in Fig. 1 and given its name of phomopoxide G.

325

Biological Activities. All compounds were tested for their enzyme inhibitory activity against

326

α-glycosidase. As a consequence, compounds 1−7 and 4a showed a promising α-glycosidase

327

inhibition with IC50 values of 1.47, 1.55, 1.83, 2.76, 2.88, 3.16, 2.94, and 2.24 mM, respectively,

328

by employing acarbose as a positive control (IC50 = 1.22 mM) (Table 3). It is noted that the

329

inhibitory effects of compounds 4−7, which forming an epoxy moiety, were weaker than those of

330

1−3, suggesting the tetrahydroxyl substitution in cyclohexene ring is likely to be crucial for α-

331

glycosidase inhibition.

332

Compounds 1−7 and 4a were further assayed in vitro for their cytotoxic activities towards

333

Hela, MCF-7, and NCI-H460 cell lines. Each of them displayed weak activities against the tested

334

tumor cell lines (Table 3).

335

Moreover, these compounds were also evaluated for their antifungal activities towards A. niger,

336

C. albicans, F. avenaceum, H. compactum, and P. oryzae by the broth microdilution method.

337

Among them, compound 4a bearing a chlorine atom possessed the most potent activity against C.

338

albicans and A. niger with MIC values of 16 and 32 µg/mL, respectively. In addition, compound

339

4 displayed moderate inhibitory activity towards C. albicans and A. niger with MIC value of 64

340

µg/mL. Compound 2 inhibited the growth of C. albicans, A. niger, and H. compactum with MIC

341

values of 32, 64, and 64 µg/mL, respectively. The remaining five compounds showed only weak

342

antifungal activities towards the tested pathogenic fungal strains.

343

Structurally, this group of new polyoxygenated cyclohexenoids is typical of fungal polyketide

344

metabolites. The cyclohexene ring seems to be formed by the cyclization of a polyketide


16

Page 17 of 26


345

intermediate. Thus far, these molecules have drawn more and more attention by chemists owing

346

to their characteristics of extensive oxygenation pattern and stereochemical variations.21 Recently,

347

several compounds with similar skeleton had also been isolated from the genus Phoma,22

348

Eupenicillium,23 and Aspergillus.16 Particularly, this group of polyoxygenated cyclohexenoids

349

also occur in genus Streptomyces.24 Nevertheless, in addition to contribute more new

350

homologues to this type of compounds, our current work indicates that these cyclohexenoids

351

might be a promising source for new α-glycosidase inhibitors.

352

Supporting Information. 1D and 2D NMR, HRESIMS, and IR spectra of compounds 1−7 and

353

4a; UV and CD spectra of compounds 1, 2, 4, and 5; X-ray crystallographic data of compounds 1

354

and 4a.

355

Funding

356

This research was financially supported by the National Natural Science Foundation of China

357

(81460545 and 81773556).

358

REFERENCES

359 360

(1) Strobel, G. M.; Daisy, B. Bioprospecting for microbial endophytes and their natural products. Microbiol. Mol. Biol. Rev. 2003, 67, 491–502.

361

(2) Gunatilaka, A. A. L. Natural products from plant-associated microorganisms: distribution,

362

structural diversity, bioactivity, and implication of their occurence. J. Nat. Prod. 2006, 69, 509–

363

526.

364 365 366 367

(3) Huang, W. Y.; Cai, Y. Z.; Hyde, K. D.; Corke, H.; Sun, M. Biodiversity of endophytic fungi associated with 29 traditional Chinese medicinal plants. Fungal Divers. 2008, 33, 61–75. (4) Gao, J. M.; Yang, S. X.; Qin, J. C. Azaphilones: chemistry and biology. Chem. Rev. 2013, 113, 4755–4811.


17


Page 18 of 26

368

(5) Li, X. J.; Zhang, Q.; Zhang, A. L.; Gao, J. M. Metabolites from Aspergillus fumigatus, an

369

endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic

370

activities. J. Agric. Food Chem. 2012, 60, 3424–3431.

371

(6) Xiao, J.; Zhang, Q.; Gao, Y. Q.; Shi, X. W.; Gao, J. M. Antifungal and antibacterial

372

metabolites from an endophytic Aspergillus sp. associated with Melia azedarach. Nat. Prod. Res.

373

2014, 28, 1388–1392.

374

(7) Udayanga, D.; Liu, X. Z.; McKenzie, E. H. C.; Chukeatirote, E.; Bahkali, A. H. A.; Hyde,

375

K. D. The genus Phomopsis: biology, applications, species concepts and names of common

376

phytopathogens. Fungal Divers. 2011, 50, 189–225.

377

(8) Wu, S. H.; Chen, Y. W.; Shao, S. C.; Wang, L. D.; Li, Z. Y.; Yang, L. Y.; Li, S. L.; Huang,

378

R. Ten-membered lactones from Phomopsis sp., an endophytic fungus of Azadirachta indica. J.

379

Nat. Prod. 2008, 71, 731–734.

380 381 382 383 384 385 386 387

(9) Wu, S. H.; Huang, R.; Miao, C. P.; Chen, Y. W. Two new steroids from an endophytic fungus Phomopsis sp. Chem. Biodivers. 2013, 10, 1276–1283. (10) Lin, H. C.; Ding, H. Y.; Wu, T. S.; Wu, P. L. Monoterpene glycosides from Paeonia suffruticosa. Phytochemistry 1996, 41, 237–242. (11) Kang, S. S.; Shin, K. H.; Chi, H. J. Galloylpaeoniflorin, a new acylated monoterpene glucoside from Paeony root. Arch. Pharm. Res. 1991, 14, 52–54. (12) Kubo, M.; Tani, T.; Kosoto, H.; Kimura, Y.; Arichi, S. Studies on Moutan Cortex (I): historical analysis. Shoyakugaku Zasshi 1979, 33, 155–157.

388

(13) Wu, S. H.; Zhao, L. X.; Chen, Y. W.; Huang, R.; Miao, C. P.; Wang, J. Sesquiterpenoids

389

from the endophytic fungus Trichoderma sp. PR-35 of Paeonia delavayi. Chem. Biodivers. 2011,

390

8, 1717–1723.

391

(14) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. 2008, A64, 112–122.


18

Page 19 of 26


392

(15) Flack, H. D. On enantiomorph-polarity estimation. Acta Crystallogr. 1983, A39, 876–881.

393

(16) Kong, F. D.; Zhao, C. Y.; Hao, J. J.; Wang, C.; Wang, W.; Huang, X. L.; Zhu, W. M. New

394

α-glucosidase inhibitors from a marine sponge-derived fungus, Aspergillus sp. OUCMDZ-1583.

395

RSC Adv. 2015, 5, 68852–68863.

396 397 398 399 400 401

(17) Wei, J.; Zhang, X. Y.; Deng, S.; Cao, L.; Xue, Q. H.; Gao, J. M. α-Glucosidase inhibitors and phytotoxins from Streptomyces xanthophaeus. Nat. Prod. Res. 2017, 31, 2062−2066. (18) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (19) Reed, L. J.; Muench, H. A simple method of estimating fifty percent endpoint. Am. J. Hyg. 1938, 27, 493–497.

402

(20) Hussain, H.; Akhtar, N.; Draeger, S.; Schulz, B.; Pescitelli, G.; Salvadori, P.; Antus, S.;

403

Kurtán, T.; Krohn, K. New bioactive 2,3-epoxycyclohexenes and isocoumarins from the

404

endophytic fungus Phomopsis sp. from Laurus azorica. Eur. J. Org. Chem. 2009, 2009, 749–756.

405

(21) Mehta, G.; Roy, S.; Davis, R. A. On the stereostructures of (+)-eupenoxide and (−)-3′,4′-

406

dihydrophomoxide: a caveat on the spectral comparisons of oxygenated cyclohexenoids. Tetra.

407

Lett. 2008, 49, 5162–5164.

408 409 410 411

(22) Liu, Z. M.; Jensen, P. R.; Fenical, W. A cyclic carbonate and related polyketides from a marine-derived fungus of the genus Phoma. Phytochemistry 2003, 64, 571–574. (23) Davis, R. A.; Andjic, V.; Kotiw, M.; Shivas, R. G. Phomoxins B and C: Polyketides from an endophytic fungus of the genus Eupenicillium. Phytochemistry 2005, 66, 2771–2775.

412

(24) Wei, J.; Liu, L. L.; Dong, S.; Li, H.; Tang, D.; Zhang, Q.; Xue, Q. H.; Gao, J. M.

413

Gabosines P and Q, new carbasugars from Streptomyces sp. and their α-glucosidase inhibitory

414

activity. Bioorg. Med. Chem. Lett. 2016, 26, 4903–4906.


19


415

Figure captions:

416

Figure 1. Chemical structures of compounds 1−7 and 4a.

417

Figure 2. Selected HMBC and 1H−1H COSY correlations of compound 1.

418

Figure 3. X-ray crystallographic structure of compound 1.

419

Figure 4. X-ray crystallographic structure of compound 4a.


Page 20 of 26

20

Page 21 of 26


Table 1. 1H NMR Data of Compounds 1− −7 and 4a (δ in ppm, J in Hz, 500 MHz) a

a

a

a

a

b

b

b

position 1

2

3

4

4a

5

6

7

3

4.22, d (6.1)

3.83, d (5.5)

4.18, d (6.7)

4.66, br s

4.20, d (7.5)

4.37, br s

4.46, br s

4.56, br s

4

3.55, dd (9.3, 6.9)

3.36, dd (10.4, 7.6) 3.46, dd (10.1, 7.2) 3.52, t (3.4)

3.57, dd (11.0, 7.5) 3.57, t (2.8)

3.57, br s

3.57, t (3.2)

5

3.48, dd (9.3, 7.4)

3.31, dd (10.4, 7.3) 3.50, dd (10.1, 7.0) 3.45, t (2.6)

3.82, dd (11.0, 8.0 ) 3.52, t (2.5)

3.54, br s

3.52, t (2.8)

6

4.26, d (6.6)

4.15, d (7.1)

4.06, d (7.0)

4.24, d (8.0)

4.49, br s

4.48, br s

7

4.37, br s

4.10, br s

4.78, dd (12.8, 3.9) 4.39, d (12.1)

4.79, dd (13.0, 4.0) 4.34, d (11.7)

4.86, br d (9.8)

4.76, ddd (9.4, 3.2, 2.4)

4.61, dd (12.8, 4.5) 4.22, d (12.1)

4.62, d (13.0)

3.99, d (11.7)

4.53, d (9.8)

4.51, d, (9.4)

5.27, br s

5.27, br s

6.14, d (11.3)

5.28, br s

5.27, br s

5.95, d (11.3)

4.58, br s

6.38, d (16.0)

4.61, br s

1'

6.45, d (15.9)

2'

5.94, dd (15.9, 6.5) 5.53, dd (11.3, 6.4) 5.46, dd (15.3, 7.5) 5.98, dd (16.0, 6.6) 5.44, dd (15.5, 7.5) 5.69, dd (11.3, 8.8) 5.39, dd (15.1, 7.8) 2.93, dd (15.8, 5.2) 2.73, dd (15.8, 5.7)

3'

4.13, q (6.5)

4.01, q (6.4)

5.82, dt (15.3, 6.8) 4.13, q (6.4)

5.81, dt (15.0, 7.0)

4.06, q (6.7)

5.84, dt (15.1, 6.4)

4'

1.55−1.59, m

1.31−1.33, m

2.08, q (6.8)

1.53−1.57, m

2.09, q (7.0)

1.53−1.57, m

2.05, d (6.7)

2.49, t (7.4)

5'

1.40−1.42, m

1.20−1.28, m

1.40−1.42, m

1.40−1.42, m

1.41−1.43, m

1.47−1.50, m

1.36−1.38, m

1.53−1.54, m

6'–10'

1.33−1.40, m

1.20−1.28, m

1.32−1.40, m

1.33−1.40, m

1.32−1.40, m

1.31−1.40, m

1.26−1.35, m

1.23−1.30, m

11'

0.92, t (5.3)

0.80, t (6.3)

0.92, t (6.3)

0.92, t (6.5)

0.92, t (6.5)

0.87, t (6.5)

0.88, t (6.6)

0.88, t (6.6)

a

Measured in CD3OD. bMeasured in CDCl3.


21


Page 22 of 26

−7 and 4a (δ in ppm, 125 MHz) Table 2. 13C NMR Data of Compounds 1− position 1a

2a

3a

4a

4a

1

136.8

135.9

136.1

134.6

2

135.9

135.6

137.5

3

74.6

76.9

4

76.7

5

a

5b

6b

7b

135.8

132.0

135.1

133.1

134.1

135.0

133.2

132.6

134.6

71.6

66.7

69.8

67.7

62.8

63.2

77.3

78.7

56.6

76.8

54.5

55.3

54.9

76.3

77.1

79.2

55.9

68.5

55.6

58.3

57.3

6

74.5

75.2

71.4

65.3

70.5

67.1

61.7

61.4

7

59.7

61.4

75.5

60.7

73.6

64.2

75.0

75.3

1'

126.3

128.4

88.4

126.1

86.7

128.5

87.3

82.5

2'

139.0

138.2

129.9

139.5

127.9

136.9

127.9

46.9

3'

74.4

69.2

135.5

74.4

134.0

68.4

135.9

211.5

4'

38.8

38.3

33.6

38.8

31.8

37.4

32.9

44.6

5'

27.0

26.9

30.6

27.0

28.7

25.9

30.1

23.9

6'

31.5

31.2

31.1

31.2

29.1

30.1

29.5

32.2

7'

31.2

31.1

31.0

31.2

29.0

30.0

29.5

29.7

8'

30.8

30.8

30.8

30.8

28.8

29.9

29.3

29.5

9'

33.5

33.5

33.5

33.5

31.6

32.3

32.8

29.5

10'

24.1

24.1

24.1

24.1

22.3

23.0

23.1

23.0

11'

14.9

14.8

14.8

14.9

13.1

14.5

14.5

14.4

a

Measured in CD3OD. bMeasured in CDCl3.


22

Page 23 of 26


−7 Table 3. α-Glucosidase Inhibitory, Cytotoxic, and Antifungal Activities of Compounds 1− and 4a α-Glucosidase

Cytotoxicity

Antifungal

Inhibitions

(IC50, µM)

(MIC, µg/mL)

Compds.

(IC50, mM)

H460

Hela

MCF-7

Caa

Anb

Poc

Fad

Hce

1

1.47

11.22

13.29

>40

256

128

256

512

128

2

1.55

19.34

9.10

>40

32

64

256 >512

64

3

1.83

6.73

>40

>40

128

256

128

512

256

4

2.76

7.37

8.15

12.77

64

64

256

512

256

4a

2.24

6.26

13.70

>40

16

32

256

256

128

5

2.88

15.11

16.14

11.88

128

256

512 >512

128

6

3.16

9.06

6.25

8.57

128

>512

256

512

256

7

2.94

15.38

8.87

14.24

64

512

256 >512

128

acarbose

1.22

NT

f

NT

NT

NT

NT

NT

NT

NT

paclitaxel

NT

0.06

0.04

0.05

NT

NT

NT

NT

NT

nystatin

NT

NT

NT

NT

8

8

8

16

8

a

Candida albicans YM 2005; bAspergillus niger YM 3029; c Pyricularia oryzae YM 3051;

d

Fusarium avenaceum YM 3065; eHormodendrum compactum YM 3077. f

NT not tested


23


Page 24 of 26

Figure 1. Chemical structures of compounds 1−7 and 4a.

Figure 2. Selected HMBC and 1H−1H COSY correlations of compound 1.


24

Page 25 of 26


Figure 3. X-ray crystallographic structure of compound 1.

Figure 4. X-ray crystallographic structure of compound 4a.


25



Page 26 of 26

Polyoxygenated Cyclohexenoids with Promising Î±-Glycosidase

Recommend Documents