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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
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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.
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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
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INTRODUCTION
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Endophytic fungi have gained much more attention from natural product chemists because of
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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
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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.
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MATERIALS AND METHODS
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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).
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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
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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
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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
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days, each 30 mL of the seed culture solution was added into 1 L Erlenmeyer flasks (×150)
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containing 300 mL of PDB medium. The following cultivation was carried out at 28 °C with
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shaking (200 rpm) for 7 days. The culture broth was filtered and concentrated to 10 L, and
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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
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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
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reversed-phase silica gel C18 (MeOH/H2O, gradiently from 2:8 to 4:6) to give compound 1 (15.5
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mg) and compopund 2 (7.4 mg).
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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,
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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;
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HRESIMS m/z 367.2094 [M + Na]+ (calcd for C18H32O6Na, 367.2097); 1H and
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spectral data, see Tables 1 and 2.
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C NMR
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Phomopoxide C (3): white powder; TLC Rf 0.48 (CHCl3/MeOH, 85:15); [α]20 D −35.1 (c 0.23,
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MeOH); IR (KBr) νmax 3405 (br), 3377, 2955, 2926, 2856, 1642, 1403, 1251, 1090, 994 cm−1;
97
HRESIMS m/z 349.1993 [M + Na]+ (calcd for C18H30O5Na, 349.1991); 1H and
98
spectral data, see Tables 1 and 2.
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C NMR
99
Phomopoxide D (4): white powder; TLC Rf 0.23 (PE/acetone, 6:4); [α] 20 D −68.3 (c 0.38,
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MeOH); UV (MeOH) λmax (log ε) 237 (4.25) nm; CD (c 0.27, MeOH) λmax (∆ε) 234 (−9.87) nm;
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IR (KBr) νmax 3366 (br), 2957, 2925, 2855, 1638, 1465, 1257, 1087, 1030, 966 cm−1; HRESIMS
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m/z 349.1985 [M + Na]+ (calcd for C18H30O5Na, 349.1991); 1H and 13C NMR spectral data, see
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Tables 1 and 2.
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Phomopoxide E (5): white powder; TLC Rf 0.37 (PE/acetone, 6:4); [α]20 D +9.1 (c 0.24, MeOH);
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UV (MeOH) λmax (log ε) 196 (4.02) nm; CD (c 0.64, MeOH) λmax (∆ε) 204 (−4.65), 233 (−1.08)
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nm; IR (KBr) νmax 3346 (br), 3194, 2962, 2932, 2858, 1633, 1495, 1252, 1098, 1012, 960 cm−1;
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HRESIMS m/z 349.1997 [M + Na]+ (calcd for C18H30O5Na, 349.1991); 1H and
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spectral data, see Tables 1 and 2.
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C NMR
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Phomopoxide F (6): colorless gum; TLC Rf 0.51 (PE/acetone, 6:4); [α] 20 D −28.7 (c 0.17,
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MeOH); IR (KBr) νmax 3446, 3367 (br), 2950, 2924, 2851, 1464, 1412, 1347, 1258, 1119, 1090,
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971 cm−1; HRESIMS m/z 331.1887 [M + Na]+ (calcd for C18H28O4Na, 331.1885); 1H and
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NMR spectral data, see Tables 1 and 2.
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C
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Phomopoxide G (7): colorless gum; TLC Rf 0.47 (PE/acetone, 6:4); [α] 20 D −15.1 (c 0.21,
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MeOH); IR (KBr) νmax 3451 (br), 3387, 2956, 2927, 2856, 1711, 1466, 1408, 1351, 1271, 1120,
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1038, 943 cm−1; HRESIMS m/z 347.1837 [M + Na]+ (calcd for C18H28O5Na, 347.1834); 1H and
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13
C NMR spectral data, see Tables 1 and 2.
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Chemical Transformation of 4 to 4a. 50 µL concentrated HCl (37%) was added into the
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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.
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The obtained solution was washed sufficiently with 10 mL saturated NaHCO3 for three times and
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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).
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Compound 4a: colorless crystals; TLC Rf 0.56 (PE/acetone, 6:4); mp 173−174 °C; [α]20 D −54.7
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(c 0.24, MeOH); IR (KBr) νmax 3358, 2953, 2916, 2852, 1629, 1469, 1217, 1128, 1100, 963 cm−1;
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HRESIMS m/z 367.1652 [M + Na]+ (calcd for C18H29O4ClNa, 367.1652); 1H and
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spectral data, see Tables 1 and 2.
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C NMR
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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.
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Crystallographic data for 1: C18H32O6·H2O, Mr = 362.45, orthorhombic, crystal dimensions
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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
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= 1908.24(3) Å3, α = 90.00°, β = 90.00°, γ = 90.00°, Z = 4, Dcalcd = 1.262 mg/m3, F(000) = 792,
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µ(CuKα) = 0.790 mm-1, Tmin/Tmax = 0.54/0.93, 2.11° ≤ θ ≤ 69.62°, 11914 reflections measured,
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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).
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Crystallographic data for 4a: C18H29ClO4· H2O, Mr = 362.88, monoclinic, crystal dimensions
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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
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= 90°, β = 93.657(2)°, γ = 90°, V = 942.9(2) Å3, Z = 2, Dcalcd=1.278 mg/m3, F(000)= 392,
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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
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and wR(F2) = 0.0811 (all data), goodness of fit on F2 = 1.133. The Flack parameter is 0.016(17).
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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.
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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
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values were calculated by using Reed and Muench’s method.19
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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.
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RESULTS AND DISCUSSION
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Structural Elucidation. The current chemical investigations on fermentation product of
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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
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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.
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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
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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
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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
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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
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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
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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
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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,
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structural diversity, bioactivity, and implication of their occurence. J. Nat. Prod. 2006, 69, 509–
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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.
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(5) Li, X. J.; Zhang, Q.; Zhang, A. L.; Gao, J. M. Metabolites from Aspergillus fumigatus, an
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endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic
370
activities. J. Agric. Food Chem. 2012, 60, 3424–3431.
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(6) Xiao, J.; Zhang, Q.; Gao, Y. Q.; Shi, X. W.; Gao, J. M. Antifungal and antibacterial
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metabolites from an endophytic Aspergillus sp. associated with Melia azedarach. Nat. Prod. Res.
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2014, 28, 1388–1392.
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(7) Udayanga, D.; Liu, X. Z.; McKenzie, E. H. C.; Chukeatirote, E.; Bahkali, A. H. A.; Hyde,
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K. D. The genus Phomopsis: biology, applications, species concepts and names of common
376
phytopathogens. Fungal Divers. 2011, 50, 189–225.
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(8) Wu, S. H.; Chen, Y. W.; Shao, S. C.; Wang, L. D.; Li, Z. Y.; Yang, L. Y.; Li, S. L.; Huang,
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R. Ten-membered lactones from Phomopsis sp., an endophytic fungus of Azadirachta indica. J.
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Nat. Prod. 2008, 71, 731–734.
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(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,
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8, 1717–1723.
391
(14) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. 2008, A64, 112–122.
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(15) Flack, H. D. On enantiomorph-polarity estimation. Acta Crystallogr. 1983, A39, 876–881.
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(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.
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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.
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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.
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−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.
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Journal of Agricultural and Food Chemistry
−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
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Figure 1. Chemical structures of compounds 1−7 and 4a.
Figure 2. Selected HMBC and 1H−1H COSY correlations of compound 1.
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Journal of Agricultural and Food Chemistry
Figure 3. X-ray crystallographic structure of compound 1.
Figure 4. X-ray crystallographic structure of compound 4a.
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