Seven new drimane-type sesquiterpenoids from a marine-derived fungus paraconiothyrium sporulosum YK-03

Article abstract of DOI:10.3390/molecules24091817

Seven new drimane-type sesquiterpenoids, namely the sporulositols A-D (1-4), 6-hydroxydiaporol (5), seco-sporulositol (6) and sporuloside (7) were isolated from the ethyl acetate extract of fermentation broth for a marine-derived fungus Paraconiothyrium sporulosum YK-03. Their structureswere elucidated by analysis of extensive spectroscopic data, and the absolute configurations were established by crystal X-ray diffraction analysis and comparisons of circular dichroism data. Among them, sporulositols A-E (1-4) and seco-sporulositol (6) represent the first five examples of a unique class of drimanic mannitol derivatives, while compounds 6 and 7 may represent two new series of natural drimanes, possessing an aromatic ring with a rare 4,5-secodrimanic skeleton and an unusual CH3-15 rearranged drimanic α-D-glucopyranside, respectively. Furthermore, the origin of mannitol moiety was investigated by reliable HPLC and NMR analyses.

Full text of DOI:10.3390/molecules24091817

molecules
Article
Seven New Drimane-Type Sesquiterpenoids from
a Marine-Derived Fungus Paraconiothyrium
sporulosum YK-03
Li-Hua Zhang 2,3, Gang Chen 2, Yi Sun 4, Hai-Feng Wang 2 , Jiao Bai 2, Hui-Ming Hua 2
and Yue-Hu Pei 1,
*
1
Department of Medicinal Chemistry and Natural Medicine Chemistry, College of Pharmacy, Harbin Medical
University, Harbin 150081, China
2
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, China; zhanglihua200061@163.com (L.-H.Z.); chengang1152001@hotmail.com (G.C.);
wanghaifeng0310@163.com (H.-F.W.); baijiao@hotmail.com (J.B.); huimhua@163.com (H.-M.H.)
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;
sylotus@hotmail.com
3
4
*
Correspondence: peiyueh@vip.163.com; Tel.: +86-0451-86685745
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 April 2019; Accepted: 30 April 2019; Published: 10 May 2019
Abstract: Seven new drimane-type sesquiterpenoids, namely the sporulositols
A–D (1–4),
6-hydroxydiaporol (5), seco-sporulositol (6) and sporuloside (7) were isolated from the ethyl acetate
extract of fermentation broth for a marine-derived fungus Paraconiothyrium sporulosum YK-03. Their
structures were elucidated by analysis of extensive spectroscopic data, and the absolute configurations
were established by crystal X-ray diffraction analysis and comparisons of circular dichroism data.
Among them, sporulositols
unique class of drimanic mannitol derivatives, while compounds
series of natural drimanes, possessing an aromatic ring with a rare 4,5-secodrimanic skeleton and an
unusual CH3-15 rearranged drimanic -D-glucopyranside, respectively. Furthermore, the origin of
A
–E
(1
–4) and seco-sporulositol (
6
) represent the first five examples of a
6
and may represent two new
7
α
mannitol moiety was investigated by reliable HPLC and NMR analyses.
Keywords: Paraconiothyrium; Paraconiothyrium sporulosum; drimane-type sesquiterpenoid;
sporulositol; seco-sporulositol; sporuloside
1. Introduction
Marine fungi afforded chemically diverse and pharmacologically active metabolites, and have
become a remarkable source of marine drugs [
by Verkley et al. through analysis of 18S rRNA and ITS sequences [
plant pathogen similar to Phoma and rarely as a human pathogen [ ]. Verkley proposed that two
Coniothyrium species (C. fuckelii and C. sporulosum) should be combined into the genus Paraconiothyrium,
and united as P. sporulosum [ ]. So far, structural diverse terpenoids including brasilamides A–N [ ],
hawaiinolides A–G [10 11], epoxyphomalins A–E [12], sporulaminals A–B [13] and six isopimarane
1–
4]. Paraconiothyrium genus is a new genus specified
5], and has been known as a
6
5
,
6
7–9
,
diterpenoid glycosides [14], and several polyketides [15–26] have been identified from the genus
Paraconiothyrium.
Drimanes, a type of sesquiterpenoid with a bicyclic scaffold, are widely distributed in plants,
liverworts, fungi and certain marine organisms (sponges), possessing diverse structural features [27 28
33],
,
]
and extensive biological activities, such as antibacterial [29,30], antifungal [31,32], antiviral [29,
Molecules 2019, 24, 1817; doi:10.3390/molecules24091817
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1817
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cytotoxic [34
,
35], antifeedant [36
,
37], plant-growth [38
,
39], and so on. Natural rearranged drimanes
only occurred with a 1,2 methyl shift from C-4 to C-3 [40]. In 2013, the first drimanic compound with
an aromatic ring was synthesized [41], while a synthesized seco-drimanic compound was reported for
the first time in 2016 [42]. Because of their interesting structural features and bioactivities, they have
attracted increasing attention of biologists and chemists for further research [36,41,43].
In course of our continuous exploration for novel marine natural products, seven new drimane-type
sesquiterpenoids (Figure 1), namely sporulositols
A–D (1–4), 6-hydroxydiaporol (5), seco-sporulositol
(6
) and sporuloside ( ), were isolated from the ethyl acetate extract of a fermentation broth of a
7
marine-derived fungus P. sporulosum YK-03 (Genbank Accession Number KC416199), which was
collected from the sea mud in the intertidal zone of Bohai Bay (Liaoning Province, China). Among them,
sporulositols A–D (1–4) and seco-sporulositol (6) represent the first five examples of unique drimanic
mannitol derivatives, seco-sporulositol (
a CH3-15 rearrangement derivative of drimanic glucoside. Compounds
new series of natural drimanes with an aromatic ring (compound ), or a drimane-type sesquiterpene
glycoside with an -D-glucose moiety (compound ). Herein, we report the isolation and structure
6
) possesses a 4,5-secodrimanic skeleton, and sporuloside (
7) is
6
and may represent two
7
6
α
7
elucidation of the isolated compounds, together with the origin of mannitol moiety.
Figure 1. The structures of 1–7.
2. Results and Discussion
The fermentation broth of P. sporulosum YK-03 was concentrated and extracted with ethyl acetate
and n-butanol, successively. Then the ethyl acetate extract of the fermentation broth of P. sporulosum
YK-03 was subjected to various modern chromatographic isolation methods (including silica
gel/Sephadex LH-20 column chromatography and reversed-phase C18 preparative high performance
liquid chromatography) to give seven new compounds 1−7 (4.3 mg, 93.3 mg, 89.8 mg, 2.6 mg, 2.3 mg,
2.5 mg and 2.0 mg). Their structures and the absolute configurations were elucidated by analysis of
HRESIMS, 1D/2D NMR, circular dichroism (CD) and X-ray diffraction analyses.
2.1. Structural Elucidation
Sporulositols
A–D (1–4) and seco-sporulositol (6) were isolated as colorless oils. Their respective
molecular formulas of C21H38O6, C21H38O7, C21H38O7, C21H38O8, and C21H34O6 were established by
analyses of their positive HRESIMS data, in which the sodium adduct ions ([M + Na]+) peaks appeared
at m/z 409.2568, m/z 425.2512, m/z 425.2512, m/z 441.2452, and m/z 405.2239, respectively. Further,

Molecules 2019, 24, 1817
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the above determination of molecular formulas was supported by analysis of the NMR data (Tables 1
and 2), indicating three, three, three, three, and five indices of hydrogen deficiency, respectively, for
1–4
and 6. The IR spectra of sporulositols A–D (1–4) and seco-sporulositol (6) showed the presence of
hydroxyl (νmax 3384.7–3416.6 cm−1) and olefinic (νmax 1632.0–1659.0 cm−1) groups in their structures,
and beyond these, there was an aromatic (νmax 1597.8, 1554.4, and 1432.7 cm−1) group in 6.
The NMR spectra (Tables 1 and 2) of
moiety (δH 3.51–3.54, 3.40–3.42/δC 62.9–63.0, δH 3.65–3.68/δC 72.6–72.8, δH 3.54–3.57/δC 77.6–77.7, δH
3.50–3.51/δC 71.0–71.1, δH 3.49–3.51/δC 71.3–71.4, and δH 3.60–3.61, 3.35–3.36/δC 63.9–64.0 for δH
3.56, 3.43/δC 63.0, δH 3.72/δC 72.3, δH 3.76/δC 78.1, δH 3.58/δC 70.9, δH 3.54/δC 71.3, and δH 3.63, 3.41/δC
63.8 for ) and the corresponding free hydroxyl groups (δH 4.20–4.77), indicating that the hexitol
moieties existed in and share the same structures with similar configurations, and the same
etherification positions with the remaining skeletons. The deduction was also supported by the HMBC
1–4 and 6 all displayed very similar signals for a hexitol
1–4;
6
1–
4
6
correlations (Figure 2) of 1
from δH 3.68 (H-20) to δC 63.0 (C-10), 77.6 (C-30) and 71.1 (C-40); from δH 4.57
(HO-10) to δC 72.8 (C-20); from δH 4.72 (HO-20) to δC 63.0, 72.8 and 77.6; from 3.57 (H-30) to δC 63.0, 72.8
and 71.4 (C-50); from δH 3.68 (H-20) to δC 63.0, 77.6 and 71.4 (C-50); from δH 4.20 (HO-40) to δC 77.6, 71.1
and 71.4; from δH 3.61, 3.35 (H2-60) to δC 71.4 and from δH 4.32 (HO-60) to δC 71.4 and 64.0 (C-60).
Figure 2. The key HMBC correlations for 1–7.
The remaining 13C-NMR data (Table 1) of
methyls (δC 19.8, 20.7, 21.5, 33.1), six methylenes (δC 18.6, 18.7, 33.3, 36.1, 41.4, 66.8), one methine
δC 51.1) and four quaternary carbons (δC 33.2, 37.5, 131.7, 138.4), supported by analyses of the 1H-NMR
1 included fifteen carbon signals, attributable to four
(
and HSQC data. Among these carbons, there were one oxygenated methylene {δH 4.00 (d, J = 15.0 Hz),
4.02 (d, J = 15.0 Hz)/δC 66.8} and one methyl {δH 1.65 (s)/δC 19.8} located at a pair of olefinic carbons (δC
131.7 and 138.4). The 11-hydroxyldrimane-8-en moiety in 1 was then established by HMBC correlations
(Figure 2) from δH 4.00, 4.02 (H2-11) to δC 131.7 (C-8), δC 138.4 (C-9) and δC 37.5 (C-10), from δH 1.65
(H3-12) to δC 33.3 (C-7), C-8 and C-9, from δH 0.92 (H3-15) to δC 36.1 (C-1), C-9 and C-10, from δH
1.05 (H-5) to C-1, C-9, C-10, δC 33.2 (C-4), δC 21.5 (C-13) and δC 18.6 (C-6), from δH 2.00, 2.01 (H2-7) to
C-6, C-8, C-9 and δC 51.1 (C-5), from δH 1.89, 1.18 (H2-1) to C-10 and δC 18.7 (C-2), from δH 1.36, 1.11
(H2-3) to C-2, C-4 and δC 21.5 (C-13), and from δH 0.81 (H3-13), 0.86 (H3-14) to C-4. Then, the HMBC
correlations from δH 3.57 (H-30) to δC 66.8 (C-11) and from δH 4.00, 4.02 (H2-11) to δC 77.6 (C-30)
established the above deduced hexitol and drimane-type sesquiterpenoid moieties to afford the planar
structure of 1 through C11-O-C3.

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Table 1. NMR spectroscopic data of 1–5 a.
2a b
δC
36.9
3a b
δC
1
2
3
4
5
No.
δC
δH, mult (J, Hz)
δC
36.7
δH, mult (J, Hz)
δH, mult (J, Hz)
δC
δH, mult (J, Hz)
δH, mult (J, Hz)
δC
δH, mult (J, Hz)
δC
δH, mult (J, Hz)
1
2
3
36.1
1.89, d (13.2);
1.18, td (13.2, 2.4)
1.55, m;
1.43, m
1.36, m;
1.84, d (13.2);
1.20, td (13.2, 3.0)
1.71, d (12.6);
1.18, td (13.2, 3.6)
1.50, qt (13.2, 3.6); 18.5
1.38, dt (13.2, 3.6)
1.29, d (13.2);
1.11, m
36.2
1.89, d (13.2);
1.19, dd (10.8, 3.6)
1.49, m;
1.38, m
1.77, d (13.2);
0.78, td (13.2, 3.6)
36.4
18.6
35.4
1.74, d (13.2);
1.16, d (13.2)
1.50, m;
1.37, m
1.77, d (13.2);
0.78, td (13.2, 3.6)
35.8
1.88, d (13.2);
1.17, td (13.2, 3.6)
1.55, m;
1.39, m
1.78, d (13.8);
0.82, td (13.2, 3.6)
37.0
18.6
38.1
1.78, m;
1.29, m
1.45, m;
1.33, m
1.58, d (13.2);
0.90, td (13.2, 4.0)
18.7
41.4
18.6
43.5
1.51, qt (13.6, 3.8); 18.9
1.37, dt (13.6, 3.5)
18.5
35.2
1.28, d (13.2);
1.10, d (10.8)
43.9
35.1
1.11, td (12.6, 3.6)
4
5
6
33.2
51.1
18.6
33.4
55.7
66.1
33.8
56.1
66.5
38.4
51.9
18.7
38.7
52.2
19.0
38.0
45.4
28.8
38.7
56.5
66.9
1.05, d (12.6)
1.61, m;
1.37, m
2.00, m;
2.01, m
1.09, d (11.1)
3.88, m
1.08, d (10.8)
3.89, m
1.14, d (12.6)
1.67, m;
1.36, m
1.94, m;
1.95, m
1.13, d (12.6)
1.68, m;
1.37, m
1.49, d (13.2)
1.68, d (12.6);
1.57, dd (12.6, 4.8)
3.69, m
1.21, d (11.2)
3.96, m
7
33.3
45.0
2.29, dd (17.8, 6.3); 45.2
2.30, dd (18.0, 6.0); 33.8
33.9
1.95, m
68.2
44.5
2.20, dd (17.2, 6.0);
1.94, dd (17.2, 9.6)
1.98, dd (17.8, 8.9)
1.96, dd (18.0, 9.0)
8
9
10
11
131.7
138.4
37.5
130.4
138.2
40.0
131.0
137.6
40.5
131.6
138.4
37.5
131.9
137.8
37.7
133.5
140.6
38.2
128.5
140.8
40.4
66.8
4.02, d (15.0);
4.00, d (15.0)
1.65, s
66.7
3.98, d (16.7);
3.97, d (16.7)
1.64, s
67.8
3.80, d (10.5);
3.69, d (10.5)
1.59, s
66.8
4.00, d (12.6);
3.99, d (12.6)
1.63, s
3.52, m;
3.14, m
0.87, s
0.89, s
67.9
3.80, d (10.2);
3.68, d (10.2)
1.57, s
3.52, m;
3.13, m
0.87, s
0.88, s
66.8
4.02, d (10.2);
3.99, d (10.2)
1.74, s
3.54, m;
3.13, dd (10.8, 5.4)
0.86, s
56.5
3.91, dd (11.6, 4.4);
3.81, dd (11.6, 4.8)
1.61, s
3.78, dd (10.4, 4.8);
3.37, dd (10.4, 4.4)
1.13, s
12
13
19.8
21.5
19.3
22.0
19.4
22.4
19.7
62.6
19.6
62.9
17.6
62.8
19.2
65.1
0.81, s
0.99, s
0.94, s
14
15
33.1
20.7
0.86, s
0.92, s
36.5
22.0
1.12, s
0.94, s
36.8
22.3
1.13, s
0.94, s
27.3
21.3
27.6
21.4
27.0
19.5
31.4
22.9
0.85, s
0.98, s
4.43, d (4.8)
4.11, t (4.8)
6-OH
7-OH
13-OH
10
4.22, d (6.6)
4.56, d (5.4)
4.16, t (5.4)
3.51, m;
3.42, m
3.67, m
3.57, d (4.8)
3.51, m
3.51, m
3.61, dd (9.6, 6.0);
3.36, m
4.61, t (5.4)
4.77, d (5.4)
4.25, br d (4.8)
4.42, br d (4.8)
4.34, t (5.4)
4.14, t (4.8)
3.53, m;
3.40, m
3.66, m
3.55, d (4.8)
3.50, m
3.50, m
3.60, m;
3.36, m
4.54, t (4.8)
4.70, d (4.8)
4.20, d (4.8)
4.36, br s
4.30, t (4.8)
63.0
3.54, dd (10.2, 4.8); 63.0
3.41, m
3.52, m;
3.40, m
3.65, m
3.54, d (4.8)
3.51, m
3.49, m
3.60, d (10.8);
3.36, m
4.57, br s
4.72, br s
4.23, d (5.4)
4.38, br s
4.35, br s
63.0
62.9
20
30
40
50
60
72.8
77.6
71.1
71.4
64.0
3.68, m
3.57, d (4.8)
3.51, m
72.6
77.7
71.0
71.3
63.9
72.7
77.6
71.0
71.3
63.9
72.8
77.6
71.1
71.3
63.9
3.51, m
3.61, dd (10.2,
4.2); 3.35, m
4.57, t (5.4)
4.72, d (4.8)
4.20, br s
1’-OH
2’-OH
4’-OH
5’-OH
6’-OH
4.37, br s
4.32, t (5.4)
a
b
The spectra were recorded at 600 (1H) and 100 MHz (13C) in DMSO-d6; Assignments were made by a combination of 1D and 2D NMR experiments. 2a and 3a was obtained by acid
hydrolysis of 2 and 3, respectively.

Molecules 2019, 24, 1817
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Table 2. NMR spectroscopic data of 6 and 7.
6 a
7 b
No.
δC
δH, mult (J, Hz)
δC
δH, mult (J, Hz)
1
2
29.2
29.5
2.71, t (8.4)
2.05, m
28.2
19.2
2.53, t (8.4)
1.72, m
1.84, m
1.44, m
3
124.3
5.25, t (6.0)
33.0
4
5
6
7
8
131.0
134.9
127.6
129.7
140.2
135.6
133.2
37.8
139.2
126.1
133.0
132.1
134.1
132.7
6.90, d (7.8)
6.98, d (7.8)
7.01, s
2.07, s
9
10
4.67, d (10.2)
4.60, d (10.2)
2.25, s
11
12
13
67.4
19.5
25.6
15.8
15.9
75.6
2.09, s
5.59, d (9.2)
3.24, d (9.2)
1.25, s
1.66, s
14
15
17.5
19.7
1.55, s
2.34, s
27.1
21.0
2.18, s
3.56, m
3.43, m
3.72, m
3.76, d (5.1)
3.58, m
10
63.0
99.3
4.64, d (3.6)
20
30
40
50
72.3
78.1
70.9
71.3
72.6
73.7
70.7
73.3
3.20, m
3.40, m
3.05, td (9.1, 5.2)
3.28, m
3.54, m
3.63, m
3.41, m
3.56, m
3.40, m
60
63.8
61.4
1’-OH
2’-OH
3’-OH
4’-OH
5’-OH
6’-OH
4.59, t (5.4)
4.75, d (5.4)
4.60, d (6.4)
4.74, d (4.8)
4.85, d (5.2)
4.30, d (5.4)
4.40, d (5.4)
4.35, t (5.4)
4.38, t (6.0)
a
b
The NMR spectra were recorded at 600 (1H) and 100 MHz (13C) in DMSO-d6. The NMR spectra were recorded at
400 (1H) and 100 MHz (13C) in DMSO-d6. Assignments were made by a combination of 1D and 2D NMR experiments.
The NMR data (Table 1) of
2.29, 1.98/45.0 (CH2-7), 0.99/22.0 (CH3-13), 1.12/36.5 (CH3-14), 0.94/22.0 (CH3-15) and δC 130.4 (C-8),
40.0 (C-10), were quite different from those of , indicating that C-6 was hydroxylated in due to
2 at δH/δC 1.28, 1.10/43.5 (CH2-3), 1.09/55.7 (CH-5), 3.88/66.1 (-O-CH-6),
1
2
the significant downfield shift of δC 66.1. The assignment was further confirmed by the key HMBC
correlations (Figure 2) from δH 4.22 (HO-6) to δC 55.7 (C-5) and 66.1 (C-6), from both δH 1.09 (H-5)
and δH 2.29, 1.98 (H-7) to C-6. Finally, the HMBC correlation from δH 3.54 (H-30) to δC 66.7 (C-11)
indicated that C-11 of the 6,11-dihydroxyldrimane-8-en moiety and C-3’ of the hexitol formed the
planar structure of 2 by an O-ether bridge.
Comparison of NMR data (Table 1) of
was hydroxylated instead of CH2-6 in , in clue of the differences at δH
1.14/51.9 (CH-5), 3.52, 3.14/62.6 (-O-CH2-13), 0.87/27.3 (CH3-14), 0.89/21.3 (CH3-15) and δC 38.4 (C-4).
The deduction was also supported by the key HMBC correlations (Figure ) from δH 4.14 (HO-13) to
3
with those of
1
and
2
revealed that CH3-13 of
3
2
/
δC 1.77, 0.78/35.1 (CH2-3),
2
δC 38.4 (C-4) and from δH 3.52, 3.14 (H2-13) to δC 35.1 (C-3) and C-4. Further, the HMBC correlation of
δH 3.55 (H-30)/δC 66.8 (C-11) suggested that the 11,13-dihydroxyldrimane-8-en (viz. diaporol [35]) and
hexitol moieties in 3 formed the planar structure in the same way as compounds 1 and 2.
Different from NMR data (Table 1) of
3, the signals in the NMR data of 4 at δH/δC 1.49/45.4 (CH-5),
1.68, 1.57/28.8 (CH2-6), 3.69/68.2 (-O-CH-7), 1.74/17.6 (CH3-12), 0.85/19.5 (CH3-15) and δC 133.5 (C-8),

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140.6 (C-9), 38.2 (C-10) suggested that CH2-7 was hydroxylated in
4
, which was also supported by
the key HMBC correlations from δH 4.56 (HO-7) to δC 68.2 (C-7) and C-8, from δH 3.69 (H-7) to C-8,
and from both δH 1.74 (H3-12) and δH 1.68, 1.57 (H2-6) to C-7. The planar structure of
4 was finally
established by HMBC correlation (Figure 2) from δH 3.57 (H-30) to δC 66.8 (C-11), combining the
7,11,13-trihydroxyldrimane-8-en and hexitol moieties in 4 at the same positions as compounds 1–3.
6-Hydroxydiaporol (
determined by analyses of its NMR data (Table 1) and positive HRESIMS (m/z 277.1814 [M + Na]+, calcd
for C15H26O3, 254.1882) data. Based on HSQC correlations, NMR spectra of showed the presence of
5) was obtained as a colorless oil. Its molecular formula of C15H26O3 was
5
three methyls (δH 1.61/δC19.2, δH 1.13/δC 31.4, and δH 0.98/δC 22.9), six methylenes (δH 1.78, 1.29/δC
37.0, δH 1.45, 1.33/δC 18.6, δH 1.58, 0.90/δC 38.1, δH 2.20, 1.94/δC 44.5, δH 3.91, 3.81/δC 56.5, and δH
3.78, 3.37/δC 65.1), two methines (δH 1.21/δC 56.5 and δH 3.96/δC 66.9) and four quaternary carbons
(δC 38.7, 40.4, 128.5, 140.8). Among the deduced groups, there were two oxygenated methylenes {δH
3.91 (d, J = 11.6, 4.4 Hz), 3.81 (d, J = 11.6, 4.8 Hz)/δC 56.5 and δH 3.78 (d, J = 10.4, 4.8 Hz), 3.37 (d,
J = 10.4, 4.4 Hz)/δC 65.1} and one oxygenated methine {δH 3.96 (m)/δC 66.9}. Then, the planar structure
of 5 was constructed by the key HMBC correlations (Figure 2) from δH 1.61 (H3-12) to δC 44.5 (C-7),
128.5 (C-8) and 140.8 (C-9), from δH 3.91, 3.81 (H2-11) to C-8, C-9 and δC 40.4 (C-10), from δH 4.11
(HO-11) to δC 140.8, from δH 0.98 (H3-13) to δC 140.8, 40.4 and 37.0 (C-1), from δH 1.21 (H-5) to δC
37.0, 40.4, 38.1 (C-3), 38.7 (C-4), and 66.9 (C-6), from δH 4.43 (HO-6) to δC 56.5 (C-5), 66.9 and 44.5,
from δH 2.20, 1.94 (H2-7) to δC 66.9 and 128.5, from δH 4.78 (HO-13) to δC 65.1 (C-13) and 38.7, from δH
1.13 (H3-14) to δC 38.7, and from δH 1.58, 0.91 (H2-3) to δC 38.7, 18.6 (C-2). Thus,
5 tuned out to be
6,11,13-trihydroxyldrimane-8-en, similar to the sesquiterpenoid cores in 1–4.
1
The relative configurations of compounds
1
–
5
were assigned by H-NMR J-values and NOE
correlations (Figure 3). In these drimane-type sesquiterpenoids, the doublet J values (11.1–13.2 Hz)
of H-5 suggested a trans-junction of the two cyclohexatomic ring system [35], H3-15 and H3-13
(or H2-13) adopt the same
β
-orientation, whereas H3-14, H-5 and HO-6 (or HO-7) oriented in the
, compounds and were
opposite -direction. To determine the absolute configurations of
α
1–
5
2
3
selected and subjected to acid hydrolysis (5% trifluoroacetic acid in methanol; Figure 4), due to their
abundant amounts.
Figure 3. Selected NOE correlations for 1–7.

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Figure 4. Acid hydrolysis of 2 and 3.
Acid hydrolysis of 2 and 3 afforded a hexitol, together with 2a and 3a, respectively, whose structures
were elucidated by extensive NMR spectroscopic data (Figure 2, Figure 3 and Figures S19–S29, S38–S45
in supplementary materials). Furthermore, NMR (Figure S28 and S29) and optical rotation data ([α]D25
+135.5 (c 0.380, CH3OH)) of the hexitol were quite similar as those reported of D-mannitol [44], which
was also isolated from the same strain P. sporulosum YK-03. NOESY spectra of 2a and 3a (Figure 3)
indicated that they share the same relative configurations as compounds
1–5. After many attempts,
crystal of 2a suitable for single-crystal X-ray diffraction (Cu K ) analysis (Figure 5) was successfully
α
obtained upon slow evaporation of the solvent mixture (methanol-water, 20:1) by keeping the sample
at room temperature for nearly one month. Thus, the absolute configuration of 2a was unambiguously
determined as 5S,6S,10S. Based on the fact that the CD patterns of 1–5 and 3a (Figure 6) were identical
to that of 2a, the absolute configurations of drimane-type sesquiterpenoid were assigned as 5S,10S in
5S,6S,10S in 2, 4S,5R,10S in 3a, 4S,5R,10S in 3, 4S,5R,7R,10S in 4, and 4S,5R,6S,10S in 5.
1,
Figure 5. Diamond plot for X-ray crystal structures of 2a and 7.

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Figure 6. Experimental CD spectra of 1–5 in CH3CN.
Besides the NMR data of the hexitol, the remaining NMR signals of
existence of a prenyl methyl {δH
(t, J = 8.4 Hz) /29.2 and δC 131.0} and a tetrasubstituted benzene {δH
(d, J = 7.8 Hz)/129.7 and δC 133.2, 134.9, 135.6, 140.2} groups, assisted by HMBC correlations (Figure 2).
Then, the HMBC correlations (Figure ) from δH 2.25 (s) to δC 129.7 (C-7) and 140.2 (C-8), and
6
(Table 2) showed the
/δC 1.55 (s)/17.5, 1.66 (s)/25.6, 5.25 (t, J = 6.0 Hz)/124.3, 2.05 (m)/29.5, 2.71
/δC 6.90 (d, J = 7.8 Hz)/127.6, 6.98
2
from δH 2.34 (s) to δC 127.6 (C-6) and 134.9 (C-5), from δH 4.67 (d, J = 10.2 Hz), 4.60 (d, J = 10.2 Hz) to
δC 133.2 (C-10), 135.6 (C-9) and 140.2, and from δH 2.71 to δC 133.2 and 134.9 led to the assignment of
the tetrasubstituted benzene with two methyls located at C-5 and C-8, an oxygenated methyl located
at C-9, and a prenyl methyl located at C-10. Further, the HMBC correlation of δH 3.76 (m, H-30) to δC
67.4 (C-11) indicated C-3’ was connected to C-11 by an ether O to afford the structure of
6
. Based on
the NMR data and biosynthetic homology, the hexitol moiety of
6 was presumed to be D-mannitol,
the same as that of 1–5.
Sporuloside (
by its positive HRESIMS data at m/z 403.2103 [M + Na]+. Analysis of the NMR data of
indicated the existence of an -glucosyl {δH δC 4.64 (d, J = 3.6 Hz < 7.0 Hz)/99.3, 3.20 (m)/72.6, 3.40
(m)/73.7, 3.05 (td, J = 9.1, 5.2 Hz)/70.7, 3.28 (m)/73.3, and 3.56 (m), 3.40 (m)/61.4} moiety, a pentasubstituted
benzene ring {δH δC 7.01 (s)/126.1 and δC 132.1, 132.7, 133.0, 134.1, 139.2}, and three adjacent aromatic
methyls {δH δC 2.07 (s)/15.8, 2.09 (s)/15.9, 2.18(s)/21.0}, supported by the key HMBC correlations
7) was obtained as colorless oil. The molecular formular of C21H32O6 was established
7
(Table 2)
α
/
/
/
(Figure 2) from δH 2.18 (H3-15) to δC 126.1 (C-6), 133.0 (C-7), 132.1 (C-8), from δH 2.09 (H3-12) to δC
133.0, 132.1, 134.1 (C-9), from δH 2.07 (H3-11) to δC 132.1, 134.1, 132.7 (C-10), and from δH 7.01(H-6) to δC
133.0, 132.1, 132.7. The rest of NMR signals attributed to four methylenes {δH
1.72 (m)/19.2, 1.84, 1.44 (each m)/33.0, 3.59, 3.24 (each d, J = 9.2 Hz)/75.6}, a methyl {δH
/
δC 2.53 (t, J = 8.4 Hz)/28.2,
δC 1.25 (s)/27.1}
/
and one quaternary carbon {δC 37.8}. The HMBC correlations of δH 2.53 (H2-1)/δC 19.2 (C-2), 33.0 (C-3),
139.2 (C-5), 134.1, 132.7, δH 1.72 (H2-2)/δC 33.0, 37.8 (C-4), δH 1.25 (H3-14)/δC 37.8, 139.2, δH 7.01/δC
37.8, and δH 3.59, 3.24 (H2-13)/δC 37.8, 99.3 (C-10) combined the abovementioned groups to afford the
planar structure of
for single-crystal X-ray diffraction (Cu K
evaporation of the solvent mixture (methanol-water, 20:1) by keeping the sample at room temperature
7, in which C-13 was glycosidated by a
α-glucose. Luckily, crystals of 7 suitable
α) analysis (Figure 5) were successfully obtained upon slow
for nearly one month, so the absolute configuration of C-4 was assigned as S, and α-glucosyl group
as D-form.
2.2. Investigation on the Origin of Mannitol Moiety
Sporulositols
A–D (1–4) and seco-sporulositol (6) represent the first examples of unique drimanic
mannitol derivatives. To find out whether their mannitol moiety was formed intrinsically or derived

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from the medium, the normal medium (mannitol-contained, control group), modified medium No.1
(no mannitol, blank group) and modified medium No.2 (mannitol replaced by sorbitol, experimental
group) were included for simultaneous cultivation of P. sporulosum YK-03 and HPLC analysis of the
metabolites. Compounds
was isolated from the extract of experimental group (Figure 7B,C), revealing that the fungus can
produce the mannitol moiety intrinsically, no matter whether the medium contains mannitol or not.
1–3 could be detected in all the three groups (Figure 7A), and compound
2
Figure 7. Analysis of the reference compounds
mediums using HPLC and NMR methods. (
and metabolites of P. sporulosum YK-03 in different mediums: (a) compound
(c) compound ; (d) metabolites from blank group (medium with-No mannitol); (e) metabolites from
1
A
–
3
and metabolites of P. sporulosum YK-03 in different
) HPLC Analysis of the reference compounds
; (b) compound 2;
1–3
1
3
control group (Normal medium, mannitol-contained medium); (f) metabolites from experimental group
(medium with mannitol replaced by sorbitol); 1H-NMR (B) and 13C-NMR (C) spectra of compound 2
isolated from the experimental and control groups: (a) experimental group; (b) control group.
Compounds
1–7 were tested for cytotoxicity against two cell lines A549 (human lung
adenocarcinoma cells) and MCF-7 (human breast cancer cells). Unfortunately, compounds
did not show any detectable cytotoxicity.
1–7
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured using a Perkin-Elmer Model 241 polarimeter (Perkin Elmer, Inc.
Waltham, MA, USA). UV spectra were obtained on a Shimadzu UV-1601 (Shimadzu Corp., Kyoto,
Japan). IR spectra were taken on a Bruker IFS-55 infrared spectrophotometer (Bruker Optik BmbH,
Ettlingen, Germany) with KBr disks. The HRESIMS data were obtained on a microTOF-Q Bruker mass
instrument (Bruker Daltonics, Billerica, MA, USA). CD spectra were recorded with a Biologic MOS-450

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spectrometer (BioLogic Science Instruments, Grenoble, French) using CH3CN as solvent. 1D and 2D
NMR spectra were recorded on Bruker ARX-400 and AV-600 spectrometers (1H/13C, 400/100 MHz
600/150 MHz, Bruker, Zurich, Switzerland) using TMS as an internal standard. Chemical shifts (δ)
were expressed in ppm. HPLC was performed using a Shimadzu LC-20AB HPLC pump equipped
with a SPD-20A detector (Shimadzu Corp.) for new compound analysis, employing a YMC-Pack
ODS-A column (250 mm
CHIRALPAK AD-H column (250 mm
Shimadzu LC-8A HPLC pump equipped with SPD-10A detector for the purification of new compounds,
employing a YMC-Pack ODS-A column (250 mm 10 mm, 5 m). Column chromatography (CC) was
×
4.6 mm, 5
µ
m), and for metabolite analysis in Figure 7, employing a
×
4.6 mm, 5 m). Reversed-phase HPLC was performed using a
µ
×
µ
carried out on silica gel (200–300/400–500 mesh, Qingdao Marine Chemical, Inc., Qingdao, China) and
sephadex LH-20 (Pharmacia, Uppsala, Sweden). Column fractions were monitored by TLC (Silica
gel GF254, 200–300 mesh, Qingdao Haiyang Chemical Factory, Qingdao, China), and the spots were
visualized by heating the plates after spraying with 10% H2SO4 in ethanol. All reagents of HPLC or
analytical grade were purchased from Shangdong Yuwang Reagent Co., Ltd. (Shangdong, China).
3.2. Fungal Material
Paraconiothyrium sporulosum YK-03 was isolated from the sea mud collected from the intertidal
zone of Bohai Bay in Liaoning Province of China. It was identified based on the analysis of ITS
sequence (GenBank accession No. KC416199) and has been deposited in the School of Traditional
Chinese Materia Medica, Shenyang Pharmaceutical University.
3.3. Fermentation
The strain was cultured on PDA (potato 20%, glucose 2% and agar 2%) medium in Petri dishes
at 28 ◦C for 3 days, and were inoculated in a 500 mL Erlenmeyer flask containing 150 mL of media
(maltose 2%, monosodium glutamate 1%, glucose 1%, yeast cream 0.3%, corn steep liquor 0.1%, maltose
.
2%, KH2PO4 0.05%, MgSO4 7H2O 0.03%). After incubation at 28 ◦C and 180 rpm for 4 days, a 5 mL
cultural solution was transferred as a seed into each of 500 mL flask containing 150 mL liquid medium
(maltose 2%, monosodium glutamate 1%, glucose 1%, yeast cream 0.5%, east cream 0.3%, corn steep
.
liquor 0.1%, mannitol 2%, KH2PO4 0.05%, MgSO4 7H2O 0.03%, CaCO3 2%, sea water element 3.3%,
pH6.5). The flasks were subsequently incubated at the same conditions for 8 days.
3.4. Extraction and Isolation
Following incubation, the fermentation broth of P. sporulosum YK-03 (70 L) was concentrated and
extracted with ethyl acetate and n-butanol, successively. The ethyl acetate extract (20 g) was subjected
to a silica gel column (10 cm
. Fraction (350 mg) was firstly subjected to a Sephadex LH-20 column (2.5 cm
with CHCl3-CH3OH (1:1) to remove pigment, then purified LPLC using a gradient of increasing
methanol (20%–100%) in water to afford three subfractions (L1 L3). Fraction L1 (48 mg) afforded
compound (4.3 mg, tR 36.4 min) and compound (2.5 mg, tR = 58.9 min) by using preparative
HPLC (CH3OH-H2O 53:47, flow rate 3 mL/min, wavelength 210 nm), employing a YMC-Pack ODS-A
column (250 mm 10 mm, 5 m). Fraction (628 mg) was subjected to Sephadex LH-20 column
(3 cm 120 cm), eluted with CHCl3-CH3OH (1:1) and preparative HPLC (CH3OH-H2O 44:56, flow
rate 3 mL/min, wavelength 210 nm) to obtain compound (93.3 mg, tR 40.2 min) compound (89.8 mg,
tR = 45.5 min), and compound (2.6 mg, tR = 58.2 min). Fraction (1.2 g) was subjected to a silica gel
column (4 cm 80 cm), eluted with CHCl3-CH3OH (100:1–0:1), yielding 7 fractions (N1 N7). Then,
fraction N4 (89 mg) afforded compound (3.2 mg) through recrystallization, and the rest solution was
×
120 cm), eluted with CHCl3-CH3OH (100:1–0:1), yielding 14 fractions
A
–N
L
×
100 cm), eluted
–
1
6
×
µ
M
×
2
3
4
N
×
–
8
purified by preparative HPLC (CH3OH-H2O 30:70, flow rate 3 mL/min, wavelength 210 nm) to obtain
compound 5 (2.3 mg, tR = 42.0 min) and compound 7 (2.0 mg, tR = 49.2 min).
Sporulositol A (
1
): colorless oil; [α]D25+101.6 (c 0.43, CH3OH); UV (CH3OH
λ
204.4 nm; IR
max
(KBr) νmax 3416.6, 2928.1, 1659.0, 1461.7, 1384.4, 1080.7 cm−1; CD (c 0.10, CH3CN)
λ(∆ε) 203 (+7.75)

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nm; 1H-NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; HRESIMS m/z
409.2568 [M + Na]+, (calcd for C21H38O6 386.2668).
Sporulositol B (
2
): colorless oil; [α]D25+113.9 (c 1.00, CH3OH); UV (CH3OH)
λ
206.4 nm; IR
max
(KBr) νmax 3396.3, 2927.4, 1632.3 1434.2, 1384.2, 1075.0, 1043.1 cm−1; CD (c 0.10, CH3CN)
λ(∆ε) 203
(+6.36) nm; 1H-NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; HRESIMS
m/z 425.2512 [M + Na]+, (calcd for C21H38O7 402.2617).
Sporulositol C (3 λmax 206.2,243.6 nm;
): colorless oil; [α]D25+101.8 (c 1.00, CH3OH); UV (CH3OH)
IR (KBr) νmax 3384.7, 2928.2, 1650.1, 1451.1, 1384.0, 1079.0, 1032.4 cm−1; CD (c 0.10, CH3CN)
λ(
∆ε
)
1
202 (+10.79) nm; H-NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1,
HRESIMS m/z 425.2512 [M + Na]+, (calcd for C21H38O7 402.2617).
Sporulositol D (
(KBr) νmax 3405.7, 2922.7, 1632.3, 1597.6, 1552.5, 1430.0, 1384.4, 1121.3, 1053.1, 1033.0 cm−1; CD (c 0.10,
CH3CN)
∆ε) 201 (+11.07) nm;1 H-NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6),
see Table 1; HRESIMS m/z 441.2452 [M + Na]+, (calcd for C21H38O8 418.2566).
6-hydroxyl diaporol (
): colorless oil; [α]D25+98.3 (c 0.23, CH3OH); UV (CH3OH)
4
): colorless oil; [α]D25+90.3 (c 0.26, CH3OH); UV (CH3OH)
λ
max
207.2 nm; IR
λ(
5
λ
max 207.2 nm; IR
(KBr) νmax 3418.1, 2924.9, 1658.1, 1554.1, 1433.8, 1384.3 cm−1; CD (c 0.10, CH3CN)
λ(∆ε) 196.5 (+6.12)
nm; 1H-NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; HRESIMS m/z
277.1814 [M + Na]+, (calcd for C15H26O3 254.1882).
seco-sporulositol (
6
): colorless oil; [α]D25+55.0 (c 0.25, CH3OH); UV (CH3OH)
λ
205.0 nm; IR
max
(KBr) νmax 3405.4, 2923.0, 1632.0, 1597.8, 1554.4, 1432.7, 1384.4, 1127.7, 1033.1 cm−1; 1H-NMR (600 MHz,
DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 2; HRESIMS m/z 405.2239 [M + Na]+, (calcd
for C21H34O6 382.2355).
Sporuloside (
7
): colorless oil; [α]D25+117.6 (c 0.26, CH3OH); UV (CH3OH)
λ
max
205.0 nm; IR (KBr)
νmax 3405.4, 2921.6, 1634.3, 1457.7, 1384.4, 1148.2, 1123.5, 1025.5 cm−1; 1H-NMR (600 MHz, DMSO-d6)
and 13C NMR (150 MHz, DMSO-d6), see Table 2; HRESIMS m/z 403.2103 [M + Na]+, (calcd for C21H32O6
380.2199).
D-mannitol (8): white powder; [α]D25+145.5 (c 0.50, CH3OH); 1H-NMR (DMSO-d6, 400 MHz), δH
3.60 (2H, m), 3.54 (2H, t, J = 8.0 Hz), 3.46 (2H, m), 3.38 (2H, m), 4.41 (2H, d, J = 5.6 Hz, HO
×
2 ), 4.33(2H,
t, J = 6.0 Hz, HO
×
2), 4.14 (2H, d, J = 7.2 Hz, HO
×
2); 13C-NMR (DMSO-d6, 100 MHz) δC 64.2
×
2, 70.1
× 2, 71.7 × 2.
3.5. X-ray Crystallographic Analysis of Compounds 2a and 7
Crystal Data of 2a: C16H28O2, M =252.38, orthorhombic, a = 10.0481 (3) Å, b = 10.4089(4) Å, c =
28.5282(9) Å, U = 2983.76(17) Å3, T = 100.6, space group P212121 (no. 19), Z = 8,
(Cu K ) = 0.554,
µ
α
10773 reflections measured, 5657 unique (Rint = 0.0286) which were used in all calculations. The final
wR (F2) was 0.1314 (all data). The crystallographic data for the structure of 2a have been deposited
with the Cambridge Crystallographic Data Centre as supplementary publication CCDC No. 1905155.
Crystal Data of
48.7570(7) Å, U = 1983.47(5) Å3, T = 102.3, space group P212121 (no. 19), Z = 4,
reflections measured, 3754 unique (Rint = 0.0252) which were used in all calculations. The finalwR (F2)
was 0.0996 (all data). The crystallographic data for the structure of have been deposited with the
7: C21H32O6, M = 380.47, orthorhombic, a = 5.39286(9) Å, b = 7.54344 (11) Å, c =
µ
(Cu K ) = 0.753, 6585
α
7
Cambridge Crystallographic Data Centre as supplementary publication CCDC No. 1905156.
CCDC-1905155 and CCDC-1905156 contain the supplementary crystallographic data, which can
be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.
ac.uk/conts/retrieving.html.
3.6. Acid Hydrolysis of Compounds 2 and 3
Compounds
2 and 3 (each 10 mg) were dissolved in CH3OH (1 mL) with 1 mL trifluoroacetic acid
(TFA), and heated in a H2O bath at 40 ◦C for 14 h to give an acid hydrolysate. The acid hydrolysate was
then vacuum evaporated to remove the residual TFA. Then, the hydrolysate was suspended in H2O,

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and extracted with CHCl3. Finally, the H2O solution afforded sugar alcohol through recrystallization,
and the CHCl3 solution was purified by preparative HPLC to obtain compound 2a (1.8 mg) and
compound 3a (2.0 mg), respectively.
3.7. Analysis of the Refrence Compounds 1–3 and Metabolites of P. Sporulosum YK-03 in Different Mediums
Using HPLC and NMR Methods
P. sporulosum YK-03 was simultaneously cultured in three following liquid media at 28 ◦C and 180
rpm for 4 days. Liquid media: (1) Normal medium (mannitol-containing, control group): maltose
2%, monosodium glutamate 1%, glucose 1%, yeast cream 0.5%, east cream 0.3%, corn steep liquor
.
0.1%, mannitol 2%, KH2PO4 0.05%, MgSO4 7H2O 0.03%, CaCO3 2%, sea water element 3.3%, pH
6.5. (2) Modified medium No.1 (no mannitol, blank group): maltose 2%, monosodium glutamate 1%,
.
glucose 1%, yeast cream 0.5%, east cream 0.3%, corn steep liquor 0.1%, KH2PO4 0.05%, MgSO4 7H2O
0.03%, CaCO3 2%, sea water element 3.3%, pH 6.5. (3) Modified medium No. 2 (mannitol replaced by
sorbitol, experimental group): maltose 2%, monosodium glutamate 1%, glucose 1%, yeast cream 0.5%,
.
east cream 0.3%, corn steep liquor 0.1%, sorbitol 2%, KH2PO4 0.05%, MgSO4 7H2O 0.03%, CaCO3 2%,
sea water element 3.3%, pH 6.5.
After incubation, their fermentation broths were concentrated and extracted with ethyl acetate.
Then their metabolites were analyzed by gradient HPLC analysis. HPLC chromatographic condition:
(1) Instrument: Shimadzu LC-20AB HPLC pump equipped with an SPD-20A detector (Shimadzu
Corp.); (2) Column: CHIRALPAK AD-H column (250 mm
×
4.6 mm, 5 µm); (3) Mobile phase: CH3OH
(B) and H2O (A); (4) Wavelength: 210 nm; (3) Flow rate: 1 mL min−1; (5) gradient elution program: 10%
B; 0–6 min, linearly changed to 24% B; 6–26 min, linearly changed to 35% B; 26–40 min, linearly changed
to 60% B; 40–60 min, linearly changed to 70% B; 60–72 min, linearly changed to 80% B; 72–82 min,
linearly changed to 100%. The sample injection volume was 5.0 µL.
The strain was incubated in twenty Erlenmeyer flasks (500 mL) containing 150 mL Modified
medium No. 2 medium at 28 ◦C and 180 rpm for 8 days. Then the fermentation broth was concentrated
and extracted with ethyl acetate. The ethyl acetate extract was subjected to a silica gel column eluted
with CHCl3-CH3OH (100:1–0:1) and preparative HPLC (CH3OH-H2O 44:56, flow rate 3 mL/min,
wavelength 210 nm) to yield a drimane-type sesquiterpenoid, guiding by TLC and HPLC. Then its
NMR spectra was compared with those of compounds 1–3.
3.8. Cytotoxic Activity Assay
The cytotoxicity was evaluated by using the MTT assay as described previously [45]. Doxorubicin
hydrochloride was used as a positive control. The A549 and MCF-7 Cells (China Infrastructure of Cell
Lines Resources, Beijing, China were cultured in McCoy’s 5A medium and DMEM basic medium (1
at 37 ◦C under an atmosphere of 5% CO2, and were seeded on each well of 96-well plates containing
200 L of tumor cell suspension (1 L of test solution and
104 cells). After 24 h, each well was added 2
incubated for another 72 h. 50 L of MTT solution (1 mg/mL, Beijing Cellchip Biotechnology Co., Ltd.,
Beijing, China was added to each well, and the plate was incubated for 3h under the same condition.
×
)
µ
×
µ
µ
Then, the plate was centrifuged and the supernatants were removed and cells were dissolved in 150
of DMSO to determine the IC50 values.
µL
4. Conclusions
Seven new drimane-type sesquiterpenoids, including sporulositols
), seco-sporulositol ( ) and sporuloside ( ), were isolated from a marine-derived fungus P. sporulosum
YK-03. Their structures were established by extensive NMR experiments, X-ray diffraction analysis
and comparisons of circular dichroism data. Compounds and are rare new drimanic hexitol
derivatives containing a D-mannitol moiety. seco-sporulositol ( ) and sporuloside ( ) may represent
two new series of natural drimanes, possessing an aromatic ring with a rare 4,5-secodrimanic skeleton
and an unusual CH3-15 rearranged drimanic -D-glucopyranside, respectively. Then, the cultivation
A–D (1–4), 6-hydroxy- diaporol
(5
6
7
1–
4
6
6
7
α

Molecules 2019, 24, 1817
13 of 15
medium was evaluated for the origin of D-mannitol moiety by HPLC and NMR analyses. These isolates
enriched the structural diversity of natural drimanic sesquiterpenoids.
Supplementary Materials: The following are available online, Figures S1–S79: HRESIMS, 1D and 2D NMR, IR,
UV and CD spectra of compounds
modified medium No.2.
1–8, 2a and 3a; Figures S80–S81: 1D NMR of compound 2 isolated from the
Author Contributions: Y.-H.P. designed and guided the experiments. L.-H.Z. conducted the isolation, structural
determination and wrote the manuscript. G.C. completed the preliminary original investigation on the mannitol
moiety of
1–4 and 6, and revised the manuscript. Y.S. contributed to the biological activity evaluation. H.-F.W.
performed the acid hydrolysis of compounds
2
and 3. J.B. and H.-M.H. performed the isolation, identification and
fermentation of the fungal species. All authors reviewed the manuscript.
Funding: Financially Supported by Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory
for Marine Science and Technology (LMDBKF201701) and National Natural Science Foundation of China
(No. 81202425).
Acknowledgments: We gratefully acknowledge Yi Sha and Wen Li, Department of Analytical Testing Center,
Shenyang Pharmaceutical University, for measurements of the NMR data. We thank Jian Hao, Department of
Analytical Testing Center, Beijing University of Chemical Technology, for the test of the X-ray diffraction.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
Blunt, J.W.; Carroll, A.R.; Copp, B.R.; Davis, R.A.; Keyzers, R.A.; Prinsep, M.R. Marine natural products.
Nat. Prod. Rep. 2018, 35, 8–53. [CrossRef] [PubMed]
Bugni, T.S.; Ireland, C.M. Marine-derived fungi: A chemically and biologically diverse group of
microorganisms. Nat. Prod. Rep. 2004, 21, 143–163. [CrossRef]
Barbero, H.; Diez-Poza, C.; Barbero, A. The Oxepane Motif in Marine Drugs. Mar. Drugs 2017, 15, 361.
[CrossRef]
Overy, D.P.; Bayman, P.; Kerr, R.G.; Bills, G.F. An assessment of natural product discovery from marine
(sensu strictu) and marine-derived fungi. Mycology 2014, 5, 145–167. [CrossRef] [PubMed]
Verkley, G.J.M.; Silva, M. d.; Wicklow, D.T.; Crou, P.W. Paraconiothyrium, a new genus to accommodate the
mycoparasite Coniothyrium minitans, anamorphs of Paraphaeosphaeria, and four new species. Stud. Mycol.
2004, 50, 323–335.
6.
Takano, K.; Itoh, Y.; Ogino, T.; Kurosawa, K.; Sasaki, K. Phylogenetic analysis of manganese-oxidizing
fungi isolated from manganese-rich aquatic environments in Hokkaido, Japan. Limnology 2006, 7, 219–223.
[CrossRef]
7.
8.
9.
Liu, L.; Gao, H.; Chen, X.; Cai, X.; Yang, L.; Guo, L.; Yao, X.; Che, Y. Brasilamides A-D: Sesquiterpenoids from
the Plant Endophytic Fungus Paraconiothyrium brasiliense. Eur. J. Org. Chem. 2010, 3302–3306. [CrossRef]
Liu, L.; Chen, X.; Li, D.; Zhang, Y.; Li, L.; Guo, L.; Cao, Y.; Che, Y. Bisabolane Sesquiterpenoids from the Plant
Endophytic Fungus Paraconiothyrium brasiliense. J. Nat. Prod. 2015, 78, 746–753. [CrossRef] [PubMed]
Guo, Z.; Ren, F.; Che, Y.; Liu, G.; Liu, L. New Bergamotane Sesquiterpenoids from the Plant Endophytic
Fungus Paraconiothyrium brasiliense. Molecules 2015, 20, 14611–14620. [CrossRef] [PubMed]
10. Chen, S.; Zhang, Y.; Zhao, C.; Ren, F.; Liu, X.; Che, Y. Hawaiinolides E-G, cytotoxic cassane and cleistanthane
diterpenoids from the entomogenous fungus Paraconiothyrium hawaiiense. Fitoterapia 2014, 99, 236–242.
[CrossRef] [PubMed]
11. Chen, S.; Zhang, Y.; Niu, S.; Liu, X.; Che, Y. Cytotoxic cleistanthane and cassane diterpenoids from the
entomogenous fungus Paraconiothyrium hawaiiense. J. Nat. Prod. 2014, 77, 1513–1518. [CrossRef]
12. Mohamed, I.E.; Kehraus, S.; Krick, A.; Konig, G.M.; Kelter, G.; Maier, A.; Fiebig, H.-H.; Kalesse, M.; Malek, N.P.;
Gross, H. Mode of Action of Epoxyphomalins A and B and Characterization of Related Metabolites from the
Marine-Derived Fungus Paraconiothyrium sp. J. Nat. Prod. 2010, 73, 2053–2056. [CrossRef] [PubMed]
13. Zhang, L.-H.; Feng, B.-M.; Chen, G.; Li, S.-G.; Sun, Y.; Wu, H.-H.; Bai, J.; Hua, H.-M.; Wang, H.-F.; Pei, Y.-H.
Sporulaminals A and B: A pair of unusual epimeric spiroaminal derivatives from a marine-derived fungus
Paraconiothyrium sporulosum YK-03. RSC Adv. 2016, 6, 42361–42366. [CrossRef]

Molecules 2019, 24, 1817
14 of 15
14. Shiono, Y.; Kikuchi, M.; Koseki, T.; Murayama, T.; Kwon, E.; Aburai, N.; Kimura, K.-i. Isopimarane diterpene
glycosides, isolated from endophytic fungus Paraconiothyrium sp. MY-42. Phytochemistry 2011, 72, 1400–1405.
[CrossRef] [PubMed]
15. Du, Y.; Chen, Z.; Li, H.; Wang, Y.; Fu, P.; Zhu, W. Pafuranones A and B, two dimeric polyketides from a rare
marine algae-derived fungus Paraconiothyrium sp. Chin. Chem. Lett. 2019. [CrossRef]
16. Suzuki, T.; Ariefta, N.R.; Koseki, T.; Furuno, H.; Kwon, E.; Momma, H.; Harneti, D.; Maharani, R.;
Supratman, U.; Kimura, K.I.; et al. New polyketides, paralactonic acids A-E produced by Paraconiothyrium sp.
SW-B-1, an endophytic fungus associated with a seaweed, Chondrus ocellatus Holmes. Fitoterapia 2019, 132,
75–81. [PubMed]
17. Zhao, C.; Fu, P.; Zhang, Y.; Liu, X.; Ren, F.; Che, Y. Sporulosol, a New Ketal from the Fungus Paraconiothyrium
sporulosum. Molecules 2018, 23, 1263. [CrossRef] [PubMed]
18. Quang, T.H.; Nhiem, N.X.; Tai, B.H.; Yen, P.H.; Dung, D.T.; Ngan, N.T.T.; Le Tuan Anh, H.; Van Minh, C.; Van
Kiem, P. Secondary metabolites from the marine-derived fungus Paraconiothyrium sp. VK-13. Vietnam J. Chem.
2018, 56, 434–439.
19. Ren, F.; Chen, S.; Zhang, Y.; Zhu, S.; Xiao, J.; Liu, X.; Su, R.; Che, Y. Hawaiienols A-D, Highly Oxygenated
p-Terphenyls from an Insect-Associated Fungus, Paraconiothyrium hawaiiense. J. Nat. Prod. 2018, 81, 1752–1759.
[CrossRef]
20. Almeida, C.; El Aouad, N.; Martin, J.; Perez-Victoria, I.; Gonzalez-Menendez, V.; Platas, G.; de la Cruz, M.;
Monteiro, M.C.; de Pedro, N.; Bills, G.F.; et al. Graminin B, a furanone from the fungus Paraconiothyrium sp.
J. Antibiot. 2014, 67, 421–423. [CrossRef]
21. Xu, G.-B.; Mi, J.; Yang, T.; Wu, L.-W.; Yuan, X.-H.; Li, G.-Y. Two new polyketide metabolites isolated from
Paraconiothyrium brasiliense. Chem. Nat. Compd. 2017, 53, 870–873. [CrossRef]
22. Cho, N.; Ransom, T.T.; Sigmund, J.; Tran, T.; Cichewicz, R.H.; Goetz, M.; Beutler, J.A. Growth Inhibition of
Colon Cancer and Melanoma Cells by Versiol Derivatives from a Paraconiothyrium Species. J. Nat. Prod. 2017
,
80, 2037–2044. [CrossRef]
23. Zhang, L.-H.; Li, S.-G.; Wu, H.-H.; Chen, G.; Li, L.; Bai, J.; Hua, H.-M.; Wang, H.-F.; Pei, Y.-H.
3,4-Dihydroisocoumarin derivatives from the marine-derived fungus Paraconiothyrium sporulosum YK-03.
Phytochem. Lett. 2017, 20, 200–203. [CrossRef]
24. De Gusmño, N.B.; Kaouadji, M.; Steiman, R.; Seigle-murandi, F.; Ulrichc, J. Coniothyriol, an uncommon
polyketide from Coniothyrium sporulosum. Nat. Prod. Lett. 1993, 2, 287–292. [CrossRef]
25. Guiraud, P.; Steiman, R.; Seigle-Murandi, F.; de Gusmao, N.B. Antimicrobial and Antitumor Activities of
Mycosporulone. J. Nat. Prod. 1999, 62, 1222–1224. [CrossRef]
26. Quang, T.H.; Kim, D.C.; Van Kiem, P.; Van Minh, C.; Nhiem, N.X.; Tai, B.H.; Yen, P.H.; Thi ThanH-Ngan, N.;
Kim, H.J.; Oh, H. Macrolide and phenolic metabolites from the marine-derived fungus Paraconiothyrium sp.
VK-13 with anti-inflammatory activity. J. Antibiot. 2018, 71, 826–830. [CrossRef]
27. Jansen, B.J.M.; De Groot, A. The occurrence and biological activity of drimane sesquiterpenoids. Nat. Prod. Rep.
1991, 8, 309–318. [CrossRef]
28. Jansen, B.J.M.; de Groot, A. Occurrence, biological activity and synthesis of drimane sesquiterpenoids.
Nat. Prod. Rep. 2004, 21, 449–477. [CrossRef] [PubMed]
29. Erkel, G.; Lorenzen, K.; Anke, T.; Velten, R.; Gimenez, A.; Steglich, W. Kuehneromycins A and B, two
new biological active compounds from a Tasmanian Kuehneromyces sp. (Strophariaceae, Basidiomycetes).
Naturforsch. C Biosci. 1995, 50, 1–10. [CrossRef]
30. Fleck, W.F.; Schlegel, B.; Hoffmann, P.; Ritzau, M.; Heinze, S.; Graefe, U. Isolation of isodrimenediol, a possible
intermediate of drimane biosynthesis from Polyporus arcularius. J. Nat. Prod. 1996, 59, 780–781. [CrossRef]
31. Derita, M.; Montenegro, I.; Garibotto, F.; Enriz, R.D.; Fritis, M.C.; Zacchino, S.A. Structural requirements for
the antifungal activities of natural drimane sesquiterpenes and analogues, supported by conformational and
electronic studies. Molecules 2013, 18, 2029–2051. [CrossRef] [PubMed]
32. De Almeida Alves, T.M.; Ribeiro, F.L.; Kloos, H.; Zani, C.L. Polygodial, the fungitoxic component from the
Brazilian medicinal plant Polygonum punctatum. Mem. Inst. Oswaldo Cruz 2001, 96, 831–833. [CrossRef]
33. Grabley, S.; Thiericke, R.; Zerlin, M.; Goert, A.; Phillips, S.; Zeeck, A. Secondary metabolites by chemical
screening. 33. New albrassitriols from Aspergillus sp. (FH-A 6357). J. Antibiot. 1996, 49, 593–595. [CrossRef]
[PubMed]

Molecules 2019, 24, 1817
15 of 15
34. Anke, H.; Sterner, O. Comparison of the antimicrobial and cytotoxic activities of twenty unsaturated
sesquiterpene dialdehydes from plants and mushrooms. Planta Med. 1991, 57, 344–346. [CrossRef] [PubMed]
35. Chen, C.-J.; Liu, X.-X.; Zhang, W.-J.; Zang, L.-Y.; Wang, G.; Ng, S.W.; Tan, R.-X.; Ge, H.-M. Sesquiterpenoids
isolated from an endophyte fungus Diaporthe sp. RSC Adv. 2015, 5, 17559–17565. [CrossRef]
36. Abad, A.; Agulló, C.; Cuñat, A.C.; González-Coloma, A.; Pardo, D. Preparation of 9α-Fluorinated Sesquiterpenic
Drimanes and Evaluation of Their Antifeedant Activities. Eur. J. Org. Chem. 2010, 2182–2198. [CrossRef]
37. Montenegro, I.; Pino, L.; Werner, E.; Madrid, A.; Espinoza, L.; Moreno, L.; Villena, J.; Cuellar, M. Comparative
study on the larvicidal activity of drimane sesquiterpenes and nordrimane compounds against Drosophila
melanogaster til-til. Molecules 2013, 18, 4192–4208. [CrossRef] [PubMed]
38. Powell, G.; Hardie, J.; Pickett, J.A. Laboratory evaluation of antifeedant compounds for inhibiting settling by
cereal aphids. Entomol. Exp. Appl. 1997, 84, 189–193. [CrossRef]
39. Ying, B.-P.; Peiser, G.; Ji, Y.-Y.; Mathias, K.; Tutko, D.; Hwang, Y.-S. Phytotoxic sesquiterpenoids from Canella
winterana. Phytochem. Lett. 1995, 38, 909–915. [CrossRef]
40. Minale, L.; Riccio, R.; Sodano, G. Avarol, a novel sesquiterpenoid hydroquinone with a rearranged drimane
skeleton from the sponge Disidea avara. Tetrahedron Lett. 1974, 3401–3404. [CrossRef]
41. Ciocarlan, A.; Edu, C.; Biriiac, A.; Lungu, L.; Aricu, A.; D’Ambrosio, M.; Shova, S.; Nicolescu, A.;
Deleanu, C.; Vornicu, N. Synthesis of Polyfunctional Drimanes from Drim-7,9(11)-diene and Drim-8-en-7-one.
Synth. Commun. 2013, 43, 3020–3033. [CrossRef]
42. Domingo, V.; Prieto, C.; Siva, L.; Rodilla, J.M.; Quilez del Moral, J.F.; Barrero, A.F. Iodine, a Mild Reagent for
the Aromatization of Terpenoids. J. Nat. Prod. 2016, 79, 831–837. [CrossRef]
43. Henquet, M.G.L.; Prota, N.; van der Hooft, J.J.J.; Varbanova-Herde, M.; Hulzink, R.J.M.; de Vos, M.; Prins, M.;
de Both, M.T.J.; Franssen, M.C.R.; Bouwmeester, H.; et al. Identification of a drimenol synthase and drimenol
oxidase from Persicaria hydropiper, involved in the biosynthesis of insect deterrent drimanes. Plant J. 1052, 90,
1052–1063. [CrossRef] [PubMed]
44. Schnarr, G.W.; Vyas, D.M.; Szarek, W.A. Carbon-13 nuclear magnetic resonance spectra of acyclic carbohydrate
derivatives: Alditols, 1,-bis(phenylhydrazones), and dithioacetals. J. Chem. Soc. Perkin Trans. 1979, 2, 496–503.
[CrossRef]
45. Ueoka, R.; Nakao, Y.; Kawatsu, S.; Yaegashi, J.; Matsumoto, Y.; Matsunaga, S.; Furihata, K.; van Soest, R.W.M.;
Fusetani, N. Gracilioethers A-C, Antimalarial Metabolites from the Marine Sponge Agelas gracilis. J. Org. Chem.
2009, 74, 4203–4207. [PubMed]
Sample Availability: Not Available.
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