Bisabolane, cyclonerane, and harziane derivatives from the marine-alga-endophytic fungus Trichoderma asperellum cf44-2

Article abstract of DOI:10.1016/j.phytochem.2018.04.017

Three undescribed bisabolane derivatives, trichaspin, trichaspsides A and B, three undescribed cyclonerane sesquiterpenes, 9-cycloneren-3,7,11-triol, 11-cycloneren-3,7,10-triol, and 7,10-epoxycycloneran-3,11,12-triol, and one undescribed harziane diterpene, 11-hydroxy-9-harzien-3-one, were obtained from the culture of Trichoderma asperellum cf44-2, an endophyte of the marine brown alga Sargassum sp. Their structures and relative configurations were assigned by analysis of 1D/2D NMR and MS data, and their absolute configurations were established by ECD or specific optical rotation data. Trichaspin features an unprecedented ethylated bisabolane skeleton, while trichaspsides A and B represent the first aminoglycosides of bisabolane and norbisabolane sesquiterpenes, respectively. Nine of the compounds were evaluated for inhibition of five marine-derived pathogenic bacteria and toxicity to a marine zooplankton.

Full text of DOI:10.1016/j.phytochem.2018.04.017

Phytochemistry 152 (2018) 45e52
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Bisabolane, cyclonerane, and harziane derivatives from the marine-
alga-endophytic fungus Trichoderma asperellum cf44-2
,
,
Yin-Ping Song a b, Xiang-Hong Liu a, Zhen-Zhen Shi a b, Feng-Ping Miao a,
Sheng-Tao Fang a, Nai-Yun Ji a
,
*
a Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China
b University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three undescribed bisabolane derivatives, trichaspin, trichaspsides A and B, three undescribed cyclo-
nerane sesquiterpenes, 9-cycloneren-3,7,11-triol, 11-cycloneren-3,7,10-triol, and 7,10-epoxycycloneran-
3,11,12-triol, and one undescribed harziane diterpene, 11-hydroxy-9-harzien-3-one, were obtained from
the culture of Trichoderma asperellum cf44-2, an endophyte of the marine brown alga Sargassum sp. Their
structures and relative configurations were assigned by analysis of 1D/2D NMR and MS data, and their
absolute configurations were established by ECD or specific optical rotation data. Trichaspin features an
unprecedented ethylated bisabolane skeleton, while trichaspsides A and B represent the first amino-
glycosides of bisabolane and norbisabolane sesquiterpenes, respectively. Nine of the compounds were
evaluated for inhibition of five marine-derived pathogenic bacteria and toxicity to a marine zooplankton.
© 2018 Elsevier Ltd. All rights reserved.
Received 23 February 2018
Received in revised form
25 April 2018
Accepted 27 April 2018
Keywords:
Trichoderma asperellum
Moniliaceae
Terpene
Bisabolane
Cyclonerane
Harziane
1. Introduction
result, three undescribed bisabolane derivatives, trichaspin (1),
trichaspsides A (2) and B (3), three undescribed cyclonerane ses-
Among the multifarious filamentous fungi, Trichoderma Pers.
(Moniliaceae) species have been regarded as the most potential
biocontrol agents in agriculture, and hundreds of specialised me-
tabolites with various bioactivities, such as antifungal, antibacterial,
weedicidal, and cytotoxic properties, have been discovered from
them so far (Reino et al., 2008; Keswani et al., 2014). Although
Trichoderma is commonly considered as a terrestrial genus, hal-
otolerant strains have been continuously reported from marine
sediments, invertebrates, and algae (Zhu et al., 2015). Moreover,
marine-derived Trichoderma strains have already contributed more
than 60 undescribed compounds, involving terpenes, polyketides,
alkaloids, and peptides (Zhu et al., 2015; Blunt et al., 2017). Of those,
only several (less than ten) were obtained from the marine algic-
olous strains of Trichoderma (Ji and Wang, 2016; Miao et al., 2012;
Liang et al., 2016a, 2016b; Yamazaki et al., 2015, 2016), but they
exhibited the high novelty due to cyclization and substitution and
then encouraged our further investigation towards them. As a
quiterpenes, 9-cycloneren-3,7,11-triol (6), 11-cycloneren-3,7,10-
triol (7), and 7,10-epoxycycloneran-3,11,12-triol (8), and one
undescribed harziane diterpene, 11-hydroxy-9-harzien-3-one (9),
together with the known (3S,6R,7S)-zingiberenol (4) (Terhune
et al., 1975; Khrimian et al., 2014), cyclonerodiol (5) (Laurent
et al., 1990; Langhanki et al., 2014), and harziandione (10) (Miao
et al., 2012; Adelin et al., 2014) were isolated and identified from
Trichoderma asperellum Samuels, Lieckfeldt & Nirenberg cf44-2
(Fig. 1), an endophyte of the marine brown alga Sargassum sp.
(Sargassaceae). Herein, the isolation, structure elucidation, and
bioactivity of these compounds are described in detail.
2. Results and discussion
Compound 1 was obtained as a white powder, and its molecular
ion peak appeared at m/z 294 in the EI mass spectrum. A molecular
formula of C17H26O4 was determined by HREIMS (m/z 294.1838
[M]þ), requiring five degrees of unsaturation. The 1H NMR spec-
trum (in CDCl3, Table 1) alongside HSQC data displayed one methyl
doublet, two methyl singlets, four double doublets assignable to
two methylenes, one broad doublet due to a hydroxy proton, one
* Corresponding author.
E-mail address: nyji@yic.ac.cn (N.-Y. Ji).
https://doi.org/10.1016/j.phytochem.2018.04.017
0031-9422/© 2018 Elsevier Ltd. All rights reserved.

46
Y.-P. Song et al. / Phytochemistry 152 (2018) 45e52
Fig. 1. Chemical structures of compounds 1e10.
Table 1
1H NMR Data for 1e3 (500 MHz, dH in ppm, J in Hz).
pos 1
2
3
in CDCl3
in acetone-d6
in CDCl3
in CD3OD
in CDCl3
in CD3OD
1
2
3.60, br d (9.1)
5.32, br s
3.56, br d (9.1)
5.31, br s
2.03, m
1.95, m
1.97, m
1.13, dddd (12.6, 12.6, 10.5,
5.8)
5.63, br d (10.9)
5.41, br d (10.2)
1.95, m
1.72, br d (12.7)
1.63, m
5.63, br d (10.3)
5.44, br d (10.4)
2.08, td (13.4, 3.1)
1.76, br d (13.0)
1.67, m
5.62, br d (10.7)
5.41, br d (10.3)
1.95, td (13.0, 2.5)
1.74, br d (12.8)
1.64, m
5.64, br d (10.3)
5.45, br d (10.3)
2.08, td (13.2, 3.2)
1.76, br d (13.0)
1.63,
4a 2.03, m
4b 1.94, m
5a 1.94, m
5b 1.13, m
1.33, m
1.39, m
1.33, m
1.38, m
6
7
0.91, m
1.39, m
0.86, dddd (12.9, 10.4, 9.1, 2.7) 2.11, m
2.13, m
1.50, m
1.37, m
2.11, m
1.47, m
1.29, m
2.13, m
1.50, m
1.33, m
1.40, m
1.48, m
1.32, m
8a 1.59, ddd (13.2, 3.8,
2.3)
1.63, ddd (13.1, 3.9, 2.3)
8b 1.13, m
1.06, ddd (13.0, 11.5, 11.5)
1.14, dtd (12.8, 9.1,
5.7)
1.17, dtd (13.3, 8.8,
5.9)
1.11, dddd (12.8, 10.4, 8.5,
5.0)
1.14, dddd (13.3, 10.5, 8.4,
5.2)
9a 3.68, ddt (11.3, 9.0, 2.2) 3.64, ddt (11.1, 8.6, 2.4)
9b
1.99, m
1.91, m
5.08, br t (7.1)
2.01, m
1.97, m
5.10, br t (7.2)
1.61, m
1.51, m
2.41, t (7.4)
1.60, m
1.51, m
2.47, t (7.2)
10a 1.97, dd (14.9, 9.1)
1.88, dd (14.6, 8.6)
1.78, dd (14.6, 2.5)
2.42, d (9.4)
2.42, d (9.4)
1.41, s
10b 1.83, dd (15.0, 2.1)
12a 2.58, dd (13.0, 9.9)
1.60, s
1.60, s
12b 2.43, dd (13.0, 8.8)
13 1.44, s
1.68, s
1.68, s
2.13, s
2.13, s
14 0.92, d (6.5)
15 1.65, br s
0.92, d (6.5)
1.63, br s
0.80, d (6.8)
1.27, s
0.83, d (6.8)
1.29, s
0.80, d (6.8)
1.27, s
0.83, d (6.8)
1.29, s
16 4.61, td (9.7, 2.6)
17
18
19
20
21a
21b
23
4.64, br t (9.4)
5.06, d (3.7)
4.00, td (9.8, 3.5)
3.72, t (10.2)
3.64, t (9.1)
3.77, dt (9.9, 3.0)
3.89, dd (11.5, 2.8)
3.73 dd (11.5, 3.3)
2.04, s
5.09, d (3.6)
3.79, dd (10.9, 3.6)
3.68, dd (10.8, 8.7)
3.35, t (9.1)
5.06, d (3.6)
3.99, td (9.8, 3.5)
3.70, t (9.7)
3.62, t (9.1)
5.09, d (3.6)
3.79, dd (10.8, 3.6)
3.67, dd (10.8, 8.7)
3.35, t (9.2)
3.76, ddd (9.7, 5.5, 2.5)
3.76, dd (12.6, 2.5)
3.69, dd (12.4, 5.7)
1.99, s
3.76, ddd (9.7, 5.2, 2.5) 3.77, dt (9.8, 3.0)
3.76, dd (12.5, 2.4)
3.69, dd (12.1, 5.9)
1.99, s
3.87, br d (10.5)
3.75, br d (10.9)
2.04, s
OH 2.67, br d (3.0)
NH
4.82, br d (4.7)
6.32, br d (8.4)
6.18, br d (8.8)
broad doublet/a batch of triplets of double doublet/one double
triplet ascribable to three oxygenated methines, and one broad
singlet attributable to an olefinic proton. The 13C NMR spectrum
(Table 2) exhibited 17 resonances, sorted into three methyls, five
methylenes, six methines, and three nonprotonated carbons by
DEPT experiments. COSY correlations of H-12/H-16/OH-16 indi-
cated the presence of a 1,2-disubstituted ethanol unit, which was
flanked by C-11 and C-17 on the basis of HMBC correlations from H-
12 to C-11 and C-17 and from H-13 to C-11 and C-12. Furthermore,
C-10 was attached to C-11 by HMBC correlations from H-10 to C-11
and from H-13 to C-10, which was then extended to C-2, C-4, and C-
14 by analysis of COSY correlations (Fig. 2). The connectivity at C-3
was established by HMBC correlations from H-15 to C-2, C-3, and C-
4, and an ether linkage between C-1 and C-9 was suggested by
comparison of NMR data with those reported for (2S,4R,6S)-6-
methyl-2,4-diphenyltetrahydropyran (Fries et al., 2014). C-11 and

Y.-P. Song et al. / Phytochemistry 152 (2018) 45e52
47
Table 2
and H-10b suggested the bond rotation at C-10 to be restricted, the
relative stereochemistry between rings B and C could not be
resolved by the observed NOE correlation between H-9 and H-12a.
The conformers with 1R, 6R, 7S, 9S, 11R, and 16R and 1R, 6R, 7S, 9S,
11S, and 16S configurations matching the above NOE correlations
were subjected to Gaussian 09 software for the computation of ECD
spectra using the time-dependent density function theory (TD-
DFT) method at the B3LYP/6-31G(d) level in MeOH with the inte-
gral equation formalism variant (IEF) of the polarizable continuum
model (PCM) (Frisch et al., 2010), and the results were depicted by
SpecDis software with sigma ¼ 0.2 (Bruhn et al., 2011). Based on the
comparison of experimental and calculated ECD spectra (Fig. 4), the
absolute configuration of 1, trivially named trichaspin, was
assigned to be 1R, 6R, 7S, 9S, 11R, and 16R.
Compound 2 was isolated as a colorless oil with a molecular
formula of C23H39NO6 given by HREIMS (m/z 425.2785 [M]þ),
implying five degrees of unsaturation. The 1H NMR spectrum (in
CDCl3, Table 1) showed one methyl doublet, four methyl singlets,
one doublet ascribable to an oxygenated methine, one broad triplet
and two broad doublets attributable to three olefinic protons, and
one broad doublet due to an exchangeable proton, with the
exception of six signals at dH 3.6e4.1 for four methines and one
methylene. The 13C NMR and DEPT spectra (Table 2) demonstrated
the presence of five methyls, five methylenes, ten methines, and
three nonprotonated carbons. HMBC correlations from H-12 and H-
13 to C-10 and C-11 established the connectivity at C-11, which was
elongated to C-2, C-4, and C-14 by COSY correlations (Fig. 2). A 1,10-
bisaboladiene moiety oxygenated at C-3 was proposed by the
deshielded signals (dC 77.6 in CDCl3 and 78.2 in CD3OD) of C-3 and
HMBC correlations from H-15 to C-2, C-3, and C-4, which was
confirmed by comparison of NMR data with those of zingiberenol
(Terhune et al., 1975; Khrimian et al., 2014). In addition, an analysis
of the other NMR chemical shifts and coupling constants indicated
13C NMR Data for 1e3 (125 MHz, dC in ppm).
pos
1
2
3
in CDCl3
in acetone-d6 in CDCl3
in CD3OD in CDCl3
in CD3OD
1
2
3
4
5
6
7
8
79.1, CH
123.4, CH 125.3, CH
137.2, C 135.9, C
79.6, CH
135.2, CH 135.7, CH 135.1, CH 135.6, CH
132.1, CH 133.8, CH 132.3, CH 133.9, CH
77.6, C
78.2, C
77.5, C
78.2, C
30.9, CH2 31.3, CH2
23.6, CH2 24.3, CH2
34.8, CH2 35.8, CH2 34.8, CH2 35.8, CH2
22.4, CH2 23.4, CH2 22.3, CH2 23.4, CH2
45.4, CH
34.5, CH
46.2, CH
35.2, CH
40.3, CH
36.4, CH
41.5, CH
37.5, CH
40.1, CH
36.7, CH
41.4, CH
37.9, CH
42.3, CH2 43.1, CH2
73.3, CH 74.1, CH
46.8, CH2 47.9, CH2
83.7, C 82.6, C
34.2, CH2 35.3, CH2 33.6, CH2 34.6, CH2
26.1, CH2 27.0, CH2 21.9, CH2 22.8, CH2
124.7, CH 125.7, CH 44.0, CH2 44.4, CH2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
131.5, C
132.2, C
209.2, C
212.0, C
40.3, CH2 41.4, CH2
28.4, CH3 27.9, CH3
18.8, CH3 19.0, CH3
23.0, CH3 23.0, CH3
17.8, CH3 17.7, CH3
25.9, CH3 25.9, CH3 30.1, CH3 29.8, CH3
15.8, CH3 16.0, CH3 15.8, CH3 16.0, CH3
27.3, CH3 27.5, CH3 27.3, CH3 27.5, CH3
68.8, CH
177.1, C
68.9, CH
176.9, C
92.9, CH
54.0, CH
73.1, CH
70.8, CH
71.4, CH
93.6, CH
55.9, CH
72.5, CH
72.5, CH
73.4, CH
92.8, CH
54.0, CH
73.3, CH
71.1, CH
71.3, CH
93.6, CH
55.9, CH
72.5, CH
72.5, CH
73.4, CH
61.8, CH2 62.7, CH2 62.0, CH2 62.7, CH2
171.9, C 173.5, C 171.8, C 173.5, C
23.5, CH3 22.6, CH3 23.5, CH3 22.6, CH3
C-17 were linked through an oxygen atom to form a g-lactone ring
to satisfy the unsaturation requirement, which was also supported
by the deshielded signals (dC 83.7 in CDCl3 and 82.6 in acetone-d6)
of C-11 (Zhang et al., 2014). Other HMBC correlations (Fig. 2) further
verified the planar structure of 1.
The relative configuration of 1 was established by analysis of
coupling constants and NOE correlations. H-1 and H-9 were ori-
ented to be axial by their respective constants, which were syn to
H-7 based on their NOE correlations (Fig. 3). H-6 was axial and
opposite to H-1, H-5b, and H-7 on the basis of its splitting pattern
and large coupling constants (in acetone-d6), and it was placed to
be syn to H-4a and H-8b by its NOE correlations with them. NOE
correlations between H-1 and H-5b and between H-8b and H-10a
further corroborated the relative configurations of rings A and B,
while those of H-13 with H-12b and H-16 located them on the same
face of ring C. Although the different coupling constants of H-10a
the presence of a 2-acetamido-2-deoxy-a-glucopyranosyl residue
(Yamaoka et al., 1974; Afiyatullov et al., 2007), and its attachment to
C-3 via a glycosidic linkage was supported by the observed HMBC
correlation from H-16 to C-3. An acidic hydrolysis of 2 produced
(3S,6R,7S)-zingiberenol and 2-N-acetylglucosamine, validated by
20
their identical specific optical rotation values ([
a
]
ꢀ46 and
D
22
20
[a
a
]
]
þ43, respectively) with those reported ([
a
]
ꢀ37.7 and
D
D
[
22 þ41.4, respectively) (Fondy and Emlich, 1981; Khrimian et al.,
D
Fig. 2. Key COSY (bold lines) and HMBC (arrows) correlations of 1ꢀ3 and 6e9.

48
Y.-P. Song et al. / Phytochemistry 152 (2018) 45e52
Fig. 4. Experimental and calculated ECD spectra of 1.
planar structure. The absolute configuration of 3, trivially named
trichaspside B, was assigned as that of 2 based on the biogenic
consideration and the same specific optical rotation data.
As a possible precursor of compounds 1e3, (3S,6R,7S)-zingi-
berenol (4) was also obtained as a colorless oil. Its structure and
absolute configuration were speculated by comparison of NMR and
specific optical rotation data with those reported (Terhune et al.,
1975; Khrimian et al., 2014). This sesquiterpene has previously
been found in some terrestrial plants and animals (as a sex pher-
omone) but never been reported from fungi (Borges et al., 2006;
Terhune et al., 1975; Khrimian et al., 2015). It is also worth to
mention that 2 and 3 represent the first aminoglycosides of bisa-
bolane and norbisabolane sesquiterpenes, respectively, especially
for the presence of an a-glycosidic linkage. As for the skeleton of 1,
it may be an ethylated sesquiterpene or a trinorditerpene, but no
related diterpenes were found herein.
Compounds 5 and 6 were purified as colorless oils, respectively,
and the former was identified to be cyclonerodiol by the identical
NMR and specific optical rotation data ([
a]
20 ꢀ21 (c 0.10, MeOH or
D
CHCl3) for 5) (Laurent et al., 1990; Langhanki et al., 2014). The
molecular formula of 6 was deduced to be C15H28O3 by analysis of
HREIMS (m/z 256.2041 [M]þ), implying two degrees of unsatura-
tion. The 1H NMR spectrum (Table 3) exhibited one methyl doublet,
four methyl singlets, one doublet ascribable to a methylene, and
one doublet and one triple doublet attributable two olefinic pro-
tons. The 13C NMR spectrum (Table 4) displayed 15 resonances,
classified into five methyls, three methylenes, four methines, and
three nonprotonated carbons by DEPT and HSQC data. An analysis
of the above NMR data revealed that 6 differed from 5 mainly at the
side chain moiety (Laurent et al., 1990; Langhanki et al., 2014).
Furthermore, HMBC correlations from Me-12 and Me-15 to C-10
and C-11 and COSY correlations of H-8/H-9/H-10 confirmed the
presence of 4-hydroxy-4-methylpent-2-enyl unit, which was sup-
ported by the similar NMR data with those of asporyzin C (Qiao
et al., 2010). Its attachment to ring A was indicated by HMBC cor-
relations from Me-14 to C-6, C-7, and C-8. Thus, 6 was identified to
be 9-cycloneren-3,7,11-triol, corroborated by the other HMBC and
COSY correlations (Fig. 2). The geometry of double bond at C-9 was
allowed to be trans by the large coupling constant between H-9 and
H-10, and the absolute configurations at C-2, C-3, C-6, and C-7 were
proposed to be the same as those of 5 based on the identical NMR
and specific optical rotation data as well as the biogenic
consideration.
Fig. 3. Key NOE correlations of 1, 8, and 9.
2014). Thus, the absolute configuration of 2, trivially named tri-
chaspside A, was established to be 3S, 6R, 7S, 16R, 17R, 18R, 19S, and
20R.
Compound 3 was purified as a colorless oil and assigned a
molecular formula of C22H37NO7 by interpretation of HREIMS (m/z
427.2568 [M]þ), resembling that of 2 except for the presence of one
additional oxygen atom and the lack of one carbon atom and two
protons. In its 1H and 13C NMR spectra (Tables 1 and 2), the signals
for a carbonyl group and a methylene group appeared, replacing
those for a methyl group and a trisubstituted vinyl group at the side
chain terminus of 2. HMBC correlations from H-10 to C-11 and C-13
and from H-13 to C-10 and C-11 and COSY correlations of H-8/H-9/
H-10 indicated the presence of a pentan-4-onyl group, which was
bonded to C-7 by HMBC correlations from H-14 to C-6, C-7, and C-8.
Other HMBC and COSY correlations (Fig. 2) further confirmed the

Y.-P. Song et al. / Phytochemistry 152 (2018) 45e52
49
Table 3
1H NMR data for 6e9 (500 MHz, dH in ppm, J in Hz).
pos
6 (in CD3OD)
7 (in CD3OD)
8 (in CD3OD)
9 (in CDCl3)
1
2
1.02, d (6.8)
1.64, m
1.63, m
1.54, m
1.82, m
1.62, m
1.81, m
1.02, d (6.8)
1.60, m
1.63, m
1.53, m
1.81, m
1.59, m
1.81, m
1.01, d (6.8)
1.53, m
1.66, m
1.57, m
1.88, m
1.50, m
1.96, m
2.20, d (7.9)
2.85, m
2.02, d (15.9)
2.83, m
4a
4b
5a
5b
6
7a
7b
8a
8b
9a
9b
10
11
12a
12b
13
14
15a
15b
16
17
18
19
20
1.82, ddd (12.8, 6.4, 1.0)
1.27, t (12.4)
2.34, t (13.7)
2.20, d (6.6)
2.20, d (6.6)
5.68, dt (15.6, 6.8)
1.52, m
1.40, ddd (12.1, 11.2, 4.0)
1.62, m
1.56, m
3.97, t (6.4)
1.71, m
1.67, m
1.98, m
1.93, m
1.86, m
5.62, d (15.6)
1.27, s
3.94, t (7.3)
4.70, br d (6.8)
1.88, m
1.64, dd (12.2, 1.4)
4.91, br s
4.81, br s
1.22, s
1.12, s
1.72, br s
3.48, d (11.1)
3.43, d (11.1)
1.22, s
1.14, s
1.10, s
1.23, s
1.12, s
1.27, s
2.27, dd (11.3, 9.4)
1.91, m
1.39, dd (14.4, 9.8)
0.97, s
0.94, s
1.08, d (7.3)
1.59, s
1.75, s
Table 4
preparing Mosher's esters and single crystals.
13C NMR data for 6e9 (125 MHz, dC in ppm).
Compound 8 was obtained as a colorless oil with a molecular
formula of C15H28O4 given by HREIMS (m/z 272.1981 [M]þ),
consistent with two degrees of unsaturation. Its 1H and 13C NMR
data (Tables 3 and 4) showed high similarities to those of cyclo-
nerodiol oxide (Fujita et al., 1984), except for the presence of signals
for an oxymethylene group and the lack of signals for a methyl
group. HMBC correlations from the oxymethylene to C-10, C-11, and
Me-15 indicated its attachment to C-11. Thus, 8 was identified to be
7,10-epoxycycloneran-3,11,12-triol, which was further verified by
the other HMBC and COSY correlations (Fig. 2). Me-1 was syn to H-6
by their NOE correlation, while Me-13 was syn to H-2 by their NOE
correlation (Fig. 3). Additionally, Me-14 and H-10 were located on
the same face by their NOE correlation. The absolute configurations
of chiral centers except for C-11 were assigned as those of cyclo-
nerodiol oxide by the similar specific optical rotation data (Fujita
et al., 1984).
Compounds 9 and 10 were purified as a colorless oil and a white
powder, respectively, and the latter was identified to be harzian-
dione by its spectroscopic data (Miao et al., 2012; Adelin et al.,
2014). As for 9, a molecular formula of C20H30O2 was established
by interpretation of HREIMS (m/z 302.2241 [M]þ), requiring six
degrees of unsaturation. The 1H NMR spectrum (Table 3) in com-
bination with HSQC data showed four methyl singlets, one methyl
doublet, and one broad doublet due to an oxymethine, while the
13C NMR and DEPT spectra (Table 4) demonstrated the presence of
five methyls, five methylenes, four methines, and six quaternary
carbons. A detailed comparison of NMR data with those of 10
revealed that their differences were mainly situated around C-11.
Replacing the conjugated carbonyl group of 10, a hydroxy group
appeared at C-11, and its connectivity was confirmed by HMBC
correlations from H-11 to C-10, C-12, and C-13, from H-19 to C-10,
C-12, C-13, and C-14, and from H-20 to C-8, C-9, and C-10. HMBC
correlations from H-2 and H-4 to C-3, from H-7 to C-5, C-6, and C-
14, from H-16 and H-17 to C-1, C-2, and C-6, and from H-18 to C-4,
C-5, and C-6 and COSY correlations of H-4/H-5/H-18, H-7/H-8, H-
11/H-12, and H-14/H-15/H-2 (Fig. 2) further verified 9 to be 11-
hydroxy-9-harzien-3-one. The relative configurations around
pos
6 (in CD3OD)
7 (in CD3OD)
8 (in CD3OD)
9 (in CDCl3)
1
2
3
4
5
6
7
8
15.4, CH3
45.4, CH
82.0, C
41.4, CH2
25.1, CH2
55.4, CH
75.7, C
45.1, CH2
123.9, CH
142.2, CH
71.2, C
29.9, CH3
26.1, CH3
25.3, CH3
29.9, CH3
15.4, CH3
45.5, CH
82.1, C
41.4, CH2
25.2, CH2
55.7, CH
75.4, C
37.9, CH2
30.2, CH2
77.4, CH
148.8, C
111.6, CH2
26.1, CH3
24.8, CH3
17.4, CH3
14.7, CH3
46.4, CH
81.9, C
41.4, CH2
26.2, CH2
55.5, CH
87.3, C
35.8, CH2
26.7, CH2
81.0 CH
74.8, C
69.2, CH2
26.1, CH3
23.2, CH3
19.2, CH3
49.5, C
59.7, CH
215.3, C
42.9, CH2
30.1, CH
51.4, C
30.2, CH2
28.0, CH2
134.8, C
142.3, C
68.7, CH
45.9, CH2
46.1, C
54.3, CH
25.9, CH2
23.5, CH3
25.2, CH3
21.0, CH3
21.7, CH3
20.8, CH3
9
10
11
12
13
14
15
16
17
18
19
20
Compound 7 was isolated as a colorless oil. Its molecular for-
mula was assigned to be C15H28O3, the same as for 6, by HREIMS (m/
z 256.2029 [M]þ), suggesting two degrees of unsaturation. The 1H
and 13C NMR data (Tables 3 and 4) closely resembled those of 5,
except for signals for the side chain terminus (Laurent et al., 1990;
Langhanki et al., 2014). HMBC correlations from Me-15 to C-10, C-
11, and C-12 suggested the connectivity at C-11, which was
extended to C-8 by COSY correlations of H-8/H-9/H-10 and then
attached to ring A via C-7 by HMBC correlations from H-14 to C-6,
C-7, and C-8. The established side chain was also supported by its
identical NMR data with chilianoside H (Jiang et al., 1999). Other
HMBC and COSY correlations (Fig. 2) further validated 5 to be 11-
cycloneren-3,7,10-triol, and its absolute configurations at C-2, C-3,
C-6, and C-7 were also proposed to be the same as those of 5 and 6
based on the biogenic consideration. Unfortunately, the absolute
configuration at C-10 was still unresolved due to the failure in

50
Y.-P. Song et al. / Phytochemistry 152 (2018) 45e52
rings C and D were deduced to be the same as those of 10 on the
basis of their identical NMR data (Adelin et al., 2014), which were
supported by NOE correlations of H-14 with H-7b, H-15a, and Me-
16 and of H-5 with H-15b (Fig. 3). Me-19 was syn to H-4a, H-5, and
H-15b by its NOE correlations with them, while the relative
configuration of H-11 was oriented by its different splitting pattern
and chemical shift from those of 9-harzien-11-ol (Adelin et al.,
2014). The ECD spectrum displayed a positive Cotton effect at
291 nm, which was then computed with the TD-DFT method at the
gas-phase B3LYP/6-31G(d) level in Gaussian 09 software (Frisch
et al., 2010). The result was drawn by SpecDis software with
sigma ¼ 0.2 (Bruhn et al., 2011), and it agreed well with the
experimental one (Fig. 5). Thus, the absolute configuration of 9 was
assigned to be 2S, 5R, 6R, 11R, 13S, and 15S.
inhibit some marine-derived Vibrio species, and the effects of 2 and
3 might relate to their aminoglycoside moiety.
4. Experimental section
4.1. General experimental procedures
Optical rotations were determined on a JASCO P-1020 polar-
imeter and an SGW-3 polarimeter. ECD spectra were measured on a
Chirascan CD spectrometer. IR spectra were obtained on a JASCO FT/
IR-4100 spectrometer. NMR spectra were recorded on a Bruker
Avance III 500 NMR spectrometer (500 and 125 MHz for 1H and 13C,
respectively) using tetramethylsilane (TMS) as an internal standard.
Low and high resolution EI mass spectra were acquired on an
Autospec Premier P776 mass spectrometer with a double-focusing
magnetic sector mass analyzer. HPLC separation was operated on
an Agilent HPLC system (1260 infinity quaternary pump, 1260 in-
In order to develop new inhibitors against pathogenic bacteria
that greatly threatened marine aquaculture, compounds 1e4 were
assayed for inhibition of five aquatic pathogens (Vibrio para-
haemolyticus, V. anguillarum, V. harveyi, V. splendidus, and Pseu-
finity diode-array detector) using an Eclipse SB-C18 (5 mm,
doalteromonas citrea) using the disk diffusion method at 20
m
g/disk
9.4 ꢁ 250 mm) column. Column chromatography (CC) was per-
formed with silica gel (200e300 mesh, Qingdao Haiyang Chemical
Co.), RP-18 (AAG12S50, YMC Co., Ltd.), and Sephadex LH-20 (GE
Healthcare). Thin-layer chromatography (TLC) was carried out with
precoated silica gel plates (GF-254, Qingdao Haiyang Chemical Co.).
Quantum chemical calculations were run with Gaussian 09 soft-
ware (IA32W-G09RevC.01).
(Miao et al., 2012), and chloramphenicol with inhibitory zone di-
ameters of 19.7,18.2,17.9,18.7, and 19.7 mm, respectively, was taken
as a positive control. Among them, only 2 and 3 showed potent
inhibition (6.1e6.4 mm zones) of the four Vibrio bacteria tested
(Table S1), which might correlate with the 2-acetamido-2-deoxy-
a-
D-glucopyranosyl group. Compounds 5e9 were assayed for inhi-
bition of V. parahaemolyticus and P. citrea, and only 8 and 9 showed
inhibition of V. parahaemolyticus, each with a 6.2 mm zone. Addi-
tionally, compounds 1e9 were evaluated for toxicity to the
zooplankton Artemia salina using K2CrO7 as a positive control (100%
lethal rate), but they exhibited only 52.2e78.7% lethal rates at
4.2. Fungal material and fermentation
Following a previous procedure (Wang et al., 2006), Trichoderma
asperellum Samuels, Lieckfeldt & Nirenberg cf44-2 (Moniliaceae) as
an endophyte was isolated from the fresh tissue of the surface-
sterilized brown alga Sargassum sp. (Sargassaceae) collected from
Zhoushan Islands (N30ꢂ0102000, E122ꢂ0501400) of China in August
2010. The species was identified by morphological taxonomy and
by analysis of the ITS regions of its rDNA, deposited at GenBank
(accession no. MG696741). Its fermentation was performed stati-
cally at room temperature for 30 days in 200 ꢁ 1 L Erlenmeyer
flasks, each containing 300 mL of media prepared by addition of
500 mL potato (200 g) broth, 20 g glucose, 5 g peptone, and 5 g
yeast extract powder into 500 mL natural seawater from the coast
of Yantai.
100 mg/mL.
3. Conclusion
Chemical investigation towards Trichoderma asperellum cf44-2,
an endophyte of the marine brown alga Sargassum sp., resulted in
the isolation and identification of ten terpenes, comprising three
undescribed bisabolane derivatives (1e3), three undescribed
cyclonerane sesquiterpenes (6e8), and one undescribed harziane
diterpene (9). Among them, compound 1 possesses an undescribed
ethylated bisabolane framework, while 2 and 3 represent the first
aminoglycosides of bisabolane and norbisabolane sesquiterpenes,
respectively. The bioassay results showed that 2, 3, 8, and 9 could
4.3. Extraction and isolation
The mycelia were collected by filtration, which were then dried
in the shade and exhaustively extracted with CH2Cl2 and MeOH
(1:1, v/v). After removing organic solvents by evaporation under
vacuum, the residue was partitioned between EtOAc and H2O to
give an EtOAc-soluble extract (52.4 g). The filtrate was directly
extracted with EtOAc and then concentrated to afford an extract
(31.3 g). In view of the identical TLC profiles, these two parts were
combined and then subjected to silica gel CC with step-gradient
solvent systems consisting of petroleum ether (PE)/EtOAc and
CH2Cl2/MeOH to yield 12 fractions (Frs. 1e12). Fr. 3 eluted with PE/
EtOAc (5:1) and was further purified by CC on RP-18 (MeOH/H2O,
3:1) and Sephadex LH-20 (MeOH) and preparative TLC (PE/EtOAc,
2:1) to produce 4 (2.6 mg). Fr. 4 eluted with PE/EtOAc (2:1) and was
further purified by CC on RP-18 (MeOH/H2O, 7:3) and Sephadex LH-
20 (MeOH) and preparative TLC (PE/EtOAc, 1:1) to yield 10
(12.8 mg). Fr. 7 eluted with PE/EtOAc (1:1) and was further purified
by RP-18 CC (MeOH/H2O, 7:3) and preparative TLC (CH2Cl2/MeOH,
30:1) as well as semipreparative HPLC (MeOH/H2O, 3:7 to 4:1) to
afford 1 (1.0 mg), 5 (4.8 mg), and 9 (3.4 mg). Fr. 9 eluted with EtOAc
and was further purified by RP-18 CC (MeOH/H2O, 3:7 to 2:3) and
preparative TLC (EtOAc) as well as semipreparative HPLC (MeOH/
Fig. 5. Experimental and calculated ECD spectra of 9.

Y.-P. Song et al. / Phytochemistry 152 (2018) 45e52
51
H2O, 3:17 to 1:1) to give 6 (1.2 mg), 7 (1.5 mg), and 8 (1.5 mg). Fr. 12
eluted with MeOH and was further purified by CC on RP-18 (MeOH/
H2O, 7:3) and Sephadex LH-20 (MeOH) and preparative TLC
(CH2Cl2/MeOH, 4:1) to obtain 2 (8.6 mg) and 3 (3.6 mg).
(1.2 mg). Their specific optical rotation values were determined to
20
22
be [
a
]
D
ꢀ46 (c 0.044, CH2Cl2) and [
a
]
þ43 (c 0.048, H2O),
D
respectively.
Acknowledgements
4.3.1. Trichaspin (1)
20
White powder; [
a
]
þ36 (c 0.060, MeOH); IR (KBr) vmax 3406,
D
Financial support from the National Natural Science Foundation
of China (31670355), Natural Science Foundation for Distinguished
Young Scholars of Shandong Province (JQ201712), Open Fund of Key
Laboratory of Experimental Marine Biology, CAS (KF2017NO4),
Youth Innovation Promotion Association of CAS (2013138), and
Self-Fund from Yantai Institute of Coastal Zone Research, CAS
(Y755031012) is gratefully acknowledged.
2920, 2858, 1774, 1631, 1446, 1381, 1304,1180, 1134, 1072, 941 cmꢀ1
;
1H and 13C NMR data, Tables 1 and 2; EIMS m/z (%) 294 [M]þ (80);
HREIMS m/z 294.1838 [M]þ (calcd for C17H26O4, 294.1831).
4.3.2. Trichaspside A (2)
20
Colorless oil; [
a
]
þ50 (c 0.26, CH2Cl2); IR (KBr) vmax 3402,
D
2962, 2931, 2881, 1655, 1547, 1439, 1377, 1099, 1030, 737 cmꢀ1 1H
;
and 13C NMR data, Tables 1 and 2; HREIMS m/z 425.2785 [M]þ
Appendix A. Supplementary data
(calcd for C23H39NO6, 425.2777).
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.phytochem.2018.04.017.
4.3.3. Trichaspside B (3)
Colorless oil; [
a
]
20 þ50 (c 0.13, CH2Cl2); IR (KBr) vmax 3367, 2931,
D
2870, 1709, 1655, 1547, 1442, 1373, 1107, 1030, 741 cmꢀ1; 1H and 13
C
References
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22H37NO7, 427.2570).
C
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]
20 ꢀ43 (c 0.10, CH2Cl2).
D
4.3.5. 9-Cycloneren-3,7,11-triol (6)
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Colorless oil; [
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2962, 1651, 1450, 1377, 1296, 1065, 1022, 895 cmꢀ1; 1H and 13C NMR
data, Tables 3 and 4; EIMS m/z (%) 256 [M]þ (29), 241 (15), 239 (25),
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4.3.7. 7,10-Epoxycycloneran-3,11,12-triol (8)
20
Colorless oil; [
a
]
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Colorless oil; [
a
]
20 þ115 (c 0.10, MeOH); IR (KBr) vmax 3436, 2931,
D
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C
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