Nectrianolins A, B, and C, new metabolites produced by endophytic fungus Nectria pseudotrichia 120-1NP

Article abstract of DOI:10.1016/j.tetlet.2017.09.032

Two sesquiterpene-epoxycyclohexenone conjugates, nectrianolins A (1) and B (2), together with a sesquiterpene, nectrianolin C (3), were isolated from the brown rice culture of Nectria pseudotrichia 120-1NP, an endophytic fungus isolated from Gliricidia sepium. Their structures were determined on the basis of 1D-/2D-NMR spectroscopy and HRESIMS data analyses in combination with chemical means. Nectrianolins A–C (1–3) exhibited cytotoxic activity against both HL60 and HeLa cells.

Full text of DOI:10.1016/j.tetlet.2017.09.032

Accepted Manuscript
Nectrianolins A, B, and C, new metabolites produced by endophytic fungus
Nectria pseudotrichia 120-1NP
Nanang Rudianto Ariefta, Priska Kristiana, Handojo Hadi Nurjanto, Hiroyuki
Momma, Eunsang Kwon, Tatsuya Ashitani, Keitaro Tawaraya, Tetsuya
Murayama, Takuya Koseki, Hiroshi Furuno, Narandulam Usukhbayar, Ken-ichi
Kimura, Yoshihito Shiono
PII:
S0040-4039(17)31159-0
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.09.032
TETL 49302
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 July 2017
10 September 2017
13 September 2017
Please cite this article as: Ariefta, N.R., Kristiana, P., Nurjanto, H.H., Momma, H., Kwon, E., Ashitani, T., Tawaraya,
K., Murayama, T., Koseki, T., Furuno, H., Usukhbayar, N., Kimura, K-i., Shiono, Y., Nectrianolins A, B, and C,
new metabolites produced by endophytic fungus Nectria pseudotrichia 120-1NP, Tetrahedron Letters (2017), doi:
http://dx.doi.org/10.1016/j.tetlet.2017.09.032
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Nectrianolins A, B, and C, new metabolites produced by endophytic fungus Nectria
pseudotrichia 120-1NP
b,
Nanang Rudianto Arieftaa, *, Priska Kristianab, c, Handojo Hadi Nurjantod, Hiroyuki
Mommae, Eunsang Kwone, Tatsuya Ashitanib, Keitaro Tawarayab, Tetsuya Murayamab,
Takuya Kosekib, Hiroshi Furunof, Narandulam Usukhbayara, Ken-ichi Kimuraa, Yoshihito
Shionoa, b, *
aThe United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate
020-8550, Japan
bDepartment of Food, Life, and Environmental Science, Faculty of Agriculture, Yamagata
University, Tsuruoka, Yamagata 997-8555, Japan
cDepartment of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah
Mada, Bulaksumur, Yogyakarta 55281, Indonesia
dDepartment of Silviculture, Faculty of Forestry, Universitas Gadjah Mada, Bulaksumur,
Yogyakarta 55281, Indonesia
eResearch and Analytical Center for Giant Molecules, Graduate School of Science, Tohoku
University, Sendai, Miyagi 980-8576, Japan
fInstitute for Materials Chemistry and Engineering (IMCE), Kyushu University, Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan
*
Corresponding authors. Tel. /Fax: +81-235-28-2873. E-mail: nanang.ariefta@gmail.com (N. R. Ariefta); e-
mail: yshiono@tds1.tr.yamagata-u.ac.jp (Y. Shiono)
1

Abstract
Two sesquiterpene-epoxycyclohexenone conjugates, nectrianolins A (1) and B (2), together
with a sesquiterpene, nectrianolin C (3), were isolated from the brown rice culture of Nectria
pseudotrichia 120-1NP, an endophytic fungus isolated from Gliricidia sepium. Their
structures were determined on the basis of 1D-/2D-NMR spectroscopy and HRESIMS data
analyses in combination with chemical means. Nectrianolins A-C (1-3) exhibited cytotoxic
activity against both HL-60 and HeLa cells.
Keywords
Endophyte; Nectria pseudotrichia; Cytotoxicity; Nectrianolins.
Introduction
Endophytes are ubiquitous microorganisms that are found in all plant species.1 These
microorganisms maintain a balance symbiotic relationship with their hosts, i.e., this
association is apparently asymptomatic and avirulent.2 Secondary metabolites produced by
endophytes are rich sources of diverse and pharmacologically active metabolites.2–4
Environmental factors (e.g., light, temperature, pH, and nutrients) play an important role in
production of secondary metabolites by endophytes. These factors are associated with
pathogenicity and fungal tolerance to stress including drought or heat stress.5 The Indonesian
tropical forest has a large diversity of plants due to its complex geological history, large
number of islands, and tropical climate. Thus, the Indonesian tropical forest is a rich potential
source for the isolation of endophytic fungi and their associated metabolites.6 Several strains
of endophytic fungi were isolated by examining different trees in the Wanagama forest of
Indonesia. One strain 120-1NP was isolated from the inner tissue of Gliricidia sepium healthy
stem, which was subsequently identified as Nectria pseudotrichia. The Nectria genus is a
2

saprophyte and considered as a phytopathogen that induced both stem-end rot of avocadoes7
and canker on trees.8 However, this genus is also a rich source of interesting secondary
metabolites with diverse structures and biological activities.8–10 Purification of the extract
derived from fungal culture of N. pseudotrichia 120-1NP led to the isolation of the following
three novel compounds: two sesquiterpene-epoxycyclohexenones 1 and 2, along with the
sesquiterpene congener 3. In this study, we also presented the comprehensive structural
elucidation and characterization of these compounds.
Results and Discussion
Compound 111 was obtained as a colorless crystal. Its molecular formula was determined
to be C22H32O5 through a combination of HRESIMS (m/z 399.2142 [M+Na]+) and NMR data.
The molecular formula of 1 revealed seven degrees of unsaturation. The IR absorptions at
3332 and 1677 cm-1 suggested the presence of hydroxyl and α,β-unsaturated carbonyl groups.
13
The C NMR results (Table 1) along with the HMQC data of 1 revealed the presence of 22
carbon resonances and confirmed the presence of four methyls, five methylenes, seven
methines, one ketone carbonyl, and five quaternary carbons. The 1H NMR spectroscopic data
(Table 1) showed proton resonances, which include a doublet methyl at δH 0.68 (3H, d, J =
6.9 Hz, H-14), two singlet methyls at δH 0.70 (3H, s, H-15) and 1.50 (3H, s, H-13), an
aliphatic methine at δH 1.53 (1H, m, H-5), two methylenes at δH 1.29 (2H, m, H-4), 1.85 (1H,
m, H-3a), and 1.89 (1H, m, H-3b), and an olefinic methine at δH 5.36 (1H, s, H-2). These
proton resonances are consistent with the presence of a substituted cyclohexene ring. The
double bond position was assigned by HMBC correlations from the olefinic methyl (H-13) to
two sp2 carbons at δC 139.4 (C-1) and 124.5 (C-2) and to the quaternary carbon at δC 40.4 (C-
6). Additional HMBC correlations from H-2, H-4, H-5, H-14, and H-15 to C-6 confirmed that
the structure of 1 contained a 1,5,6-trimethyl-cyclohexene moiety (Fig. 1). The presence of a
3

3-methyl-2-penten-1-ol moiety was clarified using HMBC correlations from Me-12 to C-8,
C-9 and C-10, and from H-11 to C-9 and C-10.
Table 1
1H (600 MHz) and 13C NMR (150 MHz) spectroscopic data of 1-3 in pyridine-d5
Nectrianolin A (1)
δC, type
Nectrianolin B (2)
δC, type
Nectrianolin C (3)
Pos.
δH (J in Hz)
δH (J in Hz)
δC, type
δH (J in Hz)
1
2
139.4, C
124.5, CH 5.36, s
139.5, C
124.5, CH
137.5, C
125.7, CH 5.69, m
5.37, s
a: 1.85, m
b: 1.89, m
1.29, m
a: 1.85, m
b: 1.89, m
1.29, m
a: 1.82, m
b: 1.88, m
1.31, m
1.62, m
3
25.7, CH2
25.7, CH2
25.8, CH2
4
5
6
27.1, CH2
33.3, CH
40.4, C
27.1, CH2
33.3, CH
40.5, C
27.2, CH2
33.4, CH
40.5, C
1.53, m
1.53, m
a: 1.36, td (5.5, 13.1)
b: 1.42, td (3.4, 13.7)
a: 1.56, m
a: 1.37, m
b: 1.42, m
a: 1.58, m
b: 1.82, m
a: 1.41, m
b: 1.46, m
a: 1.88, m
b: 1.91, m
7
34.8, CH2
34.8, CH2
34.7, CH2
35.1, CH2
34.3, CH2
8
9
34.6, CH2
141.2, C
b: 1.81, m
141.2, C
123.9, CH
63.5, CH
17.3, CH3
19.2, CH3
15.9, CH3
20.9, CH3
194.1, C
122.6, CH
154.9, C
64.3, CH
60.4, CH
61.6, C
139.6, C
10 124.1, CH 5.68, d (8.9)
11 63.4, CH a 5.63, d (8.9)
5.66, d (9.6)
5.61, d (8.9)
1.93, s
1.51, s
0.68, d (6.2)
0.71, s
124.5 CH
58.9, CH2
16.6, CH3
19.3, CH3
15.9, CH3
21.1, CH3
5.39, br s
4.39, d (6.9)
1.67, s
1.52, s
0.73, d (6.1)
0.76, s
12 17.2, CH3
13 19.2, CH3
14 15.8, CH3
15 20.9, CH3
1’
2’
3’
4’
5’
6’
1.92, s
1.50, s
0.68, d (6.9)
0.70, s
194.6, C
121.1, CH 6.78, s
162.3, C
6.49, s
63.4, CH a 5.11, s
5.13, s
5.09, s
60.4, CH
61.7, C
4.58, s
a: 4.64, d (17.2)
b: 5.02, d (17.2)
a: 5.12, d (16.8)
b: 5.38, d (16.8)
7’
1”
2”
63.4, CH2
63.4, CH2
171.5, C
a: 2.61, dd (4.8, 15.1)
b: 2.71, dd (8.2, 15.1)
4.55, m
44.7, CH2
3”
4”
63.3, CH
24.0, CH3
1.33, d (6.2)
a
Overlapped signals.
The HMBC correlation from H-7 to C-6 indicated that the pentenol was located at C-6.
Further analysis of the HMBC of 1 revealed correlations from H-2’ to C-6’, H-4’ to C-2’
along with C-5’ and H-7’ to C-3’ and C-2’. These data suggested the presence of an
oxygenated cyclohexenone skeleton as well as hydroxymethyl and hydroxyl moieties. The
13
presence of an epoxy group at C-5’ and C-6’ was deduced from the chemical shift of the C
4

NMR signals at these positions. The presence of this epoxy group was also supported by the
1H NMR data acquired in CD3OD since H-5’ appeared at the epoxy region (δH 3.73 d, J = 1.2
Hz), compared to H-4’, which appeared at the hydroxyl region (δH 4.47 s). The HMBC
correlations from H-11 to C-5’ and C-6’ indicated a link between C-11 and C-6’ of the
cyclohexenone moiety (Fig. 2).
Fig. 1. Structures of 1-3.
The relative configuration of the cyclohexene ring in 1 was determined on the basis of
13
13C NMR shifts compared to the reported compounds and 1D NOE (Fig. S8). The C NMR
shifts of C-5, C-6, C-14, and C-15 were similar with those of stelliosphaerol A isolated from
Stelliosphaera formicum12 [δC 34.6 (C-5), 41.6 (C-6), 16.3 (C-14), and 21.5(C-15)]. These
carbons have a cis orientation that is slightly different from the trans orientation in subersin13
[δC 37.8 (C-5), 39.5 (C-6), 16.0 (C-14), and 26.6 (C-15)] and oculatolide14 [δC 37.7 (C-5),
39.5 (C-6), 15.9 (C-14), and δC 26.4 (C-15)]. The NOEs data showed correlations from Me-
14 to H2-7, and Me-15 to H-4, but not from Me-15 to H-5. These data showed that the Me-15
has a pseudo-axial orientation along with the Me-14 and C-7 have a pseudo-equatorial
5

orientation. Further, NOE correlation between H-12 and H-11 indicated an E-form double
bond at C-9 and C-10. The trans orientation for both the epoxide and C-4’-OH groups were
assigned on the basis of the analysis of its 3J coupling constant of 0 Hz for H-4’ and H-5’.
Fig. 2. 1H-1H COSY and key HMBC correlations of 1-3.
The absolute configuration of epoxycyclohexenone was deduced by circular dichroism
(CD) analysis. The CD spectrum of 1 generated negative Cotton effects at 240 nm (-4.2) and
340 nm (-2.2). The CD data was compared with that of 13-hydroxylmacrophorin A (4’R, 5’R,
6’R) isolated from Microdiplodia sp. TT-1215 [CD (MeOH) ∆ε: 240 (-4.6), 334 (+2.86)]. This
comparison suggested that the C-4’-OH in 1 was β-oriented, identical to that observed in 13-
hydroxylmacrophorin A (4’R). In contrast, the epoxide at C-5’ and C-6’ in 1 was α-oriented
(5’S, 6’S), which is opposite of that observed in 13-hydroxylmacroporin A. This was
supported by a similar relationship between the CD spectra of neomacrophorin III isolated
from Trichoderma sp. 1212-0316 [ECD (MeOH) ∆ε: 243 (-6.1), 339 (-3.2)] and myrothecol B
isolated from Myrothecium fungus17 [CD (MeOH) ∆ε: 249 (-5.5), 334 (-3.8)], where the C-
4’-OH and epoxide at C-5’ and C-6’ were trans oriented (4’R, 5’S, 6’S). Furthermore, single
6

crystal was obtained from a methanol solution of 1 and was suitable for X-ray
crystallography. Final refinement of the diffraction data resulted in a small Flack parameter -
0.1(3), allowing the assignment of the absolute configuration of 1 (Fig. 3).18
Fig. 3. ORTEP drawing of 1, showing 50% probability ellipsoids.
Compound 219 was isolated utilizing a less polar eluent system than 1, which indicated
that 2 is a less polar compound compared to 1. The HRESIMS data of 2 showed a sodium
adduct ion at m/z 485.2509 [M+Na]+, suggesting the molecular formula C26H38O7 with eight
degrees of unsaturation. The molecular formula of 2 was 86 units larger than the molecular
13
formula of 1. The 1H and C NMR spectroscopic data (Table 1) of 2 closely resembled those
of 1 except for several additional resonances at δC 171.5 (C-1”), 44.7 (C-2”), 63.3 (C-3”),
24.0 (C-4”), δH 2.61 (dd, J = 4.8, 15.1 Hz, H-2a”), 2.71 (dd, J = 8.2, 15.1 Hz, H-2b”), 4.55 (m,
H-3”), and 1.33 (d, J = 6.2 Hz, H-4”), which indicated characteristic signals for a 3-
hydroxybutanoate moieties. In the HMBC analysis of 2, correlations from H-7’ to C-1”
indicated that the butanoate moiety was connected to C-7’ (Fig. 2). Compound 2 exhibited
identical 13C NMR chemical shifts (Table 1) and CD data [CD (MeOH) ∆ε: 248 (-4.2), 340 (-
1.9)] to those of 1. These data indicated that 1 and 2 have the same configuration of the
cyclohexene (C-5, C-6 and C-11) and epoxycyclohexenone (C-4’, C-5’ and C-6’) moieties.
The absolute configuration at C-3” of 2 was determined by alkaline methanolysis. The
resulting product was compared to the authentic standards by chiral GC-MS. Injection of the
pentane extract of hydroxylate into GC-MS with a CycloSil-B capillary column was
7

monitored by the 3-hydroxybutanoate ion ([M-CH3]+ m/z = 103) generating a signal at 12.937
min. This signal was verified with a mixture of standard methyl (S/R)-3-hydroxybutanoate
that appeared at 12.800 and 12.938 min, respectively, under the same conditions. The
configuration of C-3” was then determined to be the (R)-form. The structure of 2 was finally
determined as shown in Fig. 1.
The molecular formula of 320 was assigned as C15H26O by HRESIMS analysis (m/z
245.1876 [M+Na]+) in combination with NMR data, which indicated only three degrees of
unsaturation. In the NMR data of 3, characteristic upfield signals at δC 19.3 (C-13), 15.9 (H-
14), 21.1 (C-15), δH 1.52 (H-13), 0.73 (H-14), and 0.76 (H-15) revealed the presence of a
1,5,6-trimethyl-cyclohexene moiety. Furthermore, signals at δC 35.1 (C-7), 34.3 (C-8), 139.6
(C-9), 124.5 (C-10), 58.9 (C-11), δH 1.41 (H-7a), 1.46 (H-7b), 1.88 (H-8a), 1.91 (H-8b), 5.39
(H-10), and 4.39 (H-11) revealed the presence of a 2-methylbutene moiety. Intensive HMBC
analysis confirmed that the planar structure of 3 has the same sesquiterpene part of 1. The
critical difference between 1 and 3 was the absence of the epoxycyclohexenone group and the
appearance of a new oxygenated methylene at C-11 in compound 3. The absence of the
epoxycyclohexenone group in 3 was also supported by the finding that 3 lacked the IR
absorbance at νmax 1677 cm-1 that was present in 1 and 2. The relative configuration of 3 was
determined to be identical to 1 on the basis similar 13C NMR chemical shifts shown in Table
1. The structure of 3 was elucidated and is shown in Fig. 1.
We propose that the biosynthetic pathway of 3 derived from a farnesyl pyrophosphate,
which is different from the biosynthetic pathway of 1 and 2 that are derived from a farnesyl
pyrophosphate and a gentisyl alcohol. Compounds 1 and 2 have a rearranged
monocyclofarnesyl skeleton (which is uncommon to sesquiterpene-epoxycyclohexane
conjugates) instead of a bicyclofarnesyl skeleton which is present in macrophorins21,
neomacrophorins16, myrothecols17, and craterellins.22
8

Compounds 1, 2, and 3 were evaluated for their in vitro cytotoxicity against HL-60 and
HeLa cell lines by the MTT method using a published protocol.23 Compounds 1, 2, and 3
exhibited cytotoxic activity against the HL-60 cell lines with IC50 values of 1.7, 1.5 and 10.1
µM, respectively. Additionally, compound 1, 2, and 3 exhibited cytotoxicity against the HeLa
cell lines with IC50 values of 34.7, 16.6 and 52.1 µM, respectively.
The present study clarified the structures of 1 and 2 as sesquiterpene-
epoxycyclohexenone conjugates. The structure of 3 was also elucidated, and was found to be
derived from the farnesyl pyrophosphate. Ascochlorin24, cylindrols25, LL-Z1272s26,
moverastins27 ilicicolins C, and F28 have been reported to be prenylated phenolic derivatives.
All of these compounds consist of the farnesyl-derived cyclohexanone and the gentisyl-
derived methyl benzaldehyde moieties. It is of particular interest that 1 and 2 contain the
epoxycyclohexenone moiety instead of the benzaldehyde group. Further study of the
mechanisms of action by 1, 2 and 3 is underway.
Acknowledgements
We express our gratitude to Dr. Kenta Goto, Institute for Materials Chemistry and
Engineering (IMCE), Kyushu University, for supporting the measurement of CD spectrum.
One of authors (N.R. Ariefta) acknowledges Ministry of Education, Culture, Sports, Science
and Technology of Japan (MEXT) for Ph.D. scholarship.
Conflict of interest
The authors of present manuscript have declared that no competing of interests.
References and notes
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4338–4340.
11. Nectrianolin A (1): colorless crystal; [α] D25 -30.7 (c 0.1, MeOH); UV (MeOH) λmax
(log ε) 220 (3.96) nm; CD (3.7 x 10-5 M, MeOH) λmax (∆ε) 248 (-4.2), 333 (-2.2); IR
(KBr) νmax 3332, 2935, 1677, 1249, 1045, 732 cm-1; 1H NMR (600 MHz, pyridine-d5)
and 13C NMR (150 MHz, pyridine-d5) data, see Table 1; HRESIMS (positive-ion
mode) m/z 399.2142 ([M+Na]+ calcd. for C22H32NaO5, 399.2147).
12. Forcina, G. C., Castro, A., Bokesch, H. R., Spakowicz, D. J., Legaspi, M. E., Kucera,
K., Villota, S., Narváez-Trujillo, A., McMahon, J. B., Gustafson, K. R., Strobel, S. A.
J. Nat. Prod., 2015, 78, 3005–3010.
13. Carroll, J., Jonsson, E. N., Ebel, R., Hartman, M. S., Theodore R. Holman, A., Crews,
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P. J. Org. Chem., 2001, 66, 6847–6851.
14. Yu, S., Deng, Z., Proksch, P., Lin, W. J. Nat. Prod., 2006, 69, 1330–1334.
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16. Hirose, A., Maeda, H., Tonouchi, A., Nehira, T., Hashimoto, M. Tetrahedron, 2014,
70, 1458–1463.
17. Fu, Y., Wu, P., Xue, J., Wei, X. J. Nat. Prod., 2014, 77, 1791–1799.
18. Crystal data for nectrianolin A (1): C22H32O5, M = 376.49; orthorhombic system, space
group P212121, a = 8.4487(7) Å, b = 9.8313(9) Å, c = 24.200(2) Å, V = 2010.1(3) Å3, Z
= 4, d = 1.241 g/cm3, F(000) = 812. A crystal of dimensions 0.400 × 0.300 × 0.100
mm3 was used for measurement. All measurements were made on a Rigaku XtaLAB
mini diffractometer using graphite monochromated Mo-Kα radiation, with linear
absorption coefficient µ(Mo-Kα) = 0.864 cm-1. The data were collected at a
temperature of -123 1°C to a maximum 2θ value of 55.0°. Of the 21057 reflections
were collected, where 4601 were unique (Rint = 0.0322) and were collected for the
analysis. All calculations were performed using the CrystalStructure crystallographic
software package except for refinement, which was performed using SHELXL Version
2013/4. The final reliability factors are R1 = 0.0565, wR2 = 0.1756, the goodness of fit
on F2 was equal to 1.09, and Flack parameter was -0.1(3). Crystallographic data for the
structure reported in this paper have been deposited at the Cambridge Crystallographic
Data Centre under reference number CCDC 1556508. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1
EZ, UK, and (fax. +44-(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
19. Nectrianolin B (2): yellow oil; [α] D25 -43.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
219 (4.97) nm; CD (3.7 x 10-5 M, MeOH) λmax (∆ε) 248 (-4.2), 340 (-1.9); IR (KBr)
11

νmax 3444, 2962, 1677, 1407, 1110, 740 cm-1; 1H NMR (600 MHz, pyridine-d5) and
13C NMR (150 MHz, pyridine-d5) data, see Table 1; HRESIMS (positive-ion mode)
m/z 485.2509 ([M+Na]+ calcd. for C26H38NaO7, 485.2515).
20. Nectrianolin C (3): yellow oil; [α]D25 +23.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε)
222 (2.87) nm; IR (KBr) νmax 3421, 2923, 1635, 1380, 1091, 798 cm-1; 1H NMR (600
MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5) data, see Table 1; HRESIMS
(positive-ion mode) m/z 245.1876 ([M+Na]+ calcd. for C15H26NaO, 245.1875).
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12

Table 1
1H (600 MHz) and 13C NMR (150 MHz) spectroscopic data of 1-3 in pyridine-d5
Nectrianolin A (1)
δC, type
Nectrianolin B (2)
δC, type
Nectrianolin C (3)
Pos.
δH (J in Hz)
δH (J in Hz)
δC, type
δH (J in Hz)
1
2
139.4, C
139.5, C
137.5, C
124.5, CH 5.36, s
124.5, CH
5.37, s
a: 1.85, m
125.7, CH 5.69, m
a: 1.85, m
a: 1.82, m
25.8, CH2
3
25.7, CH2
25.7, CH2
b: 1.89, m
1.29, m
b: 1.89, m
1.29, m
b: 1.88, m
4
5
6
27.1, CH2
33.3, CH
40.4, C
27.1, CH2
33.3, CH
40.5, C
27.2, CH2
33.4, CH
40.5, C
1.31, m
1.62, m
1.53, m
1.53, m
a: 1.36, td (5.5, 13.1)
b: 1.42, td (3.4, 13.7)
a: 1.56, m
a: 1.37, m
b: 1.42, m
a: 1.58, m
b: 1.82, m
a: 1.41, m
b: 1.46, m
a: 1.88, m
b: 1.91, m
7
34.8, CH2
34.8, CH2
34.7, CH2
35.1, CH2
34.3, CH2
8
9
34.6, CH2
141.2, C
b: 1.81, m
141.2, C
123.9, CH
63.5, CH
17.3, CH3
19.2, CH3
15.9, CH3
20.9, CH3
194.1, C
122.6, CH
154.9, C
64.3, CH
60.4, CH
61.6, C
139.6, C
10 124.1, CH 5.68, d (8.9)
11 63.4, CH a 5.63, d (8.9)
5.66, d (9.6)
5.61, d (8.9)
1.93, s
124.5 CH
58.9, CH2
16.6, CH3
19.3, CH3
15.9, CH3
21.1, CH3
5.39, br s
4.39, d (6.9)
1.67, s
12 17.2, CH3
13 19.2, CH3
14 15.8, CH3
15 20.9, CH3
1.92, s
1.50, s
1.51, s
1.52, s
0.68, d (6.9)
0.70, s
0.68, d (6.2)
0.71, s
0.73, d (6.1)
0.76, s
1’
2’
3’
4’
5’
6’
194.6, C
121.1, CH 6.78, s
6.49, s
162.3, C
63.4, CH a 5.11, s
5.13, s
5.09, s
60.4, CH
61.7, C
4.58, s
a: 4.64, d (17.2)
b: 5.02, d (17.2)
a: 5.12, d (16.8)
b: 5.38, d (16.8)
7’
1”
2”
63.4, CH2
63.4, CH2
171.5, C
a: 2.61, dd (4.8, 15.1)
b: 2.71, dd (8.2, 15.1)
4.55, m
44.7, CH2
3”
4”
63.3, CH
24.0, CH3
1.33, d (6.2)
a
Overlapped signals.
13

Fig. 1. Structures of 1-3.
14

Fig. 2. 1H-1H COSY and key HMBC correlations of 1-3.
15

Fig. 3. ORTEP drawing of 1, showing 50% probability ellipsoids.
16

Graphical Abstract
17

Highlights
• Chemical study of metabolites produced by fungal endophyte Nectria pseudotrichia 120-
1NP.
• Isolation of two sesquiterpene-epoxycyclohexenone conjugates and its one sesquiterpene
congener from fungal endophyte.
• Their structures were elucidated on the basis of spectroscopy analyses.
18