Article

pubs.acs.org/jnp

Eremophilane Sesquiterpenes from an Endophytic Fungus Periconia

Species
Jimei Liu,† Dewu Zhang,† Min Zhang, Jinlian Zhao, Ridao Chen, Nan Wang, Dan Zhang, and Jungui Dai*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
*
S Supporting Information

ABSTRACT: Nine new polyoxygenated eremophilane ses-

quiterpenes, periconianones C−K (1−9), including one
unusual isoeremophilane sesquiterpene, periconianone C (1),
and four trinor-eremophilane sesquiterpenes, periconianones
H−K (6−9), were isolated from the endophytic fungus
Periconia sp. F-31. Compound 1 is the first furan-type
isoeremophilane reported containing a linkage of C-8/C-11
and a 7,12-epoxy moiety. These compound structures,
including absolute configurations, were elucidated through
extensive spectroscopic data analysis, electronic circular
dichroism, Mo2(AcO)4-induced circular dichroism, and single-crystal X-ray diffraction (Cu Kα). Compounds 2, 5, and 9
showed inhibition effects on lipopolysaccharide-induced NO production in BV2 cells by 10.2%, 18.3%, and 16.1% at a
concentration of 1.0 μM, respectively, which is comparable to the positive control curcumin (12.9% at 1.0 μM).

T he family of eremophilane sesquiterpenes is widely

distributed in terrestrial and marine organisms and
possesses a wide range of biological activities including anti-
inflammatory, antimicrobial, and antitumor properties.1 In
recent years, new bioactive eremophilane sesquiterpenes have
been discovered from various natural sources.2 Endophytic
fungi, a distinct group of microorganisms that asymptomatically
colonize living tissues of healthy plants, are an important source
of biologically active eremophilane sesquiterpenes.3 We
previously isolated three eremophilane sesquiterpenes (peri- Figure 1. Structures of compounds 1−9.
conianones A and B, dihydronaphthalene-2,6-dione) from the
endophytic fungus Periconia sp. F-31, which was originally
isolated from the medicinal plant Annona muricata.4 Further presence of hydroxy (3475 and 3307 cm−1) and conjugated
carbonyl (1675 cm−1) groups. The 1H NMR spectroscopic data
biological studies revealed that periconianones A and B
(Table 1) contained signals for three olefinic protons [δH 7.06
exhibited strong neural anti-inflammatory activities.4b Toward
(1H, d, J = 9.6 Hz), 6.14 (1H, s), and 5.77 (1H, d, J = 9.6 Hz)],
our goal of finding structurally novel eremophilane sesquiter-
two methylenes [δH 3.73 (1H, dd, J = 7.8, 7.8 Hz) and 3.53
penes with interesting biological activities, we have carried out a
(1H, dd, J = 11.4, 7.8 Hz), oxygen-bearing methylene; δH 1.95
further investigation of the metabolites of this strain. As a result,
(1H, d, J = 13.8 Hz) and 1.65 (1H, d, J = 13.8 Hz)], two
we have discovered one unusual furan-type isoeremophilane
methines [δH 2.16 (1H, m) and 2.07 (1H, q, J = 6.6 Hz)], and
sesquiterpene (periconianone C, 1), four new eremophilane-
three methyls [δH 1.19 (3H, s), 0.95 (3H, d, J = 6.6 Hz), and
type sesquiterpenes (periconianones D−G, 2−5), and four 0.87 (3H, d, J = 6.6 Hz)]. The 13C NMR and DEPT spectra
stereoisomeric trinor-eremophilane sesquiterpenes (periconia- (Table 1, Figures S6 and S7) displayed signals for one carbonyl
nones H−K, 6−9) (Figure 1). Herein, we report their isolation, carbon (δC 203.0), one dioxygenated secondary carbon (δC
structural elucidation, and biological activities. 103.0), one oxygenated tertiary carbon (δC 76.1), two

■ RESULTS AND DISCUSSION

Periconianone C (1) was obtained as colorless, columnar
crystals (cyclohexane−acetone), and its molecular formula was
determined as C15H20O4 according to the HRESIMS peak at
m/z 265.1439 [M + H]+ (calcd for C15H21O4, 265.1434) with
six degrees of unsaturation. The IR spectrum indicated the
© XXXX American Chemical Society and
American Society of Pharmacognosy
quaternary carbons (δC 136.7 and 40.0), five methines (δC
144.6, 136.8, 124.2, 52.4, and 44.6, including three olefinic),
two methylenes (δC 70.0 and 41.7, including one oxygenated),
and three methyls (δC 26.1, 14.2, and 9.1). Among the 15
Received: April 5, 2016
A DOI: 10.1021/acs.jnatprod.6b00299
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Table 1. 1H NMR and 13C NMR Data of Compounds 1−5

1a 2b 3b 4b 5b
δH (J in
position δC type δH (J in Hz) δC type δH (J in Hz) δC type δH (J in Hz) δC type δH (J in Hz) δC type Hz)
1 144.6, CH 7.06, d (9.6) 145.9, CH 7.07, d (9.6) 145.8, CH 7.05, d (9.6) 82.3, CH 4.32, dd (1.2, 5.4) 128.8, CH 5.65, d
(3.6)
2 124.2, CH 5.77, d (9.6) 127.5, CH 5.86, d (9.6) 125.5, CH 5.74, d (9.6) 42.1, CH2 2.58, ddd (1.2, 70.2, CH 3.87, m
5.4, 16.8, Ha);
2.41, dt (1.2, 16.8,
Hb)
3 203.0, C 200.8, C 203.7, C 210.7, C 77.1, CH 3.69, brs
4 52.4, CH 2.07, q (6.6) 54.4, CH 2.42, q (7.2) 54.8, CH 2.19, q (7.2) 49.0, CH 2.94, q (7.2) 40.6, CH 1.73, qd
(2.4, 7.2)
5 40.0, C 40.6, C 40.4, C 44.6, C 38.2, C
6 41.7, CH2 1.95, d (13.8, 40.1, CH2 2.10, d (13.2, 35.9, CH2 2.35, d (13.8, 39.7, CH2 1.66, dd (1.2, 38.6, CH2 1.55, m
Ha) Ha) Ha) 14.4, Ha)
1.65, d (13.8, 1.91, dd (1.2, 1.47, dd (1.2, 1.28, d (14.4, Hb)
Hb) 13.2, Hb) 13.8, Hb)
7 103.0, C 74.9, C 75.2, C 80.1, C 39.5, CH 2.80c
8 76.1, C 69.2, CH 4.02, m 69.1, CH 4.06, dd (5.4, 115.7, C 73.2, CH 3.87, m
5.4)
9 136.8, CH 6.14, s 132.4, CH 6.13, d (5.4) 135.4, CH 6.28, d (5.4) 37.7, CH2 2.80, Hac ; 2.06, 79.8, CH 4.06, m
Hbc
10 136.7, C 142.4, C 139.5, C 78.3, C 145.0, C
11 44.6, CH 2.16, m 151.2, C 151.1, C 39.8, CH 2.21, m 148.5, C
12 70.0, CH2 3.73, dd (7.8, 111.3, CH2 5.02, q (1.2, 111.4, CH2 5.01, s, Ha 74.7, CH2 3.95, dd (7.8, 7.8, 110.9, CH2 4.80, s
7.8, Ha) Ha) Ha)
3.53, dd (7.8, 4.88, quin 4.88, t (1.2, 3.48, dd (7.8, 4.75, s
11.4, Hb) (1.2, Hb) Hb) 10.2, Hb)
13 9.1, CH3 0.95, d (6.6) 19.4, CH3 1.89, s 19.3, CH3 1.90, s 9.1, CH3 0.91, d, (7.2) 22.7, CH3 1.79, s
14 14.2, CH3 0.87, d (6.6) 7.7, CH3 1.08, d (7.2) 14.4, CH3 0.96, d (7.2) 8.0, CH3 0.92, d (7.2) 12.3, CH3 1.07, d
(7.2)
15 26.1, CH3 1.19, s 20.8, CH3 1.19, s 27.9, CH3 1.37, s 22.1, CH3 1.32, s 21.8, CH3 1.35, s
2-OH 2.94, d
(6.0)
3-OH 3.62, d
(4.2)
7-OH 3.51, d (1.2) 3.58, s 3.59, s
8-OH 3.71, d (7.8) 3.77, d (5.4) 4.74, brs 3.75, brs
9-OH 3.97, d
(3.0)
a
Data were recorded at 600 MHz for proton and at 150 MHz for carbon in DMSO-d6. bData were recorded at 600 MHz for proton and at 150 MHz
for carbon in acetone-d6. cOverlapped with other signals.

carbons, the existence of one carbonyl and two double bonds
accounted for three degrees of unsaturation, illustrating the
presence of the tricyclic ring system in 1.
The HMBC correlations (Figure 2) of H-1/C-3, C-5, C-9,
and C-10; H-2/C-4 and C-10; H-4/C-3, C-5, C-10, C-14, and
C-15; H2-6/C-4, C-5, C-7, C-8, C-10, and C-15; and H-9/C-1,
C-5, C-7, and C-11 were observed, which indicated the
presence of a naphthalenone moiety. Furthermore, the HMBC
cross-peaks for H-11/C-12 and C-13; H-12/C-7, C-8, C-11,
Figure 2. 1H−1H COSY (blue lines), key HMBC (red →), and
NOESY (↔) correlations of compounds 1, 2, and 6.
B DOI: 10.1021/acs.jnatprod.6b00299
and C-13; and H3-13/C-8, C-11, and C-12 along with the spin
system of H3-13/H-11/H2-12 based on the 1H−1H COSY
correlations confirmed the presence of a furan ring, indicating a
novel eremophilane sesquiterpenoid bearing the linkage of C-8
and C-11 and a 7,12-epoxy moiety. In addition, the chemical
shift of C-7 (δC 103.0) indicated the existence of a furan
hemiacetal moiety. Thus, the planar structure of 1 was
elucidated as shown in Figure 1. To the best of our knowledge,
this finding is the first example with the linkage of C-11 to C-8
and a furan hemiacetal moiety in the eremophilane-type
sesquiterpenoid family.
The relative configuration of 1 was determined by NOESY
spectroscopic data analysis (Figure 2). The NOESY correla-
tions of H3-15/H-4 and H-6b and of H-6a/H-11 and H3-14
indicated that H3-15, H-4, and H3-13 were on the same face of
the ring system, whereas H3-14 and H-11 were on the opposite
face of the molecule. However, due to the lack of key definitive
correlations at 7-OH and 8-OH in the NOESY experiments,
the relative stereochemistry of C-7 and C-8 could not be
determined. To support the above deduction and determine
the absolute configuration of 1, a single-crystal X-ray diffraction
pattern was obtained using the anomalous scattering of Cu Kα
radiation. Figure 3 shows an ORTEP drawing and unambig-
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
uously demonstrates the absolute configuration of 4S, 5R, 7R,
8S, 11S for 1.
Figure 3. Structure of 1 resulting from single-crystal X-ray diffraction.
The asymmetric diene in 1 is a chromophore that can cause
the Cotton effect. Therefore, the absolute configuration of the
chiral center (C-5) in the nonplanar skeleton can also be
determined using the helicity rule.5 A positive Cotton effect at
274.5 nm for the π−π* transition (Figure S15) is indicative of
the 5R configuration, which is in accord with X-ray diffraction
data.
Periconianone D (2) was obtained as colorless needle
crystals (MeOH). The molecular formula of C15H20O3 was
determined from the HRESIMS peak at m/z 249.1479 [M +
H]+ (calcd for C15H21O3, 249.1485). The IR spectrum showed
the presence of hydroxy (3399 cm−1) and conjugated carbonyl
(1648 cm−1) groups. The 1H and 13C NMR spectroscopic data
of 2 were similar to those of periconianone B.4b The differences
include the presence of an oxymethine carbon (δC 69.2, C-8)
and a vinylidene carbon (δC 111.3, C-12 and δC 151.2, C-11) in
2 instead of a carbonyl carbon (δC 198.6, C-8) and a carboxyl
carbon (δC 176.4, C-12) in periconianone B. In addition, the
chemical shift of C-7 (δC 74.9) together with the molecular
formula of 2 indicated the attachment of a hydroxy at C-7.
These assignments were confirmed by the HMBC correlations
of C-8/H-9, 8-OH, and H2-6; C-7/H-8, H-9, H2-6, H2-12, and
H3-13; C-11/H2-6, H3-13; and C-12/H3-13. On the basis of the
above observations, the planar structure of 2 was elucidated
(Figure 1). The NOESY correlations of H3-15/H3-14, H-6b,
and 7-OH indicated their syn-orientation, whereas the
correlations of H-8/H3-13 and H-6a/H-4, H-12a, and H3-13
indicated that these protons were on the opposite face of the
ring system. In the CD spectrum, a positive Cotton effect at
278.5 nm was observed, which is similar to that of 1 (Figure
S26), indicating a 5R configuration for 2. Accordingly, the
absolute configuration of 2 was determined as 4R, 5R, 7R, 8R.
Periconianone E (3) was obtained as a colorless gum and had
the same molecular formula as 2 as determined by the
HRESIMS peak at m/z 249.1478 [M + H]+ (calcd for
C15H21O3, 249.1485). The general features of its 1H and 13C
NMR spectroscopic data closely resembled those of 2 except
those for the different chemical shifts for H-4, H2-6, H3-15, C-6,
C-14, and C-15 (Table 1). Analysis of DEPT, 1H−1H COSY,
and HMBC correlations suggested that 3 was the stereoisomer
of 2. The NOESY correlations of H3-15/H-4 and H-6b and of
H-8/H3-13 and H3-15 indicated that these protons were on the
same face of the ring system, whereas the correlations of H3-
14/H-6a implied that these protons were on the opposite face.
The absolute configuration of 3 was assigned as 4S, 5R, 7S, 8S
according to the helicity rule with a positive Cotton effect at
279.5 nm in the CD spectrum (Figure S37).
C DOI: 10.1021/acs.jnatprod.6b00299
Article
Periconianone F (4) was obtained as a colorless powder. The
molecular formula was C15H22O5, corresponding to five degrees
of unsaturation, on the basis of the HRESIMS peak at m/z
283.1533 [M + H]+. The IR spectrum showed absorption
bands at 3393 and 1701 cm−1 for hydroxy and carbonyl groups,
respectively. The general features of its 1H NMR spectroscopic
data (Table 1) were similar to those of 1 except for the absence
of three olefinic protons in 4 and the presence of two sets of
nonequivalent methylenes [δH 2.06 (1H, overlapped with
solvent peak)/2.80 (1H, overlapped with H2O peak); δH 2.41
(1H, dt, J = 1.2, 16.8 Hz)/2.58 (1H, ddd, J = 1.2, 5.4, 16.8 Hz)]
and one oxygenated methine [δH 4.32 (1H, dd, J = 1.2, 5.4
Hz)]. The 13C NMR and DEPT spectra displayed 15 carbon
resonances (Table 1), which consisted of one carbonyl carbon
(δC 210.7), one dioxygenated secondary carbon (δC 115.7), two
oxygenated tertiary carbons (δC 80.1 and 78.3), one quaternary
carbon (δC 44.6), three methine carbons (δC 82.3, 49.0, and
39.8, including one oxygenated carbon), four methylene
carbons (δC 74.7, 42.1, 39.7, and 37.7, including one
oxygenated carbon), and three methyl carbons (δC 22.1, 9.1,
and 8.0). By analyzing the HSQC, 1H−1H COSY, and HMBC
spectra, the naphthalenone moiety and the furan ring were
established (Figure 1). However, the HMBC correlations of H-
6b/C-11; H2-12/C-7 and C-8; and H3-13/C-7 and C-12
indicated the O-linkage of C-8 and C-12, which differs from
compound 1. In addition, the chemical shift values of C-1 (δC
82.3) and C-10 (δC 78.3) together with the molecular formula
indicated the presence of a 1,10-epoxide. The HMBC
correlations of 7-OH (δH 3.59)/C-6, C-7, C-8, and C-11
along with the corresponding shifts (C-8 at δC 115.7; C-7 at δC
80.1) demonstrated the presence of a 7,8-diol moiety (Figure
S43). Therefore, the planar structure of 4 was elucidated. The
relative configuration of 4 was established by NOESY
experiments. The enhancement of H3-15 when H-4 was
irradiated suggested the anti-orientation of H3-15 and H3-14.
The NOEs of H3-15/H-6a, H3-13, and H-9a; H-6a/H3-13 and
H3-15; 7-OH/H3-13, H3-15, and H-6a; and 8-OH/H3-13, H3-
15, H-9a, and 7-OH indicated the syn-orientation of H-6a, H3-
15, H3-13, 8-OH, and 7-OH. The NOEs of H-11/H-6b and H-
1/H-2a and H-9b established the syn-orientation of H-6b, H-1,
and H-11. The absolute configurations of the 7,8-diol moieties
were determined by Snatzke’s method (the dimolybdenum
method).6 In the Mo2(AcO)4-induced CD spectrum, positive
Cotton effects at 303 and 408 nm (Figure S50) supported the
7S and 8R configurations. Thus, the absolute configuration of 4
was assigned as 1S, 4R, 5S, 7S, 8R, 10R, 11S.
Periconianone G (5) was isolated as a colorless gum, and it
gave an HRESIMS ion peak at a m/z of 291.1559 [M + Na]+,
which corresponded to a molecular formula of C15H24O4. The
IR spectrum showed an absorption band at 3330 cm−1
corresponding to hydroxy group(s). The 1H and 13C NMR
spectroscopic data of 5 were similar to those of 7βH-eremophil-
1(10),11(12)-diene-2β,8β-diol.7 The obvious difference was the
presence of two additional hydroxy groups in 5 that were
located at C-3 and C-9 according to the 1H−1H COSY
correlations of 3-OH (δH 3.62)/H-3 (δH 3.69) and 9-OH (δH
3.97)/H-9 (δH 4.06). The relative configuration of 5 was
determined by NOESY experiments. The enhancement of H-3
was observed with irradiation of H-4, which indicated a syn-
orientation of H-4 and H-3. The NOEs of H3-15/H3-14, and
H-7; 9-OH/H3-15; and H-2/H3-14 and H3-15 suggested the
syn-orientation of H3-14, H3-15, 9-OH, H-2, and H-7. The
NOESY correlations of H 3 -13/H-8 indicated the syn-
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
orientation of H-8 and the isopropenyl group. The absolute
configuration of 5 was calculated using the TD-DFT method at
the B3LYP/6-31G(d) level. After analyzing the relative
configuration of 5, only two stereoisomers existed,
(2R,3R,4R,5R,7S,8S,9R)-5a and (2S,3S,4S,5S,7R,8R,9S)-5b
(Figure S3). On the basis of a comparison of the theoretically
calculated and experimental electronic circular dichroism
(ECD) curves (Figure 4), the best match of the absolute
configuration of 5 was 2R, 3R, 4R, 5R, 7S, 8S, 9R.
Figure 4. Calculated ECD spectra of 5a and 5b and the experimental
ECD spectrum of 5.
Periconianone H (6) was obtained as a pale yellow gum with
the molecular formula C12H16O3 as determined from the
HRESIMS peak at m/z 209.1168 [M + H]+. The IR spectrum
showed characteristic absorption bands of hydroxy and
conjugated carbonyl groups at 3393 and 1678 cm −1 ,
respectively. The 13C NMR spectrum showed 12 carbon
resonances, and its 1H NMR spectrum was similar to that of 2
except for the absence of the isopropenyl group, suggesting it
was a trinor-eremophilane sesquiterpene. This deduction was
confirmed by the HMBC correlations of H-1/C-3, C-5, C-9,
and C-10; H-9/C-1, C-5, and C-7; H-2/C-3 and C-4; H-8/C-7,
C-9, and C-10; H-7/C-5, C-6, and C-8; H-4/C-11, C-12, C-5,
C-6, C-10, C-2, and C-3; H2-6/C-12, C-5, C-4, C-7, C-8, and
C-10; H3-11/C-4, C-5, and C-3; and H3-12/C-4, C-6, and C-
10. The relative configuration of 6 was established by NOE
experiments (Figures S67 and S68). The NOEs of H-6a/H3-11,
H3-12, and H-7 and H-7/H-6a and H3-12 indicated the syn-
orientation of H3-11, H3-12, and H-7, whereas the NOEs of H-
6b/H-4 and H-8 implied these protons were on the opposite
face of the ring system. The absolute configuration of C-5 in 6
was determined to be R by analyzing the helicity rule in the CD
spectrum with a positive Cotton effect at 280 nm (Figure S77).
Thus, the absolute configuration of 6 was assigned as 4R, 5R,
7R, 8R.
Periconianone I (7) was given as a pale yellow gum with the
molecular formula C12H16O3 , as determined from the
HRESIMS peak at m/z 209.1168 [M + H]+. The IR and 1H
and 13C NMR spectroscopic data were very similar to those of
6 except that the oxymethine proton appeared at δH 4.07 (dd, J
= 3.6, 3.6 Hz, H-8) in 7 instead of at δH 4.11 (dd, J = 3.0, 8.4
Hz, H-8) in 6, indicating that the compounds might be a pair of
stereoisomers. The relative configuration of 7 was determined
by the coupling constants and the NOESY experiments
D DOI: 10.1021/acs.jnatprod.6b00299
Article
(Figures S82, S83, and S84). The large coupling constant of
JH‑6b,H‑7 (7.2 Hz) indicated their axial orientation, while the
small coupling constants of JH‑7,H‑8 (3.6 Hz) and JH‑8,H‑9 (3.6
Hz) implied an equatorial arrangement of H-8. The NOEs of
H-6a/H-4, H-7, and H-8 together with H-4/H-7 established
the syn-orientation of H-4, H-7, and H-8, whereas the NOEs of
H-6b/H3-11 and H3-12 indicated these protons were on the
opposite face of the ring system. Therefore, 7 was identified as a
7-epimer of 6, which was further supported by the CD
spectrum with a positive Cotton effect at 278 nm (Figure S88).
Periconianone J (8) and periconianone K (9) had the same
molecular formula as 6 as deduced from HRESIMS. The 1H
NMR spectroscopic data were similar to those of 6 except that
the methyl and olefinic protons in 8 and 9 resonated at a much
lower field [(δH 6.12, d, J = 2.4 Hz, H-9 and δH 1.18, s, H3-12 in
8) and (δH 6.01, d, J = 2.4 Hz, H-9 and δH 1.21, s, H3-12 in 9)
vs (δH 5.86, d, J = 3.0 Hz, H-9 and δH 1.03, s, H3-12 in 6)]. The
large coupling constants of JH-6b,H-7 (10.8 Hz) and JH-7,H-8 (7.2
Hz) indicated that both H-7 and H-8 were disposed in an axial
orientation and were on opposite sides in 8. In addition, the
NOEs of H3-11/H-6a and H-7 established the syn-orientation
of H3-11 and H-7; the NOEs of H3-12/H-6b and H-4 indicated
the syn-orientation of H3-12 and H-4, which further indicated
that 8 was a stereoisomer of 6. In the NOESY spectrum of 9,
the NOEs of H-6b/H3-12, H-4, and H-7 established the syn-
orientation of H3-12, H-4, and H-7; the NOEs of H3-11/H-6a
indicated the anti-orientation of H3-11 and H3-12. Further-
more, the large coupling constants of JH-6a,H-7 (12.6 Hz) and
JH-7,H-8 (8.4 Hz) proved a trans-axial orientation of H-7 and H-
8. Thus, 9 was identified as the 11-epimer of compound 6. On
the basis of CD spectral analysis, the absolute configurations of
8 and 9 were proposed to be 4S, 5R, 7S, 8S and 4S, 5R, 7R, 8R,
respectively (Figures S98 and S115).
We isolated and fully characterized nine eremophilane-type
sesquiterpenoids bearing the vic-diols moiety (1−9) from the
endophytic fungus Periconia sp. Interestingly, three different
configurations appeared at C-4 and C-5 (4S, 5R; 4R, 5R; and
4R, 5S), which might arise from the skeleton formation process
by nonstereospecific cyclization of sesquiterpene synthases.
Biogenetically, 1 might be formed from 3 via a pinacol
rearrangement, which would lead to the migration of the
isopropenyl group from C-7 to C-8, followed by hydroxylation
and intramolecular acetalization (Supporting Information,
Figure S4). In addition, the elimination of the isopropenyl
group in four trinor-eremophilanes (periconianones H−K, 6−
9) might be biosynthesized from 2 and 3 via oxidation and
decarboxylation. In summary, 1−9 were proposed to originate
from different biosynthetic intermediates 1a, 1b, and 1c.
Different types of tailoring reactions including hydroxylation,
oxidation, isomerization, acetalization, decarboxylation, and
rearrangements might create the structural diversity (Figure
S4).
All compounds were evaluated for their neural anti-
inflammatory, cytotoxic, and anti-HIV activities. Compounds
2, 5, and 9 exhibited anti-inflammatory activity indirectly by
suppressing LPS-induced NO production in BV2 cells with
inhibition rates of 10.2%, 18.3%, and 16.1% at a concentration
of 1.0 μM, respectively, which is comparable to that of
curcumin, a positive control with an inhibition rate of 12.9% (at
1.0 μM). Compound 3 exhibited weak cytotoxic activity against
the human MCF-7 with an IC50 value of 17.9 μM (paclitaxel as
the positive control, IC50 = 0.2 nM), and 6 displayed low
cytotoxic activity against the HeLa cancer cell line with an IC50
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Journal of Natural Products Article

Table 2. 1H NMR and 13C NMR Data of Compounds 6−9a

6 7 8 9
position δC type δH (J in Hz) δC type δH (J in Hz) δC type δH (J in Hz) δC type δH (J in Hz)
1 144.7, CH 7.01, dd (1.2, 9.6) 145.7, CH 7.06, d (10.2) 145.5, CH 7.06, d (10.2) 144.7, CH 6.99, d (9.6)
2 127.1, CH 5.83, d (9.6) 127.3, CH 5.84, d (10.2) 124.8, CH 5.70, d (10.2) 125.1, CH 5.71, d (9.6)
3 200.1, C 200.6, C 202.8, C 203.0, C
4 52.9, CH 2.46, q (6.6) 52.8, CH 2.44, q (6.6) 55.1, CH 2.14, q (7.2) 53.6, CH 2.14, q (7.2)
5 41.5, C 40.3, C 41.0, C 41.2, C
6 43.3, CH2 1.92, dd (4.2, 12.6, 38.7, CH2 1.97, dd (3.6, 14.4, 40.4, CH2 1.97, dd (4.8, 14.4, 39.5, CH2 1.92, dd (12.6, 12.6,
Ha) Ha) Ha) Ha)
1.63, dd (12.6, 12.6, 1.74, dd (7.2, 14.4, 1.47, dd (10.8, 14.4, 1.48, dd (4.2, 12.6,
Hb) Hb) Hb) Hb)
7 70.0, CH 3.77, ddd (4.2, 8.4, 71.1, CH 3.87, ddd (3.6, 3.6, 72.6, CH 3.63, ddd (4.8, 7.2, 69.6, CH 3.86, ddd (4.2, 8.4,
12.6) 7.2) 10.8) 12.6)
8 74.9, CH 4.11, dd (3.0, 8.4) 70.6, CH 4.07, dd (3.6, 3.6) 72.6, CH 4.06, dd (2.4, 7.2) 75.3, CH 4.12, dd (2.4, 8.4)
9 135.0, CH 5.86, d (3.0) 133.2, CH 6.02, d (3.6) 137.5, CH 6.12, d (2.4) 138.1, CH 6.01, d (2.4)
10 142.0, C 143.0, C 139.0, C 138.9, C
11 7.3, CH3 1.01, d (6.6) 7.9, CH3 1.06, d (6.6) 15.0, CH3 0.99, d (7.2) 14.5, CH3 0.97, d (7.2)
12 20.1, CH3 1.03, s 22.0, CH3 1.09, s 30.5, CH3 1.18, s 27.3, CH3 1.21, s
7-OH 4.07b and 4.30,b brs 3.98, brs 4.04, brs
8-OH 4.15, brs 4.29, brs
a
Data were recorded at 600 MHz for proton and at 150 MHz for carbon in acetone-d6. bNot assigned in acetone-d6 and assigned in DMSO- d6
(Supporting Information, Table S3 and Figures S69−73).

value of 16.5 μM (paclitaxel as the positive control, IC50 = 6.4
nM). In addition, compound 6 displayed anti-HIV activity with
an IC50 value of 11.0 μM, whereas efavirenz (positive control)
gave an IC50 of 1.4 nM.
(3.9 mg, tR 32.8 min). Purification of Fr3.4.3 (105.7 mg) by reversed-
phase semipreparative HPLC (CH3OH−H2O, 25:75, v/v) yielded 2
(4.4 mg, tR 27.4 min) and 3 (1.8 mg, tR 21.9 min).
Fr4 (3.4 g) was fractionated with Sephadex LH-20 CC by eluting
with CH3OH, yielding five fractions (Fr4.1−Fr4.5). Fr4.5 (950 mg)

■ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer model-343 digital polarimeter. The CD
spectra were recorded on a JASCO J-815 spectropolarimeter. The UV
absorption spectra were measured in MeOH on a Thermo Spectronic-
Vision32 Software V1.25. IR spectra were acquired on a Nicolet 5700
FT-IR microscope spectrometer (FTIR Microscope Transmission).
1D and 2D NMR spectra were obtained at 600 MHz for 1H NMR and
150 MHz for 13C NMR on VNOVA SYSTEM-600 and Bruker AVIIID
600 spectrometers. Chemical shifts (δ) are given in ppm, and coupling
constants (J) are given in hertz (Hz). HRESIMS data were measured
using an Agilent Technologies 6520 Accurate Mass Q-TOF LC/MS
spectrometer. Silica gel (200−300 mesh, Qingdao Haiyang Chemical
Co. Ltd., Qingdao, PR China) and Sephadex LH-20 gel (Amersham
Biosciences, Sweden) were used for column chromatography (CC).
Semipreparative reversed-phase and normal-phase HPLC were
performed on a Shimadzu HPLC instrument equipped with a
Shimadzu RID-10A detector and a Grace Adsorbosphere C18 column
(250 mm × 10 mm, i.d., 5 μm) by eluting with mixtures of methanol
and H2O at 3 mL/min or a Grace Allsphere silica column (250 mm ×
10 mm, i.d., 5 μm) by eluting with mixtures of n-hexane and isopropyl
alcohol at 4 mL/min, respectively. Analytical TLC was carried out on
precoated silica gel GF254 plates (Qingdao Marine Chemical Industry,
Qingdao, China), and spots were visualized under UV light or by
spraying with 10% H2SO4 in EtOH followed by heating at 120 °C.
Fungal Material, Fermentation, Extraction, and Isolation.
The fermentation, extraction, and isolation of the fungal strain
Periconia sp. F-31 were performed as described previously.4,8 The
EtOAc extract (25.0 g) of the culture filtrate was subjected to silica gel
CC eluting with a CH2Cl2−CH3OH gradient (100:0−0:100) to
produce eight fractions (Fr1−Fr8) on the basis of TLC analysis.
Fr3 (4.5 g) was initially subjected to Sephadex LH-20 CC by eluting
with CH3OH to give four fractions (Fr3.1−Fr3.4). Fr3.4 (1.0 g) was
then fractionated by reversed-phase semipreparative HPLC eluting
with CH3OH−H2O (65:35, v/v) to afford four fractions (Fr3.4.1−
Fr3.4.4). Purification of Fr3.4.2 (61.9 mg) by reversed-phase
semipreparative HPLC (CH3OH−H2O, 25:75, v/v) resulted in 1
was further separated via normal-phase semipreparative HPLC (n-
hexane−EtOAC, 5:1, v/v) to furnish seven fractions (Fr4.5.1−
Fr4.5.7). Purification of Fr4.5.2 (80.0 mg) through normal-phase
semipreparative HPLC (n-hexane−isopropyl alcohol, 15:1, v/v)
followed by reversed-phase semipreparative HPLC (CH3OH−H2O,
20:80, v/v) afforded 4 (1.5 mg, tR 25.8 min). Fr4.5.3 (74.8 mg) was
separated by normal-phase semipreparative HPLC (n-hexane−
isopropyl alcohol, 13:1, v/v) followed by reversed-phase semi-
preparative HPLC (CH3OH−H2O, 25:75, v/v) to yield 7 (8.5 mg,
tR 28.3 min). Purification of Fr4.5.4 (99.1 mg) by normal-phase
semipreparative HPLC (n-hexane−isopropyl alcohol, 13:1, v/v)
followed by reversed-phase semipreparative HPLC afforded 6 (21.4
mg, tR 30.3 min, CH3OH−H2O, 20:80, v/v), 5 (1.0 mg, tR 18.5 min,
CH3OH−H2O, 15:85, v/v), and 8 (6.3 mg, tR 40.5 min, CH3OH−
H2O, 15:85, v/v). Fr4.5.5 (106.0 mg) was separated by normal-phase
semipreparative HPLC (n-hexane−isopropyl alcohol, 20:1, v/v) to
give 9 (24.5 mg, tR 54.2 min).
Periconianone C (1): colorless columnar crystals (cyclohexane−
acetone); [α]20546 −45.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε)
203 (3.61), 282 (3.87) nm; CD (MeOH) λmax (Δε) 274 (8.03), 338
(−3.95) nm; IR (νmax) 3307, 2974, 1675 cm−1; 1H and 13C NMR data,
see Table 1; HRESIMS m/z 265.1439 [M + H]+ (C15H21O4, calcd [M
+ H]+ 265.1434).
Periconianone D (2): colorless needle crystals (MeOH); [α]20546
+672.0 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 279 (3.99) nm;
CD (MeOH) λmax (Δε) 278 (22.49), 338 (−3.75) nm; IR (νmax) 3399,
2924, 1649 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z
249.1479 [M + H]+ (C15H21O3, calcd [M + H]+ 249.1485).
Periconianone E (3): colorless gum; [α]20546 +342.8 (c 0.07,
MeOH); UV (MeOH) λmax (log ε) 282 (4.49) nm; CD (MeOH) λmax
(Δε) 279 (9.49), 338 (−1.59) nm; IR (νmax) 3433, 2968, 1658 cm−1;
1
H and 13C NMR data, see Table 1; HRESIMS m/z 249.1478 [M +
H]+ (C15H21O3, calcd [M + H]+ 249.1485).
Periconianone F (4): colorless powder; [α]20546 −33.4 (c 0.06,
MeOH); Mo2(AcO)4-induced CD (DMSO) λmax (Δε) 303 (0.28),
408 (0.09) nm; IR (νmax) 3393, 2921, 1701 cm−1; 1H and 13C NMR
data, see Table 1; HRESIMS m/z 283.1533 [M + H]+ (C15H23O5,
calcd [M + H]+ 283.1540).

E DOI: 10.1021/acs.jnatprod.6b00299
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Periconianone G (5): colorless gum; [α]20546 −50.0 (c 0.10,
MeOH); IR (νmax) 3331, 2940, 1644 cm−1; 1H and 13C NMR data, see
Table 1; HRESIMS m/z 291.1559 [M + Na]+ (C15H24O4Na, calcd [M
+ Na]+ 291.1567).
Periconianone H (6): pale yellow gum; [α]20546 +80.0 (c 0.15,
MeOH); UV (MeOH) λmax (log ε) 283 (5.93) nm; CD (MeOH) λmax
(Δε) 280 (7.81), 337 (−2.86) nm; IR (νmax) 3394, 2921, 1676 cm−1;
1
H and 13C NMR data, see Table 2; HRESIMS m/z 209.1168 [M +
H]+ (C12H17O3, calcd [M + H]+ 209.1172).
Periconianone I (7): pale yellow gum; [α]20546 +366.7 (c 0.21,
MeOH); UV (MeOH) λmax (log ε) 279 (4.05) nm; CD (MeOH) λmax
(Δε) 278 (17.03), 336 (−3.07) nm; IR (νmax) 3393, 2921, 1663 cm−1;
1
H and 13C NMR data, see Table 2; HRESIMS m/z 209.1168 [M +
H]+ (C12H17O3, calcd [M + H]+ 209.1172).
Periconianone J (8): pale yellow gum; [α]20546 +390.9 (c 0.11,
MeOH); UV (MeOH) λmax (log ε) 282 (5.94) nm; CD (MeOH) λmax
(Δε) 282 (21.74), 334 (−3.36) nm; IR (νmax) 3391, 2921, 1661 cm−1;
1
H and 13C NMR data, see Table 2; HRESIMS m/z 209.1168 [M +
H]+ (C12H17O3, calcd [M + H]+ 209.1172).
Periconianone K (9): pale yellow gum; [α]20546 −110.0 (c 0.10,
MeOH); UV (MeOH) λmax (log ε) 285 (3.97) nm; CD (MeOH) λmax
(Δε) 242 (3.81), 279 (3.89), 337 (−2.49) nm; IR (νmax) 3402, 2929,
1672 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z
209.1170 [M + H]+ (C12H17O3, calcd [M + H]+ 209.1172).
Anti-inflammatory Activity Assay.9 The BV2 microglia cell line
was obtained from the Cell Culture Center at the Institute of Basic
Medical Sciences, Chinese Academy of Medical Sciences. LPS (from
Escherichia coli 055:B5) was obtained from Sigma-Aldrich. After
preincubation for 24 h in a 96-well plate (at 37 °C with 5% CO2), the
cells were treated with various concentrations of the test compounds
(10−5, 10−6, 10−7, 10−8 M), followed by stimulation with LPS for 24 h.
The production of NO was determined by measuring the
concentration of nitrite in the culture supernatant. NaNO2 was
utilized to generate a standard curve. The absorbance at 550 nm was
measured. Curcumin was used as a positive control.
Cytotoxicity Bioassay.10 The cytotoxicity of the compounds
against the human cancer cell lines (HCT-8, Bel-7402, HeLa, and
MCF-7) was measured using the MTT assay. The cells were
maintained in an RRMI S7 1640 medium supplemented with 10%
(v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/
mL streptomycin. Cultures were incubated at 37 °C in a humidified
atmosphere of 5% CO2. Tumor cells were seeded in 96-well microtiter
plates at 1200 cells/well. After 24 h, compounds were added to the
wells. After incubation for 96 h, cell viability was determined by
measuring the metabolic conversion of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) into purple formazan crystals
by viable cells. The MTT assay results were read using an MK3
Wellscan (Labsystem Dragon, Helsinki, Finland) plate reader at 570
nm. All compounds were tested at five concentrations (10−5, 10−6,
10−7, 10−8, and 10−9 M) in 100% DMSO with a final concentration of
DMSO of 0.1% (v/v) in each well. Paclitaxel was used as a positive
control. Each concentration of the compounds was tested in three
parallel experiments. IC50 values were calculated using Microsoft Excel
software.
HIV-Inhibitory Bioassay. 11 293T cells (2 × 105 ) were
cotransfected with 0.6 μg of pNL-Luv-E−-Vpu− and 0.4 μg of
pHIT/G. After 48 h, the VSV-G pseudotyped viral supernatant (HIV-
1) was harvested by filtration through a 0.45 μm filter and the
concentration of viral capsid protein was determined by p24 antigen
capture ELISA (Biomerieux). SupT1 cells were exposed to VSV-G
pseudotyped HIV-1 (MOI = 1) at 37 °C for 48 h in the absence or
presence of test compounds (efavirenz was used as positive control).
The inhibition rate was determined by using a firefly luciferase assay
system (Promega).
F DOI: 10.1021/acs.jnatprod.6b00299
■
Article
ASSOCIATED CONTENT
* Supporting Information
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00299.
UV, IR, MS, 1D and 2D NMR, and CD spectra for 1−9,
ECD calculation of 5, X-ray crystallographic data for 1,
and proposed biosynthesis of 1−9. (PDF)
Crystallographic data (CIF)
■ AUTHOR INFORMATION
Corresponding Author
*Tel (J. Dai): 86-10-63165195. Fax: 86-10-63017757. E-mail:
jgdai@imm.ac.cn.
Author Contributions
†
J. Liu and D. Zhang contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was financially supported by the National High
Technology Research and Development Program of China
(2012AA091606).
■
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