Penipyridones A-F, Pyridone Alkaloids from Penicillium funiculosum

Article abstract of DOI:10.1021/acs.jnatprod.6b00218

Six new pyridone alkaloids, named penipyridones A-F (1-6), were isolated from the fermentation broth of an Antarctic moss-derived fungus, Penicillium funiculosum GWT2-24. Their structures were elucidated from extensive NMR and MS data. Although they possess the same major chromophore and some of them presented almost mirror ECD spectra, their absolute configurations were found to be uniformly S, as evidenced by X-ray single-crystal diffraction analysis, stereocontrolled total synthesis, and chemical conversions. TDDFT-ECD calculations of compounds 3 and 6 revealed that subtle conformational changes are responsible for the significantly different ECD curves. None of the compounds were cytotoxic (IC₅₀ > 50 μM), while compounds 1, 2, 5, and 7 elicited lipid-lowering activity in HepG2 hepatocytes.

Full text of DOI:10.1021/acs.jnatprod.6b00218

Article
pubs.acs.org/jnp
Penipyridones A−F, Pyridone Alkaloids from Penicillium funiculosum
Haibo Zhou,†,⊥ Liyuan Li,†,⊥ Chongming Wu,‡ Tibor Kurtan,§ Attila Mandi,§ Yankai Liu,† Qianqun Gu,†
́
́
Tianjiao Zhu,*,† Peng Guo,*,‡ and Dehai Li*,†
†Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China
‡Pharmacology and Toxicology Research Center, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences,
Peking Union Medical College, Beijing 100193, People’s Republic of China
§Department of Organic Chemistry, University of Debrecen, POB 20, 4010 Debrecen, Hungary
S
* Supporting Information
ABSTRACT: Six new pyridone alkaloids, named penipyridones
A−F (1−6), were isolated from the fermentation broth of an Ant-
arctic moss-derived fungus, Penicillium funiculosum GWT2-24.
Their structures were elucidated from extensive NMR and MS data.
Although they possess the same major chromophore and some of
them presented almost mirror ECD spectra, their absolute config-
urations were found to be uniformly S, as evidenced by X-ray
single-crystal diffraction analysis, stereocontrolled total synthesis,
and chemical conversions. TDDFT-ECD calculations of com-
pounds 3 and 6 revealed that subtle conformational changes are
responsible for the significantly different ECD curves. None of the
compounds were cytotoxic (IC50 > 50 μM), while compounds 1, 2,
5, and 7 elicited lipid-lowering activity in HepG2 hepatocytes.
tructure elucidation, especially the determination of stereo-
Schemistry, is essential to natural products research. All current
methods, X-ray diffraction, chemical synthesis, electronic circular
dichroism (ECD), and NMR, have unique advantages and limi-
tations. Among them, X-ray diffraction is the most reliable.
In recent years, ECD especially assisted by quantum mechanical
computations has found broad applicability and has been widely
used to solve the absolute configurations of complex natural prod-
ucts.1 In fact, the ECD data depend on both the conformation and
the absolute configuration.2
During our exploration of bioactive molecules from Antarctic-
derived fungi,3 a series of alkaloids with the unusual phenyl-
pyridone skeleton were isolated, including six new compounds,
which we named penipyridones A−F (1−6), and the known
berkeleyamide C (7).4 Although 1−7 have the same skeleton and
they all possess only one stereogenic center, their ECD spectra
are significantly different. In particular, 3 and 6 have the same
chromophore, yet they present almost mirror image ECD curves.
All the compounds proved to have the same absolute config-
urations by X-ray diffraction, total synthesis, and chemical con-
versions. On the grounds of time-dependent density functional
theory (TDDFT)-ECD analysis, the differences in the ECD
curves of the closely related analogues (3 and 6) were explained
by the different relative arrangement of the two isolated chro-
mophores, influenced by the different substitution pattern of
attached achiral groups. In addition, compounds 1, 2, 5, and 7
showed lipid-lowering activity. Herein, we report the isolation,
structural determination, and bioactivity of these compounds.
RESULTS AND DISCUSSION
■
The fungal Penicillium funiculosum strain GWT2-24 was
fermented (30 L) under static conditions.3e The EtOAc extract
of the fermentation was fractionated by Sephadex LH-20 chro-
matography, ODS MPLC, and finally HPLC to yield compounds 1
(49 mg), 2 (3 mg), 3 (28 mg), 4 (24 mg), 5 (31 mg), 6 (36 mg),
and 7 (25 mg).
Penipyridone A (1) was isolated as a white solid and has the
molecular formula C13H12O3N2 as evidenced by the HRESIMS
protonated ion at m/z 245.0926. The 1D NMR data (Table 1)
revealed the presence of 13 carbons, including two carbonyls
(δC 178.0, 165.9), one oxymethine (δC 71.3), and 10 olefinic
carbons with two of them (153.9, 142.0) attached to nitrogen.
These data were similar to those for aspernigrin A,5 with the
replacement of a methylene in asperningrin A by a hydroxylated
Received: March 10, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00218
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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of Compounds 1−3 in DMSO-d6
1
2
3
no.
1/5
δC
δH (Jin Hz)
δC
δH (Jin Hz)
δC
δH (Jin Hz)
127.0, CH
129.0, CH
128.5, CH
142.4, C
7.45, d (7.7)
126.8, CH
129.1, CH
128.5, CH
142.1, C
7.40, d (7.1)
126.2, CH
128.2, CH
127.6, CH
141.6, C
7.44, d (7.1)
2/4
3
7.38, dd (7.1, 7.7)
7.30, dd (7.7, 7.1)
7.33, dd (7.1, 7.7)
7.26, dd (7.7, 7.1)
7.36, dd (7.1, 7.8)
7.30, dd (7.1, 7.8)
6
7
71.3, CH
153.9, C
5.66, d (4.0)
6.35, s
72.4, CH
157.0, C
5.54, s
6.38, s
70.8, CH
155.2, C
5.67, s
6.47, s
8
9
116.5, CH
178.0, C
116.1, CH
176.2, C
115.4, CH
177.1, C
10
11
118.4, C
116.3, C
115.8, C
12
142.0, CH
165.9, C
8.29, s
145.8, CH
166.6, C
8.25, s
3.69, s
143.8, CH
162.8, C
8.40, s
13
1′
52.2, CH3
173.3, C
2′
3′
4′/5′
7-OH
8-NH
13-NH
46.3, CH2
24.2, CH
22.0, CH3
2.59, d (7.2)
2.07, m
0.92, d (6.6)
6.58, brs
6.62, d (4.0)
12.06, s
9.50, d (4.4); 7.41, d (4.4)
13.11, s
methine (δH/δC 5.66/71.3) in 1, which was further confirmed by
COSY (7-OH/H-7) and HMBC correlations from H-7 to C-1/5,
C-6, C-8, and C-9 (Figure 1). The absolute configuration of C-7
in 1 was determined as S by X-ray diffraction analysis using Cu
Kα radiation with a Flack parameter of −0.03(13) (Figure 2).
Penipyridones B (2) and C (3) were also isolated as white
solids. The 1D NMR data of 2 and 3 (Table 1) showed that
they share the same 2-benzylpyridin-4(1H)-one skeleton as 1.
The main differences between them and 1 were the replacement
of the amine group in 1 by a methoxy group (δH/δC 3.69/52.2)
in 2 and by an isovaleryl amide in 3, which were in agreement
with the 2D NMR correlations (Figure 1), as well as the chemical
shifts.4
Penipyridones D−F (4−6) were also found to have similar
NMR data (Table 2) to 1. Compared to 1, compound 4 showed
additional signals of a hydroxyethyl group, which was located on
the nitrogen of the pyridinone ring. The main difference between
4 and 5 was the presence of an additional acetate group attached
to oxygen at C-7 in 5, consistent with the significant downfield
shift of H-7 (from δH 5.91 to δH 6.95). The NMR data also
demonstrated that compound 6 was a hydroxyethylation deriv-
ative of 3. The planar structures of 4−6 were further confirmed
from the 2D NMR correlations (Figure 1). Compound 7 was
determined to be the known compound berkeleyamide C, whose
absolute configuration has not been reported yet.4
In order to determine the absolute configurations of 2−7,
comparisons of their ECD spectra and specific rotation values
were initially performed. However, their ECD curves were not
consistent, especially those of 1−3, which presented almost
opposite spectra of 6 and 7 (Figure 3). Similarly, the specific
rotations of compounds 1−3 were negative, while those of 4−7
were positive. Indeed, the occurrence of natural product ana-
logues with opposite absolute configurations has been observed
previously.3a
Figure 1. Key COSY and HMBC correlations of 1−6.
To solve the absolute configurations of 2−7 thoroughly, we
employed total synthesis and chemical interconversion. Com-
pound 2 was synthesized starting from (S)-(+)-mandelic acid.
The secondary alcohol of mandelic acid was acetylated, after
which treatment with oxalyl chloride yielded 8,6 which was
reacted with 9 to afford 10.7,8 Finally, compound (S)-(−)-2 (2a)
was obtained by deacetylation of 10 (Scheme 1). Using the same
method, enantiomer (R)-(+)-2 (2b) was also synthesized from
(R)-(+)-mandelic acid. The absolute configuration of the natural
2 was determined as 7S from its identical ECD spectra to syn-
thetic 2a (Figure S3, Supporting Information, SI). Conversion
Figure 2. X-ray ORTEP diagram of compound 1.
B
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Table 2. 1H (600 MHz) and 13C NMR (150 MHz) Data of 4−6 in DMSO-d6.
4
5
6
no.
1/5
δC
δH (Jin Hz)
δC
δH (Jin Hz)
δC
δH (Jin Hz)
a
a
a
a
127.5, CH
129.2, CH
128.6, CH
141.2, C
7.35, d (7.1)
128.3, CH
129.8, CH
129.6, CH
136.2, C
7.43
7.43
7.43
127.5, CH
129.2, CH
128.7, CH
140.9, C
7.38
a
2/4
3
7.40, dd (7.1, 7.8)
7.32, dd (7.1, 7.8)
7.38
a
7.38
6
7
70.6, CH
154.4, C
5.91, s
6.38, s
71.6, CH
150.2, C
6.95, s
6.45, s
70.5, CH
155.5, C
5.95, d (5.4)
6.53, s
8
9
120.0, CH
177.2, C
119.7, CH
177.0, C
120.3, CH
177.2, C
10
11
118.3, C
118.7, C
116.1, C
12
148.4, CH
165.8, C
8.45, s
148.6, CH
165.5, C
8.48, s
150.0, CH
163.1, C
8.58, s
13
14
55.1, CH2
60.9, CH2
4.08, m
3.48, m
55.5, CH2
60.5, CH2
169.9, C
4.05, m
55.5, CH2
60.6, CH2
4.15, m
3.48, m
15
3.61/3.52, m
16
17
21.2, CH3
2.18, s
1′
174.1, C
2′
3′
4′/5′
7-OH
13-NH
15-OH
47.1, CH2
25.1, CH
22.9, CH3
2.61, d (7.2)
2.08, m
0.92, d (6.6)
6.68, brs
6.51, d (4.0)
9.50, d (4.9); 7.46, d (4.9)
5.12, s
9.41, d (4.4); 7.51, d (4.4)
5.17, brs
12.95, s
5.20, brs
a
Signals were overlapped.
which can be derived from the intermediate (S)-10 (Scheme 1),
indicating that they shared the same 7S absolute configuration.
In order to reveal the origin of the significant differences in the
ECD spectra of the homochiral compounds 1−7 and to check
the applicability of ECD calculations for the configurational
assignment, TDDFT-ECD calculations were carried out using
both gas-phase and solution conformers of the two representa-
tive compounds 3 and 6. Compounds 3 and 6 have the same
chromophore with considerable conformational freedom around
the C-6 to C-8 single bonds. The relative arrangement of the two
isolated chromophores and hence the conformations adopted by
rotation around the above bonds were expected to govern the
ECD properties. The initial MMFF conformational search of
(S)-3 resulted in 42 conformers within a 21 kJ/mol energy window,
the reoptimization of which using both the B3LYP/6-31G(d)
in vacuo and B97D/TZVP9 with PCM MeOH solvent basis sets
afforded three (Figure 4) and seven (Figure S4, SI) low-energy
conformers over 2% population, respectively.
In the lowest energy B3LYP/6-31G(d) in vacuo conformer,
A (population 53.6%), the characteristic ω7‑H,C‑7,C‑6,C‑1 and ω7‑H,C‑7,C‑8,N
torsional angles were −3.8° and −8.9°, respectively, which implies
near coplanarity between the H-7−C-7 bond and the phenyl
ring, as well as between the O-7−C-7 bond and the pyridinone.
The experimental ECD spectrum of 3 showed negative Cotton
effects at 301 and 233 nm and positive Cotton effects at 268
and 213 nm. The ECD spectrum of the lowest energy conformer,
A, computed with different methods (BH&HLYP/TZVP,
B3LYP/TZVP, and PBE0/TZVP) did not reproduce well the
experimental ECD data, and additionally the BH&HLYP/TZVP
ECD spectrum was quite different from those from the other two
methods (Figure 5a). In conformer B (35.8%), the relative orien-
tation of the two chromophores was slightly different from that of
conformer A, with ω7‑H,C‑7,C‑6,C‑1 and ω7‑H,C‑7,C‑8,N torsional angles
of 17.4° and 17.0°, respectively. However, these small changes in
the torsional angles of conformer B made the computed ECDs
Figure 3. ECD spectra of compounds 1−7.
of 1 and 3 to 11 via acidic hydrolysis (Scheme 1) established
the absolute configuration of 3 as 7S, the same as 1. Similarly,
compounds 4−7 were also converted into the synthetic 13,
C
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Scheme 1. Synthesis of Compounds 2a and 13 and Acid Hydrolysis of Compounds 1 and 3−7
The ECD spectrum of 6 in methanol showed positive Cotton
effects at 295 (broad) and 231 (sharp) nm and negative Cotton
effect at 208 nm. The initial MMFF conformational search of (S)-6
resulted in 219 conformers, the reoptimization of which yielded
seven low-energy conformers above 2% at the B3LYP/6-31G(d)
level in vacuo (Figure S5, SI). In the lowest energy conformer A of
(S)-6, the H-7−C-7 bond is syn-coplanar with the H-9−C-9 bond,
while the O-7−C-7 bond is almost coplanar with the plane of the
benzene ring with 67.4° and 54.7° values for the ω7‑H, C‑7, C‑6, C‑1
and ω7‑H, C‑7, C‑8, N torsional angles, respectively (Figure 6a). Similar
conformations are adopted by conformers B, E, and F, and thus in
the more populated conformers of (S)-6, the relative arrangements
of the two isolated chromophores are different from that of (S)-3,
which is responsible for their markedly different ECD spectra. The
different torsional angles of conformers A and B of (S)-6, the main
contributors to the overall ECD spectrum, are stabilized by an
intramolecular hydrogen-bonding interaction between 7-OH and
15-OH, which is not possible for (S)-3. The Boltzmann-weighted
TDDFT-ECD spectra calculated for the B3LYP/6-31G(d) in vacuo
conformers of (S)-6 (Figure 6b) reproduced the main features of
the experimental ECD spectrum, confirming the S absolute config-
uration of 6. The results indicate that achiral substituents signifi-
cantly modify the electronic properties of the chromophores and
the conformational ensemble in compounds 3 and 6, resulting in
different ECD sepctra.
Compounds 1−7 are proposed to be biosynthesized from
phenylacetic acid (Scheme 2). With elongation of two acetate
units and methylation, the intermediate i is formed and then
transformed to ii by generation of a pyridine ring.4,10 Compound 2
is proposed to be generated by methylation of ii. By forming the
amide group at C-13 in ii, compounds 1, 4, and 5 are generated,
and compounds 3, 6, and 7 are formed by further modification of
isovaleryl-CoA originating from leucine.11
Figure 4. Structure, population, and characteristic torsional angles of the
low-energy B3LYP/6-31G(d) in vacuo conformers of (S)-3.
nearly congruent with the experimental curves, with all three
methods giving consistent results (Figure 5b). The geometry
of conformer C (8.9%) was markedly different from those of
the lower energy conformers, as demonstrated by the −66.6°
and −46.4° values for the ω7‑H,C‑7,C‑6,C‑1 and ω7‑H,C‑7,C‑8,N
torsional angles. In this conformer, the H-7−C-7 bond
was near coplanar with the pyridinone and the O-7−C-7
bond with the phenyl ring, which again gave rise to computed
ECD spectra completely different from the experimental one
(Figure 5c).
The comparison of the Boltzmann-weighted computed ECD
spectra of (S)-3 with the experimental ECD (Figure 5d)
suggested the S absolute configuration for 3, but the positive
Cotton effect at 268 nm was completely missing from the com-
puted spectra, and agreement was far from perfect. This could
be clearly attributed to the lower estimated population of con-
former B in vacuo, which presumably is the dominant conformer
in solution based on the closer resemblance of its calculated ECD
spectrum to that obtained experimentally. Interestingly, the
seven B97D/TZVP-reoptimized conformers with the PCM
solvent model for MeOH (Figure S5, SI) were all different from
the in vacuo conformer B, and the Boltzmann-weighted com-
puted ECD spectra of the B97D/TZVP (PCM) conformers showed
even less agreement with the experimental ECD than the in vacuo
spectra.
None of 1−7 were cytotoxic (IC50 > 50 μM) to K562, HL-60,
HeLa, or A-549 cells.12,13 Compounds 1−7 were also screened
for lowering of oleic acid (OA)-elicited lipid accumulation in
HepG2 hepatocytes. At 10 μM, compounds 1, 2,14 5, and 7 sig-
nificantly decreased intracellular lipid accumulation and both the
total cholesterol and triglyceride quantification (Figure 7).
Naturally occurring phenylpyridone alkaloids characterized by
the linkage of benzene and 4-pyridone moieties are rare, with
D
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Figure 5. Experimental ECD spectrum of 3 compared with computed ECD spectra of conformer A (53.6%) (a), conformer B (35.8%) (b), conformer C
(8.9%) (c), and the Boltzmann-weighted TDDFT-ECD spectra calculated for (S)-3 (d).
Scheme 2. Proposed Biosynthetic Pathway of 1−7
inhibiting MMP-3 and caspase-14 and aspernigrin B being
neuroprotective.17a This is the first report of a lipid-lowering
effect of this family of alkaloids. Although compounds 1−7
possess the same major chromophore and some of them
presented almost mirror ECD spectra, their absolute config-
urations were found to be uniform. Given the popular utilization
of comparing experimental and calculated ECD spectra for
the assignment of absolute configurations, it may be worth
emphasizing repeatedly that simple comparison of ECD spectra
for even closely related analogues may lead to the wrong con-
figurational assignment if conformational differences are over-
looked.
Figure 6. Structure and population of conformers A and B of (S)-6
(a) and experimental ECD spectrum of 6 compared with the calculated
one for (S)-6 (b).
only 11 compounds reported previously from the fungal genera
Penicillium,4,15,16 Aspergillus,17−19 Cladosporium,5 and Pestalo-
tiopsis.20 Limited bioactivity data are reported for structural
relatives of the penipyridones including berkeleyamide C
E
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Figure 7. Inhibitory effects of 1−7 on oleic acid-elicited intracellular lipid accumulation (a), total cholesterol (b), and triglycerides (c) (lovastatin as positive
control). Bars depict the means SEM of at least three experiments. ***p < 0.001, OA vs blank; †p < 0.05, ††p < 0.01, †††p < 0.001, test group vs OA group.
223 (−4.48), 270 (+0.17), 295 (−0.31) nm; UV (MeOH) λmax (log ε)
212 (1.40), 252 (0.57) nm; IR (KBr) vmax 3355, 1658, 1627, 1543, 1348,
1212, 1187, 1047, 696 cm−1; 1H and 13C NMR see Table 1; HRESIMS
m/z 245.0926 [M + H]+ (calcd for C13H13O3N2 245.0921).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were deter-
mined on an RY-1 micromelting point apparatus and are uncorrected.
Optical rotations were obtained on a JASCO P-1020 digital polarimeter.
UV spectra were recorded on a Waters 2487 absorbance detector. ECD
spectra were recorded on a JASCO J-815 spectropolarimeter, using
MeOH as solvent. IR spectra were taken on a Nicolet NEXUS 470
Penipyridone B (2): white solid; [α]26D −12 (c 0.1, CHCl3); ECD
(0.5 × 10−3 M in MeOH) λmax (Δε) 208 (+7.38), 234 (−2.92),
277 (+0.37) nm; UV (MeOH) λmax (log ε) 212 (0.34), 252 (0.12) nm;
IR (KBr) νmax 3353, 1715, 1658, 1533, 1350, 1207, 935, 734, 698 cm−1;
1H and 13C NMR see Table 1; HRESIMS m/z 260.0923 [M + H]+
(calcd for C14H13O4N 260.0921).
1
spectrophotometer as KBr disks. H and 13C NMR, DEPT, and 2D
NMR spectra were recorded on a JEOL JNMECP 600 spectrometer or
an Agilent 500 MHz DD2 spectrometer using tetramethylsilane (TMS)
as an internal standard, and chemical shifts were recorded as δ values.
ESIMS spectra were measured on a Micromass Q-TOF Ultima Global
GAA076 LC mass spectrometer. HRESIMS spectra were measured on a
Micromass EI-4000 (Autospec-Ultima-TOF). X-ray crystal data were
measured on an Agilent Gemini Ultra diffractometer (Cu Kα radiation).
Semipreparative HPLC was performed using an ODS column [YMC-
pack ODS-A, 10 × 250 mm, 5 μm, 3 mL/min]. MPLC was performed
using an ODS column [25 × 50 cm, 50 μm, 20 mL/min]. TLC and
column chromatography were performed on plates precoated with silica
gel GF254 (10−40 μm) and over silica gel (200−300 mesh, Qingdao
Marine Chemical Factory) and Sephadex LH-20 (GE Healthcare),
respectively. Vacuum-liquid chromatography (VLC) was carried out
over silica gel H (Qingdao Marine Chemical Factory).
Material. The fungal strain Penicillium funiculosum GWT2-24 was
isolated from an inner part of moss collected around the China Great
Wall Station in Antarctica and was identified by the ITS sequence with
GenBank accession number JQ670957.3e A voucher specimen is depos-
ited in our laboratory at −20 °C. The working strain was prepared on
potato dextrose agar slants and stored at 4 °C. Commercial reagents and
solvents were purchased from Sigma-Aldrich, Fluka, and Alfa Aesar and
used as received, without further purification.
Penipyridone C (3): white solid; [α]26D −12 (c 0.1, CHCl3); ECD
(1.0 × 10−3 M in MeOH) λmax (Δε) 213 (+8.81), 233 (−6.77),
268 (+0.88), 301 (−0.56) nm; UV (MeOH) λmax (log ε) 223 (2.26),
297 (0.42) nm; IR (KBr) νmax 3248, 2958, 1742, 1690, 1681, 1642, 1505,
1304, 1190, 1056, 698 cm−1; 1H and 13C NMR see Table 1; HRESIMS
m/z 329.1494 [M + H]+ (calcd for C20H21O4N2, 329.1496).
Penipyridone D (4): colorless oil; [α]26D +16 (c 0.1, CHCl3); ECD
(1.2 × 10−3 M in MeOH) λmax (Δε) 217 (+9.87), 280 (+0.75) nm; UV
(MeOH) λmax (log ε) 212 (0.58), 262 (0.25) nm; IR (KBr) νmax 3345,
1
1646, 1538, 1211, 1060, 700 cm−1; H and 13C NMR see Table 2;
HRESIMS m/z 289.1187 [M + H]+ (calcd for C15H17O4N2, 289.1183).
Penipyridone E (5): colorless oil; [α]26D +29 (c 0.1, CHCl3); ECD
(1.0 × 10−3 M in MeOH) λmax (Δε) 217 (+5.37), 280 (+0.43) nm; UV
(MeOH) λmax (log ε) 209 (1.98), 263 (1.45) nm; IR (KBr) νmax 3333,
2924, 1739, 1690, 1684, 1626, 1547, 1487, 1347, 1190, 746, 698 cm−1;
1H and 13C NMR see Table 2; HRESIMS m/z 331.1290 [M + H]+
(calcd for C17H19O5N2, 331.1288).
Penipyridone F (6): white solid; [α]26D +18 (c 0.1, CHCl3); ECD
(0.9 × 10−3 M in MeOH) λmax (Δε) 208 (−0.65), 231 (+7.19),
295 (+0.51) nm; UV (MeOH) λmax (log ε) 223 (2.45), 259 (1.36),
300 (0.72) nm; IR (KBr) νmax 3408, 3268, 2957, 1745, 1690, 1638, 1503,
1
1453, 1426, 1370, 1168, 1056, 883, 702 cm−1; H and 13C NMR see
Fermentation and Extraction. The fungus P. funiculosum
GWT2-24 was cultured and extracted as previously described.3e
Purification. The extract (20 g) was subjected to a Sephadex LH-20
column eluting with MeOH, to give four subfractions (Fr.1−Fr.4).
Fr.2 was then separated by medium-pressure liquid chromatography
(MPLC) with a gradient solvent system MeOH-H2O (20−100% in 2 h)
to afford three subfractions (Fr.2.1−Fr.2.3). Fr.2.3 was chromatographed
on a Sephadex LH-20 column eluting with CH2Cl2−MeOH (1:1, v/v)
and finally purified on semipreparativeHPLC (ODS; 5 μm, 250 × 10 mm;
MeOH−H2O, 50:50, v/v; 3 mL/min) to afford compounds 2 (3 mg),
6 (36 mg), and 7 (25 mg). Fr.4 was separated into three subfractions
(Fr.4.1−Fr.4.3) by MPLC with a step gradient elution with MeOH−H2O
(20−100%in2 h). Fr.4.1 was then subjectedtoa Sephadex LH-20column
eluting with CH2Cl2−MeOH (1:1, v/v) and further purified by using
semipreparative HPLC with 30% MeOH−H2O to obtain 1 (49 mg), 4
(24 mg), and 5 (31 mg). Fr.4.3 was purified on semipreparative HPLC
with 50% MeOH−H2O to give 3 (28 mg).
Table 2; HRESIMS m/z 373.1756 [M + H]+ (calcd for C20H25O5N2,
373.1758).
Berkeleyamide C (7): colorless oil; [α]26 +39 (c 0.1, CHCl3);
D
[lit. [α]25D +24.9 (c 0.007, CHCl3)];4 ECD (1.2 × 10−3 M in MeOH)
λmax (Δε) 211 (−0.21), 231 (+8.93), 264 (−0.18), 301 (+0.48) nm.
Synthesis of 2a and 2b.8 A solution of LiHMDS (1.0 M in THF,
3 mL, 3 mmol) was cooled to −78 °C under Ar, and then THF solutions
of freshly prepared (S)-O-acetylmandelic acid chloride 86 (300.0 mg,
4 mL, 1.4 mmol) and 97 (240.0 mg, 4 mL, 1.4 mmol) were added simul-
taneously over 10 min at −78 °C. After stirring for an additional 30 min,
the mixture was warmed to room temperature, and AcOH (3.0 mL) was
added. The mixture was then stirred at 60 °C for 30 min and then
concentrated under reduced pressure. The residue was diluted with
EtOAc and water. The organic phase was separated, washed with
saturated aqueous NaHCO3, and concentrated under reduced pressure.
Then the residue was purified by MPLC (20−100% MeOH−H2O in 1 h)
and semipreparative HPLC (40% MeOH−H2O) to yield compound 10
(45 mg). Compound 10 (10 mg) was hydrolyzed in 6 N HCl (1 mL)
Penipyridone A (1): white solid; mp 262−264 °C; [α]26 −22
D
(c 0.1, CHCl3); ECD (1.0 × 10−3 M in MeOH) λmax (Δε) 208 (+2.59),
F
DOI: 10.1021/acs.jnatprod.6b00218
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Journal of Natural Products
Article
at room temperature for 3 h and finally purified by semipreparative HPLC
(30% MeOH−H2O) to yield compound 2a (8 mg). By the same procedure,
the enantiomers 10 (ent-10) and 2b were obtained from (R)-(+)-mandelic
acid.
Sigma-Aldrich, Shanghai, China) for 12 h, then treated with 10 μM of
indicated compounds or the marketed antihyperlidemic drug lovastatin
(Sigma-Aldrich) in DMEM + 100 μM oleic acid or with DMEM + 100 μM
oleic acid alone for another 6 h. Subsequently, the cells were subjected to
Oil-Red O staining or TC and TG determination as described previously.21
Each experiment (n = 8 for Oil-Red O staining or n = 3 for TC and TG
determination) was repeated at least three times. Data are presented as
the means SEM. One-way ANOVA was used to determine significant
differences among groups, after which the modified Student’s t test with the
Bonferroni correction was used for comparison between individual groups.
P < 0.05 was considered statistically significant.
Computational Methods. Mixed torsional/low-mode conforma-
tional searches were carried out by means of the Macromodel 9.9.223
software22 using the Merck molecular force field (MMFF) applying a
21 kJ/mol energy window. Geometry reoptimizations of the resultant
conformers [B3LYP/6-31G(d) level in vacuo and B97D/TZVP9 with
PCM solvent model for MeOH] and TDDFT calculations were per-
formed with Gaussian 0923 using various functionals (B3LYP,
BH&HLYP, PBE0) and the TZVP basis set. DFT-optimized structures
were clustered for the heavy (non-hydrogen) atoms and OH hydrogens,
but the orientation of the terminal isopropyl group and thus conformers
differing only in the orientation of the isopropyl group were neglected.
ECD spectra were generated as the sum of Gaussians24 with 2400
and 3000 cm−1 half-height width (corresponding to ca. 16 and 20 at
260 nm, respectively), using dipole-velocity-computed rotational strengths.
Boltzmann distributions were estimated from the ZPVE-corrected B3LYP/
6-31G(d) energies in the gas-phase calculations and from the B97D/TZVP
energies in the PCM model ones. The MOLEKEL software25 package was
used for visualization of the results.
X-ray Crystallographic Analysis of 1. Single-crystal X-ray
diffraction data were collected on an Agilent Gemini Ultra diffrac-
tometer with Cu Kα radiation (λ = 1.541 84 Å). The structure was
solved by direct methods (SHELXS-97) and refined using full-matrix
least-squares difference Fourier techniques. All non-hydrogen atoms
were refined anisotropically, and all hydrogen atoms were placed in
idealized positions and refined relatively isotropically with a riding
model. A crystal suitable for X-ray diffraction of 1 was obtained by slow
evaporation of a solution in MeOH−H2O. Crystallographic data for 1
have been deposited in the Cambridge Crystallographic Data Centre
with the deposition number 968304. Copies of the data can be obtained,
free of charge, from the Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac.uk/data-request/cif.
Crystal data for 1: orthorhombic, C13H12N2O3, space group P212121
with a = 6.5201(1) Å, b = 7.2318(1) Å, c = 24.7168(4) Å, α = β = γ = 90°,
V = 1165.45 (3) Å3, Z = 4, T = 290 (2) K, μ(Cu Kα) = 0.836 mm−1, Dc =
1.392 g/mm3, and F (000) = 512.0. Crystal size: 0.36 × 0.32 × 0.30 mm3.
Independent reflections: 2246 with Rint = 0.0198. The final agreement
factors are R1 = 0.0298 and wR2 = 0.0818 [I ≥ 2σ(I)]. Flack parameter =
−0.03(13).
Compound 10: colorless oil; [α]26D +48 (c 0.1, CHCl3); 1H NMR
(500 MHz, DMSO-d6) δ 8.46 (1H, s), 7.43 (2H, d, J = 7.3 Hz), 7.38
(2H, t, J = 7.3 Hz), 7.34 (1H, t, J = 7.3 Hz), 6.69 (1H, s), 6.61 (1H, s),
3.77 (3H, s), 2.17 (3H, s); 13C NMR (125 MHz, DMSO-d6) δ 172.4,
169.4, 165.9, 152.6, 143.6, 137.7, 128.6 (2C), 128.5, 127.2 (2C), 114.2,
112.4, 74.9, 51.8, 20.8; ESIMS m/z 302.2 [M + H]+, 324.1 [M + Na]+,
340.1 [M + K]+.
1
Compound ent-10. colorless oil; [α]26 −42 (c 0.1, CHCl3); H
D
NMR (500 MHz, DMSO-d6) δ 8.44 (1H, s), 7.42 (2H, d, J = 7.3 Hz),
7.37 (2H, t, J = 7.3 Hz), 7.34 (1H, t, J = 7.3 Hz), 6.68 (1H, s), 6.60 (1H,
s), 3.77 (3H, s), 2.17 (3H, s); 13C NMR (125 MHz, DMSO-d6) δ 172.4,
169.4, 165.8, 152.6, 143.4, 137.6, 128.6 (2C), 128.5, 127.2 (2C), 114.1,
112.4, 74.8, 51.8, 20.8; ESIMS m/z 302.2 [M + H]+, 324.1 [M + Na]+,
340.1 [M + K]+.
Compound 2a: white solid; [α]26D −20 (c 0.1, CHCl3); ESIMS m/z
1
260.1 [M + H]+, 282.1 [M + Na]+; H and 13C NMR and ECD were
identical to those of compound 2.
Compound 2b: white solid; [α]26D +16 (c 0.1, CHCl3); ECD (1.0 ×
10−3 M in MeOH) λmax (Δε) 208 (−7.38), 234 (+2.92), 277 (−0.37)
nm; ESIMS m/z 260.1 [M + H]+, 282.1 [M + Na]+, 541.2 [2 M + Na]+;
1H and 13C NMR were identical to those of compound 2.
Acid Hydrolysis of Compounds 1 and 3−7. Compounds 1
and 3−7 were hydrolyzed with 1 mL of 6 M HCl for 6 h at 55 °C.
The mixture was directly prepared by semipreparative HPLC to
yield compound 11 from 1 and 3. Under the same procedure, com-
pounds 4−7 lead to 12.
Compound 11: colorless oil; [α]26D −28 (c 0.1, CHCl3); 1H NMR
(500 MHz, DMSO-d6) δ 12.95 (1H, brs) 8.40 (1H, s), 7.46 (2H, d, J =
7.5 Hz), 7.38 (2H, dd, J = 7.5, 7.2 Hz), 7.34 (1H, dd, J = 7.2, 7.5 Hz),
6.71 (1H, s), 5.78 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ 179.0,
166.0, 156.6, 142.5, 141.3, 137.7, 128.6 (2C), 128.1, 126.5 (2C), 114.5,
114.2, 70.7; HRESIMS m/z 246.0768 [M + H]+ (calcd for C13H12O4N,
246.0761).
Compound 12: colorless oil; [α]26D +20 (c 0.1, CHCl3); 1H NMR
(500 MHz, DMSO-d6) δ 8.61 (1H, s), 7.43−7.36 (5H), 6.75 (1H, s),
6.01 (1H, s), 4.20 (2H, dd, J = 14.7 Hz), 3.49 (2H, dd, J = 14.7 Hz);
13C NMR (125 MHz, DMSO-d6) δ 178.2, 165.9, 157.1, 148.8,140.0,
128.7, 128.2, 127.0, 117.4, 114.2, 70.0, 60.0, 55.3; HRESIMS m/z 290.1022
[M + H]+ (calcd for C15H16O5N, 290.1023).
Methylation of 12 with TMS−CHN2. To a solution of 12 (3 mg) in
MeOH (0.5 mL) was added 400 μL of TMS−CHN2 (2.0 M in hexanes)
until a chartreuse color persisted upon addition. After stirring at room
temperature for 10 h, the solvent was removed via a stream of N2;
then the residue was purified by reversed-phase HPLC using a gradient
solvent system of MeOH−H2O (30:70) to yield compound 13 (3 mg).
Syntheses of 13. A mixture of 10 (5 mg), K2CO3 (5 mg), and
2-bromoethanol (6 μL) in anhydrous CH2Cl2 (1 mL) was stirred for
20 h at room temperature. The reaction mixture was filtered through a
small 4.5 μm filter and concentrated under reduced pressure. Then the
residue was hydrolyzed in 6 M HCl (1 mL) at room temperature for 3 h
and directly separated by HPLC (30:70 MeOH−H2O) to yield com-
pound 13 (2 mg).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.6b00218.
■
S
Structures of compounds 1−13; COSY and HMBC corre-
lations of new compounds 1−6; ECD spectra of 2, 2a, and
2b; HRESIMS and NMR spectra of compounds 1−7, 2a,
2b, and 9−13 (PDF)
Compound 13: colorless oil; [α]26D +22 (c 0.1, CHCl3); 1H NMR
(500 MHz, DMSO-d6) δ 8.27 (1H, s), 7.32−7.40 (5H), 6.47 (1H, s),
6.21 (1H, s), 5.85 (1H, s), 5.10 (1H, s), 4.05 (1H, m), 3.97 (1H, m),
3.70 (3H, s), 3.47 (2H, m); 13C NMR (125 MHz, DMSO-d6) δ 174.0,
165.1, 153.1, 148.9, 140.6, 128.6, 128.0, 126.9, 120.4, 116.5, 70.0, 60.3,
54.2, 51.4; HRESIMS m/z 304.1173 [M + H]+ (calcd for C16H18O5N,
304.1179).
Cell-Based Lipid Accumulation Assay. HepG2 cells, which were
originally from the American Type Culture Collection (ATCC)
(Manassas, VA, USA), were obtained from the China Union Medical
University. Cells were maintained in DMEM medium (Gibco, Grand
Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco)
and penicillin/streptomycin (100 μg/mL, Gibco). When grown to
70−80% confluence, cells were incubated in DMEM + oleic acid (100 μM,
Crystallographic data (CIF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: zhutj@ouc.edu.cn (T. Zhu).
*E-mail: pguo@implad.ac.cn (P. Guo).
*E-mail: dehaili@ouc.edu.cn (D. Li).
■
Author Contributions
⊥H. Zhou and L. Li contributed equally to this work.
G
DOI: 10.1021/acs.jnatprod.6b00218
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(14) Zhu, T. J.; Zhou, H. B.; Li, D. H.; Gu, Q. Q.; Guo, P. Faming
Zhuanli Shenqing, CN 104370806A, 20150225, 2015.
(15) Ge, H. M.; Shen, Y.; Zhu, C. H.; Tan, S. H.; Ding, H.; Song, Y. C.;
Notes
The authors declare no competing financial interest.
Tan, R. X. Phytochemistry 2008, 69, 571−576.
ACKNOWLEDGMENTS
(16) Liu, Y. Y.; Yang, Q.; Xia, G. P.; Huang, H. B.; Li, H. X.; Ma, L.; Lu,
Y. J.; He, L.; Xia, X. K.; She, Z. G. J. Nat. Prod. 2014, 71, 1861−1865.
(17) (a) Hiort, J.; Maksimenka, K.; Reichert, M.; Perovic-Ottstadt, S.;
Lin, W. H.; Wray, V.; Steube, K.; Schaumann, K.; Weber, H.; Proksch,
■
This work was financially supported by the Chinese National Natural
Science Fund (41376147, 21372208, 81402983, and 81573436),
Chinese Polar Environment Comprehensive Investigation &
Assessment Programmes (CHINARE2015-01-06-06), Shandong
Provincial Natural Science Fund for Distinguished Young Scholars
(JQ201422), and Fundamental Research Funds for the Central
Universities (201564026 and 33320140185). T.K. and A.M. thank
the Hungarian National Research Foundation (OTKA K105871)
and the National Information Infrastructure Development Institute
(NIIFI 10038). We also thank Dr. R. A. Keyzers for help with prep-
aration of the manuscript.
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