Phenalenones from a marine-derived fungus Penicillium sp.

Article abstract of DOI:10.3390/md17030176

Six new phenalenone derivatives (1–6), along with five known compounds (7–11) of the herqueinone class, were isolated from a marine-derived fungus Penicillium sp. The absolute configurations of these compounds were assigned based on chemical modifications and their specific rotations. 4-Hydroxysclerodin (6) and an acetone adduct of a triketone (7) exhibited moderate anti-angiogenetic and anti-inflammatory activities, respectively, while ent-peniciherqueinone (1) and isoherqueinone (9) exhibited moderate abilities to induce adipogenesis without cytotoxicity.

Full text of DOI:10.3390/md17030176

marine drugs
Article
Phenalenones from a Marine-Derived Fungus
Penicillium sp.
Sung Chul Park 1, Elin Julianti 1,2, Sungjin Ahn 1 , Donghwa Kim 1, Sang Kook Lee 1
,
Minsoo Noh 1, Dong-Chan Oh 1, Ki-Bong Oh 3,* and Jongheon Shin 1,
*
1
Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim,
Gwanak, Seoul 151-742, Korea; sungchulpark@snu.ac.kr (S.C.P.); elin_julianti@fa.itb.ac.id (E.J.);
sungjinahn@snu.ac.kr (S.A.); dkim0719@snu.ac.kr (D.K.); sklee61@snu.ac.kr (S.K.L.);
minsoonoh@snu.ac.kr (M.N.); dongchanoh@snu.ac.kr (D.-C.O.)
School of Pharmacy, Bandung Institute of Technology, Jl. Ganesha 10, Bandung 40132, Indonesia
Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National
University, San 56-1, Sillim, Gwanak, Seoul 151-921, Korea
2
3
*
Correspondence: ohkibong@snu.ac.kr (K.-B.O.); shinj@snu.ac.kr (J.S.);
Tel.: +82-2-880-4646 (K.-B.O.); +82-2-880-2484 (J.S.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 February 2019; Accepted: 14 March 2019; Published: 18 March 2019
Abstract: Six new phenalenone derivatives (1–6), along with five known compounds (7–11) of
the herqueinone class, were isolated from a marine-derived fungus Penicillium sp. The absolute
configurations of these compounds were assigned based on chemical modifications and their specific
rotations. 4-Hydroxysclerodin (6) and an acetone adduct of a triketone (7) exhibited moderate
anti-angiogenetic and anti-inflammatory activities, respectively, while ent-peniciherqueinone (
1) and
isoherqueinone (9) exhibited moderate abilities to induce adipogenesis without cytotoxicity.
Keywords: herqueinones; phenalenones; Penicillium sp.; marine-derived fungi; adipogenesis;
anti-angiogenesis; anti-inflammatory
1. Introduction
Fungi produce a wide variety of polyketide-derived metabolites. Among these, phenalenones are
a class of hexa- or heptaketides bearing a perinaphtenone-type tricyclic system [1]. As summarized
in a recent comprehensive review, these compounds can have immense structural variations, such
as homo- and heterodimerization, the incorporation of additional carbon frameworks, and a high
degree of oxygenation and nitrogenation as well as being complexed with metals [2]. A frequently
occurring variation is the incorporation of an isoprene unit by forming either a linear ether or
a trimethylhydrofuran moiety, and this variation is well-represented in the herqueinones from
Penicillium sp. [
3–5]. Fungi-derived phenalenone compounds have attracted significant interest due
to their chemical structures, bioactivities, and biosynthesis [
1]. With their diverse phylogenic origins,
phenalenones are widely recognized as a representative group of fungal polyketides [1,2].
During the course of our search for novel compounds from marine-derived fungi, we reported the
structures of herqueiazole and herqueioxazole, unusual pyrrole- and oxazole-containing phenalenones
from a Penicillium sp. strain [6]. Herqueidiketal, a cytotoxic sortase A inhibitory congener, also
possessed a novel skeleton containing a highly oxidized naphthoquinone moiety [
6
]. Despite
its carbon skeleton being different from typical phenalenones, the presence of naphthalene and
dihydrofuran moieties in herqueidiketal may further emphasize the wide structural variations of
phenalenones. In our continuing search for such compounds, we isolated several structurally related
phenalenones from a large-scale cultivation of this Penicillium sp. strain. Here, we report the isolation
Mar. Drugs 2019, 17, 176; doi:10.3390/md17030176
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2019, 17, 176
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of eleven compounds (
in the herqueinone subclass. 4-Hydroxysclerodin (
on human umbilical vascular endothelial cells (HUVECs). The acetone adduct of a triketone (
1
–
11) as well as the structure determination of six new compounds (
1
–
6
7
)
)
6
) exhibited moderate anti-angiogenic activity
exhibited moderate anti-inflammatory activity in mouse macrophage RAW 264.7 cells. In addition,
ent-peniciherqueinone ( ) and isoherqueinone ( ) moderately induced adipogenesis in human bone
1
9
marrow-mesenchymal stem cells (hBM-MSCs). All of these bioactivities were found to occur
without cytotoxicity.
2. Results and Discussion
The molecular formula of
1 was deduced to be C20H20O8 with 11 degrees of unsaturation by
HRFABMS analysis. The 13C NMR data of this compound showed a signal of a ketone carbon
at δC 197.6 (Table 1). The signals at δC 178.1 and 174.6 could belong to either carbonyl or highly
deshielded olefinic carbons. The 13C NMR spectrum, in combination with DEPTs and HSQC spectra
(Supplementary Figure S4), displayed nine nonprotonated sp2 carbon signals in the δC 103.5–162.8
region. The deshielded carbons must be one carbonyl carbon and one olefinic carbon, accounting
therefore for seven degrees of unsaturation. The 13C NMR data also showed two oxygen-bearing
quaternary sp3 carbons (δC 89.5 and 79.0), one methoxy carbon (δC 60.9), one shielded quaternary sp3
carbon (δC 46.9), and four shielded methyl carbons (δC 16.4, 16.4, 14.9, and 13.3) (Table 2). Combining
the NMR data and the degrees of unsaturation,
class of phenalenones.
1 must possess four rings featuring the herqueinone
Table 1. 13C NMR (125 and 150 MHz) of 1–6.
δC, Type
Position
1 a
1 b
2 b
3 b
4 b
5 b
6 b
1
2
3
137.3, C
103.7, C
174.6, C
79.0, C
137.1, C
103.1, C
175.4, C
78.4, C
133.5, C
102.9, C
173.9, C
78.2, C
139.0, C
103.0, C
178.2, C
78.5, C
142.7, C
116.3, C
175.1, C
78.5, C
142.9, C
116.7, C
175.6, C
78.5, C
141.8, C
115.4, C
180.4, C
78.9, C
4
5
6
7
8
197.6, C
103.5, C
162.8, C
131.6, C
161.7, C
108.7, C
178.1, C
143.7, C
124.0, C
14.9, CH3
60.9, CH3
13.3, CH3
89.5, CH
46.9, C
198.2, C
102.7, C
161.7, C
131.0, C
161.7, C
108.6, C
178.7, C
144.0, C
124.5, C
14.6, CH3
60.0, CH3
12.9, CH3
89.3, CH
45.9, C
198.3, C
102.6, C
157.2, C
129.2, C
156.2, C
108.4, C
178.7, C
143.9, C
123.8, C
14.4, CH3
197.7, C
102.6, C
161.9, C
131.2, C
163.0, C
109.2, C
186.4, C
122.8, CH
150.9, C
23.8, CH3
60.0, CH3
12.9, CH3
90.6, CH
46.0, C
193.1, C
101.2, C
189.9, C
76.9, C
200.6, C
109.4, C
165.6, C
117.5, CH
152.3, C
23.5, CH3
193.3, C
101.4, C
189.3, C
75.8, C
200.3, C
109.4, C
165.8, C
117.5, CH
152.2, C
23.4, CH3
192.2, C
92.3, C
155.5, C
9
164.4, C
102.0, C
164.0, C
117.3, C
152.4, C
23.2, CH3
10
11
12
13
14
15
0
1
2
3
4
5
6
7
8
12.9, CH3
88.7, CH
45.9, C
15.9, CH3
16.1, CH3
12.7, CH3
88.6, CH
45.6, C
16.1, CH3
16.3, CH3
48.5, CH2
206.9, C
12.8, CH3
88.8, CH
45.3, C
16.1, CH3
16.3, CH3
48.6, CH2
207.1, C
12.8, CH3
90.8, CH
45.7, C
16.0, CH3
16.3, CH3
0
0
0
16.4, CH3
16.4, CH3
15.9, CH3
16.2, CH3
15.9, CH3
16.1, CH3
0
0
0
0
29.8, CH3
29.7, CH3
a,b
The spectra were recorded in CDCl3 and DMSO-d6, respectively.

Mar. Drugs 2019, 17, 176
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1
Table 2. H NMR (400 and 600 MHz) of 1–6.
δ , Mult. (J in Hz)
H
Position
1 a
1 b
2 b
3 b
4 b
5 b
6 b
12
6.36, s
2.48, s
3.77, s
6.75, s
2.57, s
6.76, s
2.55, s
6.81, s
2.58, s
14
15
2.47, s
3.92, s
2.39, s
3.77, s
2.39, s
1.40, d
(6.6)
1.35, d
(6.3)
1.34, d
(6.4)
1.37, d
(6.5)
1.30, d
(6.4)
1.29, d
(6.5)
1.36, d
(6.5)
0
1
4.99, q
(6.6)
4.91, q
(6.3)
4.88, q
(6.4)
4.99, q
(6.5)
4.77, q
(6.4)
4.79, q
(6.5)
4.92, q
(6.5)
0
2
0
4
1.43, s
0.86, s
1.30, s
0.75, s
1.30, s
0.75, s
0.78, s
1.32, s
0.79, s
1.25, s
3.30, s
2.09, s
7.15, s
0.78, s
1.25, s
3.38, s
2.12, s
7.23, s
0.78, s
1.26, s
0
5
0
6
0
8
OH-4
OH-7
OH-8
OH-9
OH-11
OH-12
7.23, s
7.38, s
7.28, s
13.14, s
8.96, s
7.52, s
7.41, s
13.23, s
13.30, s
13.26, s
6.68, s
6.82, s
13.99, s
14.97, s
14.55, s
15.73, s
12.80, s
12.75, s
11.43, br s
6.66, s
9.15, s
9.00, s
a,b
The spectra were recorded in CDCl3 and DMSO-d6, respectively.
0
Due to the lack of COSY correlations except for that from the methyl doublet at δH 1.40 (Me-1 ) to
the quartet at δH 4.99 (H-20), the structure determination of 1 had to be carried out through extensive
HMBC analyses under diverse measuring conditions (Figure 1). First, the long-range couplings from
OH-7 (δH 13.23) to C-6 (δC 103.5), C-7 (δC 162.8), and C-8 (δC 131.6); from OCH3-8 (δH 3.92) to C-8
(
δC 131.6); and OH-9 (δH 13.99) to C-8 (δC 131.6), C-9 (δC 161.7), and C-10 (δC 108.7) lead to a delineation
of the C-6 to C-10 fragment. Aided by the four-bond couplings from OH-7 (δH 13.23) to C-1 (δC 137.3)
and OH-9 (δH 13.99) to C-1 (δC 137.3) by decoupled HMBC (D-HMBC) [ ] experiments, the presence
7
of a hexa-substituted benzene ring (C-1, C-6–C-10; ring A) was confirmed. In addition, the combined
HMBC and D-HMBC correlations from OH-12 (δH 6.66) and H3-14 (δH 2.47) to neighboring carbons
revealed the presence of an α-hydroxy-β-methyl-α,β-unsaturated ketone group (OH-12 (δH 6.66) to
C-11 (δC 178.1), C-12 (δC 143.7), and C-13 (δC 124.0); H3-14 (δH 2.47) to C-2 (δC 103.7), C-11 (δC 178.1),
C-12 (δC 143.7), and C-13 (δC 124.0)), which was directly connected to the benzene ring based on the
D-HMBC correlation from OH-7 (δH 13.23) to C-11 (δC 178.1).
In addition to the correlation from H3-14 (δH 2.47) to C-2 (δC 102.7), a correlation from H3-14
(δH 2.47) to a highly deshielded C-3 (δC 175.4) in the D-HMBC spectrum was crucial evidence for the
attachment of an electron-withdrawing oxygen at this position. Subsequently, long-range correlations
from OH-4 (δH 7.23) to C-3 (δC 8), C4 (δC 8), and C5 (δC 8) defined not only its connectivity to the C-2
double bond but also placed a carbonyl carbon (δC 198.2) at C-5. These carbon–proton correlations
constructed an α,β-dioxycyclohexadienone moiety (C-1–C-6; ring C). The assignment of ring C also
secured the formation of the conjugated carbonyl group to another six-membered ring (C-1, C-2,
C-10–C-13; ring B).

Mar. Drugs 2019, 17, 176
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Figure 1. The structures of 1–11.
The remaining C5 fragment (C-10–C-50) of
1 was readily defined as a 2,3-disubstituted
2-methylbutane moiety by a combination of COSY and HMBC data (Figure 2 and Supplementary
Figures S3–S5). The cyclization of this moiety to the three-ring system was also accomplished by a
series of long-range carbon–proton correlations. That is, the connection between C-4 and C-50 was
confirmed by the HMBC correlations from H3-40 (δH 1.43) and H3-50 (δH 0.86) to C-4 (δC 79.0) as well
as a long-range correlation from H-20 to C-5. The diagnostic chemical shifts of the CH-20 methine
group (δC 89.3, δH 4.91) suggested its attachment to C-3 via an ethereal bridge. This interpretation was
0
corroborated by the correlation from H3-1 (δH 1.40) to C-3 (δC 174.6), which established a hydrofuran
moiety (C-3, C-4, C-20, and C-40; ring D). Thus, the structure of
1 was defined as a herqueinone-type
tetracyclic phenalenone.
The planar structure of
1 was found to be the same as that of the recently reported
peniciherqueinone from the fungus Penicillium herquei [
8
]. In our study of the configurations of the C-4
and C-20 stereogenic centers by NOESY analysis (Figure 3), the OH-4, H-20, and H3-50 protons were
oriented toward the same face of the hydrofuran ring based on their mutual cross-peaks. The opposite
0
0
face was occupied by H3-1 and H3-4 based on the cross peak between the methyl protons, suggesting
that
1
has the same relative configuration (4S* and 20S*) as peniciherqueinone. Interestingly, despite
the same signs of optical rotations, there was a remarkable difference in their values of the specific
25
rotations: [
α
]
(CHCl3) +203 (
1
) and +92 (peniciherqueinone). Since the absolute configurations at C-4
D
0
and C-2 of herqueinones have been the subject of comprehensive investigations [
9
,10], the discrepancy
in the specific rotations of
modification technique [11
(CHCl3)
1
–
and herqueinones needed to be justified. Using a pre-established chemical
25
13],
1
was reduced to 1a, which showed a negative specific rotation ([
α
]
D
−
23); thus, the 20S configuration was confirmed. The absolute configuration was further

Mar. Drugs 2019, 17, 176
5 of 16
evaluated via the acetylation of 1a to corresponding 9,11,12-triacetyl derivative 1b (Figure 4). The
sign of the specific rotation of 1b ([α]2D5 (MeOH) −42) was opposite to that of herqueinone (8) but the
same as that of isoherqueinone (
configuration of
herqueinone-type phenalenone.
9 9,10]. Therefore, the absolute
), which proved a 20S configuration [
1
was assigned as 4S and 20S. Thus,
1, designated as ent-peniciherqueinone, is a new
Figure 2. Key correlations of HMBC (arrows) and decoupled HMBC (D-HMBC) (dashed arrows) of
4, and 6.
1,
Figure 3. NOESY correlations of the hydrofuran moiety of 1.
The molecular formula of
NMR data of this compound were very similar to those of
A detailed examination of the 13C and 1H NMR data revealed that the OMe-8 of
replaced by a hydroxyl group (δH 8.96) in , and this assignment was confirmed by a combination of
2
was deduced to be C19H18O8 based on HRFABMS analysis. The
, with the absence of a methyl group.
δC 60.9, δH 3.92) was
1
1
(
2
two-dimensional (2D) NMR analyses. The NOESY data and specific rotation of the reduction product 2a
0
indicated the same 4S and 2 S configuration as in
1. Thus,
2
, designated as 12-hydroxynorherqueinone,
was determined to be 8-demethyl-ent-peniciherqueinone.

Mar. Drugs 2019, 17, 176
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Figure 4. Phenolic derivatization of herqueinones.
Compound
3 was isolated as an orange amorphous solid with a molecular formula of C20H20O7,
based on HRFABMS analysis. The 13C and 1H NMR data of this compound were similar to those
obtained for 1. The most noticeable difference was the replacement of a hydroxyl-bearing olefinic
carbon with the sp2 methine carbons (δC 122.8, δH 6.36). The structural difference was found to be
at C-12 based on the HMBC correlations from H-12 (δH 6.36) to C-2 (δC 103.0), C-10 (δC 109.2), and
C-14 (δC 23.8) as well as from H3-14 (δH 2.48) to C-2 (δC 103.0), C-12 (δC 122.8), and C-13 (δC 150.9).
However, the sign of the specific rotation of
implying a configurational difference. Since the NOESY spectrum showed the same cross-peaks for
the hydrofuran moiety as those in the congeners, was proposed to possess the opposite absolute
3
([
α
]
25 (MeOH)
−
69) was opposite to those of 1 and 2,
D
3
configuration at C-4 and C-20. As the reduction product of
3
(
3a) is dextrorotatory (specific rotation
25
0
0
([α
]
(MeOH) +39)), the configuration of C-4 and C-2 are 4R, 2 R, respectively. Thus,
3, designated as
D
ent-isoherqueinone, is a new herqueinone-type phenalenone derivative.
The molecular formula of was also established as C22H22O8 by HRFABMS analysis. Although
its spectroscopic data resembled those of
, several differences were found in both 13C and 1H NMR
data. First, aided by the HSQC data, it was found that three additional carbons, i.e., one carbonyl
δC 206.9), one methylene sp2 (δC 48.5, δH 3.30), and one methyl (δC 29.8, δH 2.09), were present in this
compound (Tables 1 and 2). In the 13C NMR spectrum, resonances of three ketone groups (δC 200.6,
193.1, and 189.9) were found for , unlike . In addition, an aromatic or olefinic carbon had been
replaced by an oxygen-bearing nonprotonated sp3 carbon (δC 76.9). A detailed examination of its
NMR data revealed that contained the same B and D rings as , and the structural differences were
located in the remaining portion of the molecule.
The planar structure of was established by extensive HMBC experiments (Figure 2). Several
HMBC correlations were found from an aromatic proton (δH 6.75, H-12) and a benzylic methyl proton
4
1–3
(
4
1–3
4
1–3
4

Mar. Drugs 2019, 17, 176
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(δH 2.57, H3-14) to their neighboring carbons (H-12 (δH 6.75) to C-2 (δC 116.3), C-10 (δC 109.4), and
C-14 (δC 23.5); H3-14 (δH 2.57) to C-2 (δC 116.3), C-12 (δC 117.5), and C-13 (δC 152.3)). Aided by
the D-HMBC correlations from H-12 (δH 6.75) to C-1 (δC 142.7) and C-11 (δC 165.6) and from H3-14
(δH 2.57) to C-1 (δC 142.7), the long-range carbon–proton correlations led to the establishment of a
hydroxyl- and methyl-bearing pentasubstituted benzene as ring B. Additional D-HMBC correlations
0
from these protons to the conspicuous H3-1 at δH 1.30 (H-12 (δH 6.75) to C-6 (δC 101.2); H3-14 (δH 2.57)
0
to C-3 (δC 175.1), C-5 (δC 193.1), and C-6 (δC 101.2); H3-1 (δH 1.30) to C-3 (δC 175.1) and C-5 (δC 193.1))
defined ring C as a hydroxyl-bearing cyclohexadienone. The ring D was found to be the same as that
in other herqueinones by a 2D NMR spectrum.
The remaining portion of 4 consists of three ketone carbonyl (δC 206.9, 200.6, and 189.9) and one
nonprotonated sp3 (δC 76.9), one methylene sp3 (δC 48.5), and one 0methyl (δC 29.8) carbons. These
0
carbons were initially assembled into a 2-keto-propyl group (C-6 –C-8 ) by the HMBC correlations from
the 0methylene and 0methyl protons to their neighboring carbons (H2-60 (δH 3.30) to C-70 (δC 206.9) and
0
0
C-8 (δC 29.8); H3-8 (δH 2.09) to C-6 (δC 48.5), and C-7 (δC 206.9)) (Figure 2). Then, this fragment was
0
0
connected to the core structure by the HMBC correlations from H2-6 (H2-6 (δH 3.30) to C-7 (δC 189.9),
C-8 (δC 76.9), and C-9 (δC 200.6)). The confirmation of this assignment as well as the linkage with
the B-ring was accomplished by the key D-HMBC correlations from OH-8 (δH 6.68) to C-7 (δC 189.9)
and C-9 (δC 200.6); from H-12 (δH 6.75) to C-9 (δC 200.6); from H3-14 (δH 2.57) to C-9 (δC 200.6);
and from H3-80 (δH 2.09) to C-8 (δC 76.9). Although it could not be confirmed by 2D-NMR-based
carbon–proton correlations, the presence of the four rings, required by the molecular formula and NMR
data, directly connected C-6 and C-7 carbonyl carbons to be part of a diketo-bearing six-membered
ring as ring A. Thus, the structure of
4
was determined to be a phenalenone related to an acetone
adduct of a triketone [14,15].
The molecular formula of
5 was the same as that of 4
, C22H22O8. Moreover, the 13C and 1H NMR
data of these compounds were very similar (Tables 1 and 2). Two-dimensional NMR analyses showed
the same carbon–proton correlations throughout the entire molecule, indicating that they have the
same planar structure. Therefore, 5 could be an epimer of 4.
In order to clarify the difference in stereochemistry between 4 and 5, NOESY experiments were
carried out. The NOESY spectra of both compounds showed the same cross-peaks around the D
ring as those observed in other herqueinones, suggesting the 4R,20R or 4S,20S configurations. Then,
by chemical conversions to remove the other two stereogenic centers, the absolute configurations at
C-4 were determined. That is,
4 and 5 were reduced to 4a and 5a, respectively (Figure 4), then the
compounds were dehydrated to yield 8,15-unsaturated derivatives 4b and 5b, respectively (Figure 5),
and the MS and NMR data of these compounds were identical. Furthermore, their specific rotations
were also very similar ([
α
]
25 (CHCl3)
−
27 and
−
26 for 4b and 5b, respectively), implying that these
D
were indeed the same compound. The negative specific rotations allow us to confidently assign the
0
2 R configuration for both natural products. Thus,
4 and 5, designated as oxopropylisoherqueinones A
and B respectively, were elucidated as new phenalenones possessing C3 side chains. These compounds
possessed 4R, 20R configurations. However, the configurations at C-8 remain unassigned despite
various chemical and spectroscopic analyses.
In order to determine the absolute configurations at C-8 of
4 and 5, a comparison of the
experimental and calculated ECD spectra was carried out. Initially, the experimental CD profiles of
these compounds showed opposite signs in the region of 285–340 nm, possibly reflecting the different
configuration at C-8 (Supplementary Figure S51). Despite all the efforts, however, the calculated ECD
profiles based on the postulated conformational populations failed to assign the absolute configurations
satisfactorily (Supplementary Figure S52). This could be due to a weak contribution of a single and
remote stereogenic center to the ECD in the molecule possessing several UV chromophores and
stereogenic centers.

Mar. Drugs 2019, 17, 176
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O
O
HO
O
6'
O
O
O
8
Na, EtOH
rt, 6 hr
HO
OH
HO
OH
O
O
4a, 5a, 7
4b, 5b, 7a
Figure 5. Dehydrations of 4a, 5a, and 7.
The molecular formula of
6
was deduced to be C18H16O7, which corresponds to 11 degrees
of unsaturation, by HRFABMS analysis. The 13C and H NMR data of this compound revealed
that it is a phenalenone derivative based on the presence of signals for two aromatic rings and
a trimethylhydrofuran moiety, which account for eight degrees of unsaturation (Tables 1 and 2).
However, only the carbon signals of two nonprotonated quaternary sp2 carbons (δC 164.4 and 155.4)
had replaced the NMR signals of the A ring of the other compounds. Therefore, in addition to satisfying
the three remaining degrees of unsaturation, the C2O3 portion must account for two carbonyls and a
cyclic ether or ester group.
1
The planar structure of
6 was determined with the aid of HMBC experiments (Figure 2). First, the
long-range couplings of key protons, such as the four methyl groups (H3-14 (δH 2.58), H3-10 (δH 1.36),
H3-40 (δH 0.78), and H3-50 (δH 1.26)), an aromatic proton (H-12 (δH 6.81)), and two hydroxy protons
(OH-4 (δH 7.41) and OH-11 (δH 11.43)), with their neighboring carbons confirmed the presence of the
same B-D polycyclic moiety as in
(
(
4
and
δC 164.4) at C-9, which was supported by the key D-HMBC correlation from H3-14 (δH 2.58) to C-9
δC 164.4). The other carbon (δC 155.5) must be located at C-7 due to the shielding of C-6 (δC 92.3).
5. An additional coupling to OH-11 placed a carbonyl carbon
Although it was not directly proved by NMR spectra, both the MS data and the shielded chemical shifts
of the C-7 and C-9 carbonyls were indicative of an oxygen bridge between these positions, leading to a
six-membered cyclic acid anhydride moiety as ring A. The NMR data of the ring portion of
6 were
similar to those of sclerodin (10), which was previously reported from the fungus Gremmeniella abietina
thus supporting the structure of 6 [14,16].
The NOESY correlations of 6
placed the OH-4, H-20, and H3-50 on one side and H3-10 and H3-40
on the other side of the hydrofuran moiety, leading to the same relative configuration (4S* and 20S*)
25
as that in
and
1–
3
. Then, the specific rotation of
6
was similar to that of
3
([
α
]
−
69 and
−
52 for 3
D
6
, respectively), suggesting they have the same absolute configuration (4R and 20R). However,
to remove the effect of structural differences in ring A, the reduction of
6 produced the 4-deoxy
derivative 6a (=10), which possessed only the C-20 stereogenic center (Figure 4). Interestingly, the
25
specific rotation of 6a showed the same sign as those of 1a and 2a but opposite to those of 3a ([
+34 and
α
]
D
−
18 for 3a and 6a, respectively), confirming the 20S configuration. Our results were in good
agreement with the specific rotations of natural 6a (
10) and 20-epi-6a, which are levorotatory and
dextrorotatory, respectively [14]. Overall, the configuration of this compound was assigned as 4S,20S.
Notably, changing the phenolic A ring to an acid anhydride inverted the sign of the specific rotation
of the herqueinone. Thus,
structurally related to sclerodin (10).
In addition to , five previously reported phenalenones (
a combination of spectroscopic analyses and a literature survey, these compounds were identified
as an acetone adduct of the triketone ( ) [14], herqueinone ( ) [ 17 18], isoherqueinone ( ) [19 20],
sclerodin (10) [14], and scleroderolide (11) [21]. The NMR data of these compounds were in good
agreement with the reported values in the literature. Compound was obtained as an unseparated
6, designated as 4-hydroxysclerodin, is a new phenalenone derivative and
1–
6
7–11) were also isolated. Based on
7
8
3
,
,
9
,
7

Mar. Drugs 2019, 17, 176
9 of 16
epimeric mixture, which was consistent with the literature [14
,
15]. Compound
7
was dehydrated to 7a
by the same method used for
epimerization of
C-8 stereogenic center.
Compounds , and
raised the hypothesis that
4
and
5
, and the 020R configuration was thus assigned. In this way, the
7
was found to occur not at C-2 (in the hydrofuran moiety) but at the hydroxy-bearing
0
0
4
,
5
7
possessed a C3 oxopropyl moiety (C-6 –C-8 ) whose structural resemblance
could be the acetone adduct formed during the separation process. This
7
hypothesis has a reliable experimental basis of chemical transformation of a triketone to
to verify if is an acetone adduct or a true natural compound biosynthesized by the fungus, the
production of these compounds was monitored by time-scale cultivation and LC-ESI-MS analysis.
Weekly mass analysis of the culture media showed that the major metabolite was clearly detected
7 [14]. In order
7
7
after 6 weeks without using acetone (Supplementary Figure S53). Thus, these compounds were
unambiguously proved to be the natural products produced by the Penicillum sp. fungus.
Although fungal phenalenones exhibit diverse bioactivities [
have not frequently shown remarkable bioactivities. The mild antioxidant and radical scavenging
activities of isoherqueinone ( ) [ ], the antibacterial activity of scleroderolide (11) [22], and human
1,2], herqueinone-type compounds
9
9
leukocyte elastase inhibition of atrovenetinone can be considered exceptions [23]. Regarding the
bioactivities of herqueinones, it is interesting to note that the presence of both OH-5 and OH-11 groups
are required for the antibacterial activity [
(IC50 > 10 M) against the K562 (human chronic myeloid leukemia) and A549 (adenocarcinomic human
alveolar basal epithelial) cancer cell lines. These compounds were also inactive (MIC > 128 M) against
1]. The cytotoxicity assay revealed that 1–11 were inactive
µ
µ
various bacterial and fungal strains, which was consistent with the report on the structure-activity
relationships of herqueinones [1].
Compound
M, while the rest of the isolated compounds were inactive (IC50 > 20
assay, inhibited tube formation in HUVECs with an IC50 of 20.9 M (Supplementary Table S1 and
Figures S54 and S55), while and induced adipogenesis through PPAR binding and adiponectin
secretion-promoting activity in hBM-MSCs and in a concentration-dependent manner, which was
determined by adiponectin secretion-promoting effects with their IC50 values of 57.5 M and 39.7 M,
7
moderately inhibited NO production in RAW 264.7 cells with an IC50 value of
3.2
µ
µM). In the angiogenesis
6
µ
1
9
γ
µ
µ
respectively (Table S1 and Figure S56). All of these bioactivities were found to occur without
significant cytotoxicity.
In summary, 11 polyketide-derived phenalenones, including six previously unreported
phenalenones, were isolated from the culture broth of a marine-derived Penicillium sp. The
absolute configurations of the stereogenic centers in the hydrofuran ring were assigned by chemical
modifications and measurements of specific rotations. Compounds 1, 6, 7, and 9 exhibited diverse
bioactivities, such as anti-inflammatory, anti-angiogenetic, and adipogenesis-inducing activities.
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 polarimeter (Easton, MD, USA) using a cell
with a 1-cm path length. UV spectra were acquired using a Hitachi U-3010 spectrophotometer (Tokyo,
Japan). CD spectra were recorded on an Applied Photophysics Ltd. Chirascan plus CD spectrometer
(Applied Photophysics Ltd., Leatherhead, Surrey, UK). IR spectra were recorded on a JASCO 4200
FT-IR spectrometer (Easton, MD, USA) using a ZnSe cell. NMR spectra were recorded in DMSO-d6 or
CDCl3 solutions on Bruker Avance-400, -500, or -600 instruments (Billerica, MA, USA). High-resolution
FABMS data were acquired using a JEOL JMS 700 mass spectrometer (Tokyo, Japan) with 6 keV-energy,
emission current 5.0 mA, xenon as inert gas, and meta-nitrobenzyl alcohol (NBA) as the matrix at the
Korea Basic Science Institute (Daegu, Korea). Low-resolution ESIMS data were recorded on an Agilent
Technologies 6130 quadrupole mass spectrometer (Santa Clara, CA, USA) with an Agilent Technologies
1200 series HPLC (Santa Clara, CA, USA). HPLC separations were performed on a SpectraSYSTEM

Mar. Drugs 2019, 17, 176
10 of 16
p2000 equipped with a refractive index detector (SpectraSYSTEM RI-150 (Waltham, MA, USA)) and a
UV-Vis detector (Gilson UV-Vis-151 (Middleton, WI, USA)). All solvents used were of spectroscopic
grade or were distilled prior to use.
3.2. Fungal Material
The fungal strain Penicillium sp. was isolated from marine sediments collected from Gagudo,
Korea, in October 2008. The isolate was identified using standard molecular biological protocols
by DNA amplification and sequencing of the ITS region. Genomic DNA extraction was performed
using Intron’s i-genomic BYF DNA Extraction Mini Kit according to the manufacturer’s protocol. The
nucleotide sequence was deposited in the GenBank database under the accession number JF901804.
The 18S rDNA sequence of this strain showed 99% identity with Penicillium herquei GA4 (GenBank
accession number EF536027).
3.3. Extraction and Isolation
The fungus was cultivated on YPG medium (5 g of yeast extract, 5 g of peptone, 10 g of glucose
in 1 L of artificial seawater) in 2.8 L Fernbach flasks at 30 ◦C under static conditions in the dark for
6 weeks. The mycelia and culture broth were separated by filtration, and the broth (20 L) was extracted
with EtOAc (20 L
×
3). The solvent was evaporated under reduced pressure to afford a crude EtOAc
extract (6.2 g), which was fractionated by C18 reversed-phase vacuum flash chromatography using
mixtures of H2O-MeOH, from 50:50 to 0:100, and acetone and EtOAc as the eluents.
Based on the 1H NMR and LC-MS analyses, the moderately polar fractions (30:70–10:90
H2O-MeOH) were chosen for further separation. The fraction (220 mg) that eluted with H2O-MeOH
(30:70) was separated by a semi-preparative reversed-phase HPLC (YMC-ODS-A column, 10
H2O-MeOH, 45:55; 1.7 mL/min) to yield (tR = 18.4 min, 5.5 mg) and (tR = 18.9 min, 7.7 mg).
The fraction (570 mg) that eluted with H2O-MeOH (20:80) was separated by a semi-preparative
reversed-phase HPLC (H2O-MeOH, 32:68; 1.7 mL/min) to afford (tR = 37.5 min), (tR = 27.8 min),
(tR = 21.8 min). Compounds (311.5 mg), (5.6 mg),
(4.4 mg) were purified by an analytical HPLC (YMC-ODS-A column, 4.6 250 mm;
×
250 mm;
4
5
1
2
3
(tR = 29.1 min),
8
(tR = 25.1 min), and
9
1
3
8
(16.5 mg), and
9
×
H2O-MeOH, 37:63; 0.7 mL/min; tR = 38.8, 34.5, 30.9, and 27.1 min, respectively). Compound
2
(1.7 mg) was also purified by an analytical HPLC (H2O-MeCN, 48:52; 0.7 mL/min; tR = 35.0 min). The
fraction (230 mg) eluted with H2O-MeOH (10:90) was separated by a semi-preparative reversed-phase
HPLC (H2O-MeOH, 22:78; 1.7 mL/min) to yield 7 (tR = 19.7 min, 73.4 mg), 10 (tR = 22.8 min), and 11
(tR = 23.5 min). Compounds 10 (3.9 mg) and 11 (3.3 mg) were further purified by an analytical HPLC
(H2O-MeOH, 26:74; 0.7 mL/min; tR = 26.8 and 30.1 min, respectively).
25
ent-Peniciherqueinone (
(MeOH) (log
1
): red, amorphous solid; [
α
]
+203 (c 1.7, CHCl3), +254 (c 1.0, MeOH); UV
D
λ
ε
) 217 (4.32), 248 (4.27), 311 (4.20), 427 (3.75) nm; IR (ZnSe)
1590, 1385 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRFABMS m/z 389.1239 [M + H]+ (calcd for
ν
3413 (br), 1629,
max
max
C20H21O8, 389.1239).
25
12-Hydroxynorherqueinone (
(log
2
): red, amorphous solid; [
α
]
+124 (c 0.1, MeOH); UV (MeOH)
λ
max
D
ε
) 217 (4.32), 248 (4.31), 311 (4.36), 430 (3.80) nm; IR (ZnSe) ν
max 3445 (br), 1629, 1579, 1461 cm−1; 1H
and 13C NMR data, Tables 1 and 2; HRFABMS m/z 375.1079 [M + H]+ (calcd for C19H19O8, 375.1080).
25
ent-Isoherqueinone (
3
): orange, amorphous solid; [
α
]
−
69 (c 0.2, MeOH); UV (MeOH)
λ
max (log
ε
)
D
1
217 (4.32), 248 (4.29), 311 (4.22), 428 (3.79) nm; IR (ZnSe)
13C NMR data, Tables 1 and 2; HRFABMS m/z 373.1285 [M + H]+ (calcd for C20H21O7, 373.1283).
ν
3422 (br), 1631, 1460 cm−1; H and
max

Mar. Drugs 2019, 17, 176
11 of 16
25
D
Oxopropylisoherqueinone A (
4
): brown, amorphous solid; [
α
]
+92 (c 0.2, MeOH); UV (MeOH) λ
max
(log
ε
) 224 (4.36), 274 (4.30), 357 (3.57) nm; IR (ZnSe)
ν
3382 (br), 1678, 1639, 1297 cm−1; 1H and
max
13C NMR data, Tables 1 and 2; HRFABMS m/z 415.1396 [M + H]+ (calcd for C22H23O8, 415.1393).
25
Oxopropylisoherqueinone B (
(log
5
): brown, amorphous solid; [
α
]
+43 (c 0.2, MeOH); UV (MeOH)
λ
max
D
ε
) 224 (4.36), 274 (4.30), 357 (3.57) nm; IR (ZnSe)
ν
3415 (br), 1679, 1640, 1297 cm−1; 1H and
13C NMR data, Tables 1 and 2; HRFABMS m/z 415.1396 [M + H]+ (calcd for C22H23O8, 415.1393).
max
25
4-Hydroxysclerodin (
213 (3.94), 280 (4.21), 312 (3.68) nm; IR (ZnSe)
6
): yellow, amorphous solid; [
α
]
−
52 (c 0.2, MeOH); UV (MeOH)
λ
max (log
ε
)
D
1
ν
3424 (br), 3069, 1729, 1460, 1286 cm−1; H and
13C NMR data, Tables 1 and 2; HRFABMS m/z 345.0977 [M + H]+ (calcd for C18H17O7, 345.0974).
max
3.4. Reduction of Herqueinones (1–6)
To a solution of 44.3 mg (114 µM) of 1 in 0.5 mL of glacial acetic acid was added 100.0 mg
(1.53 mM) of zinc dust under nitrogen atmosphere. The mixture was stirred at room temperature for
30 min and filtered through cotton with 1.0 mL of distilled water. The filtrate was left to stand for
45 min and extracted with 1.5 mL of ethyl acetate. Purification by analytical HPLC (YMC-ODS-A
column, 4.6
×
250 mm; H2O-MeCN (50:50); 0.7 mL/min) afforded the 4-deoxy derivative (1a, 6.8 mg)
(tR = 15.8 min) as a pure compound. Compounds 2–6 were reduced in a similar manner.
25
4-Deoxy-ent-peniciherqueinone (1a): [
α
]
−
23 (c 0.5, CHCl3); 1H NMR (CDCl3, 400 MHz) δH 13.14
D
(1H, s), 13.10 (1H, s), 13.07 (1H, s), 4.57 (1H, q, J = 6.5 Hz), 3.99 (3H, s), 2.71 (3H, s), 1.50 (3H, s), 1.44
(3H, d, J = 6.5 Hz), 1.25 (3H, s); ESIMS m/z 373.1 [M + H]+ (calcd for C20H21O7, 373.1).
25
4-Deoxy-12-hydroxynorherqueinone (2a): [
α
]
−
20 (c 0.3, CHCl3); 1H NMR (DMSO-d6, 400 MHz) δH
D
14.32 (1H, s), 13.52 (1H, s), 9.28 (1H, s), 8.72 (1H, s), 4.64 (1H, q, J = 6.5 Hz), 2.66 (3H, s), 1.49 (3H, s),
1.38 (3H, d, J = 6.5 Hz), 1.26 (3H, s); ESIMS m/z 359.1 [M + H]+ (calcd for C19H19O7, 359.1).
25
4-Deoxy-ent-isoherqueinone (3a): [
α
]
+34 (c 0.5, CHCl3); 1H NMR (DMSO-d6, 400 MHz) δH 13.52
D
(1H, s), 8.13 (1H, s), 7.48 (1H, br s), 7.14 (1H, br s), 4.69 (1H, q, J = 6.5 Hz), 3.13 (3H, s), 2.66 (3H, s), 1.47
(3H, s), 1.42 (3H, d, J = 6.5 Hz), 1.22 (3H, s); ESIMS m/z 357.3 [M + H]+ (calcd for C20H21O6, 357.3).
25
D
1
4-Deoxy-oxopropylisoherqueinone A (4a): [
α
]
+8 (c 0.5, CHCl3), +10 (c 0.5, MeOH); H NMR
(DMSO-d6, 400 MHz) δH 13.27 (1H, s), 8.48 (1H, s), 6.18 (1H, s), 5.73 (1H, s), 4.13 (1H, q, J = 6.5 Hz),
3.16 (2H, s), 2.80 (3H, s), 2.05 (3H, s), 1.35 (3H, s), 1.25 (3H, d, J = 6.5 Hz), 1.05 (3H, s); ESIMS m/z 399.1
[M + H]+ (calcd for C22H23O7, 399.1).
25
D
1
4-Deoxy-oxopropylisoherqueinone B (5a): [
α
]
+3 (c 0.5, CHCl3), +5 (c 0.5, MeOH); H NMR
(DMSO-d6, 400 MHz) δH 13.27 (1H, s), 8.47 (1H, s), 6.18 (1H, s), 5.71 (1H, s) 4.10 (1H, q, J = 6.5 Hz), 3.16
(2H, s), 2.80 (3H, s), 2.05 (3H, s), 1.34 (3H, s), 1.26 (3H, d, J = 6.5 Hz), 1.05 (3H, s); ESIMS m/z 399.1
[M + H]+ (calcd for C22H23O7, 399.1).
25
Sclerodin (6a
=
10): [α
]
−
18 (c 0.5, CHCl3); 1H NMR (CDCl3, 400 MHz) δH 11.5 (1H, s), 6.75 (1H, s),
D
5.06 (1H, q, J = 6.5 Hz), 2.69 (3H, s), 1.48 (3H, d, J = 6.5 Hz), 1.41 (3H, s), 0.92 (3H, s); ESIMS m/z 329.1
[M + H]+ (calcd for C18H17O6, 329.1).
3.5. Acetylation of 4-Deoxy-ent-peniciherqueinone (1a)
To a solution of 3.0 mg (2.7 mM) of 1a in 3.0 mL of pyridine was added 0.4 mL of Ac2O. After
stirring the mixture for 4 h at room temperature, the pyridine and excess Ac2O were removed under
vacuum. Purification by analytical HPLC (YMC-ODS column, 4.6
(40:60)) yielded 4-deoxy-9,11,12-triacetyl-ent-peniciherqueinone (1b) (tR = 35.8 min): [
×
250 mm; 0.7 mL/min; H2O-MeCN
25
α
]
−
35 (c 0.5,
D

Mar. Drugs 2019, 17, 176
12 of 16
CHCl3),
−
42 (c 0.5, MeOH); 1H NMR (CDCl3, 400 MHz) δH 4.72 (1H, q, J = 6.5 Hz), 4.05 (3H, s), 2.71
(3H, s), 2.404 (3H, s), 2.401 (3H, s), 2.39 (3H, s), 1.59 (3H, s), 1.50 (3H, d, J = 6.5 Hz), 1.35 (3H, s); ESIMS
m/z 499.5 [M + H]+ (calcd for C26H27O10, 499.5).
3.6. Dehydration of Herqueinones (4a, 5a, and 7)
To a solution of 0.5 mg (44 mM) of Na in 500
µL of anhydrous ethanol was added 1.5 mg (7.5 mM)
of 4a under nitrogen atmosphere. After stirring the mixture for 6 h at room temperature, the solvent
was removed under vacuum. Purification by analytical HPLC (YMC-ODS column, 4.6
0.7 mL/min; H2O-MeCN (40:60)) afforded the 8(6 )-dehydroxy derivative (4b) (tR = 12.2 min) as a pure
compound. Compounds 5a and 7 were dehydrated to 5b and 7a, respectively, in the same manner.
×
250 mm;
0
25
4-Deoxy-8(60)-dehydroxyoxopropylisoherqueinone A (4b): [
α
]
−
27 (c 0.5, CHCl3); 1H NMR (CDCl3,
D
400 MHz) δH 13.31 (1H, s), 12.79 (1H, s), 6.33 (1H, s), 5.61 (1H, s) 4.42 (1H, q, J = 6.5 Hz), 2.56 (3H, s),
2.38 (3H, s), 1.46 (3H, s), 1.38 (3H, d, J = 6.5 Hz), 1.20 (3H, s); ESIMS m/z 381.1 [M + H]+ (calcd for
C22H21O6, 381.1).
25
4-Deoxy-8(60)-dehydroxyoxopropylisoherqueinone B (5b): [
α
]
−
26 (c 0.5, CHCl3); 1H NMR (CDCl3,
D
400 MHz) δH 13.31 (1H, s), 12.79 (1H, s), 6.33 (1H, s), 5.61 (1H, s) 4.42 (1H, q, J = 6.5 Hz), 2.56 (3H, s),
2.38 (3H, s), 1.46 (3H, s), 1.38 (3H, d, J = 6.5 Hz), 1.20 (3H, s); ESIMS m/z 381.1 [M + H]+ (calcd for
C22H21O6, 381.1).
25
8(60)-Dehydroxy derivative of
7
(
7a): [α
]
−
26 (c 0.5, CHCl3); 1H NMR (CDCl3, 400 MHz) δH 13.31
D
(1H, s), 12.79 (1H, s), 6.33 (1H, s), 5.61 (1H, s) 4.42 (1H, q, J = 6.5 Hz), 2.56 (3H, s), 2.38 (3H, s), 1.46 (3H,
s), 1.38 (3H, d, J = 6.5 Hz), 1.20 (3H, s); ESIMS m/z 381.1 [M + H]+ (calcd for C22H21O6, 381.1).
3.7. ECD Calcualtions
The conformational searches for the C-8 position of 4 and 5 were performed using Macromodel
(Version 9.9, Schrodinger LLC.) software with “Mixed torsional/Low Mode sampling” in the GAFF
force field. The experiments were conducted in the gas phase with a 50 kJ/mol energy window limit
and a maximum of 10,000 steps to thoroughly examine all low-energy conformers. The Polak–Ribiere
conjugate gradient (PRCG) method was utilized for minimization processes with 10,000 maximum
iterations and a 0.001 kJ (mol Å)−1 convergence threshold on the RMS gradient. Conformers within
10 kJ/mol of each global minimum for R and S form of
4 and 5 were used for gauge-independent
atomic orbital (GIAO) shielding constant calculations without geometry optimization employing
TmoleX Version 4.2.1 (COSMOlogic GmbH & Co. KG, Leverkusen, Germany) at the B3LYP/6-31G(d,p)
level in the gas phase. The ECD spectra were simulated by overlapping each transition, where
σ is
the width of the band at 1/e height. Ei and Ri are the excitation energies and rotatory strengths,
∆
respectively, for transition i. In the current work, the value was 0.10 eV.
A
2
2
1
1
∆EiRie[−(E−∆E ) /(2σ) ]
i
∆ ∈ (E) =
√
∑
2.297 × 10−39 2πσ
i
3.8. Cytotoxicity and Antibacterial Assays
The cytotoxicity assay was performed in accordance with the published protocols [24]. The
antimicrobial assay was performed according to the method described previously [25].
3.8.1. iNOS Assay
Mouse macrophage RAW 264.7 cells obtained from the American Type Culture Collection (ATCC,
Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) with antibiotics-antimycotics (PSF; 100 units/mL

Mar. Drugs 2019, 17, 176
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penicillin G sodium, 100 ng/mL streptomycin, and 250 ng/mL amphotericin B) [26
,
27]. The cells
were seeded in 24-well plates (2
FBS-DMEM, and the samples were treated with the test compounds. After pretreatment with the drug
for 1 h, 1 g/mL lipopolysaccharides (LPS) was added to stimulate NO production. The cells were
×
105 cells/mL). The next day, the culture media was changed to 1%
µ
incubated for an additional 18 h, and the amount of NO produced in the supernatant was determined
by Griess reaction. Then, the absorbance was measured at 540 nm, and the nitrite concentration was
determined by comparison with a sodium nitrite standard curve. The percent inhibition was calculated
using the following formula: [1
100. The IC50 values were calculated through nonlinear regression analysis using TableCurve 2-D
v5.01 (Systat Software Inc., San Jose, CA, USA). At the same time, MTT assays were also performed
to test cell viability. MTT solution (final concentration of 500 g/mL) was added to the cells, and
−
(NO level of test samples/NO levels of vehicle-treated control)]
×
µ
they were incubated for 4 h at 37 ◦C. The culture media was removed, and the remaining dyes were
dissolved in DMSO. The absorbance of each well was measured at 570 nm using a VersaMax ELISA
microplate reader (Molecular Devices, Sunnyvale, CA, USA). The percent survival was determined by
comparison with a control group (LPS+).
3.8.2. Tube Formation Assay
Human umbilical vascular endothelial cells (HUVECs) were purchased from the American
Type Culture Collection (ATCC, Rockville, MD, USA), and cultured in EGM-2 (Lonza, Walkerswille,
MD, USA) supplemented with 10% FBS and antibiotics-antimycotics (PSF) [28,29]. The cells were
maintained at 37 ◦C under a humidified atmosphere containing 5% CO2. A 96-well plate was coated
◦
with Matrigel (Corning) for 30 min at 37 C under a humidified atmosphere containing 5% CO2.
HUVECs (1.8
×
104 cells/well) were mixed with the test compounds in 0.5% FBS EBM-2 medium
with VEGF (50 ng/mL) or 0.5% FBS EBM-2 medium only for the VEGF negative control. The cells
were incubated for 6 h and photographed using an inverted microscope (Olympus Optical Co. Ltd.,
Tokyo, Japan). Images were quantified with an angiogenesis analyzer using ImageJ software. Tube
formation activity was calculated using the following formula: [(Total segment # (tested compound)
−
Total segment # (VEGF )]/[Total segment # (VEGF+) Total segment # (VEGF )] 100 (# stands for
−
−
− ×
tubule segment number). The IC50 values were calculated through nonlinear regression analysis using
TableCurve 2-D v5.01 (Systat Software Inc., San Jose, CA, USA). Cell viabilities were evaluated with
the MTT assay. HUVECs (0.8
×
104 cells/well) were seeded into a 96-well plate and indicated for 1 day.
The culture medium was replaced with serum-free medium, and the cells were incubated overnight.
After starvation, the cells were treated with the test compounds and VEGF (50 ng/mL) in 2% FBS
EBM-2 medium. Cells were incubated for a further 24 h, and MTT solution (final concentration of
500 µg/mL) was added to the cells to measure the cell viability. The formazan products were dissolved
in DMSO. The absorbance of each well was measured at 570 nm using a VersaMax ELISA microplate
reader (Molecular Devices, Sunnyvale, CA, USA).
3.8.3. Adiponectin Production Assay
Human bone marrow-mesenchymal stem cells (hBM-MSCs) were purchased from Lonza,
Inc. (Walkersville, MD, USA) and cultured in low-glucose (1 g/L) DMEM supplemented with
10% FBS, penicillin-streptomycin, and GlutamaxTM (Invitrogen, Carlsbad, CA, USA). To induce
adipogenesis, the cell growth medium was replaced with high-glucose (4.5 g/L) DMEM supplemented
with 10% FBS, penicillin-streptomycin, 10
µg/mL insulin, 0.5 µM dexamethasone, and 0.5 mM
3-isobutyl-1-methylxanthine (IBMX) (IDX conditions) [30]. IBMX, pioglitazone, and aspirin were
purchased from Sigma-Aldrich (St. Louis, MO, USA). hBM-MSCs were stained with 0.2% oil red O
◦
(ORO) reagent for 10 min at 24 C, and then washed with H2O four times. Following a 10-min elution
with isopropanol, the absorbance was measured at 500 nm using a spectrophotometer. To visualize the
nucleus, the hBM-MSCs were counterstained with hematoxylin reagent for 2 min and then washed
twice with H2O. The level of adipocyte differentiation was observed and counted using an inverted

Mar. Drugs 2019, 17, 176
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phase microscope. A Quantikine immunoassay kit (R&D Systems, Minneapolis, MN, USA) was used
for quantitative determination of adiponectin in the cell culture supernatants.
3.8.4. Receptor Binding Assay
The time-resolved fluorescence resonance energy transfer (TR-FRET)-based nuclear receptor
binding assay to evaluate binding of the ligand to PPARγ
was performed using LanthanscreenTM
competitive binding assay kits (Invitrogen) [30]. All assay measurements were performed using a
CLARIOstar instrument (BMG LABTECH, Ortenberg, Germany) with the settings described in the
TR-FRET manufacturer’s instructions.
4. Conclusions
Six new phenalenone derivatives (
were isolated from a marine-derived fungus Penicillium sp. The structure elucidation of compounds
were established by combined spectroscopic methods. The absolute configurations were determined by
chemical modifications and their specific rotations. 4-Hydroxysclerodin ( ) and an acetone adduct of a
triketone ( ) exhibited moderate anti-angiogenetic and anti-inflammatory activities, respectively, while
1–6) and five known compounds (7–11) of the herqueinone class
1–6
6
7
ent-peniciherqueinone (1) and isoherqueinone (9) exhibited moderate abilities to induce adipogenesis
without cytotoxicity.
5. Patents
Shin, J.; Oh, K.-B. Phenalenone derivatives and antimicrobial composition. KR 2014112273 A
20140923, 2014.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/3/176/s1.
1
Figures S1–S41: annotated 1D NMR and selected 2D NMR spectra of
1
–
6
, Figures S42–S47: H NMR spectra of
1
1a
–
6a, Figures S48–S50: H NMR spectra of 4b
,
5b, and 7a, Figures S51 and S52: The CD and ECD spectrum of
, Figure S54: iNOS assay and MTT assay, Figure S55: Tube
4
–
5
, Figure S53: The time-scale LC-MS analysis of
7
formation assay and MTT assay, Figure S56: adiponectin production assay and PPAR
γ binding assay, Table S1:
The results of bioactivity tests.
Author Contributions: S.C.P. and E.J. carried out the isolation and structural elucidation; K.-B.O. performed the
antimicrobial bioassays; S.A. and M.N. performed the adiponectin production and receptor binding assays; D.K.
and S.K.L. performed iNOS and tube formation assays; J.S. and D.-C.O. reviewed and evaluated all data; J.S. and
K.-B.O. supervised the research work and prepared the paper.
Funding: This study was supported by the National Research Foundation (NRF, grant No. 2018R1A4A1021703)
funded by the Ministry of Science, ICT, & Future Planning, Korea.
Acknowledgments: We thank the Basic Science Research Institute in Daegu, Korea, for providing mass
spectrometric data.
Conflicts of Interest: The authors declare no conflict of interest.
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