Antiviral 4-Hydroxypleurogrisein and Antimicrobial Pleurotin Derivatives from Cultures of the Nematophagous Basidiomycete Hohenbuehelia grisea

Abstract

4-Hydroxypleurogrisein, a congener of the anticancer-lead compound pleurotin, as well as six further derivatives were isolated from the basidiomycete Hohenbuehelia grisea, strain MFLUCC 12-0451. The structures were elucidated utilizing high resolution electron spray ionization mass spectrometry (HRESIMS) and 1D and 2D nuclear magnetic resonance (NMR) spectral data and evaluated for their biological activities; for leucopleurotin, we provide Xray data. While most congeners showed moderate antimicrobial and cytotoxic activity, 4-hydroxypleurogrisein emerged as an inhibitor of hepatitis C virus infectivity in mammalian liver cells.

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Article
Antiviral 4-Hydroxypleurogrisein and Antimicrobial
Pleurotin Derivatives from Cultures of the
Nematophagous Basidiomycete Hohenbuehelia grisea
Birthe Sandargo 1,2, Benjarong Thongbai 1,2, Dimas Praditya 3,4 , Eike Steinmann 3,5,
Marc Stadler 1, 2, * and Frank Surup 1, 2, *
1Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH, Inhoffenstraße 7,
38124 Braunschweig, Germany; birthe.sandargo@helmholtz-hzi.de (B.S.);
benjarong.thongbai@helmholtz-hzi.de (B.T.)
2German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig,
38124 Braunschweig, Germany
3TWINCORE-Centre for Experimental and Clinical Infection Research (Institute of Experimental Virology)
Hannover. Feodor-Lynen-Str. 7-9, 30625 Hannover, Germany; dimas.praditya@twincore.de (D.P.);
eike.steinmann@rub.de (E.S.)
4Research Center for Biotechnology, Indonesian Institute of Science, Jl. Raya Bogor KM 46,
Cibinong 16911, Indonesia
5Department of Molecular and Medical Virology, Ruhr-University Bochum, 44801 Bochum, Germany
*Correspondence: marc.stadler@helmholtz-hzi.de (M.S.); frank.surup@helmholtz-hzi.de (F.S.);
Tel.: +49 531-6181-4240 (M.S.); +49-531-6181-4256 (F.S.)
Received: 19 September 2018; Accepted: 17 October 2018; Published: 19 October 2018


Abstract:
4-Hydroxypleurogrisein, a congener of the anticancer-lead compound pleurotin, as well as
six further derivatives were isolated from the basidiomycete Hohenbuehelia grisea, strain MFLUCC
12-0451. The structures were elucidated utilizing high resolution electron spray ionization mass
spectrometry (HRESIMS) and 1D and 2D nuclear magnetic resonance (NMR) spectral data and
evaluated for their biological activities; for leucopleurotin, we provide Xray data. While most
congeners showed moderate antimicrobial and cytotoxic activity, 4-hydroxypleurogrisein emerged as
an inhibitor of hepatitis C virus infectivity in mammalian liver cells.
Keywords:
Basidiomycota; fungi; HCV; Hohenbuehelia grisea; Pleurotin; secondary metabolites;
structure elucidation
1. Introduction
Fungi are known as talented producers of secondary metabolites [
1
]. With previously mainly
ascomycetes studied for their potential to produce antibiotic agents, in recent years, basidiomycetes
have become the center of attention in the search for new bioactive secondary metabolites [
2
] and
the last group of antibiotics entering the market were the basidiomycete-derived pleuromutilins [
3
].
Another promising basidiomycete metabolite is the anti-cancer lead compound pleurotin (
1
), first
isolated in 1947 from “Pleurotus griseus Peck” [
4
], which is now classified as Hohenbuehelia grisea (Peck)
Singer. Pleurotin (
1
; Figure 1) and its derivatives leucopleurotin (
2
) and dihydropleurotinic acid (
3
)
were shown to have activity against Gram-positive bacteria [
4
,
5
] and pathogenic fungi [
6
], as well as
to exhibit anticancerogenic effects [
5
,
7
]. Moreover, the total synthesis of (
±
)-pleurotin (
1
) [
8
] and its
production in multi-gram scale by fermentation [
9
] have already been accomplished. In recent years,
pleurotin has become the center of attention as a potential new anti-cancer lead drug for its highly
effective inhibition of the thioredoxin (Trx)–thioredoxin reductase (TrxR) system [
10
], a favorable target
in the treatment of cancer as well as mercury intoxication [11].
Molecules 2018,23, 2697; doi:10.3390/molecules23102697 www.mdpi.com/journal/molecules

Molecules 2018,23, 2697 2 of 11
In a concurrent study, searching for promising new pleurotin derivatives from submerged cultures
of H. grisea strain MFLUCC 12-0451, three cysteine-derived congeners of pleurotin, thiopleurotinic
acids A and B, and pleurothiazole have been found, indicating a potential glutathione detoxification
of basidiomycetes [
12
]. This discovery prompted us to investigate extracts of the strain more closely.
Scale up of production and extensive, challenging chromatography procedures of the resulting crude
extracts led to the isolation of seven pleurotin derivatives (Figure 2), of which three (
5
,
9
, and
11
)
are entirely unprecedented. The other four metabolites (
6
–
8
and
10
) had been mentioned in the
past as potential biosynthesis precursors of pleurotin during the course of pioneer studies on the
biosynthesis of pleurotin in the group of Arigoni (ETH Zurich), summarized in Capaul (1992) [
13
].
However, the corresponding PhD theses contain no spectral data of these compounds, and they may
vary in stereochemistry. The present paper is dedicated to the description of their isolation, structure
elucidation and biological characterization.
Molecules 2018, 23, x FOR PEER REVIEW 2 of 12
for its highly effective inhibition of the thioredoxin (Trx)–thioredoxin reductase (TrxR) system [10], a
favorable target in the treatment of cancer as well as mercury intoxication [11].
In a concurrent study, searching for promising new pleurotin derivatives from submerged
cultures of H. grisea strain MFLUCC 12-0451, three cysteine-derived congeners of pleurotin,
thiopleurotinic acids A and B, and pleurothiazole have been found, indicating a potential
glutathione detoxification of basidiomycetes [12]. This discovery prompted us to investigate extracts
of the strain more closely. Scale up of production and extensive, challenging chromatography
procedures of the resulting crude extracts led to the isolation of seven pleurotin derivatives (Figure
2), of which three (5, 9, and 11) are entirely unprecedented. The other four metabolites (6–8 and 10)
had been mentioned in the past as potential biosynthesis precursors of pleurotin during the course
of pioneer studies on the biosynthesis of pleurotin in the group of Arigoni (ETH Zurich),
summarized in Capaul (1992) [13]. However, the corresponding PhD theses contain no spectral data
of these compounds, and they may vary in stereochemistry. The present paper is dedicated to the
description of their isolation, structure elucidation and biological characterization.
OH
O
OH
H
OH
OHO
O
OOH
O
O
O
OH
OH H
O
O
O
O
OH
13
24
Figure 1. Chemical structures of previously isolated pleurotin derivatives: pleurotin (1),
leucopleurotin (2), dihydropleurotinic acid (3) [5], and pleurogrisein (4) [13].
Figure 1.
Chemical structures of previously isolated pleurotin derivatives: pleurotin (
1
), leucopleurotin
(2), dihydropleurotinic acid (3) [5], and pleurogrisein (4) [13].
Molecules 2018, 23, x FOR PEER REVIEW 3 of 12
O
O
O
OH
OH H
HO
O
OHO
OH
OH
OH
H
H
OH
O
O
OH
OH
H
H
OH
OH
OH
O
O
H
H
5: R1 = OH R2 = H
6: R1 = H R2 = OH
7: R1 = H
8: R1 = =O
910 11
R1
R2R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20 21
11
12
4
3
H
OH
O
O
O
O
H
Figure 2. Chemical structures of newly isolated compounds 5–11.
2. Results and Discussion
As reported earlier, the producing strain MFLUCC 12-0451 was identified as Hohenbuehelia
grisea (Peck) Singer [12]. H. grisea, more commonly known under its former name of the asexual
morph Nematoctonus robustus, has been reported to produce pleurotin (1), dihydropleurotinic acid
(2), and leucopleurotin (3) [5]. The group of Arigoni (ETH Zurich) has in the past extensively studied
the biosynthesis of pleurotin and in the course of their research proposed several structures [13],
which are also being reported with full NMR spectral data in this study for the first time.
3-Hydroxy-dihydropleurotinic acid (5) was isolated as an off-white to pale yellow powder with
a molecular formula of C21H24O6, retrieved from a molecular ion cluster [M + H]+ at m/z 373.1661,
indicating ten degrees of saturation. The correlation of 1H and 13C-NMR data (Table 1) in
combination with HMBC correlations (Figure 3) led to the establishment of dihydropleurotinic acid
as the underlying core structure [5,12], with the novelty of compound 5 being a hydroxyl
functionality at C-3 (δC 82.8), which leads to the absolute configuration of 3S,4S,5S,8R,9S,10R,15S.
9
11
13
14
15
OH
OH
O
O
O
O
7
OH
O
O
OH
OH
8
4
Figure 3. Selected 1H, 13C HMBC correlations of 3-Hydroxy-dihydropleurotinic acid (5) and
4-Hydroxy-pleurogrisein (11).
Figure 2. Chemical structures of newly isolated compounds 5–11.

Molecules 2018,23, 2697 3 of 11
2. Results and Discussion
As reported earlier, the producing strain MFLUCC 12-0451 was identified as Hohenbuehelia
grisea (Peck) Singer [
12
]. H. grisea, more commonly known under its former name of the asexual
morph Nematoctonus robustus, has been reported to produce pleurotin (
1
), dihydropleurotinic acid (
2
),
and leucopleurotin (
3
) [
5
]. The group of Arigoni (ETH Zurich) has in the past extensively studied the
biosynthesis of pleurotin and in the course of their research proposed several structures [
13
], which are
also being reported with full NMR spectral data in this study for the first time.
3-Hydroxy-dihydropleurotinic acid (
5
) was isolated as an off-white to pale yellow powder with
a molecular formula of C
21
H
24
O
6
, retrieved from a molecular ion cluster [M + H]
+
at m/z373.1661,
indicating ten degrees of saturation. The correlation of
1
H and
13
C-NMR data (Table 1) in combination
with HMBC correlations (Figure 3) led to the establishment of dihydropleurotinic acid as the underlying
core structure [
5
,
12
], with the novelty of compound
5
being a hydroxyl functionality at C-3 (
δC
82.8),
which leads to the absolute configuration of 3S,4S,5S,8R,9S,10R,15S.
Highly similar to
5
is compound
6
, 14-hydroxy-dihydropleurotinic acid, obtained as a bright
yellow solid with a molecular formula of C
21
H
24
O
6
as well. The
1
H and
13
C-NMR data resemble
largely those of compound
5
, with the major difference being two methines at
δC/H
63.9/4.55 and
32.4/2.2.
1
H,
1
H COSY and
1
H,
13
C-HMBC data confirmed the placement of these at positions C-14 and
C-3, respectively. The chemical shift of C/H-14 also indicated the presence of a hydroxyl group (
δH
4.16) and ROESY correlations of H-14 to H-9, as well as H-8 to H-15, and the absence of correlations
between H-9 and H-15/H-8 indicate an S-configuration at C-14, and the absolute configuration is in
line with the stereochemistry of leucopleurotin. A crystal structure with the absolute configuration of
leucopleurotin has been attached (Figure S2), and is deposited at the Cambridge Crystallographic Data
Center, CCDC 1872450. Compound 6resembles likely the cleaved ester form of pleurotin (1).
The molecular formula C
21
H
26
O
5
of leucopleurotinic acid (
7
), a white amorphous powder,
was retrieved from HRESIMS, entailing nine degrees of saturation. Again, NMR data largely resembled
those of dihydropleurotinic acid (
3
) with a major difference being upfield shifts in the
13
C-NMR
data of the olefinic carbons, suggesting a reduction of the quinone moiety to its corresponding
hydroquinone form.
Closely related to
7
is metabolite
8
, 14-oxo-leucopleurotininc acid, acquired as an off-white
amorphous powder and a molecular formula of C
21
H
24
O
6
. NMR spectral data coincide to great extent
with compound 7, yet indicate a keto group at position C-14 (δC202.7).
A light brown amorphous powder with a molecular formula of C
21
H
22
O
5
and eleven degrees of
saturation is nematoctone (
9
). Its
1
H and
13
C-NMR data (Table 2) overlap in many parts with
8
, yet an
1
H,
13
C-HMBC correlation of C-13 to H-14 (
δH
5.64) portend an ester bond between C-13 and C-14 (
δC
74.9), generating leucopleurotin as the underlying core structure. However, the usual chemical shifts of
C-4 and C-12 were not observed and instead carbon signals at
δC
110 (C-12) and 147.7 (C-4) showed up.
1
H,
13
C-HMBC correlations (see Supplementary Materials) of C/H-12 to C/H-11 and C/H-3, as well
as H-2, H-3, H-5, and H-11 to C-3 confirmed the position of a double bond between C-4 and C-12.
The interproton distance measurements confirms an absolute configuration of 3S,5S,8R,9S,10R,14S,15S
in line with leucopleurotin (Figure S2).
Di-oxo-leucopleurotinic acid (
10
) was isolated as a light brown powder. Its molecular formula
was determined by HRMS as C
21
H
24
O
7
and ten degrees of saturation. Compared to
8
, the molecular
formula includes an additional oxygen atom. A closer look at the
13
C spectra showed the appearance
of a second keto group at
δC
205.3 (C-15) in proximity to H-1 and H-9 (
1
H,
13
C-HMBC; Supplementary
Materials) and an upfield shift of C-11 to
δC
67.4. This suggested that the seven-membered cyclic ether
was cleaved, as compared to 9.

Molecules 2018,23, 2697 4 of 11
Table 1. 1
H- and
13
C-NMR spectroscopic data (
1
H 500 MHz,
13
C 125 MHz, CDCl
3
) for
5
; (
1
H 700 MHz,
13
C 176 MHz, CDCl
3
) for
8
, and (
1
H 500 MHz,
13
C 125 MHz,
acetone-d6) for 6, (1H 700 MHz, 13C 176 MHz, acetone-d6) for 7.
Pos 5 6 7 8
δC, Type δH(Jin Hz) δC, Type δH(Jin Hz) δC, Type δH(Jin Hz) δC, Type δH(Jin Hz)
131.9, CH21.50, m 36.9, CH22.27, ddd (12.2, 9, 2.6) 34.4, CH21.35, m 33.6, CH21.29, m
1.93, m 1.90, m 1.94, m
234.8, CH21.53, m 25.9, CH21.69, m 26.4, CH21.77, m 25.0, CH21.78, m
2.30, m 1.75, m 1.90, m 1.93, m
3 82.8, C 46.0, CH 2.08, m 46.3, CH 2.31, m 43.9, CH 2.34, m
4 39.1, CH 2.14, m 32.4, CH 2.20, m 34.8, CH 2.14, m 33.7, CH 2.11, m
5 59.3, CH 1.94, m 52.6, CH 1.65, m 53.3, CH 1.86, m 50.6, CH 1.81, m
619.3, CH21.78, m 22.6, CH21.78, m 1.86, 23.2, CH21.85, m 21.7, CH21.87, m
1.94, m 1.86, td (12.9, 3.9) 2.06, m 1.98, m
730.2, CH21.63, m 31.2, CH21.58, m 32.3, CH21.67, qd (12.8, 4.7) 29.5, CH21.87, m
2.20, m 2.10, m 2.15, m 2.23, m
8 42.9, CH 2.13, m 43.3, CH 1.94, td (12.2, 4.0) 44.9, CH 2.35, m 42.4, CH 2.62, m
9 43.5, CH 2.05, dd (12, 3.2) 51.4, CH 2.06, m 46.2, CH 1.99, dd (12, 5.8) 58.9, CH 2.63, m
10 44.5, C 46.1, C 48.0, C 49.8, C
11 75.9, CH23.69, dd (13.2, 3.6) 75.3, CH23.34, dd (12.3, 6.9) 77.6, CH23.91, dd (12.8, 8.0) 77.6, CH24.02, br d (12.8)
4.02, dd (13.2, 8.3) 3.96, dd (12.3, 8.6) 4.18, dd (12.8, 2.0) 4.12, dd (12.8, 8.0)
12 16.6, CH31.09, d (6.9) 21.3, CH30.93, d (7.0) 21.8, CH31.07, d (7.3) 20.7, CH31.10, d (7.3)
13 179.6, C 176.8, C 177.5, C 176.8, C
14 24.2, CH22.51, m 63.9, CH 4.55, br s 26.5, CH22.66, dd (17.5, 6.2) 202.7, C
2.74, br d (17.5)
15 74.2, CH 4.43, s 73.2, CH 4.46, s 82.9, CH 5.0, s 82.2, CH 5.21, s
16 140.7, C 141.8, C 123.8, C 113.6, C
17 139.8, C 139.9, C 121.6, C 121.6, C
18 186.3, C 187.6, C 152.8, C 149.7, C
19 137.7, CH 6.70 *, d (10.2) 138.6, CH 6.79, d (10.2) 114.9, CH 6.43, d (8.5) 128.0, CH 7.09, d (9.0)
20 135.6, CH 6.70 *, d (10.2) 136.8, CH 6.78, d (10.2) 115.8, CH 6.62, d (8.5) 119.0, CH 6.88, d (9.0)
21 186.8, C 187.8, C 148.8, C 157.4, C
14-OH
4.16, br s
18-OH
8.17, s 8.71
* Overlapping signal, C-19/20 may therefore be interchange.

Molecules 2018,23, 2697 5 of 11
Table 2. 1
H- and
13
C-NMR spectroscopic data (
1
H 700 MHz,
13
C 176 MHz, CDCl
3
) for
9
–
10
, and (
1
H
500 MHz, 13C 125 MHz, acetone-d6) for 11.
Pos 9 10 11
δC, Type δH(Jin Hz) δC, Type δH(Jin Hz) δC, Type δH(Jin Hz)
132.0, CH22.10, m 36.9, CH21.89, m 39.4, CH21.11, m
1.33, m 1.59, m 1.54, dd (11.6, 7.1)
230.0, CH21.9, m 28.5, CH21.41, m 27.22, CH21.73, m
2.12, m 1.95, m 1.83, m
3 47.3, CH 3.04, m 41.6, CH 2.11, m 50.9, CH 2.31, m
4 147.7, C 37.0, CH 2.38, m 72.8, C
5 52.5, CH 1.69, m 50.3, CH 1.90, m 54.55, CH 1.94, m
622.9, CH21.9, m 23.3, CH22.11, m 23.2, CH21.72, m
1.56, m 1.90, m 2.05, m
724.0, CH21.47, dd (12.3, 3.9)
2.27, m 30.3, CH22.14, m 39.4, CH21.23, m
1.74, m 2.29, m
8 39.3, CH 2.27, m 45.9, CH 2.54, td (12.1, 4.0) 74.8, C
9 50.4, CH 2.44, d (7.1) 60.5, CH 2.81, d (12.1) 51.3, CH 1.8, m
10 48.9, C 59.4, C 43.8, C
11 73.7, CH24.87, d (16.0) 67.4, CH23.64, dd (10.6, 3.3) 29.9, CH31.18, s
4.50, d (16.0) 3.46, dd (10.6, 6.1)
12 110.0, CH24.79, br s
4.88, br s 18.4, CH31.14, d (6.6) 31.5, CH31.26, s
13 174.3, C 174.9, C 63.9, CH23.12, dd (5, 1.7)
3.40, dd (10.6, 5.0)
14 74.9, CH 5.64, d (7.3) 202.3, C 22.3, CH22.29, m
3.03, m
15 80.0, CH 4.97, s 205.3, C 27.16 CH22.15, m
2.61, m
16 118.4, C 112.2, C 142.6, C
17 118.4, C 112.9, C 142.9, C
18 152.2, C 155.8, C 188.1, C
19 118.8, CH 6.87, d (8.8) 129.0, CH 7.24 *, d (0.6) 137.4, CH 6.73 *, s
20 119.5, CH 6.82, d (8.2) 128.5, CH 7.24 *, d (0.6) 137.2, CH 6.73 *, s
21 149.4, C 155.3, C 187.8, C
4-OH 3.14, br s
8-OH 3.48, br s
13-OH
2.84, br s
18-OH
8.61, s 11.88, s
21-OH
11.64, s
* Overlapping signal, C-19/20 may therefore be interchanged.
Lastly, metabolite
11
(4-hydroxypleurogrisein) was isolated as an amber-colored solid, possessing
the molecular formula of C
21
H
28
O
5
, calculated from the ion peak at m/z383.1825 [M + Na].
13
C-NMR
signals at
δC
187.8 (C-21) and 188.1 (C-18) disclosed the quinone being present and showing
1
H,
13
C-HMBC correlations to two methylenes H-14 (
δH
2.29, 3.03) and H-15 (
δH
2.15, 2.61), respectively.
A carbon at
δC
74.8 (C-8) was observed instead of a methine, indicating a hydroxy function attached to
C-8, and HMBC correlations of C-7 and C-9 to a methylene at
δC
63.9 (C-13) suggested the proximity
of another hydroxyl function. Another carbon at
δC
72.8 (C-4), carrying an OH, showed correlations

Molecules 2018,23, 2697 6 of 11
in the
1
H,
13
C-HMBC to two methyl groups at
δH
1.18 (H-11) and 1.26 (H-12), leading to a 4-ring
system as the underlying core structure, similar to
10
. This scaffold was confirmed by 1,1-ADEQUATE
NMR data (measured in DMSO-d
6
, see Supplementary Materials). The stereochemistry of C-8 was
assigned to be Rby ROESY correlations of 8-OH to H-9, as well as a strong correlation of H-13 to
H-15 (see Supplementary Materials), indicating an axial orientation of C/H
2
-13. The axial orientation
of C/H
2
-13 was further confirmed by a J-HMBC NMR experiment, since a large coupling constant
of 6.1 Hz was observed between H-7
ax
(
δH
1.23) and C-13 indicating an anti-periplanar orientation
between H-7
ax
and C-13 and thus confirming the 8Rconfiguration, giving the absolute configuration
of 4-hydroxypleurogrisein 3S,5S,8R,9R,10R.
Molecules 2018, 23, x FOR PEER REVIEW 3 of 12
O
O
O
OH
OH H
HO
O
OHO
OH
OH
OH
H
H
OH
O
O
OH
OH
H
H
OH
OH
OH
O
O
H
H
5: R1 = OH R2 = H
6: R1 = H R2 = OH
7: R1 = H
8: R1 = =O
910 11
R1
R2R1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20 21
11
12
4
3
H
OH
O
O
O
O
H
Figure 2. Chemical structures of newly isolated compounds 5–11.
2. Results and Discussion
As reported earlier, the producing strain MFLUCC 12-0451 was identified as Hohenbuehelia
grisea (Peck) Singer [12]. H. grisea, more commonly known under its former name of the asexual
morph Nematoctonus robustus, has been reported to produce pleurotin (1), dihydropleurotinic acid
(2), and leucopleurotin (3) [5]. The group of Arigoni (ETH Zurich) has in the past extensively studied
the biosynthesis of pleurotin and in the course of their research proposed several structures [13],
which are also being reported with full NMR spectral data in this study for the first time.
3-Hydroxy-dihydropleurotinic acid (5) was isolated as an off-white to pale yellow powder with
a molecular formula of C21H24O6, retrieved from a molecular ion cluster [M + H]+ at m/z 373.1661,
indicating ten degrees of saturation. The correlation of 1H and 13C-NMR data (Table 1) in
combination with HMBC correlations (Figure 3) led to the establishment of dihydropleurotinic acid
as the underlying core structure [5,12], with the novelty of compound 5 being a hydroxyl
functionality at C-3 (δC 82.8), which leads to the absolute configuration of 3S,4S,5S,8R,9S,10R,15S.
9
11
13
14
15
OH
OH
O
O
O
O
7
OH
O
O
OH
OH
8
4
Figure 3. Selected 1H, 13C HMBC correlations of 3-Hydroxy-dihydropleurotinic acid (5) and
4-Hydroxy-pleurogrisein (11).
Figure 3.
Selected
1
H,
13
C-HMBC correlations of 3-Hydroxy-dihydropleurotinic acid (
5
) and
4-Hydroxy-pleurogrisein (11).
Biological Activities
To evaluate the biological activity of the isolated derivatives, compounds
5
–
11
were subjected to
antimicrobial, cytotoxicity, and nematicidal activity assays. None of the isolated compounds, including
1
–
3
, displayed any signs of nematode toxicity in our assay, despite H. grisea being a nematode-trapping
fungus and injecting a substance, the color of pleurotin, into captured nematodes in a water agar test
(see Supplementary Materials, Figure S1). This is in accordance with previous results on pleurotin
and other congeners (
1
–
3
), which also did not affect C. elegans [
5
]. Metabolites
5
–
11
have also been
tested against a selection of microorganisms (Table 3). All, but
7
, showed signs of antimicrobial
activity against yeasts, such as Candida tenuis (
5
: 100
µ
g/mL;
9
: 25
µ
g/mL) and Rhodotorula glutinis
(
5: 33.3 µg/mL)
, or Gram-positive bacteria such as Bacillus subtilis (
5
and
10
: 100
µ
g/mL,
8
and
11: 50 µg/mL), Micrococcus luteus (11: 66.7 µg/mL), and Staphylococcus aureus (11: 33.3 µg/mL).
Assessing the cytotoxicity, only 4-hydroxypleurogrisein (
11
) displayed a comparable cytotoxicity
to pleurotin, with IC
50
values of 6.9
µ
g/mL for the murine fibroblast cell line L929 and IC
50
7.5
µ
g/mL
for the cervix carcinoma cell line KB3.1 (Table 3).
Next, the compounds were tested for their inhibitory effect against hepatitis C virus (HCV). HCV
infections is continuing to impose a global threat to human health with 71 million people infected
worldwide. Although various potent direct acting antiviral agents have been licensed, high costs
prevent the majority of infected individuals from having access to treatment. Out of the compounds
tested, only 4-hydroxypleurogrisein (
11
) showed significant activities
in vitro
, while compound
5
,
which was tested concurrently, was devoid of any activity on the host cells. As depicted in Figure 4,
HCV infectivity decreased in a dose-dependent manner with an IC
50
value of around 5 ng/
µ
L and
strong inhibitory effect at 10 ng/
µ
L, while at 20 ng/
µ
L cytotoxic effects were noted. The green tea
molecule epigallocatechin gallate (EGCG) was used as positive control [14].

Molecules 2018,23, 2697 7 of 11
Table 3.
Minimum inhibitory concentration (MIC) in the serial dilution assay for bacteria and fungi and
half-inhibitory concentrations (IC
50
for cell lines) in
µ
g/mL. For determination of MICs, 20
µ
L of either
1 mg/mL stock solution (67
µ
g/mL) or 1.5 mg/mL (100
µ
g/mL) of
1
–
3
,
5
–
11
were tested. Cell density
was adjusted to 6.7
×
10
5
cells/mL. Twenty microliters of Ethanol were used as negative control and
displayed no activity against the selected test organisms. For IC
50
values, 6
×
10
3
cells/well were sown
in 96-well microtiter plates and treated with 1–3,5–11 over five days.
Organism MIC (µg/mL) Reference (MIC)
1 * 2 * 3 * 5 6 7 8 9 10 11
Mucor plumbeus MUCL49355 100 - 100 - - - - - - - Nystatin (12.5)
Candida tenuis MUCL29892 25 100 100
100
- - - 25 - - Nystatin (12.5)
Bacillus subtilis DSM10 50 25 100
100
- - 50 -
100
50 Penicillin (6.3)
Pichia anomala DSM6766 66.7 66.7 66.7 - - - - - - - Nystatin (16.7)
Candida albicans DSM1665 33.3 - - - - - - - - - Nystatin (16.7)
Rhodotorula glutinis DSM10134
16.7 33.3 33.3
33.3
- - - - - - Nystatin (16.7)
Mucor hiemalis DSM2656 8.3 33.3 16.7 - - - - - - - Nystatin (16.7)
Micrococcus luteus DSM1790 66.7 66.7 - - - - - - -
66.7
Oxytetracycline (0.4)
Staphylococcus aureus DSM346 33.3 33.3 66.7 - - - - - -
33.3
Oxytetracycline (6.7)
Cell line IC50 (µg/mL) Reference (IC50)
L929 (IC50) 7.5 2.2 18 22 23 17 22 21 22
6.9
Epothilone B (0.00062)
KB3.1 (IC50) 8.5 2.8 18 22 22 20 22 18 22
7.5
Epothilone B (0.00003)
* Pleurotin (
1
), dihydropleurotinic acid (
2
), and leucopleurotin (
3
) used for comparison. - no inhibition observed
under test conditions.
Molecules 2018, 23, x FOR PEER REVIEW 8 of 12
compound 5, which was tested concurrently, was devoid of any activity on the host cells. As
depicted in Figure 4, HCV infectivity decreased in a dose-dependent manner with an IC
50
value of
around 5 ng/µL and strong inhibitory effect at 10 ng/µL, while at 20 ng/µL cytotoxic effects were
noted. The green tea molecule epigallocatechin gallate (EGCG) was used as positive control [14].
Figure 4. Antiviral activity of 4-hydroxypleurogrisin (11). NC-Negative control, EGCG-Positive
control. HCV assay was performed in triplicates and is presented as the mean ± standard deviation.
The asterisk indicates statistically significant differences (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Huh-7.5
cells were inoculated with RLuc-Jc1 reporter viruses in the presence of 4-hydroxypleurogrisein. The
inoculum was removed 4 h later and monolayers were washed three times with PBS and overlaid
with fresh medium containing no inhibitors. Infected cells were lysed three days later, and reporter
virus infection was determined by renilla luciferase activity (top). The cell viability was measured by
determination of firefly luciferase (bottom), which is stably expressed in the target cells.
3. Materials and Methods
3.1. General
1D and 2D-NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer (Bremen,
Germany) with a BBFO(plus) SmartProbe (
1
H 500 MHz,
13
C 126 MHz), and a Bruker Avance III 700
MHz spectrometer (Bremen, Germany) with a 5 mm TCI cryoprobe (
1
H 700 MHz,
13
C 175 MHz).
Chemical shifts  were referenced to the solvents: chloroform-d (
1
H, δ = 7.27 ppm;
13
C, δ = 77.0
ppm) and acetone-d
6
(
1
H, δ = 2.05 ppm; 13C, δ = 29.3 ppm). HRMS mass spectra were measured on
the Agilent 1200 series HPLC-UV system (Santa Clara, CA, USA) combined with ESI-TOF-MS
(Maxis, Bruker, Bremen, Germany), scan range 100−2500 m/z, capillary voltage 4500 V, temperature
200 °C, (column 2.1 × 50 mm, 1.7 µm, C18 Acquity UPLC BEH [Waters, MZ-Analysetechnik, Mainz,
Germany], solvent A: 95% 5 mM ammonium acetate buffer [pH 5.5, adjusted with 1 M acetic acid]
with 5% acetonitrile; solvent B: 95% acetonitrile with 5% 5 mM ammonium acetate buffer); gradient:
Figure 4.
Antiviral activity of 4-hydroxypleurogrisin (
11
). NC-Negative control, EGCG-Positive
control. HCV assay was performed in triplicates and is presented as the mean
±
standard deviation.
The asterisk indicates statistically significant differences (* p
≤
0.05, ** p
≤
0.01, *** p
≤
0.001). Huh-7.5

Molecules 2018,23, 2697 8 of 11
cells were inoculated with RLuc-Jc1 reporter viruses in the presence of 4-hydroxypleurogrisein.
The inoculum
was removed 4 h later and monolayers were washed three times with PBS and overlaid
with fresh medium containing no inhibitors. Infected cells were lysed three days later, and reporter
virus infection was determined by renilla luciferase activity (
top
). The cell viability was measured by
determination of firefly luciferase (bottom), which is stably expressed in the target cells.
3. Materials and Methods
3.1. General
1D and 2D-NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer (Bremen,
Germany) with a BBFO(plus) SmartProbe (
1
H 500 MHz,
13
C 126 MHz), and a Bruker Avance III
700 MHz
spectrometer (Bremen, Germany) with a 5 mm TCI cryoprobe (
1
H 700 MHz,
13
C 175 MHz).
Chemical shifts were referenced to the solvents: chloroform-d(
1
H,
δ
= 7.27 ppm;
13
C,
δ= 77.0 ppm
)
and acetone-d
6
(
1
H,
δ
= 2.05 ppm; 13C,
δ
= 29.3 ppm). HRMS mass spectra were measured on the
Agilent 1200 series HPLC-UV system (Santa Clara, CA, USA) combined with ESI-TOF-MS (Maxis,
Bruker, Bremen, Germany), scan range 100
−
2500 m/z, capillary voltage 4500 V, temperature 200
◦
C,
(column 2.1
×
50 mm, 1.7
µ
m, C18 Acquity UPLC BEH [Waters, MZ-Analysetechnik, Mainz, Germany],
solvent A: 95% 5 mM ammonium acetate buffer [pH 5.5, adjusted with 1 M acetic acid] with 5%
acetonitrile; solvent B: 95% acetonitrile with 5% 5 mM ammonium acetate buffer); gradient: 10%
solvent B increasing to 100% solvent B within 30 min, continuing at 100% B for further 10 min,
RF= 0.3 mL min−1
, UV detection 200–600 nm. UV spectra were recorded using a Shimadzu UV-vis
spectrophotometer UV-2450 (Shimadzu, Duisburg, Germany). Optical rotation was determined using
a PerkinElmer 241 polarimeter (PerkinElmer LAS, Rodgau Jürgesheim, Germany).
3.2. Fungal Material
Basidiomes of the nematode-trapping fungus of H. grisea were collected from decaying wood in
the tropical rainforest of Thailand near the Mushroom Research Centre, Chiang Mai Province, Thailand
(http://www.mushroomresearchcentre.com/), in August of 2012 and the corresponding culture was
obtained from basidiospores. The dried specimen and a corresponding culture are deposited at the
mycological herbarium of the Mae Fah Luang University Culture Collection, Chiang Rai, Thailand,
under the accession number MFLUCC 12-0451. Its 5.8S gene region, the internal transcribed spacer
1 and 2 (ITS) and part of the large subunit (LSU) were previously sequenced and published by
Sandargo et al.
[
12
] and the sequence data are deposited with GenBank, accession number MF150036.
3.3. Fermentation and Extraction
The strain H. grisea MFLUCC 12-0451 was cultivated in two different liquid media BAF (DSMZ
392), and Robbins medium [
4
] modified based on results of Shipley et al. [
9
] using alder extract
(100 g Alnus glutinosa dead wood and branches without leaves, collection site Helmholtz Centre for
Infection Research, campus Braunschweig, Germany, soaked for 24 h at room temperature in 1 L of
deionized water)]. A sufficiently grown culture of H. grisea MFLUCC 12-0451 on BAF (DSMZ 392)
agar was utilized to inoculate 200 mL of each medium in 500 mL Erlenmeyer flasks, incubated on
a rotary shaker at 24
◦
C and 140 rpm. After 14 days, 30 mL of these seed cultures were shifted into
6×2 L
Erlenmeyer flasks with 800 mL of the respective medium each. Incubation of the cultures at
24
◦
C and 140 rpm on rotary shakers occurred until all free glucose was consumed, after 21 days in
BAF [12] and 24 days in modified Robbin’s media. Extraction followed an earlier published protocol,
described in
Sandargo et al. (2018)
[
12
], leading to 4 g of crude extract for BAF medium and 2 g for
modified Robbins medium. All crude extracts were filtered using an SPME Strata
™
-X 33 u Polymeric
RP cartridge (Phenomenex, Inc., Aschaffenburg, Germany).

Molecules 2018,23, 2697 9 of 11
3.4. Isolation of Metabolites 5–11
The crude extracts of both media were pre-fractionated as described by Sandargo et al. (2018) [
12
]
using RP. MPLC fractions were first subjected to analytical NP-HPLC using the Orbit 100 Diol column,
250
×
4 mm, 5
µ
m (MZ-Analysetechnik, Mainz, Germany), gradient: 100% solvent A (75% n-heptane
and 25% tert.-butyl methyl ether) for 10 min, increasing over a period of 30 min to 100% solvent B (67%
tert.-butyl methyl ether and 23% n-heptane with 10% acetonitrile), staying on 100% Solvent B for 10 min;
flow rate: 2 mL/min and analytical RP-HPLC (maXis, Bruker) The isolation of compounds from MPLC
pre-fractions was then performed using preparative NP-HPLC (Orbit 100 Diol column, 250
×
20 mm,
5
µ
m [MZ-Analysentechnik, Mainz, Germany]; solvent A: 75% n-heptane + 25% tert.-butyl methyl
ether, solvent B: 67% tert.-butyl methyl ether + 23% n-heptane + 10% acetonitrile) or RP-HPLC (VP
Nucleodur 100-5 C
18
ec column, 250
×
20 mm, 5
µ
m [Macherey-Nagel, Düren, Germany], solvent
A: water [MilliQ, Darmstadt, Germany], solvent B: acetonitrile). In both cases, applying a flow rate
of 15 mL/min, UV detection at 215 and 248 nm, with an optimized gradient for each fraction minus
5–10% to plus 5–10% of the previously established eluting percentage, using the analytical NP-HPLC
or RP-HPLC, over a period of 45 min. Table S1 lists the isolated compounds with their individual
retention times using analytical RP-HPLC-MS (MaXis Bruker, Bremen, Germany) and the gradient
used with the respective preparative HPLC. While compound 5 can only be found in modified Robbins
media, all other metabolites appear in both media.
3-Hydroxy-dihydropleurotinic acid (
5
): off-white to pale yellow amorphous powder; [
α]21
D
+ 35
◦
(c1,
ACN); UV (CHCl
3
)
λmax
(log
ε
) 249 (5.0), 334 (3.7); IR: KBr,
¯
v
(cm
−1
) = 3400 s, 2950 s, 2900 m, 1700 s,
1600 vs, 1290 s, 1100 s;
1
H- and
13
C-NMR in CHLOROFORM-dsee Table 1; HRESIMS m/z373.1661
[M + H]+(calcd. for C21 H25O6+, 373.1651).
14-Hydroxy-dihydropleurotinic acid (
6
): yellow amorphous powder; [
α]21
D−
33
◦
(c1, ACN); UV (CHCl
3
)
λmax
(log
ε
) 249 (4.9), 333 (3.6);
1
H- and
13
C-NMR in ACETONE-d
6
see Table 1; HRESIMS m/z373.1651
[M + H]+(calcd. for C21 H25O6+, 373.1651).
Leucopleurotinic acid (
7
): white amorphous powder; [
α]21
D
+ 16
◦
(c1, ACN); UV (CHCl
3
)
λmax
(log
ε
)
233 (5.0), 297 (4.0);
1
H- and
13
C-NMR in ACETONE-d
6
see Table 1; HRESIMS m/z359.1868 [M + H]
+
(calcd. for C21 H27O5+, 359.1853).
14-oxo-leucopleuotinic acid (
8
): off-white amorphous powder; [
α]21
D
+ 19
◦
(c1, ACN); UV (CHCl
3
)
λmax
(log
ε
) 233 (5.0), 271 (4.0), 378 (3.8);
1
H- and
13
C-NMR in CHLOROFORM-dsee Table 1; HRESIMS m/z
373.1653 [M + H]+(calcd. for C21 H25O6+, 373.1651).
Nematoctone (
9
): light brown amorphous powder; [
α]21
D
+ 21
◦
(c1, ACN); UV (CHCl
3
)
λmax
(log
ε
) 219
(5.1), 302 (4.1); IR: KBr,
¯
v
(cm
−1
) = 3400 vs, 2950 s, 2900 s, 2850 s, 1750 s, 1600 m, 1420 s,
1290 s,
1190 m,
1150 s, 1050 s, 800 s;
1
H- and
13
C-NMR in CHLOROFORM-dsee Table 1; HRESIMS m/z355.1539
[M + H]+(calcd. for C21 H23O5+, 355.1545).
Di-oxo-leucopleurotinic acid (
10
): light brown amorphous powder; [
α]21
D
+ 11
◦
(c1, ACN); UV (CHCl
3
)
λmax
(log
ε
) 229 (5.0), 399 (3.5);
1
H- and
13
C-NMR in CHLOROFORM-dsee Table 1; HRESIMS m/z
387.1447 [M −H]−(calcd. for C21H23 O7−, 387.1444).
4-Hydroxypleurogrisein (
11
): golden yellow amorphous powder; [
α]21
D
+ 24
◦
(c1, ACN); UV (CHCl
3
)
λmax
(log
ε
) 249 (4.7) 348 (3.7); IR: KBr,
¯
v
(cm
−1
) = 3400 s, 2950 s, 2900 s, 1650 vs, 1310 s, 1200 s, 1070s,
850 m;
1
H- and
13
C-NMR in ACETONE-d
6
see Table 1and DMSO-d
6
(see Supplementary Materials);
HRESIMS m/z383.1825 [M + Na], 743.3760 [2M + Na], 721.3952 [2M + H]
+
, 343.1908 [M
−
H
2
O + H]
+
,
325.1795 [M −2 H2O + H]+, (calcd. for C21 H29O5+, 361.2169).

Molecules 2018,23, 2697 10 of 11
3.5. Biological Activities
3.5.1. Antimicrobial Activities
The minimum inhibitory concentration (MIC) for each compound was ascertained in a serial
dilution assay in 96-well microtiter plates, as previously published by Kuhnert et al. [
15
], using YM
media for yeasts and filamentous fungi and BDTM Difco™ Mueller Hinton Broth for bacteria.
3.5.2. Cytotoxicity Assay
In vitro
cytotoxicity assay was performed as described by Richter et al. [
16
] against the mouse
fibroblast cell line L929 and the cervix carcinoma cell line KB3.1.
3.5.3. Nematicidal Activity Assay
Nematicidal activity of the isolated metabolites against Caenorhabditis elegans, grown on nematode
agar (soy peptone 2 g, NaCl 1 g, agar 20 g, 1000 mL deionized water; adding 0.5 mL cholesterol
(1 mg/mL EtOH), 1 mL 1 M CaCl
2
, 1 mL 1 M MgSO
4
, and 12.5 mL 40 mM potassium phosphate buffer
after autoclaving; pH adjusted to 6.8) with living E. coli DSM498, at 20
◦
C for a week, was assessed
according to a protocol by Kuephadungphan et al. [17].
3.5.4. Inhibitory Effects on HCV Infectivity
This assay was carried out as described previously by Mulwa et al. [
18
]. Huh7.5 cells
stably expressing Firefly luciferase (Huh7.5 Fluc) were cultured in Dulbecco’s modified minimum
essential medium (DMEM, Life Technologies, Darmstadt, Germany) containing 2 mM L glutamine,
1×minimum
essential medium nonessential amino acids (MEM NEAA, Life Technologies),
100
µ
g/mL streptomycin, 100 IU/mL penicillin (Life Technologies), 5
µ
g/mL blasticidin and 10%
fetal bovine serum. Cells were maintained in a 37
◦
C environment with 5% CO
2
supply. Cells were
infected with Jc1-derived Renilla reporter viruses in the presence or absence of compounds as described
previously [
19
]. Infected cells were lysed and then frozen at
−
80
◦
C for 1 h following measurements of
Renilla and Firefly luciferase activities on a Berthold Technologies Centro XS3 Microplate Luminometer
(Bad Wildbad, Germany) as indicators of viral genome replication and cell viability, respectively.
Supplementary Materials:
The following are available online. Table S1: Retention time of isolated peaks and
gradients used for preparative HPLC, water agar test with C. elegans, Figure S1: Nematode captured by H. grisea,
HRESIMS data of 5–11, and full NMR data of 5–11.
Author Contributions:
B.T. and M.S. contributed to fungal specimen collection, isolation, and cultivation. B.S.
and B.T. contributed to species identification, fermentation, and isolation of the compounds. D.P. and E.S.
contributed to the antiviral characterization and manuscript writing. B.S. further contributed to structure
elucidation, characterization of compounds, and determination of biological activities, and wrote the paper.
F.S. contributed to experiment guidance and manuscript writing and was the project leader.
Funding:
E.S., D.P. and M.S. were supported by a grant (GINACIO, 16GW0105) of the German Ministry for
Education and Research (BMBF). Financial support by the Deutsche Forschungsgemeinschaft (DFG) for grant
SU 936/1-1, German Academic Exchange Service (DAAD) and the Thai Royal Golden Jubilee-Industry Program
(RGJ) for a joint TRF/DAAD PPP (2014-2015) academic exchange grant to M.S. and F.S. and the RGJ for a personal
grant to B.T. (No. Ph.D/0138/2553 in 4.S.MF/53/A.3) is gratefully acknowledged.
Acknowledgments:
We are grateful to W. Collisi for assisting with the bioassays; C. Kakoschke and C. Bergmann
for recording NMR and HPLC-MS data, respectivel; and Volker Huch for providing the Xray data of leucopleurotin.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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