Azaphilones from the marine sponge-derived fungus penicillium sclerotiorum OUCMDZ-3839

Article abstract of DOI:10.3390/md17050260

Four new azaphilones, sclerotiorins A-D (1-4), as well as the dimeric sclerotiorin E (5) of which we first determined its absolute configuration, and 12 known analogues (5-16) were isolated from the fermentation broth of Penicillium sclerotiorum OUCMDZ-3839 associated with a marine sponge Paratetilla sp.. The new structures, including absolute configurations, were elucidated by spectroscopic analyses, optical rotation, ECD spectra, X-ray single-crystal diffraction, and chemical transformations. Compounds 11 and 14 displayed significant inhibitory activity against α-glycosidase, with IC₅₀ values of 17.3 and 166.1 μ M, respectively. In addition, compounds 5, 7, 10, 12-14, and 16 showed moderate bioactivity against H1N1 virus.

Full text of DOI:10.3390/md17050260

marine drugs
Communication
Azaphilones from the Marine Sponge-Derived
Fungus Penicillium sclerotiorum OUCMDZ-3839
Qian Jia 1 , Yuqi Du
,2,†
1,2,†
, Chen Wang , Yi Wang , Tonghan Zhu
1
1
1,3
and Weiming Zhu 1,2,
*
1
Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy,
Ocean University of China, Qingdao 266003, China; jiaqianxiaoyugan@126.com (Q.J.);
mystagedu@foxmail.com (Y.D.); wangchen133wc@163.com (C.W.); wangyi0213@ouc.edu.cn (Y.W.);
sdueduzth@126.com (T.Z.)
2
3
Open Studio for Druggability Research of Marine Natural Products, Laboratory for Marine Drugs and
Bioproducts, Pilot National Laboratory for Marine Science and Technology (Qingdao),
Qingdao 266003, China
College of Computer Science and Engineering, Shandong University of Science and Technology,
Qingdao 266590, China
*
†
Correspondence: weimingzhu@ouc.edu.cn; Tel./Fax: +86-532-8203-1268
These authors contributed equally to this paper.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 5 April 2019; Accepted: 30 April 2019; Published: 30 April 2019
Abstract: Four new azaphilones, sclerotiorins A–D (
1–4
), as well as the dimeric sclerotiorin E (
5) of
which we first determined its absolute configuration, and 12 known analogues (
5–16) were isolated
from the fermentation broth of Penicillium sclerotiorum OUCMDZ-3839 associated with a marine
sponge Paratetilla sp.. The new structures, including absolute configurations, were elucidated by
spectroscopic analyses, optical rotation, ECD spectra, X-ray single-crystal diffraction, and chemical
transformations. Compounds 11 and 14 displayed significant inhibitory activity against
with IC50 values of 17.3 and 166.1 M, respectively. In addition, compounds 10
6 showed moderate bioactivity against H1N1 virus.
α
-glycosidase,
µ
5,
7,
,
12–14, and
1
Keywords: azaphilones; anti-virus; anti-α-glycosidase; Paratetilla sp.; Penicillium sclerotiorum
1
. Introduction
Marine fungi are important sources of bioactive natural products (NPs) [
than 3000 NPs were discovered from marine fungi in the past decades, accounting for 27% of all
the marine-derived NPs [ ]. Azaphilones are a class of biologically active metabolites of fungi
and have been reported to display antimicrobial, antiviral, antioxidant, cytotoxic, nematocidal,
and anti-inflammatory bioactivities [ ]. So far, over 430 azaphilones derived from both marine
and terrestrial fungi have been reported, such as the cytotoxic chaetomugilins A–O [10 13] and
16] and pleosporalones B–C [17],
1]. Accordingly, more
2–7
8,9
–
isochromophilones A–F [14], anti-bacterial penicilones A–H [15
,
anti-inflammatory monapilol A–D [18], and others.
The chemical investigation of a Paratetilla sp. sponge-derived fungus, Penicillium sclerotiorum
OUCMDZ-3839, led to the identification of four new azaphilones, called sclerotiorins A–D ( ),
as well as 12 known analogues (Figure 1), sclerotiorin E ( ) [19], geumsanol G ( ) [20 21], (+)sclerotiorin
) [22 25], isochromophilone I (11) [26], IV ( ) [27], VI (15) [27 28], VIII ( ) [29], and IX (16) [28],
TL-1-monoactate (10) [30], ochrephilone (12) [24 31], 8-acetyldechloroisochromophilone III (13) [32],
and scleratioramine (14) [24 33]. Although the composition of sclerotiorin E ( ) was previously reported,
its absolute configuration is determined in the current work for the first time. Compounds 10
14, and 16 showed stronger antiviral activity against H1N1 in the MDCK cell line than the positive
1–4
5
6
,
(7
–
9
,
8
,
,
5
5,
7,
,
12–
Mar. Drugs 2019, 17, 260; doi:10.3390/md17050260
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2019, 17, 260
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control ribavirin. Furthermore, compounds 11 and 14 displayed significant inhibitory activity against
α-glycosidase, with IC50 values of 17.3 and 166.1 µM, respectively.
Figure 1. Structures of compounds 1–16.
2
. Results and Discussion
Sclerotiorin A ( ) was obtained as a yellow amorphous powder. The molecular formula of
was determined to be C H O Cl by the HRESIMS (high resolution electrospray ionization mass
1
1
2
3
29
4
+
1
spectroscopy) peak at m/z 405.1834 [M + H] (Figure S1), with the 1:3 chlorine isotope peaks.
Table 1, Figure S2), 13C (Table 2, Figure S3) combined with DEPT (distortionless enhancement by
H
(
polarization transfer, Figure S4) and HSQC (heteronuclear single-quantum correlation, Figure S5)
3
NMR data of
2
1
revealed the presence of 5 singlet methyls, 1 methoxy, two methylenes, 2 sp methines,
3
heteroatom-bonded sp non-protonated carbons, 10 olefinic/aromatic carbons, and 1 carbonyl.
The HMBC (heteronuclear multiple bond correlation, Figure 2 and Figure S7) from H-1 to C-3 and C-4a,
8
H-4 to C-3, C-5, and C-8a, H-8 to C-1, C-4a, C-6, and C-7 established the core skeleton of azaphilones.
Moreover, the COSY (correlation spectroscopy) cross peaks (Figure 2 and Figure S6) from H-9 to H-10
and from H-13 to H-12, H-14, H-16, then from H-14 to H-15, along with the HMBC correlations from
H-9 to C-11, H-10 to C-17 and C-12, H-12 to C-10 and C-17 demonstrated the presence of the common
side chain of azaphilones [
by the HMBC correlations from H-9 to C-3 and C-4 along with that from H-10 to C-3. Additionally,
the other HMBC correlations from H-20a to C-7, H-20b to C-19, C-8a ,and C-8, H- -OCH to C-19, and
8]. The linkage of the unsaturated side chain to C-3 was demonstrated
β
3
H-21 to C-20 revealed a furan nucleus. The COSY correlations from H-8 to H-20, as well as the HMBC
correlations from H-20 to C-7 and C-8, confirmed the connection mode of the furan nucleus to the
fused pyrone–quinone core skeleton. Thus, the constitution of sclerotiorin A (1) was determined.
Sclerotiorin B (
2
) was obtained as a yellow amorphous powder. HRESIMS gave the peak of m/z
05.1836 [M + H] (Figure S10) and the chlorine isotope peaks; consequently, the molecular formula
was determined to be C H O Cl, the same as that of . The NMR data of ( Table 1; Table 2, Figures
, except that C-8, C-19, and C-20 were shielded and shifted from
+
4
1
2
23
29
4
S11–S14) were similar to those of
3.4, 106.0, and 45.8 of to 42.7, 105.4, and 44.2 of
triggered by the difference of the stereochemistry on C-19. Accordingly, compound
a 19-epimer of compound
and S16).
1
δ
C
4
1
δ
2
, respectively. The chemical shift changes may be
was identified as
1, which was further confirmed by the 2D NMR data (Figure 2, Figures S15
C
2

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Table 1. 13C (125 MHz) NMR data for compounds 1–4 (δ in ppm).
1 a 2 a 3 a
4 b
Position
δ
C
δ
C
δ
C
δ
C
1
3
4
144.6, CH
157.4, C
105.0, CH
138.9, C
109.0, C
187.7, C
83.5, C
43.4, CH
116.4, C
117.6, CH
140.3, CH
132.2, C
146.2, CH
34.3, CH
29.6, CH2
11.8, CH3
20.2, CH3
12.3, CH3
23.9, CH3
106.0, C
45.8, CH2
22.9, CH3
-
145.4, CH
157.7, C
104.9, CH
139.6, C
108.4, C
187.9, C
84.1, C
42.7, CH
115.7, C
117.5, CH
140.5, CH
132.2, C
146.4, CH
34.3, CH
29.6, CH2
11.8, CH3
20.2, CH3
12.3, CH3
24.4, CH3
105.4, C
44.2, CH2
22.8, CH3
-
145.4, CH
155.4, C
107.7, CH
143.0, C
106.1, CH
195.1, C
83.1, C
43.2, CH
116.4, C
117.4, CH
139.0, CH
132.0, C
145.3, CH
34.2, CH
29.6, CH2
11.9, CH3
20.3, CH3
12.3, CH3
24.3, CH3
105.1, C
44.4, CH2
23.0, CH3
-
141.2, CH
148.3, C
111.7, CH
144.9, C
102.3, C
184.5, C
84.8, C
193.9, C
115.1, C
4
a
5
6
7
8
8
a
9
114.6, CH
145.6, CH
132.2, C
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
1
1
1
1
1
1
1
1
1
2
2
2
2
2
148.4, CH
35.2, CH
30.0, CH2
12.1, CH3
20.3, CH3
12.6, CH3
23.4, CH3
170.3, C
20.4, CH3
53.4, CH2
25.4, CH2
30.2, CH2
172.6, C
-
-
-
-
-
-
OCH3
48.3, CH3
48.3, CH3
48.3, CH3
52.2, CH3
a
recorded in DMSO-d6. b recorded in CDCl3.
1
Table 2. H (500 MHz) NMR data for compounds 1–4 (δ in ppm).
1 a
2 a
3a
4 b
Position
δ
H
(J in Hz)
δ
H
(J in Hz)
δ
H
(J in Hz)
δ (J in Hz)
H
1
4
5
8
9
7.65, s
6.67, s
-
7.80, s
6.69, s
-
7.65, s
6.40, s
5.23, s
7.76, s
7.05, s
-
-
3.23, dd (10.3, 10.3) 3.42, dd (12.7, 10.3)
3.30, m
6.44, d (15.8)
6.99, d (15.8)
5.71, d (9.7)
2.46, m
1.39, m; 1.26, m
0.81, t (7.5)
0.96, d (6.6)
1.80, s
6.45, d (15.8)
7.01, d (15.8)
5.72, d (9.6)
2.46, m
1.39, m; 1.27, m
0.81, t (7.5)
0.96, d (6.6)
1.80, s
6.18, d (15.8)
6.90, d (15.8)
5.65, d (9.7)
2.45, m
1.37, m; 1.27, m
0.81, t (7.4)
0.95, d (6.6)
1.77, s
6.31, d (15.3)
6.98, d (15.3)
5.71, d (9.7)
2.48, m
1.44, m; 1.36, m
0.88, t (7.4)
1.02, d (6.6)
1.90, s
1
1
1
1
1
1
1
1
0
2
3
4
5
6
7
8
1.19, s
1.25, s
1.20, s
1.55, s
2
.36, dd (13.0,10.0)
2.18, dd (12.7,7.3)
1.87, dd (12.7,10.5)
1.25, s
2.15, dd (13.1,10.0)
1.84, m
2
0
2.16, s
1
.89, dd (13.0,10.5)
2
2
2
1
2
3
1.35, s
1.23, s
3.95, t (7.8)
2.05, m
2.43, t (6.4)
3.70, s
-
-
-
-
-
-
3.03, s
a
OCH3
3.20, s
3.19, s
recorded in DMSO-d6. b recorded in CDCl3.

Mar. Drugs 2019, 17, 260
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Figure 2. Key COSY, HMBC, and Nuclear Overhauser Effect (NOE) correlations of compounds 1–4.
Sclerotiorin C (
3
) was also obtained as a yellow powder. The molecular formula was deduced to
+
be C H O by the m/z 371.2218 [M + H] of the HRESIMS (Figure S18), without the chlorine isotope
23
30
4
peaks. The NMR data (Tables 1 and 2, Figures S19–S23) and optical rotation of
of , except for the expected extra CH signal ( C/H 106.1/5.23) on C-5 indicating that the chlorine atom
at C-5 in was substituted by a hydrogen atom. The key HMBC correlations (Figure S24) from H-5
5.23) to C-7 ( 83.1), C-8a ( 116.4), and C-4 ( C 107.7) confirmed the position of the extra H-5.
Thus, 3 was identified as the dechlorinated derivative of 2, named sclerotiorin C.
Sclerotiorin D ( ) was a red powder after purification. The peak m/z 490.1993 [M + H] of
HRESIMS (Figure S26) indicated the molecular formula was C H O NCl. When comparing the NMR
3 were similar to those
2
δ
3
(δ
H
δ
C
δ
C
δ
+
4
26
32
6
data (Tables 1 and 2, Figures S27–S30) of compound
were that compound had one more methyl group (
172.6) was deshielded. Consequently, compound
4
to those of 16
δ 52.2/3.70), and the carbonyl carbon C-24
C/H-25
was supposed to be the methyl ester derivative
[28], the most obvious differences
4
(δ
4
C
of 16. A further analysis of 1D and 2D NMR confirmed the chemical composition of compound
4
(Figure 2, Figure S31 and Figure S32).
In all new compounds
1
–
4
, the large J value between H-9 and H-10 (Table 2) and the NOESY
9
(
nuclear Overhauser enhancement spectroscopy) correlations of H-17/H-13 suggested the E-type of
∆
11
and
∆
double bonds (Figure 2, Figures S8, S17, and Figure S25). According to the common Cotton
effect of azaphilones established by Steyn and Vleggaar [25
was suggested to be R from the positive Cotton effects at 317 nm (∆ε + 2.59,
and 324 nm (∆ε + 2.09, ) (Figure 3), which were consistent with those reported for isochromophilones
C (314 nm, ∆ε + 2.85) and D (314 nm, ∆ε + 3.99) [14]. Then, key NOE (Nuclear Overhauser Effect)
enhancements of H-18 ( 1.19) and H-21 ( H 1.35) were observed after irradiation of H-8 ( H 3.23) in
(Figure 2 and Figure S9). However, only the H-8/H-18 correlations were observed on the NOESY
spectra of and , indicating the opposite configurations of C-19. Therefore, the absolute configurations
of sclerotiorin A–C ( ) were determined to be 7R/8R/19S, 7R/8R/19R, and 7R/8R/19R, respectively.
The absolute configurations of C-13 in were suggested as S by the common biosynthetic pathway of
the aliphatic side chain in reported azaphilones [ 14], which were determined by X-ray single-crystal
diffraction [35], hydrolysis [36], or ECD (electronic circular dichroism) calculation [14].
Taking into account the structural similarity of 14 with 15, and 16, the absolute configurations
,
34], the absolute configuration of C-7 in
1–3
1
), 317 nm (∆ε + 2.45,
2),
3
δ
H
δ
δ
1
2
3
1–3
1–3
9,
4, 5, 7,
of these compounds could be resolved by chemical correlation if a single crystal of 14 could be obtained.
Fortunately, a single crystal of compound 14 was obtained, and the X-ray diffraction (Figure 4) clearly
determined the absolute configuration of 14 as 7R and 13S from the absolute structure parameter
of 0.01(2). Thus, a series of reactions were carried out using (+)-sclerotiorin (
7) as a raw material
(
Scheme 1) [37]. Compounds 14 16 were directly produced after the reaction of 7
–
with ammonium

Mar. Drugs 2019, 17, 260
5 of 12
acetate, aminoethanol, and 3-aminobutyric acid, while compounds
4
and
5
resulted from the reactions of
1
4
6
,
and 14 with iodomethane and 1,4-diiodobutane (Figure S39), respectively. The synthetic compounds
5, and 14 16 were identified as the natural ones by the same retention times in their co-HPLC
profiles, as shown in Figure S37. In addition, compounds 5, and 14 16 displayed similar ECD
5, and 14 16 had the same
–
4
,
–
(
(
Figure S38) and the same sign of specific rotation. Therefore, compounds
7R,13S) configurations.
4,
–
Figure 3. Measured ECD curves of compounds 1–3.
Figure 4. ORTEP (Oak Ridge Thermal-Ellipsoid Plot) drawing of compounds 14 and 15 (Mo Kα).
Scheme 1. Chemical transformation of
7 to 4, 5, and 14–16. Reagents and conditions: a. NH OAc,
4
MeOH, THF (tetrahydrofuran), 17h, r.t (room temperature), 40.1% yield; b. 1,4-diiodobutane, K CO ,
2
3
◦
acetone, 7d, 50 C, 3.3% yield; c. aminobutyric acid, DMF (N,N-dimethylformamide), N , 5h, r.t,
2
9
9
2.7% yield; d. CH I, K CO , acetone, N , 24h, r.t, 94.1% yield; e. aminoethanol, CH Cl , N , 24h, r.t,
3 2 3 2 2 2 2
2.4% yield.
Sclerotiorin E (
5
) was obtained as a red powder. The HRESIMS peak m/z 833.3350 [M + H]+
19
was identified to be the same as that reported by NMR (Table S1 and S2, Figures S34–S37), the absolute
configuration has not been resolved yet. Expectedly, compound was the dimer of 14 linked by a
indicated the molecular formula was C H O N Cl (Figure S33). Although the constitution of
5
[
]
46
54
8
2
2
5
1
,4-butylidene bridge. As shown in Scheme 1, compound
5
could be semi-synthesized from 14. Thus,
0
the absolute configuration of compound
1
5
that we named sclerotiorin E was determined to be (7R, 7 R,
0
3S, 13 S) for the first time.

Mar. Drugs 2019, 17, 260
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The anti-H1N1-virus activity of compounds
1–
16 were examined in the MDCK cell line by the
CPE (cytopathic effect) + MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide)
method [38 39]. Compounds 10 12 13 14, and 16 showed better inhibitory activity on H1N1 than
the positive control (ribavirin), with IC50 values ranging from 78.6 to 156.8 M (Table 3). The results
,
5,
7
,
,
,
,
µ
displayed the structure–activity relationships of these azaphilones against the H1N1 virus: the chlorine
atom at C-5 was not necessary for the activity, the dimer had a stronger anti-viral activity than the
monomer, replacement of the oxygen atom connected to C-1 by a nitrogen atom did not affect the
anti-H1N1 activity. Furthermore, the
by the PNPG (p-nitrophenyl -D-glucopyranoside) method [40]. Compounds 11 and 14 displayed
good activity against -glycosidase, with IC50 values of 17.3 and 166.1 M, respectively (acarbose,
.1 mM).
α-glycosidase inhibitory activity of compounds 1–16 was assayed
β
α
µ
1
Table 3. Activity against H1N1 of compounds 5, 7, 10, 12–14, and 16. IC .
5
0
Compound
5
7
10
12
13
14
16
Ribavirin
IC50 (µM)
78.6
128.7
115.0
150.5
91.4
133.9
156.8
179.8
3
. Experimental Section
3
.1. General Experimental Procedures
All the NMR spectra were recorded on a JEOLJN M-ECP 600 spectrometer (JEOL, Tokyo, Japan)
or a Bruker Advance 500 spectrometer (Bruker, Fällanden, Switzerland) used TMS (tetramethylsilane)
1
as internal standard. H chemical shifts were referenced to the residual CDCl or DMSO-d signal
3
6
(δ
7.26 and 2.50 ppm, respectively). 13C chemical shifts were referenced to the CDCl or DMSO-d6
3
solvent peak (δ77.16 or 39.52 ppm, respectively). Optical rotations were measured on a JASCO
P-1020 digital polarimeter (JASCO Corporation, Tokyo, Japan). ECD data were acquired on a JASCO
J-815 spectropolarimeter (JASCO Corporation, Tokyo, Japan). HRESIMS spectra were collected
using the Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer (Waters Asia Ltd., Singapore).
Semi-preparative HPLC was performed using an ODS (octadecylsilyl) column (YMC-pack ODS-A,
1
0
×
250 mm, 5 µm, 4 mL/min). TLC was performed on plates precoated with silica gel GF254 (10–40 µm,
Qingdao Marine Chemical Factory, Qingdao, China). Column chromatography (CC) was carried by
silica gel GF254 and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden). Vacuum–liquid
chromatography (VLC) utilized silica gel H (Qingdao Marine Chemical Factory, Qingdao, China).
3
.2. Fungal Material and Fermentation
The fungus OUCMDZ-3839 was isolated from Paratetilla sp. sponges collected from Xisha Island
in November 2011. The sponges were firstly clipped, then ground and suspended in sterile distilled
water. The suspension was spread on a seawater starch (SWS) agar plate (10 g starch, 1 g protein
◦
powder, 20 g agar per liter of sea water). After culturing at 28 C for several days, the single colony
was purified on a potato dextrose agar plate (PDA, 200 g potato, 20 g glucose, 20 g agar per liter of tap
◦
◦
water) and maintained at
was inoculated into 200
20 g sorbitol, 20 g maltose, 10 g monosodium glutamate, 0.5 g KH PO , 0.3 g MgSO
−
80 C. After culture on the PDA plate at 28 C for 4 days, the mycelium
×
1000 mL Erlenmeyer flasks, each containing 300 mL of liquid medium
(
·
7H O, 0.5 g
2
4
4
2
◦
tryptophan, 3 g yeast extract per liter of sea water pH 6.5). The medium was incubated at 28 C under
static conditions for 41 days.
3
.3. Extraction and Purification
The mycelium and the aqueous layer of the whole culture (60 L) were separated through
cheesecloth. The mycelium was soaked by acetone for three times, and the acetone phases were
collected and then evaporated under vacuum until acetone was removed. The residue water layer and

Mar. Drugs 2019, 17, 260
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the whole aqueous layer were extracted with three volumes of ethyl acetate (EtOAc) for three times.
The EtOAc layer was collected and evaporated to dry. A total of 51.4 g of crude extract was obtained.
The EtOAc extract was subjected to a silica gel VLC column, eluting with petroleum, petroleum/CH Cl
2
2
(
v/v, 1:1), CH Cl , a stepwise gradient of CH Cl /MeOH (v/v, 100:1, 75:1,50:1, 25:1, 10:1, 5:1, 2:1, 1:1),
2 2 2 2
and MeOH to give 11 fractions. Fraction 4 (400 mg) was subjected to Sephadex LH-20 chromatography
50 1270 mm) to give two fractions (Fr. 4-1–Fr. 4-2). Fraction 4-2 was further purified by HPLC
over an ODS column (80% MeOH H O, v/v) to give compounds (t 16.6 min, 3.6 mg) and (t 20.9
min, 6.3 mg) and a mixture (20 mg) of
chromatography (30 80 mm) and further purified by HPLC over the ODS column (75% MeOH
v/v) to give compounds (t 11.1 min, 12.0 mg) and 10 (t 26.8 min, 3.0 mg). Fraction 4-1 was subjected
to Sephadex LH-20 chromatography (30
then purified by HPLC over an ODS column (25% MeOH
min, 26.0 mg). Fraction 4-1-2 was further subjected to Sephadex LH-20 chromatography (20
mm) to give 4 fractions (Fr. 4-1-2-1–Fr. 4-1-2-4). Fraction 4-1-2-1 was purified by HPLC over an ODS
column (70% MeOH H O, v/v) to give compounds (t 35.4 min, 3.0 mg), (t 42.2 min, 4.8 mg),
and (t 30.9 min, 3.0 mg). Fraction 4-1-2-2 was further subjected to a silica column, eluting with
petroleum ether/EtOAc to give eight fractions (Fr. 4-1-2-2-1–Fr. 4-1-2-2-8). Fraction 4-1-2-2-1 was
(
×
−
8
9
2
R
R
7
and 10. The mixture was then subjected to Sephadex LH-20
H O,
×
−
2
7
R
R
×
80 mm) to give Fr. 4-1-1–Fr. 4-1-2. Fraction 4-1-1 was
H O, v/v) to give compound 12 (t 15.2
−
2
R
×
78
−
1
2
2
R
R
3
R
purified by HPLC over an ODS column (70% MeOH
−
H O, v/v) to give compound 14 (t 24.0 min,
2
R
4.5 mg). Fraction 4-1-2-2-5 was purified by HPLC over an ODS column (75% MeOH
−
H O, v/v) to
2
give compound 13 (t 20.3 min, 3.0 mg). Fraction 4-1-2-2-8 was further subjected to a silica column,
R
eluting with petroleum ether/EtOAc to give compound 11 (ODS, 75% MeOH
.0 mg). Fraction 7 was further subjected to a silica column, eluting with petroleum ether/EtOAc to
give two fractions (Fr. 7-1 and Fr. 7-2). Fraction 7-1 was subjected to a silica column, eluting with
petroleum ether/EtOAc and further purified by HPLC over an ODS column (75% MeOH H O, v/v) to
−
H O, v/v, t 20.2 min,
2 R
8
−
2
give compound 15 (t 9.9 min, 100.0 mg). Fraction 7-2 was subjected to a silica LC column, eluting
R
with petroleum ether/EtOAc to give Fr. 7-2-1 and Fr. 7-2-2. Fraction 7-2-1 was further purified by
HPLC over an ODS column (75% MeOH
-2-2 was further purified by HPLC over an ODS column (75%MeOH
(t 4.2 min, 25.6 mg) and
−
H O, v/v) to give compound
4
−
(t 11.3 min, 8.6 mg). Fraction
H O, v/v) to give compounds
2
2
R
7
1
6
5
(t 5.5 min, 45.9 mg). Fraction 8 was further applied to a silica LC
R
R
column, eluting with petroleum ether/EtOAc, and further purified by HPLC over an ODS column
(
60% MeOH−H O, v/v) to give compound 6 (t 4.39 min, 20.1 mg).
2
R
25
D
Sclerotiorin A (1): yellow amorphous powder; [α] + 45.7 (c 0.2, EtOH); UV (MeOH) λmax (log
ε
) 255 (2.07), 361 (2.10), 391 (2.12); ECD (0.6 mM, MeOH) λmax
(
∆ε) 233 (+0.52), 258 (
−
0.53), 317 (+2.59,)
−1
1
13
and 394 (–0.46) nm; IR (KBr) νmax 3526, 1618, 1400 cm ; H and C NMR data, see Tables 1 and 2;
HRESIMS m/z 405.1834 [M + H] (calcd. for C H O Cl, 405.1827).
+
23
30
4
2
5
Sclerotiorin B (2): yellow amorphous powder; [α] + 6.6 (c 0.1, EtOH); UV (MeOH) λmax (log ε)
D
2
58 (1.67), 370 (1.76), 390 (1.79); ECD (0.6 mM, MeOH) λmax
(
∆ε) 228 (+0.45), 258 (
−
0.68), 317 (+2.45),
−1
1
13
and 387 (
−
0.10) nm; IR (KBr) νmax 3599, 1622, 1513, 1372 cm ; H and C NMR data, see Tables 1
+
and 2; HRESIMS m/z 405.1836 [M + H] (calcd. for C H O Cl, 405.1827).
2
3
30
4
2
5
Sclerotiorin C (3): yellow amorphous powder; [α] + 10.7 (c 0.1, EtOH); UV (MeOH) λmax (log ε)
D
2
48 (0.85), 367 (0.95), 391 (1.02); ECD (0.1 mM, MeOH) λmax
(
∆ε) 225 (+0.52), 253 (
−
0.14), 324 (+2.09),
−1
1
13
and 387 (–1.00) nm; IR (KBr) νmax 3465, 1622, 1388 cm ; H and C NMR data, see Tables 1 and 2;
HRESIMS m/z 371.2218 [M + H] (calcd. for C H O , 371.2217).
+
23
31
4
25
Sclerotiorin D (4): red powder; [α] + 207.5 (c 0.025, MeOH); UV (MeOH) λmax (log
ε
) 236(2.46),
D
3
2
4
69 (2.56); ECD (0.5 mM, MeOH) λmax
(
∆ε) 244 (+3.7), 303 (
−
5.98), and 383 (+4.6) nm; IR (KBr) νmax
−
1
1
13
921, 2365, 1739, 1591, 1502, 1240 cm ; H and C NMR data, see Tables 1 and 2; HRESIMS m/z
+
90.1993 [M + H] (calcd. for C H O NCl, 490.1991).
26
33
6
2
D
5
Sclerotiorin E (5): red powder, [α] + 143.7 (c 0.05, MeOH); ECD (0.72 mM, MeOH) λmax
(
∆ε
)
223 (−
1.43), 244 (+6.68), 305 (
−
12.21), and 385 (+9.45) nm; IR (KBr) νmax 3443, 2956, 2357, 1707, 1590,

Mar. Drugs 2019, 17, 260
8 of 12
−
509, 1236 cm ; H NMR (600 MHz, CDCl ) and C NMR (150 MHz, CDCl ) shown in Tables S1–S2;
3 3
1 1
13
1
+
HRESIMS m/z 833.3350 [M + H] (calcd. for C H O N Cl , 833.3330).
46
55
8
2
2
3
.4. Conversion from 7 to 14
To a solution of compound
added. The mixture was stirred at 25 C for 17 h. The reaction mixture was diluted with 5 mL of water
and extracted three times with equal volumes of EtOAc. The EtOAc layers were combined, washed
7
(15 mg) in 2 ml of THF, 300 mg of NH OAc and 300
µ
L of MeOH were
4
◦
with water, dried over anhydrous Na SO , filtered, and concentrated in vacuo to yield a product as an
2
4
orange oil. The orange oil was purified by semipreparative HPLC on ODS (70% acetonitrile/H O, v/v)
2
to afford compound 14 (6.0 mg, t 5.6 min, 40% yield) that was identified by the same retention time in
R
the co-HPLC (Figure S37) and the same sign of specific rotation as natural 14.
3
.5. Conversion from 7 to 15
Amino ethanol (20 L) was added to 1 mL of CH Cl solution of compound
solution was stirred at 25 C for 24 h under a nitrogen atmosphere. After CH Cl was dried in vacuo,
µ
7 (6 mg). The reaction
2
2
◦
2
2
the mixture was purified by semipreparative HPLC on an ODS column (85% acetonitrile/H O, v/v) to
2
afford compound 15 (6.1 mg, t 5.6 min, 92% yield), which was identified by the same retention time in
R
the co-HPLC (Figure S37) and the same sign of specific rotation as natural 15.
3
.6. Conversion from 7 to 16
Amino butyric acid (21.5 mg) was added to 2 mL of DMF solution of compound
7
(8 mg). The
◦
solution was stirred at 25 C for 5 h under a nitrogen atmosphere, and then 10 mL of H O was added.
2
The reaction mixture was extracted three times with equal volumes of EtOAc. The EtOAc layers were
combined, dried over anhydrous Na SO , filtered, and concentrated in vacuo to yield an orange oil.
2
4
The orange oil was purified by semipreparative HPLC on an ODS column (85% acetonitrile/H O, v/v)
2
to afford compound 16 (9.0 mg, t 7.9 min, 93% yield), which was identified by the same retention time
R
in the co-HPLC (Figure S37) and the same sign of specific rotation as natural 16.
3
.7. Conversion from 14 to 5
,4-Diiodobutane (6.96
1
µ
L) was added to the acetone solution (3 mL) of compound 14 (20 mg) and
◦
K CO (21.2 mg). The reaction solution was heated to 50 C and stirred for 7 days under a nitrogen
2
3
atmosphere. The reaction solution was concentrated in vacuo and dissolved in 10 mL of H O. The
2
obtained solution was extracted three times with equal volumes of EtOAc. The EtOAc layers were
combined, dried over anhydrous Na SO , filtered, and concentrated in vacuo to give a gum that was
2
4
purified by semipreparative HPLC on an ODS column (85% acetonitrile/H O, v/v) to afford compound
2
5
(1.41 mg, t 7.9 min, 3.3% yield), which was identified by the same retention time in the co-HPLC
R
(Figure S37) and the same sign of specific rotation as natural 5.
3
.8. Conversion from 16 to 4
A volume of 10
µ
L of CH I was added to the acetone solution of compound 16 (1.33 mg) and
3
◦
K CO (5.50 mg). The solution was stirred at 28 C for 24 h under a nitrogen atmosphere, and then
2
3
1
0 mL of H O was added. The mixture was extracted three times with equal volumes of CH Cl . After
2 2 2
CH Cl was dried in vacuo, the CH Cl extracts were applied to semipreparative HPLC on an ODS
2
2
2
2
column (80% acetonitrile/H O, v/v) to afford compound
4 (1.28 mg, t 5.40 min, 94% yield), which was
R
2
identified by the same retention time in the co-HPLC (Figure S37) and the same sign of specific rotation
as natural 4.

Mar. Drugs 2019, 17, 260
9 of 12
3
.9. X-ray Crystal Data for 14 (Mo Kα Radiation)
Red orthorhombic crystal from MeOH; molecular formula C H O NCl; space group P2 with
21
25
4
3
1
a = 8.6912 (7) Å, b = 7.4026 (6) Å, c = 16.0515 (14) Å, V = 996.39 (14) Å , absolute structure Flack
parameter: 0.01(2). Z = 2, Dcalcd = 1.299 mg/m ,
0
3
−1
µ
= 1.913 mm , and F (000) = 412; crystal size
3
◦
.35
×
0.12
×
0.07 mm . T = 293(2) K. A total of 5671 unique reflections (2
θ
< 140 ) were collected on
a Bruker Smart CCD area detector diffractometer with graphite monochromated Mo K
α radiation
(λ
= 1.54178 Å). The structure was solved by direct methods (SHELXS-97) and expanded using
Fourier techniques (SHELXL-97). The final cycle of full-matrix least-squares refinement was based on
◦
2
612 unique reflections (2
θ
< 140 ) and 249 variable parameters and converged with unweighted and
weighted agreement factors of R = 0.0426 and wR = 0.0952 for I > 2
σ
(I) data. Crystallographic data for
have been deposited in the Cambridge Crystallographic Data Centre as supplementary publication
no. CCDC 1055936. Copies of the data can be obtained, free of charge, on application to CCDC,
2 Union Road, Cambridge CB2 1EZ, U.K. (Fax: +44 (0)-1223-336033; e-mail: deposit@ccdc.cam.ac.uk).
1
2
1
4
1
3
.10. X-ray Crystal Data for 15 (Mo Kα Radiation)
Red orthorhombic crystal from MeOH; molecular formula C H O NCl; space group P2 2 2
1 1 1
23
29
5
3
with a = 6.7149(3) Å, b = 8.5574 (5) Å, c = 40.085 (2) Å, V = 2303.4 (2) Å , absolute structure Flack
3
−1
parameter:
.40 0.11
−
0.2(2). Z = 4, D
= 1.251 mg/m ,
µ
= 0.774 mm , and F (000) = 920; crystal size
calcd
3
◦
0
×
×
0.08 mm . T = 298(2) K. A total of 10,946 unique reflections (2
on a Bruker Smart CCD area detector diffractometer with graphite monochromated Mo K
θ
< 50 ) were collected
α radiation
(λ = 0.71073 Å). The structure was solved by direct methods (SHELXS-97) and expanded using
Fourier techniques (SHELXL-97). The final cycle of full-matrix least-squares refinement was based on
◦
3
965 unique reflections (2
θ
< 50 ) and 288 variable parameters and converged with unweighted and
weighted agreement factors of R = 0.1023 and wR = 0.2473 for I > 2
σ
(I) data. Crystallographic data for
have been deposited in the Cambridge Crystallographic Data Centre as supplementary publication
no. CCDC 1056013. Copies of the data can be obtained, free of charge, on application to CCDC,
2 Union Road, Cambridge CB2 1EZ, U.K. (Fax: +44 (0)-1223-336033; e-mail: deposit@ccdc.cam.ac.uk)
1
2
1
5
1
3
.11. Anti-influenza A (H1N1) Virus Bioassay
The antiviral activity against H1N1 was examined by the CPE+MTT assay [38
were cultured in RPMI-1640 medium containing 10% fetal bovine serum in 5% CO at 37 C. When
,
39]. MDCK cells
◦
2
the cells reached 70–80% confluency, they trypsinized into individual cells, and the cell suspension
5
concentration was adjusted to 1
×
10 cells/mL. The cells were seeded in a 96-well plate in 100
µL per
◦
well and cultured at 37 C, 5% CO for 18 h. After eliminating the culture medium, the influenza virus
2,
(
A/Puerto Rico/8/34 (H1N1), PR/8), diluted to 100 TCID , was added (100 µL per well) to the 96-well
50
plate; an equal amount of virus-free dilution was used as a negative control. The 96-well plate was
◦
incubated for 1 h at 37 C, 5% CO . The samples to be tested and a positive-control drug were diluted
2
in PBS buffer, and 20
µ
L of these solutions was added into the wells. PBS buffer was used as a negative
◦
control. After incubation for 48 h at 37 C, the cells were fixed with 100
µ
L of 4% formaldehyde for
L of 0.1% crystal violet
stain was added, staining for 30 min at 37 C. After the plates were washed and dried, the absorbance
OD) of each well was measured at 570 nm with a microplate reader (Bio-Rad, USA). The IC , as the
20 min at room temperature, then the formaldehyde was poured out, and 50 µ
◦
(
50
compound concentration required to inhibit influenza virus yield at 48 h post-infection by 50%, was
calculated. The IC50 value of the positive control, Ribavirin, was 179.8 µM.
3
.12. Anti-α-glycosidase Bioassay
The PNPG method [40] used to evaluate the inhibitory activity against α-glycosidase was described
in our previous work [41].

Mar. Drugs 2019, 17, 260
10 of 12
4
. Conclusions
Four new azaphilones, sclerotiorins A–D (
1–
4
), along with 12 known analogues (
5–
16) were isolated
and identified from a fermentation broth of the sponge-derived fungus, P. sclerotiorum OUCMDZ-3839.
Compounds 10 12 14, and 16 showed stronger antiviral activity against H1N1 in the MDCK cell
5
,
7,
,
–
line than the positive control Ribavirin. Additionally, compounds 11 and 14 displayed significant
inhibitory activity against α-glycosidase, with IC50 values of 17.3 and 166.1 µM, respectively.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/5/260/s1,
The ITS rRNA sequences data of Penicillium sp. OUCMDZ-3839; Tables S1–S3: NMR data of known compounds
(
5
–
16); physicochemical data of known compounds
5
–
16; specific rotation of synthetic compounds
4, 5 and 14–16;
Figure S1: HRESIM Spectrum of Compound
Figure S10: HRESIM Spectrum of Compound
Figure S18: HRESIM Spectrum of Compound
1
; Figures S2–S9: NMR Spectrum of Compound
1
in DMSO-d6;
in DMSO-d6;
in DMSO-d6;
2
3
; Figures S11–S17: NMR Spectrum of Compound
; Figures S19–S25: NMR Spectrum of Compound
; Figures S27–S32: NMR Spectrum of Compound
; Figures S34–S36: NMR Spectrum of Compound
2
3
Figure S26: HRESIM Spectrum of Compound
Figure S33: HRESIM Spectrum of Compound
Figure S37: Co-HPLC profiles of the synthetic and the natural 4, 5 and 14–16; Figure S38: Measured ECD curves of
compounds 4, 5, 7 and 14–16.
4
4
5
in CDCl3;
in CDCl3;
5
Author Contributions: Y.D. analyzed the data and prepared the draft of the manuscript; Q.J. and C.W. performed
most experiments; T.Z. and Y.W. checked the data; W.Z. designed and supervised the research and revised
the manuscript.
Funding: This work was financially supported by grants from the National Natural Science Foundation of China
(NSFC) (Nos. 41876172, U1501221 & U1606403).
Conflicts of Interest: The authors declare no conflict of interest.
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