Anti-respiratory syncytial virus prenylated dihydroquinolone derivatives from the gorgonian-derived fungus aspergillus sp. XS-20090B15

Article abstract of DOI:10.1021/np500650t

Two new prenylated dihydroquinolone derivatives, 22-O-(N-Me-l-valyl)aflaquinolone B (1) and 22-O-(N-Me-l-valyl)-21-epi-aflaquinolone B (2), and two known analogues, aflaquinolones A (3) and D (or a diastereomer of D, 4), were isolated from the mycelia of a gorgonian-derived Aspergillus sp. fungus. The structures of the new compounds were elucidated by spectroscopic methods, ECD spectra, Marfey's method, and chemical conversion. Compounds 1 and 2 display an unusual esterification of N-Me-l-Val to the side-chain prenyl group. Compound 2 exhibited outstanding anti-RSV activity with an IC₅₀ value of 42 nM, approximately 500-fold stronger than that of the positive control ribavirin (IC₅₀ = 20 μM), and showed a comparatively higher therapeutic ratio (TC₅₀/IC₅₀ = 520).

Full text of DOI:10.1021/np500650t

Note
pubs.acs.org/jnp
Anti-Respiratory Syncytial Virus Prenylated Dihydroquinolone
Derivatives from the Gorgonian-Derived Fungus Aspergillus sp. XS-
20090B15
Min Chen,† Chang-Lun Shao,† Hong Meng,‡ Zhi-Gang She,§ and Chang-Yun Wang*,†
†Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of
China, Qingdao 266003, People’s Republic of China
‡Key Laboratory of Rare and Uncommon Diseases, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan
250001, People’s Republic of China
§School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China
S
* Supporting Information
ABSTRACT: Two new prenylated dihydroquinolone deriva-
tives, 22-O-(N-Me-L-valyl)aflaquinolone B (1) and 22-O-(N-
Me-L-valyl)-21-epi-aflaquinolone B (2), and two known
analogues, aflaquinolones A (3) and D (or a diastereomer of
D, 4), were isolated from the mycelia of a gorgonian-derived
Aspergillus sp. fungus. The structures of the new compounds
were elucidated by spectroscopic methods, ECD spectra, Marfey’s method, and chemical conversion. Compounds 1 and 2 display
an unusual esterification of N-Me-L-Val to the side-chain prenyl group. Compound 2 exhibited outstanding anti-RSV activity with
an IC50 value of 42 nM, approximately 500-fold stronger than that of the positive control ribavirin (IC50 = 20 μM), and showed a
comparatively higher therapeutic ratio (TC50/IC50 = 520).
the South China Sea.5 Unfortunately, these peptides showed no
uman respiratory syncytial virus (RSV) is an enveloped,
Hnonsegmented negative-strand RNA virus of the family potent biological activities. However, screening of an extract
prepared from Aspergillus sp. XS-20090B15 cultured on a rice
medium revealed anti-RSV activity. Bioassay-guided separation
led to the isolation of two new prenylated dihydroquinolone
derivatives, 22-O-(N-Me-L-valyl)aflaquinolone B (1) and 22-O-
(N-Me-L-valyl)-21-epi-aflaquinolone B (2), and two known
analogues, aflaquinolones A and D (3 and 4). Herein, we report
the isolation, structural characterization, and anti-RSV activities
of 1−4.
Paramyxoviridae. RSV is the most important cause of viral
lower respiratory tract infections in infants and young children
and the second leading cause of death from respiratory viral
infections in the elderly.1 The only licensed antiviral treatment
available today is ribavirin, a guanosine analogue generally
administered as a small particle aerosol to immunocompro-
mised patients with lower respiratory tract disease due to RSV.2
However, the route of administration, the toxicity (risk of
teratogenicity), the cost, and the highly variable efficacy limit its
use. Given the above, developing effective anti-RSV agents with
unique structures is a pressing need. Marine organisms have
been proven to produce secondary metabolites that are
structurally distinct from those produced by terrestrial
organisms due to the special environment in the oceans, such
as high salinity, high pressure, low concentration of oxygen, and
dark conditions. Undoubtedly, marine organisms, especially
marine-derived fungi, represent a frontier source of novel
chemical entities for the discovery of new drug candidates
including antiviral agents.3 The most prominent example of
antiviral compounds isolated from marine fungi is the
hexapeptide halovir A, which exhibited potent inhibition of
herpes simplex virus 1 (HSV-1).4 So far none of natural
products isolated from marine-derived fungi have been reported
to show promising antiviral activity against RSV.
Compound 1 was obtained as a pale yellow, amorphous
powder and showed a protonated molecule in the low-
resolution positive ESIMS spectrum at m/z 551 [M + H]+.
Its molecular formula was determined as C32H42N2O6 based on
1
HRESIMS data, requiring 13 degrees of unsaturation. The H
NMR spectrum (Table 1) exhibited signals for a phenyl group,
a pair of ortho-coupled aromatic protons indicative of a 1,2,3,4-
tetrasubstituted benzene ring, a trans olefin unit, and six methyl
groups including two methyl groups bound to heteroatoms.
The 13C NMR data (Table 1) revealed the presence of two
carbonyl carbons (one amide and one ester), 14 aromatic or
olefinic carbons (corresponding to the two aromatic rings and
one double bond), and four sp3 carbons bound to O or N
atoms. These fragments accounted for 11 of the 13 degrees of
unsaturation, requiring two additional rings to be present in 1.
Detailed analysis of the above NMR data as well as 2D NMR
Recently, we obtained a series of new peptides from a potato-
based liquid culture of the fungus Aspergillus sp. XS-20090B15
derived from the gorgonian Muricella abnormaliz collected from
Received: August 16, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Note
were placed on the opposite face. This assignment was
consistent with the corresponding configuration of aflaquino-
lone A (3). As for the terpenoid portion in the side chain, the
axial proton at C-20 showed a large trans-diaxial-type coupling
(12.0 Hz) to H-21, indicating that H-21 must be axially
oriented and placing the C-21 methyl group in an equatorial
position. The oxymethine signal (H-22) showed very small
couplings (brs), indicating a large trans-diaxial coupling was
clearly absent. Thereby H-22 was placed in an equatorial
position, and consequently the N-Me-Val residue adopted an
axial orientation. The NOESY correlations of Hax-20/19-CH3,
21-CH3/19-CH3, Heq-20/H-17, and Heq-24/H-18 suggested
that 19-CH3 and the disubstituted olefin unit should be
equatorial and axial, respectively (Figure 1). On the basis of the
above evidence, the relative configurations for the 4-phenyl-3,4-
dihydroquinolin-2-one and terpenoid units were determined.
However, the stereochemical relationship between these two
units could not be correlated based on the NOESY experiment,
as no diagnostic NOE cross-peak could be detected between
them.
The absolute configuration of 1 was determined by electronic
circular dichroism (ECD) data, Marfey’s method, and chemical
conversion. The absolute configuration of the 4-phenyl-3,4-
dihydroquinolin-2-one unit in 1 was designated by comparison
of the ECD data with those of known dihydroquinolone
derivatives. It was reported that the ECD spectra of
dihydroquinolones and the related analogues are affected
largely by the absolute configuration of the dihydroquinolone
unit, regardless of the configuration of the terpenoid side chain.
The ECD spectrum of 1 (Figure 2) showed positive Cotton
effects at 224, 280, and 318 nm and a strong negative Cotton
effect at 253 nm, which were nearly identical to those observed
for aflaquinolone A (3).6 Thus, the 3S,4S-absolute config-
uration was assigned to 1. Hydrolysis of 1 with NaOH−MeOH
yielded two fragments, N-Me-Val and the secondary alcohol 1a.
The absolute configuration of N-Me-Val was determined as L
by Marfey’s method (Figure S18). Compound 1a was identified
as the known compound aflaquinolone B by the 1H NMR, MS,
correlations indicated that 1 is a prenylated dihydroquinolone
derivative and is structurally related to the known compound
aflaquinolone A (3).6 The 4-phenyl-3,4-dihydroquinolin-2-one
portion of 1 was the same as that of 3, as indicated by the
HMBC correlations from H-3 to C-2, C-4, C-5, and C-11, from
the amide NH proton H-1 to C-5, from the methoxy protons
H3-27 to C-3, and from the aromatic protons H-12/H-16 to C-
4 (Figure S19). The terpenoid portion of the side chain in 1
was similar to that of 3. The main difference was the
and specific rotation data (1a: [α]25 +28 (c 0.17, MeOH) vs
1
appearance of H and 13C NMR signals for an oxymethine
D
aflaquinolone B: [α]22 +20 (c 0.14, MeOH)), whose absolute
unit (CH-22) in 1 (Table 1) instead of the ketone carbonyl 13
C
D
configuration of the cyclohexane moiety in the side chain was
determined by Mosher’s method.6 Therefore, the absolute
configuration of the terpenoid portion in 1 was established as
19R,21S,22R, identical to that of aflaquinolone B. Thus, the full
absolute configuration of 1 was determined as
3S,4S,19R,21S,22R,29S. The name 22-O-(N-Me-L-valyl)-
aflaquinolone B is assigned to 1.
NMR signal in 3. The continuous sequence of COSY
correlations from H-20 to H-24 and the HMBC correlations
from H-23 and H3-26 to C-22 confirmed the connection of the
oxymethine unit (CH-22).
Besides the terpenoid portion, one amino acid residue was
1
found in the side chain of 1. In the H and 13C NMR spectra,
signals attributed to two methyl groups (δH 1.14 (d, J = 6.6
Hz); 1.01 (d, J = 6.6 Hz)), one methyl group bound to the N
atom (δH 2.71 (brs)), and two methine groups (δH 3.65 (m);
2.38 (m)) together with the carbonyl carbon (δC 174.5)
indicated that an N-Me-Val residue was presented in 1. The
COSY and HMBC correlations further confirmed the structure
of the N-Me-Val residue. The N-Me-Val residue was proposed
to connect at the C-22 position through an ester bond based on
the significant downfield chemical shift of H-22 (δH 5.12). This
was further confirmed by the hydrolysis product of 1, the
secondary alcohol 1a, whose chemical shift of H-22 was found
at δH 3.72 (Figure S17), shifting to the upfield significantly
compared with that of 1.
Compound 2 was also obtained as a pale yellow, amorphous
powder. The molecular formula of 2 was assigned as
C32H42N2O6 on the basis of HRESIMS data, the same as that
of 1. Detailed analysis of the 1D and 2D NMR data as well as
by comparison with the data of 1 (Table 1) indicated that the
planar structure of 2 was the same as that of 1. However, the
configuration of the terpenoid portion in 2 slightly differed
1
from that of 1 due to significant changes in H and 13C NMR
data of the cyclohexane moiety in 2. Analysis of J values and
NOESY data could assign the relative configuration of the
cyclohexane portion for 2. The axial proton at C-20 showed a
large trans-diaxial-type coupling (13.2 Hz) to H-21, thus
indicating that H-21 and the C-21 methyl group were placed
axially and equatorially, respectively. The oxymethine proton
H-22 showed large trans-diaxial couplings with both H-21 (11.6
Hz) and Hax-23 (11.6 Hz), thereby placing H-22 in an axial
The relative configuration of 1 was determined by analysis of
J values and NOESY data. The observed key NOE correlations
from H-3 to H-12/H-16 indicated the cofacial orientation of H-
3 and the phenyl ring, while the 3-OCH3 and 4-OH groups
B
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Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data (600/150 MHz, CDCl3) for 1 and 2
1
2
position
δC, type
δH, mult. (J in Hz)
δC, type
δH, mult. (J in Hz)
1-NH
2
9.06, brs
9.07, brs
166.9, C
83.4, CH
78.2, C
167.4, C
84.2, CH
78.9, C
3
3.67, s
3.67, s
4
5
110.2, C
154.0, C
121.8, C
126.4, CH
106.4, CH
133.5, C
136.6, C
128.2, CH
125.6, CH
128.5, CH
121.0, CH
135.9, CH
36.2, C
110.7, C
154.8, C
122.6, C
127.2, CH
107.4, CH
134.2, C
137.4, C
128.9, CH
126.4, CH
129.3, CH
119.1, CH
140.8, CH
36.0, C
6
7
8
7.36, d (8.4)
6.41, d (8.4)
7.35, d (8.4)
6.41, d (8.4)
9
10
11
12/16
13/15
14
7.28−7.31, m
7.28−7.31, m
7.28−7.31, m
6.60, d (16.8)
6.06, d (16.8)
7.29−7.31, m
7.29−7.31, m
7.29−7.31, m
6.57, d (16.2)
6.09, d (16.2)
17
18
19
20eq
20ax
21
39.8, CH2
1.54, brd (12.0)
1.36, t (12.0)
1.85, m
44.4, CH2
1.58, brd (13.2)
1.23, t (13.2)
1.89, m
30.5, CH
75.9, CH
26.7, CH2
32.2, CH
82.0, CH
27.5, CH2
22
5.12, brs
4.55, td (11.6, 4.2)
1.87, m
23eq
23ax
24eq
24ax
25
1.74−1.78, m
1.74−1.78, m
1.67, brd (12.6)
1.43, m
1.63, qd (11.6, 4.2)
1.48−1.53, m
1.48−1.53, m
1.14, s
30.7, CH2
35.3, CH2
31.0, CH3
16.5, CH3
58.2, CH3
174.5, C
1.04, s
22.6, CH3
19.3, CH3
59.0, CH3
174.5, C
26
0.82, d (7.2)
3.60, s
0.85, d (6.0)
3.60, s
27
28
29
66.3, CH
28.9, CH
18.4, CH3
17.4, CH3
31.9, CH3
3.65, m
67.0, CH
29.5, CH
18.5, CH3
17.2, CH3
32.6, CH3
3.64, m
31
2.38, m
2.35, m
32
1.14, d (6.6)
1.01, d (6.6)
2.71, brs
1.01, d (6.0)
1.11, d (6.0)
2.70, brs
33
34
6-OH
8.20, brs
8.55, brs
Figure 1. Key NOESY correlations for the side chains of 1 and 2.
position, and consequently the N-Me-Val residue adopted an
equatorial orientation. The NOESY correlation of Hax-20/Hax-
22 further confirmed the setting of the relative configuration at
C-22 (Figure 1). The NOESY correlations of Hax-20/H-18 and
Hax-24/H-18 indicated that the disubstituted olefin unit
occupied an equatorial orientation and 19-CH3 adopted an
axial orientation. The absolute configuration of 4-phenyl-3,4-
dihydroquinolin-2-one unit in 2 could also be assigned as 3S,4S,
as the ECD spectrum of 2 matched closely with that of 1
(Figure 2). Similar to 1, hydrolysis of 2 with NaOH−MeOH
yielded two fragments. The Marfey’s analysis showed the N-
Me-Val residue in 2 was of the L-configuration (Figure S18).
C
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Journal of Natural Products
Note
with an IC50 value of 42 nM (Table 2), approximately 500-fold
stronger than that of the positive control ribavirin (IC50 = 20
Table 2. Antiviral Activities of 1−4 against RSV
compound
IC50 (μM)
TC50 (μM)
TC50/IC50
520
1
2
3
4
>50
0.042
>50
6.6
22
27
4
a
ribavirin
20
>3000
>150
a
Ribavirin was used as a positive control.
μM). Compound 2 showed a high therapeutic ratio (TC50/IC50
= 520). These results suggest that 2 might be a promising drug
candidate against RSV. It should be noted that 2 showed more
potent anti-RSV activity than 4 (IC50 = 6.6 μM), indicating that
the additional N-Me-L-Val residue may improve the anti-RSV
activity. Compounds 1 and 3 exhibited no antiviral activity,
suggesting that the configuration of the cyclohexane unit also
plays a key role in the anti-RSV activity. In brief, the above
findings indicate that both the N-Me-L-Val residue and the
configuration of the cyclohexane unit in 2 are important for its
anti-RSV activity. This is the first report of antiviral activity for
prenylated dihydroquinolone derivatives.
In summary, two new prenylated dihydroquinolone deriva-
tives, 22-O-(N-Me-L-valyl)aflaquinolone B (1) and 22-O-(N-
Me-L-valyl)-21-epi-aflaquinolone B (2), were isolated from the
gorgonian-derived fungus Aspergillus sp. XS-20090B15. In
contrast to all known prenylated dihydroquinolone derivatives,
compounds 1 and 2 have a terpenoid side chain modified with
an N-Me-L-Val residue via an ester bond. Compound 2
displayed prominent anti-RSV activity and a high therapeutic
ratio. This is the first report of prenylated dihydroquinolone
derivatives with potent antiviral activity. Compound 2 might
have the opportunity to be further developed as a lead
compound for anti-RSV drug discovery.
Figure 2. ECD spectra of 1 and 2.
Unfortunately, the establishment of the absolute configuration
of the cyclohexane moiety in the side chain by Mosher’s
method was unsuccessful because of the limited amount of the
secondary alcohol-containing hydrolysate of 2. However, the
configuration at C-19 is presumably set by a terpene cyclase
from a biosynthetic standpoint, while it typically would have a
preference for one configuration in the same fungal strain.
Therefore, the absolute configurations at C-19 in 2 could be
tentatively assigned as R, identical to that of 1, but the other
possible diastereomer (19S,21S,22S) could not be ruled out.
The full absolute configuration of 2 is tentatively proposed as
3S,4S,19R,21R,22R,29S. Compound 2 is named 22-O-(N-Me-L-
valyl)-21-epi-aflaquinolone B.
The structure of the known compound 3 was identified as
aflaquinolone A on the basis of its NMR, ESIMS, and specific
rotation data and by comparison with the data previously
reported in the literature.6 The structure of compound 4 might
be aflaquinolone D because its NMR and MS data match well
with the published data.6 Aflaquinolone D was tentatively
assigned as 19S,21S, but the 19R,21R diastereomer could not be
ruled out. Because compounds 1 and 3 have a 19R
configuration established by Mosher’s method along with
coupling constant and NOESY data, it seems likely that 4
would have a 19R configuration based on biogenetic
considerations. Additionally, it would seem to be easier to
invert the C-21 configuration, as this stereocenter is α to the
ketone carbonyl. Therefore, compound 4 also might be the
3S,4S,19R,21R diastereomer of aflaquinolone D, or 21-epi-
aflaquinolone A.7
Compounds 1−4 are members of a known general class of
prenylated dihydroquinolone derivative fungal metabolites that
includes aflaquinolones,6 aniduquinolones,8 aspoquinolones,9
penigequinolones,10 and yaequinolones.11 All of these metab-
olites contain a 3,4-dihydroquinolin-2-one nucleus and a
terpenoid side chain. Interestingly, both 1 and 2 have an
additional N-Me-L-Val residue connected to the terpenoid
moiety via an ester bond in the side chain, which represent the
first examples of prenylated dihydroquinolone derivatives
containing an amino acid residue in the side chain.
Some prenylated dihydroquinolone derivatives have been
reported to exhibit diverse bioactivities such as lethality to brine
shrimp, cytotoxicity, and antibacterial activity.6,8−10 In the
present study, the antiviral activities of 1−4 against RSV were
evaluated by a cytopathic effect (CPE) assay. Compound 2
exhibited significant antiviral activity against RSV virus-induced
cytopathogenicity in human laryngeal carcinoma (Hep-2) cells
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-1020 digital polarimeter. UV spectra were
obtained on a Beckman DU 640 spectrophotometer. The ECD
spectrum was recorded on a JASCO J-810 circular dichroism
spectrometer. IR spectra were recorded on a Nicolet-Nexus-470
spectrometer using KBr pellets. NMR spectra were acquired on a
JEOL JEM-ECP NMR spectrometer (600 MHz for 1H and 150 MHz
for 13C) and an Agilent DD2 500 MHz NMR spectrometer (500 MHz
for H and 125 MHz for 13C), using TMS as an internal standard.
1
ESIMS and HRESIMS spectra were measured on a Micromass Q-TOF
spectrometer. Semipreparative HPLC was performed on a Waters
1525 system using a C18 (Kromasil, 5 μm, 250 × 10 mm) column
coupled with a Waters 2996 photodiode array detector. Silica gel
(Qing Dao Hai Yang Chemical Group Co.; 200−300 mesh), Sephadex
LH-20 (Amersham Biosciences), and octadecylsilyl silica gel (Unicorn;
45−60 μm) were used for column chromatography. Precoated silica
gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used
for thin-layer chromatography.
Fungal Material. The isolation and identification of Aspergillus sp.
XS-20090B15 have been described previously (GenBank
HM991281).5
Fermentation, Extraction, and Isolation. The fungus Aspergillus
sp. XS-20090B15 was cultivated in a rice medium (12 g of natural sea
salt (from Yangkou saltern, China), 100 g of rice, 0.6 g of peptone, 100
mL of H2O) in 1 L Erlenmeyer flasks for 35 days at room temperature
(rt). The fermented rice substrate (20 flasks) was extracted repeatedly
D
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Journal of Natural Products
Note
with EtOAc (3 × 300 mL for each flask), and the solvent was
combined and concentrated in vacuo to afford a residue (17 g), which
was subjected to silica gel column chromatography (CC) using a step
gradient elution with EtOAc−petroleum ether (0−100%) and then
with MeOH−CHCl3 (0−100%) to provide six fractions (Fr.1−Fr.6).
Fr.3 and Fr.4 exhibited evident antiviral activities against RSV. Fr.3 was
subjected to Sephadex LH-20 CC eluting with a mixture of CHCl3−
MeOH (v/v, 1:1) and further purified by HPLC eluting with 75%
MeOH−(H2O + 0.2% TFA) to afford 3 (17.2 mg) and 4 (10.5 mg).
Fr.4 was subjected to silica gel CC using gradient elution with
petroleum ether−EtOAc, then was subjected to an ODS column
eluting with 80% MeOH−H2O, and finally was purified by HPLC
eluting with 72% MeOH−(H2O + 0.2% TFA) to give 1 (6.5 mg) and
2 (3.6 mg).
AUTHOR INFORMATION
Corresponding Author
*(C.-Y. Wang) Tel/Fax: 86-532-82031536. E-mail: changyun@
ouc.edu.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (Nos. 41130858; 41322037; 81172977),
the NSFC-Shandong Joint Fund for Marine Science Research
Centers (U1406402), and the China Postdoctoral Science
Foundation (No. 2014M560582).
22-O-(N-Me-L-valyl)aflaquinolone B (1): pale yellow, amorphous
powder; [α]25 +50 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 212
D
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22-O-(N-Me-L-valyl)-21-epi-aflaquinolone B (2): pale yellow,
amorphous powder; [α]25 +15 (c 0.20, MeOH); UV (MeOH) λmax
D
(log ε) 214 (4.9), 234 (3.3), 278 (2.8), 325 (2.6) nm; CD (0.20 mM,
MeOH) λmax (Δε) 222 (20.0), 252 (−20.5), 280 (6.8), 320 (4.5) nm;
IR (KBr) νmax 3450, 3222, 2857, 1690, 1684, 1636, 1365, 1345, 977
cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150
MHz), Table 1; ESIMS m/z 551 [M + H]+; HRESIMS m/z 551.3111
[M + H]+ (calcd for C32H43N2O6, 551.3116).
Aflaquinolone A (3): white, amorphous powder; [α]25D +16 (c 0.20,
MeOH) (lit. [α]21 +14 (c 0.19, MeOH)).6
D
Aflaquinolone D (4): white, amorphous powder; [α]25 −18 (c
D
(6) Neff, S. A.; Lee, S. U.; Asami, Y.; Ahn, J. S.; Oh, H.; Baltrusaitis,
J.; Gloer, J. B.; Wicklow, D. T. J. Nat. Prod. 2012, 75, 464−472.
(7) Note: the authors of ref 6 were contacted, but no aflaquinolone D
was available for direct comparison.
0.15, MeOH) (lit. [α]25 −10 (c 0.10, MeOH)).6
D
Hydrolysis of 1 and 2 and Marfey’s Analysis of 1 and 2.12 To
a solution of 1 (2.0 mg) or 2 (1.0 mg) in MeOH (0.5 mL) was added
a newly prepared NaOH in MeOH solution (1.0 M, pH 9−10). The
mixture was stirred at 40 °C for 15−20 min and neutralized with
Dowex H+ resin to pH 7.0 and then filtered. The filtrate of 1 was
concentrated and divided into two parts. The major part was purified
by HPLC to afford the secondary alcohol-containing portion of 1 (1a,
1.1 mg). In addition, the other part was dissolved in H2O (50 μL), and
a 1% solution of FDAA (200 μL) and 1.0 M NaHCO3 (40 μL) were
added. The reaction mixture was heated at 45 °C for 1.0 h and then
cooled. The mixture was acidified with 2.0 M HCl (20 μL). Separately,
the standard amino acids N-Me-L-Val and N-Me-DL-Val and the
hydrolysate of 2 were derivatized with FDAA in the same manner as
that of 1. All FDAA derivatives were analyzed by HPLC using
MeCN−(H2O + 0.2% TFA) as the mobile phase (see Supporting
Information).
(8) An, C. Y.; Li, X. M.; Luo, H.; Li, C. S.; Wang, M. H.; Xu, G. M.;
Wang, B. G. J. Nat. Prod. 2013, 76, 1896−1901.
(9) Scherlach, K.; Hertweck, C. Org. Biomol. Chem. 2006, 4, 3517−
3520.
(10) Kimura, Y.; Kusano, M.; Koshino, H.; Uzawa, J.; Fujioka, S.;
Tani, K. Tetrahedron Lett. 1996, 37, 4961−4964.
(11) (a) Uchida, R.; Imasato, R.; Yamaguchi, Y.; Masuma, R.; Shiomi,
K.; Tomoda, H.; Omura, S. J. Antibiot. 2006, 59, 646−651. (b) Uchida,
R.; Imasato, R.; Tomoda, H.; Omura, S. J. Antibiot. 2006, 59, 652−658.
(c) Uchida, R.; Imasato, R.; Shiomi, K.; Tomoda, H.; Omura, S. Org.
Lett. 2005, 7, 5701−5704.
(12) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
(13) Grassauer, A.; Weinmuellner, R.; Meier, C.; Pretsch, A.;
Prieschl-Grassauer, E.; Unger, H. Virol. J. 2008, 5, 107.
Hydrolysis Product of 1 (1a): pale yellow, amorphous powder;
[α]25D +28 (c 0.17, MeOH); 1H NMR (CDCl3, 500 MHz), see Figure
S17; ESIMS m/z 460 [M + Na]+.
Antiviral Activity Assays. The antiviral activities of 1−4 against
respiratory syncytial virus were determined by the CPE inhibition
assay according to established procedures.13 The assay results were the
average mean of three replicate determinations. Ribavirin was used as a
positive control.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
The NMR and MS spectra of 1 and 2, the H NMR spectrum
of 1a, HPLC profiles of FDAA derivatives of the hydrolysates of
1 and 2, and COSY and key HMBC correlations of 1. These
materials are available free of charge via the Internet at http://
pubs.acs.org.
E
dx.doi.org/10.1021/np500650t | J. Nat. Prod. XXXX, XXX, XXX−XXX