Phenylspirodrimanes with anti-HIV activity from the sponge-derived fungus stachybotrys chartarum mxh-x73

Article abstract of DOI:10.1021/np400683h

Seven new phenylspirodrimanes, named stachybotrins D-F (1, 3, 4), stachybocins E and F (5, 6), and stachybosides A and B (7, 8), and four known compounds (2, 9-11), were isolated from the sponge-derived fungus Stachybotrys chartarum MXH-X73. Their structures were determined by detailed analysis of spectroscopic data. The absolute configurations of 1-8 were determined by chemical hydrolysis and modified Mosher's and Marfey's methods. All compounds were tested in an anti-HIV activity assay, and compound 1 showed an inhibitory effect on HIV-1 replication by targeting reverse transcriptase. Further study exhibited that 1 could block NNRTIs-resistant strains (HIV-1_RT-K103N, HIV-1_{RT-L100I,K103N}, HIV-1_{RT-K103N,V108I}, HIV-1 _{RT-K103N,G190A}, and HIV-1_{RT-K103N,P225H}) as well as wild-type HIV-1 (HIV-1_wt) with EC₅₀ values of 7.0, 23.8, 13.3, 14.2, 6.2, and 8.4 μM, respectively.

Full text of DOI:10.1021/np400683h

Article
pubs.acs.org/jnp
Phenylspirodrimanes with Anti-HIV Activity from the Sponge-
Derived Fungus Stachybotrys chartarum MXH-X73
Xinhua Ma,†,§ Letao Li,‡,§ Tianjiao Zhu,† Mingyu Ba,‡ Guoqiang Li,† Qianqun Gu,† Ying Guo,*,‡
and Dehai Li*,†
†Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China
‡Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, People’s Republic of China
S
* Supporting Information
ABSTRACT: Seven new phenylspirodrimanes, named stachy-
botrins D−F (1, 3, 4), stachybocins E and F (5, 6), and
stachybosides A and B (7, 8), and four known compounds (2,
9−11), were isolated from the sponge-derived fungus
Stachybotrys chartarum MXH-X73. Their structures were
determined by detailed analysis of spectroscopic data. The
absolute configurations of 1−8 were determined by chemical
hydrolysis and modified Mosher’s and Marfey’s methods. All
compounds were tested in an anti-HIV activity assay, and compound 1 showed an inhibitory effect on HIV-1 replication by
targeting reverse transcriptase. Further study exhibited that 1 could block NNRTIs-resistant strains (HIV-1RT‑K103N, HIV-
1
RT‑L100I,K103N, HIV-1RT‑K103N,V108I, HIV-1RT‑K103N,G190A, and HIV-1RT‑K103N,P225H) as well as wild-type HIV-1 (HIV-1wt) with EC50
values of 7.0, 23.8, 13.3, 14.2, 6.2, and 8.4 μM, respectively.
everse transcriptase (RT), a critical enzyme in the human
Rimmunodeficiency virus (HIV) life cycle, is one of the
main targets for the chemotherapeutic treatment of HIV-1
infection. RT inhibitors consist of two functionally distinct
classes: nucleoside RT inhibitors (NRTIs) and non-nucleoside
RT inhibitors (NNRTIs).1,2 NNRTIs are important compo-
nents for highly active antiretroviral therapy (HAART). All five
NNRTIs, nevirapine (NVP), delavirdine (DLV), efavirenz
(EFV), etravirine (ETR), and rilpivirine (RPV), bind to the
hydrophobic pocket in the palm domain adjacent to the thumb
domain on the RT p66 subunit, in blocking RT allostery.3−7
Drug resistance is the most important factor of the failure of
HAART. NNRTI-resistant mutations exist widely and spread in
patients since NVP, EFV, and DLV have been used in clinical
settings over the past decade. Both clinical and in vitro studies
have shown that E138K is a resistant mutation to ETR and
RPV, which were approved by the FDA in 2008 and 2012,
respectively.8−10 Therefore, there remains a strong demand for
the development of new NNRTIs that can effectively inhibit
NNRTI-resistant viruses.
derivatives (2, 9−11).12−14 In this paper, we describe the
isolation, structure elucidation, and anti-HIV activities of these
new phenylspirodrimanes. We discovered that stachybotrin D
(1) is a novel non-nucleoside reverse transcriptase inhibitor of
both wild-type HIV-1 and five NNRTI-resistant strains.
As part of our program to search for bioactive secondary
metabolites from sponge-associated microorganisms,11 the
fermentation of the fungus Stachybotrys chartarum MXH-X73,
which was isolated from the sponge Xestospongia testudinaris
collected from Xisha Island, China, was selected because of the
intriguing UV profiles of an extract. Chemical investigation of
the extract led to the isolation of seven new phenyl-
spirodrimanes, stachybotrins D−F (1, 3, 4), stachybocins E
and F (5, 6), and stachybosides A and B (7, 8), and four known
Received: August 21, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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1
Table 1. H (600 MHz) and 13C NMR (150 MHz) Data of 1, 3, and 4 in DMSO-d6
1
3
4
no.
1
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1.74, m
δC, type
δH (J in Hz)
1.74, m
24.4, CH2
1.75, m
0.90, m
1.72, m
1.35, m
3.16, brs
24.9, CH2
24.9, CH2
0.93, m
1.81, m
1.38, m
3.18, brs
0.92, m
1.82, m
1.38, m
3.18, brs
2
25.4, CH2
25.8, CH2
25.9, CH2
3
4
5
6
74.0, CH
37.8, C
74.4, CH
38.3, C
74.4, CH
38.4, C
39.6, CH
21.0, CH2
2.00, d (11.6)
1.43, m
39.2, CH
21.4, CH2
2.02, d (11.5)
1.45, m
39.2, CH
21.5, CH2
2.00, d (11.0)
1.45, m
1.39, m
1.41, m
1.41, m
7
31.4, CH2
1.51, m
31.7, CH2
1.52, m
31.7, CH2
1.52, m
1.37, m
1.40, m
1.40, m
8
36.9, CH
98.4, C
1.80, m
37.5, CH
99.0, C
1.79, m
37.6, CH
99.1, C
1.79, m
9
10
11
42.3, C
42.8, C
42.8, C
32.2, CH2
3.10, d (16.5)
2.75, d (16.5)
0.64, d (6.6)
0.86, s
32.7, CH2
3.13, d (17.0)
2.76, d (17.0)
0.66, d (6.0)
0.87, s
32.8, CH2
3.13, d (16.5)
2.76, d (16.5)
0.65, d (6.6)
0.87, s
12
13
14
15
1′
2′
3′
4′
5′
6′
7′
8′
16.0, CH3
29.2, CH3
22.9, CH3
16.3, CH3
117.2, C
16.5, CH3
29.6, CH3
23.4, CH3
16.8, CH3
117.9, C
16.5, CH3
29.7, CH3
23.4, CH3
16.9, CH3
118.1, C
0.78, s
0.79, s
0.79, s
0.93, s
0.95, s
0.95, s
154.3, C
154.7, C
154.9, C
101.4, CH
133.9, C
6.56, s
101.9, CH
134.1, C
6.57, s
102.0, CH
134.0, C
6.60, s
112.8, C
113.4, C
113.4, C
156.4, C
156.9, C
157.0, C
168.4, C
169.3, C
169.4, C
48.0, CH2
4.21, d (16.5)
4.22, d (16.5)
4.42, d (18.7)
4.33, d (18.7)
45.0, CH2
4.21, d (15.9)
45.2, CH2
4.22, d (16.5)
4.32, d (16.5)
4.29, d (15.9)
9′
52.4, CH2
204.6, C
54.1, CH
25.2, CH2
4.73, dd (11.1, 4.4)
54.1, CH
25.5, CH2
4.87, dd (10.4, 4.4)
10′
2.15, m
2.28, m
2.32, m
2.13, m
2.26, m
2.20, m
11′
12′
13′
14′
27.7, CH3
2.13, s
31.3, CH2
173.5, C
31.6, CH2
172.3, C
173.2, C
171.4, C
52.2, CH3
3.50, s
53.3, CH3
3.66, s
(Figure 1) from H3-11′ (δH 2.13) to C-9′ (δC 52.4) and C-10′
(δC 204.6) and from H2-9′ (δH 4.42, 4.33) to C-7′, C-8′ (δC
RESULTS AND DISCUSSION
■
The fermented whole broth (60 L) was obtained by culturing
the fungal strain S. chartarum MXH-X73 with shaking at 28 °C
for 11 days. The organic extracts prepared by solvent extraction
were separated by repeated silica gel column chromatography,
ODS column chromatography, LH-20 column chromatogra-
phy, and finally semipreparative ODS HPLC to yield the new
compounds 1 (28.0 mg), 3 (6.0 mg), 4 (5.0 mg), 5 (22 mg), 6
(20 mg), 7 (21 mg), and 8 (17 mg).
Compound 1 was obtained as a colorless oil, and its
molecular formula was determined as C26H35NO5 according to
the HRESIMS peak at m/z 442.2587 [M + H]+, requiring nine
degrees of unsaturation. The UV absorptions at 261 and 302
nm indicated the presence of an aromatic system. The IR
spectrum showed absorption bands for hydroxy (3420 cm−1),
ketone carbonyl (1716 cm−1), and amide carbonyl (1679 cm−1)
Figure 1. Key COSY, HMBC, and NOESY correlations and NOE
effects of 1.
48.0), and C-10′. The planar structure of 1 was further
confirmed by 2D NMR data (Figure 1). The relative
configuration of 1 was inferred to be the same as compound
2 and was further established by the NOESY and 1D NOE
experiments (Figure 1). The small coupling constant of H-3
(δH 3.16, brs) indicated that it was equatorial. Strong NOESY
1
groups. Comparison of the H and 13C NMR data (Table 1)
with those of the known compound 2 showed the presence of
the same phenylspirodrimane skeleton, except the side chain
moiety on the nitrogen was replaced by an acetonyl group in 1,
which was further confirmed by the HMBC correlations
B
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correlations of H-3 (δH 3.16) and H3-14 (δH 0.78) and NOE
correlations of H3-14 and H3-15 (δH 0.93), H3-15 and H-8 (δH
1.80), and NOESY correlations of H3-15 and H2-11 (δH 3.10,
2.75) suggested they were on the same face of the molecule. On
the other hand, NOE effects for H-5 (δH 2.00)/H3-13 (δH 0.86)
and NOESY correlations of H-5 (δH 2.00) and H-7b (δH 1.37)
indicated these protons were on the opposite face. The absolute
configuration at C-3 of 1 was assigned by the modified
Mosher’s method (Figure 2).15−17 The methylated derivative
Figure 4. Structures of compounds 3a and 4a.
and at C-12′ in 4. Thus the methyl groups were located at C-
12′ in 3 and C-9′ in 4, respectively. With the failure to make the
(R)-PGME amides of 3 and 4, the absolute configurations of C-
9′ in 3 and 4 were not determined by this method.
Compound 2 was previously isolated from the culture of a S.
chartarum strain;12 however, the absolute configuration was not
established. The similar NMR data and the similar specific
rotation values ([α]24 −24.3 for 1, [α]24 −32.3 for 2) for
Figure 2. Δδ (δS − δR) values (in ppm) for the MTPA esters of 1a.
D
D
compound 1a16 was treated with (R)- and (S)-MTPA chloride,
to afford the (S)- and (R)-MTPA esters, 1aS and 1aR,
respectively. Calculations for all of the relevant signals in the
diastereomeric esters 1aS and 1aR confirmed the R absolute
configuration at C-3 in 1.17 Accordingly, the absolute
configuration of 1, named stachybotrin D, was concluded to
be 3R, 5S, 8R, 9R, and 10S, the same as the stachybo-
trylactam.18
The molecular formulas for compounds 3 and 4 were both
deduced as C29H39NO8 by HRESIMS. Analysis of the 1D and
2D NMR data (Table 1) for 3 and 4 suggested that they were
methylated congeners of 2. The HMBC correlations (Figure 3)
compounds 1 and 2 indicated that their phenylspirodrimane
moieties shared the same absolute configuration. To determine
the absolute configuration of C-9′, compound 2 was subjected
to Jones oxidation, followed by acid hydrolysis.20 According to
Marfey’s method, the absolute configuration of C-9′ in 2 was
assigned as S based on the HPLC analysis of the FDAA
derivative, which resulted in the same retention time as the
FDAA derivative of standard L-glutamic acid. The absolute
configurations in 3 and 4 were also postulated to be the same as
2 based on the similar specific rotation values ([α]24D −34.0 for
3 and [α]24D −33.7 for 4), co-isolation of the metabolites from
the same fungus, and biosynthetic considerations. Compounds
3 and 4 were named stachybotrins E and F, respectively.
Compounds 3 and 4 could possibly be formed by reaction with
MeOH during the isolation. But when compound 2 was stirred
with silica in MeOH for 48 h, neither 3 nor 4 was detected by
HPLC-UV analysis.
Compounds 5 and 6 were obtained as white powders. Their
molecular formulas were determined as C50H68N2O8 and
C51H70N2O8 by HRESIMS data. However, only 25 carbon
signals were observed in the 13C NMR spectrum of 5,
indicating that 5 was a symmetrical dimer. The 1D NMR
data of 5 (Table 2) were very similar to those of 1, except that
the moiety at nitrogen in 1 was replaced by two connected
methylenes in 5, which was confirmed by the COSY
correlations (H-9′/H-10′) and the HMBC correlations from
H2-9′ (δH 3.48) to C-7′ (δC 167.8), C-8′ (δC 47.1), and C-10′
(δC 25.8) and from H2-8′ (δH 4.20, 4.30) to C-4′ (δC 134.5), C-
5′ (δC 112.2), C-6′ (δC 156.3), and C-7′ (Figure 5).
Considering the above information, compound 5 was suggested
to be a dimer connected by C-10′ and C-10‴. The 1D NMR
data showed that 6 was also a dimer and had one more
methylene (δH 1.25, δC 24.1) than 5. In compound 6, the
COSY correlations of H-10′/H-11′ and HMBC correlations
(Figure 5) from H2-9′ (δH 3.44) to C-7′ (δC 167.8), C-8′ (δC
47.1), C-10′ (δC 25.3), and C-11′ (δC 24.1), from H2-10′ (δH
1.63) to C-9′ (δC 42.2) and C-11′, and from H2-11′ (δH 1.25)
to C-9′ and C-10′ suggested that C-11′ connected the two
monomers. The gross structures of 5 and 6 were further
assigned by 2D NMR and combining their molecular formulas.
The relative configurations of the sesquiterpene moieties in 5
and 6 were determined to be the same as 1−4 by NOESY
Figure 3. Key COSY and HMBC correlations of 3 and 4.
from H3-14′ (δH 3.50) to C-12′ (δC 173.5) in 3 and that from
H3-14′ (δH 3.66) to C-13′ (δC 172.3) in 4 indicated the
methylation occurred at one carboxylic acid. To further
determine the methylated position, compounds 3 and 4 were
converted into the (S)-phenylglycine methyl ester (PGME)
amides 3a and 4a, respectively (Figure 4).19 PGME derivatives
have been applied to determine the absolute configuration of
carboxylic acids due to the shielding effect of the phenyl group
on both sides of the stereogenic center. Comparison of the
larger values (Δδ = δ3a − δ3) of H-8′ (Δδ = +0.24, +0.12), H-9′
(Δδ = +0.20), H-10′(Δδ = −0.10, −0.06), and H-11′ (Δδ =
−0.02) obtained from the 1H NMR data for 3a and 3, with the
smaller ones (Δδ = δ4a − δ4) [H-8′ (Δδ = −0.02, +0.01), H-9′
(Δδ = −0.08), H-10′ (Δδ = +0.01, +0.02), and H-11′ (Δδ =
+0.02)] for 4a and 4 indicated that the PGME moiety was
attached to the carbonyl at the asymmetric carbon (C-9′) in 3
C
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Table 2. 1H (600 MHz) and 13C NMR (125 MHz) Data of 5
and 6 in DMSO-d6
Phenylspirodrimane dimers are rare. Only five examples have
been reported, and all were from the genus Stachybotrys.20−22
The first dimer, stachybocin A, is presumably formed by
reaction between lysine and phenylspirodrimane monomer
derivatives.20 Compounds 5 and 6, absent of carboxylic acid
groups, might have gone through decarboxylation in a similar
biosynthetic process.
Stachybosides A (7) and B (8), were obtained as pale yellow
oils. The HRESIMS peaks at m/z 573.2661 [M + Na]+ and
589.2617 [M + Na]+ suggested the molecular formulas of
C29H42O10 and C29H42O11 for 7 and 8, respectively. Comparing
the 1D NMR data (Table 3) of 7 with those of compound 10
(Mer-NF5003E) revealed that they shared the same phenyl-
spirodrimane skeleton, except for the replacement of the 7′-
5
6
no.
1 (1″)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
24.3, CH2 1.73, m
0.90, m
24.3, CH2 0.90, m
1.73, m
2 (2″)
25.3, CH2 1.80, m
1.37, m
25.3, CH2 1.37, m
1.79, m
3 (3″)
3 (3″)-OH
4 (4″)
73.9, CH
3.17, brs
4.08
73.9, CH
3.17, brs
4.09
37.7, C
37.7, C
5 (5″)
39.4, CH
2.01, d (12.1)
39.4, CH
2.01, d (12.1)
6 (6″)
20.9, CH2 1.44, m
1.40, m
20.9, CH2 1.40, m
1.44, m
7 (7″)
31.2, CH2 1.50, m
1.38, m
31.2, CH2 1.50, m
1.39, m
Table 3. 1H (600 MHz) and 13C NMR (150 MHz) Data of 7
and 8 in DMSO-d6
8 (8″)
9 (9″)
10 (10″)
11 (11″)
36.9, CH
98.3, C
42.0, C
1.75, m
36.9, CH
98.2, C
42.0, C
1.76, m
7
8
no.
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
32.1, CH2 3.10, d (21.1)
2.73, d (21.1)
32.1, CH2 3.10, d (16.5)
2.74, d (16.5)
1
2
24.4, CH2 1.77, m
33.4, CH2 1.11, dd (11.8,
4.1)
12 (12″)
13 (13″)
14 (14″)
15 (15″)
1′ (1‴)
2′ (2‴)
2′ (2‴)-OH
3′ (3‴)
4′ (4‴)
5′ (5‴)
6′ (6‴)
7′ (7‴)
16.2, CH3 0.64, d (5.5)
29.0, CH3 0.87, s
22.8, CH3 0.78, s
15.9, CH3 0.93, s
116.8, C
16.2, CH3 0.63, d (6.6)
29.0, CH3 0.87, s
22.8, CH3 0.78, s
15.9, CH3 0.93, s
116.8, C
0.91, m
25.4, CH2 1.70, m
1.37, m
1.59, t (12.0),
65.4, CH
3.77, dt (11.8,
3.3)
3
4
5
73.9, CH
37.7, C
3.15, brs
78.1, CH
38.6, C
3.12, d (3.3)
154.1, C
154.1, C
40.0, CH
2.04, d (11.6)
39.6, CH
1.96, dd (12.2,
2.6)
9.69
9.71
101.2, CH
134.5, C
112.3, C
156.3, C
167.8, C
6.54, s
101.2, CH
134.5, C
112.2, C
156.3, C
167.8, C
6.55, s
6
7
21.1, CH2 1.45, m
1.41, m
20.8, CH2 1.46, m
1.39, m
31.5, CH2 1.53, m
1.38, m
31.4, CH2 1.55, m
1.36, m
8
9
36.8, CH
99.5, C
42.4, C
1.80, m
36.6, CH
99.2, C
43.5, C
1.81, m
8′ (8‴)
47.2, CH2 4.30, d (16.5)
4.20, d (16.5)
47.1, CH2 4.29, d (16.5)
4.19, d (16.5)
10
11
9′ (9‴)
10′ (10‴)
11′
42.2, CH2 3.48, t (6.6)
25.8, CH2 1.58, m
42.2, CH2 3.44, t (6.6)
25.3, CH2 1.63, m
24.1, CH2 1.25, m
30.9, CH2 3.03, d (16.5)
2.69, d (16.5)
31.0, CH2 3.03, d (16.5)
2.69, d (16.5)
12
13
14
15
1′
16.0, CH3 0.67, d (6.6)
29.2, CH3 0.86, s
22.8, CH3 0.77, s
16.3, CH3 0.93, s
111.7, C
15.9, CH3 0.67, d (6.5)
29.6, CH3 0.91, s
22.5, CH3 0.80, s
17.2, CH3 0.96, s
111.6, C
2′
159.6, C
159.7, C
2′-OH
3′
4′
5′
6′
10.61
10.64
107.1, CH
142.3, C
108.4, C
168.2, C
6.72, s
107.1, CH
142.3, C
108.5, C
168.1, C
6.73, s
7′
67.1, CH2 4.89, d (15.9),
4.67, d (15.9)
67.1, CH2 4.90, d (15.9),
4.68, d (15.9)
8′
1″
2″
187.2, CH
98.9, CH
72.5, CH
10.13, s
187.2, CH
98.9, CH
72.5, CH
10.11, s
4.78, d (3.3)
4.78, d (3.6)
3.28, dd (9.9,
3.3)
3.28, dd (9.9,
3.6)
3″
4″
5″
6″
73.8, CH
70.8, CH
73.6, CH
3.56, dd (9.9,
8.8)
73.8, CH
70.8, CH
73.6, CH
3.56, dd (9.9,
8.8)
3.11, dd (9.3,
8.8)
3.11, dd (9.9,
8.8)
Figure 5. Key COSY and HMBC correlations of compounds 5 and 6.
3.37, m
3.37, dt (9.9,
5.5)
experiments, and the absolute configurations were also
proposed to be the same as 1−4 based on a shared biogenesis.
The compounds were named stachybocins E (5) and F (6).
61.4, CH2 3.43, dd (12.1,
6.1)
61.5, CH2 3.43, dd (11.0,
5.5)
3.58, m
3.58, m
D
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hydroxymethyl in 10 by a glycosylated hydroxymethyl in 7. The
sugar unit was determined as α-glucopyranose in 7 by
comparison of the NMR data with reported values, especially
the anomeric methine at δH 4.78 (d, J = 3.3 Hz)/δC 98.9.23
Further HPLC comparison of the acid hydrolysis product of
compound 7 with standard D and L samples using a chiral-phase
column led to the determination of α-D-glucopyranose in 7.
The molecular formula of 8 indicated one more oxygen atom
than 7. The oxygen was located at C-2 on the basis of chemical
shifts of C-2 (δC 65.4) and H-2 (δH 3.77), together with the
COSY correlations of H-1/H-2/H-3 and the HMBC
correlations from H2-1 (δH 1.59, 1.11) to C-2 and C-10 (δC
43.5) (Figure 6). The relationship of H-2 and H-3 of 8 was
resistant HIV-1 strains. Therefore, five NNRTI-resistant HIV-1
pseudoviral strains were used. Compound 1 exhibited activity
against all five resistant strains similar to wild-type HIV-1 with
EC50 values ranging from 0.7- to 2.8-fold the value against the
wild-type strain. In contrast, the mutant strains were much less
susceptible to NVP as compared to the wild type (Table 4).
The phenylspirodrimanes, mainly isolated from Stachybotrys
spp., are meroterpenoids containing an unusual phenyl-
spirodrimane skeleton of three main classes: tetracyclic
aromatic sesquiterpenoids with an alcohol or aldehyde side
chain14,25 such as K-76,26 pentacyclic aromatic sesquiterpenoids
including the stachybotrylactams,12,13 stachybotrylactones,26,27
and dispirostachybotrylactams,20 and the stachyflins with a
pentacyclic moiety including a cis-fused decalin.21 They are
proposed to be biosynthesized by a hybrid polyketide and
isoprenoid pathway.28 These compounds display diverse
bioactivities, with targets including the complement system,14
inositol monophosphatase,27 tyrosine kinase,12 pancreatic
cholesterol esterase,13 and HIV-1 protease and endothelin,20,22
which made them attractive to pharmacologists.12,22
In summary, 11 phenylspirodrimanes, including seven new
ones, were isolated from the sponge-derived fungus S.
chartarum MXH-X73. Compound 1, discovered to be an
HIV-1 RT inhibitor, displayed inhibitory effects on HIV-1
replication against wild-type and five NNRTI-resistant HIV-1
strains. It is structurally distinct from the known non-
nucleoside reverse transcriptase inhibitors, and this discovery
provides a new class of chemotype for the search for novel
NNRTIs.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Specific rotations were
obtained on a JASCO P-1020 digital polarimeter. UV spectra were
recorded on a Beckman DU 640 spectrophotometer. IR spectra were
taken on a Nicolet Nexus 470 spectrophotometer in KBr discs. NMR
spectra were recorded on JEOL JNM-ECP 600 and Agilent 500 MHz
DD2 spectrometers using TMS as internal standard, with chemical
shifts recorded as δ values. ESIMS spectra were recorded on a Q-TOF
Ultima Global GAA076 LC mass spectrometer. HRESIMS spectra
were measured on a Micromass EI-4000 (Autospec-Ultima-TOF).
Semipreparative HPLC was performed using an ODS column [HPLC:
YMC-Pack ODS-A (5 μm, 10 × 250 mm, 3 mL/min)]. Column
chromatography (CC) was performed with silica gel (200−300 mesh,
Qingdao Marine Chemical Inc.) and Sephadex LH-20 (Amersham
Biosciences), respectively.
Figure 6. Key COSY, HMBC, and NOESY correlations of compounds
7 and 8.
established as cis by the small coupling constants (3JH‑2,H‑3 = 3.3
Hz). The relative configurations for the phenylspirodrimane
moieties of 7 and 8 were further established by NOESY
correlations (Figure 6). The absolute configuration of 7 was
postulated in analogy with 1, while the six asymmetric centers
of 8 were 2R, 3S, 5S, 8R, 9R, and10S. This is the first report of
phenylspirodrimane glucosides.
Compounds 1−11 were tested at a final concentration of 10
μM for their antiviral activities against wild-type HIV-1
replication by using a pseudotyping system. Only compound
1 (Table S1, Supporting Information) exhibited an inhibitory
effect on HIV-1 replication with an EC50 value of 8.4 μM. It
should be noted that no cytotoxicity was detected for all tested
compounds at 10 μM. The mechanism of the inhibitory effects
on HIV replication of 1 was identified using time of addition
(TOA) assays.24 Compound 1 showed a time of 50% failure
(FT50) at 12.66 h, which was between 11.59 h for the NRTI
AZT (zidovudine) and 17.78 h for the integrase inhibitor
MK0518 and the same as that of the NNRTI EFV (efavirenz,
13.06 h) (Figure 7A). The TOA results suggested that
compound 1 inhibits the HIV reverse transcription process,
and this led us to test the effect of compound 1 on reverse
transcriptase activity. Compound 1 inhibited RT RNA-
dependent DNA polymerase activity in a dose-dependent
manner with an EC50 of 50 μM (Figure 7B). As an NNRTI, an
important characteristic is the antiviral activity against NNRTI-
Fungal Material. The fungus Stachybotrys chartarum MXH-X73
was isolated from the sponge Xestospongia testudinaris derived from
Xisha Island, China. The ITS1-5.8S-ITS2 sequence of the fungus
MXH-X73 has been submitted to GenBank with the accession no.
KC295244. The voucher specimen is deposited in our laboratory at
−20 °C. The producing strain was prepared on potato dextrose agar
slants and stored at 4 °C.
Fermentation and Extraction. The fungus was grown on rotary
shakers (165 rpm) at 28 °C for 11 d in 500 mL Erlenmeyer flasks
containing liquid medium (150 mL/flask) composed of mannitol (20
g/L), maltose (20 g/L), glucose (10 g/L), monosodium glutamate (10
g/L), KH2PO4 (0.5 g/L), MgSO4·7H2O (0.3 g/L), yeast extract (3 g/
L), corn steep liquor (1 g/L), and naturally collected seawater after
adjusting its pH to 6.5. The fermented whole broth (60 L) was filtered
through cheese cloth to separate the supernatant from the mycelia.
The former was extracted three times with EtOAc to give an EtOAc
solution, while the latter was extracted three times with acetone. The
acetone solution was concentrated under reduced pressure to afford an
aqueous solution. The aqueous solution was extracted three times with
EtOAc to give another EtOAc solution. Both EtOAc solutions were
E
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Figure 7. Compound 1 is an HIV-1 reverse transcriptase inhibitor. (A) Time of addition study. The tested compounds, AZT, 1, EFV, and MK-0518,
exhibited times of 50% failure (FT50) of 11.59, 12.66, 13.06, and 17.78 h, respectively. Infectivity (%) was calculated by comparing with control. (B)
Effect of compound 1 on HIV-1 reverse transcriptase RNA-dependent DNA polymerase activity. Compound 1 showed a dose-dependent inhibitory
effect on RNA-dependent DNA polymerase activity with an EC50 at 50 μM; nevirapine (NVP), a NNRTI drug, inhibited RNA-dependent DNA
polymerase activity with an EC50 of 1.7 μM.
H2O step gradient elution to give Fr.3-2 and Fr.3-3. Fr.3-2 was purified
by semipreparative HPLC (70:30 MeOH/H2O; 3.0 mL/min) to give
compounds 11 (7 mg, tR = 27 min), 10 (8 mg, tR = 30 min), and 9 (24
mg, tR = 35 min). Fr.3-3 was further subfractionated by Sephadex LH-
20 using MeOH and then purified by semipreparative HPLC (75:25
MeOH/H2O; 3.0 mL/min) to give compound 1 (28 mg, tR = 19 min).
Fr.4 (6 g) was separated on a C18 ODS column (Fr.4-1−Fr.4-7), and
subfraction Fr.4-4 was further separated into four subfractions (Fr.4-4-
1−Fr.4-4-4) by Sephadex LH-20 using MeOH as the eluting solvent.
Subfraction Fr.4-4-2 was purified by semipreparative HPLC (75:25
MeOH/H2O; 3.0 mL/min) to give compounds 2 (36 mg, tR = 20.1
min), 3 (6 mg, tR = 23.4 min), and 4 (5 mg, tR = 24.2 min).
Subfraction Fr.4-5 was further separated by Sephadex LH-20 using
MeOH as the eluting solvent and then on a semipreparative HPLC
column (85% MeOH, 3.0 mL/min) to afford compound 5 (22 mg, tR
= 25.4 min) and compound 6 (20 mg, tR = 28.0 min). Fr.5 (4 g) was
fractionated by Sephadex LH-20 using MeOH and further purified by
semipreparative HPLC (75:25 MeOH/H2O; 3.0 mL/min) to give
compounds 7 (21 mg, tR = 29.6 min) and 8 (17 mg, tR = 26.2 min).
Table 4. Inhibitory Effects of Compound 1 on Wild-Type
and NNRTI-Resistant HIV-1 Replication
compound 1
nevirapine (NVP)
EC50 value
EC50 value
EC
compared
to wt EC50
EC50
compared
50b
(μM)
(μM)
to wt EC50
a
VSVG/HIV-1wt
8.4
7.0
1
0.023
2.3
1
99.1
VSVG/HIV-1RT‑K103N
0.8
2.8
1.6
1.7
VSVG/HIV-1RT‑L100I,K103N
VSVG/HIV-1RT‑K103N,V108I
VSVG/HIV-1RT‑K103N,G190A
VSVG/HIV-1RT‑K103N,P225H
23.8
13.3
14.2
6.2
3.3
140
7.5
317.4
2256
1013
51.9
23.3
0.7
a
b
VSV-G: vesicular stomatitis virus G protein. EC50 is the value where
infectivity is 50% that of infectivity when an equal amount of solvent is
used.
combined and concentrated under reduced pressure to give an extract
(52.0 g).
Stachybotrin D (1): colorless oil; [α]24 −24.3 (c 0.10, MeOH);
D
UV (MeOH) λmax (log ε) 216 (4.77), 261 (3.81), 302 (3.22) nm; IR
(KBr) νmax 3407, 3122, 2936, 2897, 1716, 1679, 1618, 1466, 1380,
1251, 1122, 1066, 982, 786 cm−1; 1H and 13C NMR data, see Table 1;
HRESIMS m/z 442.2587 [M + H]+ (calcd for C26H36NO5, 442.2588).
Purification. The extract (52.0 g) was applied to a silica gel (300−
400 mesh) column and was separated into six fractions (Fr.1−Fr.6)
using a step gradient elution of petroleum ether/CH2Cl2, and CH2Cl2/
MeOH (18 g) was fractionated on a C18 ODS column using MeOH/
F
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1
Stachybotrin E (3): colorless oil; [α]24D −34.0 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 218 (4.83), 261 (3.73), 301 (3.25) nm; IR
(KBr) νmax 3411, 3200, 2939, 1681, 1620, 1451, 1385, 1261, 1116,
Compound 1aS: white powder; H NMR (DMSO-d6, 500 MHz)
δH 7.25−7.48 (5H, m, Ph-MTPA), 6.73 (1H, s, H-3′), 4.79 (1H, brs,
H-3), 4.39 (1H, dd, J = 18.3 Hz, H-8′), 4.23 (1H, d, J = 18.3 Hz, H-
8′), 3.81 (3H, s, OCH3-MTPA), 3.14 (1H, d, J = 17.2 Hz, H-11), 2.79
(1H, d, J = 17.2 Hz, H-11), 2.09 (3H, s, H-11′), 2.02 (1H, s, H-2),
1.87 (1H, m, H-5), 1.79 (1H, m, H-8), 1.66 (1H, m, H-1), 1.55 (1H,
m, H-2), 1.49 (1H, m, H-7), 1.40 (1H, m, H-6), 1.38 (1H, m, H-6),
1.32 (1H, m, H-7), 1.19 (1H, m, H-1), 0.98 (3H, s, H-15), 0.90 (3H, s,
H-13), 0.68 (3H, s, H-14), 0.58 (3H, d, J = 6.5 Hz, H-12); ESIMS m/z
672.3 [M + H]+.
1
1033, 990 cm−1; H and 13C NMR data, see Table 1; HRESIMS m/z
530.2755 [M + H]+ (calcd for C29H40NO8, 530.2748).
Stachybotrin F (4): colorless oil; [α]24D −33.7 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 217 (4.79), 262 (3.77), 301 (3.20) nm; IR
(KBr) νmax 3410, 3208, 2942, 1678, 1618, 1455, 1378, 1263, 1122,
1
1030, 990 cm−1; H and 13C NMR data, see Table 1; HRESIMS m/z
530.2746 [M + H]+ (calcd for C29H40NO8, 530.2748).
Stachybocin E (5): white powder; [α]24D −5.7 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 217 (4.88), 262 (3.85), 305 (3.25) nm; IR
(KBr) νmax 3420, 2937, 2849, 1682, 1617, 1466, 1333, 1261, 1060, 945,
Preparation of (S)-PGME Amides of Compounds 3 and 4. To
a DMF solution (500 μL) of 3 (1.0 mg) and (S)-PGME (1.2 mg) were
added PyBOP (2.4 mg), HOBt (1.2 mg), and N-methylmorpholine
(20 μL), and stirring was continued at rt for 8 h. After addition of 5%
HCl (1 mL), the mixture was extracted with n-BuOH (1 mL). The
extract was washed with saturated aqueous NaHCO3 (1 mL) and brine
(1 mL) and concentrated in a vacuum to yield a residue. Then the
residue was purified by semipreparative HPLC (80% MeOH/H2O) to
yield the (S)-PGME amide (3a) of 3 (0.9 mg). The (S)-PGME amide
(4a) (0.8 mg) was prepared according to the same procedure as
described above.
1
842, 793, 754 cm−1; H and 13C NMR data, see Table 2; HRESIMS
m/z 825.5066 [M + H]+ (calcd for C50H69N2O8, 825.5048).
Stachybocin F (6): white powder; [α]24 −5.3 (c 0.10, MeOH); UV
D
(MeOH) λmax (log ε) 218 (4.69), 264 (3.72), 303 (3.12) nm; IR
(KBr) νmax 3422, 2933, 2809, 1677, 1612, 1465, 1373, 1230, 1066, 920,
1
842, 793, 753 cm−1; H and 13C NMR data, see Table 2; HRESIMS
m/z 839.5218 [M + H]+ (calcd for C51H71N2O8, 839.5205).
Stachyboside A (7): pale yellow oil; [α]24D +25.8 (c 0.10, MeOH);
UV (MeOH) λmax (log ε) 228 (4.45), 283 (3.11), 322 (3.77) nm; IR
(KBr) νmax 3425, 2938, 1696, 1650,1576, 1332, 1087, 960, 878, 772
Compound 3a: white, amorphous solid; 1H NMR (DMSO-d6, 500
MHz) δH 7.36−7.40 (5H, m, Ph-PGME), 6.54 (1H, s, H-3′), 4.93
(1H, dd, J = 9.1, 6.4 Hz, H-9′), 4.53 (1H, d, J = 16.6 Hz, H-8′), 4.34
(1H, d, J = 16.6 Hz, H-8′), 3.63 (3H, s, OCH3-PGME), 3.54 (3H, s,
H-14′), 3.19 (1H, brs, H-3), 3.11 (1H, d, J = 16.9 Hz, H-11), 2.75
(1H, d, J = 16.9 Hz, H-11), 2.30 (2H, m, H-11′), 2.19 (1H, m, H-10′),
2.08 (1H, m, H-10′), 2.04 (1H, m, H-5), 1.80 (1H, m, H-8), 1.77 (1H,
m, H-1), 1.73 (1H, m, H-2), 1.53 (1H, m, H-7), 1.47 (1H, m, H-6),
1.44 (1H, m, H-6), 1.40 (1H, m, H-7), 1.39 (1H, m, H-2), 0.95 (3H, s,
H-15), 0.92 (1H, m, H-1), 0.89 (3H, s, H-13), 0.80 (3H, s, H-14), 0.66
(3H, d, J = 6.4 Hz, H-12); ESIMS m/z 677.4 [M + H]+, 699.4 [M +
Na]+.
1
cm−1; H and 13C NMR data, see Table 3; HRESIMS m/z 573.2661
[M + Na]+ (calcd for C29H42O10Na, 573.2670).
Stachyboside B (8): pale yellow oil; [α]24D +12.2 (c 0.10, MeOH);
UV (MeOH) λmax (log ε) 229 (4.54), 286 (4.12), 325 (3.82) nm; IR
(KBr) νmax 3428, 2923, 1683, 1647,1583, 1270, 1028, 987, 843, 773
1
cm−1; H and 13C NMR data, see Table 3; HRESIMS m/z 589.2617
[M + Na]+ (calcd for C29H42O11Na, 589.2619).
Compound 2: colorless oil; [α]24 −32.3 (c 0.10, MeOH); ESIMS
D
m/z 516.2 [M + H]+.
Methylation of Compound 1. A solution of 1 (3 mg) in dry
acetone (1 mL) with anhydrous potassium carbonate (10 mg) and
methyl iodide (10 μL) was refluxed at 60−70 °C for about 6 h. The
reaction mixture was filtered, and the residue was washed thoroughly
with acetone. The combined filtrate was evaporated in a vacuum, and
the residue was purified on HPLC with 85% MeOH/H2O to afford 1a
(2.3 mg).
Compound 4a: white, amorphous solid; 1H NMR (DMSO-d6, 500
MHz) δH7.33−7.38 (5H, m, Ph-PGME), 6.59 (1H, s, H-3′), 4.79 (1H,
dd, J = 4.4, 10.6 Hz, H-9′), 4.30 (1H, d, J = 16.3 Hz, H-8′), 4.23 (1H,
d, J = 16.3 Hz, H-8′), 3.65 (3H, s, OCH3-PGME), 3.60 (3H, s, H-14′),
3.18 (1H, brs, H-3), 3.13 (1H, d, J = 16.9 Hz, H-11), 2.77 (1H, d, J =
16.9 Hz, H-11), 2.18−2.24 (2H, m, H-11′), 2.27 (1H, m, H-10′), 2.15
(1H, m, H-10′), 2.02 (1H, m, H-5), 1.82 (1H, m, H-8), 1.79 (1H, m,
H-1), 1.75 (1H, m, H-2), 1.53 (1H, m, H-7), 1.47 (1H, m, H-6), 1.44
(1H, m, H-6), 1.42 (1H, m, H-7), 1.40 (1H, m, H-2), 0.95 (3H, s, H-
15), 0.92 (1H, m, H-1), 0.88 (3H, s, H-13), 0.80 (3H, s, H-14), 0.66
(3H, d, J = 6.5 Hz, H-12); ESIMS m/z 677.4 [M + H]+, 699.4 [M +
Na]+.
Determination of the Absolute Configuration at C-9′ of
Compound 2. A solution of 2 (3 mg) in dry acetone (1 mL) with
anhydrous potassium carbonate (10 mg) and methyl iodide (10 μL)
was refluxed at 60−70 °C for about 6 h. The reaction mixture was
filtered, and the residue was washed thoroughly with acetone. The
combined filtrate was evaporated in a vacuum to dryness. The residue
was dissolved in acetone (1 mL) and then reacted with CrO3(VI) and
concentrated H2SO4 (Jones reagent, 0.3 mL) at 20 °C for 4 h. The
reaction mixture was treated with NaHSO3 and then extracted with
diethyl ether. The organic layer was washed successively with saturated
aqueous NaCl and NaHCO3 and evaporated to dryness. The oxidation
product was hydrolyzed in 6 N HC1 at 110 °C for 48 h. The solution
was evaporated to dryness and redissolved in 200 μL of H2O. Then 25
μL of 1 M NaHCO3 and 100 μL of 2.5 μM FDAA in acetone were
added, and the solution was heated for 1 h at 40 °C. The reaction was
quenched with 100 μL of 2 N HCl. The derivatized samples were
analyzed by HPLC on an analytical YMC ODS-AQ C18 column (4.6
mm, i.d. × 250 mm L, Waters Corp.) using a linear gradient from 95%
H2O (with 0.05% TFA)/5% CH3CN to 45% H2O (with 0.05% TFA)/
55% CH3CN over 55 min, and the eluent was monitored at 340 nm.
The retention time of the FDAA-derivatized hydrolysate was 34.0 min,
while those for the standard L-Glu and D-Glu were 34.3 and 35.1 min,
respectively.
1
Compound 1a: colorless oil; H NMR (DMSO-d6, 500 MHz) δH
6.72 (1H, s, H-3′), 4.45 (1H, dd, J = 18.4 Hz, H-9′), 4.35 (1H, d, J =
18.4 Hz, H-9′), 4.26 (1H, dd, J = 16.9 Hz, H-8′), 4.24 (1H, d, J = 16.9
Hz, H-8′), 4.06 (1H, d, J = 3.3 Hz, OH-3), 3.81 (3H, s, OCH3), 3.15
(1H, brs, H-3), 3.12 (1H, d, J = 17.1 Hz, H-11), 2.78 (1H, d, J = 17.1
Hz, H-11), 2.13 (3H, s, H-11′), 1.99 (1H, m, H-5), 1.78 (1H, m, H-8),
1.75 (1H, s, H-1), 1.72 (1H, m, H-2), 1.51 (1H, m, H-7), 1.43 (1H, m,
H-6), 1.39 (1H, m, H-6), 1.37 (1H, m, H-7), 1.35 (1H, m, H-2), 0.93
(3H, s, H-15), 0.90 (1H, m, H-1), 0.85 (3H, s, H-13), 0.77 (3H, s, H-
14), 0.63 (3H, d, J = 6.4 Hz, H-12); ESIMS m/z 456.3 [M + H]+.
Preparation of the (R)- and (S)-MTPA Esters of 1a. Compound
1a (1.1 mg) was dissolved in 500 μL of pyridine, and
dimethylaminopyridine (2.0 mg) and (R)-MTPACl (8 μL) were
then added in sequence. The reaction mixture was stirred for 14 h at
room temperature (rt), and 1 mL of H2O was then added. The
solution was extracted with 5 mL of CH2Cl2, and the organic phase
was concentrated under reduced pressure. Then the residue was
purified by semipreparative HPLC (95% MeOH/H2O) to yield the
(S)-MTPA ester 1aS (1.0 mg). By the same procedure, the (R)-MTPA
ester 1aR (0.9 mg) was obtained from the reaction of 1 (1.2 mg) with
(S)-MTPACl (8 μL).
1
Compound 1aR: white powder; H NMR (DMSO-d6, 500 MHz)
δH 7.24−7.48 (5H, m, Ph-MTPA), 6.71 (1H, s, H-3′), 4.78 (1H, brs,
H-3), 4.15 (1H, dd, J = 16.7 Hz, H-8′), 3.89 (1H, d, J = 16.7 Hz, H-
8′), 3.79 (3H, s, OCH3-MTPA), 3.09 (1H, d, J = 17.1 Hz, H-11), 2.74
(1H, d, J = 17.1 Hz, H-11), 2.13 (3H, s, H-11′), 1.98 (1H, s, H-2),
1.89 (1H, m, H-5), 1.77 (1H, m, H-8), 1.62 (1H, m, H-1), 1.48 (1H,
m, H-2), 1.43 (1H, m, H-7), 1.40 (1H, m, H-6), 1.36 (1H, m, H-6),
1.33 (1H, m, H-7), 1.08 (1H, m, H-1), 0.96 (3H, s, H-15), 0.93 (3H, s,
H-13), 0.89 (3H, s, H-14), 0.55 (3H, d, J = 6.5 Hz, H-12); ESIMS m/z
672.2 [M + H]+.
Acid Hydrolysis of Compound 7. Compound 7 (8 mg) was
hydrolyzed with 1 mL of 6 N HCl for 6 h at 100 °C. The mixture was
G
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extracted with EtOAc (3 × 1.5 mL), and the aqueous layer was
evaporated to dryness with H2O until neutral. The residue was
dissolved in EtOH and subjected to HPLC (Waters HPLC equipped
with a 515 HPLC pump and a Waters 2410 RID detector; column,
Chiralpak AD-H column (Daicel), 4.6 × 250 mm, 5 μm; mobile phase,
EtOH/n-hexane/TFA, 3/7/0.05 (v/v); flow rate, 0.5 mL/min).29 The
sugar was identified as D-glucose by comparison of the retention time
(tR = 12.20 min) with those of the standard samples of D-glucose (tR =
12.16 min) and L-glucose (tR = 12.84 min).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by National Natural
Science Foundation of China (Nos. 21372208, 41176120,
81273561), National High Technology Research and Develop-
ment Program of China (No. 2013AA092901), Program for
New Century Excellent Talents in University (No. NCET-12-
0499), Public Projects of State Oceanic Administration (No.
2010418022-3), Promotive Research Fund for Excellent Young
and Middle-aged Scientists of Shandong Province (No.
BS2010HZ027), and National Major Scientific and Techno-
logical Special Project for Infectious Diseases (No.
2013ZX10004601).
Anti-HIV Activity Assay by Pseudotyped Viruses. Vesicular
stomatitis virus glycoprotein (VSV-G) plasmid was cotransfected with
env-deficient HIV vector (pNL4-3.luc.R-E− or pNL4-
30
3.luc.R−E−
)
into 293T cells by using a modified Ca3(PO4)2
RT mutant
method.31 Briefly, plates were washed with PBS, and fresh media was
added 16 h after transfection. Supernatant that contains pseudotyped
virions (VSVG/HIV-wt or VSVG/HIV-RT mutant) was harvested and
filtered through a 0.45 μm filter 48 h post-transfection. Viral solution
was quantified by p24 concentrations, which were detected by ELISA
(ZeptoMetrix, cat. 0801111) and diluted to 0.2 ng p24/mL which can
be used directly or stored at −80 °C.
REFERENCES
■
One day prior to infection, 293T cells were seeded on 24-well plates
at the density of 6 × 104 cells per well. Compounds were dissolved in
DMSO and added into target cells 15 min ahead of infection. Forty-
eight hours postinfection, infected cells were lysed in 50 μL of Cell
Lysis Reagent (Promega). Luciferase activity of the cell lysate was
measured by a Sirius luminometer (Berthold Detection System)
according to the manufacturer’s instructions.
Cytotoxicity. The cytotoxicity assay was performed by incubating
compound 1 with cells for 48 h at the same culture condition as for
anti-HIV activity assay, but no viruses were added. Cytotoxicity was
measured by using Cell Proliferation Assay kit (Promega) according to
the manufacturer’s instructions.
Time of Addition Study (ref 32). HEK 293T cells were seeded at
6 × 104/well into 24-well plates and placed overnight into an
incubator at 37 °C. The following day, cells were infected with VSVG/
HIV-wt pseudoparticles. Compounds were added at a final
concentration of 10 μM respectively at different time points (0, 2, 4,
6, 8, 10, 12, 14, 16 h), with t = 0 defined as the time of pseudoparticle
addition. At 48 h postinfection, cells were lysed with 50 μL of Cell
Lysis Reagent (Promega). Luciferase activity of the cell lysate was
measured by a Sirius luminometer as mentioned before.33
RT RNA-Dependent DNA Polymerase Activity Detection
(refs 34 and 35). Tested compound was added into a mixture
containing 50 mM Tris-HCl (pH 7.8), 290 mM KCl, 30 mM MgCl2,
10 mM DTT, 11.7 ng/L poly(rA), 11.7 ng/L oligo(dT)15, 2.8 μM
dTTP, 800 nM Digoxigenin-11-dUTP, and 40 nM Biotin-11-dUTP.
The polymerization reaction was initiated by adding 0.226 ng/L
reverse transcriptase at 37 °C for 1 h. The mixture was transferred into
a streptavidin-coated microplate (Roche) and incubated at 37 °C for 1
h. Supernatant was discarded, and TMB (3,3,5,5-tetramethylbenzi-
dine) was added. After 15 min, 1 mM H2SO4 was added to terminate
the reaction, and the OD450 was measured by using a microplate reader
(Molecular Devices).
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ASSOCIATED CONTENT
■
S
* Supporting Information
The MS and NMR spectra of 1 and 3−8 and anti-HIV
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AUTHOR INFORMATION
■
Corresponding Authors
*(Y. Guo) E-mail: yingguo6@imm.ac.cn.
* (D. Li) Tel: 0086-532-82031619. Fax: 0086-532-82033054.
E-mail: dehaili@ouc.edu.cn.
Author Contributions
§X. Ma and L. Li contributed equally.
H
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