Amicoumacins from the marine-derived bacterium Bacillus sp. with the inhibition of NO production

Article abstract of DOI:10.1016/j.tetlet.2014.09.100

Chemical examination of the fermentation broth of the marine-derived bacterium Bacillus sp. resulted in the isolation of seven new amicoumacin-type isocoumarin derivatives, namely bacillcoumacins A-G (1-7), together with four known analogues. Their structures were elucidated on the basis of extensive spectroscopic analysis, while the absolute configurations of the new compounds were determined by CD, Mosher's method, and chemical conversion. Compounds 7 and 9 showed inhibitory effects against the NO production induced by lipopolysaccharide (LPS) in mouse macrophage RAW 264.7 cells.

Full text of DOI:10.1016/j.tetlet.2014.09.100

Tetrahedron Letters 55 (2014) 6286–6291
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Amicoumacins from the marine-derived bacterium Bacillus sp. with
the inhibition of NO production
Jian Bai a, Dong Liu a, Siwang Yu a, Peter Proksch b, Wenhan Lin a,
⇑
a State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China
b Institute für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, Geb.26.23, 40225 Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2014
Revised 18 September 2014
Accepted 22 September 2014
Available online 28 September 2014
Chemical examination of the fermentation broth of the marine-derived bacterium Bacillus sp. resulted in
the isolation of seven new amicoumacin-type isocoumarin derivatives, namely bacillcoumacins A–G (1–
7), together with four known analogues. Their structures were elucidated on the basis of extensive spec-
troscopic analysis, while the absolute configurations of the new compounds were determined by CD,
Mosher’s method, and chemical conversion. Compounds 7 and 9 showed inhibitory effects against the
NO production induced by lipopolysaccharide (LPS) in mouse macrophage RAW 264.7 cells.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Bacillus sp.
Isocoumarin derivatives
Bacillcoumacins
Inhibition of NO production
Marine-derived microorganisms are the prolific sources of
structurally diverse and biologically active secondary metabolites,
which are considered as a promising source for the discovery of
drug-related lead compounds.1,2 Under marine stress environ-
ment (cold, lightless, and high pressure), microorganisms
probably produce the structurally unique secondary metabolites
to play the role of chemical defense for survival.3–6 Among the
metabolites derived from marine microorganisms, amicoumacin-
based isocoumarins7 are a small group of structurally unusual
PKS-NRPS derived natural products characterized by an isopentyl
unit bonded to a dihydroisocoumarin nucleus, while the structure
variation was mainly attributed to an amide side chain.7–11 These
compounds have been reported to exhibit a range of bioactivities,
including antibacterial,11–16 cytotoxic,17 antiulcer against stress-
induced ulcers,12,13 growth inhibition against barnyard millet,10
as well as anti-inflammatory effects. Most amicoumacins were
isolated from marine-derived bacterial genus Bacillus.18 In the
course of our investigation of bioactive and structurally diverse
including seven new analogues namely bacillcoumacins A–G
(1–7). Four known analogues were identical to AI77-H (8),19
AI-77-F (9),20 AI-77-C (10),20 and AI-77-D (11),20 on the basis of
the spectroscopic analyses and comparison of their spectroscopic
data with those in literature.
natural products from marine-derived microorganisms,
a
chemical assay guided selection from our microbial library
informed that a Bacillus sp. strain from the Pacific sediments
presented the HPLC–ESIMS fingerprints with rich metabolites.
Chromatographic separation of the fermentation broth resulted
in the isolation of eleven amicoumacin derivatives (1–11),
Figure 1. CD spectra of 1–9 and 11.
⇑
Corresponding author. Tel./fax: +86 10 82806188.
E-mail address: whlin@bjmu.edu.cn (W. Lin).
http://dx.doi.org/10.1016/j.tetlet.2014.09.100
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

J. Bai et al. / Tetrahedron Letters 55 (2014) 6286–6291
6287
5'
3'
O
OH
10'
O
O
OH
H
N
5
4
8'
OH
O
8'
3
7'
N
H
1'
N
H
N
H
12'
HN
O
OH
O
O
OH
O
1
7
9
O
1
2
OH
O
3
OH
OH
OH
O
OH
O
OH
OH
O
12'
O
O
O
O
O
O
O
O
8'
N
H
N
N
10'
H
H
O
O
OH
O
OH SMe
O
O
OH SMe
O
O
4
5
6
O
OH
O
O
O
O
O
HN
O
O
H
O
N
H
N
7'
N
H
H
O
OH
R
HN
OH
10 R = CH3
11
7
8 9'R
9'S
O
O
OH
O
9
R = CH2CH3
substructure, of which six carbon resonances including two car-
bonyl carbons, a methyl carbon, two oxymethines, and a methy-
lene were observed in the DEPT spectrum. The COSY correlations
from H-90 (dH 3.69) to H-80 (dH 3.89, dd, J = 4.9, 5.8 Hz) and H2-100
(dH 3.08, 3.22) and between H-80/OH-80 (dH 5.58, d, J = 5.8 Hz),
H-90/OH-90 (dH 4.95, d, J = 5.0 Hz), and H2-100/NH (dH 7.79, t,
J = 5.5 Hz), in association with the HMBC interactions from H2-100
and NH to an acetyl carbon at dC 170.3 and from H-80 and H-90 to
the second carbonyl carbon at dC 172.5 (C-70), established an 100-
acetamido-80,90-dihydroxybutyryl moiety. In order to assign the
relative configuration of H-80 and H-90, conversion of 2 to acetonide
2a was achieved (Fig. 3). The NOE interactions between H-80 (dH
4.61) and H-90 (dH 4.33) and from the methyl protons of acetonide
at dH 1.31 to both H-80 and H-90, without the correlation of H-80 and
H-90 with the second methyl protons (dH 1.50), clarified an erythro
80,90-diol. Thus, the chiral configurations were depicted as 80R/90R
Bacillcoumacin A (1) was isolated as a colorless amorphous solid,
and its molecular formula was determined as C16H21NO5 on the
basis of the HRESIMS (m/z 308.1497 [M+H]+) and NMR data, requir-
ing seven degrees of unsaturation. The IR absorptions at 3292, 1672,
1620, and 1462 cmꢀ1 suggested the presence of hydroxy, carbonyl,
and aromatic functionalities, while the UV absorption bands at
204, 246, and 313 nm were characteristic of amicoumacins.7 The
1H NMR and COSY spectra showed an aromatic ABC spin system at
dH 6.84 (1H, d, J = 7.3 Hz, H-5), 7.50 (1H, dd, J = 7.3, 8.4 Hz, H-6),
and 6.86 (1H, d, J = 8.4 Hz, H-7) for a 1,2,3-trisubstituted aromatic
ring, as well as two methyl doublets and a number of alkyl protons.
Analyses of the COSY, HMQC, and HMBC spectra revealed 1 possess-
ing a dihydroisocoumarin nucleus, in which C-8 (dC 161.3) was posi-
tioned by a hydroxy group on the basis of a D2O exchangeable proton
at dH 10.83 (1H, s, 8-OH) correlated to C-7 (dC 115.8), C-8 and C-9 (dC
108.8) in the HMBC spectrum. The COSY couplings extended a spin
system from H3-40/H3-50 to the amide NH (dH 7.47) and H2-4 (dH
2.94), establishing an isopentyl unit linked to C-3. Thus, the partial
structure of an amicoumacin was established. In addition, the COSY
relationship between H2-80 (dH 3.86) and OH (dH 5.44) and the HMBC
correlation between H2-80 and the carbonyl carbon C-70 (dC 172.5)
led to assign a hydroxyacetyl unit. This unit was positioned at C-10
through an amide bond according to the COSY relationship between
H-10 (dH 4.21) and NH (dH 7.47, d, J = 9.5 Hz) in addition to the HMBC
interactions from C-70 to H-10 and NH. Based on the helicity rule of
the chiral benzoic ester chromophore,21 the negative sign of Cotton
effect of 1 at 259 nm for the n–p⁄ transition of the carbonyl group
reflected the half chair conformation of the heterocyclic ring
(Fig. 1), in which the side chain at C-3 is oriented equatorially.22
Thus, C-3 was determined as S configuration. Acidic hydrolysis of 1
or 80S/90S. Based on the in situ dimolybdenum CD method,23,24
a
metal complex of 2 with dimolybdenum tetraacteate Mo2(OAc)4
in DMSO was generated. The signs of the induced CD bands at
300 (band IV) reflected the O–C–C–O torsion angle, while the neg-
ative Cotton effect (CE) observed at 300 nm (Fig. 4) permitted the
assignment of 80S and 90S configurations. Apart from erythro
(1R,2S)-indane-1,2-diol,24 this is an additional example to apply
ICD data for the configurational determination of erythro diol.
Bacillcoumacin C (3) had a molecular formula of C21H28N2O7 as
determined by the HRESIMS (m/z 443.17751 [M+Na]+) data. Com-
parison of the NMR data (Tables 1 and 2) revealed the gross struc-
ture of 3 closely related to xenocoumacin.15 The distinction was
found at the terminal moiety, where a -lactam ring of 3 replaced
the pyrrolidine of the known analogue. This finding was evident
yielded a chromophoric moiety 1a (Fig. 2), whose NMR data as well
25
as the sign and the magnitude of specific rotation ([
a
]
D
ꢀ52.0) were
25
similar to those of the hydrolyzed product of AI-77-C (10) ([a]
D
O
ꢀ43.0).20 Thus, the absolute configuration of the stereogenic center
C-10 was the same as that of AI-77-C, indicating 10S configuration.
The molecular formula of bacillcoumacin B (2) was established
as C20H28N2O7 by the HRESIMS (m/z 409.1954 [M+H]+) and NMR
data. Comparison of the NMR spectroscopic data (Tables 1 and 2)
revealed both 2 and 1 sharing the partial structure of dihydroiso-
coumarin nucleus. The difference was attributed to the amide
O
N
H
R
NH2
HCl
O
+
R
HO
O
OH
O
- ,
OH
O
1a
1 8 11
Figure 2. Chemical conversion of 1–8 and 11 to 1a.

6288
J. Bai et al. / Tetrahedron Letters 55 (2014) 6286–6291
Table 1
1H and 13C NMR data for bacillcoumacins A–G (1–7)
1a
2b
3a
4a
5a
6a
7a
3
4
4.70, br t (7.2)
2.94, d (7.2)
4.71, br dd (2.5, 12.0) 4.71, br dd (2.5,12.6) 4.70, br t (7.6)
4.71, br dd (2.5,12.6) 4.71, br dd (2.5,12.6) 4.70, br dd (2.5,12.6)
2.95, dd (2.5, 16.0) 2.95, dd (2.5, 16.0) 2.90, dd (2.5, 16.0)
3.00, dd (12.6, 16.0) 2.98, dd (12.6, 16.0) 2.92, dd dd (12.6,
16.0)
2.88, dd (2.5, 16.8)
3.02 dd (12.0, 16.8)
2.87, dd (2.5, 16.3) 2.96, d (7.6)
3.04, dd (12.6, 16.3)
5
6
7
10
6.84, d (7.3)
7.50, dd (7.3, 8.4)
6.86, d (8.4)
4.21, ddd (9.5, 10.4, 4.20, ddd (9.5, 10.4,
12.8) 12.8)
1.37, dd (10.4, 12.4) 1.35, dd (10.4, 12.4)
1.70, dd (12.4, 12.8) 1.71, dd (12.4, 12.8)
6.84, d (7.3)
7.50, dd (7.3, 8.3)
6.86, d (8.3)
6.83, d (7.4)
7.50, dd (7.4, 8.3)
6.86, d (8.3)
6.85, d (7.4)
7.50, dd (7.4, 8.3)
6.87, d (8.3)
6.84, d (7.7)
7.50, dd (7.7, 8.7)
6.87, d (8.7)
6.84, d (7.8)
7.50, dd (7.8, 8.8)
6.87, d (8.8)
6.84, d (7.2)
7.49, dd (7.2, 8.3)
6.86, d (8.3)
4.20, ddd (9.5, 10.4, 4.19, ddd (9.2, 10.4, 4.21, ddd (9.5, 10.4, 4.22, ddd (9.5, 10.4, 4.21, ddd (9.2, 10.4,
12.8) 12.8) 12.8) 12.8) 12.8)
1.34, dd (10.4, 12.4) 1.31, dd (10.4, 12.4) 1.32, dd (10.4, 12.4) 1.32, dd (10.4, 12.4) 1.36, dd (10.4, 12.4)
1.71, dd (12.4, 12.8) 1.72, dd (12.4, 12.8) 1.76, dd (12.4, 12.8) 1.68, dd (12.4, 12.8) 1.64, dd (12.4, 12.8)
20
30
40
50
80
90
1.60, m
1.66, m
1.67, m
1.57, m
1.61, m
0.92, d (6.5)
0.86, d (6.5)
4.31, dd (2.7, 5.8)
4.64, dd (2.4, 2.7)
1.67, m
1.59, m
0.91, d (6.4)
0.86, d (6.4)
3.86 s
0.91, d (6.5)
0.86, d (6.5)
3.89, dd (4.9, 5.8)
3.69, dddd (2.5,
4.9,5.0,11.0)
3.08, ddd
(5.5,11.0,12.0)
3.22, ddd (2.5, 5.5,
12.0)
0.91, d (6.5)
0.87, d (6.5)
3.93, dd (4.8, 6.2)
3.58, ddd
(4.8,5.4,9.8)
3.68, m
0.91, d (6.5)
0.84, d (6.5)
4.31, dd (2.7, 5.5)
4.80, m
0.91, d (6.5)
0.85, d (6.5)
4.22, m
0.91, d (6.5)
0.86, d (6.5)
5.11, s
4.67, dd (2.4, 4.0)
100
2.06, m
2.07, m
3.50, ddd (2.4,2.7,
8.8)
3.60, m
2.77, t (7.6)
110
120
1.92, m
2.02, m
2.01, m
2.45, m
2.46, m
2.42 dd (2.7, 18.4)
3.08 dd (8.8, 18.4)
2.53, dd (4.4, 16.0) 2.38, t (7.6)
2.99, dd (9.0, 16.0)
2.09, m
NH
7.47, d (9.5)
10.83, s
5.44, br s
7.60, d (9.5)
10.84, s
5.58, d (5.8)
4.95, d (5.0)
7.79 t (5.5)
1.81, s
7.66, d (9.5)
10.84, s
5.53, d (6.2)
5.08, d (5.4)
7.35, s
7.81, d (9.2)
10.82, s
6.18, d (5.5)
7.91, d (9.5)
10.82, s
6.39, d (5.8)
7.66, d (9.5)
10.82, s
6.30, d (5.2)
7.91, d (9.2)
10.84, s
OH-8
OH-80
OH-90
NH
10.42, s
Ac
SMe
2.03, s
2.14, s
a
Recorded in DMSO-d6, at 400 MHz
Recorded in DMSO-d6, at 500 MHz.
b
Table 2
in agreement with an erythro configuration of the diol,25 the same
as that of 2. Based on the in situ dimolybdenum tetraacteate [Mo2(-
OAc)4] CD method,23,24 the negative Cotton effect around 300 nm
(band IV) (Fig. 4) was in agreement with 80S and 90S configurations.
13C NMR data for bacillcoumacins A–G (1–7)a
a
b
a
a
a
a
a
Position
1
2
3
4
5
6
7
1
3
4
5
6
7
8
9
10
10
20
30
40
50
70
80
90
100
110
120
130
Ac
169.4
81.5
169.5
81.6
169.6
81.6
169.3
81.4
169.4
81.4
29.6
119.0
136.8
115.8
161.3
108.8
140.8
48.8
39.4
24.6
23.9
21.8
170.3
72.5
85.8
40.0
36.2
169.4
81.5
29.7
119.0
136.8
115.8
161.3
108.8
140.8
48.9
39.6
24.2
23.9
21.9
171.0
71.2
85.2
41.0
35.6
169.6
81.2
29.8
119.0
136.8
115.8
161.3
108.8
140.8
22.0
23.7
24.7
39.8
48.4
168.2
92.3
154.5
25.9
27.8
177.7
The JH-9 /H-10 (9.8 Hz) value indicated both H-90 and H-100 to be in
an anti conformation. Thus, there are two possible relative confor-
mations for the chiral center C-100 (Fig. 5). Irradiation of H-80
induced the NOE enhancement of the lactam NH (dH 7.35), indicat-
ing that 3 adopted 3a confirmation. Thus, the absolute configura-
tion of C-100 was supposed to be 100R.
0
0
29.7
29.6
29.6
29.6
119.0
136.8
115.8
161.3
108.8
140.8
48.4
119.0
136.8
115.7
161.3
108.8
141.1
48.4
119.0
136.7
115.7
161.4
108.8
141.2
48.5
119.0
136.8
115.8
162.4
109.4
140.7
48.7
The molecular formula of bacillcoumacin D (4) was established
as C20H25NO7 by the HRESIMS (m/z 390.15523 [MꢀH]ꢀ) and NMR
data. Apart from the NMR data for the dihydroisocoumarin which
was identical to that of 3, the remaining NMR resonances of 4
showed six carbon signals instead of seven of 3. Interpretation of
the COSY and HMBC correlations, such as the HMBC interaction
of the oxymethine proton H-90 (dH 4.80) with the carbonyl carbon
at dC 177.7 (C-120), disclosed the side chain to be terminated by a -
lactone. This unit was bonded to the hydroxymethine C-80 (dC 72.2)
according to the HMBC interactions from OH-80 (dH 6.18, d,
J = 5.5 Hz) to C-70 (dC 170.8), C-80, and C-90. According to the bulk-
iness rule,26,27 a Rh2(OCOCF3)4 complex of 4 was generated in
CHCl3. The positive sign of Cotton effect for E band at 346 nm in
the CD spectrum (Fig 6) was in agreement with 80S configuration.
39.8
39.7
39.7
40.2
24.6
24.4
24.5
24.6
23.7
23.8
23.8
23.8
22.0
22.0
22.0
21.7
172.5
61.7
172.5
73.7
173.0
73.8
170.8
72.2
71.9
73.0
80.8
40.2
55.4
21.1
21.4
28.5
30.4
177.7
175.8
175.2
177.4
170.3
23.1
SMe
13.3
13.3
a
Recorded in DMSO-d6, at 100 MHz.
Recorded in DMSO-d6, at 125 MHz.
b
0
0
In addition, the JH-8 /H-9 (2.7 Hz) value was indicative of cis confor-
mation of H-80 and H-90. Thus, four possible relative configurations
(Fig. 7) were depicted. Irradiation of the amide NH (dH 7.81, d,
J = 9.2 Hz) induced the NOE enhancement of H-90 (dH 4.80, m)
and H2-100 (dH 2.06, 2.07, m), while these results were in agree-
ment with 4d conformation.28 Accordingly, the stereogenic center
C-90 was determined as 90S configuration.
from the COSY correlation between H-100 (dH 3.68, m) and NH (dH
7.35) in association with the HMBC interactions from the carbonyl
carbon at dC 177.4 (C-130) to H-100, NH (dH 7.35), H2-110 (dH 1.92,
2.02), and H2-120 (dH 2.01, 2.09). The JH-8 /H-9 (4.8 Hz) value was
0
0

J. Bai et al. / Tetrahedron Letters 55 (2014) 6286–6291
6289
O
O
O
OH
H
N
H
H
pyridinium
8'
p-toluene sulfonate
N
H
N
H
N
H
8O'
O
O
O
OH
O
2,2-dimethyoxypropane
NOE
OH
O
2a
OH
O
2
Figure 3. Chemical conversion of 2 to 2a and the NOE interactions of 2a.
2
1
4+
2
3
[Mo24+
[Mo2
]
]
0
H
O
H
Mol. CD
H
-1
-2
-3
8'
[Mo2OAc4]
O
O
7'
R
CH2R'
10'
H
250
300
350
Wavelength [nm]
400
450
negative chirality
Figure 4. ICD curves of 2 and 3 induced by Mo2(OAc)4 in DMSO-d6.
OH
9'
11'
10'
H
CH2
8' HC
3a
H
NH
9'
10'
8'
OH
9'
10'
H
NH
H
8' HC
3b
CH2
11'
NOE
Figure 5. Key NOE correlations and the Newman projects of 3.
instead of a methylene conducted to assign C-100 of 5 being substi-
tuted. The observation of a methyl singlet (dH 2.03, s) correlated to
C-100 and in turn H-100 (dH 3.50, ddd, J = 2.4, 2.7, 8.8 Hz) to the
methyl carbon (dC 13.3) in the HMBC spectrum deduced C-100 to
be bonded by a methylthio group. On the basis of Mosher’s
method,20 the stereogenic center at C-80 was determined as S
configuration (Fig. 8). The NOE interactions observed from the
methylthio protons (dH 2.03) to H-90 (dH 4.64, dd, J = 2.4, 2.7 Hz)
and H-11a0 (dH 2.42, dd, J = 2.7, 18.4 Hz) and between H-90 and
H-11a0 clarified the same orientation of H-90 and the methylthio
group. A cis relationship of H-80 and H-90 was recognized by the
0.08
0.05
4
(Rh)
8 (Rh)
Mol. CD
0
0
0
JH-8 /H-9 (2.7 Hz) value, which resulted in four possible relative con-
formations (5a–5d) (Fig. 7). The NOE interactions from amide NH
(dH 7.91, d, J = 9.5 Hz) to H-90 and H-100 revealed 5a to be a suitable
confirmation. Accordingly, the stereogenic center C-90 was
determined as 90R configuration, while C-100 was assigned to S
configuration.
-0.05
-0.06
300
350
400
450
Wavelength [nm]
Figure 6. ICD curves of 4 and 8 induced by Rh2(OCOCF3)4 in CHCl3.
Analysis of the NMR and MS data revealed the same gross struc-
ture of bacillcoumacin F (6) and 5. Calculation of the
Dd(dR–dS) data
of the MPA esters of 620 (Fig. 6) assigned the same configuration of
C-80 as that of 5, while the same orientation of methylthio group
toward H-90 (dH 4.67) was evident from the NOE interaction
Bacillcoumacin E (5) has a molecular formula of C21H27NO7S as
provided by the HRESIMS (m/z 897.29213 [2M+Na]+) data, contain-
ing a sulfur atom. The close similarity of the NMR data between 5
and 4 with the exception of C-100 (dC 40.0) of 5 to be a methine
between H-90 and SMe (dH 2.14, s). Similarly, the JH-8 /H-9 (2.4 Hz)
0
0

6290
J. Bai et al. / Tetrahedron Letters 55 (2014) 6286–6291
8'
H
8'
H
8'
H
8'
H
9'
H
10'
9'
H
9'
H
9'
H
10'
O
CH2
O
H2C
OH
OH
HNOC
HNOC
OH
OH
HNOC
HNOC
CH2
10'
CH2
O
O
10'
4d
4a
4b
4c
8'
8'
8'
H
8'
H
H
H
9'
H
9'
H
9'
H
10'
10'
9'
H
O
CHSMe
OH
MeSHC
O
OH
HNOC
OH
OH
HNOC
HNOC
HNOC
CHSMe
O
CHSMe
O
10'
5d
10'
5c
5a
5b
8'
H
8'
H
8'
8'
H
H
9'
9'
H
9'
H
10'
9'
H
O
H
CHSMe
10'
MeSHC
O
OH
CHSMe
HNOC
OH
OH
CHSMe
HNOC
HNOC
OH
HNOC
O
O
NOE
10'
6d
10'
6c
6a
6b
Figure 7. Newman projects for C-80/C-90 and key NOE interactions for 4–6.
dose less than 10 lM. The inhibitory effects of these compounds
O
against NO production induced by LPS in mouse macrophage
RAW 264.7 cells were tested. As shown in Table 3, compounds 7
and 9 exhibited significantly inhibitory activities toward the pro-
O
O
+0.05
-0.02
0.00
-0.14
+0.005 +0.09
-0.07
-0.02
duction of NO with the IC50 values of 27.8 and 30.9
lM, respec-
N
+0.07
H
tively, comparable to the data for positive control curcumine
+0.24
O
OMPA S
-0.05
(IC50 = 20.5 l c-lactam
M).29 Compound 7 is the only one bearing a
+region
-region
with a double bond resided at C-80/C-90, suggesting the olefin bond
C-80/C-90 in the scaffolds contributed as a functional group. For the
OH
O
5
analogues with an unsaturated c-lactone (8 and 9), the stereogenic
O
center C-90 directly affected the inhibitory effects, as evident from
O
+0.07
N
+0.03
+region
AI-77-F with 90S (9) showing stronger inhibition than AI-77-H with
O
-0.15
-0.11
+0.12
+0.04
90R (8). The NO is produced from the enzymatic oxidation of
L-argi-
-0.10
-0.14
-0.45
nine by iNOS in numerous mammalian cells and tissues, and serves
as a key signaling molecule in physiological processes, such as host
defense, neuronal communication, and vascular regulation.30,31
Amicoumacin A has been reported to exhibit potent anti-inflam-
matory activity to reduce carrageenan mice paw edema, but the
high toxicity limited its application.32,33 The potent inhibition of
7 and 9 against NO production with low cytotoxicity implied these
analogues to be possible for the development as anti-inflammatory
agents.
Biogenetically, amicoumacin-based isocoumarins are consid-
ered to be synthesized by the hybrid pathway involving non ribo-
somal peptide synthetases (NRPSs) and polyketide synthtases
(PKSs). It is noted that all amicoumacins from nature exclusively
possess a leucine unit to take part in the biosynthesis, implying
the NRPS enzymes stringently selecting amino acid. Thus,
detection of the specific NRPS synthesis gene may provide a
+0.05
H
O
OMPA S
-region
0.00
OH
O
6
Figure 8.
Dd
RS(dR–dS) data for the MPA esters of 5 and 6.
value for a cis relationship of H-80 and H-90 resulted in four possible
relative conformations (6a–6d). However, the observation of the
NOE interactions from H-80 (dH 4.22, m) to H-90 and H-100 (dH
3.60, m) and the absence of NOE interaction between amide NH
and H-100 clarified a suitable conformation of 6 to be 6b. Thus,
the absolute configurations at the lactone were assigned to 90S
and 100R.
Bacillcoumacin G (7) has a molecular formula of C20H24N2O5 as
provided by the HRESIMS and NMR data. Its NMR spectroscopic data
(Tables 1 and 2) closely resembled those of 4, except for C-80
(dC 92.3)/C-90 (dC 154.5) to be resided by a double bond. This
assignment was supported by the HMBC correlations from H-80
(dH 5.11) to C-70 (dC 168.2) and C-100 (dC 25.9). The NOE correlation
between H-80 and H2-100 (dH 2.77) was indicative of 80Z geometry.
Compounds 1–9 and the known analogue 11 were hydrolyzed
with 4 M HCl, respectively, to derive the same chromophoric prod-
uct (1a) (Fig. 2), indicating the new compounds sharing the same
absolute configurations (3S and 10S) in the nucleus part of 1-7.
Compounds 1–11 were measured against five human tumor cell
lines, including BGC-823, HCT-116, HepG2, NCI-H1650, and A2780
by the MTT method, showing weak inhibitory activities with the
Table 3
Inhibitory activities of
7 and 9 on NO production induced by LPS in mouse
macrophage RAW 264.7 cellsa,b,c
Compound
IC50 (lM)
7
9
30.9 0.8
27.8 1.6
20.5 2.0
Curcumined
a
No cytotoxicity was observed.
Data are mean SD (n = 3).
The other compounds were inactive (IC50 >50
Positive control.
b
c
l
M).
d

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