Bioactive alkaloids from the actinomycete Actinoalloteichus sp. ZZ1866

Article abstract of DOI:10.1021/acs.jnatprod.0c00588

The new alkaloids marinacarbolines E?Q (1?10, 12?14), caerulomycin N (15), and actinoallonaphthyridine A (16), together with the known marinacarboline C (11) and cyanogramide (17), were isolated from the actinomycete Actinoalloteichus sp. ZZ1866. The structures of the isolated compounds were elucidated based on their HRESIMS data, extensive NMR spectroscopic analyses, Mosher’s method, ECD calculations, single-crystal X-ray diffraction analysis, and chemical degradation studies. Marinacarbolines E?L (1? 8) share an indole-pyridone-imidazole tetracyclic skeleton, which is the first example of this kind of skeleton. Caerulomycin N (15) and cyanogramide (17) exhibited cytotoxic activity against both human glioma U251 and U87MG cells with IC₅₀ values of 2.0?7.2 μM. Marinacarbolines E (1), G (3), I (5), and M (9) showed cytotoxic activity against U87MG cells with IC₅₀ values of 2.3?8.9 μM.

Full text of DOI:10.1021/acs.jnatprod.0c00588

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Article
Bioactive Alkaloids from the Actinomycete Actinoalloteichus sp.
ZZ1866
Le Qin, Wenwen Yi, Xiao-Yuan Lian,* and Zhizhen Zhang*
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sı Supporting Information
ABSTRACT: The new alkaloids marinacarbolines E−Q (1−10, 12−14),
caerulomycin N (15), and actinoallonaphthyridine A (16), together with the
known marinacarboline C (11) and cyanogramide (17), were isolated from the
actinomycete Actinoalloteichus sp. ZZ1866. The structures of the isolated
compounds were elucidated based on their HRESIMS data, extensive NMR
spectroscopic analyses, Mosher’s method, ECD calculations, single-crystal X-ray
diffraction analysis, and chemical degradation studies. Marinacarbolines E−L (1−
8
) share an indole-pyridone-imidazole tetracyclic skeleton, which is the first
example of this kind of skeleton. Caerulomycin N (15) and cyanogramide (17)
exhibited cytotoxic activity against both human glioma U251 and U87MG cells with IC values of 2.0−7.2 μM. Marinacarbolines E
5
0
(
1), G (3), I (5), and M (9) showed cytotoxic activity against U87MG cells with IC values of 2.3−8.9 μM.
5
0
he genus Actinoalloteichus was initially described as a new
isolated from the deep sea actinomycete Marinactinospora
7a
T
taxon in 1984, and the type species was Actinoalloteichus
thermotolerans SCSIO, and their biosynthesis has been
1
7b
cyanogriseus Y388 isolated from a soil sample. Later in 2000,
Actinoalloteichus was confirmed as a new genus (family
Pseudonocardiaceae) with a new species, Actinoalloteichus
investigated. Herein, we described the isolation of strain
ZZ1866 and the structural elucidation and bioactive evaluation
of these isolated compounds.
2
cyanogriseus sp. nov. Since then, a number of Actinoalloteichus
strains have been isolated, including several new species of A.
spitiensis sp. nov. from the cold desert of the Indian
RESULTS AND DISCUSSION
■
3
a
The strain ZZ1866 (Figure S1, Supporting Information) was
classified as Actinoalloteichus sp. ZZ1866 according to its 16S
rDNA sequence, which was very close to those of several
Actinoalloteichus strains (Table S1). The EtOAc extract from
the culture of strain ZZ1866 in rice medium was separated by
column chromatography, followed by HPLC purification, to
give compounds 1−17.
Based on the NMR data (Tables S2 and S3) and specific
rotation values as well as the comparison with the data of
references, compound 11 was identified as marinacarboline
Himalayas, A. hymeniacidonis sp. nov. from the marine
3b
sponge Hymeniacidon perleve, A. nanshanensis sp. nov. from
3c
the rhizosphere of Ficus religiosa, and A. hoggarensis sp. nov.
3
d
from Saharan soil. It has been reported that the Actino-
alloteichus actinomycetes produced different types of secondary
4
metabolites. Over 60 compounds have been isolated and
identified from the wild and mutant strains of marine
sediment-originated A. cyanogriseus WH1-2216-6, including
the caerulomycin bipyridine alkaloids, the cyanogriside
bipyridine glycosides, and polycyclic tetramate macrolactams.
Some of these compounds exhibited cytotoxic, antimicrobial,
7a
C, and 17 was proved to be cyanogramide, previously
4
,5
isolated from the marine-derived Actinoalloteichus cyanogriseus
or immunosuppressive activities.
4d
WH1-2216-6.
As part of our ongoing research program to discover novel
Compound 1 was obtained as colorless orthorhombic
crystals and had a molecular formula of C H N O deduced
from its HRESIMS ion at m/z 422.1479 [M + Na] and
NMR data. The molecule 1 showed UV absorptions at 235,
94, and 363 nm for conjugated double bonds and an IR
6
bioactive agents from marine microorganisms, the actino-
2
4
21
3
3
mycete Actinoalloteichus sp. ZZ1866 was isolated from a sea
mud sample collected from the coastal area of Putuo,
Zhoushan, China. The extract prepared from a large-scale
culture of this strain in rice medium showed antiproliferative
activity against human glioma U87MG and U251 cells with
+
13
C
2
Received: May 25, 2020
1
00% inhibition at a concentration of 1.0 mg/mL. Chemical
investigation of this active extract resulted in the isolation of 17
alkaloids, including 15 new ones (1−10 and 12−16).
Compounds 1−10 and 12−14 are analogues of marinacarbo-
lines A−D and named as marinacarbolines E−Q, respectively.
Marinacarbolines A−D are antimalarial β-carboline alkaloids
©
XXXX American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c00588
A
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Chart 1
Table 1. 13C NMR Data of Compounds 1−8 (150 MHz, DMSO-d6)
no.
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
140.8, C
111.1, CH
127.3, CH
121.2, CH
121.9, CH
121.4, C
123.3, C
129.0, C
152.3, C
139.5, C
112.8, CH
127.0, CH
120.7, CH
121.6, CH
122.4, C
122.7, C
130.5, C
151.5, C
140.8, C
111.1, CH
127.4, CH
121.2, CH
121.8, CH
121.5, C
123.2, C
128.7, C
152.5, C
139.8, C
113.1, CH
127.0, CH
120.7, CH
121.7, CH
122.5, C
122.7, C
130.7, C
152.0, C
140.8, C
111.0, CH
127.2, CH
121.0, CH
121.8, CH
121.4, C
123.2, C
128.6, C
152.6, C
140.7, C
110.9, CH
127.0, CH
120.8, CH
121.8, CH
121.4, C
123.2, C
128.8, C
152.8 C
140.8, C
111.0, CH
127.3, CH
121.0, CH
121.8, CH
121.4, C
123.2, C
128.6, C
152.6 C
140.8, C
111.0, CH
127.3, CH
121.0, CH
121.8, CH
121.4, C
123.2, C
128.6, C
152.5 C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
126.4, C
100.1, CH
157.5, C
126.1, C
100.2, CH
157.6, C
127.1, C
99.8, CH
156.5, C
126.8, C
100.2, CH
156.8, C
127.4, C
98.5, CH
159.7, C
127.5, C
97.5, CH
159.1, C
127.5, C
98.4, CH
160.2, C
127.5, C
98.6, CH
160.5, C
102.2, C
101.9, C
101.5, C
101.3, C
101.8, C
99.2, C
102.1, C
101.8, C
21.4, CH3
118.6, CH
116.7, CH
135.8, C
125.9, CH
128.8, CH
127.4, CH
128.8, CH
125.9, CH
49.3, CH3
31.5, CH3
21.4, CH3
118.6, CH
116.8, CH
135.7, C
125.8, CH
128.6, CH
127.1, CH
128.6, CH
125.8, CH
49.1, CH3
22.4, CH3
116.1, CH
129.0, CH
135.1, C
128.6, CH
128.2, CH
127.8, CH
128.2, CH
128.6, CH
49.0, CH3
31.4, CH3
22.5, CH3
116.2, CH
128.9, CH
135.2, C
128.6, CH
128.2, CH
127.8, CH
128.2, CH
128.6, CH
49.0, CH3
21.4, CH3
40.3, CH2
33.7, CH2
138.6, C
128.8, CH
128.5, CH
126.5, CH
128.5, CH
128.8, CH
48.8, CH3
31.4, CH3
22.5, CH3
40.4, CH2
34.4, CH2
138.9, C
128.7, CH
128.5, CH
126.4, CH
128.5, CH
128.7, CH
22.1, CH3
47.5, CH2
69.2, CH
143.2, C
126.0, CH
128.3, CH
127.4, CH
128.3, CH
126.0, CH
48.8, CH3
31.4, CH3
21.0, CH3
47.3, CH2
69.4, CH
143.2, C
126.3, CH
128.2, CH
127.4, CH
128.2 CH
126.3, CH
48.9, CH3
31.4, CH3
31.3, CH3
−
1
absorption band at 1713 cm for a carbonyl group. Analyses
methylindole based on the COSY and HMBC correlations
(Table S4). The ring E-related structure was recognized as a
styrene group by analysis of its characteristic NMR data
(Tables 1 and 2), which was further confirmed by COSY and
HMBC correlations. The remaining structural part related to
rings C and D was proposed to be a pyridone−imidazole
fusion with methoxy and methyl substituents at C-13, as
1
13
of its H, C, and HMQC spectra indicated the presence of
two carbonyls, nine pairs of double bonds, one methoxy, one
nonprotonated carbon, and two methyls. The two carbonyls
and nine double bonds accounted for 11 out of the 16 degrees
of unsaturation required by the molecular formula, and the
remaining five suggested a structure with five rings for 1. The
1
3
1
6
/5 bicyclic fusion for rings A and B was identified as N-
determined by its C and H NMR data and HMBC
B
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Article
1
Table 2. H NMR Data of Compounds 1−8 (600 MHz, DMSO-d , J in Hz)
6
no.
1
2
3
4
5
6
7
8
2
7.73, d
7.60, d
(8.0)
7.48, t
(8.0)
7.27, t
(8.0)
8.26, d
(8.0)
7.73, d
(8.0)
7.57, t
(8.0)
7.32, t
(8.0)
8.26, d
(8.0)
7.59, d (8.1) 7.69, d (8.0)
7.46, t (8.1) 7.55, t (8.0)
7.25, t (8.1) 7.31, t (8.0)
8.20, d (8.1) 8.26, d (8.0)
7.69, d (8.0)
7.71, d (7.9)
7.56, t (7.9)
7.29, t (7.9)
8.28, d (7.9)
7.70, d (8.0)
7.56, t (8.0)
7.31, t (8.0)
8.27, d (8.0)
(
7.7)
3
4
5
7.58, t
7.7)
7.54, t (8.0)
7.29, t (8.0)
8.25, d (8.0)
(
7.34, t
7.7)
(
8.31, d
7.7)
(
1
1
1
1
4
5
8.00, s
2.32, s
7.28, d
7.99, s
2.32, s
7.29, d
(15.0)
7.04, d
(15.0)
7.54, d
(7.8)
7.35, t
(7.8)
7.25, t
(7.8)
7.35, t
(7.8)
7.54, d
(7.8)
7.88, s
2.20, s
5.96, d
(9.3)
6.65, d
(9.3)
7.34, d
(7.5)
7.28, t
(7.5)
7.22, t
(7.5)
7.28, t
(7.5)
7.34, d
(7.5)
7.86, s
2.21, s
7.82, s
2.06, s
7.72, s
2.03, s
3.58, t (8.0)
7.83, s
2.23, s
7.84, s
1.74, s
5.97, d (9.3) a: 3.63, m;
b: 3.46, m
6.64, d (9.3) 3.02, m
a: 3.51, dd (14.5, 2.6); b: 3.36, a: 3.64, dd (14.3, 7.6); b: 3.28,
dd (14.5, 9.6)
5.13, dd (9.6, 2.6)
(
15.0)
7.04, d
15.0)
7.55, d
7.7)
7.37, t
7.7)
7.25, t
7.7)
7.37, t
7.7)
7.55, d
7.7)
dd (14.3, 6.3)
5.20, br t (6.3)
1
1
1
2
2
2
6
8
9
0
1
2
3.00, t (8.0)
(
7.34, d (7.7) 7.31−7.33, m
7.28, t (7.7) 7.31−7.33, m
7.22, t (7.7) 7.22, m
7.29−7.34, m 7.47, d (7.7)
7.29−7.34, m 7.38 d (7.7)
7.41, d (7.6)
7.33 d (7.6)
7.25, t (7.6)
7.33 d (7.6)
7.41, d (7.6)
(
(
7.22, m
7.31, t (7.7)
(
7.28, t (7.7) 7.31−7.33, m
7.34, d (7.7) 7.31−7.33, m
7.29−7.34, m 7.38 d (7.7)
7.29−7.34, m 7.47, d (7.7)
2.80, s
(
(
2
2
3
4
2.99, s
4.32, s
2.97, s
2.94, s
4.31, s
2.92, s
2.81, s
4.27, s
2.84, s
4.27, s
4.30, s
4.29, s
NH
OH
12.63, s
12.58, br s
5.67, br s
5.66, br s
correlations. The HMBC correlations of H-11 with C-6 and C-
established the 5/6 bicyclic fusion for rings B and C.
skeleton as 1, with structural differences at the indole nitrogen
and the C15−16 configuration. Compound 2 was the N-
demethyl analogue of 1, because the N-CH (δ 31.5; δ 4.32,
s) in 1 was replaced by a NH group (δH 12.63, s) in 2.
Compounds 3 and 4 were 15-cis double-bond isomers of 1 and
8
Similarly, HMBC correlations of H-15 with C-12 and C-13
demonstrated the position of the styrene group. The trans-
coupling constant of 15.0 Hz between H-15 and H-16
indicated a 15E configuration. Single-crystal X-ray diffraction
analysis [Cu Kα radiation, Flack/Hooft parameter: −0.05(8)/
3
C
H
2
, respectively, as indicated by a small coupling constant (9.3
Hz) between H-15 and H-16 observed in 3 and 4. A shared
biogenesis, along with all negative specific rotations, suggested
the same 13S absolute configuration for 1−4. The structures of
−
0.01(7)] of 1 confirmed its structure and established a 13S
absolute configuration (Figure 1). Based on the foregoing
2
−4 were thus elucidated as three new β-carboline alkaloids,
named marinacarbolines F−H, respectively.
Compounds 5−8 showed similar characteristic UV
absorptions and positive specific rotation values. The
molecular formulas C H N O for 5, C H N O for 6,
2
4
23
3
3
23 21
3
3
and C H N O for 7 and 8 were deduced from their
2
4
23
3
4
1
3
13
HRESIMS and C NMR data. Further analyses of their
and H NMR data (Tables 1 and 2) and 2D NMR correlations
C
1
(
Figure 2 and Tables S8−S10, S12) demonstrated that 5−8
were analogues of 1 lacking the C-15/C-16 double bond with
differences in the substitution at C-13 and C-16.
Figure 1. X-ray crystal structure of marinacarboline E (1, Cu Kα
radiation, displacement ellipsoids are drawn at the 30% probability
level).
Compound 5 had two mass units higher than that of 1. In
the NMR spectra, the signals for the C15−16 double bond (δC
1
18.6, 116.7; δ 7.28, 7.04) in 1 were replaced by the signals
H
for a −CH CH − group (δ 40.3, 33.7; δ 3.63, 1H, 3.46, 1H,
2
2
C
H
evidence, the structure of 1 was elucidated as a new β-carboline
alkaloid with a unique indole-pyridone-imidazole tetracyclic
skeleton, named marinacarboline E.
3
.02, 2H) in 5. These data indicated the presence of a
phenylethane group in 5, instead of the styrene group in 1.
Electronic circular dichroism (ECD) calculations were used to
determine the absolute configuration of 5, and the results
Compounds 2−4 had very similar UV absorptions and
NMR spectra to those of 1, implying that they are analogues.
HRESIMS data suggested the same molecular formula
C H N O for 1 and 3 and C H N O for 2 and 4.
(
Figure 2) supported a 13S configuration for 5. The HRESIMS
+
spectrum of 6 showed a [M + Na] ion at m/z 410.1477, 14
mass units less than that of 5. Comparison of the NMR data of
2
4
21
3
3
23 19
3
3
13
1
Detailed analyses of their C and H NMR data (Tables 1 and
) and 2D NMR correlations (Tables S5−S7) revealed that
they shared the same indole-pyridone-imidazole tetracyclic
2
6 and 5 concluded that the methoxy (δ 48.8, δ 2.81, s) at C-
C H
13 in 5 was replaced by a hydroxy in 6. Therefore, the
C
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Article
Figure 2. HMBC correlations of marinacarboline I (5) and experimental ECD spectrum of marinacarboline I (5, 230−450 nm) in CHCl and
3
calculated ECD spectrum of 13R-5 and 13S-5 at the b3lyp/6-311+g(d,p) level in CHCl .
3
Figure 3. Δδ values for the MTPA esters 7s and 7r and the experimental ECD spectrum of marinacarbolines K (7, 190−450 nm) in MeOH and
S−R
calculated ECD spectrum of 13S,16S-7 and 13R,16S-7 at the b3lyp/6-311+g(d,p) level in MeOH.
Table 3. 13C NMR Data of Compounds 9, 10, and 12−16 (150 MHz, DMSO-d6)
no.
9
10
12
13
14
15
16
1
2
3
4
5
6
7
8
9
143.2, C
110.8, CH
129.5, CH
120.9, CH
122.1, CH
120.3, C
131.9, C
134.8, C
138.2, C
138.1, C
143.3, C
110.9, CH
129.6, CH
121.0, CH
122.2, CH
120.4, C
132.1, C
134.9, C
138.3, C
137.9, C
140.7, C
111.0, CH
126.9, CH
120.7, CH
120.9, CH
121.5, C
123.0, C
128.2, C
155.1, C
128.7, C
101.0, CH
161.1, C
41.0, CH2
35.0, CH2
139.3, C
128.7, CH
128.4, CH
126.2, CH
128.4, CH
128.7, CH
31.2, CH3
139.4, C
112.9, CH
126.6, CH
120.3, CH
120.9, CH
122.4, C
122.7, C
129.7, C
154.6, C
128.7, C
101.0, CH
161.5, C
41.0, CH2
35.0, CH2
139.3, C
128.7, CH
128.4, CH
126.2, CH
128.4, CH
128.7, CH
140.6, C
111.0, CH
127.1, CH
121.1, CH
121.5, CH
121.6, C
122.4, C
129.6, C
155.5, C
124.8, C
106.7 CH
160.4, C
43.7, CH2
76.0, CH
138.1, C
126.4, CH
128.5, CH
128.2, CH
128.5, CH
126.4, CH
31.3, CH3
169.6, C
161.4, C
156.9, C
105.8, CH
166.8, C
109.3, CH
150.3, C
154.5, C
144.6, C
105.0, C
116.6, CH
150.8, CH
149.8, CH
119.6, C
143.7, C
30.4, CH2
28.2, CH2
30.8, CH2
21.9, CH2
13.9, CH3
11.2, CH3
149.2, CH
124.6, CH
137.3, CH
120.6, CH
135.0, CH
55.6, CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
116.4, CH
163.8, C
200.8, C
116.5, CH
163.8, C
200.6, C
28.4, CH3
40.5, CH2
35.3, CH2
139.4, C
128.7, CH
128.4, CH
126.6, CH
128.4, CH
128.7, CH
33.5, CH3
28.3, CH3
46.9, CH2
71.1, CH2
143.6, C
126.0, CH
128.1, CH
127.1, CH
128.1, CH
126.0, CH
33.6, CH3
67.1, CH2
33.2, CH2
137.8, C
128.7. CH
128.4, CH
126.5, CH
128.4, CH
128.7, CH
22.6, CH3
structures of 5 and 6 were assigned as two new β-carboline
alkaloids, named marinacarbolines I and J, respectively.
Compounds 7 and 8 had the same molecular formula of
C H N O , one oxygen more than that of 5, implying the
Comparison of their NMR data (Tables 1 and 2) revealed that
the methylene C-16 in 5 was substituted with a hydroxy in 7
and 8. Further analysis of the NMR data suggested that the
structural difference between 7 and 8 was a different
configuration at C-16 because of the significantly different
2
4
23
3
4
presence of an additional oxygen-bearing function in 7 and 8.
D
https://dx.doi.org/10.1021/acs.jnatprod.0c00588
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Article
1
Table 4. H NMR Data of Compounds 9, 10, and 12−16 (600 MHz, DMSO-d , J in Hz)
6
no.
9
10
12
13
14
15
16
2
3
4
5
6
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
7.74, d (7.9)
7.67, t (7.9)
7.35, t (7.9)
8.44, d (7.9)
7.80, d (7.9)
7.71, t (7.9)
7.39, t (7.9)
8.50, d (7.9)
7.68, d (7.6)
7.54, t (7.6)
7.31, t (7.6)
8.02, d (7.6)
7.55, d (7.9)
7.45, t (7.9)
7.25, t (7.9)
7.99, d (7.9)
7.71, d (8.0)
7.55, t (8.0)
7.33, t (8.0)
8.23, d (8.0)
11.46, s
7.91, d (2.6)
8.76, d (2.6)
7.56, d (5.6)
8.69, d (5.6)
9.27, s
8.69, dd (4.6, 1.6)
7.47, dd (7.6, 4.6)
7.94, td (7.6, 1.6)
8.29, d (7.6)
7.98, s
0
1
2
3
4
5
6
7
8
9
0
1
2
3
9.00, s
9.05, s
7.79, s
7.80, s
8.02, s
2.58, t (7.7)
1.54, m
1.31, m
1.29, m
0.85, t (7.0)
2.16, s
3.51, dd (7.5, 5.6)
2.87, t (7.5)
3.51, dd (7.6, 5.6)
2.87, t (7.6)
3.59, t (5.9)
5.91, t (5.9)
2.92, s
3.62, dd (6.6, 6.6)
2.93, t (6.6)
2.91, s
a: 3.65, m; b: 3.50, m
4.86, dd (7.5, 4.7)
3.95, s
7.27, d (7.5)
7.30, t (7.5)
7.20, t (7.5)
7.30, t (7.5)
7.27, d (7.5)
4.26, s
7.27, d (7.5)
7.30, t (7.5)
7.20, t (7.5)
7.30, t (7.5)
7.27, d (7.5)
7.48, d (7.6)
7.38, t (7.6)
7.32, t (7.6)
7.38, t (7.6)
7.48, d (7.6)
4.31, s
4.29, t (7.5)
3.19, t (7.5)
7.31−7.32, m
7.31−7.32, m
7.22, m
7.31−7.32, m
7.31−7.32, m
3.86, s
7.44, d (7.5)
7.35, t (7.5)
7.26, t (7.5)
7.35, t (7.5)
7.44, d (7.5)
3.91, s
7.29−7.32, m
7.29−7.32, m
7.19, m
7.29−7.32, m
7.29−7.32, m
1.83, s
NH-1
NH-9
NH-12
NH-13
OH-16
12.34, s
10.76, br s
8.87, t (5.6)
10.62, br s
8.82, t (5.6)
11.41, br s
8.29, t (5.9)
8.70, t (6.6)
8.60, t (6.0)
5.73, br s
chemical shifts and splitting patterns of H-16 (δ 5.13, dd, 9.6,
was an analogue of 9 with an additional oxygen-bearing
function in 10. Detailed analyses of HMQC and HMBC
correlations (Table S14) confirmed the presence of a hydroxy
group at C-16 in 10. In order to determine the configuration at
C-16, compound 10 was hydrolyzed in 6 N HCl at 110 °C to
H
2
.6 Hz for 7; 5.20, br t, 6.3 Hz for 8). The Mosher’s ester
NMR method was first used to determine the absolute
configuration of C-16 in 7. Treatment of 7 with (R)-α-
methoxy-α-(trifluoromethyl) phenylacetyl chloride (R-MTPA-
Cl) or S-MTPA-Cl gave the S-MTPA ester (7s) or the R-
release (R)-2-amino-1-phenylethanol (t 11.0 min), which was
R
1
MTPA ester (7r), respectively. The H NMR chemical shift
compared by HPLC analysis on a chiral-phase column using
differences (ΔδS−R) (Figure 3 and Table S11) between 7s and
authentic (R)-2-amino-1-phenylethanol (t 11.0 min) and (S)-
R
7
r gave positive values for H-11, H -14, H -15, and H -23 and
2-amino-1-phenylethanol (t 13.0 min) as references. There-
3
2
3
R
negative values for H-18, H-19, H-20, H-21, and H-22,
indicating a 16S-configuration. Then, ECD calculations were
applied to determine the configuration of C-13. Two model
molecules of 13R,16S-7 and 13S,16S-7 were chosen for ECD
calculations, and the results (Figure 3) showed that the
experimental ECD spectrum of 7 was in good agreement with
the calculated ECD curve of 13S,16S-7. Thus, a 13S,16S-
configuration was determined for 7. Accordingly, the
configuration of 8 was assigned to be 13S,16R. The structures
of 7 and 8 were thus elucidated as two new β-carboline
alkaloids, named marinacarbolines K and L, respectively.
The HRESIMS spectrum of compound 9 showed an ion at
fore, a 16R-configuration was assigned for 10. The structure of
10 was identified as a new β-carboline alkaloid, named
marinacarboline N.
The molecular formulas of C H N O for 12, C H N O
2
1
19
3
2
20 17
3
2
for 13, and C H N O for 14 were established based on their
2
3
21
3
4
1
3
HRESIMS and C data. Compounds 12−14 exhibited very
1
13
similar UV absorptions. Analysis of their H, C, HMQC, and
HMBC spectra revealed that 12−14 shared the same indole-
pyridone tricyclic skeleton, which was different from the
indole-pyridine tricyclic skeleton for 9−11. The acetyl
functionality at the C-9 position in 9−11 was replaced by an
oxygen atom in 12−14. The structural difference between 12
+
m/z 394.1534 [M + Na] , corresponding to a molecular
and 13 was an indole N-CH for 12 and NH for 13, which was
3
1
13
formula of C H N O . Detailed analyses of the H, C,
confirmed by HMBC correlations (Tables S15 and S16). The
structures of 12 and 13 were named marinacarbolines O and P,
respectively, which are seco-ring D analogues of 5 and 6.
2
3
21
3
2
HMQC, and HMBC spectra demonstrated that 9 was the
indole N-methyl analogue of the known alkaloid marinacarbo-
7
a
line C (11), both of them sharing an indole-pyridine tricyclic
skeleton. The structure and assignments of the 13C and H
NMR data (Tables 3 and 4) of 9 were confirmed by HMQC
and HMBC correlations (Table S13). Compound 9 was
determined as a new β-carboline alkaloid, named marinacarbo-
line M.
Compared to 12 and 13, compound 14 had a different
1
13
substituent at the C-10 position. Detailed analyses of its
C
1
and H NMR data (Tables 3 and 4) and HMQC and HMBC
correlations (Table S17) demonstrated that the acylamino and
phenylethane groups in 12 and 13 were replaced by an ester
and N-(2-hydroxy-2-phenylethyl)acetamide groups in 14.
HMBC correlations of H-11 (δH 8.02, s) with C-10 (δC
124.8) and C-12 (δ 160.4) and H-14 (δ 5.91, t, 5.9 Hz)
Compound 10 gave an HRESIMS ion at m/z 410.1479 [M
+
+
Na] , 16 mass units higher than that of 9, suggesting that 10
C
H
E
https://dx.doi.org/10.1021/acs.jnatprod.0c00588
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Table 5. Antiproliferative Activity of Compounds 1−17 (IC : μM, n = 5)
50
compound
U251
U87MG
compound
U251
U87MG
NA
13 ± 1
11 ± 1
NA
19 ± 2
6.5 ± 0.2
NA
a
1
2
3
4
5
6
7
8
9
17 ± 1
36 ± 3
12 ± 1
NA
27 ± 1
NA
36 ± 2
19 ± 2
12 ± 1
4.5 ± 0.4
43 ± 1
2.3 ± 0.6
NA
8.9 ± 1.6
NA
29 ± 1
17 ± 1
7.4 ± 1.1
10
11
12
13
14
15
16
17
NA
12 ± 1
13 ± 1
NA
15 ± 1
2.0 ± 0.1
NA
5.8 ± 0.5
3.9 ± 0.6
7.2 ± 1.0
0.6 ± 0.2
DOX
a
NA: no activity at a concentration of 50 μM.
with C-12 established the linkage of the tricyclic skeleton,
ester, and N-(2-hydroxy-2-phenylethyl)acetamide units. For
assignment of its configuration, compound 14 was hydrolyzed
by sodium hydroxide in aqueous MeOH to produce (R)-N-(2-
hydroxy-2-phenylethyl)acetamide (14a) and N-methyldicho-
tomine E (14b). 14a was further hydrolyzed by hydrochloric
acid to give (R)-2-amino-1-phenylethanol (14c), as deter-
C-8 (δ 149.8), and C-10 (δ 143.7), and H-16 (δ 2.16, s)
C C H
with C-3 (δ 144.6), C-4, and C-10. These NMR analyses
C
indicated the existence of a pyridine-pyridone bicyclic skeleton
with a methyl at C-4 in 16. In addition, its HMBC spectrum
showed correlations of H-11 (δ 2.58, 2H, t, 7.7 Hz) with C-3,
H
C-4, C-12 (δ 28.2), and C-13 (δ 30.8), H-12 (δ 1.54, 2H,
C
C
H
m) with C-11 (δ 30.4) and C-14 (δ 21.9), H-13 (δ 1.31,
C
C
H
1
mined by its H NMR and specific rotation data as well as
chiral-phase HPLC analysis using the standard compounds
2
H, m) with C-11 and C-15 (δ 13.9), H-14 (δ 1.29, 2H, m)
C H
with C-13, and H -15 (δ 0.85, 3H, t, 7.0 Hz) with C-13 and
3
H
(
R)-2-amino-1-phenylethanol and (S)-2-amino-1-phenyletha-
C-14. A spin system of H-11/H-12/H-13/H-14/H-15 was
observed in the COSY spectrum. These COSY and HMBC
correlations determined a side chain of −(CH ) CH at the C-
nol as references. Accordingly, a 14R-configuration was
assigned for 14. The structure of 14 was named marinacarbo-
line Q.
2
4
3
3
position in 16. The structure of 16 was thus identified as a
Compound 15 was obtained as a white amorphous powder
and had a molecular formula of C H N O based on its
new 2,7-naphthyridine analogue, named actinoallonaphthyr-
idine A.
2
0
19
3
2
+
HRESIMS ions at m/z 334.1553 [M + H] and 356.1374 [M
Na] . The C NMR data at δ 166.8 (C), 156.9 (C), 154.5
+
13
All isolated compounds (1−17) were tested for their
antiproliferative activities against human glioma U87MG and
+
C
(
(
C), 150.3 (C), 149.2 (CH), 137.3 (CH), 135.0 (CH), 124.6
8
a
U251 cells through a sulforhodamine B (SRB) assay.
CH), 120.6 (CH), 109.3 (CH), 105.8 (CH), and 55.6 (CH )
3
1
Doxorubicin (DOX) was used as a positive control. The
results (Table 5 and Figure S3) showed that the new
caerulomycin N (15) and the known cyanogramide (17)
exhibited cytotoxic activity against both human glioma U251
and U87MG cells with IC values of 2.0−7.2 μM, and the new
and H NMR data at δ 8.76 (1H, d, 2.6 Hz), 8.69 (1H, dd,
H
4
7
.6, 1.6 Hz), 8.29 (1H, d, 7.6 Hz), 7.94 (1H, td, 7.6, 1.6 Hz),
.91 (1H, d, 2.6 Hz), 7.47 (1H, dd, 7.6, 4.6 Hz), 7.98 (1H, s),
and 3.96 (3H, s) suggested that 15 had a partial structure of 4-
methoxy-[2,2′-bipyridine]-6-carbaldehyde oxime (caerulomy-
5
0
4
a
cin A), which was confirmed by HMQC, COSY, and HMBC
correlations (Table S18). The remaining NMR signals were
observed at δ 67.1 (CH ), 33.2 (CH ), 137.8 (C), 128.7
marinacarbolines E (1), G (3), I (5), and M (9) showed
cytotoxic activity against U87MG cells with IC values of 2.3−
5
0
C
2
2
8.9 μM.
(
CH), 128.4 (CH), and 126.5 (CH) and δ 4.29 (2H, t, 7.5
Compounds 1−17 were also tested for their antimicrobial
activities against methicillin-resistant Staphylococcus aureus
H
Hz), 3.19 (2H, t, 7.5 Hz), 7.29−7.32 (4H), and 7.19 (H, m),
which were assigned to a phenethanol unit. HMBC
correlations of H-13 with C-16 and H-16 with C-13 as well
as NOE correlation of H-13 with H-16 established the linkage
of the two structural parts and also a 13E configuration.
Therefore, the structure of 15 was identified as a new
caerulomycin, named caerulomycin N. Its 13C and H NMR
data (Tables 3 and 4) were unambiguously assigned based on
the HMQC, COSY, and HMBC correlations.
(
MRSA), Escherichia coli, and Candida albicans using the
microbroth dilution method as described in our previous
8b
publication. However, none of the tested compounds were
active at a concentration of 50 μg/mL.
In conclusion, this study discovered and characterized 15
new alkaloids, including marinacarbolines E−Q, caerulomycin
N, and actinoallonaphthyridine A, from the rare actinomycete
Actinoalloteichus sp. ZZ1866. New marinacarbolines E−Q (1−
10 and 12−14) are β-carboline alkaloids, and this type of
alkaloid is newly reported as secondary metabolites produced
by Actinoalloteichus actinomycetes. Marinacarbolines E−L (1−
1
The HRESIMS spectrum of compound 16 displayed ions at
+
+
m/z 231.1493 [M + H] and 483.2726 [2 M + Na] ,
1
3
corresponding to a molecular formula of C H N O. Its
C
14
18
2
NMR spectrum showed one carbonyl (δ 161.4) and seven
C
8
) share a unique indole-pyridone-imidazole tetracyclic
olefinic carbons (δ 150.8, 149.8, 144.6, 143.7, 119.6, 116.6,
C
1
skeleton, which represents the first example of this type of
skeleton. Caerulomycin N (15) and cyanogramide (17)
displayed antiproliferative activity against both glioma
U87MG and U151 cells with IC50 values of 2.0 to 7.2 μM.
The data from this study enrich the chemical and bioactive
diversities of the secondary metabolites produced by the
Actinoalloteichus actinomycetes.
1
05.0), and the H NMR spectrum exhibited three olefinic
protons (δH 9.27, 8.69, 7.56) and one heteroatom-attached
proton (δ 11.46). The COSY spectrum showed correlations
H
(
5
2
Table S19) of H-5 (δ 7.56, d, 5.6 Hz) with H-6 (δ 8.69, d,
.6 Hz), and the HMBC spectrum showed correlations of NH-
H H
(δ 11.46, s) with C-4 (δ 105.0) and C-9 (δ 119.6), H-5
H
C
C
with C-4, C-6 (δ 150.8), and C-9, H-6 with C-5 (δ 116.6),
C
C
F
https://dx.doi.org/10.1021/acs.jnatprod.0c00588
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
times. The combined EtOAc extract solution was concentrated in
vacuo to give an extract (47.83 g). The EtOAc extract was fractionated
on a column (160 × 10 cm) of silica gel (800 g) eluting with a
mixture of cyclohexane and EtOAc (10:1, 5:1, 1:1, 0:1, each 1000 L)
and a mixture of EtOAc and MeOH (5:1, 1:1, each 1000 L) to afford
six fractions (Frs. 1−6).
EXPERIMENTAL SECTION
■
General Experimental Procedures. The melting point was
measured with a WRX-4 microscope apparatus (Shanghai Yice
Apparatus & Equipment Co. Ltd.) and is uncorrected. Specific
rotations, UV, ECD, and IR spectra were recorded on an Autopol I
polarimeter (Rudolph Research Analytical), a METASH UV-8000
spectrometer (Shanghai METASH Instruments Co. Ltd.), a JASCO J-
Fr. 3 was further fractionated on an ODS column eluting with 70%
and 100% MeOH to give two fractions (Frs. 3A and 3B). Fr. 3A was
separated by HPLC using an Agilent Zorbax column (column B,
8
15 spectropolarimeter, and a Nicolet IS 10 FT-IR spectrometer,
respectively. All NMR spectra were acquired on a JEOL 600
spectrometer using standard programs and acquisition parameters,
and chemical shifts were expressed in δ (ppm) relative to DMSO-d6
mobile phase: MeCN/H O, 75/25; flow rate: 1 mL/min; UV
2
detection: 210 nm) to give compounds 11 (6.6 mg, 0.014%, t 28
R
min), 5 (12.8 mg, 0.027%, t 25 min), and 9 (18.3 mg, 0.038%, t 26
R
R
(
δ 39.5, δ 2.50). HRESIMS data were acquired on an Agilent 6230
C H
min). Fr. 3B was separated by preparative HPLC using a CT-30
TOF LC/MS spectrometer. Silica gel (100−200 mesh, Qingdao
Marine Chemical Co. Ltd.) and octadecyl-functionalized silica gel
column (column A, mobile phase: MeOH/H O, 90/10; flow rate: 10
2
mL/min; UV detection: 210 nm) to give compound 1 (10.8 mg,
(
ODS, Cosmosil 75C -Prep, Nacalai Tesque Inc.) were used for
18
0.022%, t 32 min). Fr. 3C was separated on the same CT-30 column
R
column chromatography. HPLC separation was performed on a
Shimadzu LC-20AP prepared HPLC system with column A (CT-30,
with the same flow rate and a different mobile phase (MeOH/H O,
2
7
5/25) to give 17 (22.7 mg, 0.047%, t 38 min).
Fr. 4 was also separated by an ODS column eluting with 70% and
R
2
80 × 30 mm, 10 μm, Fuji-C ) and an Agilent 1260 HPLC system
18
using a Zorbax SB-C18 column (column B: 250 × 9.4 mm, 5 μm) or a
CHIRALCEL OJ-RH column (150 × 4.6 mm, 5 μm), with a DAD
detector. X-ray diffraction analysis was carried out on an Xcalibur
Atlas Gemini Ultra diffractometer (Agilent Technologies) with Cu Kα
radiation (λ = 1.541 84 Å) at 100 K. Mosher’s reagents R-MTPA-Cl
and S-MTPA-Cl were ordered from Aladdin Industrial Corporation,
and (S)-2-amino-1-phenylethanol (>98.0%) and (R)-2-amino-1-
phenylethanol (>98.0%) from J & K Scientific Ltd. All solvents
used for this study were bought from the Shanghai Lingfeng Co. Ltd.
Human glioma U251 (XB-0439) and U87MG (JDS-2568) cells were
obtained from the Cell Bank of the Chinese Academy of Sciences.
MRSA ATCC 43300, E. coli ATCC 25922, and C. albicans ATCC
100% MeOH to furnish Frs. 4A and 4B. Fr. 4B was further
fractionated on a Sephadex LH-20 column eluting with MeOH to give
three fractions, 4B −4B . Compounds 10 (1.8 mg, 0.004%, t 40
1
3
R
min), 8 (3.0 mg, 0.006%, t 66 min), and 7 (11.2 mg, 0.023%, t 70
R
R
min) were obtained from Fr. 4B through HPLC purification using
1
column B (mobile phase: MeCN/H O, 44/56; flow rate: 1 mL/min;
2
UV detection: 210 nm). Fr. 4B was separated by preparative HPLC
2
using column A (mobile phase: MeOH/H O, 82/18; flow rate: 10
2
mL/min; UV detection: 210 nm) to give compounds 6 (4.0 mg,
0
.008%, t 24 min) and 3 (5.9 mg, 0.012%, t 35 min). Similarly, by
R
R
preparative HPLC purification using the same column A and the same
flow rate, compounds 4 (1.8 mg, 0.004%, t 35 min, MeOH/H O,
R
2
1
0231 were provided by Drs. Zhongjun Ma, Pinmei Wang, and Bin
7
5/25) and 2 (18.7 mg, 0.039%, t 44 min, MeOH/H O, 82/18)
R
2
Wu, respectively. Doxorubicin (>98.0%) was ordered from Sigma-
Aldrich, and vancomycin (>98.0%), gentamicin (99.6%), and
amphotericin B (99.0%) were from Meilune Biotechnology Co. Ltd.
Gauze’s agar and rice were purchased from Guangdong Huankai
Microbial Science and Technology Co. Ltd. and Beijing Yihai Kerry
Co. Ltd., respectively. Sea water was obtained from the East China
Sea close to Zhoushan Archipelago at 29°99′79″ N and 122°17′08″
E.
were obtained from Fr. 4A and Fr. 4B , respectively.
3
Each of Fr. 5 and Fr. 6 were fractionated on an ODS column
eluting with 70% and 90% MeOH to give Frs. 5A and 5B and Frs. 6A
and 6B, respectively. Through HPLC separation by using column A at
flow rate of 10 mL/min and UV detection of 210 nm, compounds 12
(
0
9.7 mg, 0.02%, t 51 min, MeOH/H O, 80/20) and 14 (8.6 mg,
R
2
.02%, t 30 min, MeOH/H O, 75/25) were obtained from Fr. 5B
R
2
and Fr. 6B, respectively. Similarly, by using column B (flow rate: 1
mL/min; UV detection: 210 nm), compounds 13 (1.8 mg, 0.004%, tR
Isolation and Identification of Strain ZZ1866. Strain ZZ1866
was isolated from a sea mud sample collected from the coastal area of
Putuo, Zhoushan, Zhejiang, China, in July 2018. Briefly, 1 g of sea
1
6 min, MeCN/H O, 65/35) and 15 (2.4 mg, 0.005%, t 23 min,
2
R
MeCN/H O, 65/35) were purified from Fr. 5A and 16 (2.7 mg,
2
mud was dissolved in 10 mL of sterile H O in a tube and then
0.006%, t 30 min, MeCN/H O, 55/45) from Fr. 6A.
2
R
2
shocked at 28 °C overnight on a rotary shaker (180 rpm). Then the
Marinacarboline E (1): Colorless, orthorhombic crystals; mp 291−
sample was serially diluted to 10− to 10 g/mL, and 200 μL of each
dilution was spread on the surface of Gauze’s solid medium. After 7
days of incubation at room temperature, the grayish purple bacteria
colonies were picked with sterile needles and transferred to a Gauze’s
agar plate. After transferring three times, the single colony that grew
well was transferred onto Gauze’s agar slants to prepare working
stocks and stored at 4 °C until use.
2
−4
20
293 °C; [α] −90 (c 0.12, CHCl ); UV (MeOH) λ (log ε) 235
D
3
max
(3.79), 294 (3.92), 363 (3.74) nm; IR (ATR) ν 2926, 1713, 1664,
1610, 1372, 1327, 1262, 735, 692 cm ; C NMR data (150 MHz),
Table 1; H NMR data (600 MHz), Table 2; HRESIMS m/z
422.1479 [M + Na] (calcd for C H N NaO , 422.1481) and
max
−
1 13
1
+
2
4
21
3
3
+
821.3068 [2 M + Na] (calcd C H N NaO , 821.3064).
4
8
42
6
6
Crystal data of marinacarboline E (1): C H N O (M = 399.44
2
4
21
3
3
Strain ZZ1866 was identified using 16S rDNA sequence analysis by
Legenomics. The DNA sequence of strain ZZ1866 was compared to
those in GenBank using nucleotide BLAST and has been deposited in
GenBank with an accession number of MT096343. The strain
Actinoalloteichus sp. ZZ1866 is preserved at the Laboratory of Institute
of Marine Biology, Ocean College, Zhoushan Campus, Zhejiang
University, China.
Large-Scale Culture of Strain ZZ1866. Colonies of Actino-
alloteichus sp. ZZ1866 growing on Gauze’s agar medium were
incubated into a 500 mL Erlenmeyer flask containing 250 mL of
Gauze’s liquid medium and then incubated at 28 °C for 5 days on a
rotary shaker (180 rpm) to produce seed broth. The seed broth (5
mL) was covered on the surface of rice medium (40 g of rice with 60
mL of seawater). A total of 200 Erlenmeyer flasks were applied to
culture strain ZZ1866, which were incubated at room temperature for
g/mol), orthorhombic, space group P2 2 2 (no. 19), a =
1 1 1
6.30648(11) Å, b = 11.9372(2) Å, c = 26.4614(5) Å, V =
3
1992.06(6) Å , Z = 4, T = 100.01(10) K, μ(Cu Kα) = 0.723
−
1
3
mm , D = 1.332 g/cm , 9529 reflections measured (6.68° ≤ 2θ ≤
calc
147.176°), 3878 unique (Rint = 0.0201, R
used in all calculations. The final R was 0.0310 (I > 2σ(I)) and wR
= 0.0231), which were
sigma
1
2
was 0.0820 (all data); Flack/Hooft parameter: −0.05(8)/−0.01(7)
(Table S20). Crystallographic data of marinacarboline E (1) have
been deposited at the Cambridge Crystallographic Data Centre
(CCDC Number: 1999039). Copies of the data can be obtained free
of charge from Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge CB2 1EZ, U.K. [fax (+44)1223-336-033; or e-mail:
data_request@ccdc.cam.ac.uk).
2
0
D
Marinacarboline F (2): white, amorphous powder; [α] −40 (c
0.10, CHCl ); UV (MeOH) λ (log ε) 235 (3.83), 291 (3.95), 359
3
max
6
0 days in stationary state.
Isolation of Compounds 1−17. The culture of strain ZZ1866 in
(3.76) nm; IR (ATR) ν 3256, 2927, 1717, 1661, 1604, 1374, 1320,
max
13
−1
1
1272, 741, 695 cm ; C NMR data (150 MHz), Table 1; H NMR
rice medium in each flask was extracted with EtOAc (250 mL) three
data (600 MHz), Table 2; HRESIMS m/z 408.1320 [M + Na]+
G
https://dx.doi.org/10.1021/acs.jnatprod.0c00588
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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Article
+
368.1372 [M + Na]+ (calcd for C H N NaO , 368.1375) and
21 19 3 2
(
(
calcd for C H N NaO , 408.1324) and 793.2740 [2 M + Na]
23
19
3
3
calcd for C H N NaO , 793.2750).
713.2853 [2 M + Na] (calcd for C H N NaO , 713.2852).
46
38
6
6
42 38 6 4
20
Marinacarboline G (3): white, amorphous powder; [α] −42 (c
.28, CHCl ); UV (MeOH) λ (log ε) 241 (3.82), 290 (3.71), 352
3.54), 369 (3.52) nm; IR (ATR) ν 2932, 1723, 1675, 1612, 1391,
266, 747 cm ; C NMR data (150 MHz), Table 1; H NMR data
600 MHz), Table 2; HRESIMS m/z 422.1476 [M + Na] (calcd for
C H N NaO , 422.1481) and 821.3066 [2 M + Na] (calcd
Marinacarboline P (13): white, amorphous powder; UV (MeOH)
λmax (log ε) 238 (3.84), 274 (3.68), 282 (3.77), 321 (3.48), 333
(3.55), 349 (3.48) nm; IR (ATR) νmax 3339, 3300, 3157, 1655, 1631,
D
0
(
1
(
3
max
max
−1
13
1
−1 13
1542, 1329, 1271, 756, 736, 712, 695 cm ; C NMR data (150
+
1
MHz), Table 3; H NMR data (600 MHz), Table 4; HRESIMS m/z
+
+
354.1214 [M + Na] (calcd for C H N NaO , 354.1218) and
24
21
3
3
20 17
3
2
+
C H N NaO , 821.3064).
685.2538 [2 M + Na] (calcd for C H N NaO , 685.2539).
48
42
6
6
40 34 6 4
20
20
Marinacarboline H (4): white, amorphous powder; [α] −19 (c
.14, CHCl ); UV (MeOH) λ (log ε) 240 (3.88), 287 (3.82), 352
Marinacarboline Q (14): white, amorphous powder; [α] −24 (c
0.10, MeOH); UV (MeOH) λmax (log ε) 237 (3.73), 278 (3.63), 286
D
D
0
3
max
(
1
3.54), 357 (3.56) nm; IR (ATR) ν 3278, 2924, 1704, 1660, 1610,
(3.66), 335 (3.47), 344 (3.59), 361 (3.58) nm; IR (ATR) ν 3300,
max
max
−1
13
1
−1 13
393, 1266, 746 cm ; C NMR data (150 MHz), Table 1; H NMR
2923, 1646, 1657, 1456, 1339, 1260, 748 cm ; C NMR data (150
+
1
data (600 MHz), Table 2; HRESIMS m/z 408.1321 [M + Na]
(
(
MHz), Table 3; H NMR data (600 MHz), Table 4; HRESIMS m/z
+
+
calcd for C H N NaO , 408.1324) and 793.2748 [2 M + Na]
426.1428 [M + Na] (calcd for C H N NaO , 426.1430) and
23
19
3
3
23 21
3
4
+
calcd for C H N NaO , 793.2750).
Marinacarboline I (5): white solid; [α]
829.2962 [2 M + Na] (calcd for C H N NaO , 829.2962).
46
38
6
6
46 42 6 8
2
0
+66 (c 0.50, CHCl );
Caerulomycin N (15): white, amorphous powder; UV (MeOH)
λmax (log ε) 244 (3.55), 282 (3.76) nm; IR (ATR) νmax 2928, 1579,
1568, 1557, 1456, 1422, 1340, 1217, 1135, 1049, 872, 795, 745, 700
D
3
UV (MeOH) λmax (log ε) 244 (3.93), 265 (3.75), 278 (3.84), 287
3.89), 332 (3.57), 348 (3.69), 365 (3.68) nm; ECD (25 μM,
CHCl ) λ (Δε) 237 (−0.88), 252 (+9.70), 291 (−1.34), 347
(
−
1
13
1
cm ; C NMR data (150 MHz), Table 3; H NMR data (600
3
max
+
(
+3.06) nm; IR (ATR) ν 2933, 1709, 1666, 1618, 1400, 1258,
MHz), Table 4; HRESIMS m/z 334.1553 [M + H] (calcd for
C H N O ,334.1556) and 356.1374 [M + Na] (calcd for
0
max
−
1
13
1
+
1
121, 1044, 747, 698 cm ; C NMR data (150 MHz), Table 1; H
2
20
3
2
+
NMR data (600 MHz), Table 2; HRESIMS m/z 424.1633 [M + Na]
(
(
C H N NaO , 356.1375).
20 19 3 2
+
calcd for C H N NaO , 424.1637) and 825.3373 [2 M + Na]
Actinoallonaphthyridine A (16): white, amorphous powder; UV
(MeOH) λmax (log ε) 202 (3.98), 219 (3.61), 238 (3.31), 316 (3.55)
24
23
3
3
calcd for C H N NaO , 825.3377).
Marinacarboline J (6): white, amorphous powder; [α] +20 (c
48
46
6
6
20
D
nm; IR (ATR) ν 3200, 2925, 2856, 1655, 1631, 1596, 1473, 1456,
max
−
1
13
1
0
.15, CHCl ); UV (MeOH) λ (log ε) 243 (3.99), 265 (3.83), 277
1039 cm ; C NMR data (150 MHz), Table 3; H NMR data (600
MHz), Table 4; HRESIMS m/z 231.1493 [M + H] (calcd for
C H N O, 231.1497) and 483.2726 [2 M + Na] (calcd for
3
max
+
(
3.88), 286 (3.92), 332 (3.60), 347 (3.69), 364 (3.66) nm; IR (ATR)
−
1
13
+
νmax 2933, 1709, 1660, 1604, 1455, 1397, 1279, 1261, 739 cm ;
C
14
19
2
1
NMR data (150 MHz), Table 1; H NMR data (600 MHz), Table 2;
C H N NaO , 483.2736).
28 36 4 2
HRESIMS m/z 410.1477 [M + Na]+ (calcd for C H N NaO ,
Cyanogramide (17): yellowish powder; [α]
D
20
−96 (c 0.70,
2
3
21
3
3
+
13
1
4
7
10.1481) and 797.3060 [2 M + Na] (calcd for C H N NaO ,
MeOH); C NMR data (150 MHz), Table S3; H NMR data (600
MHz), Table S3.
4
6
42
6
6
97.3064).
Marinacarboline K (7): white, amorphous powder; [α]20D +158 (c
.18, MeOH); UV (MeOH) λmax (log ε) 244 (3.95), 265 (3.78), 278
MTPA Esterification of Marinacarboline K (7). Marinacarbo-
line K (7, 2.2 mg) and dimethylaminopyridine (1.1 mg) were
dissolved in anhydrous pyridine (0.5 mL), and then either (R)- or
(S)-MTPA-Cl (45 μL) was added. The mixtures were stirred at 45 °C
for 48 h until adding MeOH (1 mL) to terminate the reaction. The
reaction mixtures were dried under reduced pressure to give a residue.
(S)-MTPA ester 7s (1.5 mg, t 40.0 min, MeOH/H O, 80/20) or
0
(
3.86), 287 (3.92), 333 (3.60), 348 (3.71), 365 (3.72) nm; ECD (24
μM, MeOH) λmax (Δε) 210 (+8.05), 224 (−3.32), 260 (+4.74), 276
−3.62), 291 (−3.53), 348 (+4.99), 365 (+4.82) nm; IR (ATR) ν
(
3
max
451, 2933, 2853, 1695, 1667, 1615, 1445, 1397, 1394, 1261, 741
−
1
13
1
cm ; C NMR data (150 MHz), Table 1; H NMR data (600
MHz), Table 2; HRESIMS m/z 440.1580 [M + Na] (calcd for
C H N NaO , 440.1586) and 857.3274 [2 M + Na] (calcd for
C H N NaO , 857.3275).
R
2
+
(R)-MTPA ester 7r (0.8 mg, t 29.3 min, MeOH/H O, 85/15) was
R
2
+
obtained from the residue by HPLC purification using column B at a
flow rate of 1.0 mL/min and UV detection of 210 nm.
24
23
3
4
4
8
46
6
8
2
0
1
Marinacarboline L (8): white solid; [α]
+28 (c 0.1, MeOH);
(S)-MTPA ester 7s: H NMR data (600 MHz), Table S11;
D
+
UV (MeOH) λmax (log ε) 243 (3.66), 265 (3.48), 278 (3.56), 287
HRESIMS 656.1979 [M + Na] (calcd for C H F N NaO ,
34
30
3
3
6
(
2
3.61), 333 (3.29), 348 (3.41), 365 (3.40) nm; IR (ATR) ν 3450,
656.1984).
max
−
1
13
1
923, 1696, 1665, 1614, 1446, 1393, 1261, 740 cm ; C NMR data
(S)-MTPA ester 7r: H NMR data (600 MHz), Table S11;
HRESIMS 656.1981 [M + Na] (calcd for C H F N NaO ,
656.1984).
1
+
(
150 MHz), Table 1; H NMR data (600 MHz), Table 2; HRESIMS
3
4
30
3
3
6
+
m/z 440.1583 [M + Na] (calcd for C H N NaO , 440.1586) and
2
4
23
3
4
+
8
57.3270 [2 M + Na] (calcd for C H N NaO , 857.3275).
Hydrolysis of Marinacarbolines Q (14) and N (10).
4
8
46
6
8
Marinacarboline M (9): colorless needles; UV (MeOH) λmax (log
ε) 224 (3.94), 268 (3.80), 288 (3.95), 367 (3.10) nm; IR (ATR) νmax
Marinacarboline Q (14, 3.0 mg) was dissolved in 1.0 mL of MeOH
and 0.2 mL of H O containing 120 mg of NaOH. The mixture was
2
3
7
381, 2927, 2856, 1686, 1669, 1524, 1465, 1258, 1184, 1124, 1062,
stirred at room temperature (25 °C) overnight and then neutralized
by 1 N HCl to pH 7 and extracted with EtOAc (10 mL × 3). The
EtOAc extract was separated by HPLC on a Zorbax SB-C18 column
−1
13
1
49, 729, 703, 570 cm ; C NMR data (150 MHz), Table 3; H
+
NMR data (600 MHz), Table 4; HRESIMS m/z 394.1534 [M + Na]
(
calcd for C H N NaO , 394.1531).
(250 × 9.4 mm, 5 μm) using 30% MeCN in H O at a flow rate of 1.0
2
3
21
3
2
2
2
0
D
Marinacarboline N (10): white, amorphous powder; [α] −10 (c
mL/min and UV detection of 210 nm to afford (R)-N-(2-hydroxy-2-
phenylethyl)acetamide (14a, 1.1 mg, tR 23.7 min) and N-
0
.10, MeOH); UV (MeOH) λmax (log ε) 224 (3.91), 273 (3.76), 289
(
1
3.93), 372 (3.08) nm; IR (ATR) νmax 3379, 2923, 2853, 1690, 1652,
methyldichotomine E (14b, 1.3 mg, t 11.6 min). Compound 14a
R
−
1 13
525, 1463, 1250, 1190, 1124, 1063, 750, 737, 702, 578 cm ;
C
was heated at 110 °C in 1.0 mL of 6 N HCl for 24 h, and the
1
NMR data (150 MHz), Table 3; H NMR data (600 MHz), Table 4;
hydrolysis solution was neutralized by a saturated NaHCO solution
3
HRESIMS m/z 410.1479 [M + Na]+ (calcd for C H N NaO ,
and then extracted with EtOAc (5 mL × 3). The dried EtOAc extract
was separated by HPLC using the same column, flow rate, and UV
detection with a different mobile phase of 75% MeOH to give (R)-2-
2
3
21
3
3
4
10.1481).
Marinacarboline C (11): white, amorphous powder; 13C NMR
1
20
D
data (150 MHz), Table S2, H NMR data (600 MHz), Table S2.
Marinacarboline O (12): white, amorphous powder; UV (MeOH)
λmax (log ε) 241 (3.97), 276 (3.82), 284 (3.90), 329 (3.64), 343
amino-1-phenylethanol (14c, 0.6 mg, t 13.9 min, [α] −32). In the
R
same way, marinacarboline N (10, 0.3 mg) was hydrolyzed in 1.0 mL
of 6 N HCl to release (R)-2-amino-1-phenylethanol (10a, tR 11.0
min), which was detected by HPLC on a CHIRALCEL OJ-RH
column (150 × 4.6 mm, 5 μm; mobile phase: 40% MeCN; flow rate
0.7 mL/min; UV detection: 210 nm) using standard compounds of
(
3.73), 360 (3.68) nm; IR (ATR) ν 3346, 2926, 1645, 1621, 1461,
max
−
1 13
1
327, 1276, 745, 698, 541, 505 cm ; C NMR data (150 MHz),
1
Table 3; H NMR data (600 MHz), Table 4; HRESIMS m/z
H
https://dx.doi.org/10.1021/acs.jnatprod.0c00588
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
2
0
(
R)-2-amino-1-phenylethanol (t 11.0 min, [α]
−38) and (S)-2-
Zhizhen Zhang − Ocean College, Zhoushan Campus, Zhejiang
University, Zhoushan 316021, People’s Republic of China;
orcid.org/0000-0002-8290-3507; Phone: +86-
R
D
2
0
amino-1-phenylethanol (t 13.0 min, α] +38) as references.
R)-N-(2-Hydroxy-2-phenylethyl)acetamide (14a): [α] −26 (c
.10, MeOH); H NMR data (600 MHz) δ 7.37 (2H, d, J = 7.2 Hz,
H-2, H-6), 7.33 (2H, t, J = 7.2 Hz, H-3, H-5), 7.25 (1H, t, J = 7.2 Hz,
H-4), 4.73 (1H, dd, J = 8.2, 4.5 Hz, H-7), 3.42 (1H, dd, J = 13.6, 4.5
Hz, H-8a), 3.28 (1H, dd, J = 13.6, 8.2 Hz, H-8b), 1.93 (3H, s, H-10);
R
D
20
D
(
1
0
H
1
0
3675859706; Email: zzhang88@zju.edu.cn; Fax: +86-
580-2092891
Authors
+
HRESIMS m/z 180.1022 [M + H] (calcd for C H NO , 180.1025)
1
0
14
2
+
Le Qin − Ocean College, Zhoushan Campus, Zhejiang
and 202.0838 [M + Na] (calcd for C H NNaO , 202.0844).
N-Methyldichotomine E (14b): H NMR data (600 MHz) δ 7.60
1H, d, J = 8.0 Hz, H-2), 7.55 (1H, t, J = 8.0 Hz, H-3), 7.29 (1H, t, J
8.0 Hz, H-4), 8.09 (1H, d, J = 8.0 Hz, H-5), 7.78 (1H, s, H-11),
.31 (3H, s, H-13); HRESIMS m/z 180.1022 [M + H] (calcd for
C H NO , 180.1025) and 202.0838 [M + Na] (calcd for
1
0
13
2
1
University, Zhoushan 316021, People’s Republic of China
Wenwen Yi − Ocean College, Zhoushan Campus, Zhejiang
University, Zhoushan 316021, People’s Republic of China
H
(
=
4
+
+
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00588
1
0
14
2
C H NNaO , 202.0844).
1
0
(
13
2
2
0
R)-2-Amino-1-phenylethanol (14c): [α] −32 (c 0.10, MeOH);
D
1
Notes
H NMR data (600 MHz) δ 7.33−7.37 (4H, H-2, 3, 5, 6), 7.24 (1H,
H
H-4), 4.61 (1H, dd, J = 7.5, 4.7 Hz, H-7), 2.75−2.82 (2H, H-8);
The authors declare no competing financial interest.
+
HRESIMS m/z 138.0914 [M + H] (calcd for C H NO, 138.0919).
8
12
ECD Calculations. Monte Carlo conformational searches were
carried out by means of the Spartan’14 software using the Merck
Molecular Force Field (MMFF). The conformers with Boltzmann
populations of over 0.4% were chosen for ECD calculations, and then
the conformers were initially optimized at the B3LYP/6-31G level in
gas. The theoretical calculation of ECD was conducted in chloroform
using time-dependent density functional theory at the B3LYP/6-
ACKNOWLEDGMENTS
■
This study was supported by the National Natural Science
Foundation of China (No. 81773587), the National Key R&D
Program of China (No. 2018YFC0310600), and the HPC
Center of Zhejiang University (Zhoushan Campus).
3
1+G(d,p) level for all conformers of the model molecules. Rotatory
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AUTHOR INFORMATION
■
(
Corresponding Authors
Xiao-Yuan Lian − College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, People’s Republic of China;
Email: xylian@zju.edu.cn
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https://dx.doi.org/10.1021/acs.jnatprod.0c00588
J. Nat. Prod. XXXX, XXX, XXX−XXX