New quinazolinone alkaloids within rare amino acid residue from coral-associated fungus, aspergillus versicolor LCJ-5-4

Article abstract of DOI:10.1021/ol103164n

Cottoquinazoline D (3), a new alkaloid with a 1-aminocyclopropane-1- carboxylic acid residue rarely discovered in nature, was isolated and identified together with two new quinazolinone alkaloids, cottoquinazolines B (1) and C (2), from coral-associated fungus Aspergillus versicolor LCJ-5-4. Their structures including absolute configurations were elucidated based on spectroscopic methods, X-ray single crystal diffraction analysis, and chemical methods. A possible biogenetic pathway for them was proposed.(Figure Presented)

Full text of DOI:10.1021/ol103164n

ORGANIC
LETTERS
2011
Vol. 13, No. 5
1130–1133
New Quinazolinone Alkaloids within Rare
Amino Acid Residue from Coral-
Associated Fungus, Aspergillus
versicolor LCJ-5-4
Yibin Zhuang, Xiancun Teng, Yi Wang, Peipei Liu, Guoqiang Li, and Weiming Zhu*
Key Laboratory of Marine Drugs, Chinese Ministry of Education,
School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003,
Peoples’ Republic of China
weimingzhu@ouc.edu.cn
Received December 28, 2010
ABSTRACT
Cottoquinazoline D (3), a new alkaloid with a 1-aminocyclopropane-1-carboxylic acid residue rarely discovered in nature, was isolated and
identified together with two new quinazolinone alkaloids, cottoquinazolines B (1) and C (2), from coral-associated fungus Aspergillus versicolor
LCJ-5-4. Their structures including absolute configurations were elucidated based on spectroscopic methods, X-ray single crystal diffraction
analysis, and chemical methods. A possible biogenetic pathway for them was proposed.
Coral is one of the three main sources of marine natural
products,1 from which various kinds of compounds with
diverse bioactivities have been identified. For example,
pseudopterosin A from Pseudopterogorgia elisabethae is
now in phase II trials as a wound healing agent.2 However,
research on secondary metabolites of coral-associated
microorganisms that were regarded as the true producers
of their host-derived compounds have been rarely reported.3
During our ongoing pursuit for structurally novel and
bioactive natural products from microorganisms isolated
from unusual or specialized ecological niches, we investi-
gated the metabolites of 168 coral-associated fungal strains
by integrated chemical and bioactive screening. Among
these strains, a fungal strain LCJ-5-4 authenticated as
Aspergillus versicolor was isolated from the soft coral
Cladiella sp. collected from the South China Sea. About
150 secondary metabolites of A. versicolor were character-
ized, mainly including xanthones,4 anthraquinones,5 lacto-
nes,6 chromones,7 polyketides,8 and tryptophane-derived
alkaloids.9,10 These compounds exhibited antimicrobial
activity8,11 and cytotoxicity.9 The EtOAc extract of the
fermentation broth of A. versicolor LCJ-5-4 showed
(4) Fukuyama, K.; Tsukihara, T.; Katsube, Y.; Hamasaki, T.;
Hatsuda, Y.; Tanaka, N.; Ashida, T.; Kakudo, M. Bull. Chem. Soc.
Jpn. 1975, 48, 1639–1640.
(5) Berger, D. M.; Berger, Y. Phytochemistry 1982, 21, 1449–1451.
(6) Carvalho, M. R.; Barbosa, L. C. d. A.; de Queiroz, J. H.;
Howarth, O. W. Tetrahedron Lett. 2001, 42, 809–811.
(7) Lin, W. H.; Brauers, G.; Ebel, R.; Wray, V.; Berg, A.; Proksch,
S. P. J. Nat. Prod. 2003, 66, 57–61.
(1) John, W. B.; Brent, R. C.; Hu, W. P.; Murray, H. G. M.; Peter,
ꢀ
T. N.; Michele, R. P. Nat. Prod. Rep. 2009, 26, 170–224.
(2) Mayer, A. M. S.; Glaser, K. B.; Cuevas, C.; Jacobs, R. S.; Kem,
W.; Little, R. D.; McIntosh, J. M.; Newman, D. J.; Potts, B. C.; Shuster,
D. E. Trends Pharmacol. Sci. 2010, 31, 255–265.
(3) Anahit, P.; Staffan, K.; Suhelen, E. Mar. Drugs 2010, 8, 438–459.
r
10.1021/ol103164n
Published on Web 01/27/2011
2011 American Chemical Society

Table 1. 1H and 13C NMR Data for Cottoquinazolines B-D (1-3) (600, 150 MHz, DMSO-d6, TMS, δ ppm)
1
2
3
position
δC
δH (J in Hz)
δC
δ
H (J in Hz)
δC
δH (J in Hz)
1
169.9, qC
169.8, qC
170.5, qC
2
9.46, d (4.4)
5.49, d (5.5)
9.46, d (5.0)
5.41, d (5.0)
9.45, d (4.9)
5.11, d (4.9)
3
64.4, CH
149.4, qC
147.2, qC
127.4, CH
134.7, CH
127.0, CH
126.4, CH
120.3, qC
160.1, qC
53.3, CH
33.5, CH2
63.9, CH
149.2, qC
147.2, qC
127.3, CH
134.7, CH
126.9, CH
126.4, CH
120.3, qC
160.1, qC
53.2, CH
33.6, CH2
63.0, CH
150.3, qC
147.4, qC
127.4, CH
134.8, CH
127.1, CH
126.4, CH
120.2, qC
160.0, qC
53.8, CH
34.6, CH2
4
6
7
7.69, d (7.7)
7.64, d (7.8)
7.66, d (8.1)
8
7.83, dt (7.7, 1.5)
7.55, dt (7.7, 1.0)
8.16, dd (7.7,1.5)
7.82, dt (7.8, 1.3)
7.55, dt (7.8, 0.9)
8.16, dd (7.7, 1.3)
7.84, dt (7.7, 1.6)
7.55, dt (7.7, 1.1)
8.16, dd (7.7, 1.6)
9
10
11
12
14
15
5.37, d (6.5)
5.38, d (6.8)
5.34, d (6.6)
3.11, dd (14.3, 6.6);
2.32, d (14.3)
3.10, dd (15.1, 7.5);
2.31, d (14.6)
3.13, dd (15.3, 6.6);
2.36, d (14.8)
16
78.6, qC
79.1, qC
75.8, qC
16-OH
17
5.30, s
5.09, s
4.13, m
5.23, d (1.4)
5.07, t (1.4)
4.19, s
5.40, s
5.16, s
82.1, CH
59.2, CH
174.1, qC
139.0, qC
117.5, CH
129.5, CH
125.7, CH
123.7, CH
138.7, qC
15.8, CH3
82.8, CH
65.1, CH
174.0, qC
139.2, qC
118.3, CH
129.3, CH
125.8, CH
123.4, CH
139.0, qC
15.1, CH3
34.2, CH
25.0, CH2
12.2, CH3
79.2, qC
19
50.4, qC
20
167.1, qC
138.9, qC
114.6, CH
129.8, CH
124.9, CH
124.6, CH
136.7, qC
9.9, CH2
7.4, CH2
22
23
7.30, d (8.3)
7.29, dd (8.2)
7.32, d (7.8)
7.33, t (7.7)
24
7.31, dt (7.7, 1.1)
7.16, dt (7.7, 1.5)
7.29, d (7.8)
7.30, dt (7.8, 1.4)
7.16, dt (7.3, 1.3)
7.26, d (8.2)
25
7.11, dt (7.7, 2.2)
7.36, d (7.5)
26
27
28
1.49, d (6.6)
1.07, d (6.8)
2.11, m
1.54, m; 0.96, m
1.35, m; 1.15, m
29
30
1.67, m; 1.56, m
1.04, t (7.3)
31
significant cytotoxicity against P388 cells and different
secondary metabolites from those reported by HPLC
analysis with diode array detection. Chemical study re-
sulted in the isolation and identification of three new
tryptophane-derived quinazolinone alkaloids, named cot-
toquinazolines B-D (1-3). Compound 3 contained a
1-aminocyclopropane-1-carboxylic acid residue which
has been rarely discovered in nature.
silica gel vacuum liquid chromatography and HPLC
purification to yield compounds 1 (5 mg), 2 (5 mg), and 3
(10 mg) (Supporting Information).12
Compound 1 was obtained as colorless prisms (MeOH),
25
mp 273 °C (dec.), [R]D þ83 (c 0.5, MeOH). Its molecular
formula was determined as C23H19N5O4 according to the
HRESIMS at m/z 430.1526 [M þ H]þ (calcd for C23H20-
N5O4, 430.1515), requiring 17degreesof unsaturation. The
IR absorption bands at 3443, 3219, 1692, and 1670 cm-1
suggested the presence of hydroxy and amidocarbonyl
groups. 1H and 13C NMR spectral analysis (Table 1) and
literature retrieval revealed that 1 was the congener of
cottoquinazoline A whose absolute configuration has not
been assigned.9 The main differences occurred in rings
C-G, indicating a stereoisomer of cottoquinazoline A that
was confirmed by 2D NMR (Figure 1). The relative con-
figuration of 1 was assigned on the basis of NOE difference
experiments. When H-3 and HO-16 were irradiated, the
signals of H-17/28 and H-19 were enhanced by 8.7%,
5.3%, and 4.0%, respectively, indicating H-3/H-17/28 was
cis-configuration and CH3-28/HO-16 was trans-configura-
tion that was opposite to cottoquinazoline A9 (Figure 1).
The X-ray diffraction experiment of 1 confirmed this
A. versicolor LCJ-5-4 was grown under static conditions
at 20 °C for 30 days and then harvested by extraction
with EtOAc. The extract (140 g) was separated by repeated
(8) Lee, Y. M.; Mansoor, T. A.; Hong, J. k.; Lee, C. O.; Bae, K. S.;
Jung, J. H. Nat. Prod. Sci. 2007, 13, 90–96.
(9) Leith, J. F.; Andrew, M. P.; Ernest, L.; Robert, J. C. J. Nat. Prod.
2009, 72, 666–670.
(10) Li, G. Y.; Yang, T.; Luo, Y. G.; Chen, X. Z.; Fang, D. M.;
Zhang, G. L. Org. Lett. 2009, 11, 3714–3717.
(11) Dhar, A. K.; Bose, S. K. Tetrahedron Lett. 1969, 55, 4871–4874.
(12) Four known compounds, averufanin, versicolorin B, versicol,
and 1,3,6,8-tetrahydroxy-9,10-anthraquinone, were also isolated. These
compounds contributed to the cytotoxicity of the extract.
Org. Lett., Vol. 13, No. 5, 2011
1131

Scheme 1. Plausible Biogenetic Pathway of Quinazolinone Al-
kaloids
Figure 1. Key 2D NMR correlations and NOE effects of com-
pounds 1-3.
for an additional CH3CH2CH moiety, the NMR data
(Table 1) were almost identical to those of 1, indicating
that 2 was the congener of 1 in which the alanine was
replaced by the isoleucine. This inference was confirmed
1
1
by H-1H and H-13C correlation spectroscopy experi-
ments (Figure 1) and amino acid analysis. Acidic hydro-
lysis of 2 afforded L-Ile that was identified by the same
method as that for 1 (Figure S17). The absolute configura-
tion of 2 was thus determined to be (3S,14R,16S,17S,-
19S,29S) (Figure 1).
Compound 3 was obtained as colorless prisms (MeOH),
mp 270 °C (dec.), with a molecular formula of C24H19N5O4
according to HRESIMS at m/z 442.1501 [M þ H]þ (calcd
25
for C24H20N5O4, 442.1515), [R]D þ78 (c 0.2, MeOH).
Figure 2. X-ray crystal structures of compounds 1 and 3.
The similarity of NMR (Table 1) between 3 and 1 indicated
3 also as the congener of 1. Comparison of NMR data
revealed that two characteristic methylenes of cyclopro-
pane (δH/C 1.54 and 0.96/9.9 and 1.35 and 1.15/7.4) and
quaternary carbon (δC 50.4) in 3 replaced the methyl at
deduction and indicated a cis-configuration between H-3
and H-14 (Figure 2). Acidic hydrolysis of 1 afforded D-Ala
that was identified by amino acid analysis with authentic
samples (coinjection) on a chiral Crownpak CR(þ) HPLC
column (Figure S16).13,14 Therefore, the absolute config-
uration of 1 was unambiguously assigned as (3S,14R,16S,-
17S,19R).
δH/C 1.49/15.8 and methine at δH/C 4.13/59.2 in 1, respec-
tively. In addition, C-16, C-17, and C-20 were shifted upfield
by 2.8, 2.9, and 7.0 ppm, respectively. These data sup-
ported that D-Ala residue in 1 was replaced by 1-ami-
nocyclopropane-1-carboxylic acid residue that was
confirmed by amino acid analysis. When hydrolyzed,
3 produced 1-aminocyclopropane-1-carboxylic acid
that was identified on a chiral Crownpak CR(þ) HPLC
column by coinjection with an authentic sample (Figure
S18).13,14 An NOE difference experiment and X-ray
diffraction revealed the same relative configuration
Compound 2, wasobtainedascolorless prisms (MeOH),
mp 263 °C (dec.), witha molecular formula of C26H25N5O4
from HRESIMS at m/z 472.1984 [M þ H]þ (calcd for
25
C26H26N5O4, 472.1985), [R]D þ31(c 1.0, MeOH). Except
(13) Wang, W. L.; Lu, Z. Y.; Tao, H. W.; Zhu, T. J.; Fang, Y. C.; Gu,
Q. Q.; Zhu, W. M. J. Nat. Prod. 2007, 70, 1558–1564.
(14) Gunasekera, S. P.; Ross, C.; Paul, V. J.; Matthew, S.; Luesch, H.
J. Nat. Prod. 2008, 71, 887–890.
1132
Org. Lett., Vol. 13, No. 5, 2011

compared to those of 1 and 2. Additionally, the specific
rotation of 3 was of the same sign as those of 1 and 2,
suggesting that 3 also has the same (3S,14R,16S,17S)-
configuration (Figure 1).
Compound 3 exhibited moderate antifungal activity
against Candida albicans with an MIC value of 22.6 μM,
while other compounds did not show antimicrobial activity
at a concentration of 100 μg/mL. Compounds 1-3 showed
no cytotoxicity against the tested Hela and P388 cell lines
(IC50 > 50 μM).
The new quinazolinone alkaloids 1-3 are probably
biosynthesized via an amino acid pathway (Scheme 1).
Intermolecular condensation among anthranilic acid,
glycine, and tryptophan produced intermediate a that
further condensed with the fourth amino acid, and the
obtained product was oxidized to form the corresponding
epoxidation product b. Intramolecular nucleophilic cycli-
zation of b produced c that was oxidized to form d. The
oxidation product d further underwent an intramole-
cular reductive amination to form compounds 1-3
(Scheme 1). Compounds 1-3 were evaluated in vitro
for cytotoxicity against Hela and P388 cells using an
MTT assay with 5-FU as a positive control15 and for
antimicrobial activity against Escherichia coli, Staphy-
lococcus aureus, Enterobacter aerogenes, Bacillus sub-
tilis, and Candida albicans by an agar dilution method.16
Acknowledgment. This work was supported by grants
from the National Basic Research Program of China (No.
2010CB833800), National Natural Science Foundation of
China (Nos. 30973680 and 30670219), Special Fund for
Marine Scientific Research in the Public Interest of China
(No. 2010418022-3), and PCSIRT (No. IRT0944). The
cytotoxicity assay was performed at the Division of Mo-
lecular Pharmacology, OUC.
Supporting Information Available. The NMR spectra
and UV and IR data of 1-3, the X-ray data for 1 and 3,
HPLC profiles of acidic hydrolysates of 1-3, 18S rRNA
sequence of A. versicolor LCJ-5-4, isolation, purification,
and bioassay protocols used. This material is available
free of charge via the Internet at http://pubs.acs.org.
(15) Mosmann, T. Immunol. Methods 1983, 65, 55–63.
(16) Zaika, L. L. J. Food Safety 1988, 9, 97–118.
Org. Lett., Vol. 13, No. 5, 2011
1133