Aniquinazolines A-D, four new quinazolinone alkaloids from marine-derived endophytic fungus aspergillus nidulans

Article abstract of DOI:10.3390/md11072682

Four new quinazolinone alkaloids, namely, aniquinazolines A-D (1-4), were isolated and identified from the culture of Aspergillus nidulans MA-143, an endophytic fungus obtained from the leaves of marine mangrove plant Rhizophora stylosa. The structures of the new compounds were elucidated by spectroscopic analysis, and their absolute configurations were determined on the basis of chiral HPLC analysis of the acidic hydrolysates. The structure for 1 was confirmed by single-crystal X-ray diffraction analysis. All these compounds were examined for antibacterial and cytotoxic activity as well as brine shrimp (Artemia salina) lethality.

Full text of DOI:10.3390/md11072682

Mar. Drugs 2013, 11, 2682-2694; doi:10.3390/md11072682
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Aniquinazolines A–D, Four New Quinazolinone Alkaloids from
Marine-Derived Endophytic Fungus Aspergillus nidulans
1
,2
1
1
1,2
1
Chun-Yan An , Xiao-Ming Li , Chun-Shun Li , Ming-Hui Wang , Gang-Ming Xu and
1
,
Bin-Gui Wang *
1
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of
Sciences, Nanhai Road 7, Qingdao 266071, China; E-Mails: anchy5885@gmail.com (C.-Y.A.);
lixmqd@yahoo.com.cn (X.-M.L.); lichunshun@ms.qdio.ac.cn (C.-S.L.);
wangmh29@yahoo.cn (M.-H.W.); AericXu@gmail.com (G.-M.X.)
2
University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, China
*
Author to whom correspondence should be addressed; E-Mail: wangbg@ms.qdio.ac.cn;
Tel./Fax: +86-532-8289-8553.
Received: 11 March 2013; in revised form: 29 May 2013 / Accepted: 25 June 2013 /
Published: 23 July 2013
Abstract: Four new quinazolinone alkaloids, namely, aniquinazolines A–D (1–4), were
isolated and identified from the culture of Aspergillus nidulans MA-143, an endophytic
fungus obtained from the leaves of marine mangrove plant Rhizophora stylosa. The
structures of the new compounds were elucidated by spectroscopic analysis, and their
absolute configurations were determined on the basis of chiral HPLC analysis of the acidic
hydrolysates. The structure for 1 was confirmed by single-crystal X-ray diffraction analysis.
All these compounds were examined for antibacterial and cytotoxic activity as well as brine
shrimp (Artemia salina) lethality.
Keywords: mangrove; endophytic fungus; Aspergillus nidulans; secondary
metabolites; bioactivity
1
. Introduction
Quinazolinone derivatives generally refer to the compounds possessing a building block of
quinazolin-4-one. More than 160 naturally occurring quinazolinones have been isolated from different
organisms including plants, animals and microorganisms [1]. This family of alkaloids has attracted great

Mar. Drugs 2013, 11
2683
attention due to their diversified structures and potent biological activities such as cytotoxicity,
anti-feedant, and anti-microbial activities [2–9]. It is notable that some quinazolinone derivatives
biogenetically synthesized by nonribosomal peptide synthesis (NRPS) [10] were isolated from a diverse
group of fungi including Aspergillus fumigatus, A. flavipes, A. versicolor, Chaetomium sp., and
Penicillium thymicola. During our ongoing investigation for the bioactive secondary metabolites of
marine-derived fungi [11–15], four new quinazolinone alkaloids, namely, aniquinazolines A–D (1–4)
(Figure 1), were isolated and elucidated from the culture of the fungus Aspergillus nidulans MA-143,
which was obtained from the leaves of a mangrove plant Rhizophora stylosa. Details of the isolation,
structure elucidation, and biological activity of compounds 1–4 are reported herein.
Figure 1. Structures of the isolated compounds 1–4 and reference compounds
fumiquinazoline C and chaetominine.
2
. Results and Discussion
2
.1. Structure Elucidation of the New Compounds 1–4
Compound 1 was obtained as a colorless crystal. Its molecular formula was determined as
1
C H N O on the basis of HRESIMS, with 17 degrees of unsaturation. Analysis of the H NMR
26
25
5
4
spectrum of 1 revealed the resonances of four triple-doublets at δ 7.88 (td dd, J = 7.9, 1.3 Hz, H-8), 7.61
H
(td, J = 7.9, 0.9 Hz, H-9), 7.31 (td, J = 7.7, 0.9 Hz, H-25) and 7.04 (td, J = 7.7, 0.9 Hz, H-26),
two double-doublets at δ 7.75 (dd, J = 7.9, 0.9 Hz, H-7) and 8.18 (dd, J = 7.9, 1.3 Hz, H-10), and
H
two doublets at δ 7.38 (d, J = 7.7 Hz, H-24) and 7.25 (d, J = 7.7 Hz, H-27) (Table 1), which indicated
H
1
3
the presence of two 1,2-disubstituted phenyl groups (C-6 to C-11 and C-23 to C-28) in 1. Its C and
2
3
DEPT NMR spectra showed signals for three methyls, one methylene, eight sp and four sp methines, as
3
well as ten quaternary carbons including two oxygenated sp carbons and three ester/amide carbonyl
carbons (Table 2). A detailed NMR data comparison with those reported for fumiquinazoline C, a
quinazolinone alkaloid identified from a fungal strain of Aspergillus fumigatus originally derived from
the marine fish Pseudolabrus japonicus [2], revealed that the two compounds had the same carbon
skeleton. Compound 1 differed from fumiquinazoline C mainly at the presence of an isopropyl group
(
δ 2.09/δ 31.4, CH-29; δ 1.07/δ 17.9, CH -30; and δ 1.07/δ 18.5, CH -31) connected at C-20 in 1,
H C H C 3 H C 3
as it was a methyl group (δ 1.06/δ 18.7, CH ) in that of fumiquinazoline C. This deduction was further
H
C
3
1
1
verified by the H– H COSY correlations from H-29 to H-20 and H -30/H -31 as well as by the observed
3
3
HMBC correlations from H -31 to C-20, C-29, and C-30 and from NH-19 to C-29 as shown in Figure 2.
3

Mar. Drugs 2013, 11
2684
1
Table 1. H NMR (500 MHz) data of compounds 1–4 in DMSO-d (δ in ppm).
6
Position
1 (J in Hz)
2 (J in Hz)
8.61, br s
3 (J in Hz)
4 (J in Hz)
2
-NH
9.84, br s
9.49, br s
3
4
7
8
9
4.94, q (6.5)
8.30, s
7.75, dd (7.9, 0.9)
7.88, td (7.9, 1.3)
7.61, td (7.9, 0.9)
8.18, dd (7.9, 1.3)
5.36, dd (5.5, 1.5)
7.66, d (7.8)
7.82, t (7.8)
7.70, m
7.71, dd (7.9, 0.8)
7.87, td (7.9, 1.3)
7.59, td (7.9, 0.8)
8.18, dd (7.9, 1.3)
4.86, dd (8.1, 5.3)
7.86, td (7.8, 1.2)
7.58, td (7.8, 1.2)
8.16, dd (7.8, 1.2)
5.44, dd (7.4, 6.5)
7.53, t (7.8)
1
1
1
0
4
5
8.12, d (7.8)
5.58, dd (9.6, 4.3)
2.98, dd (14.0, 5.5) 2.59, dd (14.8, 9.6) 2.61, dd (14.5, 7.4) 3.01, dd (14.0, 8.1)
.92,dd (14.0, 1.5) 1.83, dd (14.8, 4.3) 2.20, dd (14.5, 6.5) 2.77, dd (14.0, 5.3)
1
1
6
1.91, s
1.59, d (6.5)
5.67, br s
1.76, s
5.67, br s
1
1
7-OH
5.45, br s *
5.82, s
1
8
5.70, dd (6.0, 1.5)
2.67, d (5.5)
5.26, d (5.8)
3.51, m
5.31, dd (7.3, 1.5)
3.38, dd (7.3, 2.8)
3.53, br s
9-NH
2
2
2
2
2
2
3
3
0
4
5
6
7
9
0
1
3.59, br d (5.5)
7.38, d (7.7)
3.55, br s
4.21, d (9.3)
7.47, d (7.4)
7.43, td (7.4, 1.2)
7.26, td (7.4, 1.2)
7.54, d (7.4)
2.45, m
7.35, d (7.8)
7.26, t (7.8)
7.09, t (7.8)
7.82, d (7.8)
1.99, m
7.34, d (7.6)
7.27, td (7.6, 1.0)
7.15, td (7.6, 1.0)
7.70, m
7.31, td (7.7, 0.9)
7.04, td (7.7, 0.9)
7.25, d (7.7)
2.09, dq (13.4, 6.8)
1.07, d (6.8)
1.98, m
0.93, d (6.8)
0.96, d (6.8)
0.91, d (6.6)
0.92, d (6.6)
1.15, d (6.7)
1.12, d (6.7)
1.07, d (6.8)
*
This exchangeable proton was detected in acetone-d6.
13
Table 2. C NMR (125 MHz) data of compounds 1–4 in DMSO-d (δ in ppm).
6
Position
1
2
3
4
1
3
4
6
7
8
9
168.6, C
84.2, C
168.9, C
48.5, CH
153.3, C
146.6, C
126.9, CH
134.4, CH
126.7, CH
126.4, CH
120.0, C
160.2, C
51.8, CH
35.5, CH2
16.5, CH3
80.1, C
169.7, C
84.4, C
164.8, C
151.5, C
146.6, C
127.8, CH
134.6, CH
127.5, CH
126.4, CH
120.5, C
158.8, C
52.7, CH
33.7, CH2
23.7, CH3
86.3, C
150.3, C
146.0, C
127.3, CH
134.6, CH
127.4, CH
126.4, CH
120.1, C
160.5, C
52.6, CH
39.1, CH2
20.3, CH3
80.3, C
146.7, CH
147.5, C
127.1, CH
134.6, CH
127.3, CH
126.1, CH
121.5, C
1
1
1
1
1
1
1
1
2
2
2
0
1
2
4
5
6
7
8
0
1
3
159.7, C
55.6, CH
34.1, CH2
73.8, C
83.7, CH
69.3, CH
173.8, C
140.2, C
88.4, CH
69.0, CH
171.5, C
136.6, C
88.1, CH
69.1, CH
172.1, C
137.2, C
88.1, CH
69.0, CH
172.6, C
137.7, C

Mar. Drugs 2013, 11
2685
Table 2. Cont.
2
2
2
2
2
2
3
3
4
5
6
7
8
9
0
1
114.3, CH
129.7, CH
125.1, CH
126.2, CH
137.4, C
115.1, CH
128.8, CH
124.6, CH
125.4, CH
138.7, C
115.2, CH
128.6, CH
124.6, CH
125.2, CH
138.8, C
114.2, CH
130.0, CH
125.1, CH
124.5, CH
134.9, C
31.4, CH
17.9, CH3
18.5, CH3
31.0, CH
17.6, CH3
18.6, CH3
31.0, CH
17.4, CH3
18.5, CH3
28.2, CH
18.6, CH3
20.1, CH3
1
1
Figure 2. Key H– H COSY (bold lines) and HMBC (red arrows) correlations of
compounds 1–4.
2
5
26
2
6
2
4
16
24
27
1
6
5
7
5
OH
17
6
O
3
6
22N
N
H
22N
1
N
2
8
9
8
O
O
17
18 HN
8
HN
21
2
1
N
12
N
12
N19H 15
1
1
NH 15
0
29
13
1
1
31
2
1
3
1
14
1
0
1
9
1
O
10
O
3
0
3
0
O
O
1
2
2
5
2
6
2
5
2
7
16
2
7
OH
OH3
5
5
4
22N 18
N
OH4
N
6
2
8
22N
N
O
8
6
O
HN
21
18
N
12
21
NH 15
1
1
2
9
20
N
19
3
1
19
31
11
2
9
13
14
1
0
1
0
1
O
O
3
0
O
O
3
0
3
4
The relative configuration of compound 1 was determined by NOESY spectrum and by the X-ray
experiment. The NOE correlations from H-16 to H-14 and H-18 as well as from H-18 to H-29 indicated
the same orientation of these protons (Figure 3). The structure and relative configuration of compound 1
were confirmed by the single crystal X-ray diffraction analysis (Figure 4). The absolute configuration of
1
was deduced by chiral HPLC analysis of the degradation products of the acidic hydrolysates. The
HPLC profile of the hydrolysates was compared with that of authentic standard to establish the
configuration of the amino acid-derived unit as L-valine (Figure 5), corresponding to the S-configuration
for C-20. The absolute configuration of compound 1 was therefore assigned to be 3R, 14R, 17R, 18R,
and 20S, which was identical to that of fumiquinazoline C [2]. The ECD (Electronic Circular Dichroism)
spectrum of 1 showed negative Cotton Effect (CE) at 199, 238, 258, and 303 nm, and positive CE at 216
nm (Figure 6). The structure of 1 was thus elucidated and it was named as aniquinazoline A.

Mar. Drugs 2013, 11
2686
Figure 3. NOESY correlations of compounds 1–4.
Figure 4. X-ray structure of compound 1 (Note: A different numbering system is used for
the structure in the text).

Mar. Drugs 2013, 11
Figure 5. Chiral HPLC analysis of the amino acid derivatives of compounds 1–4.
2687
Figure 6. Electronic Circular Dichroism (ECD) spectra of compounds 1–4.
Compound 2 was obtained as yellowish solid. The HRESIMS experiment established its molecular
1
13
formula C H N O (16 degrees of unsaturation), with two more H-atom than that of 1. The H and C
2
6
27
5
4
NMR data of 2 were very similar to those of 1 (Tables 1 and 2), indicating that they shared the same
3
13
carbon skeleton. However, the oxygenated sp quaternary carbon at δ 84.2 (C-3) in the C NMR
C
spectrum of 1 disappeared in that of 2. Instead, signals for a methine group (δ 4.94/δ 48.5, CH-3) were
H
C

Mar. Drugs 2013, 11
2688
observed in the NMR spectra of 2 (Tables 1 and 2). Accordingly, the singlet methyl signal at δ 1.91
H
1
(
H -16) in the H NMR spectrum of 1 was replaced by a doublet methyl group at δ 1.59
3
H
(
J = 6.5 Hz) in that of 2, and an exchangeable OH proton at δ 5.67 (br s, 17-OH) was observed (Table 1).
H
This OH group was attached at C-17 on the basis of HMBC correlations from the OH proton to C-15 (δ
C
1
1
3
5.5, CH ), C-17 (δ 80.1, C), and C-27 (δ 125.4, CH) (Figure 2). The H– H COSY and HMBC
2 C C
correlations shown in Figure 2 confirmed the planar structure of 2. The relative configuration of
1
compound 2 was determined by NOESY spectrum as well as by comparing the H-NMR J-values with
that of fumiquinazolines A and B [2]. The observed NOE correlations from H-18 to H-14 and H-29 and
from H-14 to H-3 and 17-OH, indicated them on the same face of the molecule (Figure 3). In contrast to
compound 1, no NOE correlations could be observed from H-16 to H-14 and H-18. Instead, NOE
correlation from H-14 to H-3 was observed in the NOESY spectrum of 2 (see Figure 3 as well as
Figure S12b in the Supplementary Materials). In the reference report, a large coupling constant (J = 4.9 Hz)
for H-3 and NH-2 was observed in fumiquinazoline B, whereas its C-3 epimer, fumiquinazoline A,
showed a very small coupling constant (J = 0.3 Hz) [2]. In the case of compound 2, the proton NH-2 was
observed as a broad singlet (Table 1), which means the coupling constant between H-3 and NH-2 was
very small, thus suggesting the configuration at C-3 is close to that of funiquinazoline A. The above data
suggested that the C –O–C ether ring in 1 was opening in that of 2, and the configuration of C-3
3
17
changed during the ring-opening process. To substantiate this, compound 1 was reduced with NaBH in
4
THF at room temperature for 40 min and the resulting product was revealed to be identical to 2 by
1
1
1
co-TLC analysis. The H-NMR and H– H COSY as well as NOESY spectra also showed that the
reduced product of 1 was the same as that of compound 2 (see Supplementary Material). However, it was
described in the reference that reduction of fumiquinazoline C yielded fumiquinazoline A without C-3
configuration change [2], and this differs from our observation as discussed above. Unfortunately, no
NOESY experiment was performed and described for the reducing product of fumiquinazoline C [2], which
would have helped give solid evidence to clarify the problem. Based on the above data, the relative
configurations between C-3 and C-14 of fumiquinazoline A [2] should be reconsidered.
The absolute configuration of C-20 in compound 2 was also assigned as S based on the chiral HPLC
analysis of its acidic hydrolysates (Figure 5) and the absolute configurations of other chiral centers in 2
were therefore assigned as 3R, 14R, 17R, and 18R. The structure of 2 was thus elucidated and named as
aniquinazoline B.
Compound 3 was obtained as a yellowish solid. Its molecular formula was determined to be
C H N O (16 degrees of unsaturation) on the basis of HRESIMS data, with one more O-atom than
26
27
5
5
that of 2. The assignments of the NMR data of 3 (Tables 1 and 2) matched well with that of the
corresponding signals for 2, except for the presence of an OH group at C-3 in 3, which was consistent
with the difference in molecular formula. Accordingly, the non-oxygenated CH signals resonated at δH
4
.94/δ 48.5 (CH-3) in the NMR spectra of 2 and disappeared in that of 3. Instead, an oxygenated
C
1
3
quaternary carbon at δ 84.4 (C-3) was observed in the C NMR spectrum of 3 (Table 2).
C
1
Correspondingly, the methyl doublet signal at δ 1.59 (J = 6.5 Hz, H -16) in the H NMR spectrum of 2
H
3
1
1
was replaced by a downfield singlet at δ 1.76 (H -16) in that of 3 (Table 1). The H– H COSY and
H
3
HMBC correlations shown in Figure 2 confirmed the planar structure of 3.
The relative configuration of compound 3 was determined by NOESY spectrum. The NOE
correlations from H-18 to H-29 and 17-OH and from 17-OH to H-14 revealed these protons were on the

Mar. Drugs 2013, 11
2689
same face of the molecule (Figure 3). The relative configuration at C-3 was assigned as being the same
as that of 2 from the biogenetic point of view. The absolute configuration of C-20 was also determined as
S by the chiral HPLC analysis of the acidic hydrolysates of 3 (Figure 5). Consequently, the absolute
configuration of compound 3 was assigned as 3S, 14R, 17R, 18R, and 20S. The very similar ECD spectra
for compounds 2 and 3 (Figure 6) supported the above assignment. The structure of compound 3 was
thus determined and was named aniquinazoline C.
Compound 4 was isolated as a yellowish solid. Its molecular formula was determined as C H N O
4
2
4
22
4
by HRESIMS data. Detailed comparison of NMR data of compound 4 (Tables 1 and 2) with those of
chaetominine, a cytotoxic alkaloid produced by endophytic Chaetomium sp. IFB-E015 originally
isolated from the apparently healthy Adenophora axilliflora leaves [5], suggested that they shared the
same carbon skeleton. The main difference was that the methyl group at C-20 in chaetominine was
1
1
replaced by an isopropyl group in 4. This deduction was verified by H– H COSY and HMBC
correlations as shown in Figure 2. The relative configuration of 4 was assigned by NOESY spectrum.
The correlations from H-18 to H-14, 17-OH and H-20 revealed their same orientation on the molecule
(Figure 3). The absolute configuration of C-20 was assigned as S on the basis of the chiral HPLC
analysis of the acidic degradation product of 4 (Figure 5). Thus the absolute configuration of compound
4
was determined as 14R, 17S, 18S, and 20S, and it was named as aniquinazoline D.
.2. Biological Activities of the Isolated Compounds
2
The isolated compounds 1–4 were examined for brine shrimp toxicity, antitumor, and antibacterial
activities. Compounds 1–4 showed potent lethality against brine shrimp with LD values of 1.27, 2.11,
5
0
4
.95 and 3.42 μΜ, respectively, which were stronger than that of the positive control colchicine (with
LD value of 88.4 μΜ). Compounds 1–4 were also evaluated for antitumor and antibacterial activity,
5
0
but none of them displayed inhibitory activity against four cell lines (BEL-7402, MDA-MB-231, HL-60,
and K562) and two bacteria (Escherichia coli and Staphyloccocus aureus).
3
. Experimental Section
3
.1. General
The melting point was determined with an SGW X-4 micro-melting-point apparatus. The optical
rotations were measured on an Optical Activity A-55 polarimeter. UV data were obtained on a
1
13
Lengguang Gold S54 spectrophotometer. The H, C, and 2D NMR spectroscopic data were acquired
on Bruker Advance 500 spectrometers. Mass spectra were measured on a VG Autospec 3000 mass
spectrometer. HPLC analysis was performed on a Dionex HPLC System equipped with a P680 pump, an
ASI-100 automated sample injector, a TCC-100 column oven, a UV-DAD 340U detector, and a Dionex
Acclaim ODS column (4.6 × 250 mm, 5 μm). Semi-preparative HPLC was operated on a Dionex
UltiMate U3000 system using an Agilent Prep RP-18 column (21.2 × 250 mm, 10 μm) with UV
detection. A Phenomenex-Chirex 3126 N,S-dioctyl-(D)-penicillamine column (250 × 4.60 mm, 5 μm)
was used for chiral HPLC analysis. Column chromatography (CC) was performed with silica gel
(200–300 mesh, Qingdao Marine Chemical Factory), Lobar LiChroprep RP-18 (40–63 μm; Merck), and
Sephadex LH-20 (18–110 μm, Merck).

Mar. Drugs 2013, 11
.2. Fungal Material
The fungus Aspergillus nidulans MA-143 was isolated from the leaves of marine mangrove plant
2690
3
Rhizophora stylosa. The fungus grew slowly on potato dextrose agar (PDA) plate and turned from white
to green mycelia within 5 days. The fungus was identified by sequence analysis of the ITS region of its
rDNA as described previously [16] and the sequence data obtained from the fungus have been deposited
in GenBank with accession number JQ839285. The strain is preserved at the Key Laboratory of
Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences.
3
.3. Fermentation
The fermentation was statically carried out in liquid potato-dextrose broth medium (PDB) (1000 mL
seawater, 20 g glucose, 5 g peptone, 3 g yeast extract, pH 6.5–7.0, liquid medium/flask = 300 mL) in
1
L Erlenmeyer flasks for 30 days at room temperature.
3
.4. Extraction and Isolation
The fermented whole broth (100 flasks) was filtered through cheesecloth to separate the culture broth
and mycelia, which were extracted exhaustively with EtOAc and MeOH, respectively. Since the
chemical profiles of the two extracts were almost identical, they were combined and concentrated to
afford the crude extract (31.0 g) for further separation. The crude extract was subjected to silica gel
vacuum liquid chromatography (VLC) eluting with mixed solvents of increasing polarity (petroleum
ether-EtOAc, 5:1 to 1:1, and then CHCl –MeOH, 20:1 to 0:1) to yield 7 fractions (Fr.1 to Fr.7). Fraction 2
3
(11.0 g) was further separated to seven subfractions (Fr.2.1–2.7) by CC on silica gel eluted with
CHCl –MeOH (100:1 to 5:1). Fr.2.1 (7.0 g) was subjected to CC on Lobar LiChroprep C eluting with
3
18
MeOH-H O gradient (1:9 to 1:0) and then further purified by semi-preparative HPLC (MeOH–H O 7:3,
2
2
1
6 mL/min) to afford 2 (t = 14.6 min, 5.2 mg). Fr.2.2 (3.0 g) was subjected to CC on silica gel eluted
R
with CHCl –MeOH (100:1 to 5:1) and Sephadex LH-20 (petroleum ether–CHCl –MeOH, 5:5:1), and
3
3
then further purified by semi-preparative HPLC (MeOH–H O 7:3, 16 mL/min) to obtain 1 (t = 15.8 min,
2
R
2
0.0 mg), 3 (t = 13.5 min, 5.5 mg), and 4 (t = 10.5 min, 7.7 mg).
R
R
20
Aniquinazoline A (1): colorless crystal; mp 243–245 °C; [α] : −13 (c 0.30, MeOH); UV (MeOH)
D
λmax (log ε) 200 (4.41), 218 (4.26), 245 (3.97), 270 (3.82), 297 (3.32), 305 (3.37) nm; CD λ (Δε)
max
1
13
1
99 (−11.33), 216 (+21.60), 238 (−4.92), 258 (−4.52), 303 (−2.63) nm; H and C NMR data, see
+
+
+
Tables 1 and 2; ESIMS m/z 472 [M + H] ; HRESIMS m/z 472.1982 [M + H] (calcd for C H N O ,
2
6
26
5
4
4
72.1985, Δ 0.3 ppm).
Aniquinazoline B (2): yellowish solid; [α] : −118 (c 0.28, MeOH); UV (MeOH) λ (log ε) 202
2
D
0
max
(
4.46), 225 (4.33), 243 (4.03), 304 (3.35) nm; CD λ (Δε) 197 (−10.99), 210 (+21.55), 233 (−19.63),
max
1
13
+
3
4
04 (−1.59) nm; H and C NMR data, see Tables 1 and 2; ESIMS m/z 474 [M + H] ; HRESIMS m/z
+
+
74.2143 [M + H] (calcd for C H N O , 474.2141, Δ 0.2 ppm).
2
6
28
5
4
2
D
0
Aniquinazoline C (3): yellowish solid; [α] : −19 (c 0.28, MeOH); UV (MeOH) λ
(log ε) 200
max
(4.63), 214 (4.51), 246 (4.14), 271 (4.01), 290 (3.70), 301 (3.61) nm; CD λmax (Δε) 199 (−9.37), 212

Mar. Drugs 2013, 11
2691
1
13
(
+24.53), 235 (−21.94), 303 (−2.65) nm; H and C NMR data, see Tables 1 and 2; ESIMS m/z 490
+
+
+
[
M + H] ; HRESIMS m/z 490.2084 [M + H] (calcd for C H N O , 490.2090, Δ 0.6 ppm).
2
6
28
5
5
2
D
0
Aniquinazoline D (4): yellowish solid; [α] : −33 (c 0.37, MeOH); UV (MeOH) λ
(log ε)
max
2
00 (6.06), 218 (5.91), 246 (5.61), 268 (5.52), 298 (4.98), 312 (4.92) nm; CD λ (Δε) 201 (−33.36), 218
max
1
13
(+13.62), 244 (−9.78), 276 (+1.17), 301 (−2.08) nm; H and C NMR data, see Tables 1 and 2; ESIMS m/z
+
+
+
4
31 [M + H] ; HRESIMS m/z 431.1710 [M + H] (calcd for C H N O , 431.1719, Δ 0.9 ppm).
24 23 4 4
3
.5. X-ray Crystallographic Analysis of Compounds 1
All crystallographic data [17] were collected on a Bruker Smart-1000 CCD diffractometer equipped
with a graphite-monochromatic Mo-Kα radiation (λ = 0.71073 Å) at 298(2) K. The data were corrected
for absorption by using the program SADABS [18]. The structure was solved by direct methods with the
SHELXTL software package [19]. All non-hydrogen atoms were refined anisotropically. The H atoms
were located by geometrical calculations, and their positions and thermal parameters were fixed during
the structure refinement. The structure was refined by full-matrix least-squares techniques [20].
Crystal data for compound 1: C H N O , F.W. = 503.55, one molecule containing a CH OH solvent
27
29
5
5
3
molecule in the unit, monoclinic space group P2(1), unit cell dimensions a = 11.3860(5) Å,
3
b = 7.8898(3) Å, c = 13.6963(5) Å, V = 1230.18(8) Å , α = γ = 90, β = 91.044(3), Z = 2,
3
−1
dcalcd = 1.359 mg/m , crystal dimensions 0.45 × 0.40 × 0.32 mm, μ = 0.096 mm , F(000) = 532. The
506 measurements yielded 4172 independent reflections after equivalent data were averaged, and
7
Lorentz and polarization corrections were applied. The final refinement gave R = 0.0332 and
1
wR = 0.0778[I > 2σ(I)].
2
3
.6. Amino Acid Analysis
Compounds 1 (1.3 mg), 2 (0.9 mg), 3 (0.8 mg), and 4 (1.1 mg) were each solved in 10 mL 6 N HCl
and heated at 110 °C for 24 h in sealed tubes. The solutions were then evaporated to dryness under
reduced pressure. Each sample, including the standard amino acid L-val and D-val, was solved in 1 mL of
eluting solvent (2 mM CuSO ·5H O, with 5 mL CH CN in every 100 mL solvent) and was concentrated
4
2
3
at a speed of 12,000 rpm. Chiral HPLC analysis was carried out using a Phenomenex-Chirex-3126
column at 254 nm with flow rate 1.0 mL/min at 40 °C . The HPLC analysis showed that the products of
acidic hydrolysis of 1–4 contained the identical amino acid, which have the same retention time as that
of the standard L-val. The results established the chiral center of the valine moiety in the structure of the
four compounds to be S-configuration (Figure 5).
3
.7. Reduction of Compound 1
Compound 1 (6.8 mg) was dissolved in 1.0 mL THF (tetrahydrofuran), and NaBH (5.0 mg) was
4
added to the solution. The mixture was stirred at room temperature for 40 min and then concentrated
under reduced pressure. The residue was diluted with water, extracted by CH Cl and then evaporated.
2
2
The reducing product (1.5 mg) was obtained from the extract by preparative TLC (CHCl –MeOH, 30:1).
3
Compound 2 and the reducing product were evidenced to be identical on the basis of co-TLC and NMR
data analysis (see Supplementary Material, Figures S28–S30).

Mar. Drugs 2013, 11
.8. Brine Shrimp Toxicity
2692
3
Brine shrimp (Artemia salina) toxicity of the isolated compounds was determined as described
previously [21]. Colchicine was used as a positive control.
3
.9. Cytotoxicity Assay
The cytotoxic activity against BEL-7402 (human hepatocellular carcinoma), MDA-MB-231 (human
breast carcinoma), HL-60 (human promyelocytic leukemia cells), and K562 (human acute myelocytic
leukemia cell line) cell lines were determined according to previously reported methods [22].
3
.10. Antibacterial Assay
Antibacterial assay against E. coli and S. aureus was carried out using the well diffusion method [23].
Chloromycetin was used as a positive control.
4
. Conclusions
Four new quinazolinone alkaloids (1–4) were isolated from the mangrove-derived endophytic fungus
Aspergillus nidulans MA-143. The structures and relative configurations were determined on the
interpretation of the NMR data and the absolute configuration of all compounds was determined by
chiral HPLC analysis of the acid hydrolysates. Compounds 1–4 exhibited potent brine shrimp toxicity
with LD values of 1.27, 2.11, 4.95 and 3.42 μΜ, respectively.
5
0
Acknowledgments
This work was financially supported by Ministry of Science and Technology (2013AA092901 and
2
010CB833802) and by National Natural Science Foundation of China (31270403).
Conflict of Interest
The authors declare no conflict of interest.
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