Cristatumins A-D, new indole alkaloids from the marine-derived endophytic fungus Eurotium cristatum EN-220

Article abstract of DOI:10.1016/j.bmcl.2012.05.088

Four new indole alkaloids, namely, cristatumins A-D (1-4), along with six known congeners (5-10) were identified from the culture extract of Eurotium cristatum EN-220, an endophytic fungus isolated from the marine alga Sargassum thunbergii. The structures of these compounds were established on the basis of extensive spectroscopic analysis. Each of these compounds was evaluated for antimicrobial and insecticidal activity. Compounds 1 and 9 showed antibacterial activity against Escherichia coli and Staphyloccocus aureus, respectively, while compounds 2, 6, and 7 exhibited moderate lethal activity against brine shrimp. Preliminary structure-activity relationships were also discussed.

Full text of DOI:10.1016/j.bmcl.2012.05.088

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4650–4653
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cristatumins A–D, new indole alkaloids from the marine-derived
endophytic fungus Eurotium cristatum EN-220
Feng-Yu Du a,b, Xiao-Ming Li a, Chun-Shun Li a, Zhuo Shang a, Bin-Gui Wang a,
⇑
a Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, China
b Graduate School of the Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new indole alkaloids, namely, cristatumins A–D (1–4), along with six known congeners (5–10) were
identified from the culture extract of Eurotium cristatum EN-220, an endophytic fungus isolated from the
marine alga Sargassum thunbergii. The structures of these compounds were established on the basis of
extensive spectroscopic analysis. Each of these compounds was evaluated for antimicrobial and insecti-
cidal activity. Compounds 1 and 9 showed antibacterial activity against Escherichia coli and Staphyloccocus
aureus, respectively, while compounds 2, 6, and 7 exhibited moderate lethal activity against brine shrimp.
Preliminary structure–activity relationships were also discussed.
Received 23 March 2012
Revised 18 May 2012
Accepted 24 May 2012
Available online 1 June 2012
Keywords:
Marine endophyte
Eurotium cristatum
Indole alkaloid
Ó 2012 Elsevier Ltd. All rights reserved.
Antimicrobial activity
Brine shrimp inhibition
The fungal genus Eurotium, which is the teleomorph of Aspergil-
lus, has been proved to be a rich source of novel bioactive metabo-
lites.1–3 The tryptophan-derived alkaloid, generally characterized
by a reverse isoprenic chain in the C-2 position of the indole nucleus,
is an important class of secondary metabolites found in Eurotium
species.2,3 These compounds were reported to possess radical
scavenging,4 anti HIV-1 replication,5 and antibacterial activity.6
As part of our efforts toward the chemical investigation of mar-
ine-derived fungi, a variety of structurally interesting and biologi-
cally active compounds were isolated and identified from fungal
species derived from marine algae7,8 and mangrove plants.9 In
the current study, four new indole alkaloids, cristatumins A–D
(1–4, Fig. 1),10 along with six known congeners (5–10, Fig. 1), were
isolated from Eurotium cristatum EN-220, an endophytic fungus
isolated from the marine alga Sargassum thunbergii. This Letter
describes the isolation, structure elucidation and biological activity
of these compounds (1–10).
The fungal strain E. cristatum EN-220 was cultured on rice solid
medium at room temperature under static conditions for 30 days.
The rice culture was exhaustively extracted with EtOAc to give a
crude extract, which was dried and fractionated by silica gel vac-
uum liquid chromatography (VLC) using different solvent systems
of increasing polarity from petroleum ether (PE) to MeOH to yield
12 fractions (Frs. 1–12) based on TLC analysis. Frs. 7–10 were fur-
ther purified by a combination of silica gel, Sephadex LH-20, and
Lobar LiChroprep RP-18 column chromatography to yield four
new indole alkaloids 1–4, along with six other congeners including
neoechinulin A (5),11 isoechinulin A (6),12 variecolorin G (7),4 pree-
chinulin (8),13 tardioxopiperazine A (9),14 and echinulin (10).15
Compound 1,10 a colorless amorphous powder, was detemined
to have a molecular formula C19H21N3O3 on the basis of HRESIMS
data. The 1D NMR data (Table 1) and UV absorptions at kmax 223,
284, and 333 nm suggested that 1 was an anologue of neoechinulin
A (5).11 However, the methyl signals at dC 17.9 (q, C-20) and dH 1.42
of 5 were replaced by the oxygenated CH2 signals at dC 65.3 (t, C-
20) and dH 4.00, 3.89 of 1. This deduction was supported by the
1H–1H COSY correlations from 20-OH to H-20 and from H-20 to
H-12 (Fig. 2). The above evidence indicated that the alanine residue
in the 2,5-diketopiperazine moiety of 5 was replaced by the serine
residue in 1, which was so far not found in this type of indole dike-
topiperazine. The lower-field-shifted CH at dH 7.03 (1H, s, H-8) of 1
implied that H-8 was influenced by the deshielding effect of the
C@O group, which suggested the double bond between C-8 and
C-9 to have Z-geometry.16 The absolute configuration at C-12
was tentatively assigned as S by comparing its specific rotation
(½a 2D0
ꢀ
= ꢁ17.6, c 0.34, MeOH) with that of 5 (½a D24
= ꢁ28.0, c 0.53,
ꢀ
MeOH).17 Based on the above spectral evidence, the structure of
1 was determined and it was named as cristatumin A.
Compound 2 was obtained as a colorless amorphous powder
with the molecular formula, C29H39N3O3, as established by the
HRESIMS analysis. The 1D NMR data of 2 (Table 1) revealed marked
similarities to echinulin (10)15 except that the methyl group at
C-20 (dC 19.9, dH 1.47) of 10 disappeared in that of 2. Instead, the
⇑
Corresponding author. Tel./fax: +86 532 82898553.
E-mail address: wangbg@ms.qdio.ac.cn (B.-G. Wang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.088

F.-Y. Du et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4650–4653
4651
O
O
H
1'
H
1'
H
N
15'
17'
15'
H
H
8'
N
8'
N
13'
N
13'
N
O
H
N
R1
7'a
7'a
R1
2'
20
2'
O
10 12
6'
6'
3'
3'
20
10 12
18'
HN
HN
8
3'a
3'a
9'
9'
8
N14
H
O
11'
11'
10'
10'
4
R2
19
4'
10
3
4'
10
N14
H
O
5
3a
7a
R2
5
3a
7a
3
19
O
O
O
O
18
4
11
11
18
3a
15
3a
9
9
15
NH
NH
N 1
H
3
3
7
R3
6
N
H 1
6
16
16
2
7
R3
2
1
15
1
15
N
N
17
7a
13
7a
13
8
17
2
8
N
N
30
25
H
H
26
21
H
H
17
17
1
2
O
O
2
8
, R3=
R =CH2OH, R =
1
3
23
24
28
29
3
11
1
5
R =CH2OH, R =R =H
5
1
2
3
5
O
R =CH3, R =R =H
1
2
3
O
R =CH3, R =R =H
25
8
3a
10
21
8
3a
11
3
1
2
25
OMe
O
10
6
, R3=H
R =CH3, R =
7
NH
21
23
7
19 HN14
7a
, R3= H
1
2
7a
19
9
24
R =CH3, R =
12
23
HN
HN
25
HN
14
24
25
1
O
15
1
21
18
O
4
15
7 R1=CH , R =H,
3
30
11
R =
26
21
3
2
18
O
, R3=
17
23
24
1
2
12
NH2
17
10
R =CH3, R =
28
23
29
24
12
Figure 1. The structures of isolated compounds 1–10 and the reference compounds 11 and 12.
oxymethylene signals (dC 63.9 for C-20, dH 3.94) were observed in
the 1D NMR spectrum of 2 (Table 1). These spectroscopic evidences
suggested that the C-20 Me group in 10 was replaced by CH2OH
group in 2. Further analysis of the 1H–1H COSY, HSQC, and HMBC
data verified the structure of 2 as shown in Figure 1. The NOE cor-
relations from H-9 to H-12 indicated the cis relationship of these
two protons. The absolute configuration of 2 was tentatively as-
signed as 9S and 12S by comparing its specific rotation with that
HMBC correlations (Fig. 2). The observed NOE correlations from
H-2 and H-13 to H-9, from H-9 to H-40/H-50, from H-90 to H-4,
and from H-20 and H-130 to H-90 established the relative configura-
tion of 3.19,20 Chiral HPLC analyses21 of the acid hydrolysate of 3
revealed that the valine and alanine residues were in D- and L-
configuration, respectively (Supplementary material). This result
indicated a 2S, 3S, 9R, 13R, 20R, 30R, 90S and 130S absolute configura-
tion for 3.
of 10 (½a 2D0
ꢀ
= ꢁ11.1 for 2 vs ½a D20
ꢀ
= ꢁ39.1 for 10, both determined
The HRESIMS data of cristatumin D (4) gave a molecular for-
mula of C19H21N3O4, indicating eleven degrees of unsaturation.
Its NMR data was very similar to 12 (neoechinulin E),12 except
one more methoxyl signal at dH 3.72 (3H, s, OCH3-10) and dC 51.9
was observed in the 1D NMR spectra of 4. However, the degrees
of unsaturation for 12 were twelve, one more than that of 4, indi-
cating that 4 was a ring-opened diketopiperazine derivative of 12.
This deduction was supported by the HMBC correlations from H-
OCH3 to C-10 and from 11-NH2 to C-11. The lower-field-shifted
CH at dH 7.77 (1H, s, H-8) of 4 suggested the double bond between
C-8 and C-9 to have Z-geometry.16 This is the first report of indole
alkaloid characterized by the ring open of 2,5-diketopiperazine
moiety.
in CHCl3).15,18 Based on the above spectral evidence, the structure
of 2 was determined and it was named as cristatumin B.
Cristatumin C (3) showed the molecular formula C30H32N6O4 as
determined by HRESIMS. Analysis of the 1D NMR data revealed
that, similar to 11,19 compound 3 was an almost symmetrical mol-
ecule consisting of two indole diketopiperazine moieties. The NMR
spectroscopic data of 3 suggested that the isobutyl unit in 11 was
replaced by a methyl group in 3, which was indicated by the fact
that the signals corresponding to isobutyl unit of 11 at dC 40.7 (t,
C-150), 23.5 (d, C-160), 22.6 (q, C-170), and 21.1 (q, C-180) were
not observed in that of 3. Instead, resonance for a methyl group
at dC 14.8 (q, C-150) was present. The above spectroscopic data indi-
cated that the leucine residue in 11 was replaced by an alanine res-
idue in 3. This deduction was supported by the difference in the
molecular formulae of 3 and 11, as well as the 1H–1H COSY, and
Compounds 1–10 were evaluated for the lethality against brine
shrimp (Artemia salina)22 and the results indicated that compounds
2, 6, and 7 exhibited moderate activity with the LD50 values of 74.4,
O
H
N
O
N
O
HN
OMe
O
O
NH
H
HN
O
N
H
O
O
O
HN
OH
NH
H
HN
HN
OH
N
H
NH
NH2
O
N
O
N
H
4
1
2
3
O
H
H
H
H
N
N
H
COSY
HN
O
O
H
H
O
H
H
C HMBC
O
H
NH
H
H
OH
N
H
NH
H
H NOE
N
N
H
H
O
2
3
(NOESY)
(NOESY)
Figure 2. Key HMBC, 1H-1H COSY, and NOE correlations of 1–4.

Table 1
1H and 13C NMR data of compounds 1–4
No.
1a
No.
2b
No.
3c
No.
4c
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
1-NH
2
3
3a
4
5
6
7
7a
8
9
10.28 (br s)
1-NH
2
3
3a
4
5
6
7
7a
8
9
8.05 (br s)
1-NH
2
3
3a
4
5
6
7
7a
9
10
11
12-NH
13
14
15
16
6.70 (s)
4.80 (s)
1-NH
2
3
3a
4
5
6
7
7a
8
9
11.23 (br s)
144.7 (s)
104.5 (s)
127.4 (s)
120.2 (d)
120.8 (d)
122.2 (d)
112.4 (d)
136.3 (s)
110.2 (d)
126.9 (s)
160.6 (s)
141.4 (s)
104.1 (s)
129.1 (s)
115.2 (d)
134.0 (s)
122.9 (d)
123.4 (s)
132.3 (s)
78.8 (d)
59.7 (s)
146.0 (s)
105.7 (s)
125.5 (s)
119.9 (d)
119.4 (d)
120.8 (d)
111.5 (d)
134.9 (s)
129.5 (d)
134.9 (s)
165.0 (s)
51.9 (q)
130.3 (s)
124.3 (d)
117.9 (d)
128.6 (d)
108.8 (d)
149.0 (s)
55.6 (d)
7.40 (d, 8.5)
7.02 (m)
7.09 (m)
7.15 (s)
6.80 (s)
7.39 (d, 7.3)
6.63 (m)
7.02 (m)
7.19 (d, 8.0)
6.89 (m)
7.03 (m)
7.38 (d, 8.6)
6.60 (d, 7.8)
7.37 (d, 8.0)
7.03 (s)
3.67 (dd, 14.6, 3.5) 3.23 (dd, 14.6, 11.7) 29.9 (t)
4.41 (dd, 11.7, 3.5)
4.08 (t, 9.5)
3.04 (m) 2.33 (dd, 13.9, 9.5) 37.2 (t)
168.3 (s)
8.25 (br d, 4.1)
3.37 (m)
7.77 (s)
54.6 (d)
168.4 (s)
10
10
10
11-NH 7.21 (br s)
12
13
14-NH 7.92 (br s)
15
16
17
18
19
20
11-NH 6.63 (br s)
12
13
14-NH 5.81 (br s)
15
10-OMe 3.72 (s)
11-NH2
12
13
14-NH
15
16
17
18
19
4.14 (m)
59.4 (d)
165.3 (s)
4.09 (br s)
56.2 (d)
166.0 (s)
62.4 (d)
167.4 (s)
31.8 (d)
18.0 (q)
18.9 (q)
7.97 (br s) 7.66 (br s)
158.1 (s)
161.4 (s)
1.93 (dq, 13.1, 6.7)
0.69 (d, 6.7)
0.79 (d, 6.7)
6.64 (s)
40.1 (s)
39.0 (s)
9.62 (br s)
6.14 (dd, 17.4, 10.5)
5.10 (d, 17.4) 5.08 (d, 10.5) 112.3 (t)
1.56 (s)
1.56 (s)
146.1 (d) 16
6.09 (dd, 17.4, 10.5)
5.16 (d, 17.4) 5.15 (d, 10.5)
1.51 (s)
1.51 (s)
3.94 (m)
145.8 (d) 17
39.5 (s)
145.0 (d)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
112.3 (t)
27.8 (q)
27.9 (q)
63.9 (t)
34.6 (t)
10-NH
6.14 (dd, 17.6, 10.5)
5.13 (d, 17.6) 5.12 (d, 10.5) 111.8 (t)
1.51 (s)
1.51 (s)
28.0 (q)
28.0 (q)
65.3 (t)
20
30
4.97 (s)
78.7 (d)
60.0 (s)
27.8 (q)
27.8 (q)
4.00 (m) 3.89 (m)
30a
40
130.6 (s)
124.6 (d)
118.1 (d)
128.6 (d)
108.9 (d)
149.0 (s)
57.0 (d)
20-OH 4.54 (br s)
3.39 (d, 7.2)
5.35 (t, 7.2)
7.38 (d, 7.4)
6.62 (m)
7.03 (m)
124.5 (d) 50
131.6 (s)
25.8 (q)
17.9 (q)
31.3 (t)
60
70
70a
90
1.74 (s)
1.80 (s)
3.53 (d, 7.3)
5.42 (t, 7.3)
6.61 (d, 7.7)
4.20 (t, 8.6)
3.07 (m) 2.62 (dd, 14.0, 8.6) 35.3 (t)
169.5 (s)
122.9 (d) 100
133.0 (s)
25.7 (q)
17.9 (q)
110
1.74 (s)
1.86 (s)
120-NH 8.06 (s)
130
140
150
4.00 (q, 6.9)
50.5 (d)
169.3 (s)
14.8 (q)
1.15 (d, 6.9)
a
b
c
Measured in acetone-d6.
Measured in CDCl3.
Measured in DMSO-d6, at 500 MHz for 1H and 125 MHz for 13C with reference to the solvent signals, d in ppm.

F.-Y. Du et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4650–4653
4653
16.9, and 42.6 l
g/mL, respectively. The antimicrobial activities23,24
2. Slack, G. J.; Puniani, E.; Frisvad, J. C.; Samson, R. A.; Miller, J. D. Mycol. Res. 2009,
113, 480.
3. Li, D. L.; Li, X. M.; Wang, B. G. J. Microbiol. Biotechnol. 2009, 19, 675.
4. 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.
5. Ding, G.; Jiang, L. H.; Guo, L. D.; Chen, X. L.; Zhang, H.; Che, Y. S. J. Nat. Prod.
2008, 71, 1861.
6. Naik, C.G.; Devi, P.; Rodrigues, E. U.S. Patent 0 143 392, 2005.
7. Gao, S. S.; Li, X. M.; Li, C. S.; Proksch, P.; Wang, B. G. Bioorg. Med. Chem. Lett.
2011, 21, 2894.
against two bacteria (Escherichia coli and Staphyloccocus aureus)
and five plant-pathogenic fungi (Alternaria brassicae, Valsa mali,
Physalospora obtuse, Alternaria solania, and Sclerotinia miyabeana)
were also evaluated. Compounds 1 and 9 displayed potent inhibi-
tory activity against E. coli and S. aureus with the MIC values of 64
and 8
weak activity against S. aureus, each giving the inhibition zone of
8 mm at 100 g/disk (MICs were not determined). Chlorampheni-
col was used as positive control and showed the MIC value of
g/mL against E. coli and S. aureus.
The antibacterial activity of 1 appears related to the serine res-
lg/mL, respectively, while compounds 4 and 10 showed
8. Sun, H. F.; Li, X. M.; Meng, L.; Cui, C. M.; Gao, S. S.; Li, C. S.; Huang, C. G.; Wang,
B. G. J. Nat. Prod. 2012, 75, 148.
9. Liu, D.; Li, X. M.; Meng, L.; Li, C. S.; Gao, S. S.; Shang, Z.; Proksch, P.; Huang, C. G.;
Wang, B. G. J. Nat. Prod. 2011, 74, 1787.
l
4
l
10. Physical and spectroscopic data of new compounds: (a) cristatumin A (1):
colorless amorphous powder; ½a D20
: ꢁ17.6 (c 0.34, MeOH); UV (MeOH) kmax
ꢀ
(log e
) 223 (4.28), 284 (3.69), 333 (3.79) nm; 1H and 13C NMR data, see Table 1;
idue in the 2,5-diketopiperazine moiety compared to that of 5
(with the alanine residue). The serine residue was also important
to the brine shrimp lethality of 2 compared with that of 10. The
structure difference between 6 and 9 was only at C-8/C-9, indicat-
ing that the single bond between C-8/C-9 (compound 9) was essen-
tial to its antibacterial activity, while the double bond at C-8/C-9
(compound 6) appears important for its brine shrimp lethality.
Compound 6 was more active than 5 and 7 in the brine shrimp bio-
assay, which was probably due to the number and substituted po-
sition of the isoprenic chain. This deduction was also proved by the
structure differences between compounds 8, 9, and 10 compared
with their antibacterial activities.
In summary, we identified four new indole alkaloids, cristatu-
mins A–D (1–4), along with six congeners (5–10) from E. cristatum
EN-220, an endophytic fungus isolated from the marine alga S.
thunbergii. Cristatumin A (1) displayed moderate activity against
E. coli, and tardioxopiperazine A (9) displayed potent activity
against S. aureus. This is the first report for the antibacterial activity
of 9. Cristatumin B (2), isoechinulin A (6), and variecolorin G (7)
exhibited moderate lethal activity against brine shrimp. This is also
the first report for the brine shrimp inhibition activity of 6 and 7.
ESIMS m/z 362 [M+Na]+; HRESIMS m/z 362.1477 [M+Na]+ (calcd for
C
19H21N3O3Na+, 362.1480). (b) Cristatumin
B
(2): colorless amorphous
powder; ½a 2D0
ꢀ
: –11.1 (c 0.09, CHCl3); UV (MeOH) kmax (log e) 228 (4.52), 276
(3.90) nm; 1H and 13C NMR data, see Table 1; ESIMS m/z 500 [M+Na]+;
HRESIMS m/z 500.2885 [M+Na]+ (calcd for C29H39N3O3Na+, 500.2889). (c)
Cristatumin C (3): colorless amorphous powder; ½a D20
ꢀ
: +315.7 (c 1.21, MeOH);
UV (MeOH) kmax (log e
) 204 (4.77), 242 (4.02), 301 (3.47) nm; 1H and 13C NMR
data, see Table 1; ESIMS m/z 563 [M+Na]+; HRESIMS m/z 563.2377 [M+Na]+
(calcd for C30H32N6O4Na+, 563.2382). (d) Cristatumin D (4): yellow amorphous
powder; UV (MeOH) kmax (log e
) 220 (4.41), 265 (4.02), 350 (4.11) nm; 1H and
13C NMR data, see Table 1; ESIMS m/z 378 [M+Na]+; HRESIMS m/z 378.1425
[M+Na]+ (calcd for C19H21N3O4Na+, 378.1429).
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Acknowledgments
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S. miyabeana were carried out using the well diffusion method.24
Chloramphenicol (Chl) and amphotericin B were used as positive control for
antibacterial and antifungal assay, respectively..
This work was financial supported by the Ministry of Science
and Technology (2010CB833802) and by the Natural Science Foun-
dation of China (30910103914 and 30970293).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
05.088.
24. Song, Z. W.; Liu, P.; Yin, W. P.; Jiang, Y. L.; Ren, Y. L. Bioorg. Med. Chem. Lett.
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