Isoterreulactone A, a novel meroterpenoid with anti-acetylcholinesterase activity produced by Aspergillus terreus

Article abstract of DOI:10.1016/j.bmcl.2004.10.067

A new seven-membered lactone type meroterpenoid, isoterreulactone A, was isolated from the solid state fermentation of Aspergillus terreus and its structure was established by various spectral analysis. Isoterreulactone A inhibited acetylcholinesterase with an IC₅₀ value of 2.5 μM while did not inhibit butyrylcholinesterase even at 500 μM. A new seven-membered lactone type meroterpenoid, isoterreulactone A, was isolated from the solid state fermentation of Aspergillus terreus and its structure was established by various spectral analysis. Isoterreulactone A inhibited acetylcholinesterase with an IC₅₀ value of 2.5 μM while did not inhibit butyrylcholinesterase even at 500 μM.

Full text of DOI:10.1016/j.bmcl.2004.10.067

Bioorganic & Medicinal Chemistry Letters 15 (2005) 353–356
Isoterreulactone A, a novel meroterpenoid with
anti-acetylcholinesterase activity produced by Aspergillus terreus
a
Ick-Dong Yoo, Kyung-Mi Cho, Chong-Kil Lee and Won-Gon Kim
a,b
b
a,
*
a
Korea Research Institute of Bioscience and Biotechnology, PO Box 115, Yusong, Taejon 305-600, Korea
b
Department of Pharmacy, Chung-Buk National University, Cheong-Ju 361-763, Korea
Received 20 August 2004; revised 20 October 2004; accepted 22 October 2004
Available online 11 November 2004
Abstract—Anew seven-membered lactone type meroterpenoid, isoterreulactone A, was isolated from the solid state fermentation of
Aspergillus terreus and its structure was established by various spectral analysis. Isoterreulactone Ainhibited acetylcholinesterase
with an IC50 value of 2.5lM while did not inhibit butyrylcholinesterase even at 500lM.
ꢀ
2004 Elsevier Ltd. All rights reserved.
Alzheimer disease is a neurodegenerative disorder that is
the most common cause of dementia among the elderly.
The neurophathological evidences have demonstrated
that cholinergic functions declined in the basal forebrain
tide–terpenoid structures. Especially, terreulactone Ais
a sesquiterpene lactone type meroterpenoid incorporat-
ing an uniquely fused lactone skeleton in its sesquiter-
pene moiety.
1
,2
and cortex in senile dementia of the Alzheimer type.
Accordingly, enhancement of cholinergic neurotrans-
mission have been considered as one potential therapeu-
tic approach against Alzheimer disease. One treatment
strategy to enhance cholinergic functions is the use of
acetylcholinesterase (AChE, EC 3.1.1.7) inhibitors to in-
crease the amount of acetylcholine present in the synap-
Further investigation on polar metabolites of Aspergillus
terreus Fb000501, which is the producer of terreulac-
tones A–D, has resulted in isolation of new seven-mem-
bered lactone type meroterpenoid named isoterreu-
lactone A( 1) (Fig. 1). We report here the isolation,
physico-chemical properties, structure determination,
and biological activities of 1.
1
2
3
,4
ses between cholinergic neurons. Acetylcholinesterase
inhibitors like tacrine, one of the most extensively evalu-
ated acetylcholinesterase inhibitors, have been shown to
significantly improve cognitive function in AlzheimerÕs
Fermentation of A. terreus Fb000501 was carried out in
solid state of moistured wheat-bran because iosterreu-
lactones Awas not produced in liquid culture media
containing glucose 2%, yeast extract 0.2%, polypeptone
0.5%, MgSO 0.05%, and KH PO 0.1% (pH5.7 before
5
,6
disease. Tacrine, however, has been known to cause
hepatotoxic side effects by also inhibiting butyrylcholi-
nesterase (BuChE, EC 3.1.1.8), which is found in plas-
4
2
4
7
ma. In this respect, an inhibitor selective for
sterilization). Apiece of strain Fb000501 was inoculated
from a mature plate culture into 500mL baffled
Erlenmeyer flasks each containing 100mL of a sterile
seed medium with the above composition. After incuba-
tion at 28ꢁC for 3days on a rotary shaker (150rpm),
5mL of the seed culture was transferred to 500mL
Erlenmeyer flasks containing 90g of moistured wheat-
bran. The fermentation was carried out at 28ꢁC for
acetylcholinesterase has attracted particular attention
for treatment of the Alzheimer-type dementia. In the
course of our screening for selective inhibitors of
acetylcholinesterase from microbial metabolites, we
8
–10
previously isolated terreulactones A–D
1
and quino-
Terreulactones A–D are
1
lactacins A1 and A2.
meroterpenoid type compounds that have mixed polyke-
10days under a stationary condition. The solid-state fer-
mented whole medium (1.8kg) was extracted with 80%
acetone and the extract was concentrated in vacuo to
an aqueous solution, which was then extracted with an
Keywords: Meroterpenoid; Anti-acetylcholinesterase.
*
Corresponding author. Tel.: +82 42 860 4298; fax: +82 42 860
595; e-mail: wgkim@kribb.re.kr
4
0
960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.067

354
I.-D. Yoo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 353–356
OCH3
OCH3
O
OCH3
OCH3
O
OCH3
OCH3
OCH3
O
O
O
O
O
O
O
O
O
O
O
CH3
CH3
O
O
CH3
CH3
CH3
CH3
CH3
CH3
O
CH3
OH
CH3
O
OH
O
OH
H3CO
OH
OH
OH
O
OH
3
H C
O
O
3
H CO
CH3
CH3
O
OH
CH3
H3C
OH
CH3
O
H3C
H3C
H3C
CH3
1
terreulactone A
terreulactone B
terreulactone C
terreulactone D
Figure 1. The relative structures of isoterreulactone A( 1) terreulactones A–D.
equal volume of EtOAc three times. EtOAc extract was
concentrated in vacuo to dryness. The crude extract was
subjected to SiO column chromatography followed by
two –CH –CH –, an isolated methylene, two carboxylic
2 2
2
3
carbons, five sp quaternary carbons, five sp quaternary
carbons, and two exchangeable protons. These spectral
data are similar to those of terreulactone A. The major
differences were that one more –CH –CH – group and
2
stepwise elution with CHCl –MeOH (100:1, 50:1,
3
2
0:1). The active fractions eluted with CHCl –MeOH
3
2
2
(
20:1) were pooled and concentrated in vacuo to give
one exchangeable proton appeared in 1 while the carbon-
yl carbon, one sp quaternary carbon, and one methoxy
3
an oily residue. The residue was applied again to a
Sephadex LH-20 and then eluted with MeOH. The ac-
tive fraction dissolved in MeOH was further purified
by reverse phase HPLC column (20 · 250mm, YMC
C ) chromatography with a photodiode array detector.
of terreulactone Adisappeared. This data suggested that
the structure of the ring Aof terreulactone Awas chan-
ged in 1. The structure of the ring Awas determined by
HMBC spectral data. Long-range couplings were ob-
served from the methyl protons of 13b-Me (d 1.29) to
a methylene carbon (C-1, d 29.9) of one –CH –CH –
1
8
The column was eluted with CH CN–H O (55:45) at a
3
2
flow rate of 8mL/min to afford isoterreulactone A
85mg) at a retention time of 11.9min as a white
powder.
2
2
3
(
group and three sp quaternary carbons [C-13b (d
41.9), C-13a (d 75.6), and C-5a (d 89.4)]. The methylene
protons (H -1, d 2.02 and d 2.16) of the –CH –CH –
2
2
2
The molecular formula of 1 was determined to be
C H O on the basis of high resolution FAB-MS
group were in turn long-range coupled to 13b-Me, C-
13b, C-5a, and one carboxylic carbon (C-3,
172.2). This spectral data suggested the presence of
d
2
8
32
8
+
(M+H) , 485.2177 m/z (+0.2mmu error)] in combina-
[
tion with H and C NMR data. The IR data suggested
1
13
13b
1
2
3
a
–
the ring A. On the other hand, one exchangeable proton
C(CH )– CH – CH – C(@O)–O– moiety in
3
2
2
ꢀ
1
the presence of a lactone (1696cm ) and a hydroxyl
ꢀ
1
3430cm ) moiety. The H and C NMR data (Table
1
13
(
(5a-OH, d 4.78) was long-range coupled to C-5a and one
1
) with DEPT, H– H COSY, and HMQC data sug-
1
3
3
1
sp quaternary carbon (C-5, d 78.7). The sp quaternary
carbon of C-5 was correlated with two isolated methyls
(5 -Me, d 1.34 and 5 -Me, d 1.16). This spectral data
gested the presence of an 1,4-disubstituted benzene ring,
an olefinic methine, four isolated methyls, one methoxy,
a
b
1
3
1
Table 1. The C (125MHz) and H (600MHz) NMR spectral data of 1 in DMSO-d
6
C
1
DEPT dH (J, Hz)
HMBC
C
DEPT
dH (J, Hz)
HMBC
29.9 t
H
H
a
b
2.02 (m)
2.16 (m)
C-2, C-3, C-5a, C-13a, C-13b
C-2, C-3, C-13a, C-13b, 13b-Me
C-1, C-3, C-13b
8a
9
162.8 s
96.7 d
157.1 s
163.4 s
96.7 s
0
6.74 (s)
C-8a, C-10, C-1
2
3
5
28.7 t
172.2 s
78.7 s
2.47 (m)
10
12
12a
13
5
a
-Me 29.7 q
1.34 (s)
1.16 (s)
C-5, C-5a, 5
C-5, C-5a, 5
b
-Me
-Me
25.5 t
2.50 (s)
4.64 (s)
1.29 (s)
C-7a, C-8a, C-12,
C-12a, C-13, C-13a
5
5
b
-Me 29.2 q
89.4 s
a
13a
13a-OH
75.6 s
a
C-7a, C-13, C-13a,
C-13b
5
6
a-OH
4.78
H
C-5, C-5a
C-5a, C-7, C-7a, C-13b
13b 41.9 s
13b-Me 23.2 q
27.8 t
a
1.72 (ddd, 14.6, 3.4, 3.7)
C-1, C-5a, C-13a,
C-13b
0
H
H
H
b
a
b
1.85 (ddd, 14.6, 13.6, 3.0) C-7, C-7a
1.45 (ddd, 12.1, 3.7, 3.0) C-5a, C-7a, C-13a
2.35 (ddd, 13.6, 12.1, 3.4) C-6, C-7a, 7a-Me
1
2 ,6
123.6 s
0
126.9 d 7.81 (d, 8.9) C-10, C-6 , C-4
0
0
0
0
7
27.9 t
81.3 s
0
0
0
0
0
0
3 ,5
4
4 -OMe 55.4 q
114.6 d 7.04 (d, 8.9) C-1 , C-5 , C-4
161.1 s
7
7
a
0
a-Me 22.8 q
1.39 (s)
C-7, C-7a, C-13a
3.81 (s)
C-4
1
1
The assignments were aided by H– H COSY, DEPT, NOESY, HMQC, and HMBC.

I.-D. Yoo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 353–356
355
5
'
OCH3
6
'
4
'
E
Hβ
1
1
1
'
O
O
3'
2
'
Hβ
CH3
D
Hα
13
CH3
1
2a
O
1
9
3
1
3b
1
3
8
a
Hβ
C
CH3
H
7a
O
O
Hα
1
CH3
13a
1
3a
O
OH
7
Hα
1
3b
7a
7
H
5
OH
B
5a
Hα
O
3
A
5a
CH3
.34
1
HMBC
Hβ
H3C
1.16
OH
5
O
1
1
OH
H- H COSY
NOE
CH3
H3C
Figure 2. Key HMBC and NOE correlations of Isoterreulactone A.
indicated the presence of the free hydroxyl group (5a-
OH) at C-5a. The chemical shift (d 78.7) of C-5 and
unsaturation degree of 1 requiring the existence of one
ring in the ring Asuggested that the presence of a
seven-membered lactone in the ring Athrough the
connection of the carboxylic oxygen of the
Table 2. Inhibitory activities of isoterreulactone Aand terreulactones
A–D against acetylcholinesterase and butyrylcholinesterase
IC50 (lM)
AChE
2.5
BuChE
>500
1
1
3b
1
2
3
Terreulactone A0.23
Terreulactone B
Terreulactone C
Terreulactone D
Tacrine
>200
–
C(CH )– CH – CH – C(@O)–O– moiety to C-5.
3
2
2
0.09
0.06
0.42
0.09
>200
>200
>200
0.01
This was confirmed by HMBC spectral data. The
methylene protons (H -2, 2.47) of the
C(CH )– CH – CH – C(@O)–O– moiety were
d
2
1
3b
1
2
3
–
3
2
2
long-range coupled to C-5 as well as C-13b. The remain-
ing rings B, C, D, and E were also confirmed by HMBC
data. The relative stereochemistry was determined by
NOESY spectral data. The NOEs effect were observed
formation of 5-thio-2-nitrobenzoate, yellow anion, at
412nm of UV wavelength. The inhibitory activities
against BuChE were measured as described above
for AChE by using 0.16unit BuChE and 20lM butyryl-
thiocholine iodide instead of AChE and acetylthiocho-
line iodide for enzyme and substrate, respectively
(Table 2).
among 5a-OH, 7-H , and 13a-OH. Also, the NOEs
b
effect among 1-H , 13b-Me, 6-H , 13-H , and 7a-Me
b
b
b
were observed. Thus, the relative stereochemistry of C-
a, C-7a, C-13a, and C-13b were determined to be S*,
R*, S*, and S*, respectively (Fig. 2).
5
Isoterreulactone Ais a new meroterpenoid incorporat-
ing a seven-membered lactone skeleton in its molecule.
Isoterreulactone Ainhibited acetylcholinesterase in a
dose-dependent mode with an IC50 (lM) value of 2.5.
Anti-acetylcholinesterase activity of isoterreulactone A
was 10 times weaker than that (0.23lM) of terreulac-
tone A, which suggested the important role of the ring
Ain acetylcholinesterase inhibitory activity. Isoterreu-
lactone A, however, did not inhibit butyrylcholinest-
erase even at 500lM. Therefore, isoterreulactones A
showed more than 250 times potent inhibitory activity
against AChE compared with that against BuChE while
tacrine, as a positive control, had a low selectivity with a
stronger inhibitory activity on butyrylcholinesterase
1
3
Since some derivatives of arisugacin C and territrem
1
4
B, terreulactones C and D, respectively, were also de-
tected in the same culture, isoterreulactone Aseems to
be biogenetically related to arisugacin isolated from
Penicillium sp. Interestingly, arisugacins and territrems
were, however, not detected in this study. Meroterpe-
1
5,16
noids
ven-membered lactone type terpenoids
such as pyripyropene and oxalicine, and se-
1
7
such as
andilesins, anditomin, fumigatonin, and obacunol have
been isolated from fungi.
(IC50 (lM); 0.01) rather than acetylcholinesterase (IC50
The inhibitory activity of isoterreulactone Aagainst acet-
ylcholinesterase was examined according to EllmanÕs
(lM); 0.09) in this assay system. By Lineweaver–Burk
plot analysis, isoterreulactone Aexhibited noncompeti-
tive inhibition with acetylcholine and its K and K
1
8
coupled enzyme assay with some modifications as
follows; 0.08units AChE dissolved in 0.1M potassium
phosphate buffer (pH7.4) and purified compounds dis-
solved in methanol were added to each well of a 96-well
i
m
ꢀ
6
values for acetylcholinesterase were 2.3 · 10
and
ꢀ
5
2.0 · 10 M, respectively.
0
plate. Then, acetylthiocholine iodide and 5,5 -dithio-
bis(2-nitrobenzoic acid) dissolved in 0.1M potassium
phosphate buffer (pH7.4) were added to final 20 and
Acknowledgements
3
0lM, respectively, to each well. The reaction was
This work was supported in part by the 21C Frontier
Microbial Genomics and Application Center Program
(to W.-G.K.) and National Research Laboratory grants
carried out at room temperature for 5min and the initial
rate of the enzyme was analyzed by measuring the

356
I.-D. Yoo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 353–356
(to I.-D.Y.) from the Korean Ministry of Science and
Technology.
9. Kim, W.-G.; Cho, K.-M.; Lee, C.-K.; Yoo, I.-D. J.
Antibiot. 2003, 56, 344–350.
0. Kim, W.-G.; Cho, K.-M.; Lee, C.-K.; Yoo, I.-D. J.
Antibiot. 2003, 56, 351–357.
1. Kim, W.-G.; Song, N.-K.; Yoo, I.-D. J. Antibiot. 2001, 54,
831–835.
1
1
1
References and notes
. Davies, P.; Maloney, A. J. F. Lancet 1976, 2, 1403.
. Whitehouse, P. J.; Price, D. L.; Struble, R. G.; Clarke, A.
2. Compound 1: a white powder; UV kmax nm (e) in MeOH:
215 (81,000), 251 (11,000), 330 (12,000); IR (KBr): 3430,
1
2
ꢀ
1
2932, 1696, 1571, 1514, 1258, 1181cm ; [a] +60 (c 0.1,
CHCl
requires 485.2175.
D
+
W.; Coyle, J. T.; DeLong, M. R. Science 1982, 15, 1237–
); HRFAB-MS: m/z 485.2177 (M+H) , C27H O
3 32 8
1
239.
. Davies, K. L.; Mohs, R. C. Am. J. Psychiatry 1982, 139,
421–1424.
. Thal, L. J.; Flud, P. A.; Masur, D. M.; Sharpless, N. S.
Ann. Neurol. 1983, 13, 491–496.
3
4
13. Kuno, F.; Shiomi, K.; Otoguro, K.; Sunazuka, T.; Omura,
S. J. Antibiot. 1996, 49, 748–751.
14. Ling, K. H.; Liou, H.-H.; Yang, C.-M.; Yang, C.-K. Appl.
Environ. Microbiol. 1984, 47, 98–100.
1
5
6
. Relman, A. S. N. Engl. J. Med. 1991, 324, 349–352.
. Camps, P.; Achab, R. El.; Morral, J.; Munoz-Torrero,
D.; Badia, A.; Banos, J. E.; Vivas, N. M.; Barril, X.;
Orozco, M.; Luque, F. J. Med. Chem. 2000, 43, 4657–
15. Omura, S.; Tomoda, H.; Kim, Y. K.; Nishida, H. J.
Antibiot. 1993, 46, 1168–1169.
16. Ubillas, R.; Barnes, C. L.; Gracz, H.; Rottinghaus, G. E.;
Tempesta, M. S. J. Chem. Soc., Chem. Commun. 1989,
1618–1619.
4666.
7
. Thomsen, T.; Zendeh, B.; Fischer, J. P.; Kewitz, H.
Biochem. Pharmacol. 1991, 41, 139–141.
. Kim, W.-G.; Cho, K.-M.; Lee, C.-K.; Yoo, I.-D. Tetra-
hedron Lett. 2002, 43, 3197–3198.
17. Simpson, T. J. Chem. Soc. Rev. 1987, 16, 123–160.
18. Ellman, G.-L.; Courtney, K.-D.; Andres, J. R. V.;
Featherstone, R.-M. Biochem. Pharmacol. 1961, 7, 88–
95.
8