Structure and anti-acetylcholinesterase activity of 4α- (hydroxymethyl)-4α-demethylterritrem B

Article abstract of DOI:10.1021/np9701236

The structure of a metabolite produced on incubation of territrem B (1) with rat liver microsomes has been established to be 4α-(hydroxymethyl)- 4α-demethylterritrem B (5). Compound 5 was a potent inhibitor of electric eel acetylcholinesterase (ACHE) (E.C

Full text of DOI:10.1021/np9701236

842
J . Nat. Prod. 1997, 60, 842-843
Str u ctu r e a n d An ti-Acetylch olin ester a se Activity of
4r-(Hyd r oxym eth yl)-4r-d em eth ylter r itr em B
Fu-Chuo Peng
Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
Received February 7, 1997X
The structure of a metabolite produced on incubation of territrem B (1) with rat liver microsomes
has been established to be 4R-(hydroxymethyl)-4R-demethylterritrem B (5). Compound 5 was
a potent inhibitor of electric eel acetylcholinesterase (AChE) (E.C. 3.1.1.7).
Territrems A, B, and C are secondary metabolites,
isolated from CHCl3 extracts of cultures of Aspergillus
terreus 23-1.1-3 These compounds induced whole body
tremor when injected ip into rats or mice.4 It was
demonstrated previously that the site of tremorgenic
action in animals injected with territrem B (1) was the
peripheral motor nerve ending and that functional
integrity was necessary for the tremorgenic activity.4
It was indicated that 1 might potentiate the release of
acetylcholine in the presynaptic area of the peripheral
motor nerve ending.4 However, it has also been dem-
onstrated that 1 is 20 times more potent than neostig-
mine in inhibiting AChE activity in human red blood
cells.5 On investigation of the relationship between the
structures and inhibition of AChE activity, five deriva-
tives of 1 were obtained, and their inhibitory potencies
on electric eel AChE were tested.6 It was concluded that
substitution on the aromatic ring of 1 has little effect
on anti-AChE activity.5 However, the enone and pyrone
moieties of 1 seem to play important roles in AChE
inhibitory activity.6
F igu r e 1. The structure of territrem derivatives.
was 4.23 × 10-10 M, indicating that 5 is 68 times more
potent than 1. The data (Table 2) showed that 5 is the
most potent inhibitor of eel AChE (of 1 and its deriva-
tives tested).
Previous work had shown that incubation of 1 with
rat liver microsomes produced four metabolites desig-
nated MB1-MB4. The reaction was NADPH dependent
and enhanced by pretreatment of the rats with phe-
nobarbital.7 MB2 (2) is a major metabolite that arose
from hydroxylation of the pro-S methyl group at C4 of
1. MB4 (3) was identical to territrem C.7 MB1 (4) was
shown to be the 4′-demethyl analogue of 2 and the major
metabolite obtained from incubation of territrem C with
rat liver microsome.7 The structure of MB3 (5) was not
determined previously. A large-scale incubation experi-
ment has provided sufficient 5 for elucidation of its
structure and inhibitory effect on eel AChE. The results
are described in this paper.
The 1H- and 13C-NMR spectra of 5 were similar to
those of 1 except that the signals assigned to the 4R-
methyl of the latter were absent and had been replaced
by an AB pattern at δΗ 3.27, 4.08 (J ) 10.8 Hz) and by
a triplet at δc 69.44. The HRFABMS showed an [M +
1]+ at m/z 543.2224 consistent with a hydroxylated
derivative of 1 having molecular formula C29H34O10. The
hetero-COSY spectrum revealed the relationship be-
tween the signals of a hydroxymethyl group. This
information indicates that 5 is the C-4 epimer of 2
(Figure 1).
Exp er im en ta l Section
Gen er a l Exp er im en ta l Con d ition s. The melting
point was measured on a hot-stage melting point
apparatus (Shimatzu Seisakusho Ltd.) and was uncor-
rected. The mass spectrum was recorded using a J EOL
J MS-HX 110 mass spectrometer. 1H- and 13C-NMR
spectra were recorded in CDCl3 on a Bruker AMX 400
spectrometer using solvent peaks as the reference
standard.
Extr a ction a n d Isola tion of 1. Compound 1 was
isolated from rice culture of Aspergillus terreus 23-1
according to previously published procedures.1-3 1H-
and 13C-NMR data; see Table 1.
P r ep a r a tion of 5. Male Wistar rats, weighing 200-
250 g, were fed freely with a phenobarbital-H2O
solution (sodium salt, Wako Pure Chemical Industries,
Ltd., 1 g/L H2O) for two weeks and sacrificed on the 15th
day. The preparation of the S9 fraction from the rat
livers followed the method of Maron and Ames.8 The
solution contained 20 µL S9 (4 mg/mL protein), 0.1 M
NADP (20 µL) (Sigma), 0.4 M KCl-1.65 M MgCl2
solution (10 µL), 0.1 M glucose-6-phosphate solution salt
(5 µL) (Sigma), 0.1 M sodium phosphate buffer (250 µL),
pH 7.4, and distilled H2O to make the final volume to
500 µL. After the solution was preincubated at 37 °C
for 30 min, 4 µL of 1 (1 mg/mL MeOH) was added. An
The inhibitory activity of 1 and 5 on electric eel AChE
was evaluated by the colorimetric method.9 The IC50
of 1 on eel AChE was 2.6 × 10-7 M, and the IC50 of 5
X Abstract published in Advance ACS Abstracts, J uly 15, 1997.
S0163-3864(97)00123-7 CCC: $14.00
© 1997 American Chemical Society and American Society of Pharmacognosy

Notes
J ournal of Natural Products, 1997, Vol. 60, No. 8 843
Ta ble 1. 1H- (400 MHz) and 13C- (100 MHz) NMR Data (CDCl3, δ) for 1 and 5
1
5
position
δH (J , Hz)
δC
δH (J , Hz)
δC
1
2
3
4
4a
5
204.47 s
123.30 d
153.41 d
42.59 s
79.06 s
25.75 t
203.88 s
126.68 d
151.39 d
46.53 s
79.69 s
26.19 t
5.81 (d, J ) 10.0)
6.29 (d, J ) 10.0)
6.08 (d, J ) 10.0)
6.51 (d, J ) 10.0)
5R, 5â
6
1.85-1.89 (m)
1.85-2.09 (m)
28.45 t
28.27 t
6R
1.79-1.81 (m)
2.40-2.47 (m)
1.79-1.85 (m)
2.44-2.52 (m)
6â
6a
7a
8
9
79.97 s
162.83 s
97.56 d
158.47 s
164.31 s
97.27 s
82.66 t
162.83 s
97.46 d
158.72 s
164.26 s
96.99 s
6.30 (s)
6.35 (s)
11
11a
12R
12â
12
12a
12b
4R-Me
4R-CH2
4â-Me
6a-Me
12b-Me
1′
2′,6′
3′,5′
4′
3′,5′-OMe
4′-OM4
3.41 (d, J ) 18.0)
2.84 (d, J ) 18.0)
3.41 (d, J ) 17.8)
2.83 (d, J ) 17.8)
27.86 t
76.10 s
56.29 s
23.83 q
27.79 t
77.32 s
55.81 s
1.24 (s)
3.27, 4.08 (d, J ) 10.8)
1.23 (s)
1.51 (s)
69.44 t
19.33 q
24.00 q
21.65 q
126.68 s
102.83 d
153.50 s
140.43 s
56.33 q
60.98 q
1.15 (s)
1.49 (s)
1.43 (s)
25.44 q
23.80 q
21.79 q
126.72 s
102.77 d
153.44 s
140.31 s
56.21 q
60.94 q
1.43 (s)
6.96 (s)
6.98 (s)
3.87 (s)
3.86 (s)
3.89 (s)
3.88 (s)
Ta ble 2. Inhibition of Electric Eel Acetylcholinesterase by
Territrem B (1) Derivatives
buffer, pH, 8.0. The initial rate of substrate hydrolysis
was determined at 412 nm at room temperature using
a Beckman spectrophotometer. The activity of AChE
was calculated according to Gordon et al.10
compound
in vitro I50 (M)a
1
2
5
2.60 × 10-7
7.9 × 10-7a
4.23 × 10-10
1.0 × 10-8
Ack n ow led gm en t. This work was supported by
grant NSC85-2621-B-002-026 Z. The author is indebted
to Dr. Kuo-Huang Ling, Institute of Biochemistry, and
Dr. Shoie-Sheng Lee, School of Pharmacy, College of
Medicine, National Taiwn University, for reading the
manuscript and for suggestions.
BW284C51b
a
I50 values were calculated by probit analysis from responses
obtained from eight doses of inhibitor, each differing by an order
b
of magnitude. See Chen and Ling.5
BW281C51: 1,5-bis[4-
(allyldimethylammonio)phenyl]pentan-3-one dibromide, see Chen.11
additional 60-min incubation was carried out at 37 °C
by shaking (100 oscillations/min). The reaction was
stopped by adding 1 mL MeOH. The reaction mixture
was centrifuged at 15 000 rpm, and then 100 µL of the
supernatant was taken for HPLC analysis. For isola-
tion of large quantities of the product, the amounts of
the above-described reaction mixture were scaled up to
200-fold and divided into 14 Erlenmeyer flasks. To each
flask (250 mL), 32 mg of 1 (1 mg/mL MeOH) was added.
Compound 5 (0.32 mg) was separated via preparative
TLC [C6H6-EtOAc-HOAc-HOAc (6:3:1)] and finally
purified by ODS HPLC [MeCN-H2O (6:4)].
Refer en ces a n d Notes
(1) Ling, K. H.; Yang, C. K.; Peng, F. C. Appl. Environ. Microbiol.
1979, 17, 355-357.
(2) Ling, K. H.; Liou, H. H.; Yang, C. M.; Yang, C. K. Appl. Environ.
Microbiol. 1984, 47, 98-100.
(3) Yang, C. K. Studies on territrems from Aspergillus terreus:
isolation, assay methods, molecular structure. Ph.D. Thesis,
Institute of Biochemistry, College of Medicine, National Taiwan
University, Republic of China, 1981; pp 16-39.
(4) Ling, K. H.; Liou, H. H.; Fu, T. C.; Kuo, L.; Tasi, M. C.; Lin, M.
Y. Mechansim of action of the territrem, tremorgenic mycotoxin
isolated from Aspergillus terreus. In Mycotoxins and Phycotoxins,
Steyn, P.S., Velggaar, R., Eds. 6th International IUPAC Sym-
posium on Mycotoxins and Phcotoxins. Elsevier: Amsterdam,
1985; pp 387-298.
(5) Chen, J . W.; Ling, K. H. Territrem, an Inhibitor of Acetylcho-
linesterase ROC-J apan seminar on mycotoxins, National Tai-
wan University, Taipei, 1991, Sep 22-24, p 24.
(6) Peng F. C. J . Nat. Prods. 1995, 58, 857-862.
(7) Ling, K. H.; Chiou, C. M.; Tseng, Y. L. Drug. Metab. Dispos.
1991, 19, 587.
(8) Maron, D. M.; Ames, B. Mutat. Res. 1983, 113, 171-205.
(9) Ellman, G. L.; Courtney, K. D.; Andres, V., J r.; Featherstone,
R. M. Biochem. Pharmacol. 1961, 77, 88-95.
(10) Gordon, A.; Carpenter, D. E.; Wilson, I. B. Mol. Pharmacol. 1978,
14, 266-270.
(11) Chen, J . W. Inhibition mechanism of acetylcholinesterase by
territrem. Ph.D. Thesis, Institute of Biochemistry, College of
Medicine, National Taiwan University, Republic of China, 1991;
p 17.
4r-(Hyd r oxym eth yl)-4r-d em eth ylter r itr em B (5):
mp 240-242 °C (from CHCl3); UV (MeOH) λmax (log ꢀ)
1
331 (1.1), 218 (3.2); H- and 13C-NMR data; see Table
1, FABMS m/z [M + 1]+ 543 (92), 524 (9), 509 (14), 359
(5), 291 (44), 237 (14), 214 (24), 195 (68); HRFABMS
m/z [M + 1]+ 543.2224 (calcd for C29H34O10, 542.2152).
Assa y of Acetylch olin ester a se. The AChE activity
was determined by the method of Ellman et al.9 Typi-
cally, an aliquot of 20-40 µL of the working enzyme
solution or of the inhibited specimen was added to 1 mL
of the assay system containing 4.8 × 10-4 M acetylthio-
choline and 3.2 × 10-4 M DTNB in a 0.1 M phosphate
NP9701236