Cage amines as the stopper inhibitors of cholinesterases

Article abstract of DOI:10.1016/S0960-894X(03)00599-7

Cage amines 1-4 are potent peripheral anionic site-bound reversible inhibitors of both acetylcholinesterase and butyrylcholinesterase. Cage amines 1-3 are selective butyrylcholinesterase inhibitor versus acetylcholinesterase. For both enzymes, the -log K<

Full text of DOI:10.1016/S0960-894X(03)00599-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2887–2890
Cage Amines as the Stopper Inhibitors of Cholinesterases
Gialih Lin,a,* Hou-Jen Tsaib and Yi-Hon Tsaia
aDepartment of Chemistry, National Chung-Hsing University, Taichung, Taiwan
bDepartment of Applied Chemistry, National Chung Cheng Institute of Technology, Ta-Shi, Tao-Yuan, Taiwan
Received 19 February 2003;accepted 2 June 2003
Abstract—Cage amines 1–4 are potent peripheral anionic site-bound reversible inhibitors of both acetylcholinesterase and butyryl-
cholinesterase. Cage amines 1–3 are selective butyrylcholinesterase inhibitor versus acetylcholinesterase. For both enzymes, the
ꢀlog Ki values linearly correlate with the difference of substituted phenyl radius of cage amines (ꢀlog Ki=5.4+3.4ꢀg for acetyl-
cholinesterase, ꢀlog Ki=5.9+3.2ꢀg for butyrylcholinesterase). Moreover, the relationship between the enzymes and cage amines
mimics that between bottles and stoppers.
# 2003 Elsevier Ltd. All rights reserved.
The drugs used in Alzheimer’s disease are cholinesterase
inhibitors.1 Two forms of cholinesterase coexist ubiqui-
tously throughout the body, acetylcholinesterase
(AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE,
EC 3.1.1.8), and although highly homologous, >65%,
they are products of different genes on chromosomes 7
and 3 in humans, respectively.2
hexa(3,4-methylenedioxyphenyl-methyl)-2,4,6,8,10,12-
hexaazatetracyclo [5.5.0. 05,9.03,11] dodecane (4) are
called as cage amines and available in one step reaction
(Scheme 1).5 Cage amines partially resemble the struc-
ture of physostigmine (Fig. 1) and are less possible to
enter the narrow gorge active sites of AChE6ꢀ11 and
BchE12,13 due to the bulkiness of cage amines.
The analogues of physostigmine (Fig. 1), an alkaloid
extracted from a tropical plant, have been shown to
display cholinergic activity, and have been widely stud-
ied as potential drugs for Alzheimer’s disease.3,4
2,4,6,8,10,12-Hexabenzyl-2,4,6,8,10,12-hexaazatetracy-
clo[5.5.0.05,9.03,11]dodecane (1), 2,4,6,8,10,12-hexa(2-
The active site serine, S200(or 198), of both AChE and
BChE is located at the bottom of a 20 A gorge.6ꢀ8,13
Cage amines may bind to the peripheral anionic site
(PAS) of both enzymes, which is located at the entrance
of active site gorge and is regarded as the recognition
binding site.13 Since an active site-bound reversible
inhibitor such as edrophonium (Fig. 1)7,8 further inhi-
bits both enzymes in the presence of cage amines 1–4,
cage amines 1–4 do not bound to active site but PAS of
both enzymes (Fig. 2).
chloro
phenylmethyl)-2,4,6,8,10,12-hexaazatetracy-
clo[5.5.0.05,9.03,11]dodecane (2), 2,4,6,8,10,12-hexa(3,5-
dimethoxyphenylmethyl)- 2,4,6,8,10,12 - hexaazatetracy-
clo [5.5.0.05,9.03,11]dodecane (3), and 2,4,6,8,10,12-
Figure 1. Structures of physostigmine, tacrine, and edrophonium.
*Corresponding author. Tel.: +886-930-383816;fax: +886-4-286-2547;e-mail: gilin@dragon.nchu.edu.tw
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00599-7

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G. Lin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2887–2890
Scheme 1. Synthesis of cage amines 1–4.
Table 1. ꢀg and kinetic data for AChE and BChE inhibitions by
cage amines 1–4
Inhibitors Substituents ꢀg (A)a
AChE
BChE
BChE
Ki (nM)b Ki (nM)c Selectivityd
1e
2
3
4
H
o-Cl
0
0.582
4000ꢁ1000 900ꢁ300
4.4
3.9
2.2
1.0
39ꢁ3
130ꢁ9
320ꢁ50
10ꢁ3
60ꢁ10
310ꢁ80
3,5-di-OCH3 0.468
3,4-OCH2O 0.279
TM
.
aCalculation from the MM-2 minimized 3D structure by Chem 3D
bDetermination by the Ellman assay15 as described by Radic et al.16
and Nair et al.17 The Ki values were obtained from the Lineweaver–
Burk plots. Initial rates of Electrophorus electricus AChE (Sigma)-
catalyzed hydrolysis of acetylthiocholine (0.1 mM) in the presence of
5,50-dithio-bis-2-nitrobenzoate (0.1 mM) and cage amines 1–4 were
followed continuously at 410 nm on an UV–visible spectrometer (Agi-
lent 8453).
cHorse serum BChE (Sigma)-catalyzed hydrolysis of butyrylthiocho-
line (0.1 mM). All the other procedures were the same as described in
footnote b.
dKiðAChEÞ=KiðBChEÞ.
Figure 2. Stopped-time assay for AChE inhibition by cage amine 1.
Initial rates were AChE catalyzed hydrolysis of acetylthiocholine
(0.1 mM) in the presence of 5,50-dithio-bis-2- nitrobenzoate (0.1 mM)
and cage amine 1 (0.1 mM) at 25 ꢂC. The control, squares, were initial
rates for incubation of AChE at 25 ꢂC for a period of time before the
reaction. Circles were incubation of AChE and cage amine 1 at 25 ꢂC
for a period of time before the reaction. Triangles were incubation of
AChE, cage amine 1, and edrophonium (0.1 mM) at 25 ꢂC for a period
of time before the reaction.
eSynthesis from Scheme 1.5 Characterization of 1: Yellow solid;
mp=153–157 ꢂC; 1H NMR (CDCl3) d 3.62 (2H, ring CH), 4.08 (4H,
ring CH), 4.11 (8H, benzyl CH2), 4.20 (s, 4H, benzyl CH2), 7.18–7.27
(30H, phenyl); 13C NMR (CDCl3) d 56.2, 56.8 (benzyl CH2), 76.7, 80.6
(ring), 126.6, 126.7, 128.0, 128.1, 128.3, 129.1 (phenyl);IR (KBr)
l
2853, 1637, 1351, 699 cmꢀ1;Mass spectrum 708 (M +, 10), 617(100),
341(100). Anal. calcd for C48H48N6: C, 81.32;H, 6.83;N, 11.86.
Found C, 81.31;H, 6.79;N, 12.12.
Cage amines 1–4 are reversible inhibitors of both AChE
and BChE (Table 1). Like physostigmine14 and tacrine11
(Fig. 1), cage amines 1–3 are selective inhibitors of
BChE versus AChE (Table 1).
Table 2. Correlation of the ꢀlogKi values of AChE and BChE inhib-
itions by cage amines 1–4a with ꢀg
Enzyme
h
s
R
AChE
BChE
5.4ꢁ0.1
5.9ꢁ0.3
3.4ꢁ0.3
3.2ꢁ0.3
0.992
0.957
Difference in substituted phenyl radius (ꢀg) is defined
as substituted phenyl radius minus phenyl radius (Fig. 3
and Table 1). The ꢀlogKi values for both AChE and
BChE inhibitions linearly correlate with ꢀg (Fig. 4 and
Table 2) by eq 1, where the h and s values are the cal-
culated ꢀlogKi values for cage amine 1 (ꢀlogKi0) and
the substituent steric sensitivity, respectively.
aCorrelations with eq 1, ꢀlogKi=h+sꢀg.
Straight lines are linear least-squares fits to eq 1 and
give s=3.4ꢁ0.3 and s=3.2ꢁ0.7 for AChE and BChE
inhibitions, respectively (Fig. 4 and Table 2). Since these
s values are about the same, cage amines interact with
ꢀlogKi ¼ h þ sDꢀ
ð1Þ

G. Lin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2887–2890
2889
both enzymes in a similar way for substituent steric
effect of cage amines. Positive s values indicate that
bulky substituted cage amines fit well into the entrance
of active site gorge, PAS, through the well known p–p
interaction between cage amine phenyl group and W278
of AChE or Y332 of BChE (Fig. 5).8,12 This interaction
for AChE is slightly better than that for BChE probably
because tryptophan provides more p electrons than
tyrosine.
Since the ꢀlogKi values do not correlate with Hammett
substituent constant, s, electronic effects of cage amine
substituents are unimportant. Thus, interactions
between cage amines and PAS are mostly steric effects.
On the other hand, the ionic interaction between the
aspartate anion of PAS (D72 of AChE or D70 of
BChE)13 and the protonated quaternary amine cation
may occur (Fig. 5).
Figure 3. ꢀg of cage amines 2 and 3.
Overall, the relationship between cholinesterases
and cage amines 1–4 mimics that between bottles and
stoppers.
Acknowledgements
We thank the National Science Council of Taiwan for
financial support.
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Figure 4. Correlations of the ꢀlogKi values of AChE and BChE inhib-
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Figure 5. Stopper-bottle type interaction between cage amines 1–4
(stopper) and AChE (bottle). PAS is located at the entrance of active
site gorge and mimics the bottle mouth. The p–p interaction between
W2786,7 and cage amine phenyl group and the ionic interaction
between D72 anion6,7 and the protonated quaternary amine cation of
cage amine are proposed.

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