Design, synthesis and evaluation of carbamate-modified (-)-N1-phenethylnorphysostigmine derivatives as selective butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.035

We synthesized carbamate-modified (-)-N¹-phenethylnorphysostigmine derivatives 3a-u and evaluated their anti-cholinesterase activities. In vitro evaluation showed that cyclohexylmethylcarbamate derivative 3u potently and selectively inhibits bu

Full text of DOI:10.1016/j.bmcl.2010.01.035

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1721–1723
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of carbamate-modified
(ꢀ)-N1-phenethylnorphysostigmine derivatives as selective
butyrylcholinesterase inhibitors
Jun Takahashi a, Ichiro Hijikuro d, Takeshi Kihara a, Modachur G. Murugesh d, Shinichiro Fuse c,
Yoshinori Tsumura b, Akinori Akaike b, Tetsuhiro Niidome a, Takashi Takahashi c, Hachiro Sugimoto a,
*
a Department of Neuroscience for Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
c Department of Applied Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro, Tokyo 152-8552, Japan
d Department of Development, ChemGenesis Incorporated, 4-10-1 Nihonbashi-Honcho, Chuo-ku, Tokyo 103-0023, Japan
a r t i c l e i n f o
a b s t r a c t
We synthesized carbamate-modified (ꢀ)-N1-phenethylnorphysostigmine derivatives 3a–u and evaluated
their anti-cholinesterase activities. In vitro evaluation showed that cyclohexylmethylcarbamate deriva-
tive 3u potently and selectively inhibits butyrylcholinesterase.
Article history:
Received 26 November 2009
Revised 7 January 2010
Accepted 8 January 2010
Available online 20 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Acetylcholinesterase
Butyrylcholinesterase
Carbamate
Alzheimer’s disease
Alzheimer’s disease (AD) is a progressive and neurodegenera-
tive disease that causes cognitive and memory impairment. It is
characterized by senile plaques mainly composed of amyloid beta
and neurofibrillary tangles composed of tau protein. Deficiency in
cholinergic neurotransmission is, however, the direct cause of cog-
nitive disorders, as evidenced by clinical and experimental re-
ports.1–4 Intensifying cholinergic transmission is considered to be
useful for improving memory functions. Various therapeutic
approaches based on the cholinergic hypothesis have been at-
tempted, but only drugs that inhibit the breakdown of acetylcho-
line (ACh) in the synaptic cleft are clinically approved to date.
ACh released from nerve terminals is rapidly degraded by cho-
linesterases (ChEs). Because acetylcholinesterase (AChE) is the
main player regarding degradation of ACh in normal brain, most
work aimed at developing ChE inhibitors has targeted AChE. In
AD brain, however, cholinergic neurons are lost with progression
of the disease, and AChE is also reduced.5
duced, and its contribution to degrading ACh tends to be elevated
in AD brain.
It has been reported that selective BuChE inhibitors increase
brain ACh and augment learning in rodents,7 and both BuChE
knockout mice and silent mutants in humans show no physiologi-
cal disadvantage,8,9 inspiring the hypothesis that BuChE may be a
promising target in developing anti-AD drugs without serious ad-
verse effects.
There are three important subsites in ChE; anionic site, oxyan-
ion hole, and acyl pocket. Anionic site is mainly composed of
Trp86(82) in human AChE (human BuChE), it is important to at-
tract and stabilize the quaternary nitrogen in degradation of ACh.
Oxyanion hole is composed of Gly121(116), Gly122(117) and
Ala204(199), and it stabilizes carbonyl oxygen of substrates by
hydrogen bonding and facilitates the nucleophilic attack on the
carbonyl carbon by catalytic Ser203(198). Acyl pocket interacts
with acyl moiety of ACh, and among three subsites, its spatial
capacity is the most prominent difference between the structures
of AChE and BuChE. In AChE, two aromatic amino acids, Phe295
and Phe297, locate in this pocket, but in BuChE, they are replaced
by smaller ones, Leu286 and Val288, and this enables BuChE to cat-
alyze various substrates including bulkier esters.
Vertebrates have another acetylcholine hydrolase, butyrylcho-
linesterase (BuChE). Although it is abundant in plasma, it also ex-
ists in the brain. In contrast to AChE, BuChE is mainly derived
from glia in the brain.6 BuChE is therefore believed to not be re-
Physostigmine (1) is the most classical ChE inhibitor, and its
derivatives have been reported to direct their carbamate region to-
* Corresponding author. Tel.: +81 75 753 9270; fax: +81 75 753 9269.
E-mail address: hsugimot@pharm.kyoto-u.ac.jp (H. Sugimoto).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.035

1722
J. Takahashi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1721–1723
wards the acyl pocket.10 Regarding the relationship between the
carbamate moiety of physostigmine derivatives and anti-ChE
activity, the following findings have been reported to date: (i) con-
version from methyl group to phenyl or 2-methylphenyl group
renders the compounds AChE-selective; (ii) a 4-isopropyl group
improves anti-BuChE activity; (iii) substituents on the carbamate
moiety greatly affect the duration of the anti-ChE effect.11 In addi-
tion, (ꢀ)-N1-phenethylnorcymserine (PEC, 3d), which possesses a
4-isopropylphenyl carbamate and N1-phenethyl group, has been
reported to have highly selective and potent anti-BuChE activity.7
Because physostigmine (1) and its derivatives can exploit the
acyl pocket, physostigmine derivatives are considered to be highly
useful for developing selective BuChE inhibitors. Although some
studies on the structure–activity relationship (SAR) of the carba-
mate moiety of physostigmine analogs have already been carried
out, the variety of substituents was limited to relatively simple
ones and some of them were aimed at anti-AChE activity.10,12–19
An investigation using more diverse derivatives may increase our
understanding and aid in the development of more effective com-
pounds. In this report, therefore, we investigated the SAR of the
carbamate moiety by synthesizing carbamate-modified derivatives
3a–u of (ꢀ)-N1-phenethylnorphysostigmine. The N1 moiety was
fixed in the shape of a phenethyl group because the N1-phenethyl
group increases the selectivity for BuChE.20
Table 1
Anti-ChE activity and enzyme selectivity
Compounds
R
AChEa
(nM)
BuChEa
(nM)
Selectivityb
0.14
1
Methyl (N1-methyl,
physostigmine)
Methyl
n-Hexyl
Phenyl
4-Isopropylphenyl
4-t-Butylphenyl
4-n-Butylphenyl
4-Phenylphenyl
4-Dimethylaminophenyl >100,000 79
2-Naphthyl
4-Methoxyphenyl
4-Hexanoxyphenyl
4-Phenoxyphenyl
3,4-Dimethoxyphenyl
5-Benzo[d][1,3]dioxolyl
4-Chlorophenyl
4-Acetylphenyl
4-Nitrophenyl
Benzyl
40
280
3a
3b
3c
3d
3e
3f
3g
3h
3i
170
970
10
14
340
17
69
5
>185
35
1600
>100,000 540
15,000
48,000
>100,000 1100
430
980
49
>91
>1266
76
398
>71
>50
113
19
>270
>17
>15
27
44,000
33,000
>100,000 1400
>100,000 2000
11,000
1900
>100,000 370
>100,000 5900
>100,000 6700
180
450
5600
2500
580
83
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
97
100
6.6
16
22
Phenethyl
Cyclohexyl
Cyclohexylmethyl
28
255
1388
3u
1.8
a
To estimate the enzyme activity, changes in the absorbance at 2 min intervals
As shown in Scheme 1, carbamate-modified (ꢀ)-N1-phenethyl-
norphysostigmine derivatives 3a–u were synthesized from
N1-phenethylnoreseroline (2), which was prepared from physo-
stigmine (1).21,22 The intermediate 2 was reacted with different
R–NCO compounds (a–u, Scheme 1) in the presence of catalytic
Na15,16,20,21,23 to generate 3a–u, including the reported products
(ꢀ)-N1-phenethylnorphysostigmine (3a), (ꢀ)-N1-phenethylnor-
phenserine (3c) and PEC (3d).21
were measured at 415 nm using a spectrophotometer. The enzyme activity is
expressed as a percent of the activity of the solvent, DMSO. Seven different con-
centrations of each compound were used and the IC50 values of each compound
were calculated by nonlinear regression of the sigmoidal dose–response curve using
GraphPad Prism version 4.03. Only the results with correlation coefficients of
r
2 P 0.95 were accepted. The results are represented as the mean of the IC50
obtained from at least four independent measurements.
b
The selectivity was calculated as follows; selectivity = IC50 AChE/IC50 BuChE.
Anti-ChE activities were measured using modified Ellman’s col-
orimetric method.24 Briefly, we used the extracts from mice brain
and mice serum as sources of AChE and BuChE, respectively, and
all compounds were preincubated with the enzymes for 1 h at
37 °C before the addition of acetylthiocholine or butyrylthiocholine
in combination with the coloring reagent 5,50-dithiobis-(2-nitro-
benzoic acid).
previous reports, the methyl group increased both activities,17–19
but the n-hexyl group selectively reduced the anti-AChE activity
by one-fifth (3a and 3b). Regarding substitution on the phenyl ring,
the anti-BuChE activity tended to correlate inversely with the ste-
ric bulk (3c–i).
Physostigmine (1) and its derivatives inhibit ChEs by carbamoy-
The IC50 values for AChE and BuChE are summarized in Table 1.
In all synthesized derivatives, we fixed the N1 moiety in the shape
of a phenethyl group, which is reported to increase the selectivity
for BuChE.20 In fact, the selectivity was BuChE in all derivatives.
The carbamate moiety of physostigmine derivatives is reported
to bind in the acyl pocket, and it seems to have an important role
in the acquisition of the selectivity for BuChE. Since there is a
prominent difference between AChE and BuChE in terms of their
spatial volume, we first examined the effect of steric bulk. As in
lating the c-O of the active center Ser. Since the hydroxyl group of
the Ser attacks the carbamate in this reaction, the electronic prop-
erties of the carbamate moiety may affect the anti-ChE activity.
Thus, we also investigated whether the electronic properties influ-
ence the activity. The methoxy group at the 4-position of the phe-
nyl ring greatly improved the activity, whereas the n-hexanoxy
and phenoxy group diminished it, even though they were all elec-
tron-donating (3j–l). Considering that the anti-ChE activity corre-
lated inversely with the steric bulk, these results indicate that
H
H
6 steps21,22)
HO
N
O
N
O
R-NCO
R
N
N
N
O
O
N
Na, Et2O
rt, 5 min
N
N
Physostigmine (1)
2
3a-u
R-NCO :
methyl isocyanate (a),
n
-hexyl isocyanate (b), phenyl isocyanate (c),4-isopropylphenyl isocyanate (d),
-butylphenyl isocyanate (f), 4-phenylphenyl isocyanate (g),
4- -butylphenyl isocyanate (e), 4-
t
n
4-dimethylaminophenyl isocyanate (h), 2-naphthyl isocyanate (i), 4-methoxyphenyl isocyanate (j),
4-hexanoxyphenyl isocyanate (k), 4-phenoxyphenyl isocyanate (l), 3,4-dimethoxyphenyl isocyanate (m),
5-benzo[d][1,3]dioxolyl isocyanate (n), 4-chlorophenyl isocyanate (o), 4-acetylphenyl isocyanate (p),
4-nitrophenyl isocyanate (q), benzyl isocyanate (r), phenethyl isocyanate (s), cyclohexyl isocyanate (t),
cyclohexylmethyl isocyanate (u).
Scheme 1.

J. Takahashi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1721–1723
1723
the steric factor is more influential than the electronic one.
Although the anti-BuChE activities were similar to those of the
methoxy substituent, the 3,4-dimetoxyphenyl or cyclic 5-
benzo[1,3]dioxolyl substituent enhanced the anti-AChE activity
and gave lower selectivity (3m and 3n). Whereas chloro substitu-
tion at the 4-position of the phenyl ring maintained the activity, it
was decreased by acetyl and nitro group substitution, more potent
electron-withdrawing substituents (3o–q). Overall, the electron-
donating property seemed to increase the anti-BuChE activity,
whereas the electron-withdrawing property diminished it.
In previous reports, most of the carbamoyl substituents of phy-
sostigmine were the n-alkyl, phenyl and simply substituted phenyl
groups.10,12,13,17,25 Therefore, we investigated the aralkyl and
cycloalkyl groups. The benzyl and phenethyl group greatly in-
creased the anti-BuChE activity, but they concurrently increased
the anti-AChE activity and reduced the selectivity (3r and 3s).
The cyclohexyl group reduced the anti-AChE activity, whereas high
anti-BuChE activity was sustained, resulting in a higher selectivity
(3t). Considering the results with the phenyl, benzyl and phenethyl
group, the compounds having an intervening methylene between
the carbamate nitrogen and the ring structure tended to have more
potent anti-ChE activities than the directly bound ones. When we
applied this trend to the cyclohexyl group, cyclohexylmethyl sub-
stitution showed the highest anti-BuChE activity while keeping its
high selectivity (3u).
physostigmine, and determined that the cyclohexylmethyl group
greatly increases the anti-BuChE activity and selectivity for BuChE.
Acknowledgment
This work was financially supported by Eisai Co., Ltd.
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sidered to increase the anti-AChE activity by
Phe295 and Phe297, cyclohexyl group is voluminous and does not
possess electron, hence it may be difficult to accommodate cyclo-
p–p interaction with
p
hexyl group to AChE’s strait acyl pocket and anti-AChE activity was
decreased. Although we have not conducted a detailed analysis
about how this compound interacts with the enzyme, the interven-
ing methylene may confer flexibility to the substituent and enable
the carbamate moiety to fit into the acyl pocket more easily.
In conclusion, we synthesized and biologically evaluated di-
verse carbamate-modified derivatives of (ꢀ)-N1-phenethylnor-
25. Yu, Q.; Atack, J.; Rapoport, S.; Brossi, A. J. Med. Chem. 1988, 31, 2297.