Selective acetylcholinesterase inhibitors derived from muscle relaxant dantrolene

Article abstract of DOI:10.1016/j.bmcl.2019.126888

Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC₅₀ value of 34.2 nM. Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.

Full text of DOI:10.1016/j.bmcl.2019.126888

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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Selective acetylcholinesterase inhibitors derived from muscle relaxant
dantrolene
⁎
Hiroshi Aoyamaa, , Tomohiro Douraa,b
a School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392, Japan
b Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Dantrolene, the only therapeutic agent for malignant hyperthermia, is known to have not only a muscle relaxant
effect, but also a neuroprotective effect and Alzheimer's disease improving effect. Recently, it has been reported
that dantrolene has a weak inhibitory effect on acetylcholinesterase (AChE), which is a therapeutic drug target
for Alzheimer's disease. Thus, we focused on developing of AChE inhibitors with benzylpiperidine/piperazine
moieties that are based on the dantrolene skeleton. Several derivatives showed an inhibitory activity. Among
them, ortho-nitro derivative 8c showed the most potent inhibitory activity with the IC50 value of 34.2 nM.
Furthermore, Lineweaver-Burk plot analysis indicated that 8c is AChE-selective inhibitor, which shows only a
weak inhibitory effect on butyrylcholinesterase (BuChE) and a non-competitive inhibition.
Acetylcholinesterase
Muscle relaxant
Dantrolene
Alzheimer's disease
Dantrolene is a hydantoin compound that was developed in 1967 by
Snyder and co-workers as a muscle relaxant.1 Later, its application to
treat malignant hyperthermia was studied in the late 1970s,2,3 and it
was approved by the FDA as a treatment for malignant hyperthermia in
1979, and it remains the only treatment to date.4
chemical modification of dantrolene. During our studies, various dan-
trolene derivatives were developed,13,14 and a protein related to sugar
metabolism was discovered by a photoaffinity labelled probe that
suppresses skeletal muscle contraction.13,15 In addition, we successfully
developed
a dantrolene derivative that suppresses mitochondrial
The effect of dantrolene on muscle relaxation and malignant hy-
perthermia is attributed to the suppression of Ca2+-releasing channels
that are called ryanodine receptors (RyRs).5,6 There are three subtypes
of RyR, i.e. skeletal-type (RyR1), cardiac-type (RyR2) and brain-type
(RyR3). For these subtypes, dantrolene is known to suppress the release
of Ca2+ from RyR1 and RyR3. RyRs are expressed in various tissues,
although there are differences in their distribution among subtypes.7,8
Therefore, it has been reported that dantrolene show improvement ef-
fect not only on malignant hyperthermia but also on various patholo-
gies caused by the breakdown of calcium homeostasis (e.g. stroke,
ischemia/reperfusion injury and neurodegenerative diseases).9,10
Thus, although the pharmacological effect exhibited by dantrolene
has been mainly attributed to the suppression of the function of RyRs,
recently it has been confirmed that it binds to molecules other than
RyRs. For example, dantrolene binds to the NMDA receptor in the brain
and suppresses its activation.11 In addition to receptor proteins, in-
hibitory activity of acetylcholinesterase (AChE) has also been re-
ported.12
swelling caused by Ca2+ overload, which is an in vitro model of
ischemia/reperfusion injury.16
This time, we focused on the recent report that dantrolene shows an
inhibitory effect on acetylcholinesterase.12 Thus, more useful deriva-
tives can be developed by appropriately modifying dantrolene. At first,
AChE inhibitory activity of dantrolene was confirmed according to lit-
erature information, but the inhibitory activity could not be confirmed
in our evaluation system. However, we determined that dantrolene has
the ability to recognise acetylcholinesterase from the fact that dan-
trolene has an improvement effect on Alzheimer's disease and exhibits
various neuroprotection activities. Moreover, most of the cholinesterase
expressed in the brain is AChE, but it is known that the expression level
of butyrylcholinesterase (BuChE), the other isozyme of cholinesterase,
is higher in the blood. Since a decrease in blood cholinesterase con-
centration increases the risk of causing a cholinergic crisis, it is con-
sidered important to develop compounds that selectively suppress
AChE over BuChE from the viewpoint of drug discovery targeting brain
diseases. Thus, we decided to chemically modify dantrolene with the
aim of creating a selective AChE inhibitor.
We have been studying the function of muscle contraction using
dantrolene derivatives and developing functional molecules by
The catalytic site of AChE has a narrow and deep pocket structure,
⁎ Corresponding author.
E-mail address: aoyamahr@toyaku.ac.jp (H. Aoyama).
https://doi.org/10.1016/j.bmcl.2019.126888
Received 31 October 2019; Received in revised form 3 December 2019; Accepted 3 December 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:HiroshiAoyamaandTomohiroDoura,Bioorganic&MedicinalChemistryLetters,
https://doi.org/10.1016/j.bmcl.2019.126888

H. Aoyama and T. Doura
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 1. Design of AChE inhibitors derived from muscle relaxant dantrolene.
Scheme 1. Reagents and conditions: (a) BnBr, Et3N, MeCN, rt, 52%; (b) 1, DEAD, PPh3, THF, 60 °C, 5.7%; (c) I2, PPh3, imidazole, THF, rt, 97% (n = 1), 88% (n = 2);
(d) 4a or 4b, DMF, rt, 52% (n = 1), 96% (n = 2); (e) 4.0 M HCl (1,4-dioxane soln), CH2Cl2, rt; (f) BnBr, K2CO3, DMF, rt, 75% (7, 2 steps), 55% (8a, 2 steps).
Scheme 2. Reagents and conditions: (a) 4b, K2CO3, DMF, rt, 82%-quant.; (b) 4.0 M HCl (1,4-dioxane soln), CH2Cl2, rt; (c) BnBr, K2CO3, DMF, rt, 38–63% (2 steps).
and benzylpiperidine inhibitors represented by donepezil are known to
form a cation-π interaction with Phe330 at the catalytic site when the
piperidine ring nitrogen is cationised.17,18 Therefore, a compound in
which this benzylpiperidine unit and a piperazine unit of a six-mem-
bered ring species capable of cationization were introduced into the
hydantoin portion of dantrolene was designed (Fig. 1).
condition followed by benzylation with benzyl bromide to give dan-
trolene derivatives with benzylpiperidine moiety 7 and 8a.
The effects of dantrolene sodium salt, three synthesized compounds
(i.e. 3, 7 and 8a), and Tacrine hydrochloride (positive control) on AChE
activity were determined by Ellman method21 in sodium phosphate
buffer (pH 8.0) in the absence or presence of the abovementioned
compounds. First, we measured the enzyme inhibition rate of com-
pounds at a fixed concentration of 1.0 μM against AChE, as summarized
in Table 1. Dantrolene sodium salt and dantrolene derivative with N-
benzylpiperidine moiety connected by a methylene bridge 7 did not
show the AChE inhibitory activity, whereas derivatives with N-ben-
zylpiperazine (3) and N-benzylpiperidine (8a) moieties connected by an
ethylene bridge exhibited a significant AChE inhibitory activity. Ta-
crine hyrdochloride, a positive control compound, also significantly
suppressed AChE activity.
The syntheses of piperidine/piperazine moieties introduced at hy-
dantoin of dantrolene and dantrolene derivatives are outlined in
Schemes 1 and 2, respectively. Briefly, commercially available 1-(2-
hydroxyethyl)piperazine was benzylated with benzyl bromide followed
by the introduction of a benzylpiperazine unit into hydantoin under
Mitsunobu reaction condition to give 3. The preparation of dantrolene
derivatives with benzylpiperidine moiety was carried out using dan-
trolene sodium salt and 1-[(tert-butoxycarbonyl)-4-iodomethyl]piper-
idine (4a)19 or 1-[(tert-butoxycarbonyl)-4-(2-iodoethyl)]piperidine
(4b)20 prepared from the corresponding alcohol to obtain Boc protected
compounds 5 and 6a. Then, the deprotection of Boc group under acidic
On the basis of the abovementioned result, we synthesized dan-
trolene derivatives, which are based on 8a with nitro (8b and 8c) or
2

H. Aoyama and T. Doura
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Table 1
Effect of dantrolene sodium salt and its derivatives 3, 7 and 8a on AChE in-
hibition.
Compound
X
n
Inhibition rate (%)a
Dantrolene (1)b
–
–
–
2
1
2
N.I.c
70.8d
67.9
N.I.c
91.3
Tacrine·HCl
–
3
N
7
CH
CH
8a
a
b
c
d
At 1.0 μM.
Sodium salt.
No inhibition.
At 0.10 μM.
Fig. 2. Lineweaver–Burk plot analysis for AChE using 8c. [ATCh]: concentra-
tion of acetylthiocholine, substrate as AChE. ABU: absorbance of 5-mercapto-2-
nitrobenzoic acid which generate from 5,5′-dithiobis(2-nitrobenzoic acid) as
color reagent of Ellman method. The change in absorbance (ABU) per minute
was taken as V.
Table 2
Effect of dantrolene derivatives 3, 8a-c and 11a-c on AChE and BuChE in-
hibition.
AChE inhibition activity among the synthesized compounds with the
IC50 value of 34.2 nM. Concerning BuChE inhibition activity, all com-
pounds revealed that weak or no inhibition activity in the presence of
1.0 μM inhibitor concentration. Even 8c, which showed the strongest
inhibitory activity, had an inhibition rate of 14.6% in the presence of
1.0 µM, and its IC50 value was 4.47 μM. From the above results, the
selectivity of 8c for acetylcholinesterase is estimated to be at least about
130 times.
Compound
R
X
IC50 value for AChE
(nM)
Inhibition rate for BuChE
(%)a
3
4-NO2
4-NO2
3-NO2
2-NO2
N
201
N.I.b
Finally, the inhibition mechanism of 8c, which showed the strongest
inhibitory activity, was analyzed. In the Lineweaver-Burk plot analysis
using 8c and AChE, the drawn line intersected on the horizontal axis,
indicated that the inhibition mode of 8c is non-competitive (Fig. 2).
In addition, when a docking study was performed using 8c and
AChE by Molegro Virtual Docker 6.0,23 the nitrophenyl group was ac-
commodated in the pocket consisting of His287, Trp286, and Tyr72
existing near the entrance of the ligand binding site of AChE (Fig. 3a).
This result suggested that the distance between the nitro group of 8c
and the hydroxyl group of Tyr72 is close, and an efficient interaction is
formed. On the other hand, the part corresponding to the AChE pocket
of BuChE is covered by Ala277 (Fig. 3b). Therefore, it is speculated that
the interaction formed in this pocket affects the AChE/BuChE se-
lectivity shown by 8c.
8a
CH 46.4
CH 290
CH 34.2
N.I.b
8b
N.I.b
8c
14.6 (4.47 μM)c
11a
4-MeO CH 108
3-MeO CH 47.0
2-MeO CH 90.4
4.6
11b
3.8
11c
1.6
Tacrine·HCl
–
–
106d
64.5e
a
b
c
d
e
At 1.0 μM.
No inhibition.
IC50 value in parenthesis.
Literature value in Ref. 22.
At 20 nM.
methoxy (11a–c) groups to examine the effect of substituents on the
phenyl group of 8a on AChE inhibition. At the same time, the inhibitory
effect of the compounds on BuChE was performed, and the selectivity
for the cholinesterase isozyme was also evaluated. Syntheses of these
compounds were carried out in the same manner as the synthesis of 8a,
as summarized in Scheme 2.
In summary, we successfully developed novel AChE inhibitors that
are based on muscle relaxant dantrolene. Compound with nitro group at
the ortho position 8c exhibited the most potent and selective AChE
inhibitory activity with the IC50 value of 34.2 nM. In addition,
Lineweaver-Burk plot analysis revealed that the inhibition mode of 8c
was non-competitive. Further structural development and structure-
activity relationship studies are currently underway.
As shown in Table 2, all synthesized compounds showed moderate
to potent AChE inhibition activity. Compound 8b (with an electron
withdrawing group in the meta position) and compounds 11a and 11b
(with an electron donating group in the ortho or para position) showed
lower activity than that of 8a and the same or slightly stronger activity
compared to that of piperazine compound 3. In contrast, 8c (with an
electron withdrawing group in the ortho position) and 11b (with an
electron donating group in the meta position) exhibited the same or
more potent activity than 8a. Especially, 8c indicated the most potent
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
3

H. Aoyama and T. Doura
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 3. (a) Proposed binding mode of 8c (green stick) to AChE catalytic site (PDB: 4EY7); (b) crystal structure of BuChE catalytic site (PDB: 4BDS). The molecular
mode of docked 8c with AChE was displayed using PyMOL (The PyMOL Molecular Graphics System, Version 0.99 Schrödinger, LLC).
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