Design, synthesis, and biological activity of novel semicarbazones as potent Ryanodine receptor1 inhibitors of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2020.115891

Ryanodine receptors (RyRs) are important ligand-gated Ca²⁺ channels; their excessive activation leads to Ca²⁺ leakage in the sarcoplasmic reticulum that may cause neurological diseases. In this study, three series of novel potent RyR1 inhibitors based on dantrolene and bearing semicarbazone and imidazolyl moieties were designed and synthesized, and their biological activity was evaluated. Using a single-cell calcium imaging method, the calcium overload inhibitory activities of 26 target compounds were tested in the R614C cell line, using dantrolene as a positive control. The preliminary investigation showed that compound 12a suppressed Ca²⁺ release as evidenced by store overload-induced Ca²⁺release (SOICR) (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%) at 0.1 μM, 3 μM and 10 μM, respectively. Docking simulation results showed that compound 12a could bind at the active site of the RyR1 protein. The Morris water-maze test showed that compound 12a significantly improved the cognitive behavior of AD-model mice. Further studies on the structural optimization of this series of derivatives are currently underway in our laboratory.

Full text of DOI:10.1016/j.bmc.2020.115891

Bioorg. Med. Chem. 29 (2021) 115891
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological activity of novel semicarbazones as potent
Ryanodine receptor1 inhibitors of Alzheimer’s disease☆
,
,
,
,
,
,b
Baozhu Daia b, Xingxing Maa b, Yadong Tanga b, Le Xua b, Su Guoa b, Xinyan Chena
,
,
,
,b,*
Shitong Lua b, Guangjie Wanga b, Yajing Liua
a Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, 103 Wenhua Road, Shenhe District,
Shenyang 110016, PR China
b Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China
A R T I C L E I N F O
A B S T R A C T
Ryanodine receptors (RyRs) are important ligand-gated Ca2+ channels; their excessive activation leads to Ca2+
leakage in the sarcoplasmic reticulum that may cause neurological diseases. In this study, three series of novel
potent RyR1 inhibitors based on dantrolene and bearing semicarbazone and imidazolyl moieties were designed
and synthesized, and their biological activity was evaluated. Using a single-cell calcium imaging method, the
calcium overload inhibitory activities of 26 target compounds were tested in the R614C cell line, using dan-
trolene as a positive control. The preliminary investigation showed that compound 12a suppressed Ca2+ release
as evidenced by store overload-induced Ca2+release (SOICR) (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%) at 0.1
Keywords:
RyR1 inhibitors
SOICR
Synthesis
Semicarbazones
AD
μ
M, 3 μM and 10 μM, respectively. Docking simulation results showed that compound 12a could bind at the
active site of the RyR1 protein. The Morris water-maze test showed that compound 12a significantly improved
the cognitive behavior of AD-model mice. Further studies on the structural optimization of this series of de-
rivatives are currently underway in our laboratory.
1. Introduction
cholinergic hypotheses are related to Ca2+ homeostasis disorder.11–17
Calcium ions play several roles in processes that are crucial for the
Alzheimer’s disease (AD) is a progressive neurodegenerative disease
that is clinically characterized by dementia and neurobehavioral dete-
rioration that seriously affects the quality of life and life expectancy.
Approximately 30 million people have AD worldwide1–3. There is
growing evidence that AD is a metabolic disorder due to a dysregulation
in multiple biochemical pathways4–7. Furthermore, four main hypoth-
eses have been intensively studied: the amyloid cascade hypothesis, tau
hypothesis, metal ion (Ca2+) hypothesis, and the cholinergic hypothesis.
Currently, only six drugs have been approved: (i) the cholinesterase
inhibitors: donepezil, rivastigmine, galantamine, and tacrine, (ii) the N-
methyl-d-aspartate (NMDA) receptor antagonist memantine, and (iii)
GV-971 that was approved in China in 2019.8
functioning of neuronal cells, including membrane excitability and gene
expression. Although dysregulation of intracellular Ca2+ and calcium
homeostasis in cells of AD patients has been proposed as a common
proximal cause of neurodegeneration in old age and Alzheimer’s dis-
ease, much more attention has been focused on amyloid-β (Aβ) and tau
as key causative factors for the disease. Nevertheless, there is accumu-
lating evidence pointing to the importance of disturbed calcium
signaling in AD pathogenesis.18–20
Ryanodine receptor (RyR) is a high-flux intracellular calcium chan-
nel that is responsible for the rapid and massive release of calcium into
the cytoplasm from the sarcoplasmic reticulum (SR) or endoplasmic
reticulum (ER).21–23 This process plays an indispensable role in many
cellular functions. Over-activation of the RyR channel causes leakage of
Ca2+ from the SR store.
However, these drugs have limited therapeutic effects as they can
only relieve the symptoms of AD and not stop or reverse the disease
progression.9,10 Studies have shown that the amyloid cascade, tau, and
Dantrolene (Fig. 1), a small-molecule RyRs antagonist, was used for
☆
In this study, three series of novel potent RyR1 inhibitors bearing semicarbazones and imidazoles moiety based on dantrolene were designed, synthesized and
evaluated for their biological activity, and compound 12a could improve the cognitive state in Mirror water maze test on AD mouse model.
* Corresponding author at: Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, 103
Wenhua Road, Shenhe District, Shenyang 110016, PR China.
E-mail address: lyjpharm@126.com (Y. Liu).
https://doi.org/10.1016/j.bmc.2020.115891
Received 20 August 2020; Received in revised form 18 November 2020; Accepted 19 November 2020
Available online 26 November 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

B. Dai et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115891
into the terminal N atom of the hydantoin ring to obtain Series I and II of
candidate compounds. Series III of imidazole derivatives was designed
to improve the compounds’ aqueous solubility (Fig. 2).
2. Results and discussion
Fig. 1. . Dantrolene.
2.1. Chemistry
the treatment of malignant hyperthermia (MH).24 Because of its
involvement in calcium homeostasis, dantrolene has been investigated
for its potential as a neuroprotective in several animal models of AD.25
RyRs channels govern the process of spontaneous Ca2+ release from
Ca2+-overload SR known as the store overload-induced Ca2+ release
(SOICR). Chen et al.26 reported that dantrolene could directly interact
with RyR1 and inhibit the release of calcium from the ER and SR to
reduce intracellular calcium overload.27 Furthermore, they discovered
that dantrolene could inhibit SOICR potently in HEK293 cells expressing
RyR1 but not RyR2. Therefore, we selected the RyR1 cell line R614C to
evaluate the activity of our compound in vitro. Peng et al. reported that
memory consolidation and working memory in 3xTg-AD mice were
significantly improved by treatment with dantrolene.28 Chakroborty
et al. found that Aβ deposition within the cortex and hippocampus was
reduced after the short-term administration of dantrolene in AD model
mice.29 Unfortunately, dantrolene has many adverse reactions, such as
muscle weakness, phlebitis, respiratory failure, and gastrointestinal
symptoms.30 Therefore, we used dantrolene as the lead compound and
introduced a structural modification to obtain candidate compounds
with fewer side effects and higher calcium inhibition.
2.1.1. Synthesis of target compounds 6a-6l, 10a-10j, 12a-12d
The general routes of synthesis for the target compounds are illus-
trated in Schemes 1–5. Commercially available 4-nitroaniline was
treated with NaNO2/HCl to obtain the diazonium derivative (1) that was
condensed with furan-2-carbaldehyde in the presence of copper chloride
to provide the key intermediate 5-(4-nitrophenyl) furan-2-carbaldehyde
(2).
Intermediates 5a-5l were synthesized as outlined in Scheme 2. The
substituted aniline was reacted with phenyl carbonochloridate to obtain
intermediates 4a-4l that were treated with hydrazine hydrate to obtain
intermediates 5a-5l. The synthesis method of 9a-9j was the same as that
of 5a-5l outlined in Scheme 3. The intermediates 11a-11d were syn-
thesized as profiled in Scheme 4. The substituted ethyl ester was treated
with hydrazine hydrate to obtain intermediates 11a-11d
As described in Scheme 5, intermediates 2 were treated with in-
termediates 5a-5l, 9a-9j or 11a-11d to afford the target compounds,
respectively.
2.2. Biological evaluation
The challenge in the treatment of central nervous system diseases is
mainly the inability of the drug to penetrate the blood–brain barrier
(BBB).31 In this study, we modified the lead compound by opening the
hydantoin ring and removing the acetyl group to form an acylhydrazide
structure. To increase the lipid solubility of dantrolene derivatives,
phenyl or benzyl substituents were added to the terminal N atom to
obtain two series of new compounds. Another series of derivatives were
generated by replacing phenyl with imidazolyl, expecting that com-
pounds with imidazolyl groups would have better aqueous solubility
and binding affinity.
2.2.1. Biological activity of dantrolene derivatives
R614C cells express RyR1-R614C mutation. Single-cell calcium im-
aging can be used to monitor changes in calcium ions in the cytoplasm.
Dantrolene was used as a positive control and DMSO as a blank control.
Different concentrations of the target compounds (0.1 μM, 3μM, 10 μM)
were used to evaluate the inhibitory activity on calcium channels in
vitro. The inhibition rate (%) of SOICR was used to characterize the
activity of the compounds and is shown in Table 1 as an average value
obtained from at least three independent experiments.
Consequently, a 5-(4-nitrophenyl) furan-2-methylene amino frag-
ment was retained, substituted phenyl or benzyl groups were introduced
2.2.2. In vitro SOICR inhibition activities and SARs study
As shown in Table 1, most of the target compounds exhibited
Fig. 2. . Design strategy for the dantrolene derivatives.
2

B. Dai et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115891
Scheme 1. Reagents and conditions: (i): 20%HCl, NaNO2, 0℃, 30 min; (ii): furan-2-carbaldehyde, CuCl2⋅H2O acetone, 0℃, 6 h.
Scheme 2. Reagents and conditions: (i): phenyl carbonochloridate; Na2CO3, THF: EA: H2O = 1:3:1, 25℃, 6 h; (ii): NH2NH2⋅H2O (80%), EtOH, rt, 4 h.
Scheme 3. Reagents and conditions: (i): phenyl carbonochloridate; Na2CO3, THF: EA: H2O = 1:3:1,25℃,4h; (ii): NH2NH2⋅H2O (80%), EtOH, rt, 3 h.
Scheme 4. Reagents and conditions: NH2NH2⋅H2O (80%), EtOH, rt, 3 h.
moderate or excellent inhibitory activity against SOICR at different
concentrations (0.1 M, 3 M and 10 M). Activities of compounds 6a
the activity of disubstituted compounds was significantly lower, such as
for 10b compared to 10f–10h.
μ
μ
μ
(15.90 ± 0.3%, 35.8 ± 0.2%, 93.9 ± 2.3%), 10e (23.6 ± 0.3%, 35.1 ±
0.1%, 76.1 ± 0.1%) and 12a (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%)
were equivalent to those of dantrolene (30.8 ± 0.1%,73.1 ± 0.1%,94.2
± 0.2%). Compound 12a was the best candidate and markedly sup-
pressed Ca2+ oscillations. The study has shown that these compounds
inhibit SOICR in a dose-dependent manner; we will discuss the struc-
Although compound 6a showed high inhibitory activity, its water
solubility was poor. Therefore, we replaced the phenyl with imidazolyl
to improve water solubility hoping that it would enhance the interaction
between the target compound and the receptor. Compounds 12a-12d
contain different substituents on the imidazole ring and amide nitro-
gen atom. Fortunately, compound 12a showed the same activity as
dantrolene and its solubility was better than that of dantrolene and
phenyl derivatives; it is currently the best candidate compound. For
compounds 12c, 12b, and 12d, the introduction of methyl groups into
the imidazole ring and amide nitrogen atom decreased slightly the
compounds’ activity.
ture–activity relationship at 10 μM.
The inhibitory effect of the target compounds and dantrolene on
SOICR in R614C cells is shown in Table 1. Phenyl- and benzyl-
substituted derivatives had a similar effect at different concentrations.
The imidazole derivatives were more potent.
Phenyl derivatives (Series I) containing electron-withdrawing groups
(EWG) on the benzene ring, such as bromine (6e) and chlorine (6 h),
showed higher activities than compounds containing electron-donating
groups (EDG), such as methyl (6d) and methoxy (6i). Furthermore, the
activity of meta-substituted derivatives was better than the activity of
substituted derivatives at other sites (6c vs 6f). Unexpectedly, compound
6a with no phenyl substitution was more potent than those with a
substituted phenyl group.
2.2.3. The activities of the target compounds inhibiting AChE
It has been reported that dantrolene can inhibit AChE,32,33 so we
used donepezil as a positive control to test the effect of dantrolene and
its derivatives on AChE. The effects of dantrolene, six synthesized
compounds (6a, 6b, 10d, 10 g, 12a, and 12b), and donepezil (positive
control) on AChE activity were determined using the Ellman method34
via the use of a sodium phosphate buffer (pH 8.0), with either the
absence or presence of the abovementioned compounds. First, we
measured the enzyme inhibition rate of compounds at a fixed concen-
Similar biological properties were observed for the benzyl de-
rivatives (Series II). Compounds with EWG suppressed SOICR better
than those with EDG in the benzene ring. For example, the activities of
compounds 10b (4-Cl, 70.4 ± 0.1%), 10e (4-F, 76.1 ± 0.1%), and 10i (4-
CF3, 53.1 ± 0.3%) were better than that of compound 10j (4-CH3, 35.4
± 0.3%). However, the substitution position also affected the activity,
with para-substitution being more beneficial than the other two sites as
seen in compounds 10e (4-F, 76.1 ± 0.1%) vs 10c (2-F, 44.6 ± 0.1%) and
10d (3-F, 72.1 ± 0.2%). Compared with monosubstituted compounds,
tration of 5.0 μM against AChE, as summarized in Table 2. Dantrolene
and dantrolene derivatives 10d, 12a, and 12b showed weak AChE
inhibitory activity, while 6a, 6b, and 10g showed moderate AChE
inhibitory activity. The positive control compound, donepezil, signifi-
cantly inhibited AChE activity.
3

B. Dai et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115891
Scheme 5. Reagents and conditions: EtOH, AcOH, rf, 2 h.
2.2.4. Kinetic water solubility
12a exhibited the best drug-like properties.Table5 (see Table 6).
In order to compare the solubility difference between terminal
benzene ring and imidazolyl, we tested kinetic water solubility of dan-
trolene and its derivatives 6a, 12a and 12b. We determined the kinetic
water solubility of 12a using a standard turbidimetric assay and
compared this with those of 6a and dantrolene. The aqueous solubilities
of 12a improved in comparison to 6a and dantrolene (Table 3). The
compound 12a exhibited the best kinetic water solubility (9-fold greater
than 6a, and similar to those of dantrolene and 12b), demonstrating the
introduction of an imidazolyl group at the end of the carbonyl group to
be a feasible solution to improve water solubility.
2.2.6. In vivo test
Behavioral test: Morris water maze test was used to evaluate the
spatial learning and memory ability of the animal model. In this
experiment, SPF grade FAD mice were administered at the age of 5
months, male and female at random, and the test began 30 days after
administration.36 The mice were divided into two groups: The test group
was intraperitoneally injected at a dose of 3.2 mg/kg with 12a DMSO
solution (1.12 mg/mL), and the control group was intraperitoneally
injected with a same volume of DMSO.
The results were presented in Figure 4, and the statistical significance
of each was analyzed by ANOVA versus the DMSO and compound 12a
groups. As shown in Fig. 4B, the administration of compound 12a during
the drug treatment period (30 consecutive days) did not influence the
mean daily bodyweight profile of mice. This suggested that long-term
administration of compound 12a is safe at doses of 3.2 mg/kg/day.
During the training trials, the mean escape latency for the mice in the
12a group declined progressively compared with those in the DMSO
group (Day 2, 29.89 s vs 36.72 s, p < 0.05; Day 3, 25.12 s vs 35.59 s, p <
0.05; Day 4, 16.23 s vs 31.99 s, p < 0.001) (Fig. 4C). Memory retention
was assessed by performing a spatial memory probe trial with the
platform removed from the pool 24 h after the last training trial. The
results of Fig. 4D showed that the percentage of swimming time on
target zone (southwest quadrant) in compound 12a group (35.7 ± 1.2)
was significantly higher than that in DMSO group (23.1 ± 1.1), which
indicated that the cognitive and memory functions of FAD mice in
compound 12a group were improved more obviously on the dosage of
3.2 mg/Kg/day (see Fig 5).
2.2.5. Binding model analysis
Based on the in vitro pharmacological results, we selected compound
12a, the best RyR1 inhibitor in this study, as the docking model (PDB
ID:6M7H). As shown in Fig. 3(A), ASP80 forms a hydrogen bond be-
tween the nitrogen atoms of imidazole and amide. The following reci-
procity mutual effect was noted: (1) one pi-alkyl interaction between the
imidazole ring and residue LYS75; (2) two pi-sigma interactions be-
tween the benzene ring and residue MSE71 and LEU32, respectively; (3)
one carbon-hydrogen interaction between a carbon atom of the imid-
azole ring and residue LYS75. The 3D model of the interactions between
compound 12a and the protein crystal structure shown in Fig. 3(B)
indicated that the active pockets were occupied by compound 12a.
The optimum cLogP value and molecular weight of the drug passing
through the blood–brain barrier are 2.8 and 305.3, respectively35. Some
physicochemical properties of the target compounds and dantrolene
were predicted using the free online website (http://www.molinspir
ation.com) for their adaptability with Lipinski’s rule of five and the
best value of passing through the blood–brain barrier. As shown in
Table 4, compounds 6d, 6f, and 12a were better than dantrolene, and
4

B. Dai et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115891
3. Conclusions
Table 1
The activities of the target compounds inhibiting Ca2+
.
Compd.
R
SOICR Inhibition (%) (Mean ± SDa, N = 3)
In this study, three series of novel potent RyR1 inhibitors based on
dantrolene and bearing semicarbazone and imidazole moieties were
designed, synthesized, and evaluated for their biological activity. The
results of pharmacological experiments showed that several of the new
compounds (6a, 6e, 10d, 10e, 12a, 12b) had moderate to excellent
ability to suppress SOICR in R614C cells in vitro. The primary SARs
showed that the opening of the hydantoin ring of the lead compound
could retain activity and especially the introduction of hydrophilic
imidazolyl group could improve the affinity of RyR1. Fortunately, imi-
dazolyl derivative 12a exhibited the best potency in vitro with SOICR
0.1
μ
Mb
3
μ
Mb
10
μ
Mb
6a
H
15.90 ± 0.3
35.8 ± 0.2
–
93.9 ± 2.3
–
c
6b
2-CH3
3-CH3
4-CH3
2-Br
-
6c
10.7 ± 0.1
15.2 ± 0.1
10.8 ± 0.1
38.5 ± 0.2
67.1 ± 0.1
31.3 ± 0.2
25.1 ± 0.2
–
34.6 ± 0.1
29.5 ± 0.1
67.1 ± 0.1
67.1 ± 0.1
52.9 ± 0.2
50.1 ± 0.1
30.6 ± 0.1
57.1 ± 0.1
55.5 ± 0.1
32 ± 0.1
6d
–
6e
20.1 ± 0.1
6f
3-Br
26.7 ± 0.4
6g
4-Br
13.2 ± 0.2
6h
2-Cl
–
6i
2-OCH3
2-OCF3
2-CF3
–
inhibition of 31.5 ± 0.1% at a concentration of 0.1 μM. Although 12a
6j
–
20.4 ± 0.2
30.1 ± 0.1
14.1 ± 0.2
23.1 ± 0.3
48.6 ± 0.1
26.1 ± 0.1
25.8 ± 0.3
35.1 ± 0.1
–
6k
12.6 ± 0.2
inhibited the activity of AChE, it was much weaker than donepezil.
Furthermore, the Morris water-maze test provided evidence that com-
pound 12a could significantly improve the cognitive behavior of AD-
model mice. The docking simulation results showed that compound
12a could bind well at the active site of RyR1, with imidazole providing
a much more suitable size for occupying the hydrophobic cavity
compared with dantrolene. Further studies on the structural optimiza-
tion of this series of derivatives are currently underway in our
laboratory.
6l
4-NO2
H
–
–
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
12a
12b
12c
12d
DMSOd
Dantrolenee
48.6 ± 0.1
70.4 ± 0.1
44.6 ± 0.1
72.1 ± 0.2
76.1 ± 0.1
30.6 ± 0.1
16.2 ± 0.2
37.2 ± 0.1
53.1 ± 0.3
35.4 ± 0.3
93.7 ± 0.2
82.5 ± 0.1
68.2 ± 0.1
74.9 ± 0.2
–
4-Cl
27.1 ± 0.3
2-F
–
–
3-F
4-F
23.6 ± 0.3
2,3-(Cl)2
2,4-(Cl)2
3,4-(Cl)2
4-CF3
–
–
–
–
25.3 ± 0.2
27.9 ± 0.3
–
19.7 ± 0.2
4-CH3
R1 = H; R2 = H
R1 = CH3;R2 = CH3
H
–
31.5 ± 0.1
24.5 ± 0.3
26.1 ± 0.2
23 ± 0.2
–
77.2 ± 0.1
70.5 ± 0.3
48.4 ± 0.3
53.5 ± 0.1
–
4. Experimental
4.1. Chemistry
CH3
30.8 ± 0.1
73.1 ± 0.1
94.2 ± 0.2
Unless otherwise specified, all materials were obtained from com-
mercial suppliers and were used without further purification. Reactions’
time and purity of the products were monitored by TLC on FLUKA silica
gel aluminum cards (0.2 mm thickness) with fluorescent indicator 254
nm. Column chromatography was run on silica gel (200–300 mesh) from
Qingdao Ocean Chemicals (Qingdao, Shandong, China). All melting
points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra
(MS) were taken in ESI mode on Agilent 1100 LC–MS (Agilent, Palo Alto,
CA, USA). 1H NMR and 13C NMR spectra were recorded on Bruker ARX-
400, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA)
with TMS as an internal standard.
a
Standard deviation.
b
c
d
e
The concentration of the compound.
Means no activity at the concentration.
Used as a blank control.
Used as a positive control.
Table 2
Effects of dantrolene derivatives 6a, 6b, 10d, 10 g, 12a, and 12b on AChE
inhibition.
Compd.
AChE Inhibition (%)a (Mean SDb, N ¼ 3)
6a
19.3 ± 2.56
18.2 ± 3.19
33.6 ± 1.09
8.9 ± 3.45
24.8 ± 0.64
14.3 ± 2.1
99.4 ± 0.23c
0.1 ± 2.5
6b
4.1.1. Preparation of 5-(4-nitrophenyl) furan-2-carbaldehyde (2)
10d
10g
4.1.1.1. Preparation of 1-chloro-2-(4-nitrophenyl) diazo salt (1). To a
suspension of finely powdered p-nitroaniline (2.54 g, 20 mmol) in 10 mL
of 24% aq hydrochloric acid at 0 ◦C was added a cold aqueous solution of
sodium nitrite (1.7 g, 23 mmol), and the reaction mixture was stirred for
1 h at 0–5 ◦C to obtain 4-nitrobenzenediazonium chloride (1). The re-
action solution of diazonium salt 1 was directly used for the next
reaction.
12a
12b
Donepezil
Dantrolene
a
At 5.0
μ
M.
Standard deviation.
At 1.0 M.
b
c
μ
4.1.1.2. Preparation of 5-(4-nitrophenyl) furan-2-carbaldehyde (2). 4-
Nitrobenzenediazonium Chloride (1) was added dropwise under stirring
to a solution of furfural (0.1 mol, 9.6 g) and CuCl2⋅2H2O (0.058 mol, 1 g)
in acetone (40 mL) at 20–30 ◦C. The mixture was stirred until nitrogen
no longer evolved, diluted with water (200 mL), and filtered to yield 5-
(4-nitrophenyl) furan-2-carbaldehyde (2), which was recrystallized
from DMF (17.7 g, 81.7%). Mp:202.7–204.1 ◦C, MS (ESI) m/z (%):
Table 3
Kinetic water solubility of dantrolene, 6a, 12a, and 12b.
Compound
Solubility limit a,b,c(mM)
Dantrolene
6a
0.56
0.06
0.58
0.48
12a
12b
1
218.26 [M+H]+; H NMR (400 MHz, DMSO‑d6) δ (ppm): 9.69 (s, 1H,
a
b
Precipitation was determined by turbidity at 540 nm.
CHO), 8.35 (d, J = 8.9 Hz, 2H, Ph-H), 8.13(d, J = 8.9 Hz, 2H, Ph-H), 7.72
(d, J = 3.8 Hz, 1H, furan-H), 7.59 (d, J = 3.8 Hz, 1H, furan-H).
Absorbance value > “mean + 3 standard deviations” from
the blank was considered turbid.
c
The coefficient of variation (CV) was < 1.5% for all four
compounds.
5

B. Dai et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115891
Fig. 3. . Docking mode of compound 12a. (A) 2D molecular docking model of compound 12a. (B) 3D interaction map between compound 12a and binding sites.
Table 4
Prediction of physicochemical propertiesa of the target compounds.
Compd
miLogP
TPSA
natoms
MW
nON
nOHNH
nviolations
nrotb
volume
Standard
<5
<140
112.5
112.5
129.11
120.7
<500
<5
8
<5
2
≤10
6
6d
4.7
28
28
24
23
378.69
443.26
325.28
314.26
0
1
0
0
332.17
333.5
6f
4.84
2.46
1.75
8
2
6
12a
9
2
5
267.24
252.73
Dantroleneb
9
1
4
a
miLogP: molinspiration predicted Log P; TPSA: topological polar surface area; natoms: no. of atoms; MW: molecular weight; nON: no. of hydrogen bond acceptors;
nOHNH: no. of hydrogen bond donors; nviolations: no. of violations; nrotb: no. of rotatable bonds; volume: molar volume.
b
Used as positive control.
g, 3.2 μmol) was dropped to the reaction solution at room temperature,
Table5
stirred for 6 h. When TLC showed the completion of the reaction, the
resulting precipitate was filtered, washed with H2O and dried under
reduced pressure to obtain the title compound(4a-4l).
The single-cell Ca2+ imaging time.
Ca2+ con.
Drug con.
Caffeine con.
Time (min)
End point in time (s)
0
0
0
0
0
0
3
3
3
8
8
8
8
3
180
0.1 mM
0.5 mM
1 mM
1 mM
1 mM
1 mM
1 mM
0
360
4.1.2.2. Phenyl phenylcarbamate (4a). Yellowish solid; Yield: 78.4.0%.
MS (ESI) m/z (%): 214.3 [M+H]+.
0
540
0
1020
1500
1980
2460
0.1
μ
M
0
3
μ
M
0
4.1.2.3. Phenyl o-tolylcarbamate (4b). Yellow solid; Yield: 67.8%. MS
(ESI) m/z (%): 228.2 [M+H]+.
10
0
μ
M
0
10 mM
4.1.2.4. Phenyl m-tolylcarbamate (4c). Yellowish solid; Yield: 72.6%.
MS (ESI) m/z (%): 228.2 [M+H]+.
Table 6
Mirror water maze protocol*.36
4.1.2.5. Phenyl p-tolylcarbamate (4d). Dark yellow solid; Yield: 65.6%.
MS (ESI) m/z (%): 228.2 [M+H]+.
Day 1
Day 2
Day 3
Day 4
Day 5
Platform Location: SW Starting Location as follows:
No platform.
4.1.2.6. Phenyl (2-bromophenyl)carbamate (4e). Brown-black solid ;
Yield: 64.2%. MS (ESI) m/z (%): 293.2 [M+H]+.
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
W
S
N
W
E
N
E
E
N
S
N
E
W
S
W
E
W
S
4.1.2.7. Phenyl (3-bromophenyl)carbamate (4f). Brown solid; Yield:
61.3%. MS (ESI) m/z (%): 293.2 [M+H]+.
S
N
N
* On days 1–4, the platform position remains constant while the starting di-
rection changes. On day 5, there is no platform and a single trial. The starting
direction for day 5 is farthest from the previous platform location (N) so that the
mice must travel some distance before entering the previously learned platform
quadrant.
4.1.2.8. Phenyl (4-bromophenyl)carbamate (4g). Yellowish brown solid;
Yield: 72.2%. MS (ESI) m/z (%): 293.2 [M+H]+.
4.1.2.9. Phenyl (2-chlorophenyl)carbamate (4h). Yellowish solid; Yield:
65.2%. MS (ESI) m/z (%): 248.8 [M+H]+.
4.1.2. General procedure for preparation of acylhydrazine intermediates
(5a-5l, 9a-9j)
4.1.2.10. Phenyl (2-methoxyphenyl)carbamate (4i). Milky yellow solid;
Yield: 67.6%. MS (ESI) m/z (%): 244.2 [M+H]+.
4.1.2.1. General procedure for preparation of different substitution phenyl
phenylcarbamate (4a-4l). 3 μmol substituted aniline and Na2CO3 (0.19
4.1.2.11. Phenyl (2-(trifluoromethoxy)phenyl)carbamate (4j). Yellow
solid; Yield: 62.2%. MS (ESI) m/z (%): 298.3 [M+H]+.
g, 1.8
μmol) were dissolved in the solution of 10 mL (THF: EA: H2O =
1:3:1) and stirred at 25℃ for 15 min, then phenyl chloroformate (0.499
6

B. Dai et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115891
Fig 4. Memory and learning were tested using the Morris water maze. (A) Timeline for 12a treatment and behavioral assessment. (B) Average body weight of the
mice measured daily during the administration. Data were expressed as means ± standard deviations (n = 7). Compound 12a treatment blocks memory loss in 5-
month-old FAD mice. Memory and learning were tested using the Morris water maze. (C) The mean escape latency over four consecutive days of reference memory
testing. Data are expressed as means ± standard deviations (n = 7). *p < 0.05 and ***p < 0.001, compared with the DMSO group. (D) A probe test was performed 24
h after the last training trial to determine memory retention. Data were expressed as means ± standard deviations (n = 7). *p < 0.05, compared with the
DMSO group.
4.1.2.12. Phenyl (2-(trifluoromethyl)phenyl)carbamate (4k). Yellowish
solid; Yield: 72.6%. MS (ESI) m/z (%): 282.3 [M+H]+.
4.1.3.1. Phenyl benzylcarbamate (8a). Yellow solid; Yield: 71.2%. MS
(ESI) m/z (%): 228.4 [M+H]+.
4.1.2.13. Phenyl (4-nitrophenyl)carbamate (4l). Dark green solid; Yield:
65.6%. MS (ESI) m/z (%): 259.2 [M+H]+.
4.1.3.2. Phenyl (4-chlorobenzyl)carbamate (8b). Yellowish solid; Yield:
69.8%. MS (ESI) m/z (%): 262.8[M+H]+.
4.1.3. General procedure for preparation of different substitution phenyl
benzylcarbamate (8a-8j)
4.1.3.3. Phenyl (2-fluorobenzyl)carbamate (8c). Yellow solid; Yield:
63.4%. MS (ESI) m/z (%): 246.4 [M+H]+.
The preparation of the key intermediate 8a-8j is the same as way to
preparation 4a-4l, So the synthesis method would not be listed here.
4.1.3.4. Phenyl (3-fluorobenzyl)carbamate (8d). Yellowish brown solid;
Yield: 73.5%. MS (ESI) m/z (%): 246.4 [M+H]+.
7

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Bioorganic & Medicinal Chemistry 29 (2021) 115891
DMSO
12a
12a
DMSO
Probe day
Fig 5. The representative tracks of the mice in Morris water maze during the spatial probe trial period.
Day4
4.1.3.5. Phenyl (4-fluorobenzyl)carbamate (8e). Yellowish solid; Yield:
67.4%. MS (ESI) m/z (%): 246.4 [M+H]+.
solid; Yield: 66.3%. MS (ESI) m/z (%): 182.4[M+H]+.
4.1.4.10. N-(2-(trifluoromethoxy)phenyl)hydrazinecarboxamide
(5j).
4.1.3.6. Phenyl (2,3-dichlorobenzyl)carbamate (8f). Yellow solid; Yield:
66.1%. MS (ESI) m/z (%): 297.2 [M+H]+.
Pink solid; Yield: 69.6%. MS (ESI) m/z (%):236.3 [M+H]+.
4.1.4.11. N-(2-(trifluoromethyl)phenyl)hydrazinecarboxamide
(5k).
4.1.3.7. Phenyl (2,4-dichlorobenzyl)carbamate (8g). Yellowish brown
solid; Yield: 69.7%. MS (ESI) m/z (%): 297.2 [M+H]+.
Purple and pink solid; Yield: 71.3%. MS (ESI) m/z (%): 220.3 [M+H]+.
4.1.4.12. N-(4-nitrophenyl)hydrazinecarboxamide
(5l). Pink
solid;
4.1.3.8. Phenyl (3,4-dichlorobenzyl)carbamate (8h). Yellow solid; Yield:
71.8%. MS (ESI) m/z (%): 297.2 [M+H]+.
Yield: 70.3%. MS (ESI) m/z (%): 197.2 [M+H]+.
4.1.5. General procedure for preparation of hydrazine carboxamide
intermediates (9a-9j)
4.1.3.9. Phenyl (4-(trifluoromethyl)benzyl)carbamate (8i). Yellowish
solid; Yield: 77.6.0%. MS (ESI) m/z (%): 296.4 [M+H]+.
The preparation of the key intermediate 9a-9j is the same as way to
preparation 5a-5l, so the synthesis method would not be listed here.
4.1.3.10. Phenyl (4-methylbenzyl)carbamate (8j). Yellow-red solid;
Yield: 68.4%. MS (ESI) m/z (%): 242.3 [M+H]+.
4.1.5.1. N-benzylhydrazinecarboxamide (9a). Pink solid; Yield: 63.3%.
MS (ESI) m/z (%): 166.4 [M+H]+.
4.1.4. General procedure for preparation of hydrazinecarboxamide
intermediates (5a-5l)
4.1.5.2. N-(4-chlorobenzyl)hydrazinecarboxamide (9b). Pink solid;
Yield: 70.3%. MS (ESI) m/z (%): 200.8 [M+H]+.
Substitution phenyl phenylcarbamate (1.2 μmol) and 80% hydrazine
hydrate (1.5 μmol) were added into toluene (15 mL). The mixture was
heated to 80℃ for 3 h. After being cooled to room temperature, the
precipitate was filtered and dried under reduced pressure to afford in-
termediates (5a-5l).
4.1.5.3. N-(2-fluorobenzyl)hydrazinecarboxamide (9c). Light pink solid;
Yield: 73.3%. MS (ESI) m/z (%): 184.4 [M+H]+.
4.1.5.4. N-(3-fluorobenzyl)hydrazinecarboxamide (9d). Light pink solid;
Yield: 70.1%. MS (ESI) m/z (%): 184.3 [M+H]+.
4.1.4.1. N-phenylhydrazinecarboxamide (5a). Pink solid; Yield: 66.3%.
MS (ESI) m/z (%): 152.3 [M+H]+.
4.1.5.5. N-(4-fluorobenzyl)hydrazinecarboxamide
(9e). Pink
solid;
Yield: 73.3%. MS (ESI) m/z (%): 184.4 [M+H]+.
4.1.4.2. N-(o-tolyl)hydrazinecarboxamide (5b). Light pink solid; Yield:
70.7%. MS (ESI) m/z (%): 166.4 [M+H]+.
4.1.5.6. N-(2,3-dichlorobenzyl)hydrazinecarboxamide (9f). Pink solid;
Yield: 83.3%. MS (ESI) m/z (%): 235.2[M+H]+.
4.1.4.3. N-(m-tolyl)hydrazinecarboxamide (5c). Pink solid; Yield:
66.7%. MS (ESI) m/z (%): 166.4 [M+H]+.
4.1.5.7. N-(2,4-dichlorobenzyl)hydrazinecarboxamide (9g). Pink yellow
solid; Yield: 86.7%. MS (ESI) m/z (%): 235.2 [M+H]+.
4.1.4.4. N-(p-tolyl)hydrazinecarboxamide (5d). Yellow pink solid;
Yield: 62.7%. MS (ESI) m/z (%): 166.4 [M+H]+
.
4.1.5.8. N-(3,4-dichlorobenzyl)hydrazinecarboxamide (9h). Pink solid;
Yield: 80.1%. MS (ESI) m/z (%): 235.1[M+H]+.
4.1.4.5. N-(2-bromophenyl)hydrazinecarboxamide (5e). Pink solid;
Yield: 66.7%. MS (ESI) m/z (%): 231.1 [M+H]+
.
4.1.5.9. N-(4-(trifluoromethyl)benzyl)hydrazinecarboxamide (9i). Light
pink solid; Yield: 86.6%. MS (ESI) m/z (%): 234.3 [M+H]+.
4.1.4.6. N-(3-bromophenyl)hydrazinecarboxamide (5f). Pink solid;
Yield: 62.3%. MS (ESI) m/z (%): 231.2 [M+H]+.
4.1.5.10. N-(4-methylbenzyl)hydrazinecarboxamide (9j). Pink solid;
Yield: 60.3%. MS (ESI) m/z (%): 180.4 [M+H]+
.
4.1.4.7. N-(4-bromophenyl)hydrazinecarboxamide (5g). Pink solid;
Yield: 68.4%. MS (ESI) m/z (%): 231.1 [M+H]+.
4.1.6. General procedure for preparation of imidazolyl hydrazide
intermediates (11a-11d)
4.1.4.8. N-(2-chlorophenyl)hydrazinecarboxamide (5h). Pink solid;
Yield: 60.3%. MS (ESI) m/z (%): 186.8 [M+H]+.
The preparation of the key intermediate 11a-11d is the same as way
to preparation 5a-5l, so the synthesis method would not be listed here.
4.1.4.9. N-(2-methoxyphenyl)hydrazinecarboxamide (5i). Light pink
8

B. Dai et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115891
4.1.6.1. 1H-imidazole-5-carbohydrazide (11a). White solid; Yield:
46.3%. MS (ESI) m/z (%): 128.1 [M+H]+.
3.6 Hz, 1H, furan-H), 7.04 (t, J = 7.8 Hz, 1H, Ph-H).
4.1.7.6. N-(3-bromophenyl)-2-((5-(4-nitrophenyl) furan-2-yl) methylene)
hydrazine-1-carboxamide (6f). Yellow solid; Yield: 73.6%; Mp:
252.2–254.1 ◦C; MS (ESI) m/z (%): 427.35 [Mꢀ H]-; 1H NMR (400 MHz,
DMSO‑d6) δ (ppm): 11.05 (s, 1H,CONHN), 9.05 (s, 1H,PhNH), 8.31 (d, J
= 8.9 Hz, 2H, Ph-H), 8.08 (d, J = 8.8 Hz, 2H, Ph-H), 8.02 (s, 1H, Ph-H),
7.93 (s, 1H ,N = CH), 7.66 (d, J = 3.6 Hz, 1H, furan-H), 7.48(d, J = 3.6
Hz, 1H,furan-H), 7.20 (s, 1H,Ph-H),7.21 (s, 1H,Ph-H), 7.28 (s, 1H,Ph-H).
4.1.6.2. N,1-dimethyl-1H-imidazole-5-carbohydrazide
(11b). Grayish
white solid; Yield: 39.6%. MS (ESI) m/z (%): 155.4 [M+H]+.
4.1.6.3. 2-bromo-1H-imidazole-5-carbohydrazide (11c). White solid;
Yield: 41.2%. MS (ESI) m/z (%): 205.3 [M+H]+.
4.1.6.4. 2-bromo-4-methyl-1H-imidazole-5-carbohydrazide (11d). White
solid; Yield: 44.4%. MS (ESI) m/z (%): 219.3 [M+H]+.
4.1.7.7. N-(4-bromophenyl)-2-((5-(4-nitrophenyl) furan-2-yl) methylene)
hydrazine-1-carboxamide (6g). Light yellow solid; Yield: 78.4%; Mp:
251.7–253.4 ◦C; MS (ESI) m/z (%): 427.16 [M-H]-; HRMS (ESI) m/z (%):
429.3015[M+H]+; 1H NMR (400 MHz, DMSO‑d6) δ (ppm): 11.01 (s, 1H,
NNHCO), 9.02 (s, 1H,CONHPh), 8.31 (d, J = 9.0 Hz, 2H,Ph-H), 8.08 (d,
J = 8.9 Hz, 2H,Ph-H), 7.93 (s, 1H,HC = N), 7.67 (d, J = 8.9 Hz,2H, Ph-
H), 7.52–7.45 (m, 3H, Ph-H,furan-H), 7.18 (d, J = 3.7 Hz,1H, furan-H) .
13C NMR (151 MHz, DMSO‑d6) δ 152.60, 150.51, 146.74, 143.98,
136.61(2C), 136.44, 135.46, 130.12(2C), 127.55(2C), 124.97(2C),
124.92(2C), 116.40, 112.61.
4.1.7. General procedure for preparation of target compounds (6a-6l,
10a-10j, 12a-12d)
A mixture of 2(1.5 mmol) and different substituted 5a-5l, 9a-9j or
12a-12d (1.5 mmol) in EtOH (10 mL) was stirred at for 2 h. the pre-
cipitate was filtered and washed with EtOH (5 mL) and dried under
reduced pressure to afford compounds 6a-6l, 10a-10j, 12a-12d
respectively.
4.1.7.1. 2-((5-(4-nitrophenyl) furan-2-yl) methylene)-N-phenylhydrazine-
◦
4.1.7.8. N-(2-chlorophenyl)-2-((5-(4-nitrophenyl) furan-2-yl) methylene)
hydrazine-1-carboxamide (6h). Yellowish brown solid; Yield: 70.9%;
Mp: 210.1–212.5 ◦C; MS (ESI) m/z (%): 383.30 [M-H]ꢀ ; 1H NMR (400
MHz, DMSO‑d6) δ (ppm): 11.29 (s, 1H,CONHN), 8.96 (s, 1H,PhNHCO),
8.30 (d, J = 9.0 Hz, 2H,Ph-H), 8.00 (d, J = 9.0 Hz, 2H,Ph-H), 7.93 (s, 1H,
CH = N), 7.55 (dd, J = 8.0, 1.2 Hz, 1H,Ph-H), 7.46 (d, J = 3.6 Hz, 1H,
furan-H), 7.35 (s, 1H, Ph-H), 7.33 (s, 1H, Ph-H), 7.10 (s, 1H, Ph-H), 7.09
(d, J = 3.6 Hz, 1H, , furan-H).
1-carboxamide (6a). Yellow solid; Yield: 67.1%; Mp: 239.0–241.3 C;
MS (ESI) m/z (%): 349.34 [M-H]-; 1H NMR (600 MHz, DMSO‑d6) δ
(ppm): 10.92 (s, 1H, NNHCO), 8.83 (s, 1H, CONHPh), 8.33–8.28 (m, 2H,
Ph-H), 8.11–8.05 (m, 2H, Ph-H), 7.93 (s, 1H, N = CH), 7.66 (t, J = 7.0
Hz, 2H, Ph-H),7.47(d,J = 3.6 Hz, 1H, furan-H), 7.31 (dd, J = 8.3, 7.6 Hz,
2H, Ph-H), 7.18 (d, J = 3.6 Hz, 1H, furan-H), 7.03 (t, J = 7.3 Hz, 1H, Ph-
H).
4.1.7.2. 2-((5-(4-nitrophenyl)
furan-2-yl)
methylene)-N-(o-tolyl)
4.1.7.9. N-(2-methoxyphenyl)-2-((5-(4-nitrophenyl) furan-2-yl) methy-
lene) hydrazine-1-carboxamide (6i). Yellow solid; Yield: 72.8%; Mp:
231.5–234.1 ◦C; MS (ESI) m/z (%): 379.21 [Mꢀ H]-; 1H NMR (600 MHz,
DMSO‑d6) δ (ppm): 11.11 (s, 1H, CONHN), 9.67 (s, 1H, CH = N), 8.11 (d,
J = 8.4 Hz, 2H, Ph-H), 7.99 (d, J = 8.3 Hz, 2H, Ph-H), 7.89 (s, 1H,
CONHPh), 7.70 (d, J = 3.0 Hz, 1H, furan-H), 7.57 (d, J = 3.0 Hz, 1H,
furan-H), 7.44 (s, 1H, Ph-H), 7.09–6.99 (m, 3H, Ph-H), 3.97 (s, 3H,
PhOCH3).
hydrazine-1-carboxamide (6b). Brown yellow solid; Yield: 74.1%; Mp:
210.1–213.0 ◦C; MS (ESI) m/z (%): 363.20 [M-H]-; 1H NMR (400 MHz,
DMSO‑d6) δ (ppm): 10.89 (s, 1H, NNHCO), 8.73 (s, 1H, CONHPh), 8.29
(d, J = 8.9 Hz, 2H,Ph-H), 8.06 (d, J = 8.8 Hz, 2H, Ph-H), 7.91 (s, 1H, HC
= N), 7.53 (d, J = 8.3 Hz, 2H, Ph-H), 7.46 (d, J = 3.6 Hz, 1H, furan-H),
7.17 (d, J = 3.6 Hz, 1H, furan-H), 7.11 (d, J = 8.2 Hz, 2H,Ph-H).
4.1.7.3. 2-((5-(4-nitrophenyl)
furan-2-yl)
methylene)-N-(m-tolyl)
hydrazine-1-carboxamide (6c). Yellow solid; Yield: 74.1%; Mp:
210.4–212.2 ◦C; MS (ESI) m/z (%): 363.20 [M-H]-; 1H NMR (400 MHz,
DMSO‑d6) δ (ppm): 10.93 (s, 1H, NNHCO), 8.74 (s, 1H, PhNH), 8.30 (d,
J = 9.0 Hz, 2H, Ph-H), 8.07 (d, J = 8.9 Hz, 2H, Ph-H), 7.92 (s, 1H, N =
CH), 7.50 (s, 1H, Ph-H),7.47 (d, J = 3.7 Hz, 1H, furan-H), 7.44 (d, J =
8.3 Hz, 1H, Ph-H), 7.20 (d, J = 3.7 Hz, 1H, furan-H), 7.19 (s, 1H, Ph-H),
6.85(d, J = 7.4 Hz, 1H, Ph-H), 2.30 (s, 3H, PhCH3)
4.1.7.10. 2-((5-(4-nitrophenyl)
furan-2-yl)
methylene)-N-(2-(tri-
fluoromethoxy) phenyl) hydrazine-1-carboxamide (6j). Yellow solid;
Yield: 79.7%; Mp: 230.0–232.7 ◦C; MS (ESI) m/z (%): 433.27 [Mꢀ H]-;
1H NMR (400 MHz, DMSO‑d6) δ (ppm): 11.26 (s, 1H,CONHN), 8.70 (s,
1H,PhNHCO), 8.29(dd, J = 14.8, 9.0 Hz, 3H,Ph-H), 8.00 (d, J = 9.0 Hz,
2H,Ph-H), 7.93 (s, 1H,CH = N), 7.49–7.36 (m, 4H,furan-H, Ph-H), 7.06
(d, J = 3.7 Hz, 1H,furan-H).
4.1.7.4. 2-((5-(4-nitrophenyl)
furan-2-yl)
methylene)-N-(p-tolyl)
4.1.7.11. 2-((5-(4-nitrophenyl)
furan-2-yl)
methylene)-N-(2-(tri-
hydrazine-1-carboxamide (6d). Yellow solid; Yield: 77.1%; Mp:
230.6–232.7 ◦C; MS (ESI) m/z (%): 363.26 [M-H]-; HRMS (ESI) m/z
(%):365.1569 [M+H]+; 1H NMR (400 MHz, DMSO‑d6) δ (ppm): 10.90
(s, 1H, NNHCO), 8.74 (s, 1H, CONHPh), 8.28 (t, J = 11.2 Hz, 2H, Ph-H),
8.12–8.03 (m, 2H, Ph-H), 7.92 (s, 1H, HC = N), 7.53 (d, J = 8.4 Hz, 2H,
Ph-H), 7.46 (dd, J = 8.4, 5.6 Hz, 2H, Ph-H), 7.17(d, J = 3.6 Hz, 1H,
furan-H), 6.75 (d, J = 3.6 Hz, 1H,furan-H). 13C NMR (151 MHz,
DMSO‑d6) δ 169.42, 152.56, 150.55, 146.68, 143.79, 136.51, 135.46,
132.70, 128.84(2C), 124.94(2C), 124.87(2C), 118.96(2C), 116.27,
112.57, 24.49.
fluoromethyl) phenyl) hydrazine-1-carboxamide (6k). Brown yellow
solid; Yield: 79.1%; Mp: 240.2–244.0 ◦C; MS (ESI) m/z (%): 490.46
[M+Na]+; 1H NMR (600 MHz, DMSO‑d6) δ (ppm): 11.36 (s, 1H,
CONHN), 8.94 (s, 1H, CH = N), 8.29(d,J = 8.9 Hz, 2H, Ph-H), 8.26 (d, J
= 8.3 Hz, 1H,CONHPh), 8.03 (d, J = 8.9 Hz, 2H, Ph-H),7.93 (s, 1H, Ph-
H), 7.76–7.68 (m, 2H, Ph-H), 7.48 (d, J = 3.6 Hz, 1H, furan-H), 7.31 (t, J
= 7.6 Hz, 1H, Ph-H), 7.08 (d, J = 3.6 Hz, 1H, furan-H)
4.1.7.12. N-(4-nitrophenyl)-2-((5-(4-nitrophenyl) furan-2-yl) methylene)
hydrazine-1-carboxamide (6l). Yellow solid; Yield: 78.4%; Mp:
241.3–234.1 ◦C; MS (ESI) m/z (%): 419.09 [M+Na]+; 1H NMR (400
MHz, DMSO‑d6) δ (ppm): 11.23 (s, 1H,CONHN), 9.55 (s, 1H,PhNHCO),
8.31 (d,J = 9.0 Hz, 2H,PhH), 8.22 (d, J = 9.3 Hz, 2H,PhH), 8.09 (d, J =
8.9 Hz, 2H,PhH), 7.98 (d, J = 10.2 Hz, 3H, PhH, N = CH), 7.49 (d, J =
3.6 Hz, 1H,furan-H), 7.21 (d, J = 3.6 Hz, 1H,furan-H).
4.1.7.5. N-(2-bromophenyl)-2-((5-(4-nitrophenyl) furan-2-yl) methylene)
hydrazine-1-carboxamide (6e). Light yellow solid; Yield: 72.1%; Mp:
250.2–252.8 ◦C; MS (ESI) m/z (%): 427.26 [Mꢀ H]-; 1H NMR (400 MHz,
DMSO‑d6) δ (ppm): 11.30 (s, 1H, NNHCO), 8.95 (s, 1H, CONHPh), 8.36
(d, J = 8.9 Hz, 1H,Ph-H), 8.31 (d, J = 8.9 Hz, 2H, Ph-H), 8.24 (d, J = 8.2
Hz, 1H, Ph-H), 8.01 (d, J = 8.9 Hz, 2H, Ph-H), 7.94 (s, 1H,HC = N), 7.47
(d, J = 3.6 Hz, 1H, furan-H), 7.39 (t, J = 7.8 Hz, 1H, Ph-H), 7.11 (d, J =
9

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Bioorganic & Medicinal Chemistry 29 (2021) 115891
4.1.7.13. N-benzyl-2-((5-(4-nitrophenyl)
furan-2-yl)
methylene)
4.1.7.20. N-(3,4-dichlorobenzyl)-2-((5-(4-nitrophenyl) furan-2-yl) meth-
ylene) hydrazine-1-carboxamide (10h). Yellow solid; Yield: 82.3%; Mp:
221.4–224.0 ◦C; MS (ESI) m/z (%): 431.19 [Mꢀ H]-; 1H NMR (600 MHz,
DMSO‑d6) δ (ppm): 10.72 (s, 1H, CONHN), 8.28 (d, J = 8.7 Hz, 2H, Ph-
H),8.03 (d, J = 8.7 Hz, 2H, Ph-H), 7.85 (s, 1H, CH = N), 7.63–7.54 (m,
3H, Ph-H), 7.44 (d, J = 3.4 Hz, 1H, furan-H), 7.31 (d, J = 8.0 Hz, 1H,
CH2NH), 7.05 (d, J = 3.4 Hz, 1H, furan-H),4.36 (d, J = 6.1 Hz, 2H,
PhCH2).
hydrazine-1-carboxamide (10a). Lightyellowsolid; Yield: 69.2%;
-
◦
Mp:239.2–241.1 C;MS (ESI) m/z (%):366.33[M-H] ; HRMS (ESI) m/z
(%): 365.1569[M+H]+; 1H NMR (600 MHz, DMSO‑d6) δ (ppm): 10.66
(s, 1H, CONHN), 8.30–8.26 (m, 2H, Ph-H), 8.05–8.00 (m, 2H, Ph-H),
7.85 (s, 1H, CH = N), 7.47 (t, J = 6.4 Hz, 1H, CH2NH), 7.43 (d, J =
3.6 Hz, 1H, furan-H), 7.37–7.24 (m, 5H, Ph-H), 7.05 (d, J = 3.6 Hz, 1H,
furan-H), 4.39 (d, J = 6.3 Hz, 2H, PhCH2). 13C NMR (151 MHz,
DMSO‑d6) δ 153.86, 153.29, 150.76, 146.86, 141.27, 139.54, 135.47,
128.69(2C), 128.57, 127.56(2C), 127.36, 127.26, 125.20, 124.81,
117.82, 112.75, 43.84.
4.1.7.21. 2-((5-(4-nitrophenyl)
furan-2-yl)
methylene)-N-(4-(tri-
fluoromethyl) benzyl) hydrazine-1-carboxamide (10i). Light yellow solid;
Yield: 87.4%; Mp: 208.9–210.7 ◦C; MS (ESI) m/z(%): 431.25 [M-H]-; 1H
NMR (600 MHz, DMSO‑d6) δ (ppm): 10.72 (s, 1H, CONHN), 8.29 (d, J =
2.0 Hz, 2H, Ph-H),8.04 (d, J = 1.8 Hz, 2H, Ph-H), 7.85 (s, 1H, CH = N),
7.60 (d, J = 4.2 Hz, 2H, Ph-H), 7.59 (s,1H, CH2NH), 7.54 (d, J = 4.0 Hz,
2H, Ph-H), 7.44 (d, J = 2.1 Hz, 1H, furan-H), 7.05 (d, J = 2.1 Hz, 1H,
furan-H), 4.36 (d, J = 6.3 Hz, 2H, PhCH2).
4.1.7.14. N-(4-chlorobenzyl)-2-((5-(4-nitrophenyl) furan-2-yl) methy-
lene) hydrazine-1-carboxamide (10b). Yellowish brown solid; Yield:
80.1%; Mp: 211.6–215.4 ◦C; MS (ESI) m/z (%): 397.16 [Mꢀ H]-; 1H NMR
(600 MHz, DMSO‑d6) δ (ppm): 10.69 (s, 1H, CONHN), 8.29–8.27 (m,
2H, Ph-H), 8.04–8.02 (m, 2H, Ph-H), 7.84 (s, 1H, CH = N), 7.44 (d, J =
3.6 Hz, 1H, furan-H), 7.40 (d, J = 1.8 Hz, 1H, CH2NH), 7.36 (dd, J =
29.8, 5.3 Hz, 4H, Ph-H), 7.05 (d, J = 3.6 Hz, 1H,furan-H), 4.36 (d, J =
6.3 Hz, 2H, PhCH2).
4.1.7.22. N-(4-methylbenzyl)-2-((5-(4-nitrophenyl) furan-2-yl) methy-
lene) hydrazine-1-carboxamide (10j). Brown yellow solid; Yield: 68.4%;
Mp: 231.5–233.6 ◦C; MS (ESI) m/z(%): 377.32 [Mꢀ H]-; 1H NMR (600
MHz, DMSO‑d6) δ (ppm): 10.65 (s, 1H, CONHN), 8.28 (d, J = 8.8 Hz, 2H,
Ph-H),8.03 (d, J = 8.8 Hz, 2H, Ph-H), 7.83 (s, 1H, CH = N), 7.44 (d, J =
3.6 Hz, 1H, furan-H), 7.21–7.14 (m, 4H, Ph-H), 7.13 (s, 1H, CH2NH),
7.04 (d, J = 3.6 Hz, 1H, furan-H), 4.33 (d, J = 6.2 Hz, 2H, PhCH2), 2.27
(s, 3H, PhCH3).
4.1.7.15. N-(2-fluorobenzyl)-2-((5-(4-nitrophenyl) furan-2-yl) methylene)
hydrazine-1-carboxamide (10c). Yellow solid; Yield: 77.5%; Mp:
221.3–222.8 ◦C; MS (ESI) m/z (%): 381.29 [M-H]ꢀ ; 1H NMR (400 MHz,
DMSO‑d6) δ (ppm): 10.71 (s, 1H,CONHN), 9.68 (s, 1H,CH = N), 8.27 (d,
J = 9.1 Hz, 2H,Ph-H), 8.02 (d, J = 9.0 Hz, 2H,Ph-H), 7.85 (s, 1H,
CONHCH2), 7.46 (d, J = 3.9 Hz, 1H,Ph-H), 7.43 (d, J = 3.7 Hz, 1H,
furan-H), 7.36 (t, J = 7.6 Hz, 1H,Ph-H), 7.29 (ddd, J = 9.4, 7.5, 1.6
Hz, 1H,Ph-H), 7.15–7.09 (m, 1H,Ph-H), 7.05 (d, J = 3.7 Hz, 1H,furan-
H), 4.44 (d, J = 6.2 Hz, 2H,PhCH2NH).
4.1.7.23. N’-((5-(4-nitrophenyl) furan-2-yl) methylene)-1H-imidazole-5-
carbohydrazide (12a). Light yellow solid; Yield: 78.4%; Mp:
261.9–262.8 ◦C; MS (ESI) m/z (%): 326.4 [M+H]+; HRMS (ESI) m/z (%):
1
348.0710[M+Na]+; H NMR (400 MHz, DMSO‑d6) δ (ppm): 12.69 (s,
4.1.7.16. N-(3-fluorobenzyl)-2-((5-(4-nitrophenyl) furan-2-yl) methylene)
hydrazine-1-carboxamide (10d). Yellow solid; Yield: 78.7%; Mp:
222.3–224.4 ◦C; MS (ESI) m/z (%): 381.29 [Mꢀ H]-; 1H NMR (400 MHz,
DMSO‑d6) δ (ppm): 10.69 (s, 1H, CONHN), 8.27 (d, J = 9.0 Hz, 2H, Ph-
H), 8.03 (d, J = 9.0 Hz, 2H, Ph-H), 7.85 (s, 1H, CH = N), 7.57 (dd, J =
12.1, 5.7 Hz, 1H, Ph-H),7.43 (d, J = 3.6 Hz, 1H, furan-H), 7.36 (dd, J =
7.8, 6.3 Hz, 1H, CH2NH), 7.19–7.06 (m, 3H,Ph-H), 7.05 (d, J = 3.6 Hz,
1H, furan-H), 4.40 (d, J = 6.3 Hz, 2H, PhCH2).
1H, Imidazole-NH), 11.71 (s, 1H,NNHCO), 8.51 (s, 1H, CH = N), 8.32 (d,
J = 8.9 Hz, 2H, Ph-H), 8.03 (d, J = 8.9 Hz, 2H, Ph-H), 7.86 (s, 1H,
NCHN–), 7.85 (s, 1H,C = CH-N), 7.47 (d, J = 3.6 Hz, 1H, furan-H), 7.07
(d, J = 3.6 Hz, 1H, furan-H). 13C NMR (151 MHz, DMSO‑d6) δ 152.50,
151.85, 146.61(2C), 136.40, 135.64(2C), 125.69, 124.92(4C), 115.72,
112.89(2C).
4.1.7.24. N,1-dimethyl-N’-((5-(4-nitrophenyl)furan-2-yl)methylene)-1H-
imidazole-5carbohydrazide (12b). yellow solid; Yield: 79.2%; Mp:
258.9–262.7 ◦C;MS (ESI) m/z(%): 354.2 [M+H]+; 1H NMR (400 MHz,
DMSO‑d6) δ (ppm): 8.35 (d, J = 8.6 Hz, 2H, Ph-H), 8.27 (s, 1H, CH = N),
8.06 (d, J = 8.6 Hz, 2H, Ph-H), 7.98 (s, 1H, Imidazole-N = CH), 7.75 (s,
1H, Imidazole-C = CH), 7.50 (d, J = 3.5 Hz, 1H, furan-H), 7.07 (d, J =
3.8 Hz, 1H, furan-H), 3.51 (s, 3H,–CH3), 3.44 (s, 3H, Imidazole-CH3).
4.1.7.17. N-(4-fluorobenzyl)-2-((5-(4-nitrophenyl) furan-2-yl) methylene)
hydrazine-1-carboxamide (10e). Yellowish brown solid; Yield: 77.6%;
Mp: 224.8–226.1 ◦C; MS (ESI) m/z (%): 381.29 [M-H]ꢀ ; 1H NMR (400
MHz, DMSO‑d6) δ (ppm): 10.69 (s, 1H, CONHN), 8.29–8.27 (m, 2H, Ph-
H),8.04–8.02 (m, 2H, Ph-H), 7.84 (s, 1H, CH = N), 7.38–7.32 (m, 2H,
Ph-H),7.12–7.07 (m, 2H,Ph-H), 6.96 (d, J = 3.6 Hz, 1H, furan-H), 6.64
(d, J = 3.6 Hz, 1H, furan-H), 6.12 (s, 1H, CH2NH), 4.36(d,J = 6.3 Hz,2H,
PhCH2).
4.1.7.25. 2-bromo-N’-((5-(4-nitrophenyl) furan-2-yl) methylene)-1H-
imidazole-5-carbohydrazide (12c). Light yellow solid; Yield: 81.3%; Mp:
256.8–259.7 ◦C;MS (ESI) m/z(%): 348.46 [M+Na]+;1H NMR (600 MHz,
DMSO‑d6) δ (ppm): 13.79 (s, 1H, Imidazole-NH), 12.31 (s, 1H,NH), 8.53
(s, 1H, CH = N), 8.32 (d, J = 8.9 Hz, 2H, Ph-H), 8.05 (d, J = 8.9 Hz, 2H,
Ph-H), 7.59 (s, 1H,N-CH = C), 7.49 (d, J = 3.6 Hz, 1H, furan-H), 7.14 (d,
J = 3.7 Hz, 1H, furan-H).
4.1.7.18. N-(2,3-dichlorobenzyl)-2-((5-(4-nitrophenyl) furan-2-yl) meth-
ylene) hydrazine-1-carboxamide (10f). Yellow solid; Yield: 81.1%; Mp:
226.1–229.5 ◦C; MS (ESI) m/z (%): 433.40 [M+H]+; 1H NMR (600 MHz,
DMSO‑d6) δ (ppm): 10.80(s, 1H, CONHN), 8.28 (d, J = 8.8 Hz, 2H, Ph-
H), 8.04 (d, J = 8.6 Hz, 2H, Ph-H), 7.87 (s, 1H, CH = N), 7.61 (s, 1H,
CH2NH), 7.45 (d,J = 3.5 Hz, 1H, furan-H),7.42–7.28 (m, 3H, Ph-H),
7.06 (d, J = 3.5 Hz, 1H, furan-H), 4.48 (d, J = 5.9 Hz, 2H, PhCH2).
4.1.7.26. 2-bromo-4-methyl-N’-((5-(4-nitrophenyl) furan-2-yl) methy-
lene)-1H-imidazole-5-carbohydrazide (12d). Yellow solid; Yield: 77.6%;
Mp:254.9–257.7℃; MS(ESI)m/z(%): 340.33[M+H]+; HRMS(ESI)m/z
(%):362.0903[M+Na]+; 1H NMR (600 MHz, DMSO‑d6) δ (ppm): 13.39
(s, 1H, Imidazole-NH), 11.81 (s, 1H,–NH), 8.48 (s, 1H, CH = N), 8.39 (d,
J = 8.9 Hz, 2H, Ph-H), 8.34 (d, J = 8.9 Hz, 2H, Ph-H), 7.61 (d, J = 3.8 Hz,
1H, furan-H), 7.21 (d, J = 3.8 Hz, 1H, furan-H), 2.52 (s, 3H,CH3).
4.1.7.19. N-(2,4-dichlorobenzyl)-2-((5-(4-nitrophenyl) furan-2-yl) meth-
ylene) hydrazine-1-carboxamide (10g). Yellow solid; Yield: 83.1%; Mp:
223.4–225.8 ◦C; MS (ESI) m/z (%): 433.33 [M+H]+; 1H NMR (600 MHz,
DMSO‑d6) δ (ppm): 10.79 (s, 1H, CONHN), 8.28 (d, J = 8.9 Hz, 2H, Ph-
H),8.03 (d, J = 8.9 Hz, 2H, Ph-H), 7.95 (s, 1H, CH = N), 7.87 (s, 1H, Ph-
H), 7.62 (d, J = 2.0 Hz,2H, Ph-H), 7.44 (s, 1H, CH2NH), 7.35 (d, J = 3.6
Hz, 1H, furan-H), 7.06 (d, J = 3.6 Hz, 1H, furan-H), 4.42 (d, J = 6.1 Hz,
2H, PhCH2).
10

B. Dai et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115891
4.2. Pharmacology
spectrophotometric method. Compounds 6a, 6b, 10d, 10 g, 12a, and 12b
were dissolved in DMSO solution. In a final volume of 250 μL of DMSO,
4.2.1. In vitro SOICR inhibition activities assay
60
μ
M of Ellman’s reagent or 5,5′-dithiobis(2-nitrobenzoic acid), 0.05 M
M of inhibitors, and 0.25 U/mL of AChE
The method was as follows: after induced by 1 mg/ml tetracycline,
the cells grew on glass cover slides for 20–24 h, then loaded with fluo-
rescent calcium indicator dye (fura-2AM, 5 mM) in KRH buffer (125 mm
NaH2PO4 1.2 mm glucose, 1.2 mM MgCl2 and 25 mm HEPES
Ph7.35–7.45) and kept at room temperature for 20 min. The cover slides
were installed in the perfusion chamber, and different concentrations of
CaCl2, were infused into the KRH solution to gradually increase the
calcium concentration from 0.1, 0.5 to 1 mM. Then the cells were
continuously perfused with KRH solution containing 1 mM CaCl2, and
of pH 8 phosphate buffer, 100
μ
from E. electricus were incubated for 30 min; then, 1.5 mM of ace-
tylthiocholine iodide was added to start the reaction. The assay was
carried out in three experiments in triplicate and absorbance was
measured at 412 nm for 10 min. The percentage of inhibition was
calculated by comparing the mice’s enzyme reaction rates with those of
the control group. Donepezil was used as a positive control.
4.2.5. Aqueous solubility assay
the concentration of the tested compound (0.1
μM, 3
μM, 10
μM) was
The kinetic water solubility of each tested compound was measured
using a turbidimetric assay via Cyprotex (Watertown, MA). Each test
compound was prepared as a 100-X concentrated stock solution in
DMSO, from which serial dilutions were performed to yield eight solu-
tions with final test compound concentrations of 1.6, 3.21, 6.25, 12.5,
25, 50, 100, and 200 mM. Each test compound solution was introduced
into a 96-well plate, diluted 100-fold with PBS buffer (pH 7.4), and
mixed. The solutions were incubated for two h, and absorbance was
measured at 540 nm. An absorbance value > 3X standard deviation of
the average blank absorption value was considered to exhibit turbidity.
The highest test compound concentration with no sign of turbidity was
indicative of a compound’s kinetic water solubility.
gradually increased for 8 min each time. Caffeine (10 mM) was applied
at the end of each experiment to verify that functional RyR1 channels
did exist in the cells.
At the end of the experiment, the delayed image (0.25 frame/s) was
captured by the microscope, and the cells with calcium oscillation signal
and calcium oscillation peak after adding caffeine were selected for
statistics. The number of cells without calcium oscillation signal in
different concentration time range was recorded and calculated ac-
cording to the following formula: Inhibition rate = the number of cells
without calcium oscillation signal/the total number of cells * 100%.
4.2.2. Morris water maze test
The Morris water maze test was used to evaluate the learning and
memory abilities of the animal models in terms of spatial position and
direction (spatial orientation). For the experiment proper, 5-month-old
SPF-grade FAD mice, male and female, were randomly selected as our
study subjects, and the experiment began 30 days after administration.
The animal subjects in the experiment were divided into two groups (six
mice per group): the experimental group was injected intraperitoneally
with a 3.2 mg/kg 12a DMSO solution (1.12 mg/ml), and the control
group was similarly injected with the same volume of DMSO.
4.2.6. Molecular docking
The molecular docking procedure was performed by using C-
DOCKER protocol within Accelrys Discovery Studio Visualizer 4.0. The
RyR1 models was built using PDB code: 6M7H as template. The protein
coordinates were downloaded from the Protein Data Bank (http://www.
rcsb.org/pdb/). For enzyme preparation, the hydrogen atoms were
added. The whole RyR1 enzyme was defined as a receptor and the site
sphere was selected on the basis of the ligand binding location of initial
ligand. The initial ligand was removed and compound 12a and Dan-
trolene were placed. Accelrys Discovery Studio Visualizer 4.0 was used
for graphic display.
Choose a quiet, dim light, constant temperature environment for
testing, the laboratory walls can be appropriately affixed with different
graphics to help animals determine the location. Before the experiment,
the bucket was filled with clear water to a predetermined height (about
40 cm), then an appropriate amount of milk powder was added to make
the water an opaque milky liquid, and the heater heated the water to
25℃. The platform is located in the center of southwest quadrant, about
1.5 cm below the water surface, and the position of the platform remains
unchanged throughout the experiment. During the training period, the
animals were put into the water facing the pool wall from four entry
points in turn, and the time required for the animals to find and climb
onto the platform was recorded, that is, the escape incubation period,
and stayed for 5 s for training in the next direction (by observing the
surrounding environment to locate themselves). If the platform is not
found within 60 s, the experimenter will lead it to the platform and stay
on the platform for 20 s and then train in the next direction. The first 4
days is the animal training period. The positioning navigation experi-
ment is used to evaluate the learning and memory ability of animals. On
the 5th day, the platform was removed, and the animals were placed into
the water facing the pool wall from the north point. The swimming time
in the target quadrant (the southwest quadrant) was recorded.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the Program for Innovative Research
Teams by the Ministry of Education of the People’s Republic of China
and the Program for Liaoning Innovative Research Team in University
(IRT1073), Liaoning XingLiao Talents Program (XLYC1807093), the
Student’s Platform for Innovation and Entrepreneurship Training Pro-
gram of Shenyang Pharmaceutical University, and S.R. Wayne Chen’s
lab in University of Calgary.
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