Identification and synthesis of low-molecular weight cholecystokinin B receptor (CCKBR) agonists as mediators of long-term synaptic potentiation

Article abstract of DOI:10.1007/s00044-019-02292-x

Recently, He et al. reported that CCKB receptors located in the neocortex of the brain when bound to their bound natural ligand, CCK peptides, enhance memory, bringing up the possibility that agonists targeting the CCKB receptor may act as therapeutic agents in diseases in which memory loss is marked as observed in dementia and Alzheimer’s. In this report, we describe the synthesis of novel low-molecular weight benzoamine CCKB receptor agonists. The compounds made in this series were determined to be mostly partial agonists, although some antagonists were identified, as well, capable of triggering calcium release in a cell line that overexpresses the CCKB receptor. Compound 35 demonstrated an EC₅₀ of 0.15 μM in the cell-based assay, but more importantly, several of the compounds, including 35, demonstrated a physiological effect, inducing long-term potentiation in rat brains comparable to the CCK-8 peptide albeit at much higher concentrations. Based on these findings, benzoamines may be the basis for a new series of CCKB receptor agonists in drug-discovery efforts that seek to develop therapeutics to prevent memory loss.

Full text of DOI:10.1007/s00044-019-02292-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02292-x
ORIGINAL RESEARCH
Identification and synthesis of low-molecular weight cholecystokinin
B receptor (CCKBR) agonists as mediators of long-term synaptic
potentiation
Yanmei Zhang1 Yican Wang1 Yiping Guo1 Jinxi Liao1 Zhengchao Tu1 Yongzhi Lu1 Ke Ding1
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Micky D. Tortorella1 Jufang He2
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Received: 18 September 2018 / Accepted: 11 January 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Recently, He et al. reported that CCKB receptors located in the neocortex of the brain when bound to their bound natural
ligand, CCK peptides, enhance memory, bringing up the possibility that agonists targeting the CCKB receptor may act as
therapeutic agents in diseases in which memory loss is marked as observed in dementia and Alzheimer’s. In this report, we
describe the synthesis of novel low-molecular weight benzoamine CCKB receptor agonists. The compounds made in this
series were determined to be mostly partial agonists, although some antagonists were identified, as well, capable of
triggering calcium release in a cell line that overexpresses the CCKB receptor. Compound 35 demonstrated an EC50 of 0.15
µM in the cell-based assay, but more importantly, several of the compounds, including 35, demonstrated a physiological
effect, inducing long-term potentiation in rat brains comparable to the CCK-8 peptide albeit at much higher concentrations.
Based on these findings, benzoamines may be the basis for a new series of CCKB receptor agonists in drug-discovery efforts
that seek to develop therapeutics to prevent memory loss.
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●
●
Keywords Cholecystokinin B receptor Agonist Synaptic potentiation Memory
Introduction
project to neocortical areas and the hippocampus (Canto
et al. 2008; Swanson and Kohler 1986; Li et al. 2014). CCK
is the most abundant of all neuropeptides (Rehfeld 1978),
and mice lacking the CCK gene exhibit poor performance in
a passive avoidance task and impaired spatial memory (Lo
et al. 2008). We have previously found that the local infu-
sion of CCK into the auditory cortex of anesthetized mice
enabled the auditory neurons to start responding to lights
after their pairing with an auditory stimulus (Li et al. 2014).
Activation of the entorhinal cortex potentiates neuronal
responses in the auditory cortex, and this effect is suppressed
by the infusion of a CCKB antagonist (Li et al. 2014).
Memory is stored in neural networks via changes in
synaptic strength (Kandel 2001; Martin et al. 2000). Long-
term potentiation (LTP) is considered to be the basis of
memory traces in different brains (Bliss and Lomo 1973; Bliss
and Collingridge 1993; Buonomano and Merzenich 1998;
Mulkey and Malenka 1992; Nabavi et al. 2014). In a separate
physiological experiment, we found that the administration of
a CCKB antagonist blocked high-frequency (HF) stimulation-
induced LTP in the auditory cortex, whereas the local infusion
of CCK alone induced LTP.
The entorhinal cortex in the medial temporal lobe that forms
strong reciprocal connections with the neocortex serving is
the gateway from the hippocampus to the neocortex (Brown
and Aggleton 2001; Canto et al. 2008; Swanson and Kohler
1986). Many dementia patients exhibit early neuro-atrophy
(neurodegeneration) in the entorhinal cortex (Apostolova
and Thompson 2008). Neurons in the entorhinal cortex show
heavy cholecystokinin (CCK) labeling (Greenwood et al.
1981; Innis et al. 1979; Kohler and Chan-Palay 1982) and
* Yanmei Zhang
zhang_yanmei@gibh.ac.cn
* Jufang He
jufanghe@cityu.edu.hk
1
Drug Discovery Pipeline, Guangzhou Institutes of Biomedicine
and Health, Science City, Guangzhou 510530, China
2
Division of Life Science, Hong Kong University of Science and
Technology, Clearwater Bay, N.T., Hong Kong SAR, China

Medicinal Chemistry Research
CCK-/- mice exhibited no neocortical LTP and deficits in
memory formation. HF stimulation of CCK-containing
entorhinal–neocortical projection neurons before the pairing
of an auditory stimulus and electrical stimulation of the
auditory cortex potentiated neuronal responses to the auditory
stimulus. Furthermore, when electrical stimulation was pre-
conditioned to a foot shock, mice showed a freezing response
to the auditory stimulus after the pairing of auditory and
electrical stimuli, indicating that mice had formed an asso-
ciation between auditory and electrical stimuli. These findings
demonstrate that CCK released from the entorhinal cortex
enables neocortical LTP and contributes to memory formation.
All the CCKB antagonists and agonists reported to date
tend to be large-molecular weight compounds (Kalindjian
and Iain 2007; Tikhonova et al. 2007; Wang et al. 2011;
Blommaert et al. 1997). Therefore, the goal of this study
was to develop low-molecular weight CCKB receptor
agonists capable of mediating long-term synaptic potentia-
tion in vivo. To this end, we adopted the following strategy:
first, we screened an existing compound library (>30,000)
in Guangzhou Institute of Biomedicine and Health, fol-
lowed by studying the analytical and computational chem-
istry, after which we confirmed the lead molecules in a cell
line overexpressing the CCKB receptor, and finally, tested
the most potent agonists in a rat model for measuring long-
term synaptic potentiation.
400 (at 500 and 125 MHz or 400 and 100 MHz, respec-
tively) and are reported to CDCl3 (δ 1H: 7.26 and 13C:
77.16) and 19F NMR (at 471 MHz). Coupling constants (J)
are given in Hz.
CCKBR cell-imaging assay
CHO/CCKb receptor stable cell line was developed by
lipofectin transfection. Briefly, a 1.36 kb insert containing
the full-length CDS sequence (NM_013165.2) of the rat
CCKB receptor was cloned into HindIII/EcoRI sites of the
mammalian expression vector pcDNA3.1(+) with neomy-
cin as the selection marker. G418 (1 mg/ml) was added to
the culture medium from the first passage after transfection
and resistant colonies were obtained by limiting dilution
method and were propagated in the presence of G418 (0.5
mg/ml). CCKBR, coupled to Gq protein, can induce intra-
cellular calcium mobilization after the binding of CCK-8
ligand. All the cell lines were validated using this
fluorescence-based functional assay with CCK-8s (Tocric,
UK). Before choosing to develop the high-throughput
screening (HTS) cell-based assay for screening CCKBR
agonist compounds, the cell line from Clone2 was proven to
be effective and reliable with two other commercially
available CCKb receptor agonists (BBL454 and 8b; Wuxi
Apptech, China), and we further subcultured 10 passages.
For fluorescence-based imaging assay (calcium flux
assay), the cells were seeded in a 96-well plate at a density
of 40,000 cells per well one day prior to the experiment.
Dye-loading solution (Fluo-8; AAT Bioquest, US) was
loaded for 30 min in a 37 °C incubator, followed by 15 min
in room temperature. After washing with Hanks’ Buffer
with 20mM Hepes (HHBS), the compound candidate was
added to the cells, and the fluorescence signal change was
captured with a fluorescent microscope.
Materials and methods
Materials and chemical analysis
All materials were obtained from commercial sources.
Solvents were of chromatography grade and were used
without further purification. Thin-layer chromatography
analysis was performed using Merck silica gel 60 F-254
thin-layer plates. Liquid Chromatography Mass Spectro-
meters (LC-MS) analyses were performed on Agilent 1200
HPLC/MCD electrospray mass spectrometer in positive/
negative ion mode. The scan range was 100–1000 d. High-
resolution mass spectra were obtained in ABSciex
TOF5600 + MS using electrospray ionization. Preparative
reverse-phase high-performance liquid chromatography
(HPLC) was performed on a SHIMADZU LC-20AP
equipped with a C18 column using a methanol/water gra-
dient. The purity of tested compounds was ≥ 95% as
determined by HPLC analysis conducted on the Agilent
1260 system using a reverse-phase C18 column with a
diode array detector unless stated otherwise. Chiral GC
column: CHIRALPAK OD-H, AS-H, OJ-H, 4.6 mm × 250
mm, 5 μm. Specific rotation values were recorded on
AUTOPOL® IV-T (λ = 589 nm, 50 mm cell, 25 °C). 1H and
13C NMR spectra were recorded on Bruker 500 or Bruker
96/384-well plate screening assay
Based on fluorescence-based imaging assay, the 96/384-
well plate screening assay was performed. Briefly, fluores-
cence dye was added to the cells using a bulk dispensing
system (freedom evo 150; Tecan). After 30min of incuba-
tion, the baseline was recorded prior to candidate compound
addition to the plate using the pipettor onboard the micro-
plate reader (FlexStation 3 Multi-Mode Microplate Reader;
Molecular Devices). During the first 90 s of activation, data
were acquired every 10 s. EC50 value was calculated based
on the concentration–response curve.
Homology model of CCKBR with lead agonists
Homology model of CCKBR was generated using Dis-
covery Studio. Molecular docking was carried out by the

Medicinal Chemistry Research
program Autodock Vina using the homology model as the
receptor. Three-dimensional models of compounds 31, 34,
and 35 were generated using the software ChemBio3D
Ultra from the 2D structures drawn using ChemBioDraw.
Input files of both proteins and ligands were prepared by
AutoDock Tools. The parameter values of exhaustiveness
and num_modes were set at 100 and 100, respectively.
Scheme 1 Synthesis of derivatives of 2-hydroxy-N-(4-(tri-
fluoromethyl)phenyl)benzamide
Table 1 List of derivatives made in series 1
R1
Agonist
>10 µM
Antagonist
Long-term synaptic potentiation in rat brains
Compound (EC50, µM)
1
5-Cl
Anesthesia was induced using urethane sodium (2 g/kg,
intraperitoneally) and maintained throughout the surgery and
neuronal recordings with periodic supplements. Atropine sul-
fate (0.05 mg/kg, subcutaneously) was administered 15 min
before the induction of anesthesia to inhibit tracheal secretions.
A local anesthetic (xylocaine, 2%) was applied liberally to the
incision site. Animals were prepared for surgery as previously
described. Briefly, animals were mounted on a stereotaxic
device and a midline incision was made in the scalp. A cra-
niotomy was performed at the temporal lobe (–2 to –4 mm
posterior and –1.5 to –3 mm ventral to bregma for mice) to
access the auditory cortex and the dura matter was removed. A
recording electrode array (0.5–1.0 MΩ; FHC Inc.) was stepped
into layer 2/3 of the auditory cortex of rats, and a stimulation
electrode with a low impedance (<100 kΩ; FHC Inc.) was
positioned into layer 4 of the auditory cortex. Electrode pla-
cement was confirmed by the neuronal responses to auditory
stimuli. For drug application, a glass pipette was attached to
the recording electrodes. We first recorded the baseline field
excitatory postsynaptic potential (fEPSPs) in response to
testing electrical stimulation for 15 min, and then 0.5 µl of
drug was injected at a speed of 0.1 µl/min, followed by another
60min of fEPSPs recording. Before baseline recording, an
input–output curve of fEPSPs was measured. The maximum
current was defined as that which elicited a saturated fEPSP.
We adopted 50% of the maximum current as the testing
current to examine the changes in fEPSP slope. Mean fEPSP
slopes before and after the pairings were calculated by linear
regression and analyzed using two-way ANOVA.
2
2-Cl, 5-Cl
3-Br, 5-Br
3-Cl, 5-Cl, 6-Cl
4-F
5.02 µM
9.54 µM
>10 µM
>10 µM
3
4
5
6
5-F
7
4-CF3
>10 µM
8
5-NH2
9
3-NO2, 5-NO2
5-Br
10
11
12
13
14
5-I
5-NO2
8.95 µM
5-COMe
5-COOMe
>10 µM
>10 µM
EC50 values for CCKBR agonism or antagonism
Benzoamide derivatives were normally prepared by direct
condensation using substituted benzoic acids with benzoa-
mine in the presence of EDCI and CH2Cl2 at room tem-
perature (Scheme 1). Both the benzoamines and the
substituted benzoic acids were made using commercial
chemicals as the starting points of modification. The deri-
vatives in the series were designed to be 5-substituted ben-
zamides with one 4-substituted product (Table 1). The EC50
value results showed no increase for CCKBR agonism.
Next, more analogs were designed and synthesized by
the introduction of diversified substituted groups on both
benzyl amines and benzoic acid groups as seen in Scheme
2. The derivatives in series 2 were still designed to carry 5-
substituted groups on benzoic acid and to introduce differ-
ent groups on benzoamines. To our delight, compound 21
was discovered with the highest agonism in this series
(Table 2).
Results
Chemistry
Finally, after we compared the structures of compound 1
and compound 21, a series of phenylbenzamide derivatives
from the modified benzyl amines with 5-chloro-2-hydroxy-
benzoic acid were designed and made to keep 5-chloro
substituted on benzoic acids as shown in series 3 (Scheme
3). In this series, compound 35 was observed to be the most
potent in CCKBR agonism with highest affinity and
bioactivity (Table 3).
A series of benzoamides as a new class of CCK receptor
agonists were identified through the screening of a struc-
turally diverse compound library. Compound 1 is a repre-
sentative example of this group of molecules first identified
as a CCK agonist. Based on the pharmacophore derived
from compound 1, novel benzoamide derivatives were
designed and synthesized.

Medicinal Chemistry Research
Table 3 List of derivatives made in series 3
Compound (EC50,
µM)
R4
Agonist
Antagonist
31
32
33
34
35
36
37
38
39
40
4-Br
0.65 µM
1.39 µM
10.00 µM
0.64 µM
0.49 µM
Scheme 2 Synthesis of derivatives of multiple-substituted benzamide
Table 2 List of derivatives made in series 2
4-Cl
3-Cl, 4-F
2-Cl
Compound
(EC50, µM)
R2
R3
Agonist Antagonist
4-NO2
4-CMe3
4-I
15
5-Br
3-CF3, 4-
CH3
3-OCHMe2
2-Cl, 4-Cl
16
17
5-Br
5-Br
4-CMe3
3-OMe, 4- 4.43 µM
OMe
3-COOMe, 5-
COOMe
18
5-Br
3-
41
42
43
44
45
46
47
48
3-COOH, 5-COOH
2-Cl, 4-NO2
3-OMe, 4-OMe
H
OCHMe2
6.82 µM
19
20
5-Br
5-Br
4-NO2
>10 µM
>10 µM
2-Cl, 4-
NO2
10.00 µM
>10 µM
>10 µM
2-NO2
21
22
5-Br
5-I
3-F, 5-F
0.21 µM
4-NH2
2-Cl, 4-
NO2
>10 µM >10 µM
3-F, 5-F
3-NO2
23
24
25
5-I
4-NO2
4-NO2
5-NO2
5-NO2
10.00µM
>10µM
49
3.86 µM
2-CF3, 4-
NO2
26
27
28
5-NO2
5-NO2
5-NO2
3-F, 5-F
4-Cl
EC50 values for CCKBR agonism or antagonism
3-OMe, 5-
OMe
29
30
Scheme 3 Synthesis of derivatives of 5-chloro-2-hydroxy-N-
phenylbenzamide
EC50 values for CCKBR agonism or antagonism
CCKBR cell-imaging assay
CCCKBR 96-well plate screening assay
CCKBR, coupled to Gq protein, can induce intracellular
calcium mobilization after the binding of ligand CCK-8.
Based on this, fluorescence-based imaging assay (calcium
flux assay) was established, by which a transient rise of
intracellular calcium signals upon CCKBR agonist binding
can be captured by a fluorescent microscope (Fig. 1a).
Candidate compounds from HTS 96-well plate screening
assay were further validated in this imaging assay. One
example of compound 35, which showed partial in vivo
CCKBR agonist activity, is illustrated in Fig. 1b. When
compared with the natural CCKB receptor agonist, cck-8
sulfate (cck-8s), compound 35 has nearly a 3-order mag-
nitude of lower affinity/bioactivity to CCKBR (Fig. 1).
Calcium flux assay is a well-established method suitable for
high-throughput screening, on using commercially available
specialized machines, like the FlexStation 3 Multi-Mode
Microplate Reader (Molecular Devices) used here. Its
reliability in this study was first confirmed by measuring the
EC50 value of CCK-8s (EC50 = 1.54 nM, Fig. 2 and Table
4), which is consistent with the previous reports. Then, we
screened an existing compound library (>30,000) in
Guangzhou Institute of Biomedicine and Health, and com-
pound 1 is a representative example for this HTS. Totally,
three series of compounds are described here, and their
EC50 values are listed in Tables 1–3.

Medicinal Chemistry Research
CCKBR (Fig. 3 #A). Compound 31 docked to one side of
Arg278 and compounds 34 and 35 docked to the other side.
Compound 34 formed 3 H-bonds with the residues Arg278,
Arg282, and Ser286 (Fig. 3 #C). Also, compound 35
formed 2 H-bonds with the residues Arg282 and Ser286
(Fig. 3 #B). However, no specific interaction was observed
between compound 31 and CCKBR.
Long-term potentiation assay in rat brains
In earlier studies, we have found that no LTP can be
induced with HF stimulation in the CCK-/- mouse brain, but
CCK infusion into the CCK-/- mouse brain can induce a
higher LTP in the neocortex when compared to normal
mice, indicating overly expressed CCKB receptors in the
neocortex. We used LTP induction in the CCK-/- mouse
brain as the in vivo functional assay to examine the phy-
siological effectiveness of the potential candidates popped
out from the cell-based assay. Three top candidates (com-
pound name XXX) were tested here, and two of them,
including compound 35, showed physiological functions
(Fig. 4).
Fig. 1 Cell-based calcium influx assay measuring molecules binding to
the CCKB receptors
Discussion
Targeting the CCKB receptor represents a new approach for
the development of therapeutics aimed at enhancing mem-
ory. In the past, the CCKB receptor was a hot target for
obesity as the receptor has been shown to bind to gastrin
and CCK in the gastrointestinal tract inducing one’s appe-
tite. However, the focus at that time was on the advance-
ment of antagonists and not so much on agonists. Also,
most of the effort was centered on targeting the gastro-
intestinal tract; thus, central nervous system (CNS) perme-
ability was not a priority during the development of these
new compounds. Thus, many of the reported CCKB
receptor agonists are not ideal for CNS applications. In
addition, most of the agonists targeting the CCKB receptor
tend to be high-molecular weight molecules, including 1,4-
benzodiazepines, 1,5-benzodiazepines, and pyrimidine car-
boxamides. The purpose of our study was to identify new
potent partial agonists for the CCKB receptor with a low
molecular weight and good CNS.
Fig. 2 EC50 value of CCK-8s
Homology model of CCKBR with lead agonists
As hoped, compounds targeting the CCKB receptor were
identified as partial agonists and antagonists belonging to
three chemical series, including derivatives of 2-hydroxy-N-
(4-(trifluoromethyl)phenyl)benzamide, phenylbenzamides,
To understand the potential interaction between CCKBR
and ligand compounds, we carried out docking analysis for
compounds 31, 34, and 35 using the homology model of
CCKBR as the receptor. The binding-affinity energy (BAE)
was -7.5, -8.1, and -8.6 kcal/mol for 31, 34, and 35,
respectively. The predicted binding sites of these three
compounds were located at the out-membrane entrance of
and
5-chloro-2-hydroxy-N-phenylbenzamides.
Most
importantly, several of the agonists, such as compound 35,
demonstrated long-term potentiation in rat brains, which is a
biomarker of memory enhancement, bringing up the pos-
sibility that these drugs may be used as therapeutics for

Medicinal Chemistry Research
Table 4 EC50 value of CCK-8s
at different time points
0 s
10 s
20 s
30 s 40 s
1.62 1.54
50 s
60 s
70 s
80 s
90 s
EC50 (nM) 2.21
1.90
1.73
1.65
1.64
1.53
1.37
1.53
R2
0.9927 0.987 0.9937 0.99 0.9852 0.9806 0.9825 0.9722 0.9657 0.9597
For simplifying the comparison among compounds, EC50 value at 40 s was used
Fig. 3 The predicted interaction
between ligand compounds and
CCKBR homology model. a
Docking pose of compounds 31,
34, and 35. b, c Potential
interaction between CCKBR
homology model and
compounds 34 and 35,
respectively. CCKBR homology
model is shown as a cartoon and
the residues with a distance less
than 4 Å to the ligand compound
are shown as sticks. Compounds
31, 34, and 35 are shown as
pink, yellow, and dark-blue
sticks, respectively (color figure
online)
agonists. Compound 35 demonstrated a physiological
effect, inducing long-term potentiation in rat brains com-
parable to the CCK-8 peptide, albeit at much higher con-
centrations. Therefore, this novel type of benzoamines may
be the basis for a new series of CCKB receptor agonists in
drug discovery efforts that seek to develop therapeutics to
prevent memory loss. However, more studies are required to
be conducted in the animal model of memory.
Acknowledgements Thise research work was financially supported by
the Guangzhou Science & Technology Project (2011Y2-00026 and
201508020131) to support GIBH Drug Discovery Pipeline Develop-
ment (Y.Z.), the Guangdong Science
& technology Project
Fig. 4 Time-dependent measurement of long-term potentiation in rat
brains in response to partial agonists of CCKB receptors
((2014B050505016) (M.T.), (2013B050800009) (Y.Z.), and
(2013B040200031) (Z.T.)), and CAS Key Technology Talent Program
China (2013) (Z.T.).
treating various forms of dementia. Future studies will focus
on testing these compounds in animal models of memory.
For example, it was recently reported that mice lacking the
CCK peptide (the natural ligand of the CCKB receptor) are
memory deficient in various behavioral tests, making them a
great model for testing our newly discovered partial
agonists.
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In conclusion, we designed and synthesized a series of
novel low-molecular weight benzoamine CCKB receptor

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