Phthalamide derivatives as ACE/AChE/BuChE inhibitors against cardiac hypertrophy: an in silico, in vitro, and in vivo modeling approach

Article abstract of DOI:10.1007/s00044-021-02707-8

Left ventricular hypertrophy (LVH) is a major adaptative response to the increase in the overload produced by hypertension, is a risk factor for myocardial infarction, stroke, and heart failure. Among the several factors involved in hypertension and in the progression to cardiac hypertrophy, the hyperactivity of the renin–angiotensin system (RAS) and the dysfunction of the neurovisceral/autonomic nervous system are the main mechanisms involved. Evidence demonstrates that the inhibition of RAS and the increase in parasympathetic activity reduce significantly LVH ameliorating cardiac function. The development of multi-target compounds is a relevant strategy for treating hypertension and cardiac hypertrophy. This study aimed to synthesize three phthalamide derivatives (M-01, M-02, and M-03) and evaluate them as three-target (ACE/AChE/BuChE) inhibitors with the possible dual effect of reducing hypertension and reverting cardiac hypertrophy. After in silico and in vitro experiments, one compound was tested in vivo on rats. All three phthalamides were synthesized in good yields, showing good competitive inhibition of the three-target enzymes in silico and in vitro. M-01 (10 mg/kg) significantly reversed cardiomyocite hypertrophy (by 87.3%; p < 0.001) in the heart of spontaneous hypertensive rat (SHR) model. It was at least 18-fold more potent than the reference drug (captopril), which provided only 32.7% reversion. Three-target inhibitory activity was herein demonstrated for M-01, M-02, and M-03 in vitro and in silico, each with a similar effect. The compound tested in vivo (M-01) exhibited great potency in reducing hypertension and reverting cardiomyocyte hypertrophy, making it a promising candidate for further research. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-021-02707-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:964–976
https://doi.org/10.1007/s00044-021-02707-8
ORIGINAL RESEARCH
Phthalamide derivatives as ACE/AChE/BuChE inhibitors against
cardiac hypertrophy: an in silico, in vitro, and in vivo modeling
approach
Erik Andrade-Jorge1 Jessica E. Rodríguez2,3 Jesús A. Lagos-Cruz1 Josué I. Rojas-Jiménez1
●
●
●
●
Samuel E. Estrada-Soto2 Itzell A. Gallardo-Ortíz1 José G. Trujillo-Ferrara4 Rafael Villalobos-Molina
1,5
●
●
●
Received: 22 October 2020 / Accepted: 13 January 2021 / Published online: 3 February 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Left ventricular hypertrophy (LVH) is a major adaptative response to the increase in the overload produced by hypertension,
is a risk factor for myocardial infarction, stroke, and heart failure. Among the several factors involved in hypertension and in
the progression to cardiac hypertrophy, the hyperactivity of the renin–angiotensin system (RAS) and the dysfunction of the
neurovisceral/autonomic nervous system are the main mechanisms involved. Evidence demonstrates that the inhibition of
RAS and the increase in parasympathetic activity reduce significantly LVH ameliorating cardiac function. The development
of multi-target compounds is a relevant strategy for treating hypertension and cardiac hypertrophy. This study aimed to
synthesize three phthalamide derivatives (M-01, M-02, and M-03) and evaluate them as three-target (ACE/AChE/BuChE)
inhibitors with the possible dual effect of reducing hypertension and reverting cardiac hypertrophy. After in silico and
in vitro experiments, one compound was tested in vivo on rats. All three phthalamides were synthesized in good yields,
showing good competitive inhibition of the three-target enzymes in silico and in vitro. M-01 (10 mg/kg) significantly
reversed cardiomyocite hypertrophy (by 87.3%; p < 0.001) in the heart of spontaneous hypertensive rat (SHR) model. It was
at least 18-fold more potent than the reference drug (captopril), which provided only 32.7% reversion. Three-target
inhibitory activity was herein demonstrated for M-01, M-02, and M-03 in vitro and in silico, each with a similar effect. The
compound tested in vivo (M-01) exhibited great potency in reducing hypertension and reverting cardiomyocyte hypertrophy,
making it a promising candidate for further research.
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02707-8.
* José G. Trujillo-Ferrara
3
Bioquímica Clínica, Carrera de Químico Farmacéutico Biólogo,
Facultad de Estudios Superiores Zaragoza, Universidad Nacional
Autónoma de México, Av. Guelatao con Av. Exploradores,
Ejército de Oriente, Iztapalapa, 09230 Mexico City, México
jtrujillo@ipn.mx
* Rafael Villalobos-Molina
villalobos@unam.mx
4
5
Laboratorio de Investigación en Bioquímica, Sección de Estudios
de Posgrado e Investigación, Escuela Superior de Medicina del
Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón s/n
Casco de Santo Tomás, 11340 Mexico City, México
1
Unidad de Investigación en Biomedicina y Carrera de Enfermería,
Facultad de Estudios Superiores-Iztacala, Universidad Nacional
Autónoma de México, Av. de los Barrios 1, Los Reyes Iztacala,
54090 Tlalnepantla, Estado de México, México
Departamento de Bioquímica, Facultad de Medicina, Universidad
Nacional Autónoma de México, Ciudad Universitaria, Coyoacán,
04510 Mexico City, México
2
Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad
Autónoma del Estado de Morelos, Avenida Universidad 1001,
Chamilpa, 62209 Cuernavaca, Morelos, México

Medicinal Chemistry Research (2021) 30:964–976
965
Graphical Abstract
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Keywords Hypertension Molecular docking ACE inhibitor AChE inhibitor BuChE inhibitor Cardiac hypertrophy
reversion
Highlights
●
The phthalamide derivatives are three-target inhibitors, acting on ACE, AChE, and BuChE.
●
The test compounds showed potency in treating hypertension and reverting cardiomyocyte hypertrophy.
●
M-01 was 18-fold more potent than captopril in reversing cardiomyocyte hypertrophy.
Introduction
systems can trigger changes in cardiovascular functions. For
example, high blood pressure may promote hypertensive
heart disease characterized by myocardial alterations,
structural and functional adaptations, fibrosis, and remo-
deling of the left ventricle (eventually capable of limiting
compliance) [14]. In this context, the RAS and the auto-
nomic system represent biological targets for the treatment
of hypertension and its cardiac complications [15, 16].
Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1), a
zinc dipeptidyl carboxypeptidase, plays a vital role in blood
pressure regulation. It cleaves the decapeptide angiotensin I
(Ang I) to the octapeptide Ang II and also hydrolyzes
bradykinin, a powerful vasodilator [1]. The excessive pro-
duction of Ang II and degradation of bradykinin are known
to contribute to high blood pressure and the development of
hypertension [16]. Thus, Ang II inhibition is a useful
strategy for hypertension therapy.
Hypertension is a chronic, noncommunicable disorder of
varied etiology, characterized by an increase in cardiac
output and peripheral vascular resistance. It is a primary risk
factor for heart disease (myocardial infarction and heart
failure), strokes and sudden death [1], and a secondary risk
factor for chronic kidney disease [2, 3]. Hence, lowering
blood pressure should be a priority when treating patients
with hypertension.
Despite the availability of a wide range of anti-
hypertensive drugs nowadays, only 30% of treated patients
are able to adequately control their blood pressure [4–6].
Thus, hypertension is the leading modifiable risk factor for
disability and premature death from cardiovascular disease.
Medical attention for this disease constitutes ~10% of
healthcare spending globally [5, 7].
Among the several factors associated with the develop-
ment of hypertension, two are key to its pathophysiology
and progression to heart failure: the hyperactivity of the
renin–angiotensin system (RAS) and the dysfunction of the
neurovisceral/autonomic nervous system [8, 9]. Angiotensin
II (Ang II) through its type 1 receptor (AT1R) contributes to
several physiological functions that regulate vasoconstric-
tion, fluid volume, cardiac output, cell growth, and vascular
wall integrity [10]. It also interacts with the sympathetic
nervous system [11], which regulates vital cardiovascular
functions such as heart rate acceleration, increased cardiac
contractility, reduced venous capacitance, and peripheral
vasoconstriction [12].
On the other hand, autonomic dysfunction implies a
disturbance in the capacity of the sympathetic and para-
sympathetic systems to modulate cardiovascular functions
and blood pressure. Alterations in inhibitory and excitatory
mechanisms that affect brainstem vasomotor neurons could
be involved in creating an imbalance in these two systems.
Such a disruption in homeostasis may contribute to hyper-
tension through the combination of abnormal activation of
sympathetic tone [17] and diminished vagal activity, the
latter resulting in a decline in parasympathetic control [18].
In the cardiovascular system, acetylcholine (ACh) indu-
ces vascular endothelium-dependent relaxation through the
formation of nitric oxide (NO) [19], which regulates chan-
ges in heart rate and contractility in opposition to the
influence of the sympathetic nervous system [20]. By
prolonging the availability of ACh in cholinergic nerve
Contrarily, the parasympathetic nervous system affects
the cardiovascular system by slowing the heart rate through
vagal innervation [13]. An imbalance between these

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Medicinal Chemistry Research (2021) 30:964–976
endings and enhancing the efficiency of cholinergic trans-
mission, autonomic imbalance can be avoided. This will
attenuate the inflammatory process and therefore reduce the
tendency to increased pressure overload, left ventricular
hypertrophy (LVH) and risk of heart failure [21].
Ach is hydrolyzed by cholinesterase, a specialized car-
boxylic ester hydrolase that breaks down esters of choline.
Consequently, one way to prolong the availability of ACh is
through the inhibition of cholinesterases. The two main
kinds of cholinesterases are acetylcholinesterase (AChE),
specific for ACh degradation, and butyrylcholinesterase
(BuChE), which is a nonspecific enzyme with greater
selectivity for butyrylcholine (BuCh) and/or propionylcho-
line [22].
The inhibition of AChE and/or BuChE has proven to be
effective for decreasing cardiovascular risk [23, 24]. Vagal
tone is improved and the blood pressure variability ame-
liorated [25]. Thus, drugs with high affinity for cholines-
terases are an important therapeutic tool in preventing and
treating cardiovascular disorders produced by hypertension.
However, most of the synthetic compounds currently used
to reduce hypertension and/or revert cardiac hypertrophy
have a single target, and many of them cause significant
side effects.
Therefore, hard work is being made to find novel agents
with better efficacy and lower toxicity [26]. In previous
work, our group demonstrated the antihypertensive activity
of a phthalamide derivative in an in vivo animal model. The
molecules were designed following some important char-
acteristics of the molecular targets, in order to inhibit ACE,
the molecules have phenylethylamine moieties which is
necessary to interact with the ACE, moreover to be able to
inhibit cholinesterases, the molecules possess in their
structure ethylamine moieties as well as carbonyl groups,
which allows to adequately inhibit cholinesterases. This
contribution aimed to synthesize and test three phthalamides
as possible multi-target inhibitors of ACE, AChE, and
BuChE in order to reduce hypertension and revert cardiac
hypertrophy. Additionally, the inhibitory potency and
mechanism of action of these enzymes were studied in
silico, in vitro, and in vivo.
Gasteiger charges for ligands and Kollmann partial charges
for the protein were established with Raccoon [29] and
AutoDock tools 1.5.4 [30] software. The crystal structures
of Homo sapiens butyrylcholine esterase (HuBuChE) and
Electrophorus electricus acetylcholine esterase (EeAChE)
were obtained online from the Protein Data Bank (PDB
code 4BDS and 1C2O). The grid box was set at 126 Å ×
126 Å × 126 Å, with a mesh separation of 0.375 Å and grid
center coordinates of X = 138.889, Y = 123.537, and Z =
38.703 for HuBuChE, and X = 88.481, Y = 109.042, and Z
= 83.026 for EeAChE. AutoDock4 in Fedora 22 as the
operative system was used to perform the molecular dock-
ing, finding the following values: Gibbs free energy (ΔG),
−log(10) dissociation constant (pKd), dissociation constant
(Kd), binding distance, number, and type of interactions.
Synthesis and characterization
The compounds were synthesized as previously reported by
our group [31]. Briefly, in a 100 mL round-bottom oven-
dried flask were placed 3 mmol of the corresponding amine
and 1.5 mmol of phthalic anhydride. The reaction was
gently heated to the melting point of the reagents for
10–15 min under constant stirring with a magnetic stirring
bar. After the reaction was cooled to room temperature (rt),
purification was carried out with ethyl acetate (40 mL), and
sonicated until a precipitate formed. Finally, the white
powder was purified by washing it four times with basic
water (pH 13).
All solvents and reagents were utilized as received from
Sigma-Aldrich (St. Louis, MO, USA). The compounds
were characterized by melting point (uncorrected) using a
Stuart® SMP40 automatic apparatus, infrared spectrometry
by utilizing a 100 FT-IR spectrometer (Perkin-Elmer),
nuclear magnetic resonance was recorded on a Varian
Mercury 300 spectrometer (1H, 300 MHz; 13C, 75 MHz)
with TMS as the internal reference. Finally, a Bruker
micrOTOf-Q-II instrument was used to perform the elec-
trospray ionization high-resolution mass spectrometry.
Solutions for the angiotensin-I-converting enzyme
activity assay
Materials and methods
For the phosphate buffer solution (PBS, 0.1 M, pH 8.3),
8.07 g of K2HPO4 and 0.5 g of KH2PO4 were weighed and
added to a volumetric flask. Subsequently, 8.76 g of NaCl
and 28.4 g of Na2SO4 were added to reach a 0.3 M and
0.4 M solution, respectively. Finally, distilled water was
added to give a final volume of 500 mL. ACE was obtained
from the serum of 10-week-old male Wistar rats
(250–300 g) previously euthanized under procedures
approved by the Bioethics Committee. Serum was stored at
−70 °C to await processing. Equal volumes of serum and
Docking
A semiempirical method (PM3) was employed for the
conformation analysis of all ligands. Physiological condi-
tions (pH 7.4) were assumed when determining the corre-
sponding protonation state on GaussView 5.0.9 and
Gaussian 09 [27] software. A hybrid Lamarckian Genetic
Algorithm [28] and a population size of 100 were used.

Medicinal Chemistry Research (2021) 30:964–976
967
Scheme 1 Representation of the
improved method for
determining ACE activity
carried out in a 96-well
microplate. This pattern was
followed for evaluating the
enzymatic inhibitory activity of
phthalamide derivatives
phosphate buffer solutions were mixed immediately before
use. The substrate N-Hippuryl-His-Leu (HHL code H1635,
Sigma-Aldrich, St. Louis, MO, USA.) was dissolved in
phosphate buffer to afford a 5 mM solution. For the 2,4,6-
Trichloro-1,3,5-triazine solution (TT code C95501, Sigma-
Aldrich, St. Louis, MO, USA.), 2.72 g were weighed and
added to a volumetric flask to provide a final volume of
100 mL with 1,4-dioxane (2.7%).
100 mM), numerous dilutions were made for the experiment
(Ach 48, 32, 24, 16, 12, 8, 6, and 4 mM; BuCh 32, 28, 24,
16, 12, 8, 6, and 4 mM). The stock AChE solution (1 U/mL,
Electrophorus electricus AChE, Sigma Chemical C3389),
as well as, the stock BuChE solution (0.4 U/mL, human
BuChE, Sigma Chemical C-9971) were prepared with the
corresponding freeze-dried powder. All the test and refer-
ence compounds were prepared by adding distilled water or
DMSO (<1%) as the vehicle. The solutions were kept at
−20 °C.
Solutions for the acetylcholinesterase and
butyrylcholinesterase activity assay
Angiotensin-I-converting enzyme activity assay
The PBS (0.1 M, pH 8), sodium hydroxide solution (4.2 M),
and hydroxylamine solution (2.4 M) were prepared fol-
lowing our previously reported methodology for AChE and
BuChE activity assays [32, 33]. For the alkaline hydro-
xylamine solution was prepared immediately before use,
using equal volumes of NaOH and NH2OH solution. For
ferric chloride solution (0.75 M), 101.55 g FeCl3·6H2O
were slowly mixed with an HCl solution (7.5 M) to reach a
final volume of 500 mL. After elaborating the ACh solution
(ACh iodide, 256 mM) and BuCh solution (BuCh iodide,
For enzyme kinetic experiments, twenty microliters of each
solution (HHL, inhibitor, or ACE) were placed in a 96-well
microplate following the pattern shown in Scheme 1 to
construct the standard curve for ACE and the inhibitory
curves. The microplate was incubated at 37 °C for 30 min in
a water bath. Upon completion of this period, the reaction
was stopped with 50 μL TT solution. The microplate was
then kept in the dark at rt for 5 min prior to reading optical
density at 382 nm in
a microplate reader (Epoch

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120
100
80
60
40
20
0
100
90
80
70
60
50
40
30
20
10
0
-5.0
-4.5
-4.0
-3.5
-3.0
-2.5
A
-5.0
-4.5
-4.0
-3.5
-3.0
-2.5B
Log [M]
Log [M]
100
90
80
70
60
50
40
30
20
10
0
120
100
80
60
40
20
0
-5.0
-4.5
-4.0
-3.5
-3.0
-2.5 C
-10.0
-9.5
-9.0
-8.5
-8.0
Log [M]
-7.5
-7.0
-6.5
Log [M]
-6.0D
Fig. 1 Inhibitory plots of the test and reference compounds on Rattus norvegicus ACE: (A) M-01; (B) M-02; (C) M-03, and (D) captopril.
Nonlinear regression analysis was carried out using the IC50 module
equipment). The evaluation of ACE activity was repeated in
triplicate for each test and reference compound and the
resulting data averaged to obtain the final value. The IC50
analysis was made with a nonlinear regression, a 95%
interval confidence was considered for the experiments.
to each well. The standard curve for Ach, BuCh, AChE, and
BuChE was built by following the same procedure without
the inhibitor. All assays were performed in triplicate.
In vivo evaluation
Acetylcholinesterase and butyrylcholinesterase
activity assay
Twenty-week-old male spontaneously hypertensive rats (SHR,
250–300 g) and Wistar Kyoto (WKY), acquired from the
Institute of Cell Physiology (ICP-UNAM), were used for the
cardiomyocyte hypertrophy reversion experiment. Compound
M-01 (10 mg/kg) and Captopril (40 mg/kg) were administered
intragastrically every day for 30 days, distilled water was used
as the vehicle (Veh). Four groups were formed: the normo-
tensive control (WKY + Veh), the hypertensive control (SHR
+ Veh), the reference group (SHR + captopril), and the
experimental group (SHR + M-01). Rats had food and water
ad libitum, and the conditions were 22 2 °C of temperature,
40–60% of humidity, and 12/12 h light/dark cycle. Bioethics
Committee of our institution approved all procedures with
animals, the protocol complied with the technical specifications
of the Mexican Official Norm for the production, care, and use
of laboratory animals (NOM-062-ZOO-1999, Ministry of
Agriculture).
The inhibitory effect produced on AChE and BuChE was
measured with an improved version of the corresponding
Bonting and Featherstone method previously reported by
our group [32, 34, 35]. These procedures follow the
hydroxamic acid method. Briefly, 20 μL of each solution of
AChE, BuChE, the corresponding inhibitors, ACh and
BuCh were placed in the wells (containing buffer solution)
of a 96-well microplate to afford a final volume of 160 μL.
The microplates for AChE and BuChE were incubated for
20 and 55 min, respectively, at 37 °C in a water bath
(Felisa), then stopped by adding 40 μL of alkaline hydro-
xylamine solution. A microplate reader (Accuris MR9600)
was used to read the optical density at 540 nm after 100 μL
of ferric chloride solution (premixed during 30 s) was added

Medicinal Chemistry Research (2021) 30:964–976
969
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.0 µM
200 µM
400 µM
600 µM
0.0 µM
200 µM
400 µM
600 µM
0.0
0.5
0.5
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
A
B
ACh [mM]
ACh [mM]
0.06
0.0 µM
150 µM
300 µM
400 µM
0.05
0.04
0.03
0.02
0.01
0.00
C
0.0
1.0
1.5
ACh [mM]
2.0
2.5
3.0
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.06
0.05
0.04
0.03
0.02
0.01
0 µM
1 µM
2 µM
4 µM
0.000 µM
0.195 µM
0.390 µM
0.781 µM
0.0
1.0
1.5
ACh [mM]
2.0
2.5
3.0
0.0
0.5
1.0
1.5
ACh [mM]
2.0
2.5
3.0
D
E
Fig. 2 Inhibitory plots of the test and reference compounds on Electrophorus electricus AChE: (A) M-01; (B) M-02; (C) M-03; (D) galantamine,
and (E) neostigmine. Nonlinear regression analysis was carried out using a kinetic module
Histological evaluation
minor transverse axes to estimate the cross-sectional area on
ZEN 2012 software (Carl Zeiss Laser Scanning Systems
LSM 510, Oberkochen, Germany) [36].
Histological analysis was conducted on cardiomyocytes of
the hearts of the hypertensive groups (SHR + Veh, SHR +
M-01, and SHR + captopril) and normotensive control
(WKY + Veh). Rats were euthanized with 50 mg/kg of
sodium pentobarbital (ip). Subsequently, the heart from
each animal was removed, dissected and fixed in 4%
phosphate-buffered formaldehyde solution for 24 h (n =
3–4 animals per group), and then dehydrated, cryopreserved
(with 30% sucrose during 24 h) and embedded in tissue-tek
(Sakura Finetek, USA) before being frozen with dry ice and
acetone. Hearts were sectioned in 5 µm slices and stained
with hematoxylin–eosin. The area (μm2) of cardiomyocytes
was evaluated in different fields, measuring the major and
Statistical analysis
All data from enzyme kinetic experiments are expressed
as the mean 95% confidence intervals. GraphPad Prism
software was used to carry out the statistical analysis
with the IC50 module and the enzyme kinetic module.
The results from the cardiomyocyte hypertrophy rever-
sion are expressed as the mean standard error of the
mean. One-way analysis of variance and Bonferroni post
hoc test was used to determine statistical significance
(p < 0.001).

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Medicinal Chemistry Research (2021) 30:964–976
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0 µM
150 µM
300 µM
600 µM
0.0 µM
150 µM
300 µM
600 µM
0.0
0.5
0.5
0.5
1.0
1.0
1.0
1.5
2.0
2.5
2.5
2.5
3.0
3.0
3.0
3.5
3.5
3.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
A
BuCh [mM]
BuCh [mM]
B
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0 µM
150 µM
300 µM
600 µM
0.0
1.5
2.0
C
BuCh [mM]
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0 µM
7.5 µM
15 µM
30 µM
0.0 µM
3.75 µM
7.5 µM
10 µM
0.0
1.5
2.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
D
BuCh [mM]
BuCh [mM]
E
Fig. 3 Inhibitory plots of the test and reference compounds on human BuChE: (A) M-01; (B) M-02; (C) M-03; (D) galantamine, and (E)
neostigmine. Nonlinear regression analysis was carried out using a kinetic module
Results and discussion
Enzyme activity assays (ACE, AChE, and BuChE)
Synthesis
The inhibitory capacity of the three phthalamide derivatives
(M-01, M-02, and M-03) was tested on the activity of ACE,
AChE, and BuChE (Table 2, Figs. 1–3). According to a
previous study by our group, the oral administration of
compound M-01 reduces the blood pressure of SHR in vivo
model, being ∼7-fold more potent than the reference (cap-
topril), a well-known ACE inhibitor [31]. However, it was
beyond the scope of that investigation to evaluate the
reversion of cardiac hypertrophy or explore the mechanism
of the antihypertensive effect. The phthalic anhydride
Three compounds were synthesized in good yields
according to our previously reported method without
modification [31]. The three compounds N,N′-bis(2-phe-
nylethyl)phthalamide (M-01), N,N′-bis[2-(3,4-dimethox-
yphenyl)ethyl]phthalamide
(M-02),
N,N′-bis[(1S)-1-
phenylethyl)phthalamide (M-03) were characterized by
NMR, infrared, mass spectroscopy, the results are in
agreement with our previous reported [31].

Medicinal Chemistry Research (2021) 30:964–976
971
10000
8000
6000
4000
2000
0
Table 1 Structure of the synthesized compounds evaluated as ACE,
AChE, and BuChE inhibitors in vitro and in silico
***
Structure
ID
***
O
O
+++
M-01
NH
NH
O
O
O
O
M-02
O
O
NH
NH
Fig. 4 Effect on the reversion of cardiomyocyte hypertrophy in
hypertensive rats by molecule M-01. *** Means a significant differ-
ence versus the normotensive control (WKY + Veh); +++ means a
significant difference versus the hypertensive control (SHR + Veh).
The data are expressed as the mean standard error of the mean.
Statistical significance was determined with one-way ANOVA and the
Bonferroni post hoc test (p < 0.001)
O O
NH
NH
M-03
moiety has been found to produce important biological
activity [31, 35], thought to stem from multi-target inhibi-
tion of ACE, AChE, and BuChE.
cardiovascular disease by attenuating the sympathovagal
imbalance, ischemia-induced arrhythmia, inflammation,
oxidative stress, and apoptosis [38, 39]. Moreover, the
inhibition of cholinesterase enhances the availability of
ACh in the central nervous system and the formation of NO
peripherally. The latter molecule is known to regulate vas-
cular resistance and blood pressure [40]. Cholinesterase
inhibitors provide benefits by balancing autonomic dys-
function, but only a few drugs are able to increase the
efficiency of cholinergic transmission. Since M-01 carried
out both of these functions, it is an excellent candidate for
further research into its capacity to treat and/or prevent
cardiovascular diseases.
In the current contribution, each of the derivatives
inhibited the three enzymes, showing the following order of
potency: AChE > BuChE > ACE. The type of inhibition of
AChE and BuChE is competitive for all compounds. The in
silico experiments suggest that the inhibition of ACE is also
competitive. The average Ki for AChE and BuChE was
90.2 and 314.9 μM, respectively, while the mean IC50 for
ACE was 706.5 μM. In addition to the test compounds,
some reference drugs were assessed (captopril, galantamine,
and neostigmine), not only to validate the results but also as
a point of comparison for the phthalamide derivatives.
According to the in vitro experiments, none of the test
compounds were better inhibitors than the reference drugs.
M-01 was more than a thousand-fold less potent than cap-
topril (IC50 = 1.79–15.1 nM [37] vs. 6.98 nM). Never-
theless, it provided approximately 6.85-fold greater potency
than captopril in regard to the in vivo antihypertensive
effect, presumably due to its three-target activity. On the
other hand, M-01 provided a very potent effect for cardiac
hypertrophy reversion, while captopril had no significant
effect.
Histological evaluation of cardiomyocytes
Histological analysis demonstrated a significantly larger
size of the isolated cardiomyocytes of the hypertensive
control group (SHR + Veh) than those of the normotensive
control (WKY + Veh; p < 0.001). Thus, the SHR animals
herein proved to have hypertrophied cardiomyocytes,
similar to the results found in previous studies [41]. It has
been proposed that cardiac hypertrophy may be due to an
over activation of the sympathetic nervous system and the
RAS [42–44]. The administration of M-01 in SHR rats
decreased the area of cardiomyocytes by 87.3%, reaching a
As described in earlier reports, cholinesterase inhibitors
improve autonomic and cardiac function in models of

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Medicinal Chemistry Research (2021) 30:964–976
Table 2 Inhibitory concentration 50 (IC50), inhibition constant (Ki), and type of inhibition of phthalamide derivatives and reference drugs for the
angiotensin-I-converting enzyme (ACE), acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
ACE (Rattus norvegicus)
IC50 (μM) Interval 95%
AChE (Electrophorus electricus)
BuChE (Homo sapiens)
Ki (μM) Interval 95% Type of inhibition Ki (μM) Interval 95% Type of inhibition
M-01
724.6
698.4
696.7
6.98 nM
–
538.4–899.9
465.5–1048
303.5–1090
6.03–8.08 nM
–
67.91
94.06
108.8
–
50.44–85.38
70.62–117.5
77.5–140.1
–
Competitive
Competitive
Competitive
–
324.9
307.7
312.1
–
264.4–385.3
265.3–350.0
260.3–363.9
–
Competitive
Competitive
Competitive
–
M-02
M-03
Captopril
Galantamine
Neostigmine
0.96
0.29
0.54–1.37
0.38–0.65
Competitive
Competitive
7.29
3.74
6.50–8.06
3.28–4.19
Competitive
Competitive
–
–
Table 3 Values obtained for the
main affinity parameters of the
interactions between the
phthalamides and reference
compounds with the
EeAChE
HuBuChE
ΔG0 (kcal/mol) Kd (μM) pKd
ΔG0 (kcal/mol) Kd (μM)
pKd
M-01
M-02
M-03
−9.87
−9.09
−10.72
−4.54
−6.6
0.058 7.2
M-01
−9.4
0.12855 6.89
cholinesterases (AChE
and BuChE)
0.217 6.66 M-02
0.138 6.86 M-03
−8.720
−8.960
0.407
0.270
6.39
6.57
3.34
4.84
4.14
Acetylcholine
Galantamine
Neostigmine
461.23
3.34 Acetylcholine −4.6
4.76 Galantamine −6.7
4.15 Neostigmine −5.69
453.9
17.41
70.2
14.6
73.1
−5.73
Table 4 Amino acid residues involved in the interaction inhibitor-enzyme obtained by molecular docking, as well as the type of interaction
ID
Hydrophobic
π–π interaction
H-bond
π–cation Other
HuBuChE
M-01
Leu125
Trp231, Phe329, Trp82
Trp82, Trp231, Phe329
Try128, Glu197
Glu197 (π–anion) Gly116
(π–amide)
M-02
Trp82, Phe329
Tyr128,
–
Gly116 (π–amide)
Tyr332, Thr120
M-03
Pro84, Ile69
–
Tyr440, Trp82
Trp231
Glu197, Thr120
Ser198
Gly116 (π–amide)
Glu197 (electrostatic)
Glu197 (electrostatic)
–
Butyrylcholine
Neostigmine
Galantamine
–
–
–
Glu197
Trp82
Trp82, His438
Tyr128
Tyr128
Glu197, Gly116,
Thr120
EeAChE
M-01
Trp86
Gly121
Asp74
His447 Ser203, Ser125
His447
M-02
Trp286, Trp86, Tyr332 Trp86, His447, Tyr341
Tyr124, Tyr337
Ser125
M-03
Phe338, Tyr341
His447, Tyr337, Tyr341,
Phe297, Tyr124
His447 Glu202 (π–anion)
Acetylcholine Tyr124
His447
Ser203, Trp86
Ser125
Tyr337 Glu202 (electrostatic)
Tyr337 Glu202 (electrostatic)
Neostigmine
Galantamine
Trp86
Trp86
Tyr124
Trp286, Tyr341
Ser203, Arg296
–
–
value similar to the normotensive control (WKY + Veh;
Fig. 4). Contrarily, captopril only showed a tendency, but
no significant difference, in the reduction of the area of
cardiomyocytes (32.7%) in relation to the normotensive
control. M-01 turned out to be at least 18.28-fold more
potent than captopril (mole to mole comparison), con-
sidering the captopril dose needed to reach a similar effect.
Treatments with ACE inhibitors are reported to prevent
hypertension, LVH, and vascular remodeling in young
SHRs [41]. Additionally, they lower blood pressure and

Medicinal Chemistry Research (2021) 30:964–976
973
Fig. 5 Docking simulation for the ligands on Electrophorus electricus
acetylcholine esterase: (A) binding mode of molecule M-01; (A′)
amino acid residues involved in the interaction with molecule M-01;
(B) binding mode of molecule M-02; (B′) amino acid residues
involved in the interaction with molecule M-02; (C) binding mode of
molecule M-03; (C′) amino acid residues involved in the interaction
with molecule M-03; (D) Compounds M-01, M-02, and M-03 show
similarity in their binding mode
diminish fibrosis, cardiac weight, and hypertrophy in older
SHRs [45, 46]. At the same time, cardiomyocytes have their
own functional cholinergic machinery that compensates for
adrenergic overstimulation, avoiding cardiac hypertrophy,
molecular changes, and alterations in calcium signaling.
Therefore, the inhibition of AChE, which leads to greater
bioavailability of ACh and NO, apparently modifies choli-
nergic signaling in the heart to counteract or partially neu-
tralize the hypertrophic effects of adrenergic stimulation
[47]. Consequently, the current data suggest that the pro-
nounced in vivo activity of M-01 resulted from the sum of
the inhibitory effects produced on the target enzymes (ACE,
AChE, and BuChE).
Molecular docking
To provide insights into the outcome of the present in vitro
and in vivo assays, the three phthalamide derivatives (Table
1) were modeled and docked on EeAChE and HuBuChE.
The values of ΔG, Kd, and pKd were determined for the
molecular interactions between the enzymes and the phte-
lamides (Table 2). The docking simulations demonstrated

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Medicinal Chemistry Research (2021) 30:964–976
Fig. 6 Docking simulation for the ligands on Homo sapiens butyr-
ylcholine esterase: (A) binding mode of molecule M-01; (A′) amino
acid residues involved in the interaction with molecule M-01; (B)
binding mode of molecule M-02; (B′) amino acid residues involved in
the interaction with molecule M-02; (C) binding mode of molecule M-
03; (C′) amino acid residues involved in the interaction with molecule
M-03; (D) Compounds M-01, M-02, and M-03 show similarity in their
binding mode
good affinity of the phthalamide derivatives for both
enzymes (EeAChE and HuBuChE), concurring with the
in vitro results (Table 3).
galantamine). Thus, the test compounds are capable of
binding to two important sites of the enzyme: the stearic and
the anionic site. Regarding HuBuChE, on the other hand,
the amino acid residues Gly116, Trp82, Glu 197, and
Trp231 take part in recognition (Table 4 and Fig. 6). For
both enzymes, the competitive inhibition evidenced by the
in vitro experiments is consistent with the docking
simulations.
Some amino acid residues were observed in the inter-
action ligand-receptor for both enzymes, as well as the type
of interaction, all the above is shown in Table 4. The main
interactions for EeAChE are π–π, owing to the number of
aromatic rings in the structure. The amino acids that parti-
cipated in the interactions include Trp86, His447, Glu202,
and Ser203 (Table 4 and Fig. 5), the same ones involved in
binding to the reference drugs (e.g., neostigmine and
Molecular docking for the ligands on ACE was pre-
viously reported. Briefly, the three phthalamide derivatives
showed good affinity for ACE, interacting with Lys511,

Medicinal Chemistry Research (2021) 30:964–976
975
His353, Tyr523, Glu384, His387, and Zn+2, located in the
catalytic active site of ACE. Moreover, the binding mode of
the test compounds turned out to be similar to that of the
reference drugs, suggesting competitive inhibition.
antioxidant properties of caffeic acid and chlorogenic acid:
Mechanistic role of angiotensin converting enzyme, cholinesterase
and arginase activities in cyclosporine induced hypertensive rats.
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Conclusions
The development of new multi-target molecules is a rele-
vant strategy for improving the treatment of hypertension,
especially if the effect of reverting cardiac hypertrophy is
involved. After three phthalamide derivatives (M-01, M-02,
and M-03) were synthesized, they were evaluated in vitro as
inhibitors of three-target enzymes (ACE, AChE, and
BuChE). M-01 was then tested on the SHR a model for
heart failure with the aim of reverting cardiac hypertrophy.
In old rats, the compound produced a potent effect against
cardiomyocyte hypertrophy, which was ∼18-fold greater
than that of captopril. The reversion of cardiomyocyte
hypertrophy caused by the reference drug was not sig-
nificant versus the hypertensive control. The potent effect of
M-01 is probably due to a multi-target inhibitory activity.
Since M-02 and M-03 have the same in vitro and in silico
pattern as M-01, they would likely furnish the same in vivo
effect. The apparent dual pharmacological activity against
hypertension and cardiac remodeling is promising for M-01
and perhaps the other two test compounds. Further studies
are needed to explore the potential of these molecules for
treating cardiovascular diseases.
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Acknowledgements EAJ received a postdoctoral fellowship from
DGAPA of the Universidad Nacional Autónoma de México (UNAM).
Additionally, this work was supported by CONACYT-México, SIP-
projects (M1930, and 20201031) from the Instituto Politécnico
Nacional-ESM, and RVM and IAGO received grants from PAPIIT
(IN223519 and IN226819, respectively). We thank MVZ L. Flores,
MD. F. Barrón-Moreno, C.Y. Gallegos-Quiroz, and Biol. TE
Villamar-Duque from Facultad de Estudios Superiores Iztacala (FESI),
UNAM, for their aid in animal care and housing.
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Compliance with ethical standards
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Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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