Synthesis, crystal structure, molecular docking studies and biological evaluation of aryl substituted dihydroisoquinoline imines as a potent angiotensin converting enzyme inhibitor

Article abstract of DOI:10.1016/j.molstruc.2021.130230

The different functionalities of imine group found in many compounds are of important biological activity. Since, the development of novel imines is biologically encouraged, this study aims to develop a straightforward synthesis pathway of dihydroisoquino

Full text of DOI:10.1016/j.molstruc.2021.130230

Journal of Molecular Structure 1235 (2021) 130230
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis, crystal structure, molecular docking studies and biological
evaluation of aryl substituted dihydroisoquinoline imines as a potent
angiotensin converting enzyme inhibitor
Awatef Selmia, Rihab Aydi a, Omar Kammounb, Hajer Bougatefc, Ali Bougatefc,
Nabil Miledd, Othman A. Alghamdid, Majed Kammouna,∗
a Laboratory of Medicinal and Environment Chemistry, Higher Institute of Biotechnology, University of SFAX, PB 261, 3000 SFAX, Tunisia
b Laboratoire Physicochimie de l’Etat Solide, Département de Chimie, Faculté des Sciences de Sfax, Université de Sfax, BP 1171, 3000 Sfax, Tunisia
c Laboratory of Plant Improvement and Valorization of Agroressources, National School of Engineering of Sfax (ENIS), University of Sfax, Sfax 3038, Tunisia
d Department of biological sciences, College of Science, University of Jeddah- Asfan Road, Kingdom of Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
The different functionalities of imine group found in many compounds are of important biological ac-
tivity. Since, the development of novel imines is biologically encouraged, this study aims to develop a
straightforward synthesis pathway of dihydroisoquinoline imines that could reveal antihypertensive ef-
fects. Starting from 2-méthyl-1-phényl-2-propanol, four different dihydroisoquinoline imines compounds
(2a-d) were obtained and their structures were analyzed by mass spectrum, elemental analysis and NMR
spectral studies. The crystal structure of 2b was determined from single crystal X-ray diffraction data.
Received 21 December 2020
Revised 13 February 2021
Accepted 27 February 2021
Available online 6 March 2021
Keywords:
Imines
The newly synthesized compounds were evaluated for angiotensin I-converting enzyme (ACE) inhibition,
using an enzymatic in vitro assay. The results were compared to Captopril as a reference drug. Com-
pounds 2a, 2b and 2d showed inhibition activity with IC50 values of 0.15, 0.13 and 0.40 mg/ml, re-
spectively. The docking of chemical compounds in the ACE active site explained the higher inhibitory
capability of compounds 2a and 2b on the catalytic activity of the enzyme, indicating a potential anti-
hypertensive effect of these compounds.
X-ray structure
Molecular docking
Anti-hypertensive activity
© 2021 Published by Elsevier B.V.
Introduction
Other approaches have been reported in the literature such as
the dimerization, oxidation or dehydrogenation of primary amines,
Imines (schiff bases), which were first reported by Hugo Schiff
in 1864, are compounds with the structure of azomethine group
(-C=N-) [1], and represent an important intermediates in the or-
ganic synthetic chemistry and fine chemical industry [2-6].
Also, schiff bases represent an important class of organic com-
pounds, especially in the medicinal and pharmaceutical fields.
They have shown a broad range of biological activities including
antioxidant [7], anticancer [8-9], antimalarial [10], antimicrobrial
[11], antifungal [12], antidiabetic [13], anti-obesity [14], and anti-
Leishmania tropica [15].
[17-21] and the coupling of alcohols and amines [22-23].
The imine formation is one of the most important reactions in
organic and medicinal chemistry [24]. Examples of using imines
as versatile components include the asymmetric synthesis of α-
aminonitriles [25], preparation of secondary amines by hydro-
genation [26], and cycloaddition reactions [27]. Moreover, imines
are intermediates in the synthesis of some compounds such as
isoxazoline and pyrazolines [28], quinolones [29], α-alkylidene β-
oxoamides [30], 1,5-amino/ketoalcohols [31] and oxaziridines [32-
36].
There are several methods used to prepare imines, of which is
the classical way involving the condensation of active aldehydes
and amines, whereas the synthesis of aldehydes is usually time-
consuming and expensive [16].
Owing to the interesting imines, our research group has been
working on an easy and efficient method for the synthesis of schiff
bases. Our proposed method shows distinct features such as in-
vovlving, good efficiency, short reaction times and excellent yields.
The aim of this study was twofold: (1) synthesizing four imines,
namely 2a-d with substituents in position 1, including a phenyl
and a Parachloro phenyl groups, (2) focusing on the effect of the
∗
Corresponding author.
E-mail address: majed.kammoun@isbs.rnu.tn (M. Kammoun).
https://doi.org/10.1016/j.molstruc.2021.130230
0022-2860/© 2021 Published by Elsevier B.V.

A. Selmi, R. Aydi, O. Kammoun et al.
Journal of Molecular Structure 1235 (2021) 130230
Scheme 1. Synthesis of imines 2a-d.
introduction of substituents in the aromatic ring on the ACE inhi-
bition activity.
The compounds described herein possess an electrondonor and
attracter groups which allowed evaluation of substituent effects on
1H and 13C chemical shifts.
In the 1H NMR spectra of imines (2a-d), the peak of gem-
dimethyl corresponding to the 6 protons was seen as a singlet and
the two equivalent neighboring protons of –CH2 group appeared as
a singlet.
A suitable crystal of imine 2b was obtained by recrystallization
and the molecular structure was confirmed by single X-ray analy-
sis.
The newly synthesized compounds were evaluated for their in
vitro ACE inhibition. Molecular docking studies were used to ex-
plain their ACE inhibitory power.
In the aromatic region, the proton chemical shifts evidenced
the electron-donating character of the chlorogroup, herein, chem-
ical shifts were appeared to a lower frequency compared to the
derivative with a phenyl group. In fact the aromatic protons are po-
sitioned between 7.23–7.61 ppm for 2a and between 7.16–7.51 ppm
for 2a.
2. Results and discussion
2.1. Chemistry
The chemical shifts of protons of the aromatic ring are in-
creased due to the presence of electron attracter group NO2in the
phenyl ring. Indeed the aromatic protons were appeared signals
between 7.47–8.48 ppm for 2c and 7.49–8.32 for 2d compared with
2a and 2b.
In the 13C NMR spectra of these compounds (2a-d), the chem-
ical shifts were appeared between 121.5 − 164.5 ppm correspond-
ing to the aromatic ring. And, the aliphatic carbon atoms were
shown in the range of 27.3–55.5 ppm. The two carbons of gem-
dimethyl were distinguished as one peak.
The molecules 2a-d shown in the current study were synthe-
sized starting from the commercial tertiary alcohol 1(Scheme1).
In the first step, imines 2a-b were acquired by the acid catalyzed
reaction of 2-methyl-1-phenylpropane-2-ol using Ritter–type pro-
cedure [37]. The next step included the nitration of these imines
2a-b under soft conditions [38,39] and selectively directed to their
derivatives 2c-d.
Nuclear magnetic resonance (NMR) spectral analysis is an im-
portant analytical technique used to determine the structures of
organic compounds.1H and 13C spectral data of all compounds have
been presented in SI. The NMR spectroscopy of all synthesized
compounds was registered using CDCl3 as a solvent.
Furthermore, mass spectrum showed a [M + H]+ peak at
m/z 270,10,349 corresponding to its molecular formula, C17 H16 NCl
(Fig. 1).
Fig. 1. FT-MS Spectrum of compound 2b.
2

A. Selmi, R. Aydi, O. Kammoun et al.
Journal of Molecular Structure 1235 (2021) 130230
Table 1
Crystallographic data and structure refinement parameters for 2b.
Empirical formula
Formula weight
C17 H16 Cl N
269.76
Temperature
150 K
˚
Wavelength
0.71073 A
Crystal system, space group
Unit cell dimensions
Monoclinic, P 1 21/c 1
˚
a = 8.2804(3) A, α = 90 °
˚
b = 17.5586(7) A, β = 98.6290(10) °
˚
c = 9.8062(4) A, γ = 90 °
3
˚
Volume Z,
1409.61(10) A
Calculated density
Absorption coefficient
F(000)
4, 1.271 (g.cm−3
0.256 mm−1
568
)
size
0.500 × 0.320 × 0.130 mm
colourless
Crystal color
Theta range for data collection
h_min, h_max
3.130 to 27.511 °
−10, 8
k_min, k_max
−22, 22
l_min, l_max
−12, 12
Reflections collected / unique
Reflections [I>2σ]
Completeness to theta_max
Absorption correction type
Max. and min. transmission
Refinement method
Data / restraints / parameters
bGoodness-of-fit
17,085 / 3216 [R(int)a = 0.0295]
2811
0.993
multi-scan
0.967, 0.849
Full-matrix least-squares on F2
3216 / 0 / 174
1.028
Final R indices [I>2σ]
R indices (all data)
Largest diff. peak and hole
R1c = 0.0329, wR2d = 0.0828
R1c = 0.0399, wR2d = 0.0881
Fig. 2. Crystal packing of C17 H16 NCl along the crystallographic a axis, showing its
−3
˚
0.298 and −0.295 e-.A
supramolecular structure (For clarity, intermolecular hydrogen bonds are omitted).
Table 2
˚
Selected bond distances (A) and angles (°) for 2b.
Cl – C16
1.7438(12)
1.3850(17)
1.3871(18)
1.3881(17)
1.3965(16)
1.3923(16)
1.4967(15)
1.3903(17)
1.2825(15)
1.4874(16)
1.4830(14)
121.62(11)
119.63(9)
C3 – C11
C3 - C12
C3 – C4
C4 - C10
C10 – C5
C10 – C9
C5 – C6
C6 – C7
C7 – C8
C8 – C9
1.5256(17)
1.5328(17)
1.5331(16)
1.5003(17)
1.3908(17)
1.4057(16)
1.387(2)
C16 – C17
C16 – C15
C15 – C14
C14 – C13
C13 – C18
C13 – C1
C18 – C17
1.385(2)
C1 - N
1.3925(17)
1.3950(17)
C1 – C9
N - C3
C17 – C16 – C15
C17 – C16 - Cl
C15 – C16 - Cl
C14 – C15 – C16
C15 – C14 – C13
C18 – C13 – C14
C18 – C13 – C1
C14 – C13 – C1
C17 – C18 – C13
C16 – C17 – C18
N – C1 – C9
N – C1 – C13
C9 – C1 – C13
C1 - N – C3
N – C3 - C11
N – C3 – C12
C11 – C3 – C12
N – C3 – C4
110.41(11)
111.86(9)
118.74(9)
C11 – C3 – C4
C12 – C3 – C4
C10 – C4 – C3
C5 - C10 – C9
C5 – C10 – C4
C9 - C10 – C4
C6 – C5 - C10
C7 – C6 – C5
C6 – C7 – C8
C7 – C8 – C9
C8 – C9 - C10
C8 – C9 – C1
C10 – C9 – C1
109.26(10)
111.78(10)
112.16(9)
119.03(11)
120.57(11)
119.07(11)
119.19(10)
121.74(10)
121.01(11)
118.64(11)
124.54(11)
116.52(10)
118.80(10)
119.15(10)
106.97(10)
106.42(9)
119.40(12)
123.34(11)
117.24(10)
120.62(13)
120.18(12)
119.90(12)
120.34(12)
119.54(11)
122.96(11)
117.42(10)
Fig. 3. Molecular structure of the compound 1-(4-chlorophényl) −3, 3-diméthyl
−3,4-dihydroisoquinoléine 2b with atom numbering.
2.2. Crystal structure of 2b
Crystals 2b were obtained by recrystallization from 1,4-
dichlorobutane. The molecular structure of 2b is shown in Fig. 3.
The compound with formula C17 H16 NCl crystallize in the mono-
clinic symmetry, with the centrosymmetric space group P21/c. The
crystal structure is built from only the organic imine molecules
interconnected between themselves via weak hydrogen bonding.
Such connectivity defines the supramolecular network of the struc-
ture (Fig. 2). Crystallographic data, selected bond lengths and bond
angles are listed in Tables 1-2, respectively.
The dihedral angle between the plane containing the chloro
phenyl attached to C1 and the plane formed by the atoms of the
phenyl ring coordinated at C1 and C4 is equal to 63.26° (Fig. 4).
2.3. Hirshfeld surface analysis
To more investigate intermolecular interaction in crystal pack-
ing we carry out Hirshfeld surface analysis. The Hirshfeld analysis
results are shown in Fig. 5. It is evident that in all potential molec-
ular contacts, hydrogen–hydrogen contacts predominate, contribut-
ing 54.1% of the overall intermolecular interactions. The second
most significant intermolecular interaction is HꢀC and HꢀCl, re-
In the molecular structure of 2b, the tetrahydroisoquinoline
unit is substituted by a chloro phenyl ring attached to C1, and two
methyl groups attached to C3; the one is pseudo-equatorial and
the second is pseudo-axial (Fig. 3).
3

A. Selmi, R. Aydi, O. Kammoun et al.
Journal of Molecular Structure 1235 (2021) 130230
Table 3
ACE inhibitory activities (IC50) of test compounds
and standard drug.
Compounds
IC50 (mg/ml)
2a
2b
2d
STD.
0.15
0.13
0.40
0.07
and histidyl-leucine (HL). The extent HA released is directly pro-
portional to the ACE activity.
Imines derivatives synthesized by our procedure inhibited ACE
activity, in a concentration-dependent manner (Fig. 6). Results
ACE inhibitory activities (IC50) of imines (2a-d) are presented in
Table 3.
Furthermore, the inhibitory concentration at 50% (IC50) ACE ac-
tivity for compounds 2a, 2b, 2c and 2d was calculated from dose-
response curves (Fig. 6) obtained by plotting the percentage inhi-
bition vs. the concentration.
The evaluation of IC50 data for 2a-d values ranged from 0.13 to
0.4 mg /ml, revealed 2b as the best lead compound with the low-
est IC50 (0.13 mg/ml), which was substituted with a chloro group
in position 14. This activity was conserved when the imine was
substituted with a phenyl group as in 2a (IC50 0.15 mg/ml). These
results also correlate with the IC50 values of the reference drug
Captopril (0.07 mg/ml), which was expected because Captopril is a
drug especially developed for treatment of hypertension.
The presence of a nitro group in 7 and 13 in 2d caused a reduc-
tion of ACE inhibition activity (IC50 0.40 mg/ml). While the absence
of inhibition activity from 2c.
Fig. 4. Projection on planes axes.
sulting in 25% and 16.2% of the sum of intermolecular interactions,
respectively. In addition, HꢀN, CꢀCl and CꢀC interactions also con-
tribute a little to the total intermolecular interactions. In general,
the crystal structure is stabilized by a variety of interactions.
2.4. Biological activity
The compound 2b seems therefore to be a stronger inhibitor of
ACE, followed by compound 2a. Increasing the polarity of the lig-
and by adding two NO2 group in ortho positions on the phenyl
rings (2d compound) diminished ACE inhibitory capacity. Docking
experiments were conducted in order to understand this discrep-
ancy.
Inactivation of angiotensin converting enzyme (ACE)
Hypertension is one of the largest risk factors associated with
mortality worldwide, further accounting for more than half of all
cases of stroke and coronary diseases [40]. ACE is an effective ther-
apeutic target for the treatment of hypertension, and commercial
ACE inhibitors are common [41].
The inhibition of angiotensin-converting enzyme (ACE) is an
important therapeutic approach employed in the treatment of
high blood pressure. This enzyme is a widely distributed zinc-
dependent metallopeptidase that converts angiotensin I to an-
giotensin II (vasoconstrictor octapeptide). ACE also promotes the
degradation of the vasodilator bradykinin [42].
Docking of inhibitors in the active site of ACE
All ligands were found to bind within the catalytic pocket of the
ACE with free binding energies ranging from −9 to −7.9 Kcal/mol.
All compounds were located close to the catalytic site (Fig. 7).
Compounds 2a and 2b were superimposed with an RMSD value of
˚
Several effective oral ACE inhibitors have been developed,
namely, captopril, enalapril, and lisinopril and all are currently
used as clinical antihypertensive drugs [43]. The in vitro ACE
inhibitory activity of newly synthesed compounds were mea-
sured using a colorimetric method. Most of the antihypertensive
molecules have been characterized by the rabbit lung ACE inhibitor
assay, based on the hydrolysis of the synthetic peptide hippurylhis-
tidylleucine (HHL). HHL is hydrolyzed by ACE to hippuric acid (HA)
0.5 A due to their structural similarity. Compounds 2c and 2d are
more bulky and their deviations from 2a orientation were 2.2 and
˚
3.5 A, respectively. This displacement is explained by the presence
of bulky and highly polar NO2 groups for compounds 2c and 2d
Introducing polar and bulky NO2 groups at these positions yielded
new polar interactions. Even though compounds 2c and 2d are
˚
structurally similar, their orientations deviated by 4.5 A. This is to
be explained by the introduction of chloro substituent in 2d Com-
Fig. 5. Hirshfeld surface analysis of C17 H16 NCl.
4

A. Selmi, R. Aydi, O. Kammoun et al.
Journal of Molecular Structure 1235 (2021) 130230
Fig. 6. Dose-dependent angiotensin I-converting enzyme (ACE) inhibition activity of test compounds.
Fig. 7. Complex between the ACE and the best poses corresponding to compounds 2a, 2b, 2c and 2d, generated by docking. A, Overall structure of the complex. All
compounds and residues were shown as sticks. Catalytic residues playing key role in enzymatic activity were depicted as sticks (H383 and E411). Catalytic Zinc atom bound
to H383 and E411 is shown as a red sphere. The lisinopril inhibitor in the active site of ACE (PDB code 1O86) is displayed as yellow wide sticks. 2a and 2b are represented
as wide sticks whereas 2c and 2d as thin sticks. B, Zoom view from A showing the ligands in the active site of ACE. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
˚
tively) thus creating new polar interactions. Even though they are
structurally similar, introduction of an additional chlore group in
compound 2d changed its orientation as compared to 2c (RMSD
pounds were located at distances of 3.5, 3.5, 6.7 and 4.1 A from
catalytic H383 whereas this distance was 3.5 for the lisinopril in-
hibitor. The catalytic H383 and E411 are interacting with the cat-
alytic zinc ion (Fig. 7). This proximity of imine compounds to H383
might explain their inhibitory effect through preventing the sub-
strate from acceding to the catalytic residues.
˚
of 4.4 A). This displacement created new interactions of the chloro
substituent with residues H353 and F512 (Fig. 8D and D’). Inter-
estingly, the chloro substituent in both compounds 2a and 2d is
stabilized by H353. When analyzing interactions of the native in-
hibitor lisinopril in the active site of ACE, many interactions de-
scribed for the compounds 2a-2d are reproduced, mainly interac-
tions with catalytic zinc and H353. Lisinopril is making hydropho-
bic contacts with residues V518 and favorable charge interactions
with Y523 and catalytic E411. It is hydrogen bonded to H353, cat-
alytic E411,Y520 and Y523. Its central COO group is interaction
with catalytic zinc in a similar way to NO2 group in compound
2d Compounds 2a, 2b and 2d were located at the same distance
Compound 2a was mainly establishing hydrophobic contacts
with residues H353, A354, V379, V380 and catalytic H383 (Fig. 8A
and A’). Compound 2b is superimposed to 2a and is making sim-
ilar interactions (Fig. 8B and B’). Compound 2c is also establish-
ing hydrophobic contacts with V379 and V380 but is not inter-
action with catalytic H383. Its two NO2 polar groups are hydro-
gen bonded to NH groups of residues Q281 and A354 (Fig. 8C
and C’). Regarding compound 2d, it is in hydrophobic contacts
with residues P407, H410, F512 and H353. Its NO2 groups are hy-
drogen bonded to D358 and Y523. One of its NO2 groups is in-
teracting with the catalytic zinc (Fig. 8D and D’). Orientations of
˚
range from catalytic histidine as the inhibitor lisinopril (3.5–4.1 A).
The ability of 2a, 2b and 2d inhibitors to block the access to the
catalytic histidine and zinc ion is in line with their strong in-
hibitory effect towards the ACE. Compound 2c located further from
˚
compounds 2a and 2b were similar (RMSD of 0.5 A). Introduc-
tion of polar NO2 groups for compounds 2c and 2d caused them
˚
˚
catalytic histidine (at a distance of 6.7 A) displayed a lower in-
to have different orientations (RMSDs of 2.2 and 3.5 A, respec-
5

A. Selmi, R. Aydi, O. Kammoun et al.
Journal of Molecular Structure 1235 (2021) 130230
Fig. 8. Interactions of compounds with residues from ACE. Panels A, B, C and D correspond to interacting residues with 2a, 2b, 2c and 2d, respectively. Panel E corresponds
to lisinopril inhibitor. A’, B’, C’, D’, and E’ are 2D projections from A, B, C, D and E, respectively. Compounds are shown as wide sticks whereas interacting residues are in
thin sticks. Catalytic residues H383 and E411 are depicted as red sticks. Catalytic zinc is shown as red sphere.
hibitory capacity. It is also able to prevent the access of the sub-
strate to the catalytic cavity since it is located in the substrate
binding pocket. It can therefore compete with the substrate during
binding. These compounds’ predicted orientations might be used
to design new ACE inhibitors with higher affinity through chemical
modifications.
were given in hertz (Hz) and abbreviations for multiplicity are re-
spectively called (s = singlet, d = doublet, dd = doublet of dou-
blets, t = triplet, dt = doublet triplet, m = multiplet).
All the reactions were monitored by TLC using commercial silica gel
plates and visualization was accomplished by UV light
3. Experimental section
3.1.1. General synthetic procedure of imines 2 (a-b)
To a cooled (0°) solution, 2 mL of sulfuric acid H2SO4 (95%) was
added dropwise and under magnetic stirring, to 1.25 eq of (ben-
zonitrile, p-chlorobenzonitrile) in 5 mL of hexane. Then, 500 mg
(3.33 mmol) of tertiary alcohol 1 (commercial product) in 5 mL
of hexane was added to the solution. After return to room tem-
perature, the resulting mixture was stirred at 68 °C for 2.5 h.
The solution was cooled at the room temperature, poured on ice-
cold water under magnetic stirring and alkalized with ammonia.
3.1. Chemistry
Solvents were purified by standard procedures. Melting points
(mp) were determined under a microscope with a Leitz Wet-zlair
device and uncorrected. NMR spectra were recorded on an AC 400
Bruker spectrometer at 400 MHz for 1H and 100 MHz for 13C,
chemical shifts (δ) were given in ppm and coupling constants (J)
6

A. Selmi, R. Aydi, O. Kammoun et al.
Journal of Molecular Structure 1235 (2021) 130230
The organic layer was extracted with dichloromethane, washed
with a saturated aqueous NaCl solution, dried over sodium sul-
fate, and filtered. Then, the solvent was removed in vacuo and the
obtained crude was purified by chromatography (silica gel, eluent
dichloromethane/methanol 98:2). The purity of the products was
checked by TLC.
3.2. X-ray crystallography
Suitable crystals were mounted on D8 VENTURE Bruker AXS
diffractometer. The structure was solved by direct methods using
the SIR97 program [44], and then refined with full-matrix least-
square methods based on F2 (SHELXL-97) [45]. All non-hydrogen
atoms were refined with anisotropic atomic displacement param-
eters. H atoms were finally included in their calculated positions.
A final refinement on F2 with 3216 unique intensities and 174 pa-
rameters converged at ωR(F2) = 0.0828 (R(F) = 0.0329) for 2811
observed reflections with I > 2σ(I).
Preparation
of
imine
2a:
3,3-Dimethyl-1-phenyl-3,4-
dihydroisoquinoline. The general procedure was followed using
429 mg (4.162 mmol) of benzonitrile to yield 2a as a white solid
(83%). mp: 69–71 °C. 1H NMR (400 MHz, CDCl3) δ ppm: 1.34 (s,
6H, 2CH3), 2.86 (s, 2H, CH2), 7.23–7.61 (m, 9H, H-Ar). ¹³C NMR
(100 MHz, CDCl3) δ ppm: 27.64, 38.84, 54.49, 126.43, 127.92,
128.00, 128.14, 128.22, 128.78, 128.95, 130.65, 137.54, 139.30,
164.53. HRMS (ESI) [M + H]+ calc. For C17 H18 N 236.1433; found
236.1424.
3.3.Hirshfeld. surface analysis
Hirshfeld surface analysis [46,47] is performed with CrystalEx-
plorer (Version 3.1) [48] to further understand the relative contri-
butions of intermolecular interactions by various molecular con-
tacts in the organic imine.
Preparation
of
imine
2b:1-(4-Chlorophenyl)−3,3-dimethyl-3,4-
dihydroisoquinoline. The general procedure was followed using
572 mg (4.16 mmol) of 4-chlorobenzonitrile to yield 2b as a white
solid (72%): mp 112–115 °C. 1H NMR (400 MHz, CDCl3) δ ppm:
1.27 (s, 6H, 2CH3), 2.80 (2H, CH2), 7.16 (m, 1H-Ar), 7.21 (m, 2H-Ar),
7.39 (m, 3H-Ar), 7.51 (m, 2H-Ar). 13C NMR (100 MHz, CDCl3) δ
ppm: 27.56 (2CH3), 38.84 (CH2), 54.69 (C-2CH3), 126.58, 127.67,
128.40, 130.26, 130.95, 135.10, 137.61, 137.68, 163.61. HRMS (ESI)
[M + H]+ calc. For C17 H17 ClN 270.1044; found 270.1034.
3.4. Biology
3.4.1. Determination of the angiotensin I-converting enzyme (ACE)
inhibition activity
The ACE inhibitory activity was assayed as reported by Naka-
mura et al. [49]. A volume of 80 μL containing different concentra-
tions (0.2, 0.4, 0.6 or 0.8 mg mL−1) of test compounds was added
to 200 μL of 5 mM hippuryl-l-histidyl-lleucine (HHL) and preincu-
bated for 3 min at 37 °C The test compounds and HHL were pre-
pared in 100 mM borate buffer (pH 8.3), containing 300 mM NaCl.
The reactions were then initiated by adding 20 μL of 0.1 U mL-1
ACE from rabbit lung prepared in the same buffer. After incuba-
tion for 30 min at 37 °C, the enzyme reactions were stopped by
the addition of 250 μL of 0.05 M HCl. The liberated hippuric acid
(HA) was extracted with ethyl acetate (1.7 mL) and then evapo-
rated at 90 °C for 10 min. The residue was dissolved in 1 ml of
distilled water and the absorbance of the extract at 228 nm was
determined using a UV–visible spectrophotometer (UV mini 1240,
UV/VIS spectrophotometer, SHIMDZU, China).
3.1.2. General synthetic procedure of imines 2 (c-d):
The cold imine 2(c-d) 600 mg was added dropwise to 6 mL
of sulfuric acid (95%). A solution of potassium nitrate (2,3 eq) in
2 mL sulfuric acid was added dropwise whith mainting the tem-
perature at 0 °C. The reaction medium was stirred at room tem-
perature for 2 h, then at 60 °C for 4 h. After return to room
temperature, the reaction medium was poured on ice-cold wa-
ter, and alkalized with ammonia. The organic phase was extracted
with dichloromethane, washed with a saturated aqueous solution
of chloride sodium, dried over sodium sulfate, filtered and evapo-
rated. The residue was purified by chromatography (silicgel, eluent
dichloromethane/ methanol 98:2). The purity of the products was
checked by TLC.
The average value from three determinations at each concentra-
tion was used to calculate the ACE inhibition rate as follows:
ꢀ
ꢁ
B − A
B − C
Preparation of imine 2c: 3, 3-Dimethyl-7-nitro-1-(3-nitrophenyl)−3,
4-dihydro isoquinoline. The general procedure was followed us-
ing 600 mg (2,52 mmol) of 2a to yield 2c as a yellow solid
(65%). Mp: 172–174 °C. 1H NMR (400 MHz, CDCl3) δ ppm: 1.31
ACE inhibition % =
x100
where A is the absorbance of HA generated in the presence of
ACE inhibitor, B is the absorbance of HA generated without ACE
inhibitors (100 mM borate buffer pH 8.3 was used instead of com-
pounds 2(a-d) and C is the absorbance of HA generated without
ACE (corresponding to HHL autolysis in the course of enzymatic
assay). The IC50 value, defined as the concentration of compounds
2(a-d) required to inhibit 50% of ACE activity, was calculated for
each sample using non-linear regression from a plot of percentage
ACE inhibition versus sample concentrations.
3
(s, 6H, 2CH3), 2.94 (2H, CH2), 7.47 (d, J = 8.1 Hz, 1H-Ar), 7.67
3
3
3
(t,
4
J = 8.1 Hz,
J = 7.5 Hz, 1H-Ar), 7.90 (ddd,
J = 7.5 Hz,
2.1 Hz,
2.1 Hz, 1H-Ar), 8.38
4
4
J
=
1.8 Hz,
J
=
1.2 Hz, 1H-Ar), 7.99 (d,
J
=
3
4
1H-Ar), 8.30 (dd,
3
J
=
8.1 Hz,
J
=
4
4
(ddd,
J = 8.1 Hz,
J = 2.1 Hz,
J = 1.2 Hz, 1H-Ar). 13C
NMR (100 MHz, CDCl3) δ ppm: 27.38 (2CH3), 38.68 (CH2), 55.45
(C-2CH3), 121.73, 123.79, 124.59, 126.03, 127.71, 129.75, 134.39,
145.07, 146.94, 148.49, 160.75. HRMS (ESI) [M + H]+ calc. For
C17H16N3O4 326.1135; found 326.1121.
3.4.2. Molecular docking of the chemical compounds in the ACE
binding site
Preparation of imine 2d:1-(4–chloro-3-nitrophenyl)−3, 3-dimethyl-7-
nitro-3, 4 dihydroisoquinoline. The general procedure was followed
using 600 mg (2,22 mmol) of 2b to yield 2d as a white solid
(93%). Mp: 194–196 °C. 1H NMR (400 MHz, CDCl3) δ ppm: 1.31
Docking material. Docking preparation and energy (kcal/mol) cal-
culations of compounds and Angiotensin-I converting enzyme
(ACE) were performed by MGL tool and AutoDock Vina software
with its scoring function [50-51]. MGL tool is used to generate
PDBQT files which are input files to run docking by Vina. In a
comparative assessment of scoring functions (CASF) carried out in
2018, popular and free software docking programs AutoDock and
Vina were generally in the first half and first quarter, respectively,
among all methods tested in CASF-2013. Vina was the best of all
methods in terms of docking power [52]. AutoDoc Vina uses a hy-
brid scoring function that is inspired by X-score [53], which ac-
counts for van der waals forces, hydrogen bonding, deformation
3
(s, 6H, 2CH3), 2.94 (2H, CH2), 7.49 (d, J = 8.3 Hz, 1H-Ar), 7.69
3
3
4
(d,
J = 8.3 Hz, 1H-Ar), 7.74 (dd,
J = 8.3 Hz,
J = 2.2 Hz,
J = 2.2 Hz,
4
4
1H-Ar), 8.04 (d,
1H-Ar), 8.32 (dd,
J = 2.2 Hz, 1H-Ar), 8.19 (d,
4
3
J = 8.3 Hz,
J = 2.2 Hz, 1H-Ar). 13C NMR
(100 MHz, CDCl3) δ ppm: 27.30 (2CH3), 38.69 (CH2), 55.59 (C-
2CH3), 121.54, 125.96, 126.15, 127.36, 128.50, 129.88, 132.11, 132.83,
137.78, 145.11, 147.01, 148.19, 159.77. HRMS (ESI) [M + H]+ calc. For
C17 H15ClN3O4 360.0745; found 360.0734.
7

A. Selmi, R. Aydi, O. Kammoun et al.
Journal of Molecular Structure 1235 (2021) 130230
penalty, and hydrophobic effect. In addition, it combines both the
conformational preferences of the receptor–ligand complex and ex-
perimental affinity measurements to compute its binding energy
[51]. Autodock vina allows a manual choice of the atom types for
grid maps, calculating grid map files with AutoGrid. Choosing the
”search parameters” and clustering the results after docking is no
longer necessary, as Vina calculates its own grid maps quickly and
automatically and also clusters and ranks the results [51]. Auto-
grid pre-calculation of the docking compounds was performed by
Autodock Vina. The energy grid was performed based on Lamar-
ckian genetic algorithm [53]. Discovery studio 3.5 (BIOVIA, Das-
sault Systèmes, [Discovery studio], [3.5], San Diego: Dassault Sys-
tèmes, [2020]) visualization software was used to perform the vir-
tual analysis of docking sites and to generate figures.
the most potent ACE inhibitors, hence, the introduction of a chloro
substituent in the aromatic ring and NO2 groups have been found
to affect the biological properties of Schiff base compounds. The
predicted positions of imines were close to the catalytic histidine
and zinc ion. This might highlight their efficiency as anti-ACE in-
hibitors.
The new confirmed crystal structure of compound 2b can be
considered interesting for further modification as antihypertensive
agents. Structure base chemical modification can lead to the design
of effective anti-hypertensive compounds.
Authors contributions
Author 1: Awatef SELMI
- Planned, Performed the chemistry experiments.
- analyzed spectra and designed molecules.
- Writing, editing and reviewing the manuscript.
Author 2: Rihab AYDI
Preparation of receptor and ligands. The three-dimensional struc-
ture of human ACE in complex with lisinopril (1O8A.pdb) was de-
rived from the RCSB PDB Protein Data Bank (http://www.rcsb.org/
pdb/home/home.do). Water molecules and the inhibitor lisinopril
were removed from the receptor file using Discovery Studio3.5
software. The structure was then protonated (non polar hydrogens
are merged), charges are added and structure saved in PDBQT file
format using MGL tool and Autodock 4.2 software.
-Aided in the chemistry experiments.
-Helped in writing the manuscript.
Author 3: Hajer BOUGATEF
- Processed and performed the biological part.
- Made and interpretation of the biological data.
- Helped in writing the biological part.
Author 4: Omar Kammoun
Imine compound structures were generated using ChemBio3D
Ultra 12.0 software (CambridgeSoft Co., USA), their energy was
minimized with the MM2 tools implemented to the software and
they are saved in pdb file formats. Gasteiger charges are added,
non polar hydrogens are merged and rotatable bonds detected then
files are converted to PDBQT format using MGL tool and Autodock
4.2 software.
-Verified and helped the X-ray crystallography
Author 5: Ali BOUGATEF
- Verified and helped in supervising the biological part.
Author 6: Nabil MILED
- Realized the molecular docking.
- Revision of the final manuscript.
Docking analysis. Most of docking parameters in Autodock vina are
set as default. The number of generated poses was kept 9 and the
exhaustiveness of the search was set to 10. A first docking run of
2a, 2b, 2c and 2d compounds on the whole enzyme showed their
binding into the catalytic cavity. A second docking run was carried
out in a box delimiting the catalytic cavity. The docking run was
carried out in a box centered on the macromolecule with a radius
of 1.00, center coordinates x: 42.852, y: 34.801 and z: 44.944 and
box size of x :30 ; y : 40 and z : 30.
Author 7: Othman A. Alghamdi
- Revision of the final manuscript.
Author 8: Corresponding author, Majed KAMMOUN
- Conceived the idea, encouraged to investigate, supervised the
findings of this work.
- Critical review and revision of the final manuscript.
Supplementary material
The macromolecule was set as rigid while ligand molecules
were kept flexible throughout the docking studies. Rotation was
possible for all the ligand’s rotatable bonds (One torsion for com-
pounds 2a and 2b and 3 torsions for compounds 2c and 2d). The
best ranked docking poses of each chemical compound in the ac-
tive site of ACE was obtained according to the scores and binding-
energy value. The root mean square deviations (RMSDs) between
the ligands’ orientations were calculated using ligRMSD software
[54].
Supplementary information file containing data tables of the
crystal structure of compound 2b, spectral (1 Н and
С) NMR of
13
compounds (2a-d) are available and Crystallographic data for the
structure 2b reported in this paper have been deposited with the
Cambridge Crystallographic Data centre, CCDC (2050888).
Declaration of Competing Interest
Docking validation. To check the internal variability of docking re-
sults due to random seeds of initial positions, three sets of docking
were ran for each compound. Maximal RMSD values found were
None
˚
˚
0.1 A for 2a, 2b and 2d and 0.19 A for 2c, calculated using LigRMSD
software.
Acknowledgements
Native ligand Lisinopril present in the protein structure (pdb
code 1O86.pdb) was used for re-docking experiments and the gen-
erated conformation was checked. RMSD values calculated using
ligRMSD software between the original structure and the gener-
This research was supported by the Ministry of Higher Edu-
cation, Scientific Research and Technology in Tunisia. The authors
wish to express their sincere gratitude to Dr Paul Mosset and Dr
Thierry Roisnel from ISCR-Rennes for their help to realize the X-
ray structure.
˚
˚
ated ligand best pose conformation was lower than 2 A (1.2 A).
4. Conclusion
Supplementary materials
In summary, we have described a highly efficient methodology
for the synthesis of four imines 2a-d, which we screened for their
ACE inhibition activity. Among them imines 2a and 2b exhibited
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130230.
8

A. Selmi, R. Aydi, O. Kammoun et al.
Journal of Molecular Structure 1235 (2021) 130230
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9