[(7-chloroquinolin-4-YL)amino]acetophenones and their copper(II) derivatives: Synthesis, characterization, computational studies and antimalarial activity

Article abstract of DOI:10.17179/excli2019-1805

The synthesis of the compounds [(7-chloroquinolin-4-yl)amino]acetophenones (4, 5) and their copper(II) complexes (4a, 5a) is reported. The compounds were characterized using a wide range of spectroscopic and spectrometric techniques, such as FTIR, UV-vis,

Full text of DOI:10.17179/excli2019-1805

EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
Original article:
[(7-CHLOROQUINOLIN-4-YL)AMINO]ACETOPHENONES AND
THEIR COPPER(II) DERIVATIVES:
SYNTHESIS, CHARACTERIZATION, COMPUTATIONAL STUDIES
AND ANTIMALARIAL ACTIVITY
Yonathan de J. Parraa*, Felix D. Andueza La, Rosa E. Ferrer Mb, Julia Bruno Colmenarezc,
María E. Acostad, Jaime Charrise, Philip J. Rosenthalf, Jiri Gutf
a
Escuela de Ingeniería Ambiental, Facultad de Ingeniería en Geología, Minas, Petróleos y
Ambiental, Universidad Central del Ecuador, Quito 170521, Ecuador
Departamento de Química, Facultad de Humanidades y Educación, Universidad del Zulia,
b
Apartado 526, Maracaibo, Estado Zulia, Venezuela
Centro de Investigación y Tecnología de Materiales (CITeMa), Instituto Venezolano de
c
Investigaciones Científicas, Apartado 20632, Altos de Pipe 1020-A Estado Miranda,
Venezuela
Laboratorio de Bioquímica, Facultad de Farmacia, Universidad Central de Venezuela,
d
Apartado 47206, Los Chaguaramos, 1041-A Caracas, Venezuela
Laboratorio de Síntesis Orgánica, Facultad de Farmacia, Universidad Central de
e
Venezuela, Apartado 47206, Los Chaguaramos, 1041-A Caracas, Venezuela
Department of Medicine, University of California, Box 0811, San Francisco, California
f
94143, USA
* Corresponding author: Prof. Yonathan de J. Parra, Escuela de Ingeniería Ambiental,
Facultad de Ingeniería en Geología, Minas, Petróleos y Ambiental, Universidad Central
del Ecuador, Quito 170521, Ecuador; Tel: +593-2-2550588, ext. 111;
E-mail: ydparra@uce.edu.ec
http://dx.doi.org/10.17179/excli2019-1805
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
The synthesis of the compounds [(7-chloroquinolin-4-yl)amino]acetophenones (4, 5) and their copper(II) com-
plexes (4a, 5a) is reported. The compounds were characterized using a wide range of spectroscopic and spectro-
metric techniques, such as FTIR, UV-vis, NMR, EPR, ESI-CID-MS2. The spectral results suggested that the ligand
acted as chelating species coordinating the metal through the endocyclic nitrogen of the quinoline ring in both
complexes, with general formulae expressed in two ways, according to the phase in which they are: [Cu(L)2Cl2]
for solid phase and [Cu(L)2][2Cl] for liquid phase. The EPR study of the Cu (II) complexes indicated a probable
distorted tetrahedral coordination geometry. This result was confirmed by the calculated optimized structures at
the DFT/B3LYP method with the 6-31G (d,p) basis set. The characterization of the fragmentation pattern of pro-
tonated free ligands was extended here to fragments as low as m/z 43, while for coordination complexes it extends
to fragments at m/z 80 and m/z 111. The antimalarial activity of the compounds was determined through three
different tests: inhibitory activity against in vitro growth of Plasmodium falciparum (W2), inhibition of hemozoin
formation (β-hematin) and in vitro inhibitory activity against recombinant falcipain-2, where compound 5 showed
considerable activity. However, the activity of free ligands against P. falciparum was increased by complexing
with the Cu (II) metal ion. The values of the HOMO-LUMO energy gap of 3.847 eV (4a) and 3.932 eV (5a) were
interpreted with high chemical activity and thus, could influence on biological activity. In both compounds, the
total electron density surface mapped with electrostatic potential clearly revealed the presence of high negative
charge on the Cu atom. Also, this study reported the molecular docking of free ligands (4, 5) using software pack-
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
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age ArgusLab 4.0.1. The results revealed the importance of water molecules as interaction bridges through hydro-
gen bonds between free ligands and β-hematin; at the same time, the hypothesis that π–π interaction between
quinoline derivatives and the electronic system of hematin governs the formation of adducts was confirmed.
Keywords: acetophenone; antimalarial; copper; DFT; fragmentation; quinoline
INTRODUCTION
MATERIALS AND METHODS
Malaria, a traditional infectious disease,
All reagents and solvents used were of an-
added 219 million new malaria cases world- alytical grade. Organic chemicals and metal
wide by 2017, with an estimated 435,000 salts, such as 4,7-dichloro-quinoline, amino-
deaths of which approximately 61 % were acetophenones and cupric chloride were ob-
children under five years. (WHO, 2018). tained from Aldrich Chemical Co, USA.
There are therapeutic treatments that belong
to the 4-aminoquinolines family that reduce General procedures
the mortality rate for malaria. However, many
The solvents were subjected to previous
medications related to this family have de- purification and drying processes by standard
creased in effectiveness given the ability of methods. The progress of the reactions and
Plasmodium (mainly falciparum, vivax and purity of the synthesized compounds was
malariae) to develop resistance, a major ob- evaluated by thin-layer chromatography
stacle in the fight against malaria (Parra and (TLC) using silica gel 60 plates with UV254
Ferrer, 2012).
fluorescent indicator, an ultraviolet lamp and
These facts lead to the need of develop- a suitable solvent system.
ment of new drugs to reduce resistance events
Melting points were measured with a Stu-
and increasing the effectiveness of such sub- art™ SMP3 melting point apparatus (Sigma-
stances; for this, various drug design method- Aldrich). Infrared spectra were recorded on
ologies have been proposed, including the KBr pellets with a Shimadzu FTIR 8400 spec-
synthesis of molecules based on leading com- trophotometer (400–4000 cm-1). Nuclear
pounds or pharmacophores, molecular hy- Magnetic Resonance spectra were taken using
bridization and metal complexing (Kouz- two spectrometers, one of Jeol Eclipse brand
netsov and Amado, 2008; Kouznetsov and and another Bruker Avance II brand, of 270
1
Gómez-Barrio, 2009; Camacho et al., 2005).
MHz and 300 MHz for H NMR, respec-
In this sense, as part of our research on tively. Both devices have the power of 67.9
the synthesis and biological evaluation of 4- MHz for 13C NMR.
aminoquinolines with potential antimalarial
Electron Paramagnetic Resonance (EPR)
activity, we report here synthetic, spectro- spectra were taken at 24 °C on a reconstructed
scopic, spectrometric, computational aspects and automated X-band Varian E-Line spec-
and antimalarial activity of compounds based trometer. A rectangular cavity resonating in
on the pharmacophore nucleus 7-chloro-4- the TE-102 mode was used, with modulation
aminoquinoline (antimalarial) (Egan et al., of 100 kHz, using a microwave power of 5
2000), acetophenone unit to supply other re- mW. ESI-CID-MS2 spectra were recorded us-
active sites (electrophilic unit) (Chiyanzu et ing a TSQ Quantum triple-quadrupole mass
al., 2005) and metallic centers of kind Cu(II) spectrometer (Thermo Scientific brand) cou-
to potentiate pharmacological properties, pled to a liquid chromatography (CL) system.
(Bahl et al., 2010) through possible participa-
tion in the metabolic distribution of copper in Synthesis of free ligands (4 and 5)
Plasmodium falciparum (Rasoloson et al.,
The synthesis of the free ligands was car-
2004), potential mechanism for new pathways ried out applying the protocol of Ferrer et al.
of antimalarial drugs.
(2009). In a two-necked reaction flask, a mix-
ture was prepared in a molar ratio 1:1 of the
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reactants, by dissolving 0.5 g (2.5 mmol) of flow cytometry (CellQuest software, Becton
4,7-dichloro-quinoline (1) in dry ethanol to Dickinson). Percentile plots of parasitemia
then add 0.37 g (2.75 mmol) of the 4- or 3- versus compound concentrations were used to
aminoacetophenone (2 or 3), subjecting the obtained IC50. The data were fit to a nonlinear
mixture to refluxing (80-85 °C) with constant regression analysis using GraphPad Prism
stirring for 9 hours. The resulting products 4- 3.02 software (Reinders et al., 1995). For sta-
[(7-chloroquinolin-4-yl)amino]acetophenone
tistical analyses, Student’s t-tests were per-
(4) and 3-[(7-chloroquinolin-4-yl)amino]ace- formed, and significance was set at p < 0.05.
tophenone (5) were filtered, washed with eth-
Inhibition of hemozoin formation
anol and diethyl ether. These ligands were re-
(β-hematin)
crystallized from a mixture of ethanol-metha-
The assay of inhibition of β-hematin for-
nol (2:1). The reaction route is shown in
mation (synthetic hemozoin) was developed
according to the protocol described by
Scheme 1.
Baelmans et al. (2000). Only the free ligands
were considered in the inhibitory test where
chloroquine was the control. Results are ex-
pressed as percentage of inhibition of β-hem-
atin formation (% IβHF). The significance
was set at p > 0.05 compared to chloroquine.
Synthesis of metal complexes
A solution of copper (II) chloride (0.02 M
and 25 mL MeOH) was added to a methanol
solution of free ligand (0.02 M and 25 mL
MeOH). The reaction mixture was refluxed
on a sand bath for 17 hours at 60 °C with con-
stant agitation and inert atmosphere (Ar). In
both cases, solid-colored complexes were ob-
tained and collected by filtration, washed on
paper with cold methanol and diethyl ether,
dried under vacuum in an oven at 40 °C. The
purity of the complexes was checked by TLC.
In vitro inhibitory activity against
recombinant falcipain-2
The IC50 values against the recombinant
enzyme, falcipain-2, were determined from
the plots of activity percentage over the com-
pound concentrations; the data was fit to a
nonlinear regression analysis using GraphPad
Prism 3.02 software. The controls used were
the cysteine protease inhibitor E-64 [N-(trans-
epoxysuccinyl)-1-leucine-4-guanidinobutyla-
mide] and CuCl2 (Rosenthal et al., 1996;
Shenai et al., 2000). For statistical analyses,
Student’s t-tests were performed, and signifi-
cance was set at p < 0.05.
Antimalarial activity
Inhibitory activity against in vitro growth of
Plasmodium falciparum (W2)
The antimalarial activity was evaluated
using the protocol described by Chipeleme et
al., (2007). The controls used were chloro-
quine, artemisinin and CuCl2. Parasitemia
was determined by fluorescence plots using
Scheme 1: Synthesis of [(7-chloroquinolin-4-yl)amino]acetophenones (4, 5)
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Computational section
score in kcal mol-1). Hydrogen bond interac-
tions, as well as favorable π-π interactions be-
tween the ligands and hemozoin dimer solv-
Computational details
The molecular structure of compounds 4a
and 5a have been elucidated theoretically
with Gaussian 09 program using the DFT
(B3LYP) level of theory at 6-31G (d,p) basis
ated, were explored.
RESULTS AND DISCUSSION
set (Frisch et al., 2009). The harmonic vibra- Characterization of ligands (L)
tional frequencies were calculated at the same
4-[(7-chloroquinolin-4-yl)amino]aceto-
levels of theory for the optimized structure phenone (4). Pale yellow amorphous com-
and the vibrational band assignments were pound, yield: 94.66 %, m.p. 288-290 °C, Rf
made using the Gauss-View molecular visu- (Hexane:ethyl acetate = 3:7): 0.76. UV-vis
alization program (Dennington et al., 2007). (DMSO) [λmax (nm), ε (cm-1 mol-1 L), assign-
The nonexistence of imaginary wavenumbers ment]: 261, 2100, aromatic π→π*; 362, 2792,
of the theoretically calculated vibrational π→π* and/or n→π* (conjugated carbonyl). IR
spectra affirms that these deduced structures (KBr) cm−1: 3440, 1678, 1623, 1588, 1098.
correspond to minimum energy. However, the 1H NMR (DMSO-d6): δ 2.62 (s, 3H, CH3),
wavenumber values obtained at these levels 7.04 (d, 1H, H3, J2,3: 6.7 Hz), 7.66 (d, 2H,
contain well-known systematic errors (For-
esman and Frisch, 1996). Therefore, the cal- J5,6: 9.2 Hz, J6,8: 1.7 Hz), 8.12 (d, 2H, H2´,6´
culated vibrational wavenumbers were scaled 2´,3´ = J5´,6´: 8.4 Hz), 8.22 (d, 1H, H8, J6,8: 1.7
with a scale factor of 0.9614 for B3LYP Hz), 8.62 (d, 1H, H2, J2,3: 6.9 Hz), 8.95 (d, 1H,
H3´,5´, J2´,3´ = J5´,6´: 8.4 Hz), 7.89 (dd, 1H, H6,
,
J
method (Young, 2001).
H5, J5,6: 9.2 Hz), 11.39 (broad s, 1H, NH). 13C
NMR: δ 27.21 (CH3), 102.2, 117.42, 120.73,
124.50, 126.99, 127.84, 130.44, 135.22,
138.43, 140.86, 142.67, 145.06, 154.14 (Aro-
matic-C), 197.30 (C=O). HRMS m/z (ESI):
297.00 [M+H]+, exact mass calculated for
C17H14ClN2O [M+H]+: 297.00, found:
297.00; ESI-CID-MS2: 297.00 (21) [M+H]+,
253.99 (100), 218.14 (16), 163.10 (3), 137.97
(≈ 1), 111.07 (≈ 1), 91.15 (≈ 1), 77.06 (≈ 1),
43.17 (≈ 1).
3-[(7-chloroquinolin-4-yl)amino]aceto-
phenone (5). Pale yellow crystalline com-
pound, yield: 97.92 %, m.p. 294 °C, Rf (Hex-
ane:ethyl acetate = 3:7): 0.73. UV-vis
(DMSO) [λmax (nm), ε (cm-1 mol-1 L), assign-
ment]: 259, 2351, aromatic π→π*; 347, 2335,
π→π* and/or n→π* (conjugated carbonyl). IR
(KBr) cm−1: 3440, 1685, 1611, 1581, 1100.
1H NMR (DMSO-d6): δ 2.62 (s, 3H, CH3),
6.87 (d, 1H, H3, J2,3: 6.9 Hz), 7.75 (m, 2H,
H4´,6´), 7.90 (dd, 1H, H6, J5,6: 9.2 Hz, J6,8: 1.97
Hz), 8.01 (t, 1H, H5´, J: 7.4 Hz), 8.04 (s, 1H,
H2´), 8.21 (d, 1H, H8, J6,8: 2.2 Hz), 8.55 (d, 1H,
H2, J2,3: 6.9 Hz), 8.95 (d, 1H, H5, J5,6: 9.2 Hz),
11.39 (broad s, 1H, NH). 13C NMR: δ 27.3
(CH3), 102.8, 119.0, 121.9, 124.3, 125.1,
125.8, 127.2, 127.9, 130.4, 134.8, 138.7,
141.4, 148.4, 149.8, 152.2 (Aromatic-C),
Molecular docking procedure
The ligands in protonated form (4 and 5)
were built using the molecule builder of the
software package ArgusLab 4.0.1. Geometry
optimizations and energy calculations of the
ligands were performed using the PM3 semi-
empirical quantum-mechanical method. The
binding site was the three-dimensional struc-
ture of the hemozoin dimer (β-hematin), and
it was obtained from the Cambridge Crystal-
lographic Database REFCODE: XETXUP
(Pagola et al., 2000). Hemozoin dimer was
solvated in order to simulate experimental
conditions of the in vitro evaluations, consid-
ering only those water molecules that estab-
lish H-bonds with the hemozoin dimer. The
docking program implements an efficient
grid-based docking algorithm which approxi-
mates an exhaustive search within the free
volume of the binding site solvated, around
which was built a bounding box (25 x 25 x 25
Å). It was used ArgusDock engine, and the
conformational space was explored by the ge-
ometry optimization of the flexible ligand un-
der a condition of high precision docking. The
final positions of the compounds were ranked
by lowest interaction energy values (docking
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198.2 (C=O). HRMS m/z (ESI): 296.95 trigger the processes of fragmentation (Ya-
[M+H]+, exact mass calculated for Ping, 2012). For this reason, it is convenient
C17H14ClN2O [M+H]+: 297.00, found: to differentiate between the terms protonation
296.95; ESI-CID-MS2: 297.00 (28) [M+H]+, site(s) and dissociative protonation site(s)
253.01 (100), 218.03 (33), 163.01 (5), 137.97 (Ya-Ping, 2006).
(≈ 1), 110.99 (≈ 1), 91.09 (≈ 2), 77.09 (≈ 1),
43.13 (≈ 1).
The fragments observed in the MS2 spec-
tra of compounds 4 (Figure 1) and 5 (Figure
Spectral data confirmed the proposed mo- 2) were similar, recording a signal at m/z 297
lecular and structural formula for the ligands corresponding to the precursor molecular ion
of kind acetophenone (Scheme 1). Supple- [C17H13ClN2O + H]+. This ion was probably
mentary Figures 1, 5, 7, 11 illustrate the NMR protonated in the quinolinic nitrogen, the
spectra. The spectra of compounds registered most favorable thermodynamic position ac-
signals due to the presence of ethanol used in cording to the proton affinity values (Hunter
the purification process.
and Lias, 1998). Under induced collision, the
molecular ion protonated in nitrogen at m/z
297, only released one molecule of HCl (36
Da) but did not generate any fragment indi-
cating the decomposition of the pyridine sub-
structure of the quinoline ring (Figure 3).
The main fragmentation reaction begun
with the deacylation by heterolytic cleavage
of the alpha (α) bonds to the carbonyl. This
pathway was possible if one considers the mi-
gration of a proton from the initial protonation
site (quinolinic-N) to the site of dissociative
protonation. So, proton transfer is proposed
before fragmentation occurs (Thielking et al.,
1992; Pengyuan et al., 2010; Demarque et al.,
2016).
Mechanisms of mass spectral fragmentation
The study of compounds 4 and 5 through
electrospray ionization (ESI), followed by
tandem mass experiments (MS2) with colli-
sion-induced dissociation (CID), at a voltage
of 40-45 eV and in mode positive; generated
interesting mass spectrometry results.
The elucidation of the precursor molecu-
lar ion fragmentation pathways [4 + H]+ and
[5 + H]+ allowed us to understand the dynam-
ics of the protonation of the 4-amino-7-chlo-
roquinoline nucleus in the presence of the ac-
etyl group, the protonation of the thermody-
namically favored position does not always
Figure 1: CID Product ion spectrum of protonated 4 by MS-MS
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Figure 2: CID Product ion spectrum of protonated 5 by MS-MS
After proton transfer (Figure 3), the acetyl abstraction by an intermolecular electrophilic
group underwent two routes of fragmentation reaction (Thielking et al., 1992; Wenthold and
reactions, which differed in the number of Liu, 2001). The first mechanism generated the
breaks of α-bonds to the carbonyl. Route 1 observed acyl ion at m/z 43, while the second
was given by cleavage of the methyl-carbonyl mechanism provided the main product ion at
α-bond, releasing methane (16 Da) and pro- m/z 253-254.
ducing the ion at m/z 281, in turn, this frag-
As can be seen in the spectra of com-
ment generated the ion at m/z 253-254 by loss pounds 4 (Figure 1) and 5 (Figure 2), the sig-
of a mass of 28 Da (carbon monoxide). Route nals at m/z 253, 254 and 255; suggest different
2 implies a process of fragmentation that is isotopic distributions due to the presence of
explained through the formation of a neu- heteroatoms in the structure. Figure 4 shows
tral/ion complex, but where apparently two the proposed fragmentation from the ion at
mechanisms competed, one of dissociation m/z 253-254 (Supplementary Figures 13-16),
(Thielking et al., 1992; Filges and with the probable structures of descendant
Grützmacher, 1987) and another of hydride fragments (Table 1).
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Dissociative protonation site
Favorable protonation site
H
O+
O
HN
HN
N
Proton transfer
297
m/z
Cl
N+
H
Cl
-(36)
route 2
1,4-H+ transfer
route 1
- (16)
O
No initial fragmentation of
the quinoline substructure
pyridine ring was observed.
O
C+
O
H H
C
+
HN
HN
HN
N
N+
Cl
N
Cl
H
m/z 281
m/z 261
1,2-H- transfer
O
H
H
C+
C
HN
N
Cl
Deacylation
-(28)
O
H
C+
HN
Cl
N
neutral / ion complex
Intermolecular
electrophilic reaction
Dissociation
H
C+
HN
N
HN
O
O
C+
H
+
+
Cl
Cl
N
m/z 43
m/z 253-254
Figure 3: Proposed fragmentation mechanisms for precursor molecular ion [4 + H]+ (shown) or [5 + H]+.
Note: numbers in parentheses represent mass loss due to neutral molecules.
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Figure 4: Proposed fragmentation scheme from the product ion at m/z 253-254. Note: [ ]: fragmentation
path, *: free radical path, ( ): neutral residual fragment.
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Table 1: Proposed structures for some fragments originating from the ion at m/z 253-254
Fragmentation path
Structures (m/z: relative abundance for 4/5)
C+
HN
H
H
H
a1
Cl
N
26 Da
C
(227-228: 0-1/1-2)
H
C
H
C+
HN
a2
H
Cl
N
39 Da
(214-215: 2-3/2-3)
C+
HN
HCl
36 Da
a3
a4
N
(217-219: 15/33)
N
H
C
H
N+
Cl
H
76 Da
(177-178: ˂1/1)
N
H
C+
a5
a6
N
Cl
(77: ˂1/˂1)
176 Da
C
+
C
HN
Cl
N
(91: ˂1/1-2)
(162: ˂1/1)
C+
HN
b1
HCl
36 Da
N
(191-192: ˂1/1-2)
H
N
C+
b2
b3
Cl
N
(162-163: 2-3/5)
65 Da
C+
HCN
27 Da
N
H
Cl
(150-151: ˂1/1-2)
C2 neutral
24 Da
+
N
c1
d1
Cl
H
(138: 1/1-2)
C+
HCN
27 Da
Cl
(111: ˂1/1)
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Characterization of the Cu(II) complexes
{4-[(7-chloroquinolin-4-yl)amino]ace-
Molecular structures
The computational calculations of the
tophenone}copper(II) chloride (4a). Dark compounds 4a and 5a, including geometry
green solid compound, m.p. 228-230 °C, IR optimization, single point energies and vibra-
(KBr) cm−1: 3440, 1676.8, 1619.2, 1584, tional wavenumbers evaluations; were per-
1
1094. EPR (solid): giso = 2.1658. H NMR formed by using Gaussian 09 program at
(DMSO-d6): δ 2.59 (s, 3H, CH3), 6.53 (d, 1H, B3LYP/6-31G (d,p) level. The B3LYP is a
H3, J2,3: 8.2 Hz), 7.61 (d, 2H, H3´,5´, J2´,3´
=
solid and efficient exchange correlation func-
J5´,6´: 7.8 Hz), 7.81 (dd, 1H, H6, J5,6: 8.7 Hz), tion for calculating energies and geometries;
8.08 (d, 2H, H2´,6´, J2´,3´ = J5´,6´: 7.8 Hz), 9.34 it has been proven to be able to yield reliable
(broad s, 1H, H5), 10.92 (broad s, 1H, NH). results for small molecules.
13C NMR: δ 27.23 (CH3), 112.85, 124.11,
The difference in experimental and calcu-
127.30, 128.03, 130.53, 134.99, 138.08, lated geometrical parameters came from the
142.89 (Aromatic-C), 197.25 (C=O). ESI- environment of the compound. It is clear that
CID-MS2: [M(L)2]+ = [65Cu(C17H13ClN2O)2]+ the experimental results belong to solid phase,
= 657.18 (38). Selected fragments of [M(L)2]+ and the theoretical calculations have been per-
= 378.93 (50), 360.87 (100), 280.99 (10), formed in the gas phase and the intermolecu-
260.87 (7).
{3-[(7-chloroquinolin-4-yl)amino]ace-
lar interactions are not taken into account. The
molecular structure of the compounds 4a and
tophenone}copper(II) chloride (5a). Light 5a with atom numbering scheme adopted in
green crystalline compound, m.p. 224 °C, IR the computations is shown in Figures 5a and
(KBr) cm−1: 3440, 1680, 1613, 1578, 1100. 5b, respectively.
EPR (solid): gx = 2.3436, gy = 2.1622, gz =
2.1055. H NMR (DMSO-d6): δ 2.63 (s, 3H,
CH3), 6.92 (broad s, 1H, H3), 7.74 (m, 2H,
FTIR analysis
1
The FTIR spectra of both compounds
were recorded in the range of 4000-400 cm-1
H4´,6´), 7.89 (d, 1H, H6, J5,6: 8.1 Hz), 8.00 (m,
and calculated for the optimized structures at
2H, H2´,5´), 8.16 (broad s, 1H, H8), 8.85 (d, 1H,
the DFT/B3LYP method with the 6-31G (d,p)
H5, J5,6: 9.1 Hz), 11.1 (broad s, 1H, NH). 13C
basis set. Both compounds have 71 atoms
NMR: δ 27.4 (CH3), 100.0, 125.2, 126.6,
with 207 normal modes of vibrations.
127.8, 128.1, 130.3, 131.1, 138.2, 139.1 (Ar-
In the experimental FTIR spectra of both
omatic-C), 197.8 (C=O). ESI-CID-MS2:
compounds was possible to observe the ab-
[M(L)2]+ = [65Cu(C17H13ClN2O)2]+ = 657.44
sorption band 3440 cm-1 due to NH stretching,
(19). Selected fragments of [M(L)2]+ = 378.94
whereas this vibration corresponded to
(9), 360.92 (100).
3635.40 cm-1 and 3637.59 cm-1 in the calcu-
lated FTIR spectra of the compounds 4a and
5a, respectively.
Figure 5: Optimized structures of the compounds 4a (a) and 5a (b) on B3LYP/6–31G(d, p) basis set
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
The absorption band corresponding to indicated the cationic fragment of the coordi-
C=O stretching vibration of the ketone group nation complexes was the [Cu(L)2]+ type, reg-
was observed at 1676.8 cm-1 in the experi- istering a signal at m/z 657; they corresponded
mental IR spectrum and 1616.79 cm-1 in the to
the
calculated IR spectrum for compound 4a. In [Cu(C17H13ClN2O)2]+.
compound 5a, this stretching was also present The center of positive charge was on the
at 1680 cm-1 and 1661.13 cm-1 in the experi- metal, but contrary to the expected according
mental and theoretical spectra, respectively. to the electronic paramagnetic resonance data
precursor
molecular
ion
The absorption band associated with C=N (Figures 10 and 11), where the paramagnetic
bond stretching, for compound 4a, appears at character of the complex was shown, and
1584 cm-1 in the experimental IR spectrum therefore, the oxidation state Cu(II) was con-
and 1551.63 cm-1 in the theoretical spectrum; firmed. In gaseous phase, the metal is in the
whereas for compound 5a, these are observed Cu(I) form; this is indicated by the determina-
at 1577.6 cm-1 and 1562.81 cm-1, respectively. tion of the state of charge of the signals in the
The C-N stretching band appeared at 1356 spectra where different charge distributions
cm-1 and 1351.15 cm-1 in the experimental are not observed. These electronic differences
and theoretical spectra of compound 4a, and suggest that copper underwent a reduction
in the compound 5a, 1360 cm-1 and 1359.94 during ionization by electrospray, which is
cm-1.
reasonable considering the experimental con-
Finally, the absorption band correspond- ditions.
ing to C-Cl stretching, for compound 4a, ap-
The solutions of these complexes were
peared at 1094 cm-1 in the experimental spec- prepared in mixtures of dimethylsulfox-
trum and 1091.88 cm-1 in the theoretical spec- ide:methanol (v/v 1:2) and dispersed by ap-
trum. In compound 5a, the same signal was plying a very high voltage (2-6 kV), this
observed at 1100 cm-1 in the experimental strong electric field exceeded the ionization
spectrum and 1090.32 cm-1 in the theoretical energy of the molecules of DMSO of 9.1 eV
spectrum.
(Kimura et al., 1981) and MeOH of 10.85 eV
(Tao et al., 1992). Thus, an electron could be
transferred from the neutral solvent molecules
to the ion of the doubly charged metal Cu(II).
This is a favorable-energetically reduction
process because this metal has high second
ionization energy (20.3 eV) (Haynes, 2012;
Shi et al., 2006; Park et al., 2007), which is
higher than all the transition metals.
On the other hand, copper has two iso-
topes (63Cu and 65Cu) with a natural abun-
dance ratio of 69.09:30.91 (Haynes, 2012). In
this sense, Figures 6 and 7 show the CID-MS2
spectra specifically for the precursor molecu-
lar ion [65Cu(C17H13ClN2O)2]+. However, as
noted below, additional signals were indicat-
ing the presence of the 63Cu isotope.
Mechanisms of mass spectral fragmentation
of the Cu(II) complexes
The experimental conditions during the
electrospray ionization process (ESI) favored
the partial or complete dissociation of the un-
coordinated counterions, but also of those
weakly coordinated to the metal, registering
in the spectrum only the cationic fragment
(Wikstrom et al., 2010; Underwood et al.,
2013).
The MS2 spectra of compounds 4a (Figure
6) and 5a (Figure 7) showed slight differences
that reveal changes in fragmentation patterns,
which is inferred to be a consequence of isom-
erism between ligands 4 and 5. However, both
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
Yonathan2 #597 RT: 14.42 AV: 1 NL: 3.96E6
T: + c ESIFull ms2 657.000@cid35.00 [30.000-750.000]
360.87
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
358.81
378.93
657.18
280.99
10
260.87
282.63
342.85
538.20
538.20
5
0
639.10
252.52
391.26 431.72
400
m/z
603.07
540.21
80.08 126.91 178.53
100 200
684.65 729.41
700
300
500
600
gbfb
Figure 6: Positive ion mode ESI-CID-MS2 spectrum of the ion [65Cu(C17H13ClN2O)2]+ at m/z 657.18
(cationic unit of 4a complex)
Yonathan1 #267 RT: 9.42 AV: 1 NL: 8.16E5
T: + c ESIFull ms2 657.000@cid35.00 [30.000-750.000]
360.92
100
95
90
85
80
75
70
65
60
55
50
45
358.83
40
35
30
25
657.44
20
15
10
5
378.94
323.08
282.97
252.94
36.03
110.55 137.03
138.47
435.11
526.14 575.42 637.86
639.05
723.16
700
0
100
200
300
400
m/z
500
600
Figure 7: Positive ion mode ESI-CID-MS2 spectrum of the ion [65Cu(C17H13ClN2O)2]+ at m/z 657.44
(cationic unit of 5a complex)
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
The pathways of fragmentation observed
The fragment at m/z 359-361 underwent
for the cationic unit of the complex 4a and its three dissociation pathways and a reversible
product ions are shown in Figure 8. The dis- re-coordination/de-coordination of a water
sociation
induced
at
35
eV
of molecule (Supplementary Figures 18 and 19).
[65Cu(C17H13ClN2O)2]+ (m/z = 657) resulted A dissociative path occurred by homolytic
in two channels. The minority channel (Sup- cleavage of the methyl-carbonyl α-bond of
plementary Figure 17) begun with the tautom- the ligand, releasing the neutral ligand and a
erization of each ligand up to its enol form, methyl-copper at m/z 80. Another dissociation
with subsequent inter-ligand proton transfer proceeded by heterolytic cleavage of the
accompanied by dissociation, promoting the chloro-quinoline bond, generating the chlo-
loss of an H2O molecule to generate the ion at ride anion (Cl-) which was spontaneously
m/z 639. This ion lost HCl to give the frag- driven up to the metal ion Cu(I) finally losing
ment at m/z 603, which released neutral cop- CuCl and producing the ion at m/z 261. This
per by charge reduction through the transfer product was also generated from the fragment
of an electron from the ligand to the Cu(I) at m/z 341 (63Cu) - 343 (65Cu) by liberation of
metal ion, producing the fragment at m/z 538 CuOH.
(35Cl ) - 540 (37Cl).
The last dissociation pathway, character-
This mechanism of dissociation by tau- ized by its extensive fragmentation, was trig-
tomerization and subsequent loss of H2O, gered by the heterolytic cleavage of the me-
suggests that the carbonyl groups did not act thyl-carbonyl α-bond of the ligand, generat-
as coordination sites to the metal, a property ing the methyl carbanion (-CH3) that reacts
1
that is in agreement with that found by H with the Cu(I) ion, releasing the neutral
NMR and 13C NMR.
CuCH3 and yielding the cation at m/z 281
The dominant channel (Supplementary (35Cl) - 283 (37Cl). This fragment formed the
Figure 18) involved the loss of a neutral lig- ion at m/z 253 by the loss of a molecule of CO,
and molecule to generate the ion at m/z 359 which in turn, lost the neutral 1,2,3,4-tetrade-
(63Cu) - 361 (65Cu). This ligand released with- hydrobenzene, and formed the ion at m/z 179.
out prior inter-ligand proton transfer or elec- Finally, this ion underwent a double simulta-
tron transfer from the ligand to the metal, sug- neous dissociation releasing HCl and NH3 to
gesting that the driving force of the dissocia- produce the cation at m/z 127, corresponding
tion was different from the coulombic repul- to 7,8-didehydroquinolin-4-ilium.
sions. Consequently, it is proposed that steric
The fragmentation mechanism of the cat-
repulsions promoted dissociation; these inter- ionic unit of the coordination complex 5a
actions were more intense if it is considered (Figure 9) it is not discussed in detail, due to
that both ligands were coordinated to the the presence of some dissociations, equivalent
metal by means of the quinolinic nitrogen at- to those shown for the cationic fragment of
oms.
the complex 4a.
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
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Figure 8: Proposed fragmentation for the ion [65Cu(C17H13ClN2O)2]+ at m/z 657 (cationic unit of 4a com-
plex)
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
Figure 9: Proposed fragmentation for the ion [65Cu(C17H13ClN2O)2]+ at m/z 657 (cationic unit of 5a com-
plex)
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
Electronic paramagnetic spectra of the
Cu(II) complexes
NMR spectral studies of the Cu(II)
complexes
The 1H NMR spectra of the complexes 4a
The EPR spectrum at room temperature
for coordination complex 4a (Figure 10)
showed an isotropic signal without any hyper-
fine splitting, due to dipolar interactions be-
tween magnetic moments (electronic and nu-
clear) (Zhidomirov et al., 1969) and increased
spin-red relaxation (Hoffmann et al., 1996),
with a value of g o giso = 2.1658. The value of
giso tensor obtained in this study, indicated an
increase in the covalence of a bond between
the metal ion Cu(II) and the ligand molecules,
when compared with the value of g for the
free electron (ge- = 2.0023) (Angelusiu et al.,
2008)
The EPR spectrum at room temperature
for coordination complex 5a (Figure 11)
shows a rhombic symmetry, with three values
of the tensor g of, gx = 2.3436, gy = 2.1622
and gz = 2.1055, where the lowest value of g
is higher than 2.04 (gz > 2.04). The geometric
parameter G for this complex shows a value
of less than 4; this suggests significant ex-
change interactions between centers metal in
the solid state, which is probably a conse-
quence of the closeness between independent
molecules of the complex (Hathaway and
Billing, 1970).
(Supplementary Figure 2) and 5a (Supple-
mentary Figure 8) indicate that the quinoline
ring protons were significantly affected by the
effect of the paramagnetic metal ion. For the
complex 4a, the signals assignable to H2 and
H8 were absent, while the complex 5a did not
show H2 signal, but if the one assigned to H8
with a pronounced widening and diminution
of its intensity (Table 2). The protons of the
phenyl ring and the methyl do not undergo
significant changes; for these reasons, the
quinolinic nitrogen atom must be involved in
the coordination. The above statement is con-
firmed by the study of 13C NMR spectra (Sup-
plementary Figures 6 and 12), where both
compounds recorded spectra with disappear-
ances of signals due to resonances of carbon
atoms of the quinoline ring (Table 2).
Table 2: Comparison of NMR signals of the quinoline ring between ligands (4, 5) and complexes (4a,
5a)
Signals
Compounds
4
4a
5
5a
Quinoline protons
n.d.
H2
H3
H5
H6
H8
8.62 (d)
7.04 (d)
8.95 (d)
7.89 (dd)
8.22 (d)
8.55 (d)
6.87 (d)
8.95 (d)
7.90 (dd)
8.21 (d)
n.d.
6.53 (d)
9.34 (bs)
7.81 (dd)
n.d.
6.92 (bs)
8.85 (d)
7.89 (d)
8.16 (bs)
Quinoline carbons
n.d.
C2
C3
C4
C4a
C5
C6
C7
C8
C8a
154.14
102.2
152.2
102.8
149.8
119.0
121.9
125.1
134.8
125.8
148.4
n.d.
100.0
n.d.
n.d.
n.d.
125.2
n.d.
126.6
n.d.
n.d.
n.d.
112.85
n.d.
127.30
134.99
128.03
142.89
145.06
117.42
120.73
126.99
135.22
127.84
142.67
n.d., not detected, d: doublet, dd: doublet of doublets, bs: broad singlet (widened and less intense
singlet peak).
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
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Figure 10: X-band EPR spectrum of the polycrystalline complex 4a at 298 K
Figure 11: X-band EPR spectrum of the polycrystalline complex 5a at 298 K
The spectral studies revealed that Cu(II) Hathaway, 2003) with the unpaired electron
ion in both complexes was in a tetragonal in the orbitals dx2-y2 and dxy, respectively;
field, with the following probable stereo- for complex 5a, rhombic square coplanar with
chemistries: for complex 4a, square planar the unpaired electron in the dx2-y2 orbital.
(Sreekanth and Prathapachandra Kurup, However, steric and electronic factors associ-
2003) or distorted tetrahedron (Murphy and ated with the presence of four bulky ligands
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
and sigma (σ) donors, make strictly planar or solid state, but dissociated in solution. In this
square coplanar stereochemistries almost im- sense, the general formulae for these com-
possible (Hathaway and Billing, 1970).
plexes can be expressed in two ways, accord-
ing to the phase in which they are:
[Cu(L)2Cl2] for solid phase and [Cu(L)2][2Cl]
for the liquid phase.
Relationship between spectroscopy, spectro-
metry and structure of Cu(II) complexes
The structure-spectroscopy relationship
for the compounds 4a and 5a indicates that
the atom involved in the coordination corre-
sponded to the endocyclic nitrogen of the
Computational analysis of the proposed
molecular structures of Cu(II) complexes
quinoline ring, according to the infrared re- Analysis of frontier molecular orbitals
gion; the assignment was linked to the dis- (FMOs)
placement of the aromatic C=C vibrations
The highest occupied molecular orbital
[1622 cm-1 (4) → 1619 cm-1 (4a); 1613 cm-1 (HOMO) and the lowest unoccupied molecu-
(5) → 1608 cm-1 (5a)] which suggested that lar orbital (LUMO) are useful descriptors in
the Cu(II) ion must be associated with a ring studying chemical stability of molecules
member atom rather than the carbonyl group. (Pearson, 1989). The energy of HOMO and
The spectra of 1H NMR and 13C NMR showed LUMO characterize the ability of electron do-
widening and/or decrease of the signal inten- nating and accepting, respectively. Both
sity of the protons and carbon atoms of the HOMO and LUMO were calculated at the
quinoline ring, while the signals of the phenyl B3LYP/6-31G(d,p) level in order to evaluate
ring and the acetyl group remained un- the energetic behavior of both title molecule.
changed.
The energies and the pictorial illustration of
The ESI-CID-MS2 experiments reveal an HOMO and LUMO frontier molecular orbit-
ion of formula [65Cu(C17H13ClN2O)2]+, whose als are shown in Figures 12 and 13; the posi-
fragmentation pattern was following the pre- tive and negative phase is represented in red
vious assignments, in addition to suggesting a and green color, respectively.
coordination number of two and an oxidation
The HOMO-LUMO plot clearly eluci-
state 1+ for the metal. These last two observa- dates the eventual charge transfer taking place
tions are opposed to the solid phase EPR stud- in the crystal structure. In the HOMO plot of
ies, which highlights the paramagnetic nature compound 4a, the charge was localized on
of the compound and a tetragonal environ- C13, N37, N4, C56, O62, C9, N3, N35, C46,
ment around the Cu(II) ion, with square pla- O60, Cu71, Cl1 and Cl2; and localized on the
nar geometries or distorted tetrahedron (4a) bonds C10-C20-C11, C39-C40-C42, C14-
and square rhombic coplanar (5a). Therefore, C25-C15, C50-C49-C51, C19-C27 and C29-
these remarkable differences point to a com- C24. The LUMO was spread over the entire
plete dissociation of counterions and a reduc- molecule except for the hydrogen atoms and
tion of Cu(II) → Cu(I) during the ESI process; principally localized on the bonds C59-C46,
the identity of counterions can be assigned to C44-C41, C42-C40, C39-N35, C11-C6, C27-
chloride anions since the salt used was in the C17, C8-C15, C22-C29, N37-C49, C51-C54,
form of CuCl2.
C50-C52, C56-C61 and the atoms N3, C20,
The spectroscopic correlations show that O60, C19, C9, N4, C25, C13, C24 and O62.
these coordination complexes were of the In the HOMO plot of compound 5a, the
mononuclear-tetracoordinate type, with a charge was located over the bonds C40-C39,
metal-ligand stoichiometry of 1:2, where both C19-C27, C25-C15, C48-C49, C20-C11-C10
ligand molecules behaved as monodentate in and the atoms C46, C41, N35, C9, N3, C14,
neutral form. The charge of Cu(II) ion was N4, C13, N37, C50, C55, Cu57, Cl2 and Cl1;
neutralized by two chloride anions which whereas the LUMO was located over the
were semi-coordinated (Hathaway and Bill- bonds C11-C6, C27-C17, C39-N35 and C60-
ing, 1970), in such a way that they affected C63 and the atoms C20, C22, C8, C9, C40,
the coordination sphere of metallic ion in the C49, C55, C25, C13, C24, N3, O59, O61, N4.
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Figure 12: Molecular orbitals and energies for the HOMO and LUMO of compound 4a
Figure 13: Molecular orbitals and energies for the HOMO and LUMO of compound 5a
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
The HOMO-LUMO gap, predicting the HOMO and LUMO orbital energies, ioniza-
energy difference between HOMO and tion energy (I), electron affinity (A), global
LUMO is an essential stability index (Reed et hardness (η), chemical potential (μ) and elec-
al., 1988). A large HOMO-LUMO gap of a trophilic index (ω) (Parr et al., 1999) of the
molecule implied greater stability and lesser compounds 4a and 5a can be calculated and
reactivity in chemical reactions. On calculat- is shown in Table 3.
ing the HOMO-LUMO energy gap for com-
Thermodynamic parameters
pounds 4a and 5a, the values were 3.847 eV
Thermodynamic parameters, namely,
and 3.932 eV, respectively. The conjugated
SCF energy, entropy (S), enthalpy (H), Gibbs
molecules are characterized by a highest oc-
free energy (G), zero-point energy (E0) are
cupied molecular orbital-lowest unoccupied
determined using DFT method employing
molecular orbital (HOMO–LUMO) separa-
B3LYP functional with 6-31G (d,p) basis set.
tion, which is the result of a significant degree
All the thermodynamic calculations were
of intermolecular charge transfer (ICT) from
done in the gas phase (Table 3). These calcu-
the end-capping electron-donor to the effi-
lated thermodynamic data are useful in pre-
cient electron acceptor group through p con-
dicting the reactivity of chemical reactions
jugated path. These values explained the
and judging the feasibility of different reac-
eventual charge-transfer interaction within
tion pathways.
the molecule, which influenced the biological
Determination of molecular electrostatic
potential (MEP)
activity of the compound. The HOMO-
LUMO energies and related parameters gov-
erning chemical reactivity and molecular sta-
bility are shown in Table 3.
For understanding the various aspects of
the characteristics of the bioactive molecules,
several new chemical reactivity descriptors
have been proposed. Conceptual DFT based
descriptors have helped in many ways to un-
derstand the structure of molecules and their
reactivity by calculating the chemical poten-
tial, global hardness and electrophilicity. The
Recently, the MEPs have been used for in-
terpreting and predicting relative reactivities
sites for electrophilic and nucleophilic attack,
investigation of biological recognition, hy-
drogen bonding interactions, studies of zeo-
lite, molecular cluster and crystal behavior,
and the correlation and prediction of a wide
range of macroscopic properties (Pirnau et al.,
2008; Murray and Sen, 1996).
Table 3: Thermodynamic parameters of the compounds 4a and 5a
Molecular properties
Total energy, E (kcal/mol)
EHOMO (eV)
Compound 4a
Compound 5a
-3239213.6917
-6.1564
-3239216.4434
-6.2698
-2.4226
3.8472
ELUMO (eV)
-2.2240
Energy gap (eV)
3.9323
Ionization potential (eV)
Electron affinity (eV)
Electronegativity, χ (eV)
Global hardness, η (eV)
Global softness, σ (eV)
Electrophilic index (ω)
Chemical Potential (μ)
Enthalpy, H (kcal/mol)
Gibbs free energy, G (kcal/mol)
Entropy, S (cal/mol K)
6.2698
6.1564
2.4226
2.2240
4.3462
4.1902
1.9236
1.9662
0.2599
0.2543
4.9099
4.8037
-4.3462
357.5452
281.7082
254.3585
-4.3462
362.2478
282.1036
268.8049
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
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In order to grasp the molecular interac- Antimalarial activity
tions, molecular electrostatic potentials The comparison of biological results on
(MEPs) of compounds 4a and 5a have been the growth of P. falciparum, assays on inhibi-
determined. It gives an idea about the charge tion of formation of hemozoin/β-hematin and
distribution and the relationship between di- falcipain-2, suggested that several biochemi-
pole moments, electronegativity, partial cal mechanisms are involved in the antipara-
charges and chemical reactivity of both mole- sitic action of the compounds evaluated. All
cules. The electron acquiring and electron do- the compounds had good activity (˂ 10 μM)
nating reactive sites for the investigated mol- against the W2 strain of P. falciparum (Table
ecules were found. The electron rich and par- 4). The compounds 4 and 5 were shown to be
tially negative charge of the MEP surface is active with IC50 values less than 10 μM, being
shown in red color; the blue region reveals the compound 5 the most potent. However, the
electron deficiency and the partially positive activity of compound 4 was markedly in-
charge; the light blue color indicates the creased by complexing with Cu(II) metal ion.
slightly electron deficient region; yellow
Of the two free ligands, the compound 5
color shows the slightly electron rich region was the best inhibitor, probably due to a
and, the neutral charge/electron area appears stronger interaction with the core of ferripro-
in green color.
toporphyrin IX (FPIX or hematin), which
It can be seen from Figures 14a and 14b might be mediated by the acetyl group in po-
that the region around oxygen atoms linked sition 3´ on the phenyl ring. The compound 5
with carbon through double bond and chlorine inhibited the parasite growth possibly by act-
atoms linked to copper atom specified the ing on the blockade of the polymerization (bi-
most negative potential region (red). The hy- omineralization) of hematin to hemozoin, the
drogen atoms attached to the amino group had process of detoxification of free heme by the
the maximum concentration of positive parasite.
charge (blue). The predominance of the green
The mechanism that causes the death of
region in the MEP surfaces corresponded to a the parasite is probably modulated by the in-
potential halfway between the two extremes teraction of these compounds with the for-
red and dark blue color. In both compounds, mation of hemozoin. However, it can also be
total electron density surface mapped with associated with the intraparasitic accumula-
electrostatic potential revealed the presence tion of copper in its chelated form, which is
of high negative charge on the cooper atom very active when it undergoes oxidation-re-
while more positive charge around the amino duction reactions that promote parasite death
region. The electrostatic potential of the mol- due to oxidative rupture of the cell membrane.
ecules was in the range of - 6.482e-2 (red) to
+ 6.482e-2 (blue).
Figure 14: Molecular Electrostatic Potential surface map of the compounds 4a (a) and 5a (b)
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
Table 4: In vitro activity results for synthesized compounds against growth of P. falciparum, Falcipain-
2 and hemozoin formation
Compound
Chloroquine-resistant strain (W2)
IC50 (µM)*
Falcipain-2
IC50 (µM)*
> 50
β-hematin
% IβHF**
15.55 ± 0.213
n.d.
91.83 ± 0.105
n.d.
4
4a
5
6.17 ± 0.4038
1.95 ± 0.0283
1.71 ± 0.0247
1.23 ± 0.0643
0.16 ± 0.0107
0.018 ± 0.0106
> 10
> 50
> 50
> 50
n.d.
n.d.
> 50
5a
Chloroquine
Artemisinin
CuCl2
91.44 ± 0.02
n.d.
n.d.
E64
n.d.
0.07 ± 0.0411
n.d.
The results are expressed as the mean ± standard deviation of two* and three** independent
experiments, n.d., not determined
Molecular docking studies
favorable π–π interactions. The quinoline ring
The quinoline derivatives are believed to in ligand 5 was oriented toward the planar and
form complexes with dimeric hematin (β- electron-rich periphery region of the iron–
hematin), avoiding the formation of hemo- porphyrin system, thus establishing effective
zoin. This mechanism, as well as the struc- π–π interaction. While ligand 4 showed a
ture-function relationship of established and pose, which does not favor such interaction,
developing new antimalarial drugs interfering explaining the observed decrease of activity.
hemozoin formation, have been described ex- This analysis seems to support the hypothesis
tensively. Recently it has been determined that π–π interaction between quinoline deriv-
that chloroquine (CQ) forms complexes with atives and the electronic system of hematin
both monomeric and μ-oxo-dimeric FPIX governed the formation of adducts (Kumar et
(Leed et al., 2002; de Dios et al., 2003; al., 2007; Webster et al., 2009).
Gildenhuys et al., 2013; Singh et al., 2014).
Therefore, we decided to evaluate the
mode of interaction of free ligands (4, 5) in
CONCLUSIONS
In this report, we present the results of
spectroscopic, spectrometric studies and anti-
malarial activity carried out on two types of
[(7-chloroquinolin-4-yl)amino]acetopheno-
nes ligands and its corresponding mono-che-
lated complexes. Also, DFT studies and mo-
lecular docking were performed to comple-
ment experimental studies of structural char-
acterization and biological functionality.
A detailed investigation on the gas phase
chemistry of the protonated compounds using
CID mass spectrometry, allowed to identify
and follow the changes in structural popula-
tions for better understand their possible dis-
sociation pathways. The characterization of
the fragmentation pattern of protonated free
ligands was extended here to fragments as
low as m/z 43, while for coordination com-
plexes it extended to fragments at m/z 80 and
m/z 111. Almost all the high and low m/z, as
the protonated form with hemozoin dimer (β-
hematin) solvated, in order to inquire about
the difference of both ligands in the inhibition
of hemozoin formation. For this purpose, mo-
lecular docking of free ligands in the proto-
nated form with β-hematin solvated as the
binding site was performed, using ArgusLab
4.0.1. As shown in Figure 15, the carbonyl
groups of both ligands formed hydrogen
bonds with the free propionic acid functional
groups of the dimer, using water molecules as
an interaction bridge, which could prevent
possible interactions with another dimer and
avoid the growth of the hemozoin polymer.
Additionally, ligand 5 showed an interac-
tion with the propionate coordinated to
Fe(III), mediated by a water molecule. How-
ever, the marked difference of both ligands in
the ability to inhibit hemozoin formation
seemed to be related to the establishment of
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
well as the high and low abundance frag- contributed to this ion to be the most abun-
ments, were identified and, dissociation path- dant.
ways were proposed to form them. We have
In the case of the complexes, the experi-
shown that CID of protonated [(7-chloroquin- ments reveal an oxidation state 1+ for the
olin-4-yl)amino]acetophenones ligands (4 metal, these suggest that copper undergoes a
and 5) generated a dominant product ion at reduction of Cu(II) → Cu(I) during ionization
m/z 253-254 through two probable routes, one by electrospray; the main pathway of frag-
involving an intermediate of neutral/ion com- mentation involves the loss of a ligand mole-
plex type that undergoes an intermolecular cule in its free form, dissociation promoted
electrophilic reaction; and the other more di- perhaps by steric repulsions; generating the
rect one that involves the consecutive release dominant product ion at m/z 359 (63Cu) - 361
of methane and carbon monoxide. Both routes (65Cu).
Figure 15: Docking poses of the ligands 4 and 5 in protonated form with hemozoin dimer (β-hematin)
solvated
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EXCLI Journal 2019;18:962-987 – ISSN 1611-2156
Received: September 11, 2019, accepted: October 17, 2019, published: October 28, 2019
All the spectral results suggest that the lig- Venezuelan Institute of Scientific Research
and acts as chelating species coordinating the (IVIC), and to Dr. Anibal Sierralta, Labora-
metal through the endocyclic nitrogen of the tory of Physical Chemistry and Computa-
quinoline ring in both complexes, with gen- tional Catalysis, Venezuelan Institute of Sci-
eral formulae expressed in two ways, accord- entific Research for his valuable collaboration
ing to the phase in which they are: in the use and management of structural cal-
[Cu(L)2Cl2] for the solid phase and culation programs.
[Cu(L)2][2Cl] for the liquid phase. The EPR
study of the Cu(II) complexes indicates a Declaration of conflicting interests
probable distorted tetrahedral coordination
The authors declare no potential conflicts
geometry. This result confirmed by calculated of interest with respect to the research, author-
optimized structures at the DFT/B3LYP ship, and/or publication of this article.
method with the 6-31G (d,p) basis set.
The analysis of frontier molecular orbitals Appendix A. Supplementary material
of the complexes, with HOMO-LUMO en-
Supplementary data associated with this
ergy gap of 3.847 eV (4a) and 3.932 eV (5a), article can be found, in the online version, at
is interpreted as with high chemical activity, www.excli.de/vol18/Parra_28102019_sup-
which can explain the good biological activity plementary_material.pdf.
shown on the inhibition of the growth of P.
falciparum. In general, the activity of com-
pounds 4 and 5 was increased by complexing
with the Cu(II) metal ion. The molecular
docking study carried out to understand the
reason for the biological activity of free lig-
ands, showed the participation of water mole-
cules as an interaction bridge through hydro-
gen bonds between free ligands (4, 5) and β-
hematin; at the same time, more efficient π-π
interactions were observed between 5 and the
electronic hematin system. This analysis
seems to support the hypothesis that π–π in-
teraction between quinoline derivatives and
the electronic system of hematin governs the
formation of adducts.
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