3-aryl-indolinones derivatives as antiplasmodial agents: synthesis, biological activity and computational analysis

Article abstract of DOI:10.1080/14786419.2021.1895149

Malaria is an infectious illness, affecting vulnerable populations in Third World countries. Inspired by natural products, indole alkaloids have been used as a nucleus to design new antimalarial drugs. So, eighteen oxindole derivatives, aza analogues were obtained with moderate to excellent yields. Also, the saturated derivatives of oxindole and aza derivatives via H₂/Pd/C reduction were obtained in good yields, leading to racemic mixtures of each compound. Next, the inhibitory activity against P. falciparum of 18 compounds were tested, founding six compounds with IC₅₀ < 20 μM. The most active of these compounds was 8c; however, their unsaturated derivative 7c was inactive. Then, a structure-activity relationship analysis was done, founding that focused LUMO lobe on the specific molecular zone is related to inhibitory activity against P. falciparum. Finally, we found a potential inhibition of lactate dehydrogenase by oxindole derivatives, using molecular docking virtual screening.

Full text of DOI:10.1080/14786419.2021.1895149

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
3-aryl-indolinones derivatives as antiplasmodial
agents: synthesis, biological activity and
computational analysis
Ayelen Luczywo, Lucía G. González, Anna C. C. Aguiar, Juliana Oliveira de
Souza, Guilherme E. Souza, Glaucius Oliva, Luis F. Aguilar, Juan J. Casal,
Rafael V. C. Guido, Silvia E. Asís & Marco Mellado
To cite this article: Ayelen Luczywo, Lucía G. González, Anna C. C. Aguiar, Juliana Oliveira
de Souza, Guilherme E. Souza, Glaucius Oliva, Luis F. Aguilar, Juan J. Casal, Rafael V. C.
Guido, Silvia E. Asís & Marco Mellado (2021): 3-aryl-indolinones derivatives as antiplasmodial
agents: synthesis, biological activity and computational analysis, Natural Product Research, DOI:
10.1080/14786419.2021.1895149
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1895149
3-aryl-indolinones derivatives as antiplasmodial agents:
synthesis, biological activity and computational analysis
a
a
b
ꢀ
ꢀ
Ayelen Luczywo , Lucıa G. Gonzalez , Anna C. C. Aguiar ,
Juliana Oliveira de Souzab, Guilherme E. Souzab, Glaucius Olivab, Luis F. Aguilarc,
Juan J. Casala,d, Rafael V. C. Guidob , Silvia E. Asıs
and Marco Melladoc
ꢀ a
a
ꢀ
ꢀ
ꢀ
Departamento de Quımica Organica, Facultad de Farmacia y Bioquımica, Universidad de Buenos
b
ꢀ
~
~
~
Aires, Buenos Aires, Argentina; Instituto de Fısica de Sao Carlos, Universidade de Sao Paulo, Sao
c
ꢀ
ꢀ
ꢀ
ꢀ
Carlos, Brasil; Instituto de Quımica, Pontificia Universidad Catolica de Valparaıso, Valparaıso, Chile;
d
ꢀ
ꢀ
Facultad de Medicina, Laboratorio de Biomembranas, Instituto de Fisiologıa y Biofısica Bernardo
Houssay (IFIBIO Houssay), Universidad de Buenos Aires-CONICET, Buenos Aires, Argentina
ABSTRACT
ARTICLE HISTORY
Received 8 October 2020
Accepted 21 February 2021
Malaria is an infectious illness, affecting vulnerable populations
in Third World countries. Inspired by natural products, indole
alkaloids have been used as a nucleus to design new antimalarial
drugs. So, eighteen oxindole derivatives, aza analogues were
obtained with moderate to excellent yields. Also, the saturated
derivatives of oxindole and aza derivatives via H2/Pd/C reduction
were obtained in good yields, leading to racemic mixtures of
each compound. Next, the inhibitory activity against P. falciparum
of 18 compounds were tested, founding six compounds with IC50
< 20 mM. The most active of these compounds was 8c; however,
their unsaturated derivative 7c was inactive. Then, a structure-
activity relationship analysis was done, founding that focused
LUMO lobe on the specific molecular zone is related to inhibitory
activity against P. falciparum. Finally, we found a potential
inhibition of lactate dehydrogenase by oxindole derivatives,
using molecular docking virtual screening.
KEYWORDS
Oxindole derivatives;
synthesis; structure-activity
relationship; antimalarial
activity
ꢀ
CONTACT Rafael V. C. Guido
rvcguido@usp.br; Silvia E. Asıs
elizabet@ffyb.uba.ar; Marco Mellado
marco.mellado@pucv.cl
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2021.1895149.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
A. LUCZYWO ET AL.
Figure 1. Natural and synthetic indole alkaloids with antiplasmodial activity.
1. Introduction
Malaria is caused by Plasmodium parasites which are transmitted to people
through the bites of infected female Anopheles mosquitoes, and the children are
the group most affected by this disease. World Health Organization highlights
the critical importance of keeping efforts to prevent, detect, and treat malaria
(WHO 2020), owing to this infectious disease affects vulnerable populations in
the Third World countries.
In the Third World countries, nature has been used to treat different diseases,
for example, Cryptolepis sanguinolenta, a medicinal plant frequently used in
Central and West Africa, this plant has indole alkaloids with antiplasmodial activ-
ities (Forkuo et al. 2017). In this respect, from Aristolochia cordigera were isolated
indole alkaloids such as 3,4-dihydro-hyrtiosulawesine (i) and 6-O-(b-glucopyrano-
syl)hyrtiosulawesine (ii) (Figure 1), which exhibited antiplasmodial activity in the
low micromolar range against P. falciparum strain (3D7 strain), and low cytotox-
icity on hepatic cells (Pereira et al. 2017). Moreover, in South America
Aspidosperma olivaceum is used to treat fever and malaria in some regions of
Brazil. From A. olivaceum has been isolated aspidoscarpine (iii) a promising com-
pound against P. falciparum and low cytotoxicity in hepatic cells (Chierrito et al.
2014). Inspired by the antimalarial alkaloids, have been synthesized several indole
alkaloids with in vitro activity against P. falciparum, among them an indole-pyr-
imidine hybrid (iv) (Agarwal et al. 2005), and 3-methylene-indolinone (v) (Kumar
et al. 2011). Then, indole alkaloids represent an interesting class of natural prod-
ucts with antimalarial activity and high similarity to our quinoline derivatives
(Muscia, Bollini, Bruno, et al. 2006; Muscia, Bollini, Carnevale, et al. 2006).
Based on all the active compounds i-v, the common shared structural feature is the
indole moiety linked to an aromatic ring or an aliphatic group by a single or double
bond in position 3 of indole or oxindole nucleus (Figure 1). So, according to this
information, we report the synthesis and antiplasmodial activity of thirty-one oxindole
(indolin-2-one) derivatives and aza analogues. Furthermore, we conducted
computational studies, based on a Quantitative Structure-Activity Relationship (QSAR)
and docking methods, to investigate both the structure-activity relationships and
the identification of the potential molecular target underlying this series
of P. falciparum inhibitors.

NATURAL PRODUCT RESEARCH
3
Scheme 1. Synthesis route to obtain of oxindole derivatives
2. Results and discussion
2.1. Chemistry
The condensation reactions of isatin (1) with arylamines (2) yielded 3-arylimino-indoli-
nones (3a-b) under acid catalysis in moderate yields (30–63%), while the oxindole (5)
with arylaldehydes (6) yielded 3-arylidene-indolinones (7a-g), through Knoevenagel
synthesis using an alkaline catalyst with yields between 45–92% (see Scheme 1).
1
The H-NMR spectra of the compounds 3 and 7 exhibit the typical signals for aro-
matic hydrogens and the NH signal of the oxindole core (between d ¼ 9.45–11.03 and
d ¼ 8.68–10.66 ppm, respectively, details in Supplementary Material). In addition, prod-
ucts 7 show the CH signal shift which varies depending on the nature of the aryl moi-
ety next to it. The E and Z isomer ratio was studied by the chemical shifts of the
aromatic ‘ortho’ protons on the benzylidene ring. Particularly, the Z isomer showed
these hydrogens to downfield according to a previous report (Zhang and Go 2009).
Moreover, 3-oxindole saturated analogues 4 and 8 were synthesized by palladium-
catalyzed hydrogenation from compounds 3 and 7, respectively (see Scheme 1). The
reduction of the exocyclic double bond afforded the desired products 4 and 8 in
racemic forms, with moderate to excellent yields. The 1H-NMR spectra of the com-
pounds 4a-b showed two signals at d ꢀ 5.97 and 4.00 ppm of the NH and CH frag-
ments. While the compounds 8a-g were confirmed by signals to high-field d ꢀ 3.50
and 3.00 ppm corresponding to the CH2 group bonded to the oxindole nucleus, as
well as the two signals d ꢀ 47.0 and d ꢀ 35.0 ppm in the 13C-NMR spectra.
2.2. Antiplasmodial activity
The inhibitory activity of the eighteen oxindole derivatives was tested against the P. fal-
ciparum (3D7 strain) using the SYBR Green assay. Several oxindole derivatives exhibited
poor inhibitory activity between 10–40 mM (see Table S1), thus the IC50 values greater
than 20 lM were considered inactive. Interestingly, only six compounds (3a, 7b, 7e, 7f,
7g, and 8c) showed inhibitory activity between IC50 ¼ 19 to 5.8 lM (see Table 1).
The compound 3a (4-CO2Et) showed decreased inhibitory activity amongst the
active compounds. Three 3-aryliden-indolin-2-ones derivatives possess one or more
methoxy groups (7e-g), and compound 7b (4-Cl) has an electron-attracting group, and
they showed modest antiplasmodial activity (IC50 ¼ 13–18 mM). Interestingly, the

4
A. LUCZYWO ET AL.
Table 1. Half inhibitory concentration (IC50) against P. falciparum (3D7 strain).
3D7
HepG2
3D7
HepG2
IC50
(Avg SD)
19
>20
>20
13
(mM)
IC50
(Avg SD)
(mM)
IC50
(Avg SD)
(mM)
IC50
(mM)
Comp.
Comp.
(Avg SD)
3a
3b
7a
7b
7c
7d
7e
7f
1
ND
ND
ND
ND
ND
ND
ND
ND
ND
4a
4b
8a
8b
8c
8d
8e
8f
>20
>20
>20
ND
ND
ND
ND
>100
ND
ND
ND
ND
2
>20
>20
>20
5.8 0.3
>20
>20
>20
>20
18
15
14
1
2
1
7g
C1
8g
0.006 0.002
305 24
ND ¼ not determinate, Cþ ¼ Artesunate
compound 8c was the most potent among the tested compounds (IC50 ¼ 5.8 lM),
however, its unsaturated analogue 7c (4-NMe2) was inactive (IC50 > 20 lM). This find-
ing suggests that the bond between the oxindole core and the aromatic substituent
is important to the antiplasmodial activity. Additionally, the cytotoxicity of com-
pound 8c was determined against Human Hepatoma cells (HepG2), because this cell
line acts as a surrogate for effects of toxicity on human liver (Hughes et al. 2011).
The inhibitor exhibited a very low cytotoxic effect on the cells (IC50 > 100 mM) and a
reasonable selectivity index (SI > 18).
2.3. Quantitative structure-activity relationship (QSAR)
The multilinear equations between the activity and various descriptors were developed
according to our previous reports (Mellado et al. 2018, 2020). The QSAR model
showed a correlation of the LUMO and x descriptors show with the biological activity
(r2 ¼ 0.96, see Tables S2 and S3).
ð
Þ
ð
Þ
ð
Þ
pIC50 ¼ 5:22 0:07 þ 0:41 0:12 LUMO þ 0:11 0:07 x
(1)
The descriptor LUMO (Lowest Unoccupied Molecular Orbital) is the equivalent to
Lewis acid (Lopez et al. 2013). This descriptor has been used to explain the inhibitory
activity of several compounds against P. falciparum. For instance, the antiplasmodial
activity of tryptanthrin derivatives was related to the focused LUMO lobe of the mol-
ecule (Olson et al. 2018). On the other hand, the electrophilic global index (x), is a
concept of measure for energy stabilization, such as when systems receive additional
electronic charges from underground (Pearson 1993). However, there is no information
about x related to the inhibitory activity of molecules against P. falciparum.
The Equation (1) showed the LUMO descriptor weight is ꢀ3.7-fold greater than x
descriptor. Thus, the modification of LUMO descriptor is gravitating for modulating
the inhibitory activity of oxindole derivatives against P. falciparum. For example, the
LUMO plot of compounds 7c (IC50 > 20 mM) and 8c (IC50 ¼ 5.8 mM) shows a LUMO
concentrated on the oxindole nucleus in the compound 8c (Figure S1B);
however, the LUMO in the compound 7c is expanded on the entire structure
(Figure S1A). These results are in the way of the focused LUMO on the specific
molecular zone (Olson et al. 2018).

NATURAL PRODUCT RESEARCH
5
2.4. Molecular docking study
Since the molecular mechanism of antiplasmodial action of oxindole derivatives is still
unknown, the molecular docking technique was used to predict their possible target
(Ła˛czkowski et al. 2017). Then, six enzymes crucial for the parasite development were
selected: falcipain-2, falcipain-3, dihydroorotate dehydrogenase, lactate dehydrogenase,
macrophage migration inhibitory factor, and SUB1 protease (Njogu et al. 2016).
The calculated affinity energies of all active compounds are summarized in Table
S4. The results on the SUB1 Protease showed a positive value of docking score, thus
this molecular target was ruled out. The results on falcipain-2 showed a narrow energy
affinity between selected compounds and native ligand (DE ¼ 0.5 kcal/mol), thus the
displacement of native ligand by active compounds is unlikely. In the case of the pro-
teins dihydroorotate dehydrogenase, falcipain-3, and lactate dehydrogenase, the native
ligand has decreased affinity energy than all compounds. Finally, based on the linear
relationship between the affinity energy and the experimental IC50 values, lactate
dehydrogenase stood out as a promising molecular target of oxindole derivatives
(r ¼ 0.777), while the dihydroorotate dehydrogenase (r ¼ 0.158) and falcipain-3
(r ¼ 0.100) were ruled out due to low significance.
The analysis of the modelled binding mode of 3a, 7b, 7e, 7f, 7g, and 8c to the
lactate dehydrogenase indicated that the oxindole nucleus of all compounds binds in
the same site of the pyruvate (native ligand). The oxindole core is bound between the
ASN-126 and ALA-79 residues at the catalytic binding site (see Figure S2). In this bind-
ing mode, the oxindole ring of compound 8c forms a hydrogen-bond with ALA-79
backbone (blue dashes, Figure S3), while the electron-rich 4-NMe2-Ph substituent
undergoes p-stacking with the amide side-chain of ASN-126 (blue dashes, Figure S3).
These findings are in agreement with the results obtained from the QSAR study, which
indicated that the increment of the LUMO electronic density on the oxygen atom
of the oxindole nucleus is favourable for the inhibitory activity of this series, due to
the hydrogen bonding acceptor role. In addition, a 100 ns molecular dynamic
simulation was carried out, finding stable hydrogen bond interactions during the
simulation time, between the compound 8c and the residues ALA-79 and ASN-126.
3. Experimental
Experimental details relating to this paper are in the supplementary material.
4. Conclusion
Some oxindole derivatives synthesized showed antimalarial activity. The best inhibitor,
8c, showed antiplasmodial activity in the low micromolar range (IC50 ¼ 5.8 mM) and
low cytotoxic effect on HepG2 cells (IC50 > 100 mM). The computational studies sug-
gested that the focused LUMO properties of the oxindole ring are important for anti-
parasitic activity and the P. falciparum lactate dehydrogenase might be a molecular
target underlying oxindole derivatives inhibitory activity. Our findings suggest that 8c
is an attractive candidate for hit-to-lead development.

6
A. LUCZYWO ET AL.
Acknowledgments
ꢀ
ꢀ
The authors thank to Bioq. M. Belen Iglesias Garcıa, Farm. Juan Pablo Carnevale, and to Bioq.
Felipe Stambuk for technical assistance. Also, this work was supported by [Universidad de
Buenos Aires] under Grant [UBACyT 20020170200206BA]; [Sao Paulo Research Foundation—
FAPESP] under Grant [CEPID 2013/07600-3, 18/07287-7, and 17/26679-0]; [National Council for
Scientific and Technological Development – CNPq] under Grant [405330/2016-2]; [Instituto
ꢀ
Serrapilheira] under Grant [Serra-1708-16250]; [Agencia Nacional de Investigacion y Desarrollo
ꢀ
(ANID)] under Grant [Fondecyt Postdoctorado 3180408]; and [Pontificia Universidad Catolica de
ꢀ
Valparaıso] under Grant [VRIEA-PUCV 37.0/2017].
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Rafael V. C. Guido
http://orcid.org/0000-0002-7187-0818
http://orcid.org/0000-0002-3978-8638
http://orcid.org/0000-0001-9577-5180
ꢀ
Silvia E. Asıs
Marco Mellado
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