European Journal of Medicinal Chemistry

Pyridine and Pyrimidine hybrids as privileged scaffolds in antimalarial drug discovery: A

recent development
--Manuscript Draft--

Manuscript Number: EJMECH-D-24-00905

Article Type: Review Paper

Keywords: Pyridines; pyrimidines; Plasmodium falciparum; antimalarial activities; structure-

activity relationship

Corresponding Author: Nurul Hassan

National University of Malaysia
MALAYSIA

First Author: Nurul Hassan

Order of Authors: Nurul Hassan

Lekkala Ravindar

Siti Aishah Hasbullah

K. P. Rakesh

Saki Raheem

Norzila Ismail

Lau Yee Ling

Abstract: Malaria continues to pose a significant threat to global health, which is exacerbated by
theemergence of drug-resistant strains, necessitating the urgent development of new
therapeuticoptions. Due to their significant bioactivity in the treatment of malaria,
pyridine and pyrimidine have become the focal point of drug research. Hybrids of
pyridine and pyrimidine offer a novel and promising avenue for the development of
effective antimalarial agents. The ability of these hybrids to overcome drug resistance
is highlighted, offering a potential solution to this critical obstacle in the treatment of
malaria. By targeting multiple pathways, these hybrid compounds reduce the likelihood
of resistance development, providing a promising strategy for combating drug-resistant
strains of malaria. The review focuses on the most recent developments as of 2018 in
the structural optimization of pyridine and pyrimidine hybrid compounds, highlighting
key modifications that have been shown to improve antimalarial activity. Structure-
activity studies have elucidated the essential characteristics required for potency,
selectivity, and pharmacokinetics. Molecular docking and virtual screening expedite the
identification of novel compounds with enhanced activity profiles. This analysis could
aid in the development of the most effective pyridine and pyrimidine hybrids as
antimalarial agents.

Suggested Reviewers: Oleksandr G. Drushlyak

National University of Pharmacy
aldry18@hotmail.com
We would like to choose this reviewer since their research group has been conducting
a wide range of investigation on triazolo-pyridine sulfonamides as potential antimalarial
agents.

Hitesh D patel
Gujarat University
drhiteshpatel1@gmail.com
We would like to choose this reviewer since their research interest in design, synthesis
and biological evaluation including antimalarial activities of pyrazolopyrano-pyrimidine
derivatives.

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Cover Letter

FAKULTI SAINS DAN TEKNOLOGI • FACULTY SCIENCE AND TECHNOLOGY

Date : 1 APRIL 2024

European Journal of Medicinal Chemistry

Editor in Chief

SUBMISSION OF REVIEW –PYRIDINE & PYRIMIDINE HYBRIDS AS PRIVILEGED

SCAFFOLDS IN ANTIMALARIAL DRUG DISCOVERY: A RECENT DEVELOPMENT

Dear Editor,

I am writing to submit a review paper entitled 'Pyridine and Pyrimidine Hybrids as privileged
scaffolds in antimalarial drug discovery: A recent development' for consideration for publication in the
European Journal of Medicinal Chemistry. The review will contribute significantly to understanding and
advancing malaria treatment strategies.
This manuscript comprehensively reviews recent developments using pyridine and pyrimidine
hybrids as privileged scaffolds in antimalarial drug discovery. We review the synthetic strategies
employed for preparing pyridine and pyrimidine hybrid compounds, discuss the key structural
modifications used to enhance these compounds' antimalarial activity and highlight the importance of
structure-activity relationship studies in guiding the design and synthesis of more potent and selective
compounds. The current challenges and limitations in developing pyridine and pyrimidine hybrids have
been addressed to discuss opportunities for further optimization and improvement of these compounds.
We have carefully followed the submission guidelines, and the review has been prepared
following the journal's formatting requirements. We confirm that this work has not been published
elsewhere and is not currently under consideration for publication in any other journal.
The authors of this manuscript have no conflict of interest to disclose, and all listed authors have
approved the submission. We believe that critical analysis within our recent and comprehensive literature
research will be of significant interest to the readership of European Journal of Medicinal Chemistry, and
we kindly request that it be considered for publication.
Thank you for your consideration of our manuscript. We look forward to receiving your
favourable response.

Sincerely,
Nurul Izzaty Hassan, PhD
Department of Chemical Sciences
Faculty of Science & Technology Sincerely,

CERTIFIED TO MS ISO 9001:2015 Cert. No. QMS 02549

PUSAT BAHAN TERMAJU & SUMBER KETERBAHARUAN, FAKULTI SAINS DAN TEKNOLOGI
Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan Malaysia
Telefon: +603-8921 5412/5424Faksimili: +603-89215410
Laman Web: http://www.ukm.my
Mengilham Harapan, Mencipta Masa Depan • Inspiring Futures, Nurturing Possibilities
Graphical Abstract (for review) Click here to access/download;Graphical Abstract (for
review);graphical abstract.docx

Pyridine and Pyrimidine hybrids as privileged scaffolds in antimalarial

drug discovery: A recent development
Lekkala Ravindar 1, Siti Aishah Hasbullah1, K. P. Rakesh2, Saki Raheem3, Norzila
Ismail4, Lau Yee Ling5 and Nurul Izzaty Hassan1*
Highlights (for review)

Review Highlights:

1. One of the most serious issues in malaria control is the development of drug
resistance in malaria parasites.
2. Among the N-heterocycles, pyridine and pyrimidine derivatives have been
broadly studied for antimalarial activity in recent years.
3. Electron-withdrawing group substituted pyridine and pyrimidine hybrids showed
superior activities against the plasmodium parasites.
4. This review covered the antimalarial activity, SAR, cytotoxicity, and docking
study of pyridine and pyrimidine hybrids developed by researchers since 2018.
Manuscript Click here to view linked References

Pyridine and Pyrimidine hybrids as privileged scaffolds in antimalarial

drug discovery: A recent development
Lekkala Ravindar 1, Siti Aishah Hasbullah1, K. P. Rakesh2, Saki Raheem3, Norzila
Ismail4, Lau Yee Ling5 and Nurul Izzaty Hassan1*
1. Department of Chemical Sciences, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia (UKM), Bangi 43600, Selangor, Malaysia
e-mail: drizz@ukm.edu.my
2. Department of Radiology, Biomedical Research Imaging Center, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
3. School of Life Sciences, University of Westminster, 115 New Cavendish Street,
W1W6 UW, London, United Kingdom
4. Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia,
16150 Kubang Kerian, Kelantan, Malaysia
5. Department of Parasitology, Faculty of Medicine, University of Malaysa, 50603
Kuala Lumpur, Malaysia

Abstract
Malaria continues to pose a significant threat to global health, which is exacerbated by the
emergence of drug-resistant strains, necessitating the urgent development of new therapeutic
options. Due to their significant bioactivity in the treatment of malaria, pyridine and
pyrimidine have become the focal point of drug research. Hybrids of pyridine and pyrimidine
offer a novel and promising avenue for the development of effective antimalarial agents. The
ability of these hybrids to overcome drug resistance is highlighted, offering a potential
solution to this critical obstacle in the treatment of malaria. By targeting multiple pathways,
these hybrid compounds reduce the likelihood of resistance development, providing a
promising strategy for combating drug-resistant strains of malaria. The review focuses on the
most recent developments as of 2018 in the structural optimization of pyridine and
pyrimidine hybrid compounds, highlighting key modifications that have been shown to
improve antimalarial activity. Structure-activity studies have elucidated the essential
characteristics required for potency, selectivity, and pharmacokinetics. Molecular docking
and virtual screening expedite the identification of novel compounds with enhanced activity
profiles. This analysis could aid in the development of the most effective pyridine and
pyrimidine hybrids as antimalarial agents.

1
Keywords: Pyridines, pyrimidines, Plasmodium falciparum, antimalarial activities, structure-
activity relationship

1. Introduction
Malaria remains a global public health problem, with more than 200 million cases and
400,000 deaths annually [1]. P. falciparum causes the majority of malaria-related deaths [2-
3]. The truth of the destruction caused by malaria continues to haunt humanity, with most
children under five and immune-deficient pregnant women being the victims in sub-Saharan
Africa [4]. Diverse antimalarial drugs have been used to control malaria, such as quinolines,
napthoquinones, antifolates, sulfonamides, antibiotics, endoperoxides, and other antimalarial
drugs (Figure 1). Increasing resistance to existing frontline antimalarial drugs, such as
artemisinin derivatives and insecticides used to control the mosquito vector, threatens to
undermine recent progress, thus requires the development of new antimalarial drugs that
target unique therapeutic pathways counter to the multidrug-resistant Plasmodium parasites
[5-8]. Furthermore, cost-effectiveness, mode of action, bioavailability, safety profile,
compatibility with other drug partners, and compliance with absorption, distribution,
metabolism, and excretion (ADME) profile are some of the additional attributes that guide
the development of novel antimalarial agents.

2
 Figure 1. Antimalarial drugs in current clinical use and under development
Nitrogen-containing heterocyclic compounds have been studied for various biological
applications, including antimalarial efficiency [9-15]. Pyridine and pyrimidine moieties,
sixmembered N-heterocycles, are pharmacological targets' most common structural motifs
[16-23]. The widespread use of pyridine-based ring systems in drug design is largely
attributable to their significant impact on pharmacological activity, which has resulted in the
identification of various broad-spectrum therapeutic drugs [24-26]. A broad range of
synthetic molecules containing heterocyclic motifs has been identified with privileged

3
pyridine ring connected to a wide variety of bioactivity [27-30]. A plethora of natural
products with pyridine ring counting antibiotics (collismycin A and nikkomycin Z), alkaloids
(cystine, paecilomide, huperzine A, [+]-anabasine, [–]-oxirene, and trigonelline), coenzymes
(NADP and NAD), vitamins (vitamin B6 and niacin), and many other compounds (Figure 2)
[31]. There is a large number of commercially available drugs in the market which contain
pyridine rings, for example, delavirdine [32], abiraterone [33], enpiroline [34], piroxicam
[35], tropicamide [36], doxylamine [37], tacrine [38], omeprazole [39], isoniazid [40],
pyridostigmine [41], nikethamide [42], nicotinamide [43], and enisamium iodide for
influenza [44] (Figure 3). In recent years, many antimalarial evaluations have been reported
on pyridine hybrids [45-46].

Figure 2. Pyridine ring system in medicinally important natural products

4
 Figure 3. Some commercially available drugs containing the pyridine scaffold
Due to their therapeutic applications, pyrimidine compounds are distinguished in
medicinal chemistry [47-50]. Pyrimidine derivatives are a fundamental building block in
many bioactive molecules such as the bases of nucleic acids, thymine, cytosine, and uracil
[51]. The pharmacological properties of pyrimidine moiety were remarkable [52-56].
Compounds with a pyrimidine core have a wide range of pharmaceutical applications, such as
anticancer [57-58], anti-Alzheimer [59], anticonvulsant [60], antimalarial [61], anti-
inflammatory [62], antihypertensive [63], antithyroid [64], antidepressant [65], antidiabetic
[66], antitumor [67], antibacterial [68], antitubercular [69], antiviral [70], antiangiogenic [71],
anti-HIV [72], antihepatitis [73], antimicrodial [74], sedatives and hypnotics (Figure 4). The
structural motifs of pyrimidine hybrids containing diverse heterocyclic moieties have been
evaluated for antmalarial efficacy [75-79].

5
 Figure 4. Clinically used drugs containing pyrimidine moiety
Amongst the diverse chemical structures discussed in the past reviews [80-81],
pyridine and pyrimidine-based antimalarials have gained only scant attention, and a complete
review of the antimalarial properties of the hybrids containing pyridine and pyrimidine
moieties is awaited. Herein, we have endeavoured to assemble the complete data since 2018
on the antimalarial potencies, cytotoxicity, structure-activity relationship (SAR), and
molecular docking studies of pyridine and pyrimidine derivatives hybridized with various
heterocyclic compounds that derived from the laboratory of the authors and others covering
publications. Varied important N-consisting heterocyclic scaffolds are shown in Figure 5.

Figure 5. Diverse heterocyclic moieties present in antimalarial agents

6
2. Pyridine derivatives with antimalarial potency:
Pyridine is a well-known N-heterocyclic compound that is present in a variety of
natural substances. A variety of significant pharmacological properties, including antimalarial
activity, are exhibited by pyridine compounds. The antimalarial potency of the pyridine
scaffold hybridized with various heterocyclic moieties is described.
1.1.Pyrazole containing pyridines:
A series of novel pyrazolopyrano-pyridines was synthesized by Vekariya et al. in
2018 and screened for antimalarial activity against the pf3D7 CQ-sensitive strain by
employing chloroquine (CQ; IC50 = 0.02 µg/mL) and quinine (QN; IC50 = 0.268 µg/mL) for
comparison. All the twelve tested hybrids displayed in vitro potencies with the IC50 values
ranging between 0.8 and 1.85 µg/mL. Among the para-halo substituted hybrids, the best
potency was displayed by para-chloro substituted hybrid (1d) compared to the para-fluoro
(1f) and para-bromo (1g) substituted hybrids. The second best antimalarial potency was
observed in the case of hybrid 1k (R = 3,4-diOMe) with an IC50 0.96 µg/mL against the
pf3D7 strain (Figure 6) [82].

Figure 6. In vitro potency of pyrazolopyrano-pyridines against the pf3D7 CQ-sensitive strain

Considering the ongoing resistance to conventional antimalarial drugs, Verma and co-
workers reported the construction and antimalarial potency of pyridine-conjugated 1,3,4-
oxadiazole-pyrazoles (2). Schizont maturation inhibition assay was utilized to evaluate in
vitro efficacy of the constructed hybrids. On examination, the most promising hybrid 2a
containing unsubstituted phenyl rings on pyrazole unit was found to have higher potency than
the standard CQ (IC50: 0.405 µg/mL) with an IC50 0.322 µg/mL counter to the CQ-sensitive
strain (Pf3D7) of the parasite. These two potent hybrids were also examined towards the CQ-
resistant PfRKL9 strain and displayed lower IC50 values than the CQ (IC50: 2.154 µg/mL).
The in vitro cytotoxicity of 2a and 2b was screened in Vero cells, and displayed SI of 22.5
and 17.6, respectively (Figure 7) [83].

7
 Figure 7. Antimalarial effects of pyrazole-based 1,3,4-oxadiazole-pyridines towards Pf3D7
CQ-sensitive and PfRKL9 CQ-resistant clones
In 2020, Eagon and co-workers constructed a broad range of forty-two different
substituted pyrazolo[3,4-b]pyridine derivatives and tested for their anti-P. falciparum
efficacy against the pf3D7 CQ-sensitive strain and the cytotoxicity in the human fibroblast BJ
and liver HepG2 cells. All the hybrids examined showed in vitro potency in sub-micromolar
range of IC50s counter to the pf3D7 strain with little to no cytotoxicity towards the BJ and
HepG2 cells. According to the SAR, the hybrids with o-chloro (3g, IC50: 0.363 μM), o-
methyl sulfone (3j, IC50: 0.258 μM), o-trifluoromethoxy (3n, IC50: 0.278 μM), and o-methyl
ester (3q, IC50: 0.242 μM) substituent displayed superior activities whereas the unsubstituted
hybrid (3a, IC50: 1.79 μM), o-carboxylic acid (3k, IC50: 1.41 μM), and o-ethynyl substituted
(3r, IC50: 1.33 μM) hybrids were lower in potency among the series of 3a-r. When compared
hydroxy group substituted hybrids, 3b with o-hydroxy group (IC50: 0.397 μM) was more
potent than 3o (IC50: 2.04 μM) and 3p (IC50: 1.80 μM) with m-hydroxy and p-hydroxy groups
respectively. Among the hybrids with alkyl substituents (4a-f and 4o) in the case of 4a-s, p-
tertbutyl substituted hybrid 4e displayed higher potency with an IC50 of 0.0692 μM. Other
para-substituted hybrids (4g-n) gave no substantial change in potency, except the hybrid with
methyl sulfone (4m). In addition, large aromatic and heteroaromatic ring systems (4p-s) were
also tolerated with negligible change in relative activity. Among the series of 5a-e, hybrid 5d
containing methyl group at the meta position of phenyl group (R2 = 3-Me) was identified as a
promising agent with 0.094 μM of IC50 value against the pf3D7 strain (Figure 8) [84].

8
 Figure 8. Antimalarial effects of pyrazolo[3,4-b]pyridines against the Pf3D7 CQ-sensitive
strain and the cytotoxicity against the BJ cells
1.2.Quinoline containing pyridines:
In 2019, the research group of de Silva [85] employed mefloquine (MQ),
amodiaquine (AQ), and CQ for comparison during the antiplasmodial evaluation of the
furnished pyridine-linked trifluoromethylquinoline hybrid compounds (6, Figure 9) counter
to the CQ-resistance PfW2 strain. Pyridine-4-yl substituted trifluoromethylquinoline analog
6b was found to be 2.5-fold higher potent anti-P. falciparum agent (IC50: 8.4 μM) than the
pyridine-2-yl substituted trifluoromethylquinoline analog 6a and proved to be non-cytotoxic
against the BGM cell line displaying EC50 >560 μM. The LogD, LogP (pH 5.2 and 7.4), and
pKa were tested for all hybrids. However, it fails to correlate with the biological potency.
Similarly, Patel’s group [86] very recently synthesized pyridine-linked 7-chloroquinoline

9
hybrids (7, Figure 9) and tested for in vitro potency against the P. falciparum parasite by
using QN and CQ as reference drugs. Compared to the -NH- linked conjugates (7a and 7b),
the -NHNH- linked conjugates (7c and 7d) possessed lower IC50 values. When the outcomes
of conjugates 6b containing trifluoromethylquinoline moiety and 7b having 7-
chloroquinoline ring were compared, 7b was 2-fold higher potent than 6b representing that
the type of substituent on the quinoline moiety was significant for in vitro potency. According
to the molecular docking results, analog 7d showed least (-9.2 kcal/mol) binding affinity
among the series 7.

Figure 9. Antiplasmodial potency of 4-aminoquinoline-pyridine hybrids against the CQ-

resistance PfW2 strain
A novel family of pyridine-conjugated quinoline derivatives (8) was constructed by
Huang et al. and screened for in vitro activity against CQ, a clinical candidate with an IC50
value of 0.172 μM, used as reference drug against the CQ-resistant PfD2 strain. The SAR
investigations revealed that most of the 4-aminoquinoline hybrids (8a-h) improved in vitro
potency as compared to other quinoline hybrids (8i-j). Among the amino group incorporated
series (8a-h), the hybrids containing secondary amine at the C4 position of quinoline unit
(8a-c) displayed superior activities than the hybrids having tertiary amine at the same
position (8d-h). Among all, analog 8c with dimethylamino group at the C4 position was
higher active (IC50 = 0.003 μM) than the standard CQ (IC50 = 0.172 μM) against the CQ-
resistant Pf D2 strain (Figure 10) [87].

10
 Figure 10. Antimalarial potency of pyridine-4-aminoquinolines against the CQ-resistant
PfDd2 strain
1.3.Triazole containing pyridines:
For the treatment of malaria, a small library of diverse substituted triazole containing
pyridine hybrids (9) was furnished by dos Santos et al. and tested for anti-P. falciparum
efficacy against CQ-sensitive 3D7 clone. All the hybrids were proved to be anti-P.
falciparum agents displayed a 6.96 to 23.20 µM range of inhibitory activities against the
Pf3D7 clone. The SAR revealed that the meta-substituted phenyl ring on triazole core (9e)
improved the in vitro potency of the hybrid as compared to the unsubstituted (9a-b) and
para-substituted (9c-d) phenyl ring counterparts against the Pf3D7 clone. The most active
hybrid 9e towards the Pf3D7 clone (IC50 = 6.96 µM) among the series was also examined for
in vitro activity counter to the PfSR25 clone and was found to be active displaying 9.91 µM
of IC50 value and was non-cytotoxicic against HEK293 cell line at the tested concentration
(Figure 11) [88].

11
Figure 11. In vitro efficacy of diverse substituted triazole containing pyridines counter to the
CQ-sensitive Pf3D7 and CQ-resistant PfSR25 strains
Brando and colleagues [89] 2018 synthesized novel pyridine-1,2,3-triazole hybrids
(10, Figure 12) by the chemical modification of lapachol via click chemistry and evaluated
their anti-P. falciparum efficacy against the CQ-resistant W2 clone to discover potent
antimalarial drugs. Two of the tested hybrids were 14 to 24-fold higher potent than the
standard lapachol (IC50 value of 123.5 µM), demonstrating inferior cytotoxicity with a CC50
value of >600 µM against HepG2 cells. Concerning the position of the nitrogen atom on the
pyridine ring, when the nitrogen was in the 3-position, as in 10b (IC50: 5.2 µM), the hybrid
was nearly 2-fold potent than when the nitrogen was in the 2-position, as in 10a (IC50: 8.7
µM). Docking examination of conjugates 10a and 10b to P. falciparum dihydroorotate
dehydrogenase (PfDHODH) anticipated the compound 10b as a lead hybrid (-14.55
kcal/mol). Subsequently, Theeramunkong and co-workers [90] synthesized an isoquinoline-
based 1,2,3-triazole-pyridine hybrid (11, Figure 12) through the Sharpless-Fokin reaction
and tested for antimalarial activity against CQ-sensitive and CQ-resistant Pf3D7 and PfK1
strains, respectively. Hybrid 11 had moderate activity against the 3D7 (IC50: 62.02 μM) and
K1 (IC50: 47.72 μM) strains. A novel pyridine substituted 1,2,4-triazole hybrid (12, Figure
12) furnished by Meva et al. [91] in 2022 displayed lower anti-P. falciparum potency (IC50 =
176 µM) against CQ-sensitive Pf3D7 strain. Moderate antimalarial activity with an IC50 of
5.50 μM was observed towards the PfNF54 CQ-sensitive clone for the pyridine-containing
glycyrrhetinic acid-1,2,3-triazole hybrid (13, Figure 12) synthesized in 2023 by Kapkoti et
al. [92] via click chemistry. The cytotoxicity of hybrid 13 was tested in Vero cells and
displayed 289.83 μM of CC50 value.

12
Figure 12. Anti-P. falciparum efficacy of pyridine-triazoles against CQ-sensitive pf3D7 and
pfNF54, CQ-resistant pfK1 and pfW2 strains and cytotoxicity in Vero and HepG2 cells
To discover the potent antimalarial agents, Batra and co-workers in 2018 [93]
synthesized a new pyridine-containing benzenesulfonamide-based triazole-berberine hybrid
(14, Figure 13) and tested it for antimalarial potency against CQ-sensitive Pf3D7 strain.
Hybrid 14 displayed promising activity with an IC50 value of 1 μg/mL and was non-toxic
against PC-3 cancer cells at the tested concentrations. To combat the Pf3D7 CQ-sensitive
strain, in 2020, Batra and co-workers [94] also synthesized a small library of novel pyridine
containing benzenesulfonamide-triazole hybrids (15, Figure 13) via a click reaction and
tested for in vitro activities. The SAR anticipated that the analog 15f bearing para-iodo (R =
4-I) substituted phenoxymethyl group on triazole ring significantly enhances the antimalarial
potency (IC50 = 12 µg/mL) than all other halogenated (15c-e) and unsubstituted (15b)
counterparts. Interestingly, the hybrid 15a having a more electron-withdrawing nitro group at
the para-position (R = 4-NO2) of phenoxymethyl unit displayed lower activity with IC50 >50
µg/mL against Pf3D7 strain. Towards CQ-sensitive Pf3D7 strain, moderate inhibitory
potencies with IC50 values ranging from 59.12 μM to >73.52 μM have been shown by a few
novels pyridine-containing benzenesulfonamide-based triazole-coumarin derivatives (16,
Figure 13) synthesized by Batra and co-workers in 2021 [95]. According to the SAR studies,
the pyridine hybrids containing substituted coumarin moiety (16c-d) displayed better
inhibitory activities than unsubstituted coumarin ring counterparts (16a-b) against CQ-
sensitive Pf3D7 strain.

13
 Figure 13. In vitro potency of pyridine containing hybrids towards the CQ-sensitive Pf3D7
clone
Karpina et al. in 2020 reported discovering and derivating the virtual library of novel
sulfonamide-based 1,2,4-triazolopyridines (17-19) utilizing CQ (IC50 = 0.02 μM) as the
standard drug. All the tested hybrids were found to be anti-plasmodial agents with IC50 values
ranging from 2.24 μM to >64 μM. Two of the twenty-five hybrids were potent against the
CQ-resistant PfK1 strain at a concentration of IC50 = 2.24 μM for hybrid 18a and 4.98 μM for
hybrid 17e. Seven hybrids demonstrated an 8 to 10 μM range of IC50 values and six hybrids
displayed 10 to 14 μM ranges of IC50 values. Two of the twenty-five hybrids were cytotoxic
towards MRC-5 cell lines at a concentration of 48.50 μM for 17b and 18.19 μM for 17e.
Other twenty-three hybrids were non-cytotoxic towards MRC-5 cell lines (IC50 > 64 μM),
resulting in a high selectivity. Among all, hybrids 18e and 18g were possessed higher binding
energies of -15.9 and -15.6 kcal/mol, respectively (Figure 14) [96].

14
Figure 14. Anti-P. falciparum efficacy of sulfonamide-based 1,2,4-triazolopyridines against
the CQ-resistant K1 strain and cytotoxicity in MRC-5 cell lines
1.4.Tetrazole containing pyridines:
Lawong and co-workers conducted a phenotypic evaluation to identify novel and
active antimalarial drugs and identified a series of tetrazole-based pyridine derivatives (20) as
potent antimalarial agents. All the hybrids tested for in vitro potency displayed 0.23 to 1.7
μM range of IC50 values against the pf3d7 CQ-sensitive strain and 0.41 to 3.0 μM range of
IC50 values against the pfDd2 CQ-resistant strain. The SAR showed that the replacement of

15
N-methyl (20a and 20e, R2 = CH3) with cyclopropyl (20b-d, R2 = cPr) substantially improves
the in vitro activity against two strains of P. falciparum in the case of the hybrids containing
o-tolly group on trtrazole unit (20a-e). No significant change in the antimalarial potency was
observed towards both the strains when the methoxy group (20b, R1 = 2-OMe) on benzyl ring
switched with fluorine (20c, R1 = 2-F) and hydrogen atom (20d, R1 = H). The introduction of
cyclohexyl (20f) and cyclopropyl (20g) rings on trtrazole unit decreased activities against
both strains. Among all, hybrid 20h with N-Propyl substituent and o-tolly group on trtrazole
unit was the most promising agent against the pf3d7 (EC50 = 0.23 μM) and pfDd2 (EC50 =
0.41 μM) strains. The most potent hybrids, 20b, 20d, and 20h, were also screened for
cytotoxicity against HepG2 cell lines and exhibited 16, 15 and 10 μM of CC50 values,
respectively (Figure 15) [97].

Figure 15. Anti-P. falciparum potency of tetrazole-based pyridines against the CQ-sensitive
pf3d7 and CQ-resistant pfDd2 strains and cytotoxicity against HepG2 cell lines
1.5.The metal complex containing pyridines:
A small family of novel imidazolopiperidine and 4-aminoquinoline-based ligands
possessing pyridine-linked 1,2,3-triazole unit (21-24), and their resultant rhenium metal
complexes (25-28) were furnished by Sovari et al. and their anti-P. falciparum potencies
examined counter to the CQ-sensitive (PfNF54) and multidrug(MD)-resistant (PfK1) clones
by using chloroquine diphosphate (CQDP; IC50: 0.014 μM (PfNF54) and 0.247 μM (PfK1))
as reference drug. According to the SAR investigations, imidazolopiperidine-based ligands
(23-24) and their resultant rhenium metal complexes (27-28) showed a minimum six-fold
reduced potencies compared to the 4-aminoquinolin-based ligands (21-22) and their metal
complexes (25-26) towards both the PfNF54 and PfK1 clones. 4-Aminoquinoline-based
metal complex hybrid 25 demonstrated significantly higher inhibitory activity than all the
metal complex hybrids against the PfNF54 (0.356 μM) and PfK1 (0.611 μM) clones. The

16
docking investigations showed that all rhenium metal complex hybrids, except 26 (binding
affinity: -12.62 kcal/mol), possessed superior binding affinities as compared to the reference
drug (CQDP; -13.56 kcal/mol). In addition, the ligand hybrids displayed significantly higher
binding affinity than their resultant rhenium metal complex hybrids (Figure 16) [98].

Figure 16. Anti-P. falciparum potency of pyridine-tethered Re-complex hybrids against the
CQ-resistant PfK1 and CQ-sensitive PfNF54 clones
In 2020, Melis's research team [99] used CQDP as a reference drug for antiplasmodial
testing of a synthesized quinoline-triazole half-sandwich Ir(III) metal complex (30) against
CQ-sensitive (PfNF54) and CQ-resistant (PfK1) strains. The Ir(III) metal complex hybrid
(30) demonstrated 100-fold superior antiplasmodial activity (IC50 = 0.25 μM) than its
corresponding ligand (29, IC50 = 28.02 μM) against the PfNF54 strain and was found to be
non-cytotoxic against CHO cell line at the tested concentration displaying 123.4 μM of IC 50
value (Figure 17). The following year, the same research group [100] constructed a novel
fluorescent Ir(III) 2-phenylpyridyl complex 31 via a one-pot procedure and tested inhibitory
activity against the PfNF54 strain using CQDP for comparison. The antiplasmodial activity

17
of the fluorescent Ir(III) complex hybrid 31 (IC50 = 0.82 μM) was found to be 35-fold higher
than the corresponding ligand 29 (Figure 17).

Figure 17. Antiplasmodial activity against CQ-sensitive (PfNF54) and CQ-resistant (PfK1)
strains and cytotoxicity of pyridine-tethered Ir metal-complexes
1.6.Miscellaneous pyridines:
Meyers et al. [101] utilized a hybrid target-phenotype approach to identify and
examine new chemotypes for malaria. Out of four pyrrolidine-linked pyridine hybrids (32a-d,
figure 18), only hybrid 32b having trifluoromethyl pyridine ring on pyrrolidine ring was
identified as a lead compound with an IC50 of 250 nM against the pf3D7 CQ-sensitive strain.
Against the pfNF54 CQ-sensitive strain, the antimalarial activity with an IC50 of 14.88 μM
was observed in the case of hybrid 33 synthesized by Hochegger et al.[102]. Moreover, it was
proved non-cytotoxic against L-6 cells with 53.53 μM of IC50 value (figure 18). Attram and
co-workers [103] evaluated the antimalarial activity of benzimidazole containing pyridine
hybrid compound (34, figure 18) against pfNF54 CQ-sensitive and pfK1 CQ-resistant strains
by employing CQ and artesunate (ARS) as control drugs. They examined the cytotoxicity
against the CHO cell line utilizing emetine for comparison. Hybrid 34 displayed an IC50 >10
against both the pfNF54 and pfK1 strains and showed 50 μM of IC50 value against the CHO
cell line. Promising antimalarial activity (IC50: 0.37 μg/mL), equipotent to the standard CQ,
was observed for benzenesulfonamide-based pyridine hybrid compound (35, figure 18) of
Meena et al. [104] against the P. falciparum parasite.

18
Figure 18. Antimalarial properties of various pyridine hybrid compounds against different P.
falciparum parasites and cytotoxicity against varied cells
In 2021, Lawong and co-workers designed and synthesized amide-based pyridine
derivatives (36) and tested them for in vitro potency against the pf3d7 CQ-sensitive and
pfDd2 CQ-resistant strains. According to the SAR, the hybrid containing ethyl group (36a, R2
= Et) on the phenyl ring was approximately 2-fold higher in potency than the methyl group
counterparts (36b-e, R2 = Me) against the pf3d7 strain. The replacement of CF3 group (36d
and 36e) with SCF3 (36b) and OCF3 (36c) on the benzyl ring gave similar antimalarial
activity against both strains. Compare to the N-cyclohexyl hybrid (36d), N-propyl hybrid
(36f) displayed superior activity against two strains of P. falciparum. Among all, hybrid 36g
with piperidine ring showed lower potency against the pf3d7 (EC50 = 1.8 μM) and pfDd2
(EC50 = 4.2 μM) strains. All the hybrids were also examined for cytotoxicity towards HepG2
cell lines and most of the hybrids displayed CC50 values >30 μM (Figure 19) [97].

19
 Figure 19. Anti-P. falciparum potency of amide-based pyridines against the CQ-sensitive
pf3d7 and CQ-resistant pfDd2 strains and cytotoxicity against HepG2 cell lines
3. Pyrimidine derivatives with antimalarial potency:
Developing the most promising antimalarial hybrids is a significant strategy to
combat drug resistance. In light of the significance of pyrimidine hybrid compounds as
antimalarials [105-107], we describe the antimalarial activity of the pyrimidine moiety
hybridized with various heterocyclic scaffolds.
3.1.Pyrazole containing Pyrimidines:
In 2018, Silvira and co-workers [108] designed and synthesized a library of diverse
para-substituted benzenesulfonamide and phenyl-appended pyrazolopyrimidines (37-38,
Figure 20) and examined for their antimalarial efficacy against PfW2 CQ-resistant strain by
using sulfadoxine (IC50: 15.0 µM) and CQ (IC50: 0.55 µM) for comparison. Three of the
twelve hybrids (37c, 37i, and 38b) were more potent than the control sulfadoxine drug. The
SAR showed that the para-fluoro substituent (37a-c, R1 = F) on the phenyl ring improved the
antimalarial activity as compared to the para-chloro (37d-f, R1 = Cl) and para-methyl (37g-i,
R1 = Me) substituents among the benzenesulfonamide series. In contrast, the para-chloro
substituent (38b, R1 = Cl) exhibited better potency than the para-fluoro (38a, R1 = F) and
para-methyl (38c, R1 = Me) substituents in the case of amine series. Among all, conjugate
37c with para-fluoro substituted phenyl ring and para-methyl substituted
benzenesulfonamide group was the most promising anti-P. falciparum agent displaying the
IC50 value of 5.13 µM. Most conjugates were found to be lower cytotoxic to BGM cell lines
and showed superior SI values compared to the reference drug sulfadoxine. Continuing their
interest in discovering novel antimalarials, Silveira et al. [109] furnished another small family
of benzenesulfonamidelinked pyrazolopyrimidines (39, Figure 20) and tested for anti-P.
falciparum efficacy against the CQ-sensitive Pf3D7 strain by employing CQ (IC50 = 0.03
µM) as control drug. Two of the seven hybrids displayed an IC50 <10 µM. The hybrids (39b-

20
f) containing para-substituted phenyl ring on benzenesulfonamide moiety were
comparatively less potent than unsubstituted phenyl ring counterpart (39a), the most active
antimalarial agent among the series. The naphthalene ring (39g) hybrid was lower potent with
an IC50 >10 µM against the Pf3D7 strain.

Figure 20. Anti-P. falciparum potency of benzenesulfonamide-appended

pyrazolopyrimidines against the CQ-sensitive pf3D7 and CQ-resistant PfW2 strains
A broad range of novel pyrazole-based imidazolopyrimidines 40-41 was constructed
in 2018 and examined for anti-P. falciparum efficacy utilizing QN (IC50: 0.268 µg/mL) and
CQ (IC50: 0.20 µg/mL) as control drugs by the research group of Prasad. All fourteen
examined analogues were found to be promising anti-P. falciparum agents demonstrating
0.030 to 1.45 μg/mL range of IC50 values. The SAR anticipated that the para-fluoro
substituted phenyl ring on imidazolopyrimidine moiety (40e and 41e) improves the in vitro
antimalarial potency of the hybrids, whereas the para-methyl substituted phenyl ring (40b
and 41b) reduces the anti-P. falciparum potency of the conjugates. When comparing the
antimalarial efficacy of the hybrids possessing heterocyclic substituents on
imidazolopyrimidine core, the N-heterocycles (40g and 41g) were significantly higher potent
than S-heterocycles (40f and 41f). The most promising antimalarial agent, 40e (IC50: 0.030
µg/mL), displayed 10-fold higher potency than the control drug QN and comparable activity
to CQ (Figure 21) [110].

21
Figure 21. Anti-P. falciparum efficacy of pyrazole-based imidazolopyrimidines against CQ-
sensitive pf3D7 strain
3.2.Quinoline containing pyrimidines:
4-Aminoquinoline-based piperazine-pyrimidines (42, Figure 22) were identified as
promising anti-P. falciparum agents by the research group of Kondaparla in 2018 [111] and
evaluated for in vitro efficacy counter to CQ-sensitive pf3D7 and CQ-resistant pfK1 strains.
The hybrids 42a and 42b showed higher inhibitory activities (IC50) than the control drug CQ
against both the strains and were found to be less cytotoxic against the Vero cell lines,
demonstrating SI 979.10 and 90.90, respectively. In 2019, de Silva's group [85] synthesized a
pyrimidine-linked trifluoromethyl quinoline hybrid compound (43, Figure 22). It tested it for
antiplasmodial activity against AQ, CQ, and MQ used as reference drugs against the CQ-
resistant strain of P. falciparum W2 and antimalarial efficacy againist P. berghei. Hybrid 43
was found to be a potent anti-P. falciparum agent (IC50: 8.4 μM) was non-cytotoxic against
the BGM cell line with EC50 >559 μM. Pyrimidine-linked trifluoromethyl quinoline analog
43 was also the most potent analog against P. berghei on the fifth day after infection. The
LogD, LogP (pH 5.2 and 7.4), and pKa were tested for these hybrids and failed to correlate
with the biological potency.

Figure 22. Anti-P. falciparum efficacy of 4-Aminoquinoline-based pyrimidines against CQ-

sensitive pf3D7 and CQ-resistant pfK1 and pfW2 clones

22
 Tripathi et al. hybridized 4-fluoro-amodiaquine with various substituted pyrimidines
in promising antimalarial hybrids synthesis. All the synthesized hybrids (44-47) were
examined for their in vitro properties counter to the pfNF54 (CQS) and pfDd2 (CQR) strains
by employing ART and CQ as the control drugs. Out of eighteen synthesized hybrids, only
47e displayed lower IC50 value than the control drug ART (10.0 nM) towards pfNF54 strain
while twelve hybrids possessed superior activity than the standard CQ (IC50 = 27.0 nM).
Against the pfDd2 strain, only four conjugates showed better antimalarial activities compared
to ART (IC50 = 10.0 nM), whereas all the hybrids, except 44b, exhibited lower inhibitory
activities (IC50) than the control drug CQ (222.0 nM). The SAR revealed that replacing –Cl
substituent of pyrimidine with the cycloamino substituents significantly improves the
activities towards both strains. The majority of hybrids containing pyrimidin-4-yl on fluoro-
amodiaquine moiety (44a, 46a, 46c and 46f-h) were more potent as compared to the
pyrimidin-2-yl counterparts (45a, 47a, 47c and 47f-h) against both the pfNF54 and pfDd2
strains. Two promising analogues (46f and 47b) were examined in vivo counter to a P.
berghei-mouse malaria model, and 46f displayed lower activity than 47b. All the hybrids
were low to moderate cytotoxic against the CHO cells. Mechanistic heme-binding
evaluations exhibited better binding interactions than the control drug CQ (Figure 23) [112].

Figure 23. Antiplasmodial activity of pyrimidine-4-aminoquinolines towards CQ-sensitive

PfNF54 and CQ-resistant PfDd2 strains
Against the pfD6 CQ-sensitive and pfW2 CQ-resistant strains, excellent inhibitory
potencies (IC50) in the range of 0.027-1.661 μM and 0.0189-1.443 μM respectively have been

23
showed by a broad range of pyridine and thiophene-based various substituted 4-
aminoquinoline-pyrimidine hybrids (48-51) synthesized by Maurya et al. in 2019. Towards
pfD6 strain, no conjugate showed better antimalarial activity than the control drugs ART
(IC50 = 0.0171 μM) and CQ (IC50 = 0.0204 μM). Out of thirty-five synthesized hybrids, only
51e and 51f exhibited comparable IC50 values to the ART (0.0162 μM) against pfW2 strain,
while twenty-three conjugates possessed superior activity than that of CQ (IC50 = 0.4215
μM). Amongst conjugate 48f containing pyridine moiety, they displayed superior activity
against the pfD6 strain with an IC50 of 0.027 µM. In contrast, conjugate 51f with a thiophene
core possessed promising antimalarial activity against the pfW2 strain with the IC50 value of
0.0189 µM, 22 times higher than the control drug CQ. All the promising conjugates were
non-cytotoxic against the Vero cell lines. Docking investigations carried out with Pf-DHFR
showed stronger binding interaction in the active site (Figure 24) [113].

24
 Figure 24. Anti-P. falciparum efficacy of 4-aminoquinoline-pyrimidines against the CQ-
sensitive PfD6 and CQ-resistant PfW2 clones
Utilizing QN (an IC50 of 0.108 μM) and CQ (an IC50 of 0. 0.012 μM) as control drugs,
Kayamba et al. in 2021 examined the antimalarial properties of a small library of fourteen
new synthesized chloroquine-pyrimidine conjugates (52-53) against the pfNF54 CQ-sensitive
strain. The SAR showed that the piperazine or 1,4-diaminoalkane linker between pyrimidine
and 7-chloroquinoline cores enhances the antimalarial activity, and the nature of the
substituent on phenyl rings attached to the pyrimidine moiety was also significantly
influenced the potency. Among all, conjugate 52f having electron-donating hydroxyl group at
the para-position of both the phenyl rings attached to the pyrimidine core and 1,4-
diaminobutyl spacer between two pharmacophores exhibited promising antimalarial activity
(IC50: 0.32 μM) with a good safety profile of 9.79 to the HEK293 cell lines. Hybrid 52f

25
demonstrated the maximum binding score towards both the PfHsp70s displaying KD ranging
from 4.4 to 11.4 nM. Furthermore, docking investigations revealed that conjugates 52g, 52f,
53d, and 53f exhibited improved fitness towards PfHsp70-1, with analog 52f performing the
lowest and highest binding affinities of 9.8 kcal/mol (Figure 25) [114].

Figure 25. Anti-P. falciparum efficacy of 4-aminoquinoline-pyrimidines against the CQ-

sensitive PfNF54 strain
The following year, Chowdhary and co-workers utilized a multitargeted procedure to
furnish a small family of novel chloroquinoline-based 1,2,4-triazolopyrimidine derivatives
(54). All the furnished analogues were screened for their anti-P. falciparum properties
counter to the CQ-sensitive pf3D7 and CQ-resistant pfW2 clones. All the tested analogues
displayed moderate to good inhibitory activities (IC50) counter to both the pf3D7 (0.17-12.6
μM) and pfW2 (0.20-12.9 μM) clones. The SAR revealed that the alkyl chain length and
piperazine moiety between triazolopyrimidine and chloroquinoline cores significantly
influences the antimalarial activity towards both the clones. Among all, analog 54e with n = 7
displayed higher potency towards both the pf3D7 (IC50: 0.17) and pfW2 (IC50: 0.20 μM),
whereas the hybrid 54f containing piperazine moiety showed lower potency with the IC50
values 12.6 μM (pf3D7) and 12.9 μM (pfW2). Notably, hybrid 54e was three-fold higher in

26
potency than the control drug CQ (IC50 0.53 μM) counter to the pfW2 clone. The cytotoxicity
of the most active analogues (54b-e) was examined in Vero cells and showed 180 to 350 µM
ranges of IC50 values. The most active agent (54e) surpassed the control drug CQ by
inhibiting the parasite’s cellular process and parasitic enzymes (PfDHODH and PfCRT), as
confirmed by UV-vis and molecular modelling studies (Figure 26) [115].

Figure 26. Antimalarial activity of triazolopyrimidine-4-aminoquinolines against the CQ-

sensitive Pf3D7 and CQ-resistant PfW2 strains
3.3.Triazole containing pyrimidines:
Chopra and co-workers furnished a family of eighteen novel quinoline-based 1,2,3-
triazole-pyrimidines (55-56) to examine their anti-P. falciparum activity against the pfNF54
CQ-sensitive strain. Compare to the conjugates (55a-l) having alkyl spacer between quinoline
and 1,2,3-triazole rings, the analogues (56a-f) in which quinoline ring directly linked with
1,2,3-triazole ring displayed higher inhibitory activities (IC50) against the pfNF54 strain. The
most promising analogues were then tested against the pfDd2 CQ-resistant strain and also
screened for cytotoxicity against the Vero cell lines, and the majority of them were found to
be non-cytotoxic with 145 to >200 µM range of IC50 values. According to the SAR, the alkyl
spacer length between quinoline and 1,2,3-triazole rings enhances the in vitro potency against
the pfNF54 and pfDd2 strains. Switching of methyl group (R1 = CH3) with ethyl or isopropyl
groups resulted in enhanced activities. Among the eighteen analogues, 55d and 55j
containing more electron-withdrawing nitro group at the para-position of phenyl ring
attached to the pyrimidine unit were the most promising agents against the pfNF54 and pfDd2
strains, respectively. The majority of promising hybrids with three carbon linker (55a-f)
displayed higher clog P and log D values as compared to the hybrids with two carbon linker
(55g-l, Figure 27) [116].

27
 Figure 27. Antiplasmodial activity of quinoline-1,2,3-triazoles against the CQ-sensitive
PfNF54 and CQ-resistant PfDd2 strains and cytotoxicity against Vero cells
The research group of Chan furnished a small family of 1,2,3-triazole conjugated
pyrimidine derivatives (57-59) and tested as anti-P. falciparum agents at diverse thiamine
concentrations. Most of the conjugates exhibited an IC50 of >60 μM counter to the pf3D7 CQ-
sensitive strain. According to the SAR, position of the substituent on the phenyl ring
significantly influences the antimalarial activity. Among the esters (57a-g), meta-chloro
(57c), para-bromo (57e), and para-nitro (57f) substitutions substantially increased the
potency. Among all the ten synthesized hybrids, only one hybrid (59) containing para-bromo
substituted benzyl group showed better activity with an IC50 of 2.2 μM and SI of 45. The
cytotoxicity of the conjugates 57a, 57c, and 59 was tested against HFF cells, and hybrid 59
was found to be toxic, while analogues 57a and 57c were demonstrated no cytotoxicity at the
tested concentrations (Figure 28) [117].

28
 Figure 28. Anti-P. falciparum activity of 1,2,3-triazole-pyrimidines towards Pf3D7 CQ-
sensitive strain and cytotoxicity in HFF cell
Aiming to explore potentially improved antimalarial agents, Adigun and co-workers
very recently constructed a small library of quinoline-based triazole-dihydropyrimidinones
(60-61) through the click chemistry and screened in vitro properties against CQ-sensitive
pfNF54 and CQ-resistant pfK1 strains by using CQ (IC50: 4.2 nM (pfNF54) and 35.7 nM
(pfK1)) for comparison. The conjugates possessing 7-chloroquinoline moiety (60a-d) showed
higher potency against the pfNF54 strain with the IC50 values ranging between 138 and 245
nM and displayed superior activity along with improved sensitivity counter to the pfK1 strain
with 421 to 567 nM range of IC50 values. In contast, the conjugates having MQ unit (61a-c)
showed an IC50 >5 μM against the pfK1 strain. In the case of 7-chloroquinoline hybrids (60a-
d), replacement of the N1-hydrogen (R2 = H) of dihydropyrimidinone with a methyl group
(R2 = Me) substantially improved the potency against both the pfNF54 and pfK1 strains. The
docking results showed that some of the conjugates possessed higher binding affinity when
compared to the standard CQ, however, no one of them exhibited increased binding affinity
compared to the FAD cofactor (Figure 29) [118].

29
 Figure 29. Antiplasmodial activity of dihydropyrimidinone-1,2,3-triazoles against the CQ-
sensitive PfNF54 and CQ-resistant PfK1 clones
Against the CQ-sensitive Pf3D7 strain, significant inhibitory activity with 4 µg/mL of
IC50 value has been shown by a novel pyrimidine containing benzenesulfonamide-based
triazole-berberine hybrid (62) synthesized by Batra and co-workers in 2018 [93]. The
cytotoxicity of hybrid 62 was tested in PC-3 cancer cell and found to be non-toxic
demonstrating >200 μg/mL of CC50 value (Figure 30). Batra and colleagues continued their
interest in antimalarial drug discovery in 2020 [94] by synthesizing a few new pyrimidine
containing benzenesulfonamide-triazole hybrids and testing their antimalarial efficacy against
the CQ-sensitive Pf3D7 strain. Among all, the hybrid 63a having a more electron-
withdrawing nitro group at the para-position (R = 4-NO2) of phenoxy methyl unit,
demonstrated superior activity with an IC50 value of 6.2 µg/mL. In contrast, the analog 63b
bearing unsubstituted phenoxy methyl group (R = H) on triazole ring displayed lower activity
with IC50 >50 µg/mL against the Pf3D7 strain (Figure 30). Batra and co-workers [95] also
synthesized a small library of novel pyrimidine-containing benzenesulfonamide-based
triazole-coumarin derivatives (3) and evaluated their in vitro activities in 2021 to combat the
Pf3D7 CQ-sensitive strain. The SAR revealed that the pyrimidine hybrids containing a
substituted coumarin moiety (64c-f) had a significant effect on the inhibitory activity
compared to their counterparts with an unsubstituted coumarin ring (64a-b) against the CQ-
sensitive Pf3D7 strain (Figure 30).

30
 Figure 30. In vitro potency of pyrimidine containing hybrids towards the CQ-sensitive
Pf3D7 clone
Brandão and co-workers constructed a lapachol-based pyrimidine-triazole hybrid (65)
via click chemistry and examined it for antimalarial potency against the pfW2 CQ-resistant
clone to provide a significant antiplasmodial drug. Hybrid 1 displayed nine fold superior
activity than the standard lapachol (IC50 value of 123.5 µM) with lower cytotoxicity (CC50
60.7 µM) against the HepG2 cell line at the tested concentration (Figure 31) [89].

Figure 31. Anti-P. falciparum efficacy of pyrimidine-triazole against the CQ-resistant PfW2
strain and cytotoxicity in HepG2 cell
Silveira et al. constructed a vast array of benzenesulfonamide-appended 1,2,4-
triazole-pyrimidines (66-67) as anti-P. falciparum agents. All of the twenty-one tested
conjugates were anti-P. falciparum agents, with the IC50 values ranging from 0.030 to 9.1
µM, and four of them (66o, 66p, 66r, and 67c) possessed similar inhibitory activities (IC50) to
the reference drug CQ (IC50 = 0.03 µM) towards the CQ-sensitive pf3D7 strain. According to

31
the SAR studies, the conjugates with naphthalene ring on benzenesulfonamide unit (67a-c)
were more active than the hybrids (66a-r) containing para-substituted phenyl ring at the
same position. In the case of 66a-r, the antimalarial activities were substantially influenced
by the electron-donating and electron-withdrawing groups (R and R1). Compare to
unsubstituted (66a-f, R = H) and electron-donating methyl group (66g-l, R = Me) substituted
analogues, electron-withdrawing trifluoro methylated counterparts (66m-r, R = CF3)
displayed higher potencies. Anti-P. falciparum activities of para-trifluoro methylated
conjugates (R1 = CF3, 66f, 66l, and 66r) were higher as compared to the unsubstituted
analogues (R1 = H, 66a, 66g, and 66m), which were 39-fold lower potent than the
naphthalene hybrids (67a-c). Remarkably, all the conjugates were moderate to non-cytotoxic
in the HepG2 cells with 46 to 367 µM ranges of IC50 values (Figure 32) [109].

Figure 32. Anti-P. falciparum activities of 1,2,4-triazole-pyrimidines against the CQ-

sensitive Pf3D7 strain and cytotoxicity in HepG2 cells
Mokariya et al. furnished an array of novel 1,2,3-triazole-based 1,2,4-triazole/
benzimidazole-pyrimidine derivatives (68-69) as potent anti-P. falciparum agents against the
malaria parasite. Most conjugates demonstrated lower IC50 values (0.82-2.24 μM) than the
control drug QN (IC50: 2.71 µM). According to the SAR, the hybrid 68c having
trifluoromethyl group at the para-position of benzamide unit (R = 4-CF3) was the most
effective anti-P. falciparum agent (IC50: 0.082 µM) among the series of 1,2,4-triazole-
pyrimidines 68a-h, whereas analog 69b containing fluoro substituent at the same position (R

32
= 4-F) displayed higher potency (IC50: 0.99 µM) in the case of benzimidazole-pyrimidines
69a-h (Figure 33) [119].

Figure 33. Anti-P. falciparum activity of various pyrimidine derivatives

3.4.Tetrazole containing pyrimidines:
To address the resistance of malaria parasite towards pyrimidine-based antimalarials,
Lawong and co-workers designed and synthesized a series of tetrazole-based pyrimidine
derivatives (70) and tested for their in vitro antimalarial activity against the pf3d7 CQ-
sensitive and pfDd2 CQ-resistant strains. According to the SAR, the antimalarial potency of
the hybrids containing N-cyclopropyl group (70a-d, R2 = cPr) was significantly higher as
compared to N-acetyl counterpart (70e, R2 = COCH3) against both the strains in the series of
analogues containing o-tolly group on trtrazole moiety (70a-e). Switching in the position of
fluoro substituent on benzyl ring (70b, R = 3-F; 70d, R = 4-F; 70c, R = 6-F) gave no
significant change in the antimalarial potency against two strains of P. falciparum.
Introducing a nonaromatic cyclohexyl ring (70f) on trtrazole core reduced the potency of both
strains. Among all, hybrid 70g with o-tolly group on trtrazole unit and N-propyl substituent
was found to be the most active against the pf3d7 (EC50 = 0.31 μM) and pfDd2 (EC50 = 0.52
μM) strains. Hybrids were also examined for cytotoxicity towards HepG2 cell lines, and the
majority of the hybrids displayed EC50 values >30 μM (Figure 34) [97].

33
Figure 34. Anti-P. falciparum potency of tetrazole-based pyridines against the CQ-sensitive
pf3d7 and CQ-resistant pfDd2 strains and cytotoxicity against HepG2 cell lines
3.5.Thiophene containing pyrimidines:
In 2021, a small family of novel thiophene and furan-based pyrimidine hybrid
compounds was constructed by Alidmat and co-workers and examined for in vitro properties
against the pf3D7 CQ-sensitive and pfRKL9 CQ-resistant strains by using CQDP (IC50: 0.02
µg/mL (pf3D7) and 36.31 µg/mL (pfRKL9)) and ART (IC50: 4.51 µg/mL (pf3D7) and 8.46
µg/mL (pfRKL9)) as control drugs. All the hybrids were significantly active than the control
drug CQDP against both the P. falciparum clones, and all were found to be more potent,
except 1c, than ART against pfRKL9 clone. The SAR proved that the thiophene-based
pyrimidine hybrids were more potent than the furan-based pyrimidine hybrids (71a-b vs 71c-
d and 72a-b vs 72c-d) against both clones. Concerning the position of the benzyloxy group
on the methoxy phenyl ring attached to the pirimidine core, when the benzyloxy group was in
the 3-position, the hybrids were more potent than when the benzyloxy group was in the 4-
position (71a vs 71b, 71c vs 71d, 72a vs 72b, and 72c vs 72d) against both the P. falciparum
parasites. All the thio-substituted pirimidine hybrids, except 1a, were higher in potency when
compared to amino-substituted counterparts (71b vs 71b, 71c vs 72c, and 71d vs 72d) against
both the pf3D7 and pfRKL9 clones. According to the docking results, all the hybrids
examined exhibited a good affinity to the active site of PfATP4 with free binding energy
ranging from -10.35 to -8.56 kcal mol-1 (Figure 35) [120].

34
 Figure 35. Anti-P. falciparum potency of thiophene and furan-based pyrimidine against the
CQ-sensitive pf3D7 and CQ-resistant pfRKL9 strains
A new class of piperazine-linked tetrahydrobenzothieno[2,3-d]pyrimidine hybrid
compounds (73-74), synthesized by Pal and co-workers through the multicomponent Petasis
reaction as the key step, was identified as potential antimalarials very recently. Most
conjugates displayed promising antimalarial potency counter to the pf3D7 CQ-sensitive and
pfW2 CQ-resistant clones. Out of sixteen hybrids, three hybrids (74d, 74e, and 74h) were
higher potent and two hybrids (74c and 74f) were equipotent to the control drug CQ (IC50:
0.1269 μM) counter to the pfW2 clone. The SAR anticipated that replacing the carboxylic
acid group (73a-h) with the acetamide group (74a-h) significantly improves all the hybrids'
in vitro antimalarial inhibitory potencies against the P. falciparum parasites. In case of the
carboxylic acid (73a-h) and acetamide (74a-h) series, hybrids 73h (IC50: 0.1269 μM) and
74h (IC50: 0.0557 μM) containing trifluoromethyl substituted (R2 = CF3) phenyl ring were
identified as better antimalarial agents counter to the pfW2clone. In contrast, the hybrid with
methyl-substituted phenyl ring (74b, R2 = Me) displayed superior potency (IC50: 0.0557 μM)
counter to the pf3D7 clone among the acetamide series 74a-h. The cytotoxicity of the most
active conjugates (74c-f and 74h) was examined counter to the HPL1D cell lines,
demonstrating low cytotoxicity with SI >100. The analogues with promising in vitro potency

35
demonstrated substantial binding affinities evident from their Glide energies obtained on
docking with the wild Pf-DHFR-TS (glide energy range -61.294 to -50.645 kcal/mol) and
quadruple mutant Pf-DHFR-TS (glide energy range -61.294 to -50.645 kcal/mol). The in
silico ADMET profiling also showed good pharmacokinetic and physicochemical parameters
for the higher potent analogues (Figure 36) [121].

Figure 36. Anti-P. falciparum potency of tetrahydrobenzothieno[2,3-d]pyrimidines against

the CQ-sensitive pf3D7 and CQ-resistant pfW2 strains
3.6.Miscellaneous pyrimidines
A small library of amide-based pyrimidine derivatives (75) were synthesized by
Lawong et al. and screened for antimalarial activity against the pf3d7 CQ-sensitive and pfDd2
CQ-resistant strains. The SAR revealed that the replacement of hydrogen atom (75e, R2 = H)
with ethyl (75d, R2 = Et) and methyl (75a-c, R2 = Me) groups on phenyl ring resulted in
approximately 2 to 4-fold higher antimalarial activity against both the strains. The switching
of CF3 group (75a) with SCF3 (75b) and OCF3 (75c) on the benzyl ring gave substantially
higher potency against the pf3d7 strain. Introducing a methyl group on pyrimidine ring led to
a significant 20-fold loss in potency (75f vs 75a). All the hybrids were further examined for
cytotoxicity towards HepG2 cell lines and displayed CC50 values >30 μM (Figure 37) [97].

36
Figure 37. Anti-P. falciparum potency of amide-based pyrimidines against the CQ-sensitive
pf3d7 and CQ-resistant pfDd2 strains and cytotoxicity against HepG2 cell lines

4. Conclusion
In conclusion, pyridine and pyrimidine have emerged as privileged scaffolds with
immense potential in antimalarial drug discovery. These compounds offer a unique
combination of structural features that allow them to target multiple P. falciparum strains,
making them promising candidates for developing new and effective antimalarial agents.
Researchers have enhanced the potency, selectivity and pharmacokinetic properties of
pyridine and pyrimidine hybrid compounds through careful synthesis, structural optimization
and understanding of the structure-activity relationships. The integration of computational
approaches such as virtual screening and molecular docking has facilitated the identification
of novel compounds with improved antiplasmodial profiles. Electron-withdrawing group
substituted pyridine and pyrimidine hybrid compounds displayed superior activities counter
to the plasmodium parasites. Most potent conjugates were proved less to no cytotoxic against
the HepG2, CHO, and Vero cell lines. Nonetheless, challenges remain in developing these
hybrids as further exploration of their pharmacokinetic properties, including bioavailability,
is necessary to ensure their effective translation into clinical use. Future research directions in
this field should include the exploration of new synthetic methodologies, investigation of
additional targets within the malaria parasite, and the development of combination therapies
involving pyridine and pyrimidine hybrids to combat emerging drug resistance. With
continued research and collaboration, these compounds have the potential to significantly
contribute to the global efforts to eradicate malaria and improve public health worldwide.

Acknowledgments
We thank the Ministry of Higher Education (FRGS/1/2019/STG01/UKM/02/3) for its
financial support and Universiti Kebangsaan Malaysia for awarding the UKM-Vice
Chancellor Ph.D. scholarship for Ravindar Lekkala.

37
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Declaration of Interest Statement

Declaration of interests

☒The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: