Design and synthesis of quinoline-pyrimidine inspired hybrids as potential plasmodial inhibitors

European Journal of Medicinal Chemistry 217 (2021) 1 13330

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Design and synthesis of quinoline-pyrimidine inspired hybrids as

potential plasmodial inhibitors

,

Francis Kayamba ^a, Teboho Malimabe ^{b c}, Idowu Kehinde Ademola ^d, Ofentse Jacob Pooe ^e,

,

Narva Deshwar Kushwaha ^a, Mavela Mahlalela ^a, Robyn L. van Zyl ^{b c}, Michelle Gordon ^d,

,

Pertunia T. Mudau ^f, Tawanda Zininga ^f^g, Addmore Shonhai ^g, Vincent O. Nyamori ^h,

,

*

Rajshekhar Karpoormath ^a

^aDepartment of Pharmaceutical Chemistry, College of Health Sciences, University of KwaZulu-Natal, Durban, 4000, South Africa

^bPharmacology Division, Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of Witwatersrand, Johannesburg, 2193, South

Africa

^cWITS Research Institute for Malaria (WRIM), Faculty of Health Sciences, University of Witwatersrand, Johannesburg, 2193, South Africa

^dSchool of Laboratory Medicine and Medical Sciences, College of Health Science, University of KwaZulu-Natal, Durban, 4000, South Africa

^eDiscipline of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, Durban, 4000, South Africa

^fDepartment of Biochemistry University of Venda, School of Mathematical and Natural Sciences, Thohoyandou, 0950, South Africa

^gDepartment of Biochemistry, Stellenbosch University, Stellenbosch, 7600, South Africa

^hSchool of Chemistry and Physics, University of KwaZulu-Natal, Westville Campus, Durban, 4000, South Africa

a r t i c l e i n f o

a b s t r a c t

Article history:

Presently, artemisinin-based combination therapy (ACT) is the ﬁrst-line therapy of Plasmodium falcipa-

rum malaria. With the emergence of malaria parasites that are resistant to ACT, alternative antimalarial

therapies are urgently needed. In line with this, we designed and synthesised a series of novel N-(7-

chloroquinolin-4-yl)-N’-(4,6-diphenylpyrimidin-2-yl)alkanediamine hybrids (6a-7c) and evaluated

their inhibitory activity against the NF54 chloroquine-susceptible strain as a promising class of anti-

malarial compounds. The antiplasmodial screening revealed that seven analogues showed promising to

Received 29 October 2020

Received in revised form

18 February 2021

Accepted 24 February 2021

Available online 3 March 2021

good activity with half-maximal inhibitory concentration (IC₅₀) ¼ 0.32

1,4-diamine butyl linker and 4-hydroxyl phenyl on fourth and sixth position of pyrimidine core showed

the most prominent activity with an IC₅₀value of 0.32 0.06 M, with a favourable safety proﬁle of 9.79

to human kidney epithelial (HEK293) cells. The remaining six analogues showed moderate activity with

IC₅₀values ranging from 7.50 M to 83.01 M. We further investigated the binding afﬁnities of the

molecules to two essential cytosolic P. falciparum heat shock protein 70 homologues; PfHsp70-1 and

PfHsp70-z. Compound 7a exhibited the highest binding afﬁnity for both PfHsp70s with K_Din a lower

nanomolar range (4.4e11.4 nM). Furthermore, molecular docking revealed that compounds 6, 6k, 7b and

7a exhibited better ﬁtness in PfHsp70-1 with compound 7a showing the highest and lowest binding

scores of ꢀ9.8 kcal/mol. Therefore, we speculate that PfHsp70-1 is one of the targets of these inhibitors.

The bioisoteric replacement of the groups at phenyl ring at the fourth and sixth position of the pyrim-

idine core had a constructive association with antiplasmodial activity. The promising antiplasmodial

activity of the synthesised analogues illustrates how crucial molecular hybridisation is as a strategy in the

development of quinoline-pyrimidine hybrids as prospective antiprotozoal agents.

mMe4.30 mM. Compound 7a with

Keywords:

Molecular hybrids

Quinoline

Pyrimidine

Antimalarial agents

Malaria

Antiprotozoal

Piperazine

m

PfHsp70

1. Introduction

a result of the advent of resistance on current treatment such as

artemisinin combination therapies (ACTs) and chloroquine (CQ) [1].

The evolution of Plasmodium strains has caused uncertainty in

the treatment of malaria, and thousands of people are still dying as

Malaria is predominately spread by P. falciparum which is more

prevalent in Africa. An estimate of 228 million people were infected

with malaria in 2018, killing approximately 405 000 people, of

which over 90% were in Sub-Saharan Africa. Children under the age

* Corresponding author.

E-mail address: karpoormath@ukzn.ac.za (R. Karpoormath).

https://doi.org/10.1016/j.ejmech.2021.113330

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

of ﬁve and pregnant women are the major victims of the scourge,

especially those in poor tropical regions. About 3.2 billion people

remain at risk of the epidemic [2,3]. Thus, necessitating the need for

alternative therapy in the absence of an efﬁcient antimalarial vac-

cine [4].

viral infections that is gaining more attention [14e16]. SAR studies

of CQ revealed that the basic chain is crucial for antimalarial activity

by improving the drug’s lipophilic, which allows easy passage

through the malarial plasma membrane [2]. In light of this, the

incorporation of various linkers such as piperazine and different

alkyl diamine holds promise towards making effective antimalarial

agents. In view of the aforementioned, we synthesised a hybrid

molecule, as illustrated in Fig. 2, by a molecular hybridisation

approach using two robust moieties viz pyrimidine and quinoline,

with the aim of presenting a safer and alternative therapy for ma-

laria. We anticipated presenting an antimalarial candidate effective

against both the liver and blood-stages of Plasmodium; for potential

use for both treatment and prophylaxis.

An audit of antimalarial pharmacophores revealed that, in

addition to other vast medicinal properties, the pyrimidine moiety

displayed admirable antimalarial properties. It has proven to be key

in some drugs and lead antimalarial candidates at an advanced

stage of clinical trials. Amongst the leading antimalarial pharma-

cophores are proguanil, pyrimethamine, P218 and WR9920. Most

antimalarial pyrimidine-based drugs hold prospects as prophylac-

tic agents. These compounds target P. falciparum dihydrofolate

reductase, which is implicated in the folic acid pathway that is

essential for DNA biosynthesis [5]. Chalcones are another class of

analogues well-known for excellent antimalarial therapy; where

for example, licochalcone, a natural ﬂavonoid product was active

against CQ-susceptible (3D7) and CQ-resistance (Dd2) strains [6,7].

The phenyl groups of chalcone were important for antiplasmodial

activity as indicated by SAR studies, and as such, we tried to inte-

grate these pharmacophores into the modiﬁed potent molecule [8].

Given this context, we expected that a promising antiplasmodial

agent would be the combination of active pharmacophores

inspired by pyrimethamine and chalcone to produce a 4,6-

diphenylpyrimidine. Fig. 1 shows CQ and other potential antima-

larial compounds that were crucial in designing this novel series.

While antimalarial drugs, such as piperaquine and CQ, based on

a quinoline backbone are inexpensive with a robust therapeutic

index, they have lost efﬁcacy as stand-alone drugs due to the

emergence of resistance [9,10] mediated by a mutated form of the

Plasmodium falciparum chloroquine resistance "transporter" [1 1,12].

Although prospects for CQ reapplication as an antimalarial remain

[13], it is its roles as an agent against avian inﬂuenza H5N1 and ZIKV

2. Chemistry

The novel library of quinoline-pyrimidine hybrids 6(a-t) was

synthesised using a versatile synthetic pathway. The intermediate

(E)-chalcones 1(aee) were prepared by aldol condensation reaction

as previously reported, using assorted commercially available

benzaldehydes and acetophenones using sodium hydroxide basic

condition in a good yield of 95% [1 7]. The cyclisation of chalcone

using guanidine hydrochloride and sodium hydroxide base affor-

ded numerous 4,6-diphenylpyrimidin-2-amine 2(aee) analogues.

Known Sandmeyer reaction applies to the substitution of an aro-

matic amine of compound 2 to hydroxyl (4,6-diphenylpyrimidin-2-

ol 3(aee) via diazonium salt intermediate formation. Sodium ni-

trate in acetic acid initiates the diazonium salt formation and then

substitution of diazo salt with water. 2-Chloro-4,6-

diphenylpyrimidine analogues 4(aee) were synthesised from

assorted 3 analogues using POCl₃with catalytic DMF as illustrated

in Scheme 1.

Fig. 1. Marketed drugs and chalcone based potential antimalarial compounds.

2

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

Fig. 2. Rational design of pyrimidine-quinoline hybrid molecules.

The synthesis and characterisation of a series of quinoline-

diamine compounds 5(aed) were as per reported methods [1].

Various 5(aed) compounds were further coupled with various

4(aee) compounds using K₂CO₃to afford a library of the targeted

pyrimidine-quinoline hybrids analogues (6a-7c). The methoxy

substituents on the phenyl group of ﬁnal compounds 6g, 6i and 6j

were further demethylated using HBr acid in a microwave to afford

compounds 7a, 7b and 7c, respectively, in good yield as depicted in

Scheme 2.

Scheme 1. Reaction conditions: (i) EtOH, NaOH, sonicate, 35 ^ꢁC, 1 h; (ii) Guandine hydrochloride, NaOH, EtOH, reﬂux, 24 h; (iii) Acetic acid, NaNO₂, H₂O, rt, 3 h; (iv) POCl₃, DMF,

100 ^ꢁC, reﬂux, 6 h.

3

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

a-d

a-f

a-c

a-k

Scheme 2. Reaction conditions: (i) Isopropanol (IPA), reﬂux, 100 ^ꢁC, 16 h; (ii) K₂CO₃, Dimethylformamide (DMF), Microwave (MW), 120 ^ꢁC, 40 min; (iii) HBr, MW, 100 ^ꢁC, 20 min.

3. Results and discussion

respectively. The methoxy (MeO) peak signal resonates at

55.40 ppm, and the remaining carbon signals correspond to the

aromatic carbon. Fig. 3 represents the structures of the ﬁnal com-

pounds annotated as compound 6 (a-k) and 7 (a-c).

3.1. Synthesis and spectral studies

Structural analysis based on proton nuclear magnetic resource

(¹H NMR) spectrum of compound 1b revealed two characteristic

doublets resonating at chemical shifts (d) 7.80e7.77 ppm (Ph-CH]

3.2. Antiplasmodial activity

CH) and 7.52e7.49 ppm (Ph-CH]CH) with J ¼ 15.62 Hz and

15.61 Hz, respectively, signifying the formation of a trans-oleﬁnic

conformation. Other resonating signals were observed on the

The synthesised novel analogues of (6a-k and 7a-c) were

assessed for potency against the NF54 CQ-susceptible strain of

P. falciparum maintained in vitro at the blood stage. Table 1 presents

the antiplasmodial activity of the hybrid analogues (6a-k and 7a-c),

and standard reference drugs (CQ and quinine). Compound 7a

spectrum at d 3.89 and 2.39 ppm representing methoxy and methyl

substituents on the phenyl groups of the (E)-chalcone. The two

doublet signals of the oleﬁn disappear on the ¹H NMR spectrum of

showed promising activity with IC₅₀values less than 0.5 mM, while

compound 2b, and two distinctive singlets at signal

d

7.35 ppm

the remaining analogues displayed moderate activity in micro-

molar levels against this CQ-susceptible strain (NF54). The IC₅₀

values of the compounds were determined from log sigmoid dose-

response curves using GraphPad Prism® 5.0.0 (GraphPad Software,

Inc San Diego, CA). Given the antiplasmodial activity, we assessed a

reasonable number of analogues to establish a brief structure-

activity relationship (SAR) towards identifying the groups ac-

counting for the activity. The bioisosteric replacement on the

phenyl rings on the fourth and sixth position of pyrimidine core,

and linker coupling the pyrimidine and quinoline positively

correlated with the antiprotozoal activity as shown in Table 1.

From the screened compounds, only compounds 7a showed

(pyr-H) in the aromatic region and the amino signal at

d 5.22 ppm

(pyr-NH₂) appeared indicating the formation of a pyrimidine core.

The structure of 2-hydroxyl pyrimidine analogue is signiﬁed by the

vanishing of the amino signal and presence of hydroxyl (OH) signal

downﬁeld at

The spectrum of the chlorinated compound 4 is characterised by

d

11.89 ppm on the H¹NMR spectrum of compound 3.

the disappearance of OH signal and minor shifts on pyr-H

d from

7.35 ppm to 7.60 ppm. The ¹H NMR spectrum (400 and 600 MHz,

DMSO‑d₆) of the targeted compound(s), as presented by compound

7d, is characterised by the presence of two individual peaks; broad

singlet resonating at

d 5.04 ppm (-NH) and triplet at 5.55 ppm

(-NH) of an aliphatic alkane diamine linker signiﬁed coupling of

quinoline and pyrimidine motifs. Other distinctive peaks, as

substantial plasmodial inhibitory activity with IC₅₀¼ 0.32 0.06

mM,

while the other six promising hybrids namely compounds 6d, 6e, 6f,

6i, 7b and 7c portrayed moderate activity with IC₅₀ranging from

aforementioned, included

d 2.46 ppm, 3.52 ppm, 4.06 ppm and

4.07 ppm representing a methyl, methylene, methoxy and methy-

lene, respectively. The outstanding signals from 6.47 to 8.44 ppm

represent the aromatic protons for phenyl and piperazine moieties.

The spectral properties of the ﬁnal compound 7g were conﬁrmed

by 2D NMR and further supported by their respective ¹³C NMR. The

most noticeable peak for ¹³C NMR of the ﬁnal compound for pyr-H

(C9) and methylene for diamine butane line resonate at signal

0.50 to 4.30

6h, 6j and 6k) displaying IC₅₀values < 5

tive. Compound 6i had a moderate activity with IC₅₀value of

2.61 1.00 M, and compounds 6g and 6j appraised inactive with

IC₅₀values of 83.01 1.46 M and 17.81 1.18 M, respectively.

However, demethylation of the methoxy groups from these com-

pounds resulted into improved aqua solubility that consequently

increased the antiplasmodial activity of 7a, 7b and 7c with IC₅₀

m

M (Table 1). The remaining compounds (6a, 6b, 6c, 6g,

mM were considered inac-

m

d

ranging from 100.53 to 99.89 pm and 27.42e42.99 ppm,

4

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

Fig. 3. Structures of the ﬁnal compounds 6 (aek) and 7 (aec).

Table 1

In vitro antiplasmodial activity of compounds (6a-6k) and (7a-7c).

Compound No

n

R

R⁰

Antimalarial

Cytotoxicity

Selective Index

IC₅₀

(

mM)

SD

IC₅₀

(m

M)

SD

6a

6b

6c

6d

6e

6f

6g

6h

6i

6j

6k

7a

7b

7c

p

4

p

2

3

p

4

p

4

p

4

p

4-H

4-Me

4-MeO

3,4-MeO

4-OH

4-H

4-Cl

7.50

9.49

1.49

1.12

1.28

1.01

1.22

1.28

1.46

1.42

1.00

1.18

1.22

0.06

1.14

1.04

0.003

0.002

nd

3.16

nd

0.75

nd

Nd

9.79

Nd

13.12

4.30

2.71

2.63

83.01

7.69

2.61

17.81

6.17

0.32

1.62

2.66

0.108

0.012

nd

4-MeO

3,4-MeO

4-OH

3,4-OH

4-OH

nd

Nd

Quinine

Chloroquine

Camptothecin

134.35

101.19

nd

1.45

25.72

0.20

1243.98

8432.50

0.01

IC₅₀: Concentration at 50% inhibition of the parasite’s growth.

Selective Index: IC₅₀values of cytotoxic activity/IC₅₀values of antimalarial activity.

nd: not determined.

p: Piperazine.

values of 0.32

0.06

m

M, 1.62

1.14

m

M and 2.66

1.04

m

M,

compound 6c yielded improved activity four-fold as illustrated by

compound 6f. Insigniﬁcant variation in activity is observed as more

methoxy groups are added as shown by compound 6h and 6k with

respectively of which compound 7a had the most prominent activity.

As observed, compound 6a, 6c, 6h and 6k owning a piperazine

linker were considered inactive with IC₅₀values < 5

compounds 6g and 7c with piperazine linker revealed moderate

activity with IC₅₀values of 2.63 1.28 M and 2.66 1.04 M,

respectively. It can be suggested that the piperazine linker could

have been signiﬁcant and the difference in activity could allude to

bioisosteric replacements at phenyl groups of the 4,6-

diphenylpyrimidine core. Of the piperazine linker-based com-

pounds, 6j had the least activity with an IC₅₀value of

m

M. However,

IC₅₀values of 7.69

Compounds 6d and 6e respectively bearing 1,3-diamino propane

and 1,2-diamino ethane, and both containing 4-(4-

methoxyphenyl)-6-(p-tolyl)pyrimidine motif only displayed mod-

erate activity with IC₅₀values of 4.30 1.01 M and 2.71 1.22 M.

Compounds 6b, 6g, 6i, 7a and 7b having a 1,4-diamino butane

linker presented a broader variation in the activity. Note that

compound 6g bearing two methoxy substituents on fourth posi-

tions of the phenyl groups of the 4,6-diphenylpyrimidine core had

the least activity (Table 1). Adding an extra methoxy group on the

third position increased the activity by nine-fold as deﬁned by

1.42

m

M and 6.17

1.22 mM individually.

m

a

m

17.81

almost seven-fold as illustrated by compound 7c with IC₅₀of

2.66 1.04 M. In addition, substituting chloro with a methoxy

group of the 4-(4-chlorophenyl)-6-(p-tolyl)pyrimidine core of

1.18 mM, but its demethylation boosted the activity by

m

compound 6i (IC₅₀¼ 2.61

1.00 mM). Compound 6b housing a

5

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

methyl and chloro substituents on 4-(4-chlorophenyl)-6-(p-tolyl)

pyrimidine core was nine times more active than compound 6g,

though four times less active than 6i despite both having methoxy

substituents on the 4,6-diphenypyrimindine nucleus. The deme-

thylation of methoxy groups of 4,6-bis(4-methoxyphenyl)pyrimi-

dine of compound 6g core heightened the activity by over two

hundred-fold as demonstrated by 7a, the most active compound.

Although compound 6i showed moderate activity, demethylation

of the methoxy groups of the 4-(3,4-dimethoxyphenyl)-6-(4-

methoxyphenyl)pyrimidine core compounds only mildly

PfHsp70-z, which exhibited a four and two orders of magnitude

reduction in afﬁnity for the same compounds, respectively

(Table 2). Compound 7a exhibited higher binding afﬁnity for

PfHsp70-z with one order of magnitude difference in comparison to

its afﬁnity for PfHsp70-1 (Table 2). On the other hand, compound

7b had a lower afﬁnity (K_D¼ 0.281

mM) for PfHsp70-1; and higher

afﬁnity (K_D¼ 4.75 M) for PfHsp70-z, respectively. Altogether, this

m

suggests that compound 7a showed the highest propensity to

recognize both proteins. Notably, the observed varied preference of

some of the compounds for the two PfHsp70 proteins highlights

that despite their apparent sequence conservation, PfHsp70s

possess unique motifs for selective targeting.

improved the activity of compound 7b (2.61 vs 1.62 mM).

It should be noted that, in contrast to other substituents such as

methyl, chlorine and methoxy, the hydroxyl signiﬁcantly inﬂu-

enced the antiplasmodial activity as deﬁned in the activity of

compounds 6b, 6g, 6i, 7a and 7b analogues with the same 1,4-

diamino butane linker (Fig. 4). Given the above, it can be

concluded that diamino alkane linkers, particularly 1,4-diamino

butane and piperazine were crucial for the activity. Also, substitu-

tion on the phenyl groups of 4, 6-diphenylprimidine core with

electron-donating groups, particularly hydroxyl groups, positively

correlated with antiplasmodial activity.

Shown in Fig. 5 are the representative SPR sensorgrams repre-

senting direct protein-compound interaction. The sensorgrams

were analysed to generate the kinetics in Table 2. Binding afﬁnity

was evaluated as follows: (A) PfHsp70-1-6h (K_D¼ 2.19 nM) and

PfHsp70-z-6h (K_D¼ 0.137

m

M); (B) PfHsp70-1-6k (K_D¼ 0.261 nM)

and PfHsp70-z-6k (K_D

¼

2.05 M); (C) PfHsp70-1-7a

m

(K_D¼ 0.114 nM) and PfHsp70-z-7a (K_D¼ 4.43 nM); (D) PfHsp70-1-

7b (K_D¼ 0.281

mM) and PfHsp70-z-7b (K_D¼ 4.75

mM); ,

respectively.

Pyrimidinones have previously been shown to possess the

antiplasmodial activity and target the essential parasite protein

folding pathway of P. falciparum Hsp70 [18]. As such, we investi-

gated the binding afﬁnities of these compounds against the re-

combinant forms of two essential cytosolic P. falciparum Hsp70s;

PfHsp70-1 and PfHsp70-z [1 7]. To determine binding kinetics, we

employed surface plasmon resonance (SPR) analysis, as previously

conducted [19,20]. The SPR sensorgrams generated (Fig. 5), were

ﬁtted to a simple 1:1 Langmuir kinetic binding model (Table 2).

Generally, both parasite Hsp70s interacted with the four molecules

in a concentration-dependent manner (Fig. 5). The rates of disso-

ciation were comparable for all the molecules. Estimated K_Dvalues

were within the lower micromolar to the upper nanomolar range,

suggesting a fairly high afﬁnity of the compounds for the two

essential cytosol localized P. falciparum Hsp70s (Table 2). Interest-

ingly, PfHsp70-1 exhibited higher binding afﬁnity (K_Dvalues within

lower nanomolar range) for compounds 6h and 6k compared to

SPR based kinetics for the direct interaction of PfHsp70-1 and

Hsp70-z with the various compounds are represented by the rate of

association (Ka), rate of dissociation (Kd) and the steady-state af-

ﬁnity (K_D), respectively. The ligand represents the respective

immobilised protein on the CMD 3D 500L chip surface to which the

analyte was injected at a ﬂow rate of 50 ml/min in triplicate. Data

was analysed after double referencing (refractive index changes on-

chip surface without protein immobilised and for buffer injected

without analyte) as

mean standard error of measurement. Chi-square (

show the 1:1 Langmuir curve ﬁtting residuals.

a

baseline. Data are represented as

c

²) values

Furthermore, the cytotoxicity screening of the most potent

molecule, 7a, against human embryonic kidney epithelial (HEK-

293) cells was studied (Table 1). The selective index (SI) of 9.79 was

calculated for the most potent compound by dividing the IC₅₀value

of the cytotoxic activity divided by the IC₅₀value of the antimalarial

activity. The observed activity suggests that while selectively killing

Fig. 4. Structure Activity Relationship (SAR) for quinioline-pyrimidine derivatives.

6

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

Fig. 5. Selected synthetic compounds bind P. falciparum Hsp70s directly. Plots represent interaction of PfHsp70-1 or PfHsp70-z) as investigated against varying levels of the selected

compounds (6h, 6k, 7a or 7b).

the parasite, compound 7a has a limited cytotoxic effect against

mammalian cells at therapeutic dosages; where a safety index of 10

is regarded as safe.

4. Molecular docking scores

To measure the compounds’ ﬁtness at the active catalytic sites of

the two enzymes, molecular docking analysis was performed. The

four compounds were docked into the ATPase active sites of the

Table 2

Kinetics data for the interaction of molecules with P. falciparum recombinant Hsp70 proteins.

Analyte

Ligand

K_a(1/(M*s)) est. error

K_d(1/s) est. error

K_D(M) est. error

X²

6h

PfHsp70-1

PfHsp70-z

PfHsp70-1

PfHsp70-z

PfHsp70-1

PfHsp70-z

PfHsp70-1

PfHsp70-z

3.30 ( 0.67) e^þ06

5.99 ( 0.90) e^þ03

2.68 ( 0.68) e^þ06

1.34 ( 0.99) e^þ04

7.73 ( 0.54) e^þ06

2.70 ( 0.53) e^þ07

2.79 ( 0.67) e^þ05

1.91 ( 0.77) e^þ04

1.32 ( 0.12) e^ꢀ02

8.24 ( 0.76) e^ꢀ02

7.00 ( 0.11) e^ꢀ02

7.48 ( 0.60) e^ꢀ02

8.81 ( 0.24) e^ꢀ02

1.20 ( 0.44) e^ꢀ01

7.84 ( 0.92)e^ꢀ02

9.06 ( 0.83)e^ꢀ02

2.19 ( 0.70) e^ꢀ09

1.37 ( 0.89) e^ꢀ05

2.61 ( 0.57) e^ꢀ08

2.05 ( 0.59) e^ꢀ06

1.14 ( 0.44) e^ꢀ08

4.43 ( 0.92) e^ꢀ09

2.81 ( 0.35) e^ꢀ07

4.75 ( 0.55) e^ꢀ06

1.1

7.1

2.4

6.9

4.7

3.2

8.8

7.9

6k

7a

7b

7

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

Fig. 6. Binding modes and docking scores of the molecules to PfHsp70-1 and PfHsp70-z.

8

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

enzymes (PfHsp70-1 and PfHsp70-z) to calculate and examine the

ﬁtness/afﬁnity of the compounds. Based on the computational data,

it is likely that the compounds bind to the ATPase domain for both

proteins in a similar fashion to ATP binding. Suggesting that they

could be competitive inhibitors to ATP binding for PfHsp70-1 and

PfHsp70-z. Fig. 6 represents the binding modes and docking results

of the compounds for each enzyme. Notably, the four compounds

showed relatively good binding scores for PfHsp70-1. Compounds

7a and 6k showed the highest and lowest binding scores

of ꢀ9.8 kcal/mol and ꢀ8.7 kcal/mol, respectively. The docking result

for PfHsp70-z revealed that compound 7b exhibited the highest

docking score of ꢀ8.9 kcal/mol; while, compounds 7a and 6k

showed the lowest values of ꢀ8.1 kcal/mol. The results suggest that

the four compounds exhibited better ﬁtness in PfHsp70-1 than

PfHsp70-z. To better understand the binding afﬁnity and structural

mechanisms of inhibition of the four compounds against the two

enzymes, molecular dynamics study on the compounds’ best

binding modes to the enzymes were investigated since molecular

docking scores might give a false positive results.

binding energy (ꢀ49.182 kcal/mol) than 6h (ꢀ46.752 kcal/mol).

This result is in consistent with the docking results indicating that

compounds 7a and 7b had the highest docking score for PfHsp70-1.

In the PfHsp70-z systems, compounds 7b and 6k showed the

highest binding afﬁnities of ꢀ52.097 kcal/mol and ꢀ49.122 kcal/

mol. This is also in consistent with the docking result that showed

compound 7b to have the highest docking score.

Furthermore, as shown in Table 3, compounds 6k and 7b

showed no difference in their binding afﬁnities for the two en-

zymes. Generally PfHsp70-1 showed strong binding energies for

compounds, 6h, 6k, 7a and 7b, than PfHsp70-z. The high binding

afﬁnities recorded suggest possible inhibitory activities of com-

pounds (6h, 6k, 7a and 7b) against the protein folding role of

PfHsp70-1.

4.2. Ligand-receptor interaction proﬁles of the molecules

The receptor-ligand interaction plots examined the molecular

interactions between the bound compounds and the amino acid

residues at the active sites of an enzyme [21]. To examine the

mechanistic inhibitory characteristics of the four compounds, the

interaction between the compounds and the active site amino

residues of the enzymes were assessed. Fig. 7 and Fig. 8 shows the

2D representation of the interactions plots between the com-

pounds and the different amino acid residues of PfHsp70-1 and

PfHsp70-z, respectively. Van der Waals, H-bond, pi-anion and pi-

akyl are the interactions observed in the PfHsp70-1 complexes.

Structurally related compounds 6k and 6h showed pi-anion in-

teractions with cysteine residues, Cys206 and Cys77, respectively

(Fig. 7).

4.1. Binding energies/afﬁnities of the molecules to PfHsp70-1 and

PfHsp70-z

To calculate the binding free energies of all the ﬁnals com-

pounds to the enzymes, Molecular Mechanics/Generalized Born

Surface Area (MMGBSA) computational technique was employed.

Table 3 shows the results of the thermodynamic binding free en-

ergy proﬁles for the compounds to the two enzymes. All the

compounds showed high binding energies/afﬁnity for PfHsp70-1,

ranging from ꢀ46.752 to ꢀ56.270 kcal/mol, with compounds 7a

and 7b having the highest free energies of ꢀ56.270 kcal/mol

and ꢀ53.968 kcal/mol, respectively. Compound 6k exhibited higher

While compound 7a had pi-anion interaction with Asp5, how-

ever, compound 6h showed no pi-anion interaction, but had a pi-

Table 3

Thermodynamic binding free energy proﬁles for the compounds.

Energy Components (kcal/mol)

Complex

D

E_vdW

D

E_elec

D

G_gas

D

G_solv

DG_bind

PfHsp70e1

6a

6b

6c

6d

6e

6f

6g

6h

6i

ꢀ48.805 4.167

ꢀ33.255 4.705

ꢀ37.742 4.235

ꢀ38.188 7.884

ꢀ42.131 7.134

ꢀ35.777 6.155

ꢀ37.903 3.241

ꢀ58.681 4.387

ꢀ35.113 4.585

ꢀ35.989 5.100

ꢀ59.352 5.864

ꢀ65.317 4.753

ꢀ58.507 4.406

ꢀ60.688 3.181

ꢀ10.005 3.324

ꢀ8.775 2.751

ꢀ9.155 1.545

ꢀ9.288 6.002

ꢀ38.322 10.231

ꢀ19.578 3.688

ꢀ21.588 2.411

ꢀ8.298 6.244

ꢀ22.200 3.471

ꢀ24.681 3.812

ꢀ21.389 6.159

ꢀ36.744 10.146

ꢀ42.327 13.445

ꢀ31.581 8.435

ꢀ46.179 8.117

ꢀ39.685 2.216

ꢀ41.331 4.114

ꢀ42.078 4.555

64.111 9.313

ꢀ58.124 4.574

ꢀ60.004 2.660

ꢀ66.978 7.441

ꢀ51.745 3.747

ꢀ57.818 5.558

ꢀ80.741 7.015

ꢀ102.061 12.488

100.834 12.429

ꢀ87.481 7.700

20.574 1.104

15.111 2.212

17.008 2.101

21.000 2.414

40.120 4.223

25.435 2.585

27.747 2.575

20.226 4.431

24.155 3.886

24.988 4.515

31.559 5.613

45.791 5.010

46.867 6.614

39.928 6.781

ꢀ41.485 7.544

ꢀ37.323 4.245

ꢀ40.137 3.038

ꢀ36.685 5.422

ꢀ47.363 4.205

ꢀ34.898 5.985

ꢀ35.999 4.257

ꢀ46.752 5.806

ꢀ33.219 3.888

ꢀ34.910 4.378

ꢀ49.182 6.582

ꢀ56.270 9.413

ꢀ53.968 7.005

ꢀ42.823 7.880

6j

6k

7a

7b

7c

PfHsp70-z

6a

6b

6c

6d

6e

6f

6g

6h

6i

ꢀ40.243 2.723

ꢀ34.123 4.111

ꢀ41.272 3.928

ꢀ37.223 3.983

ꢀ35.245 4.145

ꢀ21.113 3.112

ꢀ22.343 4.422

ꢀ47.397 3.501

ꢀ23.111 2.165

ꢀ42.785 3.423

ꢀ61.985 4.948

ꢀ48.129 4.947

ꢀ60.543 3.675

ꢀ29.105 5.500

ꢀ23.212 3.104

ꢀ14.423 3.933

ꢀ23.092 7.834

ꢀ21.223 7.215

ꢀ23.254 5.444

ꢀ17.723 2.863

ꢀ19.424 2.128

ꢀ16.487 5.949

ꢀ22.214 3.819

ꢀ18.447 4.551

ꢀ23.126 9.433

ꢀ28.574 12.986

ꢀ33.094 9.310

ꢀ20.117 2.234

ꢀ72.126 4.332

ꢀ44.133 2.811

ꢀ73.023 4.933

ꢀ51.443 4.113

ꢀ47.982 2.254

ꢀ32.141 4.223

ꢀ28.321 3.252

ꢀ63.884 7.430

ꢀ19.231 4.122

ꢀ55.957 5.541

ꢀ85.111 11.737

ꢀ76.703 14.429

ꢀ93.636 8.713

ꢀ45.777 424

31.432 3.424

21.331 5.121

34.422 5.474

30.551 5.441

32.268 4.141

19.321 2.113

20.441 3.938

28.353 5.285

17.654 4.525

25.415 5.998

35.990 8.178

41.877 7.891

41.539 5.474

23.425 4.646

ꢀ32.073 3.023

ꢀ30.332 1.932

ꢀ41.324 3.339

ꢀ37.525 5.251

ꢀ33.125 3.331

ꢀ29.222 3.432

ꢀ26.234 2.224

ꢀ35.531 4.910

ꢀ24.212 2.333

ꢀ31.258 4.233

ꢀ49.122 6.131

ꢀ34.826 9.645

ꢀ52.097 5.016

ꢀ30.411 4.008

6j

6k

7a

7b

7c

D

Eele electrostatic energy,

D

EvdW van der Waals energy,

DGbind total binding free energy,

DGsol solvation free energy,

D

Egas gas-phase free energy.

9

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

Fig. 7. A depiction of protein-ligand interactions plots with different amino acid residues.

sigma interaction with Cys206. These data revealed that binding

energies are proportional to the number of H-bond and the total

number of interactions observed in the four compounds, where

compounds 7a, 7b, 6k and 6h have total interaction numbers of 29,

23, 17 and 15, respectively. A slightly different interaction was

observed in PfHsp70-z complexes. Unlike the pi-anion interaction

observed in PfHsp70-1, a pi-cation interaction was observed in

PfHsp70-z. Furthermore, pi-pi stacked and donor-donor in-

teractions were also observed. All the compounds showed a sig-

niﬁcant number of H-bond interactions required for stability. A

donor-donor interaction was only observed in compound 7b. The

difference in the number and type of interactions observed for the

two proteins (PfHsp70-1 and PfHsp70-z) could explain the higher

binding energies observed in the PfHsp70-1 complexes.

reduction in average RoG values after the binding of the Brath goo

compounds (7a; 28.272 Å, 7b; 28.231 Å, 6h; 28.354 Å, 6k; 27.642 Å)

was also observed in PfHsp70-z complexes when compared with

the average value of the unbound PfHsp70-z system, 28.321 Å

(Fig. 9e1). This ﬁnding suggests the binding of the compounds

stabilizes the enzymes.

Fig. 9e2 shows the plot of the RMSD values, which is a mea-

surement of the enzyme complexes’ convergence and stability [22].

The plot shows that all the four compounds and the unbound

systems reach convergence after about 8 ns. The RMSD plots for the

complexes revealed that the four complexes are more stable (lower

RMSD values) than the unbound systems of PfHsp70-1 and

PfHsp70-z. This also suggests that the alpha carbon ﬂuctuation of

the compounds converged well during MD simulation. Likewise,

the RMSF of the complexes were studied to examine the ﬂexibility

and stability of individual amino residues of the proteins. RMSF

monitors the behaviour of the active residues of a protein. Higher

and lower ﬂuctuation values indicated more and less ﬂexible

movements, respectively. As shown in Fig. 9e2. c, residues

200e300, 305e345 and 5525 - 600 ﬂuctuate more in all the four

compounds and the unbound PfHsp70-1 systems. While most of

the residues (150e250) involved in ligand binding ﬂuctuate less.

The plot further revealed that the average RMSF values of the

compounds (7b (3.743 Å), 7a (3.771 Å), 6h (3.989 Å) and 6k

(4.023 Å)) were lower (less ﬂuctuation) than the unbound system

with average RMSF value of 4.212 Å. For the PfHsp70-z systems,

more ﬂexibility was observed at residues 230e260, 375e400 and

450e475. Unlike the PfHsp70-1 system, compounds 7a and 7b

4.3. Stability and ﬂexibility of the enzymes bound complexes

To examine the stability and dynamics of enzymes-ligand

complexes, 100 ns molecular dynamic (MD) simulations were

performed. Root means square deviation (RMSD), radius of gyration

(RoG), root means square ﬂuctuation (RMSF) and solvent accessible

surface area (SASA) of alpha carbon (Ca) atoms were monitored and

analysed along with the entire duration of 100 ns of the MD

simulation for the unbound and bound enzyme systems. For

PfHsp70-1, the RoG plots revealed the binding of the four com-

pounds lowers the mean RoG values of the four compounds (7a;

35.243 Å, 7b; 34.553 Å, 6h; 34.183 Å, 6k; 31.432 Å) when compared

to the unbound PfHsp70-1 systems; 35.973 Å (Fig. 9e1a). A similar

10

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

Fig. 8. A depiction of protein-ligand interactions plots with different amino acid residues.

exhibited higher average RMSF values (2.942 Å and 2.729 Å,

respectively) than registered by unbound PfHsp70-1 (2.547 Å).

Compounds 6h and 6k exhibited less ﬂuctuation than the unbound

enzyme with average values of 2.4675 Å and 2.501 Å, respectively.

SASA is another parameter to quantify the enzymes’ exposure to

the solvent environment [23]. The four compounds exhibited an

increase in the exposure of the enzyme to solvent molecules, as

their respective bindings raised the average SASA values when

compared to the unbound PfHsp70-1 (Fig. 9e2d). SASA mean

values of 35100 Å², 34200 Å², 34550 Å², 33800 Å²and 33700 Å²

were recorded for compounds 7a, 7b, 6h, 6k and unbound

PfHsp70-1. The average SASA values for compounds 7a, 7b, 6h, 6k

and the unbound PfHsp70-z systems are 25900 Å², 25100 Å²,

25600 Å², 24700 Å²and 24800 Å²(Fig. 9e2d). The increased SASA

values reported for the compounds showed the binding of the

compounds further exposed the enzyme to a solvent molecule. This

suggests that the structural integrity of the enzyme was not dis-

torted after 100 ns.

low [26]. Compounds 7a and 7b were predicted to be lowly

absorbed in the gastrointestinal tract (GIT), while compounds 6h

and 6k were predicted to be highly absorbed. This suggests that the

highly absorbed compounds would attain optimal concentrations

in the systemic circulation than the lowly absorbed compounds.

The blood-brain barrier (BBB) is a protective gate that prevents the

passage of toxic compounds into the brain or the central nervous

system [24]. For the four test compounds (6h, 6j, 7a and 7b) the

number of hydrogen donors ranged between 0 and 5 while

hydrogen acceptors 5 to 7. Thus, indicating that all the test com-

pounds could be considered orally active. None of the four com-

pounds were predicted to permeate the BBB. Bioavailability score is

a measurement of the rate of absorption and quantity of a given

amount of unchanged drug that goes to the systemic circulation

[27]. The four compounds showed a relatively high score of 0.55,

which suggests they all might reach their sites of active with higher

concentrations required for therapeutic effect.

5. Conclusion

4.4. Assessing the drug-likeness of the compounds

In summary, pyrimidine-quinoline analogy inspired chloro-

quine and pyrimethamine antimalarial drugs were designed, syn-

thesised by molecular hybridisation approach and screened for

their antiplasmodial activity. The facile preparation with excellent

yields of these compounds makes synthetic pathway attractive and

versatile. Among the tested compounds, only compound 7a dis-

Pharmacokinetic properties of the compounds were predicted

using the SwissADME serve [24]. The Lipinski’s rule of ﬁve is a set of

rules that assess both the physical and chemical properties of po-

tential drugs to ascertain its safety as an orally active drug [25]. As

shown in Table 4, the four compounds (6h, 6k, 7a and 7b) had no

more than 5 hydrogen bond donors, no more than 10 hydrogen

bond acceptors, thus passed the drug-likeness test and are poorly

soluble in water. The result showed that the four compounds’

partition coefﬁcients (logP) are around 5. Greater logP usually in-

dicates the probability of a drug to permeate the lipid membrane is

played the best antiplasmodial activity (IC₅₀¼ 0.32

0.06 mM),

with a selectivity index of 9.79, which is considered safe. Interest-

ingly, compound 7a bound to both parasite Hsp70 chaperones with

signiﬁcant afﬁnity, suggesting that one possible mechanism of ac-

tion of this compound could be to abrogate the protein folding

11

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

Fig. 9-1. Plots of alpha C atoms of the PfHsp70-1, 7a, 7b, 6h and 6k, a) RoG plots b) RMSD, c) RMSF and d) SASA plots systems calculated throughout 100 ns MD simulations.

pathway of the parasite. This hypothesis was further supported by

the observed molecular docking scores. Brief SAR studies revealed

that strong activating or electron-donating groups on the meta and

para position of the aromatic group enhanced the activity. Similarly

piperazine or 1,4-diamino butane linkers were essential for the

activity. The authors believe that the outcome or observation of this

research work would assist in the researchers in designing potent

antimalarial agents.

internal standard. HRMS spectra were recorded on an Autospec

mass spectrometer with electron impact at 70 eV.

6.1. A typical procedure for the synthesis of (E) chalcone (1b-1f)

6. Experimental

All the chemicals used in this research work were purchased

from Sigma-Aldrich and Merck Millipore, South Africa. The

commercially available chemicals benzaldehyde and acetophenone

were purchased from Sigma-Aldrich (South Africa). All the solvents,

except those of laboratory-reagent grade, were dried and puriﬁed

when necessary according to previously published methods. The

progress of the reactions and the purity of the compounds were

monitored by thin-layer chromatography (TLC) on pre-coated silica

gel plates procured from E. Merck and Co. (Darmstadt, Germany)

using 36% ethyl acetate in n-hexane as the mobile phase and iodine

vapour as the visualising agent.

The melting points of the synthesised compounds were deter-

mined using a Thermo Fisher Scientiﬁc (IA9000, UK) digital melting

point apparatus and are uncorrected. The IR spectra were recorded

on a Bruker Alpha FT-IR spectrometer (Billerica, MA, USA) using the

ATR technique. The ¹H NMR and ¹³C NMR spectra were recorded on

a Bruker AVANCE 400 and 600 MHz (Bruker, Rheinstetten/Karls-

ruhe, Germany) spectrometers using CDCl₃and DMSO‑d₆. The

chemical shifts are reported in units with respect to TMS as an

To a stirring solution of acetophenone (5 g, 32.34 mmol) in EtOH

(30 ml), a 60% NaOH solution (12 g NaOH/H₂O (20 mL)) was added.

The reaction was stirred at room temperature for 30 min. Benzal-

dehyde (4.7 g, 38.81 mmol) was then added and the reaction

sonicated for 1 h at 35 ^ꢁC. The progression of the reaction was

monitored by TLC. Excess ethanol was removed under vacuum, and

the reaction mixture was added into 100 g of ice and stirred. The

precipitates formed were ﬁltered, washed with excess water and

dried under vacuum. The crude product was puriﬁed by recrystal-

lized in EtOH to obtain the following pure compounds in good

yield.

(E)-1-(4-chlorophenyl)-3-(p-tolyl)prop-2-en-1-one (1b): Yellow

solid, yield: 90%, mp ¼ 162e164 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d,

ppm): 7.96 (d, J ¼ 8.57 Hz, 2H, ArH), 7.78 (d, J ¼ 15.65 Hz, 1H, ph,-

CH]CH), 7.54 (d, J ¼ 8.08 Hz, 2H, ArH), 7.47 (d, J ¼ 8.36 Hz, 2H,

ArH), 7.46-7.42 (d, J ¼ 15.65 Hz, 1H, ph,-CH]CH), 7.23 (d,

12

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

Fig. 9-2. Plots of alpha C atoms of the PfHsp70-z, 7a, 7b, 6h and 6k, a) RoG plots b) RMSD, c) RMSF and d) SASA plots systems calculated throughout 100 ns MD simulations.

Table 4

Pharmacokinetic and physicochemical properties of the molecules.

Comp M. Formula M. Weight (g/mol) H-bond donar H-bond acceptor Lipophilicity (iLOGP Water

GIT

BBB

Bioavailability Drug likeness (Lipinski)

Solubility Absorption Permeability Score

7a

7b

6h

6k

C

₂₉H₂₆ClN₅O₂512.00

₂₉H₂₆ClN₅O₃528.00

₃₁H₂₈ClN₅O₂538.04

₃₃H₃₂ClN₅O₄598.09

4

5

0

5

6

5

7

5.03

4.69

5.20

5.16

Poor

Low

High

No

0.55

Yes

J ¼ 7.96 Hz, 2H ArH), 2.40 (s, 3H, CH₃); ¹³C NMR (400 MHz, CDCl₃,

d

,

(E)-3-(3,4-dimethoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-

ppm): 189.28, 145.48, 141.38, 139.09, 136.09, 136.65, 131.96, 129.90,

129.77, 128.92, 128.58, 120.46, 21.59 (CH₃).

one (1e): Yellow solid, yield: 80.6%, mp ¼ 92e94 ^ꢁC; ¹H NMR

(400 MHz, CDCl₃,

d

, ppm): 8.03 (d, J ¼ 8.81 Hz, 2H, ArH), 7.75 (d,

(E)-1-(4-methoxyphenyl)-3-(p-tolyl)prop-2-en-1-one (1c): Yel-

J ¼ 15.56 Hz, 1H, ph,-CH]CH), 7.40 (d, J ¼ 15.56 Hz, 1H, ph-CH]

CH), 7.23 (dd, J ¼ 8.31, 1.64 Hz, 1H, ArH), 7.16 (s, 1H, ArH,), 6.98 (d,

J ¼ 8.81 Hz, 1H, ArH), 6.89 (d, J ¼ 8.27 Hz, 1H, ArH), 3.94 (s, 3H,

OCH₃), 3.92 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃); ¹³C NMR (400 MHz,

low solid, yield: 96.3%, mp ¼ 125e128 ^ꢁC; ¹H NMR (400 MHz,

CDCl₃,

d

, ppm): 8.04 (d, J ¼ 8.76 Hz, 2H, ArH), 7.89 (d, J ¼ 15.60 Hz,

1H, ph-CH]CH), 7.54 (d, J ¼ 7.95 Hz, 2H, ArH), 7.51 (d, J ¼ 15.63 Hz,

1H, ph-CH]CH), 7.22 (d, J ¼ 7.97, Hz, 2H ArH), 6.98 (d, J ¼ 8.77 Hz,

2H, ArH), 3.88 (s,3H, CH₃), 2.39 (s, 3H,CH₃); ¹³C NMR (400 MHz,

CDCl₃, d, ppm): 189.37, 163.86, 151.83, 149.80, 144.71, 131.89, 131.29,

128.65, 123.53, 120.42, 114.36, 111.70, 110.68, 56.56 (OCH₃), 56.54

(OCH₃), 56.05 (OCH₃).

CDCl₃, d, ppm): 189.43, 163.92, 144.64, 141.40, 132.92, 131.81, 131.34,

130.24, 128.96, 121.46, 114.39, 56.06(OCH₃), 22.09 (CH₃).

(E)-1,3-bis(3,4-dimethoxyphenyl)prop-2-en-1-one (1f): Yellow

(E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one (1d): yellow solid,

solid, yield: 99%, mp ¼ 109e111 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d,

yield: 85.7%, mp ¼ 102e104 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d

, ppm):

ppm): 7.76 (d, J ¼ 15.54 Hz, 1H, ph,-CH]CH), 7.69 (d, J ¼ 8.36 Hz,

1H, ArH), 7.62 (s, 1H, ArH) 7.41 (d, J ¼ 15.54 Hz, 1H, ph-CH]CH),

7.24 (d, J ¼ 8.44 Hz, 1H, ArH), 7.16 (s, 1H, ArH), 6.92 (dd, J ¼ 11.73,

8.31 Hz, 2H, ArH), 3.89e3.95 (m, 9H, CH₃), 3.92 (s, 3H, CH₃); ¹³C

8.03 (d, J ¼ 8.75 Hz, 2H, ArH), 7.78 (d, J ¼ 15.58 Hz, 1H, ph-CH]CH),

7.60 (d, J ¼ 8.66 Hz, 2H, ArH), 7.43 (d, J ¼ 15.58 Hz, 1H, ph-CH]CH),

6.96 (dd, J ¼ 17.58, 8.72 Hz, 4H, ArH), 3.89 (s,3H, OCH₃), 3.85 (s, 3H,

OCH₃); ¹³C NMR (400 MHz, CDCl₃,

d

, ppm): 189.39, 163.84, 162.09,

NMR (400 MHz, CDCl₃, d, ppm): 189.26, 153.70, 151.86, 149.81,

162.09, 144.39, 131.95, 131.28, 130.67, 128.40, 120.15, 114.96, 114.36,

56.05 (OCH₃), 55.98 (OCH₃).

144.73, 132.13, 128.64, 123.46, 123.43, 120.25, 111.72, 111.43, 110.81,

110.50, 56.65 (2OCH₃), 56.57 (2OCH₃).

13

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

6.2. A typical procedure for the synthesis of 4,6-diphenylpyrimidin-

2-amine(2a-2f)

4,6-Diphenylpyrimidin-2-amine (2) (4 g, 12.76 mmol) was dis-

solved in acetic acid (100 mL), and whilst stirring a solution of 10 eq

NaNO₂dissolved in H₂O (50 ml) was slowly added at room tem-

perature. The reaction mixture was further stirred for 3 h. The

progression of the reaction was monitored using TLC. The precipi-

tate formed was ﬁltered and washed with excess water and dried

under vacuum. The crude product was recrystallized in EtOH to

obtain the following pure compounds in good yield.

The reaction mixture of (E)-chalcone (1) (8 g, 31.16 mmol),

guanidine hydrochloride (4.8 g, 49. 86 mmol) and 60% NaOH (12 g

NaOH/H₂O (20 mL)) in EtOH (30 mL) was reﬂuxed for 24 h. The

progression of the reaction was monitored using TLC. Excess

ethanol was removed under vacuum using rotavapor, and the re-

action mixture was then added in 100 g ice water. The precipitate

formed was ﬁltered, washed with excess water and dried. The

crude compound was puriﬁed by column chromatography using

EtOAc/hexane (1:4) eluent to obtain the following pure

compounds.

4,6-diphenylpyrimidin-2-ol (3a): Yellow solid, yield: 85%,

mp ¼ 231e234 ^ꢁC; , ¹H NMR (400 MHz, DMSO‑d₆,

d, ppm): 11.96 (s,

1H, Pyr-OH), 8.17 (d, J ¼ 6.56 Hz, 4H, ArH), 7.57-7.55 (m, 7H, ArH);

¹³C NMR (400 MHz, DMSO‑d₆,

d, ppm): 134.78, 131.42, 128.79,

4,6-diphenylpyrimidin-2-amine (2a): Yellow solid, yield: 65%,

127.54, 100.40.

mp ¼ 136e138 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d

, ppm): 8.07-8.05 (m,

4-(4-chlorophenyl)-6-(p-tolyl)pyrimidin-2-ol (3b): Yellow solid,

4H, ArH), 7.51-7.49 (m, 6H, ArH), 7.46 (s, 1H, ArH), 5.35 (s, 2H, NH₂).

yield: 89%, mp ¼ <300 ^ꢁC; ¹H NMR (400 MHz, DMSO‑d₆,

d, ppm):

4-(4-chlorophenyl)-6-(p-tolyl)pyrimidin-2-amine (2b): Yellow

11.83 (s, 1H, Pyr-OH), 8.14 (d, J ¼ 8.56 Hz, 1H, ArH), 8.13 (d,

J ¼ 8.76 Hz, 2H, ArH), 7.79-7.77 (m, 1H, ArH), 7.69 (d, J ¼ 1.56 Hz, 2H,

ArH), 7.08 (d, J ¼ 8.84 Hz, 2H, ArH), 3.88 (s, 3H, OCH₃), 3.85 (s, 6H,

2OCH₃).

solid, yield: 62%, mp ¼ 162e165 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d,

ppm): 8.00 (d, J ¼ 8.36 Hz, 2H, ArH), 7.95 (d, J ¼ 7.96 Hz, 2H, ArH),

7.46 (d, J ¼ 8.36 Hz, 2H, ArH), 7.369 (s,1H, ArH), 7.30 (d, J ¼ 7.380 Hz,

2H, ArH), 5.31 (s, 2H, NH₂), 2.43 (s, 3H, CH₃); ¹³C NMR (400 MHz,

4-(4-methoxyphenyl)-6-(p-tolyl)pyrimidin-2-ol (3d): Yellow

CDCl₃,

d, ppm): 166.40, 164.76, 163.63, 140.93, 136.52, 136.25,

solid, yield: 91%, mp ¼ 235e237 ^ꢁC; , ¹H NMR (400 MHz, DMSO‑d₆,

134.74, 129.53, 128.95, 128.42, 127.04, 103.64, 21.44 (CH₃).

d

, ppm): 11.85 (s, 1H, Pyr-OH), 8.16 (d, J ¼ 8.74 Hz, 2H, ArH), 8.04 (d,

4-(4-methoxyphenyl)-6-(p-tolyl)pyrimidin-2-amine (2c): Yellow

solid, yield: 58%, mp ¼ 127e129 ^ꢁC; 8.03 (d, J ¼ 8.64 Hz, 2H, ArH),

8.00 (d, J ¼ 8.36 Hz, 2h, ArH), 7.46 (d, J ¼ 8.36 Hz, 2H, ArH), 7.37 (s,

1H, Pyr-H), 7.00 (d, J ¼ 8.56 Hz, 2H, ArH) 5.20 (s, 2H, NH₂), 3.88 (s,

J ¼ 7.97 Hz, 2H, ArH), 7.44 (s, 1H, pyr-H), 7.35 (d, J ¼ 7.98 Hz, 2H,

ArH), 7.09 (d, J ¼ 8.97 Hz, 2H, ArH), 3.85 (s, 3H, OCH₃), 2.39 (s, 3H,

CH₃); ¹³C NMR (400 MHz, DMSO‑d₆,

d, ppm): 162.05, 141.51, 129.39,

129.35, 127.46, 114.18, 55.46 (OCH₃), 55.99 (CH₃).

3H, CH₃); ¹³C NMR (400 MHz, CDCl₃,

d

, ppm): 165.88, 164.64,

4,6-bis(4-methoxyphenyl)pyrimidin-2-ol (3d): Yellow solid,

163.55, 161.76, 136.47, 136.47, 129.91, 128.96, 128.64, 128.40, 114.14,

103.18, 55.43 (OCH₃).

yield: 90%, mp ¼ <300 ^ꢁC; ¹H NMR (400 MHz, DMSO‑d₆,

d, ppm):

11.82 (s, 1H, Pyr-OH), 8.14 (d, J ¼ 8.53 Hz, 2H, ArH), 7.41 (s, 1H, pyr-

4,6-bis(4-methoxyphenyl) pyrimidin-2-amine (2d); Yellow solid,

H), 7.09 (d, J ¼ 8.91 Hz, 2H, ArH), 3.85 (s, 6H, 2OCH₃), ¹³C NMR

yield: 65%, mp ¼ 171e173 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d

, ppm):

(400 MHz, DMSO‑d₆, d, ppm): 162.00, 129.32, 114.18, 55.46 (2OCH₃).

8.04 (d, J ¼ 8.66 Hz, 4H, ArH), 7.36 (s, 1H, Pyr-H) 7.00 (d,

4-(3,4-dimethoxyphenyl)-6-(4-methoxyphenyl)pyrimidin-2-ol

J ¼ 8.69 Hz,4H, ArH), 5.36 (s, 2H, NH₂), 3.87 (s, 6H, 2CH₃); ¹³C NMR

(3e): Yellow solid, yield: 89%, mp ¼ 235e237 ^ꢁC; ¹H NMR

(400 MHz, CDCl₃,

d

, ppm): 165.19, 162.97, 161.77, 129.78, 128.69,

(400 MHz, DMSO‑d₆, d, ppm): 11.83 (s, 1H, Pyr-OH), 7.79 (d,

114.15, 102.64, 55.43 (OCH₃).

J ¼ 7.29 Hz, 2H, ArH), 7.70 (d, J ¼ 7.71 Hz, 2H, ArH), 7.41 (s, 1H, ArH),

4-(3,4-dimethoxyphenyl)-6-(4-methoxyphenyl)

pyrimidin-2-

7.10 (d, J ¼ 8.2 Hz, 2H, ArH), 3.88 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃);

amine (2e): Yellow solid, yield: 61%, mp ¼ 157e159 ^ꢁC; ¹H NMR

¹³C NMR (400 MHz, DMSO‑d₆,

d, ppm): 162.00, 151.74, 148.77,

129.37,129.31,121.10,114.7,111.47,110.43, 97.91, 55.71 (OCH₃), 55.45

(2OCH₃).

(400 MHz, CDCl₃,

d

, ppm): 8.04 (d, J ¼ 8.86 Hz, 2H, ArH), 7.71 (d,

J ¼ 1.92 Hz, 1H, ArH), 7.63 (dd, J ¼ 8.55, 2.0 Hz,1H, ArH), 7.36 (s, 1H,

Pyr-H), 7.00 (d, J ¼ 8.84 Hz, 1H, ArH), 6.96 (d, J ¼ 8.45 Hz, 1H, ArH),

5.35 (s, 2H, NH₂), 4.00 (s, 3H, CH₃), 3.95 (s, 3H, CH₃), 3.87 (s, 3H,

CH₃).

4,6-bis(3,4-dimethoxyphenyl)pyrimidin-2-ol (3f): Yellow solid,

yield: 93%, mp ¼ <300 ^ꢁC; ¹H NMR (400 MHz, DMSO‑d₆,

d, ppm):

11.83 (s, 1H, Pyr-OH), 7.79 (d, J ¼ 7.29 Hz, 2H, ArH), 7.70 (d,

J ¼ 7.71 Hz, 2H, ArH), 7.41 (s, 1H, ArH), 7.10 (d, J ¼ 8.2 Hz, 2H, ArH),

3.88 (s, 6H, 2OCH₃), 3.85 (s, 6H, 2OCH₃); ¹³C NMR (400 MHz,

4,6-bis(3,4-dimethoxyphenyl)pyrimidin-2-amine (2f): Yellow

solid, yield: 67%, ¹H NMR (400 MHz, CDCl₃,

d, ppm): 7.71 (s, 2H,

ArH), 7.62 (dd, J ¼ 8.45, 1.69 Hz, 2H, ArH), 7.37 (s, 1H, Pyr-H), 6.96 (d,

J ¼ 8.40 Hz, 1H, ArH), 5.25 (s, 2H, NH₂), 4.00 (s, 6H, 2CH₃), 3.95 (s,

6H, 2CH₃).

DMSO‑d₆, d, ppm): 151.73, 148.72, 121.18, 111.42, 110.43, 55.66

(4OCH₃); HRMS (ESI, m/z) [MþNa] calculated for C₂₀H₂₀N₂O₅,

391.1270; found 391.1279.

6.3. A typical procedure for the synthesis of 4,6-diphenylpyrimidin-

2-ol (3a-3f)

6.4. A typical procedure for the synthesis of 2-chloro-4,6-

diphenylpyrimidine

14

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

6.5. A typical procedure for the synthesis of 4,6-diphenyl-2-

(piperazin-1-yl)pyrimidine

A solution of 4,6-diphenylpyrimidin-2-ol (4g, 16.11 mmol) and

0.4 mL DMF in POCl₃(20 mL) was reﬂuxed for 6 h. The reaction

completion was monitored by TLC. The reaction was added into ice

water, and the precipitate formed was ﬁltered, washed with water

and dried under vacuum. The crude compound was puriﬁed by

column chromatography using EtOAc/hexane (1:9) eluent to obtain

pure compounds.

A reaction mixture of 2-chloro-4,6-diphenylpyrimidine (4.0 g,

15 mmol) and piperazine (13.92 g, 150 mmol) in isopropanol

(20 mL) was reﬂuxed for 20 h. The progression of the reaction was

monitored by TLC. The reaction mixture was added to 2 M NaOH

(100 ml) solution, and the precipitate formed was ﬁltered, washed

with water and diethyl ether, and dried under vacuum. The crude

product was puriﬁed by recrystallized in EtOAc to obtain the

following pure compounds.

2-chloro-4,6-diphenylpyrimidine (4a): yellow solid, yield: 93%,

mp ¼ 115e116 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d, ppm): 8.07-8.05 (m,

4H, ArH), 7.51-7.49 (m, 6H, ArH), 7.46 (s, 1H, ArH), 5.35 (s, 2H, NH₂).

2-chloro-4-(4-chlorophenyl)-6-(p-tolyl)pyrimidine (4b): Yellow

solid, yield: 89%, ¹H NMR (400 MHz, CDCl₃,

d, ppm): 8.09 (d,

J ¼ 8.52 Hz, 2H, ArH), 8.04 (d, J ¼ 8.16 Hz, 2H, ArH), 7.94 (s, 1H, Pyr-

H), 7.50 (d, J ¼ 8.52 Hz, 2H, ArH), 7.33 (d, J ¼ 8.00 Hz, 2H, ArH), 2.44

N¹-(7-chloroquinolin-4-yl)ethane-1,2-diamine (5a): Yellow solid,

(s, 3H, CH₃); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 166.77, 165.13,

yield: 97%, mp ¼ 137e139 ^ꢁC; ¹H NMR (400 MHz, DMSO‑d₆,

d,

161.05, 141.46, 136.88, 133.16, 131.65, 128.83, 128.30, 127.68, 126.37,

109.20, 20.51(CH₃).

ppm): 8.33 (d, J ¼ 5.48 Hz, 1H, ArH), 8.20 (d, J ¼ 9.01 Hz, 1H, ArH),

7.76 (d, J ¼ 1.84 Hz, 1H, ArH), 7.40 (dd, J ¼ , 9.00 Hz, 1.85 Hz, 1H,

ArH), 7.26 (s, 1H, -NH), 6.50 (d, J ¼ 5.56 Hz, 1H, ArH), 3.27 (br.s, 2H,

2-chloro-4-(4-chlorophenyl)-6-(4-methoxyphenyl)pyrimidine

(4c): Yellow solid, yield: 95%, ¹H NMR (400 MHz, CDCl₃,

d

, ppm):

CH₂), 2.79 (t, J ¼ 6.38 Hz, 2H, CH₂); ¹³C NMR (400 MHz, DMSO‑d₆,

d,

8.11 (d, J ¼ 8.84 Hz, 2H, ArH), 8.07 (d, J ¼ 8.56 Hz, 2H, ArH), 7.88 (s,

ppm): 152.35, 152.27, 150.06, 149.02, 134.24, 127.41, 124.78, 117.80,

99.25, 45.58.

1H, Pyr-H), 7.49 (d, J ¼ 8.68 Hz, 2H, ArH), 7.02 (d, J ¼ 8.92 Hz, 2H,

ArH), 3.89 (s, 3H, OCH₃); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 167.27,

N¹-(7-chloroquinolin-4-yl)propane-1,3-diamine (5b): Yellow

165.95, 162.75, 162.02, 137.82, 134.25, 129.30, 129.16, 128.67, 127.81,

114.46, 109.62, 55.50 (OCH₃).

solid, yield: 98%, mp ¼ 88e90 ^ꢁC; ¹H NMR (400 MHz, DMSO‑d₆,

d,

ppm): 8.32 (d, J ¼ 5.06 Hz, 1H, ArH), 8.17 (d, J ¼ 8.92 Hz, 1H, ArH),

7.75 (d, J ¼ 4.92Hz, 1H, ArH), 7.68 (br.s, 1H, -NH), 7.40 (d, J ¼ 7.20 Hz,

1H, ArH), 6.48 (d, J ¼ 5.54 Hz, 1H, ArH), 3.30 (t, J ¼ 6.90Hz, 2H, ArH),

2.66 (t, J ¼ 6.60 Hz, 2H, CH₂), 1.74 (q, J ¼ 6.75 Hz, 2H, CH₂), ¹³C NMR

2-chloro-4,6-bis(4-methoxyphenyl)pyrimidine (4d): Yellow solid,

yield: 93%, mp ¼ 187e190 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d, ppm):

8.10 (d, J ¼ 8.48 Hz, 3H, ArH), 7.87 (d, J ¼ 8.84 Hz, 1H, ArH), 7.85 (s,

1H, Pyr-H), 7.01 (dd, J ¼ 8.50 Hz, 1.74 Hz, 4H, ArH), 3.88 (s, 6H,

(400 MHz, DMSO‑d₆, d, ppm): 151.83, 151.70, 150.36,148.45, 133.64,

2OCH₃); ¹³C NMR (400 MHz, CDCl₃,

d

, ppm): 166.74, 166.52, 162.51,

126.84, 126.84, 124.22, 123.89, 122.04, 98.61, 40.20.

161.84, 161.58, 131.61, 129.10, 128.19, 127.90, 114.39,113.67, 109.01,

55.49(OCH₃), 55.44 (OCH₃).

N¹-(7-chloroquinolin-4-yl)butane-1,4-diamine (5c): White solid,

yield: 99%, mp ¼ 121e124 ^ꢁC; ¹H NMR (400 MHz, DMSO‑d₆,

d,

2-chloro-4,6-bis(4-methoxyphenyl)pyrimidine (4e): Yellow solid,

ppm): 8.32 (d, J ¼ 5.48 Hz, 1H, ArH), 8.19 (d, J ¼ 9.01 Hz, 1H, ArH),

7.76 (d, J ¼ 1.64 Hz, 1H, ArH), 7.39 (dd, J ¼ 8.94, 1.64, Hz, 1H, ArH),

7.36 (br.s,1H, -NH), 6.44 (d, J ¼ 5.56 Hz,1H, ArH), 3.23 (t, J ¼ 6.90 Hz,

2H, CH₂), 2.54 (t, J ¼ 6.98 Hz, 2H, CH₂), 1.62 (q, J ¼ 6.98 Hz, 2H, CH₂),

yield: 93%, mp ¼ 187e190 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d, ppm):

8.10 (d, J ¼ 8.48 Hz, 3H, ArH), 7.87 (d, J ¼ 8.84 Hz, 1H, ArH), 7.85 (s,

1H, Pyr-H), 7.01 (dd, J ¼ 8.50 Hz, 1.74 Hz, 4H, ArH), 3.88 (s, 6H,

2OCH₃); ¹³C NMR (400 MHz, CDCl₃,

d

, ppm): 166.74, 166.52, 162.51,

1.44 (p, J ¼ 7.35 Hz, 2H, CH₂); ¹³C NMR (400 MHz, DMSO‑d₆,

d,

161.84, 161.58, 131.61, 129.10, 128.19, 127.90, 114.39, 113.67, 109.01,

55.49(OCH₃), 55.44 (OCH₃).

ppm): 151.25, 149.91, 148.02, 133.19, 126.38, 123.72, 123.43, 116.77,

98.13, 41.79(CH₂), 40.88 (CH₂), 29.49 (CH₂), 24.67 (CH₂).

2-chloro-4-(3,4-dimethoxyphenyl)-6-(4-methoxyphenyl)pyrimi-

7-chloro-4-(piperazin-1-yl)quinoline (5d): Yellow solid, yield:

dine (4f): Yellow solid, yield: 89%, mp ¼ 138e141 ^ꢁC; ¹H NMR

100%, mp ¼ 113e115 ^ꢁC;¹H NMR (400 MHz, DMSO‑d₆,

d, ppm): 8.68

(400 MHz, CDCl₃,

d

, ppm): 8.12 (d, J ¼ 8.88 Hz, 2H, ArH), 7.87 (d,

(d, J ¼ 5.00 Hz, 1H, ArH), 8.01 (d, J ¼ 9.00 Hz, 1H, ArH), 7.96 (d,

J ¼ 2.16 Hz, 1H, ArH), 7.53 (dd. J ¼ 8.98 Hz, 2.26 Hz, 1H, ArH), 3.09-

3.07 (m, 4H, 2CH₂), 2.97-2.94 (m, 4H, 2CH₂); ¹³C NMR (400 MHz,

J ¼ 8.84 Hz, 1H, ArH), 7.85 (s, 1H, Pyr-H), 7.01 (d, J ¼ 8.88, 2H, ArH),

6.96 (d, J ¼ 8.36, 1H, ArH), 4.00 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃),

3.88 (s, 3H, OCH₃); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 164.28,

DMSO‑d₆, d, ppm): 156.42, 151.17, 151.88, 149.61, 148.78, 141.29,

164.24, 162.78, 161.40, 150.90, 149.06, 131.44, 131.04, 120.01, 113.92,

110.87, 110.05, 99.65, 56.00 (OCH₃), 55.95 (OCH₃), 55.39 (OCH₃).

2-chloro-4,6-bis(3,4-dimethoxyphenyl)pyrimidine (4g): Yellow

135.36, 133.53, 128.69, 128.11, 128.02, 126.04, 125.80, 125.69, 124.25,

122.03, 121.31, 109.37, 109.37, 51.86, 44.69.

solid, yield: 78%, mp ¼ 180e183 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d,

6.6. A typical procedure for the synthesis of N¹-(7-chloroquinolin-

4-yl)-N2-(4,6-diphenylpyrimidin-2-yl) ethane-1,2-diamine

ppm): 7.86 (s, 1H, ArH), 7.74 (s, 2H, ArH), 7.69 (d, J ¼ 7.12 Hz, 2H,

ArH), 6.97 (d, J ¼ 7.76 Hz, 2H, ArH), 4.00 (s, 6H, 2OCH₃), 3.96 (s, 6H,

2OCH₃).

15

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

ArH), 8.09 (d, J ¼ 1.99 Hz, 1H, ArH), 7.91 (t, J ¼ 16.44 Hz, 5H, ArH),

7.42 (d, J ¼ 8.73 Hz, 1H, ArH), 7.28 (s, 2H, ArH), 7.00-6.94 (m, 2H,

ArH), 6.37 (d, J ¼ 4.89 Hz, 1H, ArH), 5.69 (br.s, 1H, -NH), 5.48 (t,

J ¼ 4.89 Hz, 1H, -NH), 3.96 (s, 3H, OCH₃), 3.81 (q, J ¼ 6.16 Hz, 2H,

CH₂), 3.52-3.48 (m, 2H, ArH), 2.44 (s, 3H, CH₃), 2.10 (q, J ¼ 6.09 Hz,

2H, CH₂); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 166.06, 164.01, 163.17,

156.82, 140.94, 135.07, 129.58, 128.99, 127.06, 126.68, 125.26, 122.97,

121.37, 111.83, 102.59, 56.32 (OCH₃), 40.59 (CeNH₂), 38.75(CeNH₂),

28.89(CH₂), 21.44 (CH₃).

7-chloro-4-(4-(4-(4-methoxyphenyl)-6-(p-tolyl)pyrimidin-2-

yl)piperazin-1-yl)quinoline (6f): Yellow solid, yield: 83%,

nmax, cm-1): 3038 (CeH), 2998 (CeH

of CH₃), 2956 (CeH of CH₃), 1438e1565 (C]C of Ar), 1237 (CeO),

A solution of 2-chloro-4,6-diphenylpyrimidine (6) (120.65 mg,

0.45 mmol), N¹-(7-chloroquinolin-4-yl) ethane-1,2-diamine (5)

(100 mg, 0.45 mmol) and K₂CO₃(125.03 mg, 0.91 mmol) in DMF

(2 mL) was discharged to a microwave at temperature 120 ^ꢁC,

pressure 150 Psi and power 150 Watts for 40 min. The progression

of the reaction was monitored using TLC. The reaction mixture was

added to ice water. The precipitate formed was ﬁltered, washed

with water and dried under vacuum. The compound was puriﬁed

using silica gel in a chromatographic column with eluent 1% MeOH

in DCM to afford the following pure compounds.

mp ¼ 191e193 ^ꢁC; FTIR (ATR,

773 (CeCl), ¹H NMR (400 MHz, CDCl₃,

d

, ppm): 8.74 (d, J ¼ 4.96 Hz,

1H, ArH),), 8.12 (d, J ¼ 8.80 Hz, 2H, ArH), 8.08 (s, 1H, ArH), 8.06 (d,

J ¼ 5.69 Hz,1H, ArH), 8.04 (d, J ¼ 8.09 Hz, 3H, ArH) 7.47 (dd, J ¼ 8.99,

1.99 Hz, 1H, ArH), 7.40 (s, 2H, CH₂), 7.30 (d, J ¼ 8.04 Hz, 2H, ArH),

7.01 (d, J ¼ 8.79 Hz, 2H, ArH), 6.88 (d, J ¼ 5.02 Hz, 1H, ArH), 4.31-

4.29 (m, 4H, 2CH₂), 3.89 (s, 3H, OCH₃), 3.35 (t, q ¼ 4.83 Hz,4H, 2CH₂)

2.43 (s, 3H, CH₃); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 164.71, 162.12,

7-chloro-4-(4-(4,6-diphenylpyrimidin-2-yl)piperazin-1-yl)quino-

157.13, 151.96, 151.25, 150.17, 149.17, 135.06, 130.93, 128.94, 126.35,

125.19, 122.00, 120.24, 110.96, 110.13, 109.14, 101.48, 56.11(2OMe),

56.04 (2OMe), 52.38 (2CH₂), 44.03 (2CH₂), 21.44 (CH₃).

line (6a): Yellow solid, yield: 65%, mp ¼ 169e172 ^ꢁC; ¹H NMR

(400 MHz, CDCl₃,

d

, ppm): 8.76 (d, J ¼ 4.35 Hz, 1H, ArH), 8.14 (d,

J ¼ 3.63 Hz, 3H, ArH), 8.08-8.07 (m, 2H, ArH), 7.82 (d, J ¼ 3.44 Hz,

1H, ArH), 7.51-7.48 (m, 8H, ArH), 6.89 (d, J ¼ 8.45 Hz, 1H, ArH), 4.33-

4.19 (m, 4H, 2CH₂), 3.36-3.29 (m, 4H, 2CH₂); ¹³C NMR (400 MHz,

N¹-(4,6-bis(4-methoxyphenyl)pyrimidin-2-yl)-N⁴-(7-

chloroquinolin-4-yl)butane-1,4-diamine (6g): Yellow solid, yield:

83%, mp ¼ 144e145 ^ꢁC; FTIR (ATR,

nmax, cm-1): 3256 (NeH), 3069,

CDCl₃,

d

, ppm): 163.76, 157.09, 151.13, 150.64, 414.49, 134.83, 129.77,

2932 (CeH, CH₃), 1573-1450 (C]C, Ar) 1239 (CeO), 763 (CeCl); ¹H

129.11, 127.48, 127.28, 126.89, 124.60, 123.09, 122.09, 111.96, 103.09,

98.16, 56.37, 40.20, 21.47.

NMR (400 MHz, CDCl₃,

d

, ppm): 8.38 (d, J ¼ 5.57 Hz, 1H, ArH),), 8.03

(d, J ¼ 8.81 Hz, 3H, ArH), 7.91 (d, J ¼ 2.10 Hz, 1H, ArH), 7.76 (d,

J ¼ 8.69 Hz, 1H, ArH), 7.62 (d, J ¼ 8.97 Hz, 1H, ArH), 7.30 (s, 1H, ArH),

7.21 (dd, J ¼ 7.20, 2.04 Hz, 1H, ArH) 6.96 (d, J ¼ 8.72 Hz, 5H, ArH),

6.35 (d, J ¼ 5.61 Hz, 1H, ArH), 5.55 (br.s, 1H, NH), 5.40 (t, J ¼ 6.01Hz,

1H, NH), 3.85 (s, 6H, 2OCH₂), 3.66 (m, 1H, NH),), 5.56- 5.48 (m, 1H,

NH), 3.19 (s, 6H, 2CH₃), 3.81-3.60 (m, 2H, CH₂), 3.51 (q, J ¼ 6.27 Hz,

N¹-(4-(4-chlorophenyl)-6-(p-tolyl)pyrimidin-2-yl)-N⁴-(7-

chloroquinolin-4-yl)butane-1,4-diamine (6b): Yellow solid, yield:

84%, mp ¼ 192e194 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d, ppm): 8.50 (d,

J ¼ 5.32 Hz, 1H, ArH), 7.94 (d, J ¼ 1.99 Hz, 1H, ArH), 7.70 (d,

J ¼ 8.43 Hz, 2H, ArH), 7.65 (d, J ¼ 7.97 Hz, 2H, ArH), 7.54 (d,

J ¼ 8.96 Hz,1H, ArH), 7.41 (d, J ¼ 8.44 Hz, 2H, ArH), 7.29 (dd, J ¼ 8.92,

2.07 Hz, 1H, ArH), 7.24 (d, J ¼ 8.24 Hz, 3H, ArH), 6.36 (d, J ¼ 5.34 Hz,

1H, ArH), 5.36 (t, J ¼ 5.95 Hz, 1H, NH), 5.03 (br.s, 1H, NH), 3.55 (q,

J ¼ 3.55 Hz, 2H, CH₂), 3.55 (q, J ¼ 5.55 Hz, 2H, CH₂), 2.40 (s, 3H, CH₃),

2H, CH₂), 3.51 (q, J ¼ 6.10 Hz, 2H, CH₂), 1.91-1.82 (m, 4H, 2CH₂); ¹³

C

NMR (400 MHz, CDCl₃, d, ppm): 164.91, 162.99, 161.58, 150.40,

135.35, 130.97, 130.41, 128.45, 127.51, 125.47, 212.41, 116.86, 114.03,

113.37, 101.53, 99.89, 55.40 2(OCH₃), 42.99 (CeNH), 40.84 (CeNH),

27.42 (CH₂), 25.96 (CH₂); HRMS (ESI, m/z) [MþNa]^þ; calculated for

1.85-1.79 (m, 4H, 2CH₂) ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 160.03,

151.87, 149.66, 148.95, 139.99, 135.92, 135.76, 134.91, 134.46, 130.69,

129.15, 128.76, 128.29, 125.81, 117.08, 114.71, 99.03, 42.98(CH₂),

41.16(CH₂), 27.45(CH₂), 25.93 (CH₂), 21.44. (CH₃).

7-chloro-4-(4-(4-(4-chlorophenyl)-6-(p-tolyl)pyrimidin-2-yl)

piperazin-1-yl)quinoline (6c): Yellow solid, yield: 82%,

C

₃₁H₃₀ClN₅O₂, 562.1972, found 562.1986.

4-(4-(4,6-bis(4-methoxyphenyl)pyrimidin-2-yl)piperazin-1-yl)-

7-chloroquinoline (6h): Yellow solid, yield: 87%, mp ¼ 191e195 ^ꢁC;

FTIR (ATR, nmax, cm-1): 3037, 2996 (CeH, CH₃), 1604-1419 (C]C,

Ar) 1242 (CeO), 812. (CeCl); ¹H NMR (400 MHz, CDCl₃,

d, ppm):

mp ¼ 215e217 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d

, ppm): 8.74 (d,

8.74 (d, J ¼ 4.98 Hz,1H, ArH), 8.11 (d, J ¼ 8.85 Hz, 3H, ArH), 8.13-8.01

(m, 2H, ArH), 7.86 (d, J ¼ 8.84 Hz,1H, ArH) 7.47 (dd, J ¼ 9.05, 2.01 Hz,

1H, ArH), 7.37 (s, 1H, ArH), 7.01 (d, J ¼ 8.81 Hz, 4H, ArH), 6.88 (d,

J ¼ 5.03 Hz, 1H, ArH), 4.31-4.29 (m, 4H, 2CH₂), 3.88 (s, 6H, 2CH₃),

3.35 (t, J ¼ 4.88 Hz, 4H, 2CH₃), 1.29 (s, 3H, CH₃); ¹³C NMR (400 MHz,

J ¼ 4.90 Hz, 1H, ArH), 8.07 (m, 4H, ArH), 8.04 (d, J ¼ 8.16 Hz, 2H,

ArH), 7.47 (d, J ¼ 8.49 Hz, 3H, ArH), 7.40 (s, 1H, ArH), 7.31 (d,

J ¼ 8.04 Hz, 2H, ArH), 6.88 (d, J ¼ 5.00 Hz, 1H, ArH), 4.31 (t,

J ¼ 4.58 Hz, 4H, 2CH₂), 3.35 (t, J ¼ 4.86 Hz, 4H, 2CH₂), 2.44 (s, 3H,

CH₃); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 163.96, 162.21, 157.09,

CDCl₃, d, ppm): 164.71, 162.12, 157.13, 151.96, 151.25, 150.17, 149.17,

151.99, 150.21, 140.96, 136.56, 136.49, 135.05, 129.47, 128.98, 128.39,

127.03, 126.35, 125.15, 122.02, 1032.16, 101.90, 52.33, 44.00, 21.46.

N¹-(7-chloroquinolin-4-yl)-N²-(4-(4-methoxyphenyl)-6-(p-tolyl)

pyrimidin-2-yl)ethane-1,2-diamine (6d): Yellow solid, yield: 88%,

135.06, 130.93, 128.94, 126.35, 125.19, 122.00, 120.24, 110.96, 110.13,

109.14, 101.48, 56.11(2OMe), 56.04 (2OMe),52.38 (2CH₂), 44.03

(2CH₂); HRMS (ESI, m/z) [MþH]^þ; calculated for C₃₁H₂₈ClN₅O₂,

538.2010, found 538.2030.

mp ¼ 154e157 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d

, ppm): 8.43 (d,

N1-(7-chloroquinolin-4-yl)-N4-(4-(3,4-dimethoxyphenyl)-6-(4-

methoxyphenyl)pyrimidin-2-yl)butane-1,4-diamine (6i); Yellow

J ¼ 3.67 Hz, 1H, ArH), 8.15 (s, 1H, ArH), 8.01 (br, s, 4H, ArH), 7.81 (s,

1H, ArH), 7.40 (s, 1H, ArH), 7.34 (d, J ¼ 7.09 Hz, 3H, ArH), 7.11-7.02

(m, 3H, ArH), 6.46 (br, s, 1H, NH), 6.30 (d. J ¼ 5.28 Hz, 1H, ArH), 5.90

(s.br, 1H, NH), 4.05-4.04 (m, 2H, CH₂), 3.97 (s, 3H, CH₃), 3.51-3.50

solid, yield: 93% mp ¼ 130e132 ^ꢁC; FTIR (ATR,

n

max, cm^ꢀ1): 3080

(CeH), 2935 (CeH of CH₃), 2956 (CeH of CH₃), 1438e1601 (C]C of

Ar), 1179, 1260 (CeO), 773 (CeCl), ¹H NMR (400 MHz, CDCl₃,

d,

(m, 2H, CH₂), 2.46 (s, 3H, CH₃); ¹³C NMR (400 MHz, CDCl₃,

d, ppm):

ppm): 8.39 (d, J ¼ 5.52 Hz, 1H, ArH), 7.99 (d, J ¼ 8.77 Hz, 3H, ArH),

7.89 (brs.1H, ArH), 7.73 (d, J ¼ 8.72 Hz, 1H, ArH), 7.46-7.42 (m, 1H,

ArH), 7.28 (s, 1H, ArH) 6.96 (d, J ¼ 8.80 Hz, 1H, ArH), 6.35 (t,

J ¼ 4.47 Hz, 1H, ARH), 5.83-5.76 (m, 1H, NH), 5.56-5.49 (m, 1H, NH),

3.87 (s, 6H, 2OCH₃), 3.85 (s, 3H, OCH₃), 3.78 (t, J ¼ 5.76 Hz, 2H,

2CH₂), 3.48 (t, J ¼ 5.81 Hz, 2H, CH₂), 2.07 (q, J ¼ 5.81 Hz, 2H, CH₂);

163.89, 157.23, 151.26, 150.77, 134.97, 129.90, 129.24, 127.62, 127.42,

127.02, 124.74, 112.10, 103.23, 98.29, 56.50, 40.33, 21.60, 14.25.

N¹-(7-chloroquinolin-4-yl)-N³-(4-(4-methoxyphenyl)-6-(p-tolyl)

pyrimidin-2-yl)propane-1,3-diamine (6e): Yellow solid, yield: 76%,

mp ¼ 97e99 ^ꢁC; ¹H NMR (400 MHz, CDCl₃,

d, ppm): 8.42 (s, 1H,

16

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

¹³C NMR (400 MHz, CDCl₃,

151.25, 150.17, 149.17, 135.06, 130.93, 128.94, 126.35, 125.19, 122.00,

120.24, 110.96, 110.13, 109.14, 101.48, 56.11 (2OMe), 56.04 (OMe),

52.38 (2CH₂), 44.03 (2CH₂).

d

, ppm): 164.71, 162.13, 157.13, 151.96,

158.96, 155.90, 143.31, 138.84, 138.55, 134.17, 131.27, 129.70, 127.36,

127.33, 126.01, 119.51, 116.74, 115.83, 115.03, 114.89, 102.66, 100.54,

99.19, 99.13, 43.45, 40.95, 26.55, 25.43; HRMS (ESI, m/z) [MþH]^þ;

calculated for C₂₉H₂₆ClN₅O₂, 512.1853; found 512.1844.

7-chloro-4-(4-(4-(3,4-dimethoxyphenyl)-6-(4-methoxyphenyl)

pyrimidin-2-yl)piperazin-1-yl)quinoline (6j): Yellow solid, yield:

4-(2-((4-((7-chloroquinolin-4-yl)amino)butyl)amino)-6-(4-

hydroxyphenyl)pyrimidin-4-yl)benzene-1,2-diol (7b): Yellow solid,

79% mp ¼ 201e204 ^ꢁC; FTIR (ATR,

n

max, cm-1): 3276 (CeN), 3073,

yield: 89%, mp ¼ 221e223 ^ꢁC; FTIR (ATR,

n

max, cm^ꢀ1): 3418 (OeH),

2839 (CeH of CH₃), 1572-1430 (C]C Ar) 1298 (CeO), 763 (CeCl), ¹H

3228 (NeH), 2927 (CeH of CH₃), 1439e1568 (C]C of Ar), 1273

NMR (400 MHz, CDCl₃,

d

, ppm): 8.74 (d, J ¼ 4.97 Hz, 1H, ArH), 8.12

(CeO), 759 (CeCl); ¹H NMR (400 MHz, CDCl₃,

d, ppm): 9.90 (br.s,

(d, J ¼ 8.81 Hz, 2H, ArH), 8.08 (m, 2H, ArH), 7.74-7.70. (m, 2H, ArH),

7.47 (dd. J ¼ 9.01, 1.99 Hz, 1H, ArH), 7.36 (s, 1H, ArH), 7.02 (d,

J ¼ 8.77 Hz, 2H, ArH), 6.98 (d, J ¼ 8.27 Hz, 1H, ArH), 6.89 (d,

J ¼ 5.02 Hz, 1H, ArH), 4.31 (t, J ¼ 4.40 Hz, 4H, 2CH₂), 4.01 (s, 3H,

CH₃), 3.96 (s, 3H, CH₃), 3.89 (s, 3H, CH₃), 3.36 (t, J ¼ 4.40 Hz, 4H,

2H, 2ph-OH), 8.80 (br, s, 1H, ph-OH) 8.50 (d, J ¼ 9.13 Hz, 1H, ArH),

8.41 (d, J ¼ 6.64 Hz, 1H, ArH), 8.03 (d, J ¼ 8.66 Hz, 4H, ArH), 8.24-

8.05 (m, 4H, ArH), 7.87 (d, J ¼ 1.96 Hz, 1H, ArH), 7.64 (dd, J ¼ 9.12,

2.12 Hz, 1H, ArH), 7.44 (s, 1H, ArH), 7.09 (t, J ¼ 5.87 Hz, 1H, NH), 6.82

(br, s, 4H, (1NH, 3ArH)), 6.76 (d, J ¼ 6.66 Hz,1H, ArH), 3.59-3.194 (m,

6H, 2CH₂), 2.06 (t, J ¼ 6.60 Hz, 2H, ArH); ¹³C NMR (400 MHz, CDCl₃,

2CH₂); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 161.64, 157.15, 151.98,

151.19, 150.20, 149.15, 135.04, 130.99, 130.56, 128.95, 128.58, 126.33,

125.20, 122.03, 120.22, 114.04, 110.96, 110.07, 109.15, 101.25, 56.08,

56.04, 52.38, 44.03.

d, ppm): 163.08, 160.17, 154.32, 145.95, 137.11, 128.97, 128.78, 126.47,

125.67, 122.67, 116.60, 115.78, 114.92, 99.91, 99.15, 41.49, 40.61,

38.98, 28.23.

4-(4-(4,6-bis(3,4-dimethoxyphenyl)pyrimidin-2-yl)piperazin-1-

4-(2-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)-6-(4-

hydroxyphenyl)pyrimidin-4-yl)benzene-1,2-diol (7c): Yellow solid,

yl)-7-chloroquinoline

(6k):

Yellow

solid,

yield:

88%,

mp ¼ 249e251 ^ꢁC; FTIR (ATR,

n

max, cm-1): 3080 (CeH), 2935 (CeH

yield: 94%, mp ¼ < 300 ^ꢁC; FTIR (ATR,

n

max, cm^ꢀ1): 3418 (OeH),

of CH₃), 2956 (CeH of CH₃), 1438e1601 (C]C of Ar), 1179, 1260

3228 (NeH), 2927 (CeH of CH₃), 1439e1568 (C]C of Ar), 1273

(CeO), 773 (CeCl), ¹H NMR (400 MHz, CDCl₃,

d

, ppm): 8.74 (d,

(CeO), 759 (CeCl); ¹H NMR (400 MHz, CDCl₃,

d, ppm): 9.93 (s, H,

J ¼ 4.96 Hz, 1H, ArH), 8.08 (m, 2H, ArH), 7.75-7.70 (m, 4H, ArH) 7.47

(dd, J ¼ 9.01, 1.99 Hz, 1H, ArH), 7.36 (s, 1H, ArH), 6.98 (d, J ¼ 8.40 Hz,

2H, ArH), 6.89 (d, J ¼ 5.01 Hz,1H, ArH), 4.30 (t, J ¼ 4.10Hz, 4H, 2CH₂),

4.01 (s, 6H, 2OCH₃), 3.96 (s, 6H, 2OCH₃), 3.36 (t, J ¼ 4.73 Hz, 4H,

ph-OH), 9.43 (br, s, 1H, ph-OH),), 9.08 (s, H, 2ph-OH), 8.73 (d,

J ¼ 4.96 Hz, 1H, ArH), 8.20 (d, J ¼ 9.00 Hz, 1H, ArH), 8.13 (d,

J ¼ 8.55 Hz, 2H, ArH), 8.01 (d, J ¼ 1.97 Hz, 1H, ArH), 7.73 (d,

J ¼ 2.04 Hz, 1H, ArH), 7.62-7.56 (m, 2H, ArH), 7.53 (s, 1H, ArH), 7.10

(d, J ¼ 5.12 Hz, 1H, ArH), 6.90 (d, J ¼ 8.56 Hz, 2H, ArH), 6.87 (d,

J ¼ 8.28 Hz, 1H, ArH), 4.19 (br.s, 4H, 2CH₂); ¹³C NMR (400 MHz,

2CH₂); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 164.71, 162.13, 157.13,

151.96, 151.25, 150.17, 149.17, 135.06, 130.93, 128.94, 126.35, 125.19,

122.00, 120.24, 110.96, 110.13, 109.14, 101.48, 56.11(2OMe), 56.04

(2OMe), 52.38 (2CH₂), 44.03 (2CH₂).

CDCl₃,

d, ppm): 164.63, 164.30, 162.03, 160.36, 157.13, 148.64,

145.95, 134.42, 129.81, 129.30, 129.13, 128.01, 126.86, 128.01, 126.86,

126.37, 121.73, 119.50, 116.06, 116.00, 115.89, 115.24, 114.85, 109.92,

100.28, 52.22, 43.99, 40.53.

6.7. A typical procedure for the synthesis of 4-(2-(4-(7-

chloroquinolin-4-yl)piperazin-1-yl)-6-(4-hydroxyphenyl)

pyrimidin-4-yl)benzene-1,2-diol

7. Pharmacological evaluation

7.1. Antiplasmodial activity

The chloroquine-sensitive strain of P. falciparum (NF54) was

maintained continuously in vitro in supplemented RPMI-1640

culture media at 37 ^ꢁC and gassed with a mixture of 5% CO₂, 3%

O₂and 92% N₂[28]. 5% D-sorbitol was used to synchronise the

culture in the ring stage [29]. To determine the antimalarial activity

of the compounds, the synchronised ring-stage parasites were

adjusted to a ﬁnal parasitaemia of 2% and 2% haematocrit, to which

serial dilutions of the compounds and positive control, quinine

were added after a 24 h incubation. Negative controls included

uninfected erythrocytes and drug-free parasitised erythrocytes.

Following a further 48 h incubation period, the plates were frozen

7a (50 mg, 0.10 mmol) in 48% aqueous HBr (2 mL) was dis-

charged into a microwave and radiated at temperature 100 ^ꢁC,

pressure 300 PSI and power 300 W for 40 min. Using TLC, the

development of the product was monitored. The precipitate formed

was ﬁltered and washed with water and diethyl ether. The crude

product was recrystallized in ethanol to afford the following pure

compounds in good yield.

at ꢀ70 ^ꢁC for 1 h and thawed for 1 h. Lysate (25

ml) were transferred

4,4’-(2-((4-((7-chloroquinolin-4-yl)amino)butyl)amino)pyrimi-

to a second plate, to which 100 ml Malstat™ and 20 ml nitroblue

dine-4,6-diyl)diphenol

(7a);

Yellow

solid,

yield:

93%,

tetrazolium-phenazine ethosulphate (1:1) mixture was added to

each well and incubated for 40 min at 37 ^ꢁC to quantify the parasite

lactate dehydrogenase (pLDH) activity [30]. Thereafter, 5% acetic

acid was added to each well, and the absorbance of the formazan

products read at 620 nm, as an indicator of parasite viability. The

percentage parasite growth, taking the appropriate controls into

account, were calculated and used to determine the concentration

required to inhibit parasite growth by 50% (IC₅₀value) from log

sigmoid dose-response curves using the GraphPad Prism® 5.0.0

software. Each experiment was repeated in triplicate [31,32].

mp ¼ 214e216 ^ꢁC; FTIR (ATR, n_max, cm^ꢀ1): 3351 (OeH), 3256 (NeH),

3061, 2944 (CeH, CH₃), 1443e1576 (C]C, Ar), 1281 (CeO), 791

(CeCl);¹H NMR (400 MHz, CDCl₃,

d, ppm): 13.06 (s, 1H, ph-OH),

10.37 (br, s, 1H, ph-OH), 9.39-9.37 (m, 1H, NH), 8.55 (d, J ¼ 9.21 Hz,

ArH), 8.52 (br, s,1H, NH), 8.24-8.05 (m, 4H, ArH), 7.90 (d, J ¼ 2.05 Hz,

1H, ArH) 7.71 (dd, J ¼ 9.08, 2.07 Hz, 1H, ArH), 7.66 (s, 1H, ArH), 7.30-

7.23 (m, 4H, ArH), 6.36 (d, J ¼ 5.32 Hz, 1H, ArH), 5.36 (t, J ¼ 5.94 Hz,

1H, ArH), 6.93 (d, J ¼ 7.16Hz, 5H, ArH), 5.62-2.61 (m, 4H, 2CH₂), 1.83

-1.80 (m, 4H, 2CH₂); ¹³C NMR (400 MHz, CDCl₃,

d, ppm): 160.68,

17

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

8. Toxicity assays

of 1 Å and size of 35 ꢂ 43 ꢂ 45 and 43 x 40 x 32 pointing in x, y and z

directions respectively). The four compounds’ systems were then

subjected to molecular dynamics simulations.

8.1. Cell viability assay

Human embryonic kidney epithelial (HEK-293) cells were

maintained at 37 ^ꢁC in a humidiﬁed environment with a 5% CO₂as a

monolayer in Dulbecco’s modiﬁed Eagle’s medium (DMEM) sup-

plemented with 10% fetal bovine serum, 100 IU/ml penicillin and

8.4. Molecular dynamic (MD) simulations

The MD simulation was performed as described by Idowu et al.

(2019) with little modiﬁcation. The simulations were performed

using the GPU version provided with the AMBER package (AMBER

18), in which the FF18SB variant of the AMBER force ﬁeld [38] was

used to describe the systems.

ANTECHAMBER was used to generate atomic partial charges for

the compounds by utilizing the Restrained Electrostatic Potential

(RESP) and the General Amber Force Field (GAFF) procedures. The

Leap module of AMBER 18 allowed for the addition of hydrogen

atoms, as well as Na^þcounter ions for neutralization all (both

PfHsp70-1 and PfHsp70-z) systems. The amino acids were

numbered, numbering residues 1e658 for PfHsp70-1 and 1e567

for PfHsp70-z. The systems were then suspended implicitly within

an orthorhombic box of TIP3P water molecules such that all atoms

were within 8 Å of any box edge [39].

100 mg/ml streptomycin. A cell suspension (10 000 cells/well) was

incubated at 37 ^ꢁC for 48 h with serial dilutions of compounds/

positive control. A ﬁnal concentration of less than 1% DMSO had no

effect on the viability of the cells. Thereafter, 40

ml of (3-(4, 5-

dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT;

5 mg/ml in phosphate buffer saline (pH 7.3)) was added to each well

and incubated for a further 2 h. DMSO was used to dissolve the

formazan crystals and then quantiﬁed spectrophotometrically at an

absorbance of 540 nm with a reference wavelength of 690 nm

(Labsystems Multiskan RC) [33]. Percent cellular viability was

determined using the appropriate controls and used to calculate

the IC₅₀values which were compared to the positive control,

camptothecin. The experiment was repeated in triplicate [34,35].

An initial minimization of 2000 steps were carried out with an

applied restraint potential of 500 kcal/mol for both solutes, were

performed for 1000 steps using the steepest descent method fol-

lowed by 1000 steps of conjugate gradients. An additional full

minimization of 1000 steps were further carried out using the

conjugate gradient algorithm without restraint. A gradual heating

MD simulation from 0 K to 300 K was executed for 50 ps, such that

the systems maintained a ﬁxed number of atoms and ﬁxed volume.

The solutes within the systems were imposed with a potential

harmonic restraint of 10 kcal/mol and collision frequency of 1.0 ps.

Following heating, an equilibration estimating 500 ps of each sys-

tem was conducted; the operating temperature was kept constant

at 300 K. Additional features such as several atoms and pressure

were also kept constant mimicking an isobaric-isothermal

ensemble. The system’s pressure was maintained at 1 bar using

the Berendsen barostat [40,41].

The total time for the MD simulations conducted were 100 ns. In

each simulation, the SHAKE algorithm was employed to constrict

the bonds of hydrogen atoms [42]. The step size of each simulation

was 2 fs, and an SPFP precision model was used. The simulations

coincided with the isobaric-isothermal ensemble (NPT), with ran-

domized seeding, the constant pressure of 1 bar maintained by the

Berendsen barostat [40], a pressure-coupling constant of 2 ps, a

temperature of 300 K and Langevin thermostat [43] with a collision

frequency of 1.0 ps.

8.2. Determination of binding afﬁnity of select synthetic compounds

to P. falciparum Hsp70

Recombinant forms of two essential cytosol localized

P. falciparum Hsp70s; PfHsp70-1 and PfHsp70-z were expressed in

E. coli and puriﬁed using nickel afﬁnity chromatography following a

previously described approach [20]. The binding afﬁnities of the

proteins for four selected compounds were determined by SPR

analysis using the BioNavis™ 420A ILVES MP-SPR (BioNavis, Tam-

pere, Finland) at 25 ^ꢁC as previously described [20]. A running

buffer, degassed PBS Tween 20 (4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄,

137 mM NaCl, 3 mM KCl, 0.005% (v/v) Tween 20, and 20 mM EDTA;

pH 7.4) was used. The recombinant proteins were immobilised as

ligands at 0.5 mg/ml onto functionalized 3D carboxymethyl dextran

sensors (CMD 3D 500L; BioNavis, Tampere, Finland). Immobiliza-

tion of ligands was achieved through amine coupling after 1-ethyl-

3-(3-dimethylaminopropyl) carbodiimide (EDC) [Sigma Aldrich,

Germany] and N-hydroxy-succinimide (NHS) [Sigma Aldrich, Ger-

many] activation following a protocol provided by the manufac-

turer (BioNavis, Tampere, Finland) to achieve <200 RUs. A reference

channel without immobilised protein served as a control for non-

speciﬁc binding and changes in refractive index. As analyte, com-

pounds (6h, 6k, 7a, and 7b) were prepared into aliquots of 0, 1.25,

2.5, 5 and 10 nM injected three times at a ﬂow rate of 50 ml/min into

each ﬂow cell. Injections with buffer only were used as controls.

Association between ligand and analyte was allowed for 3 min, and

dissociation was monitored for a total of 10 min. Kinetics steady-

state equilibrium constant data was processed after double refer-

encing of the sensorgrams and concatenating the responses of all

ﬁve analyte concentrations by global ﬁtting using TraceDrawer

software version 1.8 (Ridgeview Instruments, Sweden).

8.5. Post-dynamic analysis

Analysis of Root Mean Square Deviation (RMSD), Root Means

Square Fluctuation (RMSF), Solvent accessible surface area (SASA)

and Radius of Gyration (RoG) was done using the CPPTRAJ module

employed in the AMBER 18 suit. All raw data plots were generated

using the Origin data analysis software [44].

8.3. Molecular docking

The molecular docking software utilized in this study was the

Autodock Vina Plugin available on Chimera [36], with default

docking parameters. Homology model of PfHsp70-1 (PlasmoDB

accession number: PF3D7_0818900) and PfHsp70-z (PlasmoDB

accession number: PF3D7_0708800.1) were also built using

chimera software [37]. Gasteiger charges were added to the com-

pounds, and the non-polar hydrogen atoms were merged to carbon

atoms. The PCs were then docked into the active site pocket of

PfHsp70-1 and PfHsp70-z (by deﬁning the grid box with a spacing

8.6. Binding free energy calculations

To estimate and compare the binding afﬁnity of the systems, the

free binding energy was calculated using the Molecular Mechanics/

GB Surface Area method (MM/GBSA) (Ylilauri, M., Pentikainen,

2013). Binding free energy was averaged over 100000 snapshots

extracted from the 100 ns trajectory. The free binding energy (DG)

computed by this method for each molecular species (complex,

ligand, and receptor) can be represented as:

€

18

F. Kayamba, T. Malimabe, I.K. Ademola et al.

European Journal of Medicinal Chemistry 217 (2021) 113330

M. Singh, Synthesis and evaluation of antiplasmodial activity of 2,2,2-

triﬂuoroethoxychalcones and 2-ﬂuoroethoxy chalcones against plasmodium

falciparum in culture, Molecules 23 (2018) 1e28, https://doi.org/10.3390/

molecules23051174.

D

G_bind¼ G_complexꢀ G_receptorꢀ G_ligand

G_bind¼ E_gasþ G_solꢀ TS

(1)

(2)

(3)

(4)

(5)

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2.

E_gas¼ E_intþ E_vdwþ E_ele

G_sol¼ G_GBþ G_SA

G_SA

¼

gSASA

The term E_gasdenotes the gas-phase energy, which consists of

the internal energy E_int; Coulomb energy E_eleand the van der Waals

energies E_vdw. The E_gaswas directly estimated from the FF14SB

force ﬁeld terms. Solvation free energy, G_sol, was estimated from

the energy contribution from the polar states, GGB, and non-polar

states, G. The non-polar solvation energy, SA. GSA, was determined

from the solvent-accessible surface area (SASA), using a water

probe radius of 1.4 Å. In contrast, the polar solvation, GGB, the

contribution was estimated by solving the GB equation. S and T

denote the total entropy of the solute and temperature,

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Declaration of competing interest

The authors declare that they have no known conﬂicting

ﬁnancial or personal interests that could have appeared to inﬂu-

ence the work reported in this paper.

~

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E.C. Loiola, A.A. Dias, F.J.M. Silva, M.T. Aliota, E.A. Caine, J.E. Osorio, M. Bellio,

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8 (2016) 1e15, https://doi.org/10.3390/

Acknowledgment

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The authors would like to express the heartﬂet gratitude to the

Discipline of Pharmaceutical Sciences, College of Health Sciences,

University of KwaZulu-Natal (UKZN), Durban, South Africa, for

providing all the necessary facilities. R.K. gratefully acknowledges

National Research Foundation-South Africa (NRF-SA) for funding

this project (grant nos. 103728, 112079 and 129247). The authors

would also like to acknowledge Dr. Vuyisa Mzozoyana (UKZN) for

NMR spectroscopic experimental data.

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Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.1 13330.

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