Structure-activity relationship studies of antiplasmodial cyclometallated ruthenium(II), rhodium(III) and iridium(III) complexes of 2-phenylbenzimidazoles

Article abstract of DOI:10.1016/j.ejmech.2018.10.019

Benzimidazoles, such as albendazole, thiabendazole and omeprazole have antiplasmodial activity against Plasmodium falciparum and are widely used as scaffolds for metal-based drug research. Incorporating substituents with various lipophilic and electronic

Full text of DOI:10.1016/j.ejmech.2018.10.019

Accepted Manuscript
Structure-activity relationship studies of antiplasmodial cyclometallated ruthenium(II),
rhodium(III) and iridium(III) complexes of 2-phenylbenzimidazoles
Laa-iqa Rylands, Athi Welsh, Keletso Maepa, Tameryn Stringer, Dale Taylor, Kelly
Chibale, Gregory S. Smith
PII:
S0223-5234(18)30881-X
10.1016/j.ejmech.2018.10.019
EJMECH 10805
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 August 2018
Revised Date: 3 October 2018
Accepted Date: 9 October 2018
Please cite this article as: L.-i. Rylands, A. Welsh, K. Maepa, T. Stringer, D. Taylor, K. Chibale, G.S.
Smith, Structure-activity relationship studies of antiplasmodial cyclometallated ruthenium(II), rhodium(III)
and iridium(III) complexes of 2-phenylbenzimidazoles, European Journal of Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.ejmech.2018.10.019.
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Structure-activity relationship studies of antiplasmodial cyclometallated
ruthenium(II), rhodium(III) and iridium(III) complexes of
2-phenylbenzimidazoles
Laa-iqa Rylandsa, Athi Welsha, Keletso Maepab, Tameryn Stringera, Dale Taylorb, Kelly Chibalea,c,
Gregory S. Smitha,∗
aDepartment of Chemistry, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa
bDepartment of Medicine, Division of Clinical Pharmacology, University of Cape Town, Observatory, Cape Town, 7925, South
Africa
cSouth African Medical Research Council Drug Discovery and Development Research Unit, Department of Chemistry
and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, Cape Town, 7701, South
Africa
Abstract
Benzimidazoles, such as albendazole, thiabendazole and omeprazole have antiplasmodial activity against
Plasmodium falciparum and are widely used as scaffolds for metal-based drug research. Incorporating
substituents with various lipophilic and electronic properties can influence trans-membrane interactions and
concomitantly improve the biological activity. To study structure-activity relationships, a series of 2-
phenylbenzimidazole and their corresponding Ru(II), Ir(III) and Rh(III) cyclometallated complexes were
synthesized and evaluated for antiplasmodial activity against the chloroquine-sensitive (NF54) strain of the
human malaria parasite Plasmodium falciparum. Selected metal complexes were further screened against the
multidrug-resistant (K1) strain. In general, the 2-phenylbenzimidazole ligands showed weak antiplasmodial
activities (IC50 ~ 17.66 – 22.32 µM) while an enhancement of antiplasmodial activity was observed upon
coordination of the ligands with either ruthenium, iridium or rhodium. The new cyclometallated complexes
were found to be active against both parasite strains, with IC50 values in the low to submicromolar range
(0.12 - 5.17 µM). In addition, the metal complexes have relatively low cytotoxicity against mammalian
Chinese Hamster Ovarian (CHO) cells.
Keywords: Bioorganometallic chemistry, Benzimidazoles, Half-sandwich complexes, antimalarials
However, reports of decreased efficacy, reduced
1. Introduction
Malaria is one of the most widespread parasitic parasite clearance time in case of the ACT regimen,
diseases, largely affecting developing countries of and widespread resistance by Plasmodium parasites
sub-Saharan Africa. According to the World Health to previously effective drugs, necessitates the
Organisation, over 216 million cases and a resultant exploration of new chemotypes which are chemically
445 000 deaths were reported in 2016 [1]. The and structurally diverse. In this regard, the
current recommended treatment for malaria is the benzimidazole chemotype has been identified as a
artemisinin-based combination therapy (ACT) promising drug development scaffold. Compounds
regimen, which is based on combining a short containing the benzimidazole moiety have
duration acting artemisinin derivative with along previously displayed interesting biological activities
acting partner drug [2].
as antitumor agents, antiretrovirals and antimalarials
[3-6]. Several recent publications reported various
benzimidazole derivatives with different substitution
∗Corresponding author.
Emailaddress: gregory.smith@uct.ac.za

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patterns, possessing potent antiplasmodial activities [4, 7, 8]. The mechanism of action of these
benzimidazoles is still unclear, with no conclusive mode of action reported. However, there are reports
of benzimidazoles and related compounds targeting the haemoglobin degradation pathway, [9]
dihydroorotate dehydrogenase (DHODH) [10] and plasmepsins [11]. In addition to promising biological
activities, benzimidazoles also provide the prospect for structural diversification through metal
complexation, by virtue of the presence of several donor atoms.
Over the past decade, the field of bioorganometallic chemistry has provided promising alternatives to
traditional organic drugs. The success of cisplatin and related platinum-based drugs has demonstrated
that the use of metals in medicine may be a useful strategy in drug design and development [12].
Consequently, this sparked research efforts into the synthesis of new transition metal complexes with
interesting and occasionally, multifunctional biological properties [13-16]. It has been well-documented
that the biological activity of an organic compound may be enhanced upon conjugation of a metal with a
particular pharmacophore [17, 18]. With regard to antimalarial drugs, several research articles have
reported the synthesis and promising antimalarial activity of various metal complexes of chloroquine
[19, 20]. This is exemplified by ferroquine, an organometallic ferrocenyl analogue of chloroquine that is
currently one of the pipeline candidates in the Medicines for Malaria Venture (MMV) portfolio and is in
phase IIb combination clinical trials with artefenomel, a trioxalane [21]. There is clear evidence that the
introduction of a metal can be advantageous, through which various oxidation states, geometries and
reactivities can be accessed [19]. Indeed, exploring the effect of metal complexation remains one of the
major driving forces for bioorganometallic studies [21].
In recent years, the synthesis and biological evaluation of cyclometalated complexes have been
extensively investigated [22-24]. The strong M-C σ bond contained within the chelating ring of a
cyclometalated complex may help prevent biological reduction and ligand exchange reactions [25].
Moreover, structural and electronic properties can be fine-tuned by modifying the bidentate (C,N-)
ligand and/ or other ancillary ligands [25], which may result in the enhancement of the overall activity of
the compound. Thus, cyclometalated complexes may aid in stabilising the metal complex under
biological conditions while also introducing chemical diversity.
Herein we report on the structure-activity relationship studies of 2-phenylbenzimidazole ligands and
their cyclometalated complexes, varying the electron-withdrawing/ donating/hydrophobicity properties
at the 5-position of the benzimidazole ring. 2-Phenylbenzimidazoles were chosen specifically because of
their known ability to readily form stable metal complexes through cyclometallation, which further
enabled an investigation into the effects of metal complexation on bioactivity.
2. Results and discussion
2.1 Synthesis and characterisation
Earlier work performed in our labs and those of others [4,7,8] have revealed benzimidazoles as
promising scaffolds to target Plasmodium falciparum. In this study, we focused on halogen, methyl
trifluoromethyl and cyano-substituted 2-phenylbenzimidazoles. These were chosen from the Craig Plot
which is often used in rational drug design, and which allows for probing variation in terms of
hydrophobicity and electron-withdrawing/donating properties. The general synthetic strategy towards
the phenylbenzimidazole ligands involved the condensation and cyclisation of substituted 1,2-

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diaminobenzenes with various aromatic benzaldehydes [26], to produce the required 2-
phenylbenzimidazoles, as outlined in Scheme 1. The first step towards the synthesis of the 2-
phenylbenzimidazole ligands involved the formation of an appropriate nitroaniline derivative, that was
reduced using a zinc in the presence of ammonium chloride to afford the required substituted 1,2-
diaminobenzenes. The diamines were reacted with benzaldehyde, in a condensation/cyclisation reaction,
to afford the substituted 2-phenylbenzimidazole ligands (L1-L4) in moderate yields (37 – 62%).
Scheme 1 General synthetic approach to 2-phenylbenzimidazole ligands. Reagents and conditions: (i) n-PrNH2/ DMF/ rt; (ii) Zn/ NH4Cl/
MeOH/ rt; (iii) Benzaldehyde/ TFA/ MgSO4/ EtOH/ rt.
The 2-phenylbenzimidazole ligands (L1 – L4) were cyclometallated via sodium acetate-mediated C-H
activation reaction to produce the corresponding cyclometalated complexes (1 – 3, Scheme 2). To
achieve this transformation, the 2-phenylbenzimidazole ligands were dissolved in dichloromethane and
reacted with 1.2 equivalents of NaOAc and 0.5 equivalents of either the dichloro(p-
cymene)ruthenium(II) dimer or the pentamethylcyclopentadienyliridium(III) /rhodium(III) dimer. The
ruthenium, iridium and rhodium complexes (1-3) were isolated as either green, yellow or red solids in
good to excellent yields (40 – 90%) and were characterized using standard spectroscopic and analytical
techniques.
Scheme 2 Transformation of 2-phenylbezimidazoles to Ru, Ir and Rh cyclometallated complexes. Reagents and conditions: (i) [RuCl(p-
cymene)]2/ NaOAc/ DCM/ rt; (ii) [RhCl(η5-C5H5)]2 or [IrCl(η5-C5H5)]2 / NaOAc/ DCM/ rt.

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1
Evidence of metal coordination is provided in the aromatic region of the H NMR spectra, integrating
for a total of seven protons confirming that cyclometallation had indeed taken place. From the 1H NMR
spectra of the ruthenium complexes, four separate doublets between 6.02 – 5.29 ppm, a multiplet at 2.15
ppm, a singlet at 1.96 ppm and two doublets between 0.82 and 0.72 ppm were observed. These
resonances correspond to the p-cymene ancillary ligand and confirmed the formation of the ruthenium
complex. Interestingly, as a result of the p-cymene ligand having less freedom of rotation in the metal-
benzimidazole complex, the aromatic protons of the p-cymene moiety appeared as four separate doublets
(6.02, 5.79, 5.59, 5.29 ppm) as opposed to two doublets observed in the precursor metal dimer.
Similarly, the methyl protons of the isopropyl group appear as two distinct doublets (0.82 and 0.72
1
ppm), due to the chirality induced by the metal centre. In the H NMR spectrum of the Ru and Ir
complexes, two distinct peaks were also observed at 4.45 and 4.31 ppm and were subsequently assigned
to the methylene protons adjacent to the tertiary amine. Heteronuclear single quantum coherence
spectroscopy (HSQC) confirmed that the two proton signals are assigned to one carbon peak. The
change from an apparent triplet before complexation to two distinct signals is likely due to the
methylene protons being diastereotopic, because of the stereogenic centre induced at the metal after
complexation.
1
Similarly, in the H NMR spectrum of the Ir/Rh Cp* cyclometallated benzimidazole complexes, the
absence of one aromatic proton, relative to the benzimidazole starting material, supported metal
complexation. Additionally, the appearance of a singlet at 1.76 ppm, which integrates for fifteen protons
was due to the Cp* ligand and further confirmed that the metal was coordinated and that the Ir/Rh
complexes were successfully synthesised. In the 13C{1H} NMR spectra of the Ir(III) complexes, a
distinct peak assigned to the methyl substituents on the Cp* moiety was observed at approximately 9.00
ppm. IR spectroscopic data was obtained for the metal complexes, which revealed a weak absorption
band around 1586 – 1577 cm-1, corresponding to the C=N stretching frequencies. Normally, the C=N
absorption band of the ligand is observed at ~1627 cm-1, after complexation, however, a shift to ~1586
cm-1 is observed. A frequency shift of the C=N absorption band observed for the corresponding
benzimidazoles to lower frequencies was a strong indicator of successful coordination of the nitrogen to
the metal centre, a phenomenon explained by the synergistic effect.
2.2 Crystallography
The molecular structures of the Ir(III) complexes (2c, 2d) and Rh(III) complexes (3a, 3c) were
elucidated by single crystal X-ray diffraction. Single crystals of each complex were obtained by the slow
diffusion of a concentrated chloroform/ethyl acetate mixture (Figures 1-4). The crystallographic data and
refinement parameters for the four complexes are presented in Table 1 and selected bond distances and
angles are listed in Table 2. The iridium complexes, 2c and 2d crystallised as red blocks in the
ꢀ
monoclinic space group P21/c and as orange blocks in the triclinic space group P1, respectively. The
rhodium complexes 3a and 3c crystallised as red blocks in the monoclinic space group C2/c and P21/c,
respectively. Figures 2-5 show the molecular structures of the complexes (2c, 2d, 3a and 3c). The Ir(III)
and Rh(III) complexes adopt a “three-legged piano-stool” structure, which is commonly observed in
many previously reported literature articles involving rhodium and iridium half-sandwich complexes
[22, 27]. In this case, the pentamethylcyclopentadienyl (Cp*) ligand forms the seat of the piano-stool
and the three legs of the stool are formed from the chloride and chelating (C,N-) benzimidazole ligand.

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The crystallographic data shows that the Rh atom (3a) is disordered over two positions and the Cp*
ligand disordered over two places (Figure 3). The crystallographic data further suggests that the
geometry around the metal centre is pseudo-octahedral, which is typically observed in piano-stool
structures. The bond distances obtained for complexes 2c, 2d, 3a and 3c are comparable. The distance
between the metal centre and the carbon atoms of the Cp* ligand ranges between 2.143 and 2.265 Å.
The bond distance between the metal centre and the chloride ligand is approximately 2.4 Å, whereas the
distance between the metal centre and the two chelating atoms ranges between 2.050 and 2.079 Å.
Table 1
Crystallographic data and refinement parameters for complexes 2c, 2d, 3a and 3c.
Complex
2c
2d
3a
3c
Chemical formula
Formula weight
Crystal system
Space group
C27H32ClIrN2
612.22
monoclinic
P21/c (No. 14)
10.9895(8),
12.3159(9),
18.2121(13)
90, 99.465(2), 90
C27H29ClF3IrN2
666.19
C26H30ClN2Rh
508.88
monoclinic
C2/c (No. 15)
14.6558(10),
17.8972(13),
17.6608(11)
90, 96.436(1), 90
C27H32ClN2Rh
522.90
triclinic
monoclinic
P21/c (No. 14)
10.9353(6),
12.3347(7),
18.2520(11)
90, 99.3740(10),
90
2429.0(2)
4
1.430
ꢀ
P1 (No. 2)
a, b, c (Å)
8.9926(7), 11.817(1),
12.0582(10)
α, β, γ (˚)
82.592(1), 77.641(1),
84.652(1)
1238.38(18
2
1.787
5.540
V (Å3)
2431.4(3)
4
1.673
5.618
4603.2(5)
8
1.469
0.873
Z
D (g.cm-3)
µ (mm-1)
0.830
F (000)
1208
652
2096
1080
Crystal size (mm)
T (K)
0.21 x 0.26 x 0.32
173
0.21 x 0.26 x 0.31
173
0.12 x 0.14 x 0.18
173
0.03 x 0.07 x 0.18
173
Scan range/ ˚
Unique reflections
Rint
1.9 < ϴ < 28.4
6077
0.073
1.7 < ϴ < 28.3
5949
0.039
2.1 < ϴ < 28.3
5742
0.079
1.9 < ϴ < 28.2
6004
0.063
Reflections used [I
> 2σ(I)]
5003
5248
4122
4757
R indices (all data)
R
0.0253,
wR2
R 0.0275, wR2 0.0517,
S 1.02
1.021
R
0.0600, wR2
R 0.0328, wR2
0.0855, S 1.04
1.043
0.0532, S 1.04
1.040
-0.93, 1.62
0.1541, S 1.04
1.042
-1.47, 0.89
Goodness-of-fit
Max, Min ꢀρ (e Å-3)
-1.30, 1.00
-0.55, 1.05

Fig. 1 Molecular structure complex 2c, where thermal ellipsoids are drawn at 50% probability level.
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Fig. 2 Molecular structure of complex 2d, where thermal ellipsoids are drawn at 50% probability level.
Fig. 3 Molecular structure of complex 3a, where thermal ellipsoids are drawn at 50% probability level.
Fig. 4 Molecular structure of complex 3c, where thermal ellipsoids are drawn at 50% probability level.

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Table 2
Selected bond distances and angles.
Complex
2c (Ir)
Selected bond distances (Å)
2.4114(9) 2.4054(10) 2.4000(19) 2.4089(8)
2d (Ir)
3a (Rh)
3c (Rh)
M(1) – Cl(1)
M(1) – N(1)
M(1) – C(1)
N(1) – C(7)
M(1) – C(18)
M(1) – C(19)
M(1) – C(20)
M(1) – C(21)
M(1) – C(22)
2.072(2)
2.062(3)
1.345(4)
2.173(3)
2.143(3)
2.177(4)
2.205(4)
2.257(3)
2.079(2)
2.050(4)
1.337(4)
2.157(4)
2.159(4)
2.144(4)
2.247(4)
2.265(4)
2.082(5)
2.029(6)
1.327(6)
2.070(2)
2.043(2)
1.345(3)
2.207(4)
2.183(4)
2.140(3)
2.182(2)
2.260(3)
_
_
_
_
_
Selected bond angles (˚)
Cl(1) – M(1 )– C(1) 89.04(9) 87.40(10)
N(1) – M(1) – C(1) 77.09(12) 77.05(12)
Cl(1) – M(1) – N(1) 88.86(7) 86.40(7)
92.56(16)
77.6(2)
90.01(12)
90.72(7)
77.78(9)
91.85(6)
2.3 In vitro antiplasmodial activity and cytotoxicity evaluation
The synthesised C5-substituted 2-phenylbenzimidazole ligands were initially screened in vitro
against the chloroquine-sensitive (NF54) strain of Plasmodium falciparum (Table 3), using the
pLDH (Plasmodium lactate dehydrogenase) assay. Based on the antiplasmodial activity
observed for the 2-phenylbenzimidazole ligands, the corresponding metal complexes (Ru, Ir
and Rh) were also evaluated against the chloroquine-sensitive (NF54) strain and selected
complexes were then further screened against the chloroquine-resistant (K1) strain (Table 4).
Table 3
In vitro antiplasmodial evaluation of 2-phenylbenzimidazole ligands (L1-L4) against the chloroquine-sensitive
(NF54) strain of P. falciparum.
Compound
R
-
H
Cl
CH3
CF3
IC50 (µM) ± SD
0.044
22.32 ± 0.16
20.64 ± 0.81
17.66 ± 1.30
20.08 ± 1.59
CQ
L1
L2
L3
L4
NA = Not active at the tested concentration
The 2-phenylbenzimidazole ligands (L1 – L4) generally show weak to moderate antiplasmodial
activity against the chloroquine-sensitive (NF54) strain of P. falciparum with IC50 values in the
same range. Although variation of the substituents in the 5-position did not have a significant
effect on the antiplasmodial activity of the ligands, it is worth noting that the electron-donating
methyl substituent does slightly improve the antiplasmodial activity, as L3 shows slightly
enhanced antiplasmodial activity across the studied ligand series.

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Table 4
In vitro antiplasmodial data for the ruthenium (1), iridium (2) and rhodium (3) cyclometallated benzimidazole
complexes against the chloroquine-sensitive (NF54) and chloroquine-resistant (K1) strains of P. falciparum as well
as cytotoxicity against CHO cells.
IC50 (µM) ± SD
Compound
R
_
M/ Ln
_
RIa
SIb
NF54
K1
0.29 ± 0.04
2.68 ± 0.15
1.85 ± 0.27
0.97 ± 0.12
1.04 ± 0.14
4.31 ± 0.27
4.20 ± 0.18
2.25 ± 0.15
1.20 ± 0.23
_
CHO
6.59
22.33
3.14
0.84
0.34
22.68
2.16
1.30
0.74
_
_
424
_
CQ
1a
0.044
50.92 ± 0.81
H
Cl
0.12 ± 0.01
0.59 ± 0.04
1.16 ± 0.02
3.02 ± 0.15
0.19 ± 0.01
1.94 ± 0.04
1.73 ± 0.13
1.62 ± 0.04
5.00 ± 1.53
5.17 ± 0.58
2.26 ± 0.20
2.04 ± 0.03
_
_
1b
Ru/ p-
cymene
_
_
1c
CH3
CF3
H
_
_
1d
20.71 ± 0.23
109
_
2a
_
2b
Cl
Ir/ Cp*
_
_
2c
CH3
CF3
H
_
_
2d
_
_
3a
_
_
_
_
3b
Cl
Rh/ Cp*
_
_
_
_
_
3c
CH3
CF3
_
_
_
_
_
3d
_
0.05 ± 0.03
_
_
Emetine
a (IC50 K1/ IC50 NF54). b (IC50 CHO/ IC50 NF54)
The biological data obtained for the cyclometallated complexes reveal good antiplasmodial
activity in the low micromolar range and there are a few discernible trends. Firstly, the
antiplasmodial activity was significantly enhanced upon complexation of the either the
ruthenium, iridium or rhodium metal centres with the 2-phenylbenzimidazole ligands, which
suggests that the metal entity plays a vital role in improving the antiplasmodial activity. The
enhanced activity may be attributed to increased trans-membrane interactions brought about by
the presence of the biofriendly metal entities, or possibly through the interaction of the metal
complex with the one or more targets via yet unknown mechanisms of action. Secondly, the
Ru(II) cyclometallated complexes are more active in comparison with the Ir(III) and Rh(III)
complexes. This is consistent acroos all three series of cyclometallated complexes. For example,
in the chloroquine-sensitive strain (NF54), the IC50 value for the chloro-substituted ruthenium
complex (1b) was found to be 0.59 µM, whereas that for the corresponding iridium (2b) and
rhodium complex (3b) was found to be 1.94 and 5.17 µM, respectively. Ruthenium-based drugs
are thought to mimic iron and interact with proteins such as serum albumin and transferrin
[28,29]. However, the nature of the substituents does not seem to greatly influence the
antiplasmodial activity. In general, the rhodium complexes are the least active compared to both
the ruthenium and iridium cyclometallated complexes.
Consequently, only the Ru(II) and Ir(III) metal complexes were evaluated further against the
chloroquine-resistant (K1) strain, a multi-drug resistant strain. The antiplasmodial data obtained
for the K1 strain emphasizes the superiority of the Ru(II) complexes over the analogous Ir(III)
complexes, where the antiplasmodial activity of the Ru(II) complexes was approximately 2-
fold greater than the corresponding Ir(III) complexes. The low activity observed in the K1
strain compared to the NF54 strain for 1a, 1b, 2a and 2b suggests that the unsubstituted Ru/Ir

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complexes (1a and 2a) may be significantly less effective at targeting resistant strains of P.
falciparum when compared with compounds 1b and 2b, which lose activity slightly. The
relatively low resistance indices of the trifluoromethyl-substituted complexes 1d (RI = 0.34)
and 2d (RI = 0.74) implies that these compounds maintain their antiplasmodial activity in the
resistant strain and show no cross resistance. They are more active in the chloroquine-resistant
strain relative to the chloroquine-sensitive strain. Both RI values are significantly lower than
that of the chloroquine standard (6.59). On the contrary, the unsubstituted complexes 1a and 2a
have relatively large RI values (22.33 and 22.68, respectively), which infers that the
introduction of an appropriate substituent to the phenylbenzimidazole scaffold may be
important to target resistant strains of the malaria parasite.
Cytotoxicity studies were performed using complexes 1a and 2a to determine whether the
synthesised metal complexes are toxic to mammalian cells and to gain insight into the
selectivity of the compounds towards the malaria parasites. The compounds were tested against
the mammalian Chinese Hamster Ovarian (CHO) cell-line and the results are also shown in
Table 4, where emetine was used as the standard control. The results indicate that the tested
compounds are relatively non-cytotoxic compared to the emetine standard. The Ru(II) complex
(1a) displayed the highest selectivity, as evidenced by its relatively high IC50 value (50.92 µM),
and the compound’s SI value (424). This highlights the selectivity of the Ru(II) complexes
towards the malarial parasites over non-parasitic mammalian cells.
2.4 β-Haematin inhibition studies
The mechanism of action for quinoline-based antimalarial drugs, such as chloroquine, is thought
to target the erythrocytic stage of the malaria parasites life cycle in which these quinoline-based
drugs target and prevent the detoxification of the host’s hematin (a toxic product of the
hemoglobin degradation process) to hemozoin in the parasite’s food vacuole. Planar compounds,
such as chloroquine, bond to hematin via π-stacking interactions [28]. Preventing further
conversion of hematin to hemozoin results in the death of the parasite due to the toxic build-up
of free hematin. The inhibition of the formation of hemozoin or its synthetic counterpart, β-
hematin, is therefore regarded as an attractive approach to preliminarily elucidate a possible
antiplasmodial mechanism of action of metal-based compounds. The NP40 detergent-mediated
assay, developed by Sandlin et al., was used in this study to determine β-haematin inhibition
activity [29]. This method involves the use of NP40, a neutral detergent, which mimics lipids
and mediates the formation of β-haematin in this assay.
Since the benzimidazole-based compounds have moderate antiplasmodial activity against both
strains of P. falciparum and the mechanism of action of benzimidazoles remains unknown, we
thought it important perform β-haematin inhibition studies using selected compounds (L1, 1a
and 1b) to shed light on a potential contributing mode of action of the synthesised
2-phenylbenzimidazole-based compounds. Figure 5 shows the respective log-based dose-
response curves of the tested compounds and chloroquine (CQ), which were screened to a
concentration of 500 µM.

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Fig. 5 β-Haematin inhibition studies of selected compounds (L1, 1a, 1b and CQ).
The results reveal that the compounds (L1, 1a, 1b) do not show any inhibition b-hematin at
the tested concentrations. Since the absorbance is directly proportional to the concentration of
free haem, CQ, as anticipated, was effective at inhibiting β-haematin formation over the tested
range. However, in the case of the benzimidazole compounds, the presence of β-haematin was
still observed over the tested concentration range (low absorbance, hence little to no free haem
present). These results suggest that the compounds are not β-haematin inhibitors using this
cell-free assay and may therefore exert their antiplasmodial effect via a different mechanism
of action in the parasite, which may include, amongst others, the disruption of the Reactive
Oxygen Species (ROS) balance in the parasite and/or parasite Dihydroorotate dehydrogenase
(DHODH) inhibition.
3. Conclusions
A series of C5 substituted 2-phenylbenzimidazole ligands (L1
– L4) and corresponding
Ru(II), Ir(III) and Rh(III) cyclometallated complexes were successfully synthesised and fully
characterised using spectroscopic and analytical techniques. The substituted 2-
phenylbenzimidazole ligands displayed very weak activity against the chloroquine-sensitive
(NF54) strain of Plasmodium falciparum. On the other hand, the antiplasmodial activity of 2-
phenylbenzimidazoles were significantly enhanced upon cyclometallation with ruthenium,
iridium and rhodium. The Ru(II) complexes generally displayed better antiplasmodial activity
compared to both the Ir(III) and Rh(III) complexes. Since the rhodium complexes generally
displayed weaker activity compared to both the ruthenium and iridium complexes, rhodium
complexes were excluded from further screening against the chloroquine-resistant (K1) strain.
A similar trend was observed in the CQR K1 strain, where the Ru(II) complexes were found
to display better antiplasmodial activity than the Ir(III) complexes. Interestingly, the
complexes also displayed lower resistance index values relative to CQ which, by extension,
suggests that the metal complexes do not show cross-resistance with CQ. In light of their
promising activity, select compounds were screened against Chinese Hamster Ovarian (CHO)
cells to determine selectivity. The complexes displayed low cytotoxicity and generally high
selectivity indices, supporting that the compounds are selective towards the malaria parasites.

ACCEPTED MANUSCRIPT
In terms of a potential mechanism of action, the benzimidazole compounds did not show β-
haematin inhibition activity in the cell-free assay at the tested concentration of 500 µM,
suggesting that the compounds are unlikely candidates for hemozoin inhibition in the parasite
and may therefore exert their pharmacological effect by a different mechanism. In conclusion,
the compounds presented here, particularly the metal complexes, have been found to display
promising antiplasmodial activity over the metal-free ligands, however, the nature of the
substituent does not seem to influence the antiplasmodial effect. The striking activity
maintained in the resistant strain for the ruthenium complexes warrant further investigations
as a potential new class of antimalarials whose unique structure may aid in overcoming
resistance mechanisms.
4. Experimental
4.1. Materials and methods
All reagents and solvents were purchased from commercial sources (Sigma Aldrich, Combi
blocks, Kimix and Merck) and were used without further purification. Reactions were carried
out under an inert nitrogen or argon atmosphere, unless otherwise stated. The [RuCl(p-
cymene)]2, [IrCl(
following literature methods [30, 31]. The ligand precursors, 2-Nitro-
Chloro-2-nitro- -propylaniline [33], 4-Methyl-2-nitro- -propylaniline [34], 3-Nitro-
η η
5-C5H5)]2 and [RhCl( 5-C5H5)]2 dimeric precursors were prepared
N-propylaniline [32], 4-
N
N
4(propylamino)benzonitrile [35], 2-Nitro-N
-propyl-4-(trifluoromethyl)aniline [36], N1
Propylbenzene-1,2-diamine [32], 4-Methyl-N1-propylbenzene-1,2-diamine [35], 3-Amino-4
(propylamino)benzonitrile [35], N1-Propyl-4-(trifluoromethyl)benzene-1,2-diamine [37], L1
[38] and L3 [39] were synthesised following methods adapted from the literature.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker XR600 MHz
spectrometer (1H at 599.95 MHz and 13C{1H} at 151.0 MHz), a Bruker XR400 MHz
spectrometer (1H at 399.95 MHz and 13C{1H} at 100.6 MHz) or a Bruker XR300 MHz (1H at
299.95 MHz) using tetramethylsilane (TMS) as the internal standard. Infrared (IR)
spectroscopy was performed on a Perkin-Elmer Spectrum 100 FT-IR spectrometer using
Attenuated Total Reflectance (ATR) with vibrations measured in units of cm-1. Mass
Spectrometry (MS) determinations were carried out using Electron Impact (EI) on a JEOL
GCmateII instrument or Electrospray Ionisation (ESI) on a Waters API Quattro Micro triple
quadrupole mass spectrometer with data recorded using the positive mode. Elemental analysis
(C, H, N) were performed using a Fissions EA 110 CHNS apparatus or an Elementar Vario
EL Cube Analyser. Purity was also determined using an analytical Agilent HPLC 1260
equipped with an Agilent infinity diode array detector (DAD) 1260 UV-Vis detector, with an
absorption wavelength range of 210 – 640 nm. Melting points were obtained using a Büchi
Melting Point Apparatus B-540 and are uncorrected.
4.2. General synthetic procedure for the synthesis of 1,2-diamine derivatives
Nitroaniline compounds (1.eq.) were dissolved in anhydrous MeOH, under an argon
atmosphere at room temperature. Ammonium chloride (10 eq.) was then added to the solution
and left to stir for 5 min. Thereafter, Zinc dust (20 eq.) was added to the reaction flask. The

ACCEPTED MANUSCRIPT
reaction was allowed to stir vigorously for 30 – 45 min. After the reaction was complete, the
reaction mixture was filtered through Celite, and washed with copious amounts of MeOH.
Excess solvent was concentrated in vacuo and saturated NaHCO3 (aq.) was added to the
resultant residue and extracted with EtOAc (2 x 30 mL). The combined organic layers were
collected, dried over anhydrous Na2SO4 and the solvent removed to afford the product or
purified further via column chromatography using EtOAc:Petroleum ether as an eluent to
afford the desired products.[40]
4.2.1. 4-Chloro-N1-propylbenzene-1,2-diamine
4-Chloro-2-nitro-N-propylaniline (0.739 g, 3.44 mmol) was dissolved in MeOH (100 mL). NH4Cl
(1.84 g, 34.4 mmol) and Zinc (4.52 g, 69.1 mmol) was then added to the solution. Product: Brown
oil (0.214 g, 34%); Rf (EtOAc: Pet. Ether, 2:8): 0.39. Elemental Analysis for C9H13ClN2 (184.67
g.mol-1): Found (%) C, 57.86; H, 6.78; N, 14.02; Calcd. (%) C, 58.54; H, 7.10; N, 15.17. 1H NMR
(300 MHz, CDCl3) δ (ppm): 6.76 (dd, 3
J
HH = 8.4 and 4
J
HH = 2.4 Hz, 1H, ArH), 6.69 (d, 4
HH = 6.9 Hz,
JHH = 2.4
Hz, 1H, ArH), 6.54 (d, 3JHH = 8.4 Hz, 1H, ArH), 3.37 (br s, 3H, NH, NH2), 3.04 (t, 3
J
3
13
2H CH2), 1.02 (t, JHH = 7.5 Hz, 3H, CH3), 1.79 – 1.58 (m, 2H, CH2). C{1H} NMR (101 MHz,
CDCl3) δ (ppm): 136.5, 135.5, 123.1, 120.0, 116.1, 112.6, 46.2, 22.8, 11.7
. MS (EI, m/z): 184.0871 (80%, [M]+).
4.3. General synthetic procedure for the synthesis of 2-phenylbenzimidazole ligands (L1
–
L4)
The synthesised 1,2-diamines (1 eq.) were dissolved in EtOH. Benzaldehyde (1.2 eq.), TFA
(0.1 eq.) and MgSO4 (6 eq.) was added to the reaction flask, under an argon atmosphere at 25
ᵒC. The reaction was then left to stir, open to air, for 24 h. The reaction mixture was filtered
and the solvent reduced in vacuo. The resultant residue was dissolved in DCM (30 mL) and
washed with H2O (2 x 20 mL) and sat. NaCl (20 mL). The organic layers were collected,
dried with anhydrous Na2SO4 and concentrated in vacuo. The crude product was then purified
via column chromatography using EtOAc:Petroleum ether as the eluent to afford the desired
compounds.[23]
4.3.1. 5-Chloro-2-phenyl-1-propyl-1H-benzo[d]imidazole (L2
)
4-Chloro-N
1-propylbenzene-1,2-diamine (0.190 g, 1.03 mmol) was dissolved in EtOH (10 mL).
Benzaldehyde (0.13 mL, 1.23 mmol), TFA (11.7 mg, 0.103 mmol) and MgSO4 (0.743 g, 6.17
mmol) was then added to the reaction flask. Product: Brown solid (0.174 g, 62%); Rf (EtOAc:Pet.
Ether, 5:5): 0.63. Mp: 84.8 – 88.6 ᵒC. FT-IR (ATR)
v
(cm-1) = 1595 (imine, C=N). 1H NMR (300
4
MHz, CDCl3) δ (ppm): 7.79 (d, JHH = 1.8 Hz, 1H, ArH), 7.72 – 7.65 (m, 2H, ArH), 7.56 – 7.48
(m, 3H, ArH), 7.32 (d, 3
J
HH = 8.6 Hz, 1H, ArH), 7.26 (dd, 3
J
HH = 8.6 and 4
J
HH = 1.8 Hz, 1H, ArH),
4.26 – 4.08 (m, 2H, CH2), 1.93 – 1.71 (m, 2H, CH2), 0.85 (t, JHH = 8.6 Hz, 3H, CH3).13C{1H}
NMR (101 MHz, CDCl3) δ (ppm): 155.1, 144.1, 134.4, 130.4, 130.1, 129.4, 128.9, 128.1, 123.2,
119.8, 111.0, 46.6, 23.3, 11.3. Purity 100% by LC (tR 3.50 min); MS (EI, m/z): 270.01 (100%,
[M]+).
3
4.3.2. 2-Phenyl-1-propyl-5-(trifluoromethyl)-1H-benzo[d]imidazole (L4
)
N
1-propyl-4-(trifluoromethyl)benzene-1,2-diamine (1.26 g, 5.79 mmol) was dissolved in EtOH
(10 mL). Benzaldehyde (0.71 mL, 6.95 mmol), TFA (65.9 mg, 0.579 mmol) and MgSO4 (4.15 g,
34.7 mmol) was then added to the reaction flask. Product: Off-white solid (0.814 g, 46%); Rf

ACCEPTED MANUSCRIPT
(EtOAc:Pet. Ether, 2:8): 0.33. Mp: 64.7 – 66.0 ᵒC. FT-IR (ATR) v
(cm-1) = 1627 (imine, C=N),
1
1323 (Ar amine, C-N). H NMR (300 MHz, CDCl3) δ (ppm): 8.12 – 8.08 (m, 1H, ArH), 7.75 –
3
7.66 (m, 2H, ArH), 7.60 – 7.52 (m, 4H, ArH), 7.49 (d, JHH = 8.4 Hz, 1H, ArH), 4.27 – 4.19 (t,
3
13
3JHH = 7.8 Hz, 2H, CH2), 1.94 – 1.76 (m, 2H, CH2), 0.87 (t, JHH = 7.2 Hz, 3H, CH3). C{1H}
NMR (101 MHz, CDCl3) δ (ppm): 155.9, 142.8, 136.8, 130.3, 129.5, 129.0, 126.4 – 123.7 (d, 1JCF
= 270.1 Hz, Ar-CF3), 125.2 – 124.9 (d, JCF = 32.1 Hz), 119.7, 117.9, 110.6, 46.7, 23.3, 11.3. F
NMR (377 MHz, CDCl3) δ (ppm): -60.67. Purity 100% by LC (tR 3.56 min); MS (m/z): 305.1
(100%, [M+H]+).
2
19
4.4. General synthetic procedure for synthesis of Ru, Ir and Rh cyclometalated complexes
The selected 2-phenylbenzimidazole (1 eq.) was dissolved in dry DCM (6 mL) under argon.
Sodium acetate (1.2 eq.) was then added, and the reaction mixture left to stir at rt for 10 min.
Thereafter, the appropriate metal dimer (2 eq.) was added, in one portion. The reaction
mixture was allowed to stir at rt, under argon, for 24 h, after which the reaction mixture was
filtered through Celite and washed with DCM. The solvent was concentrated and diethyl ether
(10 mL) was added to the residue. The mixture was then cooled to 0 ᵒC and left to stir for 10
min. The precipitate that formed was filtered by suction and dried under vacuum.[23]
4.4.1. N-Propyl Ru cyclometalated complex (1a
)
2-Phenyl-1-propyl-1H-benzo[ ]imidazole L1 (77.2 mg, 0.327 mmol) was dissolved in DCM
d
(6 mL). Sodium acetate (32.1 mg, 0.392 mmol) and dichloro( -cymene)ruthenium(II) dimer
p
(100 mg, 0.0.163 mmol) was then added to the solution. Product: Green solid (77.0 mg, 46%).
Mp: 239.3 – 247.7 ᵒC. Elemental Analysis for C26H29ClN2Ru (506.054 g.mol-1): Found (%) C,
60.73; H, 5.46; N, 5.78; Calcd. (%) C, 61.70; H, 5.20; N, 5.50. FT-IR (ATR)
v
(cm-1) = 1577
(imine, C=N). 1H NMR (300 MHz,CDCl3) δ (ppm): 8.34 (d, 3JHH = 7.5 Hz, 1H, ArH), 7.93 (d,
3
3JHH = 7.8 Hz, 1H, ArH), 7.63 (d, JHH = 7.8 Hz, 1H, ArH), 7.45 – 7.29 (m, 3H, ArH), 7.20
3
3
(td, 3JHH = 7.2, 1.2 Hz, 1H, ArH), 7.12 – 7.03 (td, JHH = 7.8, 1.2 Hz, 1H, ArH), 5.86 (d, JHH
3
3
= 5.7 Hz, 1H, Arp-cy), 5.67 (d, JHH = 5.4 Hz, 1H, Arp-cy), 5.37 (d, JHH = 6.0 Hz, 1H, Arp-cy),
3
5.14 (d, JHH = 5.7 Hz, 1H, Arp-cy), 4.49 (m, 1H), 4.36 (m, 1H), 2.33 – 2.21 (m, 1H), 2.10 (s,
3H), 1.98 (m, 2H, CH2), 1.03 (t, 3JHH = 7.5 Hz, 3H, CH3), 0.90 (d, 3JHH = 6.9 Hz, 3H), 0.76 (d,
3JHH = 6.9 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ (ppm): 183.8, 157.8, 141.5, 140.7,
136.1, 133.9, 128.8, 124.0, 123.2, 123.0, 122.5, 117.2, 110.0, 101.5, 98.5, 89.3, 89.1, 82.0,
81.3, 46.2, 31.2, 23.1, 22.6, 21.7, 18.9, 11.2. MS (HR-ESI, m/z): 471.1377 (100%, [M-Cl]+),
requires 471.1374.
4.4.2. N-Propyl (chloro-) Ru cyclometalated benzimidazole complex (1b)
5-Chloro-2-phenyl-1-propyl-1H-benzo[
d]imidazole L2 (93.5 mg, 0.345 mmol) was dissolved
in DCM (6 mL). Sodium acetate (33.9 mg, 0.414 mmol) and dichloro(
p
-
cymene)ruthenium(II) dimer (106 mg, 0.173 mmol) was then added to the solution. Product:
Orange solid (77.5 mg, 42%). Mp: Decomp. 197.7 ᵒC. Elemental Analysis for C26H28Cl2N2Ru
(540.49 g.mol-1): Found (%) C, 57.18; H, 5.32; N, 5.12; Calcd. (%) C, 57.78; H, 5.22; N, 5.18.
1
FT-IR (ATR)
v
(cm-1) = 1578 (imine, C=N). H NMR (300 MHz, DMSO-d6) δ (ppm): 8.32
3
4
3
(d, JHH = 7.2 Hz, 1H, ArH), 7.88 (d, JHH = 1.8 Hz, 1H, ArH), 7.83 (d, JHH = 8.7 Hz, 1H,
ArH), 7.79 (d, JHH = 8.7 Hz, 1H, ArH), 7.42 (d, JHH = 8.7, 1.8 Hz, 1H, ArH), 7.11 (m, 2H,
3
3
3
3
3
ArH), 6.04 (d, JHH = 6.0 Hz, 1H, Arp-cy), 5.76 (d, JHH = 6.0 Hz, 1H, Arp-cy), 5.63 (d, JHH
=
6.0 Hz, 1H, Arp-cy), 5.30 (d, 3JHH = 6.0 Hz, 1H, Arp-cy), 4.62 (t, 3JHH = 6.9 Hz 2H), 2.19 – 2.04
(m, 1H), 1.97 (s, 3H), 1.80 (m, 2H, CH2), 0.87 (t, 3JHH = 7.2 Hz, 3H, CH3), 0.78 (d, 3JHH = 6.9

ACCEPTED MANUSCRIPT
Hz, 3H), 0.69 (t, 3JHH = 6.9 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ (ppm): 184.2, 159.1,
142.3, 140.9, 134.9, 133.6, 129.2, 129.1, 124.3, 123.6, 122.8, 117.0, 111.1, 102.0, 98.5, 89.7,
89.1, 82.3, 81.3, 46.5, 31.1, 23.1, 22.7, 21.9, 19.1, 11.3. MS (HR-ESI, m/z): 541.0596 (50%,
[M+H]+), requires 541.0751.
4.4.3. N-Propyl (methyl-) Ru cyclometalated benzimidazole complex (1c
)
5-Methyl-2-phenyl-1-propyl-1H-benzo[ ]imidazole L3 (102 mg, 0.406 mmol) was dissolved
d
in DCM (6 mL). Sodium acetate (39.0 mg, 0.487 mmol) and dichloro(p-
cymene)ruthenium(II) dimer (124 mg, 0.202 mmol) was then added to the solution. Product:
Green solid (0.101 g, 48%). Mp: Decomp. 212.0 – 214.9 ᵒC. Elemental Analysis for
C27H31ClN2Ru (520.08 g.mol-1): Found (%) C, 62.01; H, 6.04; N, 5.37; Calcd. (%) C, 62.36;
H, 6.01; N, 5.39. 1H NMR (400 MHz, CDCl3) δ (ppm): 8.33 (d, 3JHH = 7.6 Hz, 1H, ArH), 7.70
(s, 1H, ArH), 7.62 (d,3 JHH = 8.0 Hz, 1H, ArH), 7.24 (d, 3J = 8.4 Hz, 1H, ArH), 7.22 – 7.13
3
3
(m, 2H, ArH), 7.06 (t, JHH = 7.6 Hz 1H, ArH), 5.86 (d, JHH = 5.6 Hz, 1H, Arp-cy), 5.66 (d,
3JHH = 6.0 Hz, 1H, Arp-cy), 5.39 (d, 3JHH = 6.0 Hz, 1H, Arp-cy), 5.13 (d, 3JHH = 5.6 Hz, 1H, Arp-
cy), 4.47 (m, 1H), 4.32 (m, 1H), 2.59 (s, 3H, Ar-CH3), 2.26 (m, 1H), 2.11 (s, 3H), 1.96 (m, 2H,
3
3
3
CH2), 1.02 (t, JHH = 7.6 Hz, 3H, CH3), 0.89 (d, JHH = 7.2 Hz, 3H), 0.76 (d, JHH = 6.8 Hz,
3H). C{1H} NMR (101 MHz, CDCl3) δ (ppm): 183.5, 157.6, 141.7, 140.6, 134.2, 134.0,
13
133.0, 128.6, 124.5, 123.8, 122.5, 117.0, 109.6, 101.7, 98.1, 89.5, 88.9, 82.2, 81.1, 46.2, 30.9,
29.7, 22.6, 21.9 (CH3), 21.8, 19.0, 11.3. MS (HR-ESI, m/z): 485.1545 (70%, [M-Cl]+),
requires 485.1530.
4.4.4. N-Propyl (trifluoromethane-) Ru cyclometalated benzimidazole (1d
)
2-Phenyl-1-propyl-5-(trifluoromethyl)-1H-benzo[ ]imidazole L4 (95.0 mg, 0.312 mmol) was
d
dissolved in DCM (6 mL). Sodium acetate (38.1 mg, 0.464 mmol) and dichloro(p-
cymene)ruthenium(II) dimer (95.6 mg, 0.156 mmol) was then added to the solution. Product:
Green solid (71.2 mg, 40%). Mp: Decomp. 214.4 – 218.7 ᵒC. Elemental Analysis for
C27H28ClF3N2Ru (574.05 g.mol-1): Found (%) C, 56.32; H, 5.15; N, 4.94; Calcd. (%) C,
1
56.49; H, 4.92; N, 4.88. FT-IR (ATR)
v
(cm-1) = 1580 (imine, C=N). H NMR (400 MHz,
3
CDCl3) δ (ppm): 8.36 (m, 1H, ArH), 8.19 (s, 1H, ArH), 7.63 (m, 1H, ArH), 7.55 (dd, JHH
8.8, 1.2 Hz, 1H, ArH), 7.44 (d, JHH = 8.4 Hz, 1H, ArH), 7.23 (td, JHH = 7.6, 1.2 Hz 1H,
=
3
3
3
3
ArH), 7.09 (td, JHH = 7.2, 1.2 Hz, 1H, ArH), 5.86 (dd, JHH = 6.0, 0.8 Hz, 1H, Arp-cy), 5.73
(dd, 3JHH = 6.0, 1.2 Hz, 1H, Arp-cy), 5.33 (dd, JHH = 6.0, 1.2 Hz, 1H, Arp-cy), 5.12 (dd, 3JHH
=
3
6.0, 1.2 Hz, 1H, Arp-cy), 4.50 – 4.26 (m, 2H), 2.31 (m, 1H), 2.10 (s, 3H), 1.96 (m, 2H, CH2),
3
3
3
1.01 (t, JHH = 7.6 Hz, 3H, CH3), 0.95 (d, JHH = 7.2 Hz, 3H), 0.79 (d, JHH = 7.2 Hz, 3H).
13C{1H} NMR (101 MHz, CDCl3) δ (ppm): 184.8, 160.0, 141.1, 141.0, 138.3, 133.4, 129.5,
126.2, 126.0, 125.6, 124.6, 122.9, 120.1, 114.6, 110.8, 101.4, 100.1, 89.7, 89.4, 82.0, 81.4,
46.7, 31.1, 23.1, 22.6, 21.9, 19.1, 11.3. MS (HR-ESI, m/z): 539.1236 (100%, [M-Cl]+),
requires 539.1248.
4.4.5. N-Propyl Ir cyclometalated benzimidazole complex (2a
)
2-Phenyl-1-propyl-1H-benzo[ ]imidazole L1 (59.3 mg, 0.251 mmol) was dissolved in DCM
d
(6 mL). Sodium acetate (24.7 mg, 0.301 mmol) and pentamethylcyclopentadienyliridium(III)
chloride dimer (100 mg, 0.126 mmol) was then added to the solution. Product: Yellow solid
(76.0 mg, 51%). Mp: 312.2 – 315.9 °C. Elemental Analysis for C26H30ClIrN2 (598.212 g.mol-
1): Found (%) C, 51.73; H, 4.51; N, 4.21; Calcd. (%) C, 52.20; H, 5.05; N, 4.73. FT-IR (ATR)
v
(cm-1) = 1578 (imine, C=N). 1H NMR (300 MHz, CDCl3) δ (ppm): 8.00 (dd, 3JHH = 7.5, 0.9
3
Hz, 1H, ArH), 7.71 – 7.62 (m, 2H, ArH), 7.40 – 7.27 (m, 3H, ArH), 7.19 (td, JHH = 7.5, 1.2

ACCEPTED MANUSCRIPT
3
Hz, 1H, ArH), 7.06 (td, JHH = 7.5, 1.2 Hz, 1H, ArH), 4.60 – 4.35 (m, 2H, CH2), 2.14 – 1.95
3
(m, 2H, CH2), 1.76 (s, 15H, Cp*CH3), 1.09 (t, JHH = 7.5 Hz, 3H, CH3). 13C{1H} NMR (101
MHz, CDCl3) δ (ppm): 165.8, 161.9, 139.8, 137.4, 136.3, 134.3, 130.6, 123.8, 123.2, 123.0,
122.0, 116.9, 110.2, 46.4, 23.4, 11.5, 9.8. MS (HR-ESI, m/z): 563.2047 (100%, [M-Cl]+),
requires 563.2038.
4.4.6. N-Propyl (chloro-) Ir cyclometalated benzimidazole complex (2b
)
5-Chloro-2-phenyl-1-propyl-1H-benzo[ ]imidazole L2 (93.7 mg, 0.346 mmol) was dissolved
d
in DCM (6 mL). Sodium acetate (36.5 mg, 0.445 mmol) and
pentamethylcyclopentadienyliridium(III) chloride dimer (138 mg, 0.173 mmol) was then
added to the solution. Product: Yellow solid (0.198 g, 90%). Mp: 304.2 – 305.3 ᵒC. Elemental
Analysis for C27H32Cl2IrN2 · H2O: Found (%) C, 48.21; H, 4.58; N, 4.41; Calcd. (%) C, 48.71;
H, 5.15; N, 4.21. FT-IR (ATR)
v
(cm-1) = 1579 (imine, C=N). 1H NMR (600 MHz, CDCl3) δ
(ppm): 8.00 (dd, 3JHH = 7.8, 1.2 Hz, 1H, ArH), 7.67 – 7.65 (m, 1H, ArH), 7.63 (dd, 3JHH = 7.8,
0.6 Hz, 1H, ArH), 7.28 – 7.25 (m, 1H, ArH), 7.24 (m, 1H, ArH), 7.20 (td, 3JHH = 7.8, 1.2 Hz,
3
1H, ArH), 7.10 – 7.03 (td, JHH = 7.8, 1.2 Hz, 1H, ArH), 4.48 – 4.40 (m, 1H), 4.36 (m, 1H),
2.06 – 1.97 (m, 2H, CH2), 1.76 (s, 15H, Cp*CH3), 1.06 (t, JHH = 7.8 Hz, 3H, CH3). 13C{1H}
3
NMR (151 MHz, CDCl3) δ (ppm): 166.3, 163.1, 140.5, 137.4, 134.9, 133.7, 131.0, 129.1,
124.0, 123.4, 122.2, 116.7, 111.2, 88.3, 46.5, 23.3, 11.4, 9.8. MS (HR-ESI, m/z): 670.1050
(10%, [M-Na]+), requires 670.1470.
4.4.7. N-Propyl (methyl-) Ir cyclometalated complex (2c
)
5-Methyl-2-phenyl-1-propyl-1H-benzo[ ]imidazole L3 (62.8 mg, 0.251 mmol) was dissolved
d
in DCM (6 mL). Sodium acetate (26.8 mg, 0.327 mmol) and
pentamethylcyclopentadienyliridium(III) chloride dimer (100 mg, 0.126 mmol) was then
added to the solution. Product: Yellow solid (0.126 g, 80%). Mp: 280.1 –
281.5 ᵒC. Elemental Analysis for C28H35ClIrN2 (627.27 g.mol-1): Found (%) C, 53.29; H,
5.31; N, 4.68; Calcd. (%) C, 53.61; H, 5.62; N, 4.47. FT-IR (ATR)
v
(cm-1) = 1580 (imine,
C=N). 1H NMR (600 MHz, CDCl3) δ (ppm): 7.99 (dd, 3JHH = 7.8, 1.2 Hz, 1H, ArH), 7.64 (d,
3JHH = 7.8 Hz, 1H, ArH), 7.48 (s, 1H, ArH), 7.24 (d, 3JHH = 8.4 Hz, 1H, ArH), 7.17 (td, 3JHH
=
=
3
3
7.2, 0.6 Hz, 1H, ArH), 7.14 – 7.10 (dd, , JHH = 7.8, 1.2 Hz, 1H, ArH), 7.08 – 7.01 (td, JHH
7.8, 1.2 Hz, 1H, ArH), 4.50 (m, 1H), 4.40 (m, 1H), 2.53 (s, 3H, Ar-CH3), 2.04 (m, 2H, CH2),
1.76 (s, 15H, Cp*CH3), 1.07 (t, 3JHH = 7.8 Hz, 3H, CH3). 13C{1H} NMR (151 MHz, CDCl3) δ
(ppm): 165.5, 161.6, 140.0, 137.3, 134.4, 133.0, 130.5, 124.4, 123.6, 122.0, 116.8, 109.8,
88.1, 46.4, 23.4, 21.8 (Ar-CH3), 11.5, 9.9. MS (HR-ESI, m/z): 577.2216 (100%, [M-Cl-
CH3]+), requires 577.2195.
4.4.8. N-Propyl (trifluoromethyl-) Ir cyclometalated benzimidazole complex (2d
)
2-Phenyl-1-propyl-5-(trifluoromethyl)-1H-benzo[ ]imidazole L4 (76.5 mg, 0.251 mmol) was
d
dissolved in DCM (6 mL). Sodium acetate (25.3 mg, 0.308 mmol) and
pentamethylcyclopentadienyliridium(III) chloride dimer (100 mg, 0.126 mmol) was then
added to the solution. Product: Yellow solid (0.112 g, 67%). Mp: 309.4 – 310.2 ᵒC. Elemental
Analysis for C28H32Cl2F3IrN2 (681.24 g.mol-1): Found (%) C, 49.05; H, 4.48; N, 4.26; Calcd.
1
(%) C, 49.37; H, 4.73; N, 4.11. FT-IR (ATR)
v
(cm-1) = 1586 (imine, C=N). H NMR (300
3
MHz, CDCl3) δ (ppm): 8.03 (dd, JHH = 7.8, 0.9 Hz, 1H, ArH), 7.99 (s, 1H, ArH), 7.65 (dd,
3
3
3JHH = 8.1, 1.2 Hz, 1H, ArH), 7.50 (dd, JHH = 8.7, 1.2 Hz, 1H, ArH), 7.43 (d, JHH = 8.7 Hz,

ACCEPTED MANUSCRIPT
3
3
1H, ArH), 7.23 (td, JHH = 7.5, 1.2 Hz, 1H, ArH), 7.13 – 7.05 (td, JHH = 7.8, 1.2 Hz, 1H,
ArH), 4.40 (m, 2H, CH2), 2.14 – 1.91 (m, 2H, CH2), 1.77 (s, 15H, Cp*CH3), 1.06 (t, 3JHH = 7.5
Hz, 3H, CH3). 13C{1H} NMR (101 MHz, CDCl3) δ (ppm): 166.6, 164.1, 139.4, 138.2, 137.4,
133.5, 131.3, 126.1, 125.8, 125.5, 124.3, 122.3, 120.0, 114.4, 110.9, 88.4, 46.6, 23.3, 11.4,
9.8. MS (HR-ESI, m/z): 707.2049 (45%, [M-Cl+IsoProp+H]+), requires 707.2800.
4.4.9 N-Propyl Rh cyclometalated benzimidazole complex (3a
)
2-Phenyl-1-propyl-1H-benzo[ ]imidazole L1 (77.0 mg, 0.320 mmol) was dissolved in DCM
d
(8 mL). Sodium acetate (32.0 mg, 0.390 mmol) and pentamethylcyclopentadienylrhodium(III)
chloride dimer (100 mg, 0.160 mmol) was then added to the solution. Product: Orange solid
(0.070 g, 42%). Mp: 255.4 – 260.0 ᵒC. Elemental Analysis for C26H30ClN2Rh (508.895 g.mol-
1): Found (%) C, 57.87; H, 5.69; N, 4.19; Calcd. (%) C, 61.40; H, 5.90; N, 5.50. FT-IR (ATR)
v (cm-1) = 1574 (imine, C=N). 1H NMR (400 MHz, CDCl3) δ (ppm): 7.98 (dd,
J
= 7.6, 1.2 Hz,
= 7.6, 0.8 Hz, 1H, ArH),
= 7.6 Hz, 1H, ArH), 4.49 – 4.34 (m, 2H), 2.03 (m,
1H, ArH), 7.76 (dd,
7.40 – 7.23 (m, 4H, ArH), 7.12 – 7.06 (t,
2H), 1.67 (s, 15H), 1.07 (t,
= 7.2 Hz, 3H). C{1H} NMR (101 MHz, CDCl3) δ (ppm):
J = 7.2, 1.2 Hz, 1H, ArH), 7.62 – 7.58 (dd, J
J
13
J
180.8, 157.7, 140.2, 138.1, 136.5, 134.7, 129.8, 123.6, 123.0, 123.0, 122.8, 117.4, 110.2, 46.5,
23.3, 11.4, 9.9. MS (HR-ESI, m/z): 473.1463 (100 %, [M-Cl]+), requires 473.1464.
4.4.10. N-Propyl (chloro-) Rh cyclometalated benzimidazole complex (3b
)
5-Chloro-2-phenyl-1-propyl-1H-benzo[ ]imidazole L2 (57.0 mg, 0.187 mmol) was dissolved
d
in DCM (6 mL). Sodium
pentamethylcyclopentadienylrhodium(III)
acetate
(15.7
mg,
chloride
0.191
mmol)
and
dimer
(56.0 mg, 0.0906 mmol) was then added to the solution. Product: Orange solid (39 mg, 88%),
Mp: 257.3 ᵒC (decomp.). Elemental Analysis for C26H29Cl2N2Rh (542.08 g.mol-1): Found (%)
C, 54.80; H, 6.03; N, 3.37; Calcd. (%) C, 57.48; H, 5.38; N, 5.16. FT-IR (ATR) v
(cm-1): 1605
1
(imine, C=N). H NMR (300 MHz, CDCl3) δ (ppm): 7.97 (d,
J
= 6.8 Hz,1H, ArH), 7.74 (s,
= 7.2 Hz, 1H,
= 7.4 Hz,
1H, ArH), 7.58 (d, = 6.8 Hz,1H, ArH), 7.30 – 7.23 (m, 3H, ArH), 7.10 (t,
J
J
ArH), 4.44 – 4.37 (m, 2H), 2.05 – 1.97 (m, 2H), 1.68 – 1.62 (s + s, 15H), 1.07 (t,
J
3H). C{1H} NMR (101 MHz, CDCl3) δ (ppm): 181.1 (d, JC-Rh = 31.4 Hz), 158.9, 140.9,
138.0, 134.8, 130.0, 128.8, 123.6, 123.3, 122.8, 117.0, 111.0, 95.8, 94.2, 46.5, 23.3, 11.0, 9.7.
MS (HR-ESI, m/z): 507.1074 (100 %, [M-Cl]+), requires 507.1074.
13
4.4.11. N-Propyl (methyl-) Rh cyclometalated benzimidazole complex (3c
)
5-Methyl-2-phenyl-1-propyl-1H-benzo[d]imidazole L3 (39.0 mg, 0.156 mmol) was dissolved
in DCM (4 mL). Sodium acetate (40.0 mg, 0.488 mmol) and
pentamethylcyclopentadienylrhodium(III) chloride dimer (39 mg, 0.0631 mmol) was then
added to the solution. Product: Orange solid (26 mg, 71%). Mp: 235.1 ᵒC (decomp.).
Elemental Analysis for C27H32ClN2Rh (522.13 g.mol-1): Found (%) C, 59.60; H, 7.90; N,
1
3.97; Calcd. (%) C, 62.00; H, 6.17; N, 5.36. FT-IR (ATR) v (cm-1): 1575 (imine, C=N). H
NMR (300 MHz, CDCl3) δ (ppm): 7.96 (d, J = 6.7 Hz, 1H, ArH), 7.59 – 7.55 (m, 2H, ArH),
7.23 (m, 2H, ArH), 7.12 – 7.05 (m, 2H, ArH), 4.44 – 4.37 (m, 2H), 2.52 (s, 3H), 2.05 – 1.98
13
(m, H). 1.67 (s, 15H, Cp*), 1.06 (t, J = 7.3 Hz, 3H). C{1H} NMR (101 MHz, CDCl3) δ
(ppm): 180.77 (d, J C-Rh = 31.3 Hz), 137.91, 134.80, 132.64, 129.48, 124.33, 123.25, 122.61,
117.24, 109.62, 95.63, 46.36, 23.14, 21.60, 11.40, 9.74. MS (HR-ESI, m/z): 487.1623 (100 %,
[M-Cl]+), requires 487.1621.

ACCEPTED MANUSCRIPT
4.4.12. N-Propyl (trifluoromethyl-) Rh cyclometalated benzimidazole complex (3d
)
2-Phenyl-1-propyl-5-(trifluoromethyl)-1H-benzo[d]imidazole L4 (0.208 g, 0.682 mmol) was
dissolved in DCM (10 mL). Sodium acetate (73.0 mg, 0.890 mmol) and
pentamethylcyclopentadienylrhodium(III) chloride dimer (0.181 g, 0.293 mmol) was then
added to the solution. Product: Orange solid (23.6 mg, 50%). Elemental Analysis for
C27H29ClF3N2Rh (576.10 g.mol-1): Found (%) C, 58.20; H, 7.94; N, 3.40; Calcd. (%) C,
1
56.21; H, 5.07; N, 4.86. FT-IR (ATR)
CDCl3) δ (ppm): 8.06 (s, 1H, ArH), 8.00 (d,
ArH), 7.47 – 7.39 (m, 2H, ArH), 7.30 (t, = 7.8 Hz, 1H, ArH), 7.13 (t,
v
(cm-1): 1546 (imine, C=N). H NMR (300 MHz,
J
= 7.7 Hz, 1H, ArH), 7.62 (d,
J = 7.8 Hz, 1H,
J
J
= 7.8 Hz, 1H,ArH),
4.48 – 4.43 (m, 2H), 2.08 – 2.01 (m, 2H), 1.69 – 1.62 (s + s, 15H), 1.11 (t,
J = 7.5 Hz, 3H).
13C{1H} NMR (101 MHz, CDCl3) δ (ppm): 181.7 (d, JC-Rh = 33 Hz), 139.7, 138.3, 138.0,
133.9, 130.3, 123.9, 122.9, 119.8, 114.8, 110.6, 95.9, 46.7, 23.1, 11.1, 9.2.
4.5. Single-crystal X-ray structure analysis
Suitable single crystals of complexes 2c, 2d, 3a and 3c were grown from a mixture of
chloroform and EtOAc which were allowed to evaporate over 3 – 5 days. Single-crystal X-ray
data were collected on a Bruker KAPPA APEX II DUO diffractometer using graphite-
monochromated Mo-Kα radiation (χ = 0.71073 Å). Data collection was carried out at 173(2)
K. Temperature was controlled by an Oxford Cryostream cooling system (Oxford Cryostat).
Cell refinement and data reduction were performed using the program SAINT. The data were
scaled and absorption correction performed using SADABS.[41] The structure was solved by
direct methods using SHELXS-97 and refined by full-matrix least-squares methods based on
F using SHELXL-2014 and using the graphics interface program X-Seed.[41, 42] The
programs X-Seed and POV-Ray were both used to prepare molecular graphic images. CCDC
1850798 (2c), 1850799 (3a), 1850800 (2d) and 1850801 (3c).
4.6. In vitro antiplasmodial assay
The test samples were tested in triplicate on one or two separate occasions against
chloroquine-sensitive (NF54) and chloroquine-resistant (K1) strain of P. falciparum
.
Continuous in vitro cultures of asexual erythrocyte stages of P. falciparum were maintained
using a modified method of Trager and Jensen.[43] Quantitative assessment of antiplasmodial
activity in vitro was determined via the parasite lactate dehydrogenase assay using a modified
method described by Makler.[44] The test samples were prepared to a 20 mg/mL stock
solution in 100% DMSO. Samples were tested as a suspension if not completely dissolved.
Stock solutions were stored at -20 °C. Further dilutions were prepared on the day of the
experiment. Chloroquine (CQ) was used as the reference drug in all experiments. A full dose-
response was performed for all compounds to determine the concentration inhibiting 50% of
parasite growth (IC50 value). Test samples were tested at a starting concentration of 100
ꢁg/mL, which was then serially diluted 2-fold in complete medium to give 10 concentrations;
with the lowest concentration being 0.2 ꢁg/mL. The same dilution technique was used for all
samples. The IC50 values were obtained using a non-linear dose response curve fitting analysis
via Graph Pad Prism v.4.0 software.
4.7 In vitro cytotoxicity

ACCEPTED MANUSCRIPT
Compounds were tested for in vitro cytotoxicity against the mammalian cell-line, Chinese
Hamster
Ovarian
(CHO),
using
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) assay.[45, 46] The MTT assay is used as a colorimetric
assay for cellular growth and survival, and compares well with other available assays. The test
samples were tested in triplicate on one occasion. The test samples were prepared to a 2
mg/mL stock solution in 10% MeOH or 10% DMSO and were tested as a suspension if not
completely dissolved. Test compounds were stored at -20°C until use. Emetine was used as
the reference drug in all experiments. The initial concentration of emetine was 100 ꢁg/mL,
which was serially diluted in complete medium with 10-fold dilutions to give 6
concentrations, the lowest being 0.001 ꢁg/mL. The same dilution technique was applied to all
the test samples. The highest concentration of solvent to which the cells were exposed had no
measurable effect on cell viability. IC50 values were obtained from full dose-response curves
using a non-linear dose-response curve fitting analysis via GraphPad Prism V.4 software.
4.8 β-Hematin inhibition assay
The β-haematin inhibition assay was modified from the method described by Sandlin et
al.[29] 10mM Stock solutions of the compounds were in DMSO. The compounds were
delivered to a 96-well plate in triplicate and were tested at a starting concentration of 500 ꢁM,
where the lowest drug concentration was 5 ꢁM. The stock solution was serially diluted to give
12 concentrations in the 96-well flat-bottom assay plate. NP-40 detergent was then added to
mediate the formation of β-haematin (30.55 ꢁM, final concentration). A 25mM stock solution
of haematin was prepared by dissolving hemin (16.3 mg) in dimethyl sulfoxide (1 mL). A
177.76 ꢁL aliquot of haematin stock was suspended in 20 mL of a 2 M acetate buffer, pH 4.7.
The haematin suspension was then added to the plate to give a final haematin concentration of
100 ꢁM. The plate was then incubated for 16 h at 37 °C. The compounds were analysed using
the pyridineferrochrome method developed by Ncokazi and Egan.[47] 32 ꢁL of a solution of
50% pyridine, 20% acetone, 20% water, and 10% 2 M HEPES buffer (pH 7.4) was added to
each well. To this, 60 ꢁL acetone was then added to each well and mixed. The UV-Vis
absorbance of the resulting complex was measured at 405 nm on a SpectraMax 340PC plate
reader. The IC50 values were obtained using a non-linear dose-response curve fitting
analysis via Graph Pad Prism v.5.0 software.[48]
Acknowledgments
Financial support from the University of Cape Town, the National Research Foundation
(NRF) and the South African Medical Research Council (SAMRC) is gratefully
acknowledged. The SAMRC and South African Research Chairs initiative of the Department
of Science and Technology, administered through the NRF, are gratefully acknowledged for
support (K.C.).
Abbreviations used
CQ, chloroquine; CHO, Chinese hamster ovarian; NMR, nuclear magnetic resonance; TLC,

ACCEPTED MANUSCRIPT
thin layer chromatography; RI, resistance index; SI, selectivity index
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Highlights
ꢀ 2-Phenylbenzimidazole ligands and their cyclometallated Ru(II), Rh(III) and
Ir(III) complexes have been prepared.
ꢀ The compounds were characterized using several spectroscopic and analytical
techniques.
ꢀ The compounds were evaluated as antiplasmodial agents against Plasmodium
falciparum.
ꢀ The metal complexes were more active than the ligands and are selective to
malaria parasites over mammalian cells.