Half-Sandwich Cyclopentadienylruthenium(II) Complexes: A New Antimalarial Chemotype

Article abstract of DOI:10.1021/acs.inorgchem.0c01795

A small library of half-sandwich cyclopentadienylruthenium(II) compounds of the general formula [(η5-C5R5)Ru(PPh3)(N-N)][PF6], a scaffold hitherto absent from the toolbox of antiplasmodials, was screened for activity against the blood stage of CQ-sensitive 3D7-GFP, CQ-resistant Dd2, and artemisinin-resistant IPC5202 Plasmodium falciparum strains and the liver stage of Plasmodium berghei. The best-performing compounds displayed dual-stage activity, with single-digit nanomolar IC50 values against blood-stage malaria parasites, nanomolar activity against liver-stage parasites, and residual cytotoxicity against HepG2 and Huh7 mammalian cells. The parasitic absorption/distribution of 7-nitrobenzoxadiazole-appended fluorescent compounds Ru4 and Ru5 was investigated by confocal fluorescence microscopy, revealing parasite-selective absorption in infected erythrocytes and nuclear accumulation of both compounds. The lead compound Ru2 impaired asexual parasite differentiation, exhibiting fast parasiticidal activity against both ring and trophozoite stages of a synchronized culture of the P. falciparum 3D7 strain. These results point to cyclopentadienylruthenium(II) complexes as a highly promising chemotype for the development of dual-stage antiplasmodials.

Full text of DOI:10.1021/acs.inorgchem.0c01795

pubs.acs.org/IC
Article
Half-Sandwich Cyclopentadienylruthenium(II) Complexes: A New
Antimalarial Chemotype
Sofia A. Milheiro, Joana Gonca̧ lves, Ricardo M. R. M. Lopes, Margarida Madureira, Lis Lobo,
́
̂
́
Andreia Lopes, Fatima Nogueira, Diana Fontinha, Miguel Prudencio, M. Fatima M. Piedade,
Sandra N. Pinto, Pedro R. Florindo,* and Rui Moreira
Cite This: https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: A small library of “half-sandwich”
cyclopentadienylruthenium(II) compounds of the general formula
[(η5-C5R5)Ru(PPh3)(N-N)][PF6], a scaffold hitherto absent from
the toolbox of antiplasmodials, was screened for activity against the
blood stage of CQ-sensitive 3D7-GFP, CQ-resistant Dd2, and
artemisinin-resistant IPC5202 Plasmodium falciparum strains and
the liver stage of Plasmodium berghei. The best-performing
compounds displayed dual-stage activity, with single-digit nano-
molar IC50 values against blood-stage malaria parasites, nanomolar
activity against liver-stage parasites, and residual cytotoxicity
against HepG2 and Huh7 mammalian cells. The parasitic
absorption/distribution of 7-nitrobenzoxadiazole-appended fluo-
rescent compounds Ru4 and Ru5 was investigated by confocal fluorescence microscopy, revealing parasite-selective absorption in
infected erythrocytes and nuclear accumulation of both compounds. The lead compound Ru2 impaired asexual parasite
differentiation, exhibiting fast parasiticidal activity against both ring and trophozoite stages of a synchronized culture of the P.
falciparum 3D7 strain. These results point to cyclopentadienylruthenium(II) complexes as a highly promising chemotype for the
development of dual-stage antiplasmodials.
INTRODUCTION
■
Malaria is a public health concern of tragic proportions, with
an estimated 228 million new cases and 405000 deaths globally
in 2018, 67% of whom were children under 5 years of age.1
This disease is caused by parasites of the genus Plasmodium
that, upon injection in a mammalian host through the bite of
an infected Anopheles mosquito, travel to the liver, initiating the
hepatic stage of infection. This culminates in the release of
blood-infectious parasites that lead to the blood stage of
infection and the clinical symptoms that are associated with
malaria. Very few drugs tackle the asymptomatic but obligatory
liver-stage bottleneck of infection, creating a gap in effective
prophylactic measures against malaria.2 Furthermore, the
effectiveness of available antiplasmodial drugs against the
blood stage of infection is increasingly threatened by the
emergence and spread of drug-resistant parasites. Chloroquine
[CQ (Figure 1)], the cornerstone antiplasmodial drug for
many decades, is now largely ineffective against Plasmodium
falciparum (Pf), the deadliest malaria parasite, due to
widespread resistance to this drug.3,4 Currently, artemisinin-
based combination therapies (ACTs), in which artemisinin or
its derivatives (artesunate, artemether, and dihydroartemisinin)
are combined with longer-acting antiplasmodial drugs with
distinct modes of action to prevent resistance development, are
Figure 1. Antimalarials chloroquine, artemisinin, and chloroquine−
metalocene hybrids ferroquine and ruthenoquine.
the front-line malaria therapeutics, with treatment success rates
of >95%.1 Nonetheless, resistance to ACTs appears to be
Received: June 17, 2020
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
© XXXX American Chemical Society
Inorg. Chem. XXXX, XXX, XXX−XXX
A

Inorganic Chemistry
pubs.acs.org/IC
Article
emerging in some regions of the world, as Pf Kelch13
mutations, associated with artemisinin resistance, are wide-
spread in Southeast Asian countries and have also been
detected in some African countries.1,3,4 Thus, the discovery of
new antiplasmodial drugs that tackle both stages of
Plasmodium infection in the mammalian host remains an
urgent clinical need.
of FQ, displaying in vitro, but not in vivo, antiplasmodial
activity.29 Ruthenium(II) arene compounds were also reported
as antiplasmodials, in combination with chloroquine or other
active antiplasmodial scaffolds (Figure 3), displaying in
vitro30−38 and in vivo39 antiplasmodial activity.
Metal complexes are especially attractive for drug design, as
they offer structural and physicochemical possibilities that
cannot be attained by organic molecules.5 The clinical success
of platinum anticancer drugs6 and of the antirheumatic agent
auranofin7 has inspired bioinorganic/organometallic drug
development over the past several decades, leading to
promising lead drugs against cancer and other diseases.8−14
In malaria, ferroquine [FQ (Figure 1)], an organometallic
ferrocenyl−chloroquine hybrid molecule, is active against both
CQ-sensitive and CQ-resistant P. falciparum strains in vitro and
CQ-resistant Plasmodium vinckei vinckei strains in vivo and is
currently undergoing phase IIb clinical trials in combination
with artefenomel, a trioxalane.15 The success of FQ led to
intense research of metal-based antiplasmodial drugs, mostly
focused on ferrocenyl and other metallic hybrids with CQ or
artemisinin derivatives.16−18
Figure 3. Ruthenium(II)-arene compounds with antiparasitic activity
(P. falciparum).
Prompted by the early success of ruthenium(III) com-
pounds NAMI-A {(ImH)[trans-RuCl4(dmso-S)(Im)], where
Im = imidazole} and KP1019/1339 {KP1019 = (IndH)[trans-
RuCl4(Ind)2], where Ind = indazole and KP1339 = Na[trans-
RuCl4(Ind)2]}, which entered clinical trials as antimetastatic
and cytotoxic agents, respectively, ruthenium(II) compounds
emerged as the most studied and promising among non-
platinum chemotherapeutic alternatives.19−21 Compounds
[(η6-C6H5C6H5)RuCl(H2NCH2CH2NH2-N,N′)][PF6]
(RM175),22,23 [(η6-C6H5Me)RuCl2(PTA)] [PTA = 1,3,5-
triaza-7-phosphoadamantane (RAPTA-T)],24,25 and [Ru-
(Phen)(κ-C,N-(2-phenyl-pyridine))(NCMe)2][PF6]
(RDC11)26,27 entered preclinical evaluation as anticancer
(RM175 and RDC11) and antimetastatic agents (RAPTA-
T), and recently, compound [Ru(dmb)2(IP-TT)]Cl2 [dmb =
4,4′-dimethyl-2,2′-bipyridine; IP-TT = 2-(2′,2″:5″,2′″-terthio-
phene)-imidazol[4,5-f][1,10]phenanthroline] (TLD1433) en-
tered a phase II clinical trial as a cancer photodynamic therapy
photosensitizer (PDT PS) for the treatment of non-muscle
invasive bladder cancer (Figure 2).28
The design of metal-based antiplasmodial drugs has been
driven by the hypothesis that conjugation of organic ligand
scaffolds with pharmacological activity with an (organo)-
metallic moiety can lead to new drugs with improved
pharmacological profile, which act on multiple targets. In
contrast, attempts to uncover new metal-based antiplasmodial
lead scaffolds are scarce. Challenging the “mainstream” design
rationale, we decided to assess the antiplasmodial activity of
five half-sandwich cyclopentadienylruthenium(II) complexes
of the general formula [(η5-C5R5)Ru(PPh3)(N-N)][PF6], a
scaffold previously featured in anticancer studies,40−43 but
hitherto absent from the toolbox of antiplasmodial com-
pounds. To gain visual insight into the parasitic/cellular
distribution of organometallic complexes, we developed two 7-
nitrobenzoxadiazole (NBD)-appended fluorescent compounds.
EXPERIMENTAL SECTION
■
Chemistry. General Procedures. Reagents were bought from
commercial sources and used without further purification. Com-
pounds Ru1 and Ru2 were previously reported40−42 but were
obtained for this study using a different synthetic methodology.
Reaction/crystallization solvents were dried according to standard
procedures.44 1H nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker Ultra-Shield 300 MHz spectrometer operating
at 300 MHz, at probe temperature. Chemical shifts are quoted on the
δ scale, in parts per million, using residual solvent peaks as the internal
standard. Coupling constants (J) are reported in hertz with the
following splitting abbreviations: s, singlet; br., broad; d, doublet; t,
triplet; q, quartet; p, quintet; m, multiplet. Thin layer chromatography
(TLC) was carried out using Merck aluminum-backed sheets coated
with 60 F254 silica gel. Flash column chromatography was performed
using Merck 60 (230−400 mesh ASTM) silica gel. Ultraviolet (UV)
spectra were traced in a Thermo Scientific Evolution 201 UV−visible
spectrophotometer. Fluorescence measurements were taken on a
SHIMADZU spectrofluorophotometer RF-6000 instrument. LC-MS
analysis was performed in an HPLC Dionex Ultimate 3000
instrument composed of a model HPG3200 binary pump, a model
WPS300 autosampler, and a model TCC3000 column oven coupled
in-line to a LCQ Fleet ion trap mass spectrometer equipped with an
ESI ion source (Thermo Scientific). Aliquots of 10 μL were injected
In the field of malaria chemotherapy, ruthenium was
employed as ruthenoquine (Figure 1), a ruthenium analogue
Figure 2. Anticancer ruthenium(II) compounds RAPTA-T,24,25
RM175,22,23 and RDC1126,27 and PDT photosensitizer TLD1433.28
B
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
into the column via a Rheodyne injector with a 25 μL loop, in the
partial injection mode. Separations were carried out with a Luna C18
100 Å column (150 mm × 2 mm, 5 μm) at 30 °C, using a flow rate of
0.2 mL min−1, a mobile phase of 0.1% acid formic in water (v/v,
eluent A) and acetonitrile (eluent B), and a 30 min linear gradient
from 5% A to 70% B, followed by a 2 min a linear gradient to 100% B,
followed by a 8 min isocratic gradient to 100% B, and then the
column was re-equilibrated with 5% B for 10 min. The mass
spectrometer was operated in the ESI positive and negative ion
modes, with the following optimized parameters: ion spray voltage,
4.5 kV; capillary voltage, 16/−18 V; tube lens offset, −70/58 V;
sheath gas (N2), 40 arbitrary units; auxiliary gas (N2), 20 arbitrary
units; capillary temperature, 300 °C. Spectra typically correspond to
the average of 20−35 scans and were recorded between 100 and 850
Da. C/H/N elemental analysis was determined using a FLASH 2000
Series CHNS/O Analyzer (Thermo Scientific) equipped with a
thermal conductivity detector. Eager Xperience software was used for
data acquisition and processing.
Ru5, 2,2′-bipyridyl; Ru2, 1,10-phenanthroline; Ru4, compound 1),
and the mixture was stirred overnight at room temperature. The
solutions were then filtered via canula and pumped to dryness. Crude
products were washed with n-hexane (10 mL) and ethyl ether (10
mL) and recrystallized by slow diffusion of n-hexane in acetone or
dichloromethane solutions.
Ru1, [(η5-C5H5)Ru(2,2′-bipyridyl)(PPh3)][PF6]. η = 82%. Orange.
1H NMR (300 MHz, DMSO): δ 9.36 (d, 2H, J = 5.5 Hz, H2), 8.17
(d, 2H, J = 8.1 Hz, H5), 7.84 (t, 2H, J = 7.8 Hz, H4), 7.44−7.35 (m,
3H, PPh3), 7.35−7.24 (complex, 8H, H3 + PPh3), 6.95 (m, 6H,
PPh3), 4.86 (s, 5H, η5-C5H5). Anal. Calcd for C33H28F6N2P2Ru: C,
54.33; H, 3.82; N, 3.84. Found: C, 54.16; H, 3.76; N, 3.61.
Ru2, [(η5-C5H5)Ru(1,10-phenanthroline)(PPh3)][PF6]. η = 87%.
1
Red. H NMR (300 MHz, DMSO): δ 9.73 (d, 2H, J = 5.3 Hz, H2),
8.45 (d, 2H, J = 8.1 Hz, H4), 7.96 (s, 2H, H6), 7.73 (dd, 2H, J = 8.1,
5.3 Hz, H3), 7.32−7.20 (m, 3H, PPh3), 7.13 (td, 6H, J = 7.6, 1.8 Hz,
PPh3), 6.89−6.74 (m, 6H, PPh3), 4.95 (s, 5H, η5-C5H5). MS (ESI):
m/z [M − PF6]+ calcd for C35H28N2PRu 609.10, found 609.15. Anal.
Calcd for C35H28F6N2P2Ru: C, 55.78; H, 3.75; N, 3.72. Found: C,
55.95; H, 3.60; N, 3.38.
Synthesis and Characterization. Compound 1. 4-Chloro-7-
nitrobenzo[c][1,2,5]oxadiazole (400 mg, 2 mmol) was added to a
solution of 3-azidopropan-1-amine (203 mg, 2 mmol) in methanol
(10 mL). After the mixture had been stirred for 30 min at room
temperature, the solvent was evaporated under reduced pressure. The
crude product was purified by flash chromatography (1:1 AcOEt/n-
hexane eluent), affording N-(3-azidopropyl)-7-nitrobenzo[c][1,2,5]-
oxadiazol-4-amine (195 mg, 0.75 mmol, η = 37%). The product was
dissolved in THF (5 mL) and CuI (141 mg, 0.75 mmol); 2-
ethynylpyridine (114 μL, 1.1 mmol) and DIPEA (260 μL, 1.5 mmol)
were added, and the mixture was stirred at 80 °C for 2 h. The solvent
was then removed under reduced pressure, and the crude product was
purified by flash chromatography (eluent of 0−5% MeOH in AcOEt),
affording 7-nitro-N-{3-[4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl]propyl}-
benzo[c][1,2,5]oxadiazol-4-amine (1) as an orange solid (178 mg, η =
65%). 1H NMR (300 MHz, DMSO): δ 9.47 (br. s, 1H, H2), 8.67 (s,
1H, H8), 8.58 (br. s, 1H, H5), 8.49 (d, 1H, J = 8.9 Hz, H6′), 7.99 (br.
s, 1H, H4), 7.89 (t, 1H, J = 7.2 Hz, NH), 7.33 (br. s, 1H, H3), 6.40
(d, J = 9.0 Hz, 1H, H5′), 4.58 (t, J = 6.7 Hz, 2H, H10), 3.55 (br. s,
2H, H12), 2.41−2.26 (m, 2H, H11). MS [ESI]: m/z [M + H]+ calcd
for C16H14N8O3 367.12, found 367.13. Anal. Calcd for C16H14N8O3:
C, 52.46; H, 3.85; N, 30.59. Found: C, 52.87; H, 4.07; N, 28.83.
Compound 2. 4-Chloro-7-nitrobenzo[c][1,2,5]oxadiazole (400
mg, 2 mmol) was added to a solution of tert-butyl (3-aminopropyl)-
carbamate (348 mg, 2 mmol) in methanol (10 mL), and the mixture
was stirred for 30 min at room temperature. The solvent was removed
under reduced pressure. The crude product was dissolved in CH2Cl2
(3 mL); trifluoroacetic acid (2 mL) was added, and the mixture was
stirred at room temperature for 20 min. The volatiles were evaporated
under reduced pressure, and the crude product was purified by flash
chromatography (eluent of 0−5% MeOH in AcOEt), affording N1-(7-
nitrobenzo[c][1,2,5]oxadiazol-4-yl)propane-1,3-diamine (91 mg, 0.38
mmol, η = 19%). The product was dissolved in DMF (2 mL) and
added to a solution of TBTU (134 mg, 0.42 mmol), DIPEA (139 μL,
0.8 mmol), and 4-diphenylphosphine benzoic acid (260 μL, 1.5
mmol) in DMF (5 mL), and the mixture was stirred overnight at
room temperature. The volatiles were then removed under reduced
pressure, and the crude product was purified by flash chromatography
(AcOEt eluent), affording 4-(diphenylphosphanyl)-N-{3-[(7-
nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]propyl} benzamide as a
1
Ru4, [(η5-C5H5)Ru(1-N,N)(PPh3)][PF6]. η = 74%. Orange. H NMR
(300 MHz, DMSO): δ 9.51 (br. s, 1H, H2), 9.30 (d, 1H, J = 5.6 Hz,
H5), 8.72 (s, 1H, H8), 8.55 (d, 1H, J = 8.9 Hz, H6′), 7.86 (t, 1H, J =
7.7 Hz, NH), 7.73 (d, 1H, J = 7.4 Hz, H4), 7.47−7.15 (m, 10H, PPh3
+ H3), 7.15−6.90 (m, 6H, PPH3), 6.42 (d, 1H, J = 8.9 Hz, H5′), 4.67
(s, 5H, η5-C5H5), 4.58 (t, 2H, J = 7.0 Hz, H10), 3.54 (br. s, 2H, H12),
2.31−2.15 (m, 2H, H11). MS (ESI): m/z [M − PF6]+ calcd for
C39H36N8O3PRu 795.15, found 795.11. Anal. Calcd for
C39H36F6N8O3P2Ru: C, 49.85; H, 3.65; N, 11.92. Found: C, 49.86;
H, 3.88; N, 11.94.
Ru5, [(η5-C5H5)Ru(2,2′-bipyridyl)(2-P)][PF6]. η = 71%. Reddish
1
orange. H NMR (300 MHz, DMSO): δ 9.53 (s, 1H, NHCO), 9.37
(d, 2H, J = 5.5 Hz, H2 bipy), 8.59 (t, 1H, J = 5.6 Hz, NH), 8.51 (d,
1H, J = 9.0 Hz, H6′), 8.18 (d, 2H, J = 8.1 Hz, H5 bipy), 7.84 (t, 2H, J
= 7.7 Hz, H4 bipy), 7.71 (d, 2H J = 7.1 Hz, H3), 7.41 (dd, 2H, J =
7.6, 6.1 Hz, H3 bipy), 7.36−7.23 (m, 6H, PPh2 + H2), 7.06 (t, 2H, J
= 9.0 Hz, PPh2), 6.98−6.83 (m, 4H, PPh2), 6.42 (d, 1H, J = 9.0 Hz,
H5′), 4.89 (s, 5H), 3.55 (s, 2H, H7), 3.43−3.32 (m, 2H, H9), 2.01−
1.86 (m, 2H, H8). MS (ESI): m/z [M − PF6]+ calcd for
C43H37N7O4PRu 848.17, found 848.20. Anal. Calcd for
C43H37F6N7O4P2Ru: C, 52.02; H, 3.76; N, 9.88. Found: C, 52.23;
H, 3.58; N, 9.34.
Synthesis of Ru3, [(η5-C5Me5)Ru(1,10-phenanthroline)(PPh3)]-
[PF6]. CH2Cl2 (15 mL) was added to a Schlenk tube charged with
[(η5-C5Me5)RuCl(PPh3)2][PF6] (80 mg, 0.10 mmol) and AgPF6 (27
mg, 0.11 mmol). After the mixture had been stirred for 30 min, 1,10-
phenanthroline (20 mg, 0.11 mmol) was added, and the mixture was
stirred overnight at room temperature. The solution was then filtered
and pumped to dryness, and the crude was washed with n-hexane (10
mL) and ethyl ether (10 mL). The crude product was dissolved in
acetone (5 mL), filtered, and pumped to dryness. The product was
recrystallized by slow diffusion of ethyl ether in a dichloromethane
solution. η = 73%. Red. 1H NMR (300 MHz, DMSO): δ 9.36 (d, 2H,
J = 5.0 Hz, H2), 8.46 (d, 2H, J = 8.1 Hz, H3), 7.94 (s, 2H, H5), 7.90
(dd, 2H, J = 8.0, 5.3 Hz, H2), 7.24 (br. m, 3H, PPh3), 7.12 (br. m,
6H, PPh3), 6.74 (br. m, 6H, PPh3), 1.39 (s, 15H). MS (ESI): m/z
[M-PF6]+ calcd for C40H38N2PRu 679.18, found 679.06. Anal. Calcd
for C40H38F6N2P2Ru: C, 58.32; H, 4.65; N, 3.40. Found: C, 58.08; H,
5.03; N, 3.01.
1
yellow solid (147 mg, η = 74%). H NMR (300 MHz, DMSO): δ
9.48 (s, 1H, NHCO), 8.56−8.48 (complex, 2H, H6′ + NH), 7.81 (d,
2H, J = 7.1 Hz, H2), 7.48−7.35 (m, 6H, PPh2), 7.34−7.18 (complex,
6H, PPh2 + H3), 6.42 (d, J = 9.0 Hz, 1H, H5′), 3.55 (s, 2H, H9), 3.39
(m, 2H, H7), 2.03−1.87 (m, 2H, H8). MS [ESI]: m/z [M + H]+
calcd for C28H24N5O4P 542.15, found 542.25. Anal. Calcd for
C28H24N5O4P: C, 64.00; H, 4.60; N, 13.33. Found: C, 63.81; H, 4.82;
N, 13.20.
Synthesis of Ru1, Ru2, Ru4, and Ru5. CH2Cl2 (15 mL) was
added to a Schlenk tube charged with [(η5-C5H5)Ru(NCCH3)][PF6]
(0.20 mmol), phosphine (0.21 mmol; Ru1, Ru2, and Ru4, PPh3; Ru5,
compound 2), and the N−N bidentate ligand (0.21 mmol; Ru1 and
X-ray Diffraction Studies of Ru3. Single-crystal X-ray diffraction
(SCXRD) experiments were performed with a Bruker D8 Quest area
detector diffractometer. The crystal was mounted on a Kaptan loop. A
graphite-monochromated Mo Kα (λ = 0.71073 Å) radiation source
running at 50 kV and 30 mA was used. An empirical absorption
correction was enforced using Bruker SADABS,45 and data were
reduced with the Bruker SAINT program.46 The structure was
determined by direct methods with Bruker SHELXS47 and refined by
full-matrix least-squares on F2 using SHELXL47 programs within
WINGX version 2014.1.48 Non-hydrogen atoms were refined with
anisotropic thermal parameters. Hydrogen atoms were placed in
C
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
calculated positions and allowed to be refined in the parent carbon
dye. Specifically, 1 mL of the incubated solution was centrifuged, the
supernatant was discarded, and cells were washed once in 1 mL of
PBS. After the last centrifugation, the supernatant was discarded, the
cell pellet was resuspended in 1 mL of PBS, and 200 μL of the cell
suspension was placed in eight-well chambered coverslips (Ibidi
GmbH, Munich, Germany). The cells were then imaged by a CLSM
using the 488 nm Ar+ laser line (Ru4 and Ru5 images) and the 633
nm He−Ne laser line (Alexa 633 images). Hoescht 3342 images were
recorded in the multiphoton mode under 780 nm excitation, and
hemozoin was visualized using the laser reflection mode. A Leica TCS
SP5 inverted microscope with a 63× water (1.2 numerical aperture)
apochromatic objective was used. Images were collected with 512 ×
512 pixels at a scan rate of 100 Hz laser. Images were processed with
LAS AF Lite, Fiji, and Wright Cell Imaging Facility (WCIF) ImageJ
software.
HepG2 Dose−Response Assay. The viability of HepG2 cells was
determined after their exposure to compounds for 48 h by the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(Sigma)] assay.55 Briefly, 5000 HepG2 cells per well (ATCC HB-
8065) were cultured in 96-well plates (Corning Primaria) and allowed
to attach for 24 h before the addition of several concentrations of
compounds (20 and 50 μM for preliminary screening and 0, 2, 5, 8,
10, 12, 15, 18, and 20 μM for CC50 determination). Compounds were
incubated for 48 h at 37 °C. MTT was added to plates at a final
concentration of 400 mg/mL per well, followed by incubation at 37
°C for 3 h. After incubation, medium was removed, and formazan
crystals were dissolved in a 4:1 dimethyl sulfoxide/glycine buffer (pH
10.5). Plates were shaken for 15 min, and cell viability was then
assessed by measuring formazan absorption at 550 nm. Nonlinear
regression analysis was employed to fit the normalized results of the
dose−response curves, and CC50 values were determined using
GraphPad Prism 5 (trial version).
Compound Activity against the Hepatic Stage of Plasmodium
Infection In Vitro. Huh-7 cells, a human hepatoma cell line, were
cultured in 1640 rpmI medium supplemented with 10% (v/v) fetal
calf serum (FCS), 1% (v/v) non-essential amino acids, 1% (v/v)
penicillin/streptomycin, 1% (v/v) glutamine, and 10 mm HEPES (pH
7) and maintained at 37 °C with 5% CO2. Inhibition of Plasmodium
berghei hepatic infection was determined by measuring the
luminescence of Huh-7 cell lysates 48 h after infection with a firefly
luciferase-expressing P. berghei line, PbGFP-Luccon, as previously
described.56,57 Briefly, Huh-7 cells (1 × 104 cells per well) were
seeded in 96-well plates the day before drug treatment and infection.
Stock solutions (10 mM) of the compounds were obtained by
dissolving in DMSO, and dilutions were subsequently performed
employing infection medium (culture medium supplemented with 50
μg/mL gentamicin and 0.8 μg/mL amphotericin B) to the desired
concentration. One hour prior to infection, the culture medium was
replaced with fresh infection medium containing the appropriate
concentration of each compound. Sporozoites (1 × 104 per well),
freshly obtained through disruption of salivary glands of infected
female Anopheles stephensi mosquitoes, were added to the wells 1 h
after compound addition. Sporozoite addition was followed by
centrifugation at 1800g for 5 min. The effect of the compounds on the
viability of Huh-7 cells was assessed 46 h post-infection by the
AlamarBlue assay (Invitrogen), according to the manufacturer’s
protocol. The parasite load was determined 48 h after infection by
a bioluminescence assay (Biotium), according to the manufacturer’s
recommendations. Nonlinear regression analysis was employed to fit
the normalized results of the dose−response curves, and IC50 values
were determined using GraphPad Prism 8.0 (GraphPad Software, La
Jolla, CA).
atoms. Structural representation was made using Mercury 3.8.49
A
summary of the crystal data, structure solution, and refinement
parameters is given in Table S1. Collected data are poor due to weak
diffraction of the crystal. CCDC 1996562.
Biology. Blood-Stage Antiplasmodial Activity. Sample
Preparation. Each compound was solubilized in 100% DMSO
(Sigma-Aldrich) to obtain a final concentration of 5 mM.
Intermediate dilutions were then prepared as appropriate in parasite
culture media.
P. falciparum In Vitro Cultures. Laboratory-adapted P. falciparum
lines 3D7 (chloroquine-, mefloquine-, and artemisinin-susceptible;
IHMT-NOVA culture collection), 3D7-GFP (chloroquine-, meflo-
quine-, and artemisinin-susceptible; MRA-1029, MR4, ATCC,
Manassas, VA), Dd2 (chloroquine- and mefloquine-resistant and
artemisinin-susceptible; IHMT-NOVA culture collection), and
IPC5202 (artemisinin-resistant; MRA-1240, MR4, ATCC) were
continuously cultivated at 5% hematocrit and 37 °C in a 5% CO2
atmosphere, and human serum was replaced with 0.5% AlbuMAXII
(Invitrogen) in the culture medium (RPMIc) and sorbitol
synchronized, as previously described.50
Dose−Response Assay for IC50 Estimation. An unsynchronized
culture with 1% hematocrit and 1% parasitemia was incubated with
the tested compounds in 5-fold serial dilutions ranging from 10000 to
0.64 nM. After 72 h at 37 °C in 5% CO2, cells were diluted to achieve
a 0.25% hematocrit and parasite growth was assessed by flow
cytometry (Beckman Coulter, Cytoflex) with a 96-well plate reader
for each parasite strain as follows. For 3D7-GFP, read directly (green;
excitation wavelength, 488 nm); for Dd2 and IPC5202, parasites were
stained with 0.5× SYBR green I (Invitrogen, Thermo Fisher
Scientific) in PBS for 30 min in the dark, at 37 °C, washed once
with PBS, and then read as for 3D7-GFP.51,52 To estimate the IC50
values of fluorescent compounds Ru4 and Ru5, the 3D7 strain (not
expressing GFP) was used; after the 72 h incubation, parasites were
stained with 0.5 μM SYTO61 (Invitrogen, Thermo Fisher Scientific)
for 45 min in the dark, at 37 °C, and washed once with PBS, and
parasite growth was assessed by flow cytometry (red; SYTO-61; 633
nm, 660/20 nm bandpass).53 Typically, 20000−40000 red blood cells
(RBCs) were counted, and samples were analyzed using FlowJo
software (Tree Star Inc.). Half-maximal inhibitory concentrations
(IC50) were determined with GraphPad Prism 5 (trial version).
Values are the average of at least three experiments, each conducted in
duplicate.
In Vitro Stage-Dependent Antimalarial Activity. Stage-specific
compound treatment effects were elucidated using synchronized (6 h
window) parasites in 96-well plates with a starting parasitemia of 1%
and a hematocrit of 2%. Parasites were treated with 10-fold IC50 (80
nM) concentrations of Ru2 or with RPMIc (with <0.0016% DMSO;
no drug control), at 3 or 6 h during the intraerythrocytic life cycle:
ring stage (6−10 h post-injection) and trophozoite stage (22−24 h
post-injection). After being incubated, washed parasites were also
diluted 1:16 and allowed to grow without the drug for two additional
cell cycles (96 h). Parasitemia after reinvasion or after growth for 96 h
was quantified. Cells were stained with 0.5× SYBR green I
(Invitrogen, Thermo Fisher Scientific) and washed with PBS, and
parasitemia was assessed by flow cytometry (Cytoflex, Beckman-
Coulter; green; 488 nm excitation, emission 530/30 nm bandpass)
collecting data from 20000−40000 RBCs. Values are the average of at
least three experiments, each conducted in duplicate.
Confocal Microscopy Studies. One milliliter of a P. falciparum 3D7
asynchronous cell suspension (2% hematocrit, ≅3% parasitemia) was
stained with 0.5 μM Ru4 or Ru5 in PBS. After a 5 min incubation (at
37 °C, 5% humidity, and 5% CO2), ≤1 μg/mL WGA-Alexa 633 (1:2
dilution from the original stock) and 5 μg/mL Hoechst 33342
(Invitrogen, Thermo Fisher Scientific) dye were added and the
mixture was incubated for 10 min. Wheat germ agglutinin-Alexa 633
was purchased from Thermo Fisher Scientific.54 Hoechst 33342
(Invitrogen, Thermo Fisher Scientific) is a lipophilic DNA-binding
fluorescent stain. After WGA-Alexa 633 and Hoechst 33342 co-
staining protocols, the samples were washed to remove the unbound
RESULTS AND DISCUSSION
■
Synthesis and Characterization. 7-Nitrobenzoxadiazole
(NBD)-appended fluorescent ligands [1 and 2 (Scheme 1A)]
were obtained via 4-propylamine-NBD derivatives function-
alized with either azide for (3+2) Cu(I)-catalyzed cyclo-
addition with 2-ethynylpyridine (1) or amine for condensation
D
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
[(η5-C5H5)Ru(NCCH3)3][PF6] (Ru1, Ru2, Ru4, and Ru5)
and [(η5-C5Me5)Ru(PPh3)2Cl)][PF6] (Ru3) (Scheme 1B).
Compounds 1, 2, and Ru1−Ru5 were characterized by H
Scheme 1. Synthesis of NBD-Labeled Ligands 1 and 2, with
(A) the Numbering Scheme and (B) Organometallic
Compounds Ru1−Ru5
1
NMR, LC-MS, and elemental analysis. 1H NMR spectra of the
organometallic complexes display a sharp singlet attributed to
the cyclopentadienyl ring, between 4.67 and 4.95 ppm for η-
C5H5 (Cp) derivatives Ru1, Ru2, Ru4, and Ru5 and at 1.39
ppm for η-C5Me5 (Cp*) compound Ru3, multiplets in the
aromatic region attributed to the triphenylphosphine ligands
(6.70−7.30 ppm), and the signals corresponding to the
nitrogen quelate ligands, in agreement with proposed
structures of the complexes. Mass spectra of the compounds
confirmed the complex formulations, with complex spectra
displaying the distinctive isotopic pattern of ruthenium.
Elemental analyses of compounds are also in agreement with
the proposed formulations, except for compounds 1 and Ru5,
for which the nitrogen values differ by 1.76% and 0.54%,
respectively. Nonetheless, LC-MS analysis supports the purity
of both compounds (Figures S8 and S9). Fluorescent
complexes Ru4 and Ru5, developed to visually probe parasitic
absorption/distribution (vide infra), were further studied by
fluorescence spectroscopy, revealing λem values of 553 nm (λex
= 462 nm) and 555 nm (λex = 474 nm), respectively.
Compound Ru3 was further characterized by X-ray
diffraction. The asymmetric unit includes a cationic complex
and a PF6 anion that is severely disordered. The cation of Ru3
(Figure S10) adopts a “three-legged piano stool” geometry,
with the ruthenium atom bonded to the phenanthroline
nitrogen and the PPh3 phosphorus atom (piano stool legs),
and the η-coordinated pentamethylcyclopentadienyl (piano
stool seat). The Ru−N bond lengths for Ru3 [2.115(4) and
2.124(4) Å (Table S2)] are in the upper range of Ru−N bond
lengths found for similar ruthenium complexes (1.992−2.176
Å).58 The Ru−P and Ru−Cp*c (superscript c = centroid)
bond lengths [2.3484(13) and 1.8420(5) Å, respectively] are
also within the ranges of similar compounds of this type
(2.277−2.359 Å for Ru−P bond lengths and 1.854−1.84 Å for
Ru−Cp*c bond lengths). The N−Ru−N angle (Table S2),
dependent on phenanthroline bite, is 77.24(18)° for the Ru3
cation, similar to those of other ruthenium phenanthroline
complexes (77.137−77.787°), whereas the P−Ru−N angles
are 88.08(11)° and 90.54(12)°, which lie at the bottom of the
array found for other complexes found in the CCSD (87.669−
97.297°).58 Structural inspection of Ru3 revealed steric
hindrance between Cp* methyl groups and PPh3 phenyl
rings, with H−H interligand contacts as short as 2.007 Å,
with 4-diphenylphosphine benzoic acid (2).
Organoruthenium(II) compounds of the general formula
[(η5-C5R5)Ru(PPh3)(N-N)][PF6] were obtained in one step
from commercially available ruthenium parent compounds
a
Table 1. In Vitro Antiplasmodial Activity (blood stage) and Cytotoxicity (HepG2) of Ruthenium Compounds
P. falciparum (blood stage) IC50 (nM)
b
3D7-GFP
3D7
Dd2
IPC5202
83
68 14
78
HepG2 CC50 (μM)
RI
6, 9
8, 9
SI
Ru1
Ru2
Ru3
Ru4
Ru5
DHA
CQ
9.6 3.0
7.69 0.22
nd
nd
nd
53 25
60 30
71 16
9
10.8 0.6
6.1 0.7
1125, 204, 130
793, 101, 89
583, 99, 90
12
6
4
7
1
6, 6
219 62
139 54
14.40 0.01
15.8 0.8
244 15
464 35
167 14
nd
nd
nd
325 116
174 34
939 299
209 64
46 12
>50
>50
>1000
>200
nd
1.5, 4.2
1.2, 1.5
2.0, 3.2
22, 3.4
2.4, 3.6
>228, >154, >53
>360, >299, >239
>71 × 103, >34 × 103, >21 × 103
>14 × 103, >7 × 103, >4 × 103
nd
29
9
340 21
585 42
54
3
PQ
880 116
a
Ligands 1, 2, and PPh3 were evaluated against the 3D7-GFP Pf strain at 1 μM, revealing inhibitions of 0%, 49.9%, and 2.7%, respectively. RI
b
(resistance index) = IC50(Dd2, IPC5202)/IC50(3D7-GFP). SI (selectivity index) = IC50(HepG2)/IC50(3D7-GFP, Dd2, IPC5202). Fluorescent
compounds Ru4 and Ru5 were also studied in the nonfluorescent 3D7 strain. nd = not determined.
E
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 4. CLSM-based distribution analysis of Ru4 and Ru5 (green channel). (A) Representative CLSM images of RBCs and iRBCs stained with
Ru4 or Ru5, Hoescht (blue channel), and WGA-Alexa 633 dyes (red channel). The parasite’s hemozoin (purple channel) was visualized using laser
reflection. Overlay images are presented in the right panels. (B) Co-localization images, highlighting the positive signals for both channels (white),
and PCC values.
despite the staggered conformation adopted by PPh3, explain-
ing the broad shape of PPh3 1H NMR signals (Figure S5). A
comparison of the crystal structures of Ru3 and Ru241 (see
Table S2), which differ only in the cyclopentadienyl ligand
(C5Me5 and C5H5, respectively), shows longer ruthenium−
ligand bond distances for Ru3. The larger Cpc−Ru−P angle of
Ru3 [129.85(4)°] relative to that of Ru2 [125.48(3)°]
evidences the steric hindrance between the bulky Cp* and
PPh3 ligands, and concomitantly, the quasi-planar phenanthro-
line ligand is “pushed” toward the Cp*, as indicated by the
smaller Cp*c−Ru−N(1) angle [127.89(11)° in Ru3 vs
131.34(9)° in Ru2], thus minimizing steric clashes within
the Ru3 coordination sphere.
Blood-Stage Antiplasmodial Activity. Dose−Response
Studies. We determined dose−response profiles of compounds
Ru1−Ru5 against the blood stage of CQ-sensitive 3D7-GFP,
CQ-resistant Dd2, and artemisinin-resistant IPC5202 Pf
strains. The antiplasmodial activity (IC50) and cytotoxicity
(CC50) of compounds in human hepatic cells (HepG2) as well
as their selectivity indices were determined (Table 1).
Reference antiplasmodial compounds dihydroartemisinin
(DHA), CQ, and primaquine (PQ) were included as controls
in these experiments.
Inspection of Table 1 reveals that compounds Ru1−Ru3
were highly potent against the 3D7-GFP Pf strain, with IC50
values ranging from 7.69 to 12 nM. To the best of our
knowledge, Ru1 and Ru2 are the first ruthenium-arene
compounds displaying single-digit nanomolar IC50 values
against this strain. This result contrasts with those reported
for hybrid ruthenium(II) complexes of chloroquine, which
were shown to be significantly less active against the 3D7-GFP
Pf strain (IC50 values ranging from 0.02 to 0.34 μM).32,39
NBD-appended complexes Ru4 and Ru5 were less active, with
IC50 values of 219 and 139 nM, respectively. The IC50 values of
Ru4 and Ru5 against a nonfluorescent 3D7 Pf strain confirmed
that NBD fluorescence does not markedly influence Pf 3D7
fluorescence-based analysis of compound activity and,
consequently, the results observed with the GFP-expressing
3D7 Pf strain. All compounds revealed lower activity against
the CQ-resistant Dd2 Pf strain than against the nonresistant
3D7-GFP Pf strain, but with IC50 values and resistance indices
lower than those of CQ. Compounds Ru1−Ru3 display the
first two-digit nanomolar IC50 values against the Dd2 Pf strain
reported so far within the ruthenium-arene chemotype (IC50
values ranging from 234 nM to 14.1 μM)31,33−35,37,38 that are
similar to those found for a CQ-ruthenocene-trioxane hybrid
(62.00 and 51.16 μM with 10% and 17% O2 atmospheres,
respectively).59 Despite being less active than compounds
Ru1-Ru3, compounds Ru4 and Ru5 exhibited lower RIs in the
Dd2 Pf strain. In the artemisinin-resistant Pf strain IPC5202,
compounds Ru1-Ru3 showed nonsignificant differences in
IC50, presenting high potency but being slightly less active and
exhibiting higher RIs than DHA. As observed for the Dd2
strain, Ru4 and Ru5 exhibit higher IC50 values but lower RIs
compared to those of Ru1−Ru3. In fact, compound Ru5
exhibits similar potency against all Pf strains tested, with RIs of
1.2 and 1.5 for the Dd2 and IPC5202 Pf strains, respectively.
Structurally, these compounds differ from previously
reported ruthenium-arene antimalarials in the arene (cyclo-
pentadienyl vs benzene derivatives), the referenced non-
hybridization with organic antimalarial motifs, and the
coordinated PPh3, a ligand known to play a key role in the
anticancer activity of cyclopentadienylruthenium(II) com-
pounds.40 The lipophilicity induced by the triphenylphosphine
in the complex’s coordination sphere may have an important
role in their high antiplasmodial activities.33 Nonetheless, it is
worth noticing that the higher lipophilicity of Ru3, when
compared to that of Ru2, induced by the bulkier Cp* ligand,
F
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
Figure 5. Effect of Ru2 on the intraerythrocytic cycle of P. falciparum. (A) Synchronized 3D7 cultures, with a 6 h window, in ring and trophozoite
(Troph.) stages were exposed to drug pulses of 10-fold IC50 Ru2 (80 nM) for 3 and 6 h. The bars represent means and standard errors of the mean
of parasite growth under each condition, relative to no drug control, quantified after one cycle (48 h, white bars) and three cycles (96 h, gray bars).
(B) Photomicrographs of Giemsa-stained blood smears illustrating parasite morphology before a 6 h pulse of Ru2 and after 48 and 96 h, in both
ring- and trophozoite-stage parasites. RPMIc culture medium.
led to neither higher antiplasmodial activity nor higher
cytotoxicity.
compounds must be selectively absorbed via a Plasmodium-
specific uptake mechanism, without affecting erythrocytes at
the tested concentration.
Compounds Ru1−Ru3 revealed low cytotoxicity against
HepG2 cells, as indicated by their CC50 values in the low
micromolar range (6.1−10.8 μM), with Ru1 showing an
improved cytotoxicity profile when compared to those of Ru2
and Ru3. This was expected, given the previous results of
anticancer activity of compounds Ru1 and Ru2,40,41 and other
compounds within the same scaffold.43 Selectivity indices (SIs)
show nonetheless large therapeutic windows for these
compounds, even against the least sensitive strain
(IPC5202), for which SIs ranged between 89 and 130.
Compounds Ru4 and Ru5 exhibited less cytotoxicity in
HepG2 cells than their Ru1−Ru3 counterparts did, with CC50
values of >50 μM, indicating good SI values also for these
compounds.
Cellular Localization Studies. Exploiting the intrinsic
fluorescence of NBD-appended compounds, we next evaluated
cellular localization of Ru4 and Ru5 in P. falciparum using a
co-staining protocol together with confocal microscopy images.
Images from confocal laser scanning microscopy (CLSM)
reveal that Ru4 and Ru5 accumulate only inside infected red
blood cells (iRBCs) and are largely dispersed throughout the
parasite’s cytoplasm after incubation for 15 min (Figure 4A).
The degree of co-localization of Ru4 and Ru5 in the nucleus
and hemozoin-containing digestive vacuole, determined by
calculation of Pearson’s correlation coefficients (PCCs)
(Figure 4B),60 shows that both compounds accumulate mainly
inside the parasite’s nucleus (Figure 4B, Hoescht), with high
PCCs obtained in both cases (0.578 and 0.501 for Ru4 and
Ru5, respectively). Nuclear accumulation is further supported
by intensity profile analysis (Figure S11), which showed a
similar spatial distribution of Ru4 and Ru5 with the nucleus.
Importantly, in addition to the nucleus, Ru5 significantly
accumulates inside the hemozoin-containing digestive vacuole
[PCC = 0.336 (Figure 4B)]. The fact that the NBD derivative
ligand 2, but not ligand 1 or PPh3 (Table 1), showed
significant blood-stage antiplasmodial activity (49.9% inhib-
ition at 1 μM) suggests ligand 2-induced digestive vacuole
accumulation and indicates tunability within the
cyclopentadienylruthenium(II) scaffold by complex ligand-
sphere variation. Interestingly, compounds within the [(η5-
C5H5)Ru(PPh3)(N-N)]+ scaffold are known to accumulate
mostly in mammalian cancer cell membranes,43 indicating that
Antiplasmodial Activity in a Synchronized P. falciparum
Culture. Following the establishment of the compound’s
activity against Pf blood stages (Table 1), the activity of
compound Ru2 against the erythrocytic stage of the parasite’s
life cycle was investigated in a tightly synchronized Pf culture
(3D7). Parasitemia and parasite development were analyzed by
flow cytometry and by Giemsa-stained thin blood smears
(Figure 5). The in vitro speed of action of Ru2 was assessed by
incubation of synchronized parasites at the ring and
trophozoite stages in the presence of a 10-fold IC50
concentration of the compound. Parasites were challenged
for 3 and 6 h periods and, following drug removal, were
allowed to resume growth in drug-free culture medium. After
completion of one maturation cycle (48 h), Ru2 induced a
modest decrease in total parasitemia of <50%, indicating only a
moderate impairment of reinvasion against both ring and
trophozoite stages (Figure 5A). In contrast, after growing for
96 h in the absence of the drug, the parasites were still unable
to resume growth, with an inhibition effect of almost 100%
under all conditions (Figure 5A). This indicates that,
regardless of the stage of development, Ru2 can induce
irreversible damage to the parasite cell after contact with the
parasite for only 3 or 6 h, strongly suggesting a cytocidal mode
of action. A considerable fraction of parasites, both during the
initial 48 and 96 h of recovery, lacked the characteristic
morphology of rings or trophozoites and were instead severely
perturbed, with a vast majority showing a lack of DNA and/or
organized nucleus, and hyaline, vacuolated and dispersed
cytoplasm (Figure 5B).61 Particularly at 96 h, the very few
parasites observed were debris-like forms or very condensed
crisis forms (Figure 5B). These results indicate that Ru2
impairs asexual parasite differentiation, exhibiting fast para-
siticidal activity against both the ring and trophozoite stages of
Pf.
Liver-Stage Antiplasmodial Activity. Compounds were
further evaluated for their ability to inhibit the invasion and
development of Plasmodium parasites in liver cells, using an in
vitro infection model that employs a human hepatoma cell line
(Huh7) infected with firefly luciferase-expressing P. berghei
rodent malaria parasites.56,57 IC50 values are listed in Table 2,
G
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
and representative dose−response curves are shown in Figure
S12.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01795.
Table 2. In Vitro Antiplasmodial Activity (liver stage) of
Ruthenium Compounds
1H NMR spectra of compounds 1, 2, and Ru1−Ru5; X-
ray crystal data, structural refinement parameters, and
molecular diagram for Ru3; and confocal microscopy
intensity profile analysis of Ru4 and Ru5 (PDF)
P. berghei IC50 (μM)
Ru1
Ru2
Ru3
Ru4
0.33 0.14
0.451 0.010
1.33 0.25
2.68 0.69
4.20 1.47
0.0011
Accession Codes
Ru5
CCDC 1996562 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
ATV62
PQ62
9.5
Notably, ruthenium compounds were able to significantly
affect hepatic infection, with IC50 values within the range of
0.33−4.20 μM. Ru1 and Ru2 presented the best activities,
displaying IC50 values of 0.33 and 0.451 μM, respectively
(nonsignificant difference). In contrast to what was observed
against the blood-stage infection of Pf strains, Ru3 was found
to be less active against the liver stage of infection than its Ru1
and Ru2 counterparts, with Ru4 and Ru5 again displaying the
lowest activity. While all compounds are thus much less active
than atovaquone (ATV), an inhibitor of the parasite’s
cytochrome bc1 with potent liver-stage activity,63 they were
shown to be significantly more effective than primaquine, the
gold standard for targeting liver-stage parasites, which requires
metabolic activation to exert its effect.64 Compounds are thus
more active than other ruthenium-arene compounds against
the liver stage of P. berghei (50% inhibition at 10 μM).39 The
confluency of Huh7 hepatoma cells was not affected within the
compound concentration ranges used for IC50 determinations,
demonstrating the absence of cytotoxicity under the
experimental conditions employed (Figure S12).
AUTHOR INFORMATION
■
Corresponding Author
Pedro R. Florindo − Instituto de Investigaca̧ o do Medicamento
̃
́
(iMed.ULisboa), Faculdade de Farmacia, Universidade de
Lisboa, 1649-003 Lisboa, Portugal; orcid.org/0000-0002-
8582-7036; Email: pedrorrflorindo@ff.ulisboa.pt
Authors
Sofia A. Milheiro − Instituto de Investigaca̧ o do Medicamento
̃
́
(iMed.ULisboa), Faculdade de Farmacia, Universidade de
Lisboa, 1649-003 Lisboa, Portugal
Joana Gonca̧ lves − Instituto de Investigaca̧ o do Medicamento
̃
́
(iMed.ULisboa), Faculdade de Farmacia, Universidade de
Lisboa, 1649-003 Lisboa, Portugal
Ricardo M. R. M. Lopes − Instituto de Investigaca̧ o do
̃
́
Medicamento (iMed.ULisboa), Faculdade de Farmacia,
Universidade de Lisboa, 1649-003 Lisboa, Portugal
CONCLUSIONS
Margarida Madureira − Instituto de Investigaca̧ o do
̃
■
́
Herein, we report “half-sandwich” cyclopentadienylruthenium-
(II) compounds of the general formula [(η5-C5R5)Ru(PPh3)-
(N-N)][PF6], a scaffold tested for the first time as an
antiplasmodial agent. Compounds displayed very high potency
against the blood stage of P. falciparum, with the most potent
compounds revealing single-digit nanomolar activity (IC50) in
3D7-GFP and low nanomolar activity in CQ-resistant Dd2 and
artemisinin-resistant IPC5202 strains, while showing residual
cytotoxicity in hepatic cells (HepG2). In the liver stage of P.
berghei, the best compounds (Ru1 and Ru2) exhibited
submicromolar antiplasmodial activity with no toxicity to
hepatic host cells (Huh7). Confocal microscopy studies
uncovered parasite-selective absorption of NBD-labeled
compounds Ru4 and Ru5 in 3D7-infected erythrocytes, and
parasite nuclear accumulation of both compounds, with
accumulation of Ru5 also in the hemozoin-containing digestive
vacuole. Lead compound Ru2 revealed impairment of asexual
differentiation in a blood-stage 3D7 P. falciparum-synchronized
strain, and fast parasiticidal activity against both ring- and
trophozoite-stage parasites. These compounds rank among the
best metalloantimalarials18 reported so far16−18,29−39 and are
especially relevant considering the nonhybridization with
organic antimalarial drugs. Overall, these results highlight the
cyclopentadienylruthenium(II) scaffold as a promising anti-
plasmodial chemotype and are expected to inspire others in the
discovery of new, “out-of-the-box” metallo-antiplasmodials.
Medicamento (iMed.ULisboa), Faculdade de Farmacia,
Universidade de Lisboa, 1649-003 Lisboa, Portugal
Lis Lobo − Department of Medical Parasitology, Global Health
and Tropical Medicine, GHTM, Instituto de Higiene e Medicina
Tropical, IHMT, Universidade Nova de Lisboa, 1349-008
Lisboa, Portugal
Andreia Lopes − Department of Medical Parasitology, Global
Health and Tropical Medicine, GHTM, Instituto de Higiene e
Medicina Tropical, IHMT, Universidade Nova de Lisboa,
1349-008 Lisboa, Portugal
́
Fatima Nogueira − Department of Medical Parasitology, Global
Health and Tropical Medicine, GHTM, Instituto de Higiene e
Medicina Tropical, IHMT, Universidade Nova de Lisboa,
1349-008 Lisboa, Portugal; orcid.org/0000-0003-0313-
0778
Diana Fontinha − Instituto de Medicina Molecular Joao Lobo
̃
Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-
028 Lisboa, Portugal
̂
Miguel Prudencio − Instituto de Medicina Molecular Joao Lobo
̃
Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-
028 Lisboa, Portugal
́
́
M. Fatima M. Piedade − Centro de Quimica Estrutural,
́
Instituto Superior Tecnico, Universidade de Lisboa, 1049-001
Lisboa, Portugal; Departamento de Quimica e Bioquimica,
́
́
̂
Faculdade de Ciencias da Universidade de Lisboa, 1749-016
Lisboa, Portugal
H
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
advances in delivery of photosensitive metal-based drugs. Coord.
Chem. Rev. 2019, 387, 154−179.
Sandra N. Pinto − iBB-Institute for Bioengineering and
Biosciences, Department of Bioengineering, Instituto Superior
(14) Imberti, C.; Sadler, P. J. 150 years of the periodic table: New
medicines and diagnostic agents. In Medicinal Chemistry; Sadler, P. J.,
van Eldik, R., Eds.; Academic Press (Elsevier): Cambridge, U.K.,
2020; pp 3−56.
́
Tecnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
do Medicamento
Rui Moreira − Instituto de Investigaca̧ o
̃
́
(iMed.ULisboa), Faculdade de Farmacia, Universidade de
Lisboa, 1649-003 Lisboa, Portugal; orcid.org/0000-0003-
0727-9852
(15) Patra, M.; Gasser, G. The medicinal chemistry of ferrocene and
its derivatives. Nat. Rev. Chem. 2017, 1, 0066.
(16) Xiao, J.; Sun, Z.; Kong, F.; Gao, F. Current scenario of
ferrocene-containing hybrids for antimalarial activity. Eur. J. Med.
Chem. 2020, 185, 111791.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c01795
́
(17) Biot, C.; Castro, W.; Botte, C. Y.; Navarro, M. The therapeutic
potential of metal-based antimalarial agents: implications for the
mechanism of action. Dalton Trans. 2012, 41, 6335.
Notes
The authors declare no competing financial interest.
(18) Salas, P. F.; Herrmann, C.; Orvig, C. Metalloantimalarials.
Chem. Rev. 2013, 113, 3450−3492.
(19) Bergamo, A.; Gaiddon, C.; Schellens, J.; Beijnen, J. H.; Sava, G.
Approaching tumour therapy beyond platinum drugs: status of the art
and perspectives of ruthenium drug candidates. J. Inorg. Biochem.
2012, 106, 90−99.
(20) Li, X.; Gorle, A. K.; Sundaraneedi, M. K.; Keene, F. R.; Collins,
J. G. Kinetically-inert polypyridylruthenium(II) complexes as
therapeutic agents. Coord. Chem. Rev. 2018, 375, 134−147.
(21) Meier-Menches, S. M.; Gerner, C.; Berger, W.; Hartinger, C.
G.; Keppler, B. K. Structure-activity relationships for ruthenium and
osmium anticancer agents - towards clinical development. Chem. Soc.
Rev. 2018, 47, 909−928.
ACKNOWLEDGMENTS
■
̂
The authors thank Fundaca
̧
o
̃
para a Ciencia e Tecnologia
(FCT) for financial support, through Projects PTDC/MED-
QUI/31468/2017 (P.R.F., S.A.M., J.G., M.F.M.P., and R.M.),
PTDC/MED-QUI/30021/2017 (R.M., F.N., L.L., and A.L.),
UIDB/04565/2020, SAICTPAC/0019/2015, PPBI-POCI-01-
0145-FEDER-022122 (S.N.P.), UID/Multi/04413/2013
(F.N., L.L., and A.L.), and PTDC/SAU-INF/29550/2017
(M.P.) and Grant SFRH/BD/121664/2016 (R.M.R.M.L.).
The authors thank MR4 for providing malaria parasites
contributed by Didier Menard, Institut Pasteur du Cambodge.
(22) Aird, R. E.; Cummings, J.; Ritchie, A. A.; Muir, M.; Jodrell, D.
I.; Morris, R. E.; Chen, H.; Sadler, P. J. In vitro and in vivo activity
and cross resistance profiles of novel ruthenium (II) organometallic
arene complexes in human ovarian cancer. Br. J. Cancer 2002, 86,
1652−1657.
(23) Hayward, R. L.; Schornagel, Q. C.; Tente, R.; Macpherson, J.
S.; Aird, R. E.; Guichard, S.; Habtemariam, A.; Sadler, P.; Jodrell, D. I.
Investigation of the role of Bax, p21/Waf1 and p53 as determinants of
cellular responses in HCT116 colorectal cancer cells exposed to the
novel cytotoxic ruthenium(II) organometallic agent, RM175. Cancer
Chemother. Pharmacol. 2005, 55, 577−583.
(24) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto,
M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. In Vitro and
in Vivo Evaluation of Ruthenium(II)−Arene PTA Complexes. J. Med.
Chem. 2005, 48, 4161−4171.
(25) Bergamo, A.; Masi, A.; Dyson, P. J.; Sava, G. Modulation of the
metastatic progression of breast cancer with an organometallic
ruthenium compound. Int. J. Oncol. 2008, 33, 1281−1289.
(26) Meng, X.; Leyva, M. L.; Jenny, M.; Gross, I.; Benosman, S.;
REFERENCES
(1) World Health Organization. World Malaria Report 2019.
(2) Rodrigues, T.; Prudencio, M.; Moreira, R.; Mota, M. M.; Lopes,
F. Targeting the Liver Stage of Malaria Parasites: A Yet Unmet Goal.
J. Med. Chem. 2012, 55, 995−1012.
(3) Capela, R.; Moreira, R.; Lopes, F. An Overview of Drug
Resistance in Protozoal Diseases. Int. J. Mol. Sci. 2019, 20, 5748.
(4) Mathenge, P. G.; Low, S. K.; Vuong, N. L.; Mohamed, M. Y. F.;
Faraj, H. A.; Alieldin, G. I.; Al Khudari, R.; Yahia, N. A.; Khan, A.;
Diab, O. M.; Mohamed, Y. M.; Zayan, A. H.; Tawfik, G. M.; Huy, N.
T.; Hirayama, K. Efficacy and resistance of different artemisinin-based
combination therapies: a systematic review and network meta-
analysis. Parasitol. Int. 2020, 74, 101919.
(5) Gambino, D.; Otero, L. Perspectives on what ruthenium-based
compounds could offer in the development of potential antiparasitic
drugs. Inorg. Chim. Acta 2012, 393, 103−114.
(6) Dilruba, S.; Kalayda, G. V. Platinum-based drugs: past, present
and future. Cancer Chemother. Pharmacol. 2016, 77, 1103−1124.
(7) Mirzadeh, N.; Reddy, T.; Bhargava, S. Advances in diphosphine
ligand-containing gold complexes as anticancer agents. Coord. Chem.
Rev. 2019, 388, 343−359.
(8) For recent advances in metal drug discovery, see: Franz, K.;
Metzler-Nolte, N. Introduction: Metals in Medicine. Chem. Rev. 2019,
119, 727−729.
(9) Yufanyi, D. M.; Abbo, H. S.; Titinchi, S. J. J.; Neville, T.
Platinum(II) and Ruthenium(II) complexes in medicine: Antimyco-
bacterial and Anti-HIV activities. Coord. Chem. Rev. 2020, 414,
213285.
(10) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen,
Z.-S. The development of anticancer ruthenium(II) complexes: from
single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017,
46, 5771−5804.
(11) Brabec, V.; Kasparkova, J. Ruthenium coordination compounds
of biological and biomedical significance. DNA binding agents. Coord.
Chem. Rev. 2018, 376, 75−94.
■
̂
́
Fricker, B.; Harlepp, S.; Hebraud, P.; Boos, A.; Wlosik, P.; Bischoff,
P.; Sirlin, C.; Pfeffer, M.; Loeffler, J.-P.; Gaiddon, C. A ruthenium-
containing organometallic compound reduces tumor growth through
induction of the endoplasmic reticulum stress gene CHOP. Cancer
Res. 2009, 69, 5458−5466.
(27) Klajner, M.; Hebraud, P.; Sirlin, C.; Gaiddon, C.; Harlepp, S.
DNA Binding to an Anticancer Organo-Ruthenium Complex. J. Phys.
Chem. B 2010, 114, 14041−14047.
́
(28) Monro, S.; Colon, K. L.; Yin, H.; Roque, J.; Konda, P.; Gujar,
S.; Thummel, R. P.; Lilge, L.; Cameron, C. G.; McFarland, S. A.
Transition Metal Complexes and Photodynamic Therapy from a
Tumor-Centered Approach: Challenges, Opportunities, and High-
lights from the Development of TLD1433. Chem. Rev. 2019, 119,
797−828.
(29) Dubar, F.; Egan, T. J.; Pradines, B.; Kuter, D.; Ncokazi, K. K.;
Forge, D.; Paul, J.-F.; Pierrot, C.; Kalamou, H.; Khalife, J.; Buisine, E.;
Rogier, C.; Vezin, H.; Forfar, I.; Slomianny, C.; Trivelli, X.;
Kapishnikov, S.; Leiserowitz, L.; Dive, D.; Biot, C. The antimalarial
ferroquine: role of the metal and intramolecular hydrogen bond in
activity and resistance. ACS Chem. Biol. 2011, 6, 275−287.
(30) Glans, L.; Ehnbom, A.; de Kock, C.; Martínez, A.; Estrada, J.;
(12) Ko, C.-N.; Li, G.; Leung, C.-H.; Ma, D.-L. Dual function
luminescent transition metal complexes for cancer theranostics: The
combination of diagnosis and therapy. Coord. Chem. Rev. 2019, 381,
79−103.
(13) Yu, Y.; Xu, Q.; He, S.; Xiong, H.; Zhang, Q.; Xu, W.; Ricotta,
V.; Bai, L.; Zhang, Q.; Yu, Z.; Ding, J.; Xiao, H.; Zhou, D. Recent
́
Smith, P. J.; Haukka, M.; Sanchez-Delgado, R. A.; Nordlander, E.
Ruthenium(II) arene complexes with chelating chloroquine analogue
I
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
ligands: Synthesis, characterization and in vitroantimalarial activity.
Dalton Trans. 2012, 41, 2764−2773.
(PF6]2·CH2Cl2] [dppm = bis(diphenylphosphino)methane; phen =
phenanthroline]. Organometallics 1987, 6, 2179−2189.
(43) Morais, T. S.; Valente, A.; Tomaz, A. I.; Marques, F.; Garcia, M.
H. Tracking antitumor metallodrugs: promising agents with the
Ru(II)- and Fe(II)-cyclopentadienyl scaffolds. Future Med. Chem.
2016, 8, 527−544.
(44) Perrin, D. D.; Amarego, W. L. F.; Perrin, D. R. Purification of
Laboratory Chemicals, 2nd ed.; Pergamon: New York, 1980.
(45) SABADS. Area-detector Absorption Correction; Bruker AXS Inc.:
Madinson, WI, 2004.
(46) SAINT, Area-detector Integration Software, ver. 7.23; Bruker AXS
Inc.: Madinson, WI, 2004.
(47) Sheldrick, G. M. A Short History of SHELX. Acta Crystallogr.,
Sect. A: Found. Crystallogr. 2008, 64, 112−122.
(48) Farrugia, L. J. WinGX and ORTEP for Windows: an update. J.
Appl. Crystallogr. 2012, 45, 849−854.
(31) Stringer, T.; Quintero, M. A. S.; Wiesner, L.; Smith, G. S.;
Nordlander, E. Evaluation of PTA-derived ruthenium(II) and
iridium(III) quinoline complexes against chloroquine-sensitive and
resistant strains of the Plasmodium falciparum malaria parasite. J.
Inorg. Biochem. 2019, 191, 164−173.
(32) Rajapakse, C. S. K.; Martínez, A.; Naoulou, B.; Jarzecki, A. A.;
́
́
Suarez, L.; Deregnaucourt, C.; Sinou, V.; Schrevel, J.; Musi, E.;
́
Ambrosini, G.; Schwartz, G. K.; Sanchez-Delgado, R. A. Synthesis,
Characterization and in vitro Antimalarial and Antitumor Activity of
New Ruthenium(II) Complexes of Chloroquine. Inorg. Chem. 2009,
48, 1122−1131.
(33) Martínez, A.; Rajapakse, C. S. K.; Jalloh, D.; Dautriche, C.;
́
Sanchez-Delgado, R. A. The antimalarial activity of Ru-chloroquine
complexes against resistant Plasmodium falciparum is related to
lipophilicity, basicity, and heme aggregation inhibition ability near
water/n-octanol interfaces. JBIC, J. Biol. Inorg. Chem. 2009, 14, 863−
871.
(49) Macrae, C. F.; Edgington, P. R.; Mccabe, P.; Pidcock, E.;
Shields, G. P.; Taylor, R.; Towler, M.; van de Streek, J. Mercury:
visualization and analysis of crystal structures. J. Appl. Crystallogr.
2006, 39, 453−457.
(34) Nkoana, W.; Nyoni, D.; Chellan, P.; Stringer, T.; Taylor, D.;
Smith, P. J.; Hutton, A. T.; Smith, G. S. Heterometallic half-sandwich
complexes containing a ferrocenyl motif: Synthesis, molecular
structure, electrochemistry and antiplasmodial evaluation. J. Organo-
met. Chem. 2014, 752, 67−75.
(35) Ekengard, E.; Glans, L.; Cassells, I.; Fogeron, T.; Govender, P.;
Stringer, T.; Chellan, P.; Lisensky, G. C.; Hersh, W. H.; Doverbratt, I.;
Lidin, S.; de Kock, C.; Smith, P. J.; Smith, G. S.; Nordlander, E.
Antimalarial activity of ruthenium(II) and osmium(II) arene
complexes with mono- and bidentate chloroquine analogue ligands.
Dalton Trans. 2015, 44, 19314−19329.
(36) Rylands, L.-I.; Welsh, A.; Maepa, K.; Stringer, T.; Taylor, D.;
Chibale, K.; Smith, G. S. Structure-activity relationship studies of
antiplasmodial cyclometallated ruthenium(II), rhodium(III) and
iridium(III) complexes of 2-phenylbenzimidazoles. Eur. J. Med.
Chem. 2019, 161, 11−21.
(37) Adams, M.; de Kock, C.; Smith, P. J.; Land, K. M.; Liu, N.;
Hopper, M.; Hsiao, A.; Burgoyne, A. R.; Stringer, T.; Meyer, M.;
Wiesner, L.; Chibale, K.; Smith, G. S. Improved antiparasitic activity
by incorporation of organosilane entities into half-sandwich
ruthenium(II) and rhodium(III) thiosemicarbazone complexes.
Dalton Trans. 2015, 44, 2456−2468.
́
(50) Nogueira, F.; Diez, A.; Radfar, A.; Perez-Benavente, S.; do
Rosario, V. E.; Puyet, A.; Bautista, J. M. Early transcriptional response
to chloroquine of the Plasmodium falciparum antioxidant defence in
sensitive and resistant clones. Acta Trop. 2010, 114, 109−115.
(51) Aguiar, L.; Biosca, A.; Lantero, E.; Gut, J.; Vale, N.; Rosenthal,
̀
P. J.; Nogueira, F.; Andreu, D.; Fernandez-Busquets, X.; Gomes, P.
Coupling the Antimalarial Cell Penetrating Peptide TP10 to Classical
Antimalarial Drugs Primaquine and Chloroquine Produces Strongly
Hemolytic Conjugates. Molecules 2019, 24, 4559.
(52) Wilson, D. W.; Crabb, B. S.; Beeson, J. G. Development of
fluorescent Plasmodium falciparum for in vitro growth inhibition
assays. Malar. J. 2010, 9, 152.
(53) Fu, Y.; Tilley, L.; Kenny, S.; Klonis, N. Dual labeling with a far-
red probe permits analysis of growth and oxidative stress in P.
falciparum-infected erythrocytes. Cytometry, Part A 2010, 77, 253−
263.
(54) Model, M. A.; Reese, J. L.; Fraizer, G. C. Measurement of wheat
germ agglutinin binding with a fluorescence microscope. Cytometry,
Part A 2009, 75, 874−881.
(55) Carvalho, C. M. L.; Chew, E.; Hashemy, S. I.; Lu, J.; Holmgren,
A. Inhibition of the Human Thioredoxin System: a Molecular
Mechanism of Mercury Toxicity. J. Biol. Chem. 2008, 283, 11913−
11923.
(38) Adams, M.; Li, Y.; Khot, H.; De Kock, C.; Smith, P. J.; Land,
K.; Chibale, K.; Smith, G. S. The synthesis and antiparasitic activity of
aryland ferrocenyl-derived thiosemicarbazone ruthenium(II)−arene
complexes. Dalton Trans. 2013, 42, 4677−4685.
(39) Macedo, T. S.; Colina-Vegas, L.; da Paixao, M.; Navarro, M.;
̃
Barreto, B. C.; Oliveira, P. C. M.; Macambira, S. G.; Machado, M.;
̂
(56) Prudencio, M.; Mota, M. M.; Mendes, A. M. A toolbox to study
liver stage malaria. Trends Parasitol. 2011, 27, 565−574.
(57) Ploemen, I. H. J.; Prudencio, M.; Douradinha, B. G.; Ramesar,
J.; Fonager, J.; van Gemert, G.-J.; Luty, A. J. F.; Hermsen, C. C.;
Sauerwein, R. W.; Baptista, F. G.; Mota, M. M.; Waters, A. P.; Que, I.;
Lowik, C. W. G. M.; Khan, S. M.; Janse, C. J.; Franke-Fayard, B. M. D.
Visualisation and Quantitative Analysis of the Rodent Malaria Liver
Stage by Real Time Imaging. PLoS One 2009, 4, e7881.
̂
Prudencio, M.; D’Alessandro, S.; Basilico, N.; Moreira, D. R. M.;
Batista, A. A.; Soares, M. B. P. Chloroquine-containing organo-
ruthenium complexes are fast-acting multistage antimalarial agents.
Parasitology 2016, 143, 1543−1556.
̂
(40) Compound Ru1: Corte-Real, L.; Robalo, M. P.; Marques, F.;
(58) Groom, C. R.; Bruno, I. J.; Lightfoot, M. P.; Ward, S. C. The
Cambridge Structural Database. Acta Crystallogr., Sect. B: Struct. Sci.,
Cryst. Eng. Mater. 2016, B72, 171−179.
Nogueira, G.; Avecilla, F.; J. L. Silva, T.; C. Santos, F.; Tomaz, A. I.;
Garcia, M. H.; Valente, A. The key role of coligands in novel
ruthenium(II)-cyclopentadienyl bipyridine derivatives: Ranging from
non-cytotoxic to highly cytotoxic compounds. J. Inorg. Biochem. 2015,
150, 148−159.
(59) Martínez, A.; Deregnaucourt, C.; Sinou, V.; Latour, C.; Roy, D.;
́
́
Schrevel, J.; Sanchez-Delgado, R. A. Synthesis of an organo-ruthenium
aminoquinoline-trioxane hybrid and evaluation of its activity against
Plasmodium falciparum and its toxicity toward normal mammalian
cells. Med. Chem. Res. 2017, 26, 473−483.
(41) Compounds Ru1 and Ru2: Florindo, P. R.; Pereira, D. M.;
Borralho, P. M.; Costa, P. J.; Piedade, M. F. M.; Rodrigues, C. M. P.;
Fernandes, A. C. New [(η5-C5H5)Ru(N−N)(PPh3)][PF6] com-
pounds: colon anticancer activity and GLUT-mediated cellular uptake
of carbohydrate-appended complexes. Dalton Trans. 2016, 45,
11926−11930.
(42) Compound Ru2 was first synthesized in 1987: Albers, M. O.;
Liles, D. C.; Robinson, D. J.; Singleton, E. The chemistry of
Cyclopentadienyl-Ruthenium and -Osmium Complexes. 5. The
systematic synthesis of novel bridged diruthenium cations containing
cyclopentadienyl and bidentate amine ligands. The crystal and
molecular structure of [{(η5-C5H5)Ru(1,10-phen)}2(μ-dppm)]-
(60) PCC values range from 1 to −1, with 1 corresponding to a
perfect correlation of the two images and −1 to uncorrelation: Dunn,
K. W.; Kamocka, M. M.; McDonald, J. H. A practical guide to
evaluating colocalization in biological microscopy. Am. J. Physiol.
2011, 300, C723−C742.
(61) Although Ru4 and Ru5 nuclear co-localization (confocal
microscopy studies) is consistent with parasitic nuclear damage
induced by Ru2, fluorophores are known to redirect compounds to
different cellular organelles: Puckett, C. A.; Barton, J. K. Fluorescein
Redirects a Ruthenium−Octaarginine Conjugate to the Nucleus. J.
J
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
Am. Chem. Soc. 2009, 131, 8738−8739. Conclusions about the
cellular distribution of compounds Ru1−Ru3 would require parasitic
cellular fractionation and analysis by ICP-MS or AAS.
(62) Rodrigues, C. A. B.; Frade, R. F. M.; Albuquerque, I. S.; Perry,
̂
M. J.; Gut, J.; Machado, M.; Rosenthal, P. J.; Prudencio, M.; Afonso,
C. A. M.; Moreira, R. Targeting the Erythrocytic and Liver Stages of
Malaria Parasites with s-Triazine-Based Hybrids. ChemMedChem
2015, 10, 883−890.
̂
(63) Derbyshire, E. R.; Prudencio, M.; Mota, M. M.; Clardy, J. Liver-
stage malaria parasites vulnerable to diverse chemical scaffolds. Proc.
Natl. Acad. Sci. U. S. A. 2012, 109, 8511−8516.
(64) Camarda, G.; Jirawatcharadech, P.; Priestley, R. S.; Saif, A.;
March, S.; Wong, M. H. L.; Leung, S.; Miller, A. B.; Baker, D. A.;
Alano, P.; Paine, M. J. I.; Bhatia, S. N.; O’Neill, P. M.; Ward, S. A.;
Biagini, G. A. Antimalarial activity of primaquine operates via a two-
step biochemical relay. Nat. Commun. 2019, 10, 3226.
K
https://dx.doi.org/10.1021/acs.inorgchem.0c01795
Inorg. Chem. XXXX, XXX, XXX−XXX