Conjugates containing two and three trithiolato-bridged dinuclear ruthenium(Ii)-arene units as in vitro antiparasitic and anticancer agents

pharmaceuticals

Article

Conjugates Containing Two and Three

Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene

Units as In Vitro Antiparasitic and Anticancer Agents

Valentin Studer ^1,†, Nicoleta Anghel ^2,†, Oksana Desiatkina ¹, Timo Felder ¹, Ghalia Boubaker ²,

Yosra Amdouni ^2,3, Jessica Ramseier ², Martin Hungerbühler ^4,5, Christoph Kempf ^4,5

,

Johannes Thomas Heverhagen ^4,5, Andrew Hemphill ^2,

* *

, Nico Ruprecht ^4,5,*, Julien Furrer ^1,

and Emilia Păunescu ^1,

*

1

Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland;

valentin.studer@dcb.unibe.ch (V.S.); oksana.desiatkina@dcb.unibe.ch (O.D.); timofelder@hotmail.com (T.F.)

Vetsuisse Faculty, Institute of Parasitology, University of Bern, Länggassstrasse 122,

CH-3012 Bern, Switzerland; nicoleta.anghel@vetsuisse.unibe.ch (N.A.);

2

ghalia.boubaker@vetsuisse.unibe.ch (G.B.); amdouniyosra.ay@gmail.com (Y.A.);

jessica.ramseier@vetsuisse.unibe.ch (J.R.)

3

4

Laboratoire de Parasitologie, Institution de la Recherche et de l’Enseignement Supérieur Agricoles,

École Nationale de Médecine Vétérinaire de Sidi Thabet, University of Manouba, Sidi Thabet 2020, Tunisia

Department of BioMedical Research, Experimental Radiology, University of Bern,

CH-3008 Bern, Switzerland; martin.hungerbuehler@dbmr.unibe.ch (M.H.);

christoph.kempf@dbmr.unibe.ch (C.K.); johannes.heverhagen@dbmr.unibe.ch (J.T.H.)

Department of Diagnostic, Interventional and Pediatric Radiology, Bern University Hospital,

University of Bern, CH-3010 Bern, Switzerland

5

*

Correspondence: andrew.hemphill@vetsuisse.unibe.ch (A.H.); nico.ruprecht@dbmr.unibe.ch (N.R.);

julien.furrer@dcb.unibe.ch (J.F.); paunescu_emilia@yahoo.com (E.P.)

†

These authors contributed equally to this work.

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

Received: 11 November 2020; Accepted: 11 December 2020; Published: 16 December 2020

ꢀꢁꢂꢃꢄꢅꢆ

Abstract: The synthesis, characterization, and in vitro antiparasitic and anticancer activity evaluation

of new conjugates containing two and three dinuclear trithiolato-bridged ruthenium(II)-arene units

are presented. Antiparasitic activity was evaluated using transgenic Toxoplasma gondii tachyzoites

constitutively expressing β-galactosidase grown in human foreskin ﬁbroblasts (HFF). The compounds

inhibited T. gondii proliferation with IC₅₀values ranging from 90 to 539 nM, and seven derivatives

displayed IC₅₀values lower than the reference compound pyrimethamine, which is currently used for

treatment of toxoplasmosis. Overall, compound ﬂexibility and size impacted on the anti-Toxoplasma

activity. The anticancer activity of 14 compounds was assessed against cancer cell lines A2780,

A2780cisR (human ovarian cisplatin sensitive and resistant), A24, (D-)A24cisPt8.0 (human lung

adenocarcinoma cells wild type and cisPt resistant subline). The compounds displayed IC₅₀values

ranging from 23 to 650 nM. In A2780cisR, A24 and (D-)A24cisPt8.0 cells, all compounds were

considerably more cytotoxic than cisplatin, with IC₅₀values lower by two orders of magnitude.

Irrespective of the nature of the connectors (alkyl/aryl) or the numbers of the di-ruthenium units

(two/three), ester conjugates

more cytotoxic than amide analogues 11

6

–

10 and 20 exhibited similar antiproliferative proﬁles, and were

14 23, and 24. Polynuclear conjugates with multiple

–

,

trithiolato-bridged di-ruthenium(II)-arene moieties deserve further investigation.

Keywords: bioactive organometallics; polynuclear ruthenium compounds; antiparasitic; anticancer

Pharmaceuticals 2020, 13, 471; doi:10.3390 /ph13120471

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1. Introduction

The recent high interest in the development of therapeutic transition metal complexes

is in part driven by the clinical success of the platinum(II) anticancer drug, cisplatin (cis-

diamminedichloroplatinum(II)). Cisplatin has become an important component in chemotherapy

regimens for the treatment of ovarian, testicular, lung and bladder cancers, as well as lymphomas,

myelomas and melanomas [

1

–

4]. The use of known platinum drugs is limited by the incidence of

intrinsic or acquired drug resistance [

5–

8

] and by their high toxicity and severe side-eﬀects [ 12].

9–

Currently, there are six platinum drugs with marketing approval in various regions throughout the

world: cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, and heptaplatin. Important research

eﬀorts are directed towards the development of other platinum(II) or (IV)-based drugs that overcome

these drawbacks [13–17]. Over the last 40 years, numerous platinum drugs have entered clinical trials,

but only two (carboplatin and oxaliplatin) have gained international marketing approval, and another

four have obtained regulatory approval in individual countries: heptaplatin (Korea), lobaplatin (China),

miriplatin and nedaplatin (Japan) [10].

Along with platinum, other transition metals including gold, titanium, rhodium, iridium,

ruthenium and osmium, as well as essential metals such as iron, copper, zinc and cobalt, inspired the

development of numerous organometallic compounds as potential anticancer candidates due to

their variable coordination modes, redox activity and reactivity towards biomolecules [18

–23].

Parallel studies were focused on the identiﬁcation of other pharmacological applications of metalorganic

compounds, e.g., diagnostic tools as biomolecular and cellular probes [24

and antiparasitic therapeutics [27–33].

–26] or antibacterial, antifungal,

Among the numerous inorganic and organometallic compounds presenting anticancer properties,

ruthenium(II) and (III) complexes were identified as some of the most promising alternatives for the

replacement of platinum drugs [23 ,34 –40 ]. For these ruthenium compounds, the oxidation state of

the ruthenium atom, their solubility and stability in physiological media, greatly influence their

activity against cancer cells. Three ruthenium(III) metal based complexes, NAMI-A ((ImH)[trans-

RuCl₄(dmso-S)(Im)], Im = imidazole) and KP1019/1339 (KP1019 = (IndH)[trans-RuCl₄(Ind)₂], Ind = indazole;

KP1339 = Na[trans-RuCl₄(Ind)₂]), were subjected to clinical tests [41 –44 ], while the most advanced

ruthenium(II) complex is TLD1433 ((Ru(4,4⁰-dimethyl-2,2⁰-bipyridine)2(2-(2⁰,2⁰⁰:5⁰⁰,2⁰⁰⁰-terthiophene)-

imidazo [4,5-f])]Cl₂), a photosensitizer that has recently entered clinical trials for non-muscle

invasive bladder cancer [45 –47 ]. Several ruthenium(II)-arene compounds including RAPTA-C

([Ru(II)(

η

⁶- p-MeC₆H₄Prⁱ)(PTA)Cl₂], PTA = 1,3,5-triaza-7-phosphatricyclo-[3.3.1]decane) and RM175

([Ru(II)(

η

⁶-biphenyl)(en)(PF₆)], en = ethylenediamine) (Figure 1 ) were submitted to advanced in vitro

and in vivo studies [48 –52 ].

This opened the door for the development of a myriad of organometallic complexes bearing the

half-sandwich ruthenium(II)-arene scaffold [53

bridged dinuclear ruthenium(II)-arene compounds of general formula [(

SR)₃]⁺[55 ⁶-p-MeC₆H₄Prⁱ)₂Ru₂(µ₂-SR¹)(µ₂-SR²)₂]⁺[57 ] (Figure 1 ) have shown high

56 ] and, respectively, [(

,

54 ]. Among those, symmetric and mixed cationic trithiolato-

η

⁶-p-MeC₆H₄Prⁱ)₂Ru₂(µ₂

-

,

η

cytotoxicity against human cancer cells (IC₅₀values as low as 30 nM against A2780 human ovarian cancer

cells and the cisplatin-resistant variant A2780cisR) [58 ]. This encouraged the synthesis and evaluation of

various libraries of complexes based on the trithiolato-bridged di-ruthenium scaﬀold in the pursuit

of improved anticancer properties [55

assessment of alternative biological applications such as antiparasitic activity [60–62].

Several symmetric complexes [(

⁶-p-MeC₆H₄Prⁱ)₂Ru₂(µ₂-S-p-C₆H₄R)₃]⁺with R = p-Me (

p-Bu^t(

), p-OH ( ) (Figure 1 ) presenting interesting in vitro cytotoxicity against cancer cells (IC₅₀values

of 30 nM against A2780 and A2780cisR cells for ) were also assessed in mice [63 65]. Mixed complexes

,58,59], but also opened the door for a broader scope, with the

η

A),

B

C

B

–

(as

D, [(η ,

⁶-p-MeC₆H₄Prⁱ)₂Ru₂(µ₂-SR¹)(µ₂-SR²)₂]⁺with R¹= CH₂-C₆H₄-p-Bu^t, R²= C₆H₄-p-OH

X = Cl^-, Figure 1) also exhibited cytotoxicity against A2780 and A2780cisR cells in the nanomolar

range [57].

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Figure 1. Structures of the ruthenium(II)-arene complexes RAPTA-C and RM175, of selected symmetric

and mixed trithiolato-bridged ruthenium(II)-arene complexes, and of the trinuclear platinum compound

BBR 3464.

Despite their unusual high potency, this class of ruthenium complexes was not selective for cancer

cells and displayed a low water solubility [58]. Unlike other Ru(II)-arene complexes presenting labile

chlorine or carboxylate ligands, they were stable in the presence of water, amino acids and DNA [58]

and interacted weakly in a noncovalent manner with proteins, including HSA (human serum albumin),

Tf (transferrin), Cytc (cytochrome c), Ub (ubiquitin) and Mb (myoglobin) [58]. Only the oxidation

of cysteine (Cys) and glutathione (GSH) to form cystine and GSSG, respectively, was observed in

the presence of this type of complexes [55

and the catalytic activity on the glutathione oxidation was observed [55

plasma mass spectrometry (ICP-MS) experiments proved that complexes

,

58], but no correlation between the in vitro cytotoxicity

58]. Inductively coupled

and speciﬁcally target

,

A

B

the mitochondrion in A2780 ovarian cancer cells, with up to 97% of the Ru content found in the

mitochondrial fraction [61].

Recently it was shown that some cationic trithiolato-bridged dinuclear ruthenium(II)-arene

compounds display interesting in vitro activities against two apicomplexan parasites Toxoplasma

gondii [60] and Neospora caninum [61], which cause abortions and fetal malformations in humans

(T. gondii) and animals (T. gondii and N. caninum), and against Trypanosoma brucei [62], the causative

agent of African sleeping sickness. In addition, several compounds derived from a library of coumarin

conjugates of trithiolato di-ruthenium complexes showed promising activity against T. gondii [66].

Thus,

did not aﬀect HFF host cells at dosages of 200

Transmission electron microscopy (TEM) detected ultrastructural alterations in the matrix of the parasite

A

and

B

inhibited T. gondii proliferation with IC₅₀values of 34 and 62 nM, respectively, and they

µM or above, resulting in high selectivity indices.

mitochondria at the early stages of treatment with

destruction of tachyzoites. However, compounds

D

A

(X⁻= BF₄⁻), followed by a more pronounced

, or applied at 250 nM did not act in a

, and also inhibited N. caninum proliferation

with low IC₅₀values of 15, 5 and 1 nM, respectively. [60 62] TEM also indicated that the parasite

mitochondrion was the primary target, but parasiticidal activity was also not detected. Complexes

, and applied orally in a neosporosis mouse model did not reduce parasite load in the central

,

B

D

parasiticidal manner. Furthermore, complexes

A

,

B

D

–

A

,

B

D

nervous system. These encouraging results stimulated us to develop a new library of complexes based

on the trithiolato-bridged di-ruthenium scaﬀold and evaluate them as antiparasitic agents.

Polynuclear compounds are a relatively new and successful approach in metal-based cancer

chemotherapy as exempliﬁed by the trinuclear platinum compound BBR 3464 [{trans-PtCl(NH₃)₂}-

µ

-{trans-Pt(NH₃)₂(H₂N(CH₂)₆NH₂)₂}][NO₃]₄, (Figure 1 ) which was evaluated in clinical trials [67

–70].

BBR 3464 exhibited interesting biological activity and a potentially diﬀerent mode of action compared to

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mononuclear cisplatin. The introduction of two additional metal fragments to the platinum scaﬀold led

to a higher activity in cisplatin-resistant cell lines and tumour models for the trinuclear complex [71 75].

–

The high cytotoxicity of the polynuclear platinum compound against cisplatin resistant cells was

attributed to the diﬀerent type of DNA-adducts formed compared to those of cisplatin, but also to the

increased number of platinum moieties [71,76–78].

Despite BBR 3464 failing clinical trials due to limited response in patients [68,79], the polynuclear

strategy was considered promising and an increased interest in the development of other compounds

with two or more metal centres emerged. Various homo-polynuclear ruthenium, osmium and gold

complexes were developed, in the quest of anticancer chemotherapeutics [78

were shown to exert their anticancer activity by diﬀerent modes of action compared to established drugs,

including enzyme inhibition or crosslinking of DNA or proteins [78 80 81]. Numerous polynuclear

,80–82]. Some compounds

,

half-sandwich metal-arene (ruthenium, osmium, rhodium, iridium) complexes have been investigated

for their anticancer, antibacterial or antiparasitic properties [83–97].

In some reports, the biological activity of polynuclear compounds was compared to that of the

corresponding mononuclear analogues [88,92,95,98]. However, no general trend can be determined

regarding a direct correlation between the number of the organometallic units and the measured

biological properties. The results remained strongly speciﬁc to the various types of compounds and

each case should be considered individually. Apart the number of the metal centres, other parameters

play an important role with regard to the stability and eﬃciency of the polynuclear complexes: (i) the

nature of the metal, (ii) the type of ligands and the coordination mode (mono- or bidentate), (iii) the

length, stereochemistry and steric hindrance of the linking units, (iv) the hydrophobicity and other

physico-chemical properties such as the total size and charge.

Numerous homo-polynuclear Ru(II)-arene complexes with various type of connections disposed

at the level of the arene or on leg ligands were previously evaluated. For example, the concept of

polynuclearity was applied to RAPTA complexes by connecting two ruthenium(II) metalorganic units

at the level of the arene ligand (Figure 2) [92

as well as 1,2-diphenylethylenediamines with controlled conﬁguration, were chosen as linkers [92

,

93]. Flexible alkyl or polyethylene glycol (PEG) diamines,

93].

,

In this case, dinuclear compounds presented increased cytotoxicity compared to the corresponding

mononuclear derivatives when assessed in A2780, A2780cisR and HEK293 (human embryonic kidney)

cells, but no important selectivity towards cancer cells could be observed. The conformation had a

strong impact on the cytotoxicity and the compounds were shown to crosslink the two dominant

RAPTA-C histone sites on the acidic patch of the NCP (Nucleosome Core Particle) [93]. This type

of dinuclear compounds induced aberrant chromatin condensation, due to intra-nucleosomal and

inter-nucleosomal cross-linking [93].

Another example is that of a series of structurally related mono- (

) compounds containing {( O)-2-methyl-4-pyridinonato-

⁶-p-MeC₆H₄Prⁱ)RuCl[3-oxo-

(Figure 2) evaluated for cytotoxicity against various cancer cells [96 98]. The units in the dinuclear

compounds were linked via ﬂexible alkyl chains of varying lengths and a correlation between

the linker size, lipophilicity, and cytotoxicity was observed. Whilst the mononuclear was inactive

against A2780 cells at concentrations up to 50 M [99], the dinuclear compound bearing the longest

chain (n = 12) exhibited an IC₅₀value of <2 M [96]. In addition, the dinuclear compound bearing the

G

), di- (

H

) and trinuclear

(I

η

κ

O4]} units

–

H

G

µ

longest spacer (n = 12) displayed a diﬀerent mode of binding to the rest of the series and could form a

high degree of DNA–protein and DNA duplex crosslinking. The extent of DNA–protein crosslinks

was shown to be dependent on the spacer length [97,100]. The number of metal centres was found

to be important, with the dinuclear compound being more active than the analogous mono- (G) and

trinuclear (I) compounds.

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Figure 2. Structure of dinuclear complexes with RAPTA units connected at the level of the arene

ligands ( ) and corresponding mononuclear derivative ( ); structure of mono-, di- and trinuclear

ruthenium(II)-arene complexes with {( O)-2-methyl-4-pyridinonato- O4]}

⁶-p-MeC₆H₄Prⁱ)RuCl[3-oxo-

units ( and ); structure of di- and mononuclear ruthenium(II)-arene complexes with phosphine

ligands (J and K).

E

F

η

κ

G

,

H

I

A lipophilicity-cytotoxicity correlation was also observed in a series of dinuclear ruthenium(II)-

p-cymene (cymene = p-MeC₆H₄Prⁱ) complexes connected at the level of the phosphine leg ligands with

ﬂexible PEG chains (J, Figure 2). For this library of compounds the cytotoxicity was mildly inﬂuenced

by the length of the linker, the conjugates with ﬁve and six ethylene glycol units being almost 2-fold

more cytotoxic compared to the other compounds on the tested cell lines [95]. The length of the

linker seemed to aﬀect to a lesser extent the selectivity of the compound for a speciﬁc cell line [95].

Nonetheless, a distinct increase in cytotoxicity was observed for the dinuclear compounds compared

with the mononuclear ruthenium control complexes K (Figure 2) [95].

An interesting example is that of the star-shape trinuclear ruthenium(II)-p-cymene complexes

with Schiﬀ-base ligands, tris-2-(salicylaldimine ethyl)amine and tris-2-(2-pyridylimine ethyl)amine,

for which the anticancer activities were compared to those of the corresponding mononuclear analogues

and (Figure 3) [88]. The measured IC₅₀values for compounds L–O showed that the increase in

the number of metal centres was correlated to a higher cytotoxicity against cancer cells.

L and

M

N

O

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Figure 3. Structure of star-shape trinuclear

Ru(II)-p-cymene complexes with Schiﬀ-base ligands; structure of di- (

L

and

M

, and of the mononuclear

and ) and trinuclear (

and T) ruthenium(II)-p-cymene complexes with polypyridyl ester and ether ligands.

N and O

P

R

S

The antiparasitic activity of some polynuclear organometallic compounds was also studied [83

For example, polynuclear pyridyl ester ( and ) and ether ( and ) complexes (Figure 3) were

screened for antimalarial activity against Plasmodium falciparum [83 84]. The trinuclear complexes

and were more active than the dinuclear derivatives and and showed superior activity compared

to the metal precursors, suggesting a cooperative eﬀect between the ligand and the metal. Compounds

were screened against the NF54 CQ (chloroquine) sensitive strain of P. falciparum and the most

active complex was also evaluated against the Dd2 CQ resistant strain [83]. Trinuclear ruthenium

,84].

P

R

S

T

,

R

T

P

S

P-T

T

complex T exhibited potent activity in both the NF54 and Dd2 strains.

Even though no general trend can be identiﬁed, several comparative studies of di- and trinuclear

vs. mononuclear organometallic complexes have shown a noteworthy increase of cytotoxicity/eﬃcacy

with the number of metal units [88,95–97]. In this study, we aim to extend the concept of polynuclearity,

and describe the synthesis and characterization of conjugates containing two or three covalently

inter-connected trithiolato dinuclear ruthenium moieties (see Supplementary Figure S1 for a schematic

representation of the compounds). Since complexes with one trithiolato-bridged ruthenium(II)-arene

unit were highly cytotoxic against cancer cells [58], and also showed promise as antiparasitic drug

candidates [60–62], the bioactivity potential of the new conjugates containing two or three di-ruthenium

units was also explored and a comparative evaluation of the eﬃcacies is presented.

2. Results and discussion

2.1. Chemistry

The compounds used in this study not only have a higher charge, but also an important

augmentation in size/molecular weight compared to each individual component. Various structural

modiﬁcations were investigated. In the case of the conjugates with two units, the type of the bonding

(ester vs. amide) as well as the nature and length of the linker were systematically varied. In the

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case of the conjugates containing three units, two topologies were considered: a tripodal star-shape

and a linear ‘beads-on-a-string’ arrangement. The nature of the central moiety was also customized

(see Supplementary Figure S1 for a schematic representation of the compounds).

Trithiolato-bridged dinuclear ruthenium(II)-arene compounds are stable, which allows the

synthesis of more sophisticated molecules by applying ‘chemistry on the complex’. First investigations

in this direction were reported in studies on conjugates with peptides [101], the anticancer drug

chlorambucil [102] and the ﬂuorophore coumarin [66] designed to improve the water solubility and to

promote the anticancer and antiparasitic activities. In general, the synthesis of both the symmetric and

mixed trithiolato complexes is straightforward and eﬃcient [57,66].

The mixed intermediate compounds

hydroxy or amino groups were synthesized following the reactions presented in Scheme 1 by adapting

previously described procedures [57 66]. A two-step reaction pathway was used, starting from

the [Ru(

⁶-p-MeC₆H₄Prⁱ)Cl]₂Cl₂dimer [103]. In the ﬁrst step, the reaction of two equivalents of

(4-(tert-butyl)phenyl)methanethiol and, respectively, 4-mercaptophenol with the ruthenium dimer in

EtOH (0 ^◦C), under inert atmosphere (N₂) aﬀorded the symmetric neutral dithiolato intermediates

and isolated with 91% yield each. The reactivity of the thiols strongly inﬂuences the recovery of the

dithioato intermediates, being easily controlled stoichiometrically for when a benzyl thiol was used,

but not for , based on a phenyl thiol, for which the respective monothiolato and trithiolato compounds

were also formed. The dithiolato intermediates and were further reacted with a third thiol in excess

3–5 containing one di-ruthenium unit and one or two

,

η

1

2

1

2

1

2

in reﬂuxing EtOH under inert atmosphere (N₂) to obtain the mixed complexes

yields (82%, 84%, and 96%, respectively).

3–5 isolated in good

Scheme 1. Synthesis of the mixed trithiolato-bridged ruthenium(II)-p-cymene intermediates 3–5.

The diester and diamide conjugates containing two di-ruthenium units were obtained by reacting

intermediates 3 and 4 with suitably difunctional compounds bearing two acyl chloride or carboxylic

acid groups using appropriate reaction conditions (Schemes 2–4). With few exceptions, the syntheses per

se did not impose particular issues and the conversions were generally good. Nevertheless, the isolation

of the pure compounds proved in some cases less straightforward (yields ranged from 7% to 50%).

These puriﬁcation issues were translated in some cases in poor yields of isolated pure dicationic and

tricationic conjugates (see full details in Supporting information). However, the quantities of the

isolated products were suﬃcient for the biological activity screening.

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Scheme 2. Synthesis of the dicationic conjugates 6–10 presenting diester alkyl linkers of various lengths.

Scheme 3. Synthesis of the dicationic conjugates 11–14 presenting alkyl diamide linkers of

various lengths.

Scheme 4. Synthesis of the dicationic conjugates 15–18 presenting meta and para substituted diester

and diamide linkers.

The diester conjugates

with a yield ranging from 7% to 50% by reacting two equivalents of the hydroxy di-ruthenium

intermediate with various commercially available bis-acyl chloride compounds in basic conditions

(TEA, triethylamine), at low temperature (0 ^◦C) under inert atmosphere (N₂) (Scheme 3).

The diester conjugates from the reaction of with oxalyl chloride (n = 0) and malonyl chloride

(n = 1) could not be isolated due to the degradation of the acyl chloride reagents in the used reaction

6–10, presenting alkyl linkers of diﬀerent lengths (n = 2–6) were obtained

3

conditions. For example, the reaction of

side product.

3 with malonyl chloride led to the isolation of the acetyl ester

Similar reaction conditions were used for the synthesis of the alkyl diamide conjugates 11

, 12,

and 14 isolated in 44%, 37%, and 34% yield, respectively. Two equivalents of amino intermediate

4

were reacted with the corresponding bis-acyl chloride compounds (n = 0, 4, and 6) in basic conditions

(TEA) at low temperature (0 ^◦C) under inert atmosphere. Diamide conjugate 13 (n = 5) was obtained

with 43% yield using heptanedioic acid, in the presence of HOBt (1-hydroxybenzotriazole) and EDCI

(N-(3-dimethylaminopropyl)-N⁰-ethylcarbodiimide hydrochloride) as coupling agents and DIPEA

(N,N-diisopropylethylamine) as base (Scheme 3, full experimental details are given in Supporting

information).

Diamide conjugates in which the two di-ruthenium units are connected through succinic,

malonic and glutaric linkers could not be obtained, neither using the respective bis-acyl chlorides or

the corresponding dicarboxylic acids. Degradation of the malonyl chloride reagent was observed in

the used reaction conditions. In the cases of the succinyl and glutaryl chlorides, the high reactivity of

the di-ruthenium amine

4 favored the formation of the corresponding 5 and 6 atom cyclic amide side

products with the 1-pyrrolidine-2,5-dione and 1-piperidine-2,6-dione rings.

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The diester 15

–

16 and diamide 17

–

18 conjugates with aromatic linkers were recovered with 32%,

82%, 50%, and 21% yield, respectively using similar conditions, by the reaction of two equivalents

of di-ruthenium hydroxy

3 or amino intermediate 4 with commercially available meta and para

substituted bis-acyl chloride compounds in basic conditions (TEA) at low temperature (0 ^◦C) under

inert atmosphere (Scheme 4).

For the obtainment of conjugate 20 containing three thiolato-bridged dinuclear ruthenium(II)-arene

units in a linear ‘beads-on-a-string’ distribution (Figure S1), two types of ruthenium intermediates

were necessary: (i) compound

and (ii) the dihydroxy compound

3

containing one free hydroxy group used for the two capping units,

(Scheme 1) playing the role of core for connecting two units of

5

3

via appropriately difunctionalized linkers. Two strategies were considered for the synthesis of this

type of conjugates with three di-ruthenium units in a linear arrangement. In one approach, the linkers

were anchored on the central dinuclear ruthenium unit in a ﬁrst step followed by the attachment of the

remaining two dinuclear units in a second step. Alternatively, the order of the reactions was reversed,

thus ﬁrst the linkers were reacted with the ‘cap’ ruthenium dinuclear units followed by the connection

on the central dinuclear ruthenium moiety. A two-step synthetic pathway was used (Schemes 5 and 6).

First, a glutaric acid linker was attached to the mono-hydroxy di-ruthenium intermediate

3 with

the formation of precursor 19 presenting a free carboxylic acid group (Scheme 5). Compound 19

was obtained in 96% yield, by reacting

3 with glutaric anhydride in basic conditions (TEA) at low

temperature (0 ^◦C) under inert atmosphere (N₂).

Scheme 5. Synthesis of precursor 19 presenting a free carboxylic acid group and of the tricationic

conjugate 20 presenting a linear distribution of the three di-ruthenium units.

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Scheme 6. Synthesis of the tripodal core linkers 21 and 22.

In the second step, two equivalents of precursor 19 were coupled in the presence of EDCI and

DMAP (4-dimethylaminopyridine) with dihydroxy di-ruthenium intermediate 5 to obtain conjugate

20 isolated with 26% yield (Scheme 5).

Two tricationic compounds presenting a star-like architecture were synthesized by attaching

three units of the mixed amino di-ruthenium intermediate on two symmetric tripodal core linkers

4

(Supplementary Figure S1, Schemes 6 and 7). To minimize steric hindrance for the attachment of the

bulky di-ruthenium complexes, central core linkers 21 and 22 with glutaric acid pendant arms were

used. These intermediates were obtained with quantitative yield starting from commercially available

tris(2-hydroxyethyl)amine, and, respectively, 1,3,5-benzenetrimethanol, following the reactions

presented in Scheme 6.

Scheme 7. Synthesis of the tricationic conjugates 23 and 24 presenting a star-like distribution of the

three di-ruthenium units.

The amide coupling of the core linkers 21 and 22 with mixed amino trithiolato-bridged dinuclear

ruthenium(II)-p-cymene compound

4 aﬀorded the star-like compounds 23 and 24 (Scheme 7 ).

The reactions were performed in the presence of coupling agents EDCI and HOBt, in basic conditions

(DIPEA), under inert atmosphere (N₂) at room temperature (Scheme 7).

All compounds were characterized by ¹H and ¹³C NMR spectroscopy, mass spectrometry and

elemental analysis (see Materials and Methods—Chemistry section in Supporting information for

full details). For diester alkyl linker conjugates

the resonances corresponding to the aromatic protons in

respectively with ∆δ_H0.12 ppm highﬁeld, and ∆δ_H0.41 ppm lowﬁeld. Likewise, the aromatic

6

–10, ester bond formation led to important shifts of

α

and position to the former OH group,

β

≈

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protons of the p-cymene rings were lowﬁeld shifted with about ∆δ_H≈ 0.05–0.26 ppm, and the resonance

of the aromatic carbon connected to the ester group (C-O-(C=O)) shifted from 160 ppm to 151 ppm.

Similarly, the formation of the amide bond in alkyl connected conjugates 11

with important shifts of speciﬁc resonances in the ¹H-NMR spectra, which facilitated the monitoring

of the reactions’ evolution. The resonance corresponding to the amide hydrogen atom (N -(C=O))

appeared at around 11 ppm. Amide bond formation led to important shifts of the resonances

corresponding to the aromatic protons in and position to the former NH₂group, respectively with

∆δ_H≈ 1.36 ppm lowﬁeld, and ∆δ_H≈ 0.23 ppm lowﬁeld. Compared to the case of the diester conjugates

10, the resonances corresponding to the aromatic protons of the p-cymene rings were less aﬀected

(lowﬁeld shift ∆δ_H= 0.01–0.02 ppm). In conjugates 11 14, the resonance of the aromatic carbon

connected to the amide group (C-NH-(C=O)) shifts from 149 ppm to 130 ppm.

In the diester (15 16) and diamide (17 18) conjugates with aromatic linkers, the resonance of the

aromatic protons in and position to the newly formed ester or amide group also shifts compared to

corresponding hydroxy and amine di-ruthenium intermediates and . Ester bond formation in 15

and 16 led to shifts of the resonances corresponding to the aromatic protons in and position to

the former OH group, with ∆δ_H0.02 ppm lowﬁeld and, respectively, with ∆δ_H0.51 ppm lowﬁeld.

Amide bond formation in 17 and 18 led to shifts of the resonances corresponding to the aromatic

protons in and position to the former NH₂group, respectively with ∆δ_H1.48 ppm lowﬁeld,

and ∆δ_H0.31 ppm lowﬁeld. Nevertheless, the nature of the linker (aliphatic vs. aromatic) had less

–14 was associated

H

α

β

6

-

–

,

α

β

3

4

α

β

≈

α

β

≈

inﬂuence on the NMR resonances compared to the type of bond (ester vs. amide). On the other hand,

the resonances of the aromatic carbon connected to the heteroatom of the ester and amide groups

(C-X-(C=O)) shifted highﬁeld with ∆δ_C≈ 9 ppm for the esters and ∆δ_C≈ 18 ppm for the amides.

For 20, the tricationic conjugate in which the di-ruthenium units are distributed in a linear

arrangement, two sets of resonances could be easily identiﬁed corresponding to the two types of

thiolato-bridged dinuclear ruthenium(II)-p-cymene moieties positioned as core or on the end of

the side-arms.

The formation of compound 23 was evidenced by important lowﬁeld shifts of the resonances

corresponding to the aromatic protons in

α and β position to the amide (NH-(C=O)) of ∆δ_H= 1.31 ppm

and ∆δ_H= 0.15 ppm, respectively. For the respective carbon atoms in the ¹³C-NMR spectrum of 23

a shift toward low ﬁeld of ∆δ_C= 4.7 ppm and highﬁeld of ∆δ_C= 0.8 ppm of the aromatic carbons

in

α and respectively β position to the amide (NH-(C=O)) was observed. In addition, an important

highﬁeld shift (∆δ_C= 19 ppm) of the aromatic quaternary carbon connected to the nitrogen was also

noticed. A similar eﬀect was also observed in the ¹H-NMR and ¹³C-NMR spectra of triamide 24.

Electrospray ionization mass spectrometry (ESI-MS) corroborated the spectroscopic data with

the intermediates

[M-Cl]⁺ions, conjugates

conjugates 20, 23, and 24 with three di-ruthenium units as [M-3Cl]³⁺ions.

Intermediate complexes and as well as newly obtained polynuclear conjugates 6–18 and

3

–

5

with one di-ruthenium unit exhibiting molecular ion peaks corresponding to

6–

18 with two di-ruthenium unit as [M-2Cl]²⁺ions, and linear and tripodal

3

4

20–22 were stable in DMSO-d₆at 0 ^◦C. (Supplementary Figures S4−S8). Of note, two new very small

resonances emerged at ~5.8 and ~8.4 ppm in some spectra recorded after 30 days (for instance for

15). Yet, although the exact origin of these resonances and the compound(s) they characterize remain

unknown, they do not denote a sign of decomposition or formation of a new complex, otherwise new

resonances would have appeared in the aromatic region and in the p-cymene region around 5.5 ppm

and between 0.5 and 3 ppm. Very importantly, recent studies by some of us have shown that dinuclear

arene ruthenium complexes are inert to ligand substitutions and remain stable for long period in water

solutions or in organic solvents [55–58].

2.2. In Vitro Activity against the Apicomplexan Parasite Toxoplasma gondii

The new conjugates presented in this study were investigated to assess the impact of compound

exposure upon T. gondii β-gal grown in HFF and non-infected HFF. Cultures exposed to concentrations

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of 1 and 0.1

ruthenium(II)-arene complexes

linkers 18, and the conjugates with three di-ruthenium units and diﬀerent architectures 20

µ

M of each compound of interest (e.g., hydroxy and amino trithiolato-bridged dinuclear

, the conjugates with two di-ruthenium units and various types of

23, and 24,

3–5

6

–

,

Supplementary Table S1 and Figure 4). The results obtained for two other compounds, acetyl ester

25 and amide 26 [104] (see structures in Supporting information) are also presented for comparison.

As a measure of parasite proliferation, β-galactosidase activity was determined, while the impact on

non-infected HFF was assessed using the AlamarBlue assay.

Figure 4. Clustered column chart showing the in vitro activities at 1 (A) and 0.1 (B) µM of the

21 compounds on HFF viability and T. gondii

only with 0.1% DMSO exhibited 100% viability and 100% proliferation was attributed to T. gondii

tachyzoites treated with 0.1% DMSO only. For each assay, standard deviations were calculated from

β-gal proliferation. Non-infected HFF monolayers treated

β-gal

triplicates. Data for compounds

part of another study [104].

3, 4 and 5 were previously reported [66] and data for 25 and 26 are

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Compound

proliferation more eﬀectively compared to compounds

group. Acetyl ester 25 and acetyl amide 26 were also more active against the parasite compared to

free OH and NH₂analogues and , indicating that the increase in hydrophobicity appears to favor

5, possessing two polar hydroxy groups, inhibited T. gondii

β-gal tachyzoite

3

and

4 possessing only one OH or NH₂

3

4

antiparasitic activity. However, 26 is also more cytotoxic against HFF host cells.

In the series of the compounds with two di-ruthenium units and diester alkyl connectors,

the derivatives with medium long chain

tachyzoites but also exhibited increased toxicity against HFF host cells. From this ﬁrst screening,

the most interesting analogue with an alkyl diester linker was compound (n = 5). Diester compounds

and were more eﬃcient against T. gondii -gal tachyzoites compared to the hydroxy di-ruthenium

compound 3 and exhibited a rather close activity/toxicity proﬁle to acetyl compound 25.

The diamide derivatives 11 14, with two di-ruthenium units and alkyl linkers, were less cytotoxic

in HFF host cells, but displayed also reduced activity with respect to T. gondii tachyzoite inhibition

compared to the diester compounds 10. Derivatives 11 14 were also less cytotoxic and less eﬃcacious

7 and 8 (n = 3 or 4) were active against T. gondii β-gal

9

6

9

β

–

6–

–

against T. gondii than the acetyl amide 26 or the amino di-ruthenium compound 4. In this small alkyl

diamide set of conjugates, increasing the length of the spacer between the two di-ruthenium units had

a favorable eﬀect on the antiparasitic activity.

Interestingly, a similar structural eﬀect (namely amide compounds less active against T. gondii/less

cytotoxic for HFF) when compared to ester analogues, could be observed in the case of the derivatives

15–18 presenting two di-ruthenium units and aryl connectors. Thus, diester compounds 15 and 16 were

signiﬁcantly more active against T. gondii tachyzoites compared to their respective diamide analogues

17 and 18. This might be the consequence of the more important conjugation of the amide bond

compared to the ester bond, which can lead to increased rigidity of the molecule, or to the diﬀerence of

chemical stability of the ester and amide bonds in the cellular environment.

Compound 20 with three di-ruthenium units in a linear distribution displayed an increased

activity against T. gondii

more cytotoxic in HFFs. Similar to the di-hydroxy analogue

showed increased activity against the parasite than the mono-hydroxy di-ruthenium compound

β

-gal compared to the two star-shape compounds 23 and 24 but was also

5

and the acetyl ester 25, compound 20

3.

It is important to note that in compound 20 the three di-ruthenium units were connected exclusively

through ester bonds, while in compounds 23 and 24 the three units were connected to the central core

through amide bonds. The increased molecular volume and rigidity might be responsible for the poor

antiparasitic properties of compounds 23 and 24.

Based on this preliminary screening, twelve compounds exhibiting favorable antiparasitic activity

and low or intermediate impairment of HFF viability were selected for the determination of the IC₅₀

values in T. gondii

IC₅₀value of a compound, two criteria had to be simultaneously satisﬁed: (i) T. gondii

was inhibited by 90% or more compared to an untreated control when the compound was applied at

M, and (ii) HFF host cell viability was not impaired by more than 50% for a compound applied

at 1 M. Pyrimethamine, currently used for the treatment of toxoplasmosis, and which inhibited the

proliferation of T. gondii -gal tachyzoites with an IC₅₀value of 0.326 M and did not aﬀect HFF

viability at 2.5 M (Table 1), was used as reference compound. The results are summarized in Table 1,

and dose response curves are shown in Supplementary Figure S3. The selection included the three

intermediate compounds , and containing one di-ruthenium unit and free OH or NH₂groups,

along with three alkyl diester compounds 8, and , the two aryl diester compounds 15 and 16, as well

as compound 20 presenting three di-ruthenium units in a linear arrangement.

β-gal and assessment of HFF viability after exposure to 2.5

µM. To determine the

β-gal growth

1

µ

β

µ

3,

4

5

6

,

9

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Table 1. Half maximal inhibitory concentration (IC₅₀) values (µM) on T. gondii β-gal for twelve selected

compounds and pyrimethamine (used as standard), and their eﬀect at 2.5 µM on HFF viability.

T. gondii β-gal

IC₅₀(µM)

HFF

Viability at 2.5 µM (%) ^e

99

Compound

[LS; LI] ^c

SE ^d

SD ^f

Pyrimethamine ^a

0.326

[0.288; 0.396]

0.051

6

Compounds with one trithiolato-bridged ruthenium(II)-p-cymene unit

3 ^a

4 ^a

0.117

0.153

0.115

0.065

[0.098; 0.139]

[0.127; 0.185]

[0.098; 0.135]

[0.042; 0.101]

0.051

0.049

0.045

0.092

56

51

2

6

5

4

5

5 ^a

25 ^b

16

Compounds with two trithiolato-bridged ruthenium(II)-p-cymene units and alkyl diester linkers

6

9

10

0.060

0.159

0.123

[0.045; 0.080]

[0.139; 0.183]

[0.088; 0.172]

0.076

0.064

0.084

0

50

27

0

1

Compounds with two trithiolato-bridged ruthenium(II)-p-cymene units and alkyl diamide linkers

12

13

0.397

0.531

[0.315; 0.501]

[0.491; 0.575]

0.072

0.019

65

74

1

Compounds with two trithiolato-bridged ruthenium(II)-p-cymene units and meta and para substituted diester linkers

15

16

0.373

0.185

[0.342; 0.407]

[0.167; 0.204]

0.034

0.032

49

53

1

Compounds with three trithiolato-bridged ruthenium(II)-p-cymene units in a star-shape arrangement

24

0.033

and

[0.014; 0.076]

0.178

55

1

a

c

b

Data for pyrimethamine,

3,

4

5

were previously reported [66]. Data for 25 are part of another study [104].

Values at 95% conﬁdence interval (CI); LS is the upper limit of CI and LI is the lower limit of CI. ^dThe standard

error of the regression (SE), represents the average distance that the observed values fall from the regression line.

e

f

Control HFF cells treated only with 0.25% DMSO exhibited 100% viability. The standard deviation of the mean

(six replicate experiments).

Interestingly, the more rigid and sterically hindered aryl diester analogues 15 and 16 were less

active in inhibiting T. gondii tachyzoite proliferation compared to the alkyl diester compounds

6, 9 and

10, and they were also less cytotoxic for HFF at 2.5 M. Compound 24 with three di-ruthenium units

µ

exhibited the highest antiparasitic activity, but was also highly cytotoxic in HFF. Mono- and di-hydroxy

compounds 3 and 5 had slightly lower IC₅₀values compared to the amino compound 4.

The ﬂexibility of the compounds and the molecular volume seemed to have an important impact

on the measured IC₅₀values against the apicomplexan parasite T. gondii, while the global charge

appeared to inﬂuence the biological activity to a lesser extent.

2.3. In Vitro Anticancer Activity

The cytotoxicity of the di-ruthenium hydroxy and amino intermediates

two trithiolato-bridged dinuclear ruthenium(II)-p-cymene complexes linked with alkyl diester (

and ), alkyl diamide (11 13), or aryl linkers (15 17 18), and of the conjugates with three di-ruthenium

3

–5

, of the conjugates with

6,

7,

9

–

,

units 20 and 24 was assessed in the human embryonic kidney cell line HEK293 used as a model for

non-cancer cells, in the human ovarian carcinoma cell lines A2780 and A2780cisR, with the latter

exhibiting acquired resistance to cisplatin, and in two cell lines A24 and (D-)A24cisPt8.0 that originate

from human lung adenocarcinoma (Supplementary Table S2, Figures 5 and 6). The A24 cells and its

subline (D-)A24cisPt8.0 were described previously [105]. The (D-)A24cisPt8.0 subline is approximately

30-fold more resistant to cisplatin (cisPt) compared to the A24 cells [105]. The cytotoxicity values of

cisplatin (cisPt), used as control, are also presented.

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Figure 5. Comparison of the IC₅₀values (µM, 72 h) determined for cisplatin (cisPt) and selected

trithiolato-bridged dinuclear ruthenium(II)-p-cymene compounds toward human ovarian A2780 and

A2780cisR (cisplatin sensitive and resistant) cancer cells and human embryonic kidney HEK293 cells.

Figure 6. Comparison of the IC₅₀values (µM, 72 h) determined for cisplatin (cisPt) and selected

trithiolato-bridged dinuclear ruthenium(II)-p-cymene compounds toward human lung adenocarcinoma

wild type A24 and de-induced (D-)A24cisPt8.0 subline cells and human embryonic kidney HEK293 cells.

Apart from compounds 17 and 18 assessed in A2780 human ovarian cells, all ruthenium compounds

were more cytotoxic than cisplatin on all cell lines tested in this study (Figure 5 and Table S2). In terms of

selectivity, compounds

non-cancer HEK293 cells. Nevertheless, hydroxy and amine compounds with only one di-ruthenium

unit ( ) and ester bonded conjugates ( 15, and 20) present similar cytotoxicity against A2780 and

3–5 and 11 assessed in cancer cells showed only very reduced selectivity to the

3

–5

6, 7, 9,

A2780cisR human ovarian cells, and A24 and (D-)A24cisPt8.0 human lung adenocarcinoma cells,

irrespectively of the cisplatin resistance proﬁle of the cell lines. This is indicative for a possible diﬀerent

mechanism of action compared to cisplatin, and the potential to circumvent cisplatin cross-resistance.

Compounds 3 and 4, containing only one polar OH and NH₂group, respectively, were less cytotoxic

on all tested cell lines compared to compound 5 presenting two hydroxy groups.

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Irrespective of the nature of the connectors (alkyl or aryl) or of the number of di-ruthenium

units (two or three), conjugates with ester bonds presented rather similar antiproliferative proﬁles,

namely they were more cytotoxic compared to conjugates in which the di-ruthenium units were

anchored via amide bonds (11–13, 17, 18, and 22).

Diester compounds

were among the most cytotoxic derivatives of the series in all tested cell lines. No or very little selectivity

against cancer cells compared to HEK293 cells could be observed for , and . These compounds

6, 7, and 9, containing two di-ruthenium units and alkyl connectors,

6,

7

9

appeared to be slightly more cytotoxic on the human lung adenocarcinoma A24 and (D-)A24cisPt8.0 cell

lines, compared to the human ovarian cancer cells A2780 and A2780cisR. Almost no inﬂuence of the

linker length was observed for these conjugates with alkyl diester connectors.

Compounds 11, 12, and 13 were less active in the cisplatin resistant cell lines A2780cisR and

(D-)A24cisPt8.0 compared to A2780 and A24 cell lines. If these compounds with alkyl diamide linkers

were more cytotoxic then cisplatin, they presented lower antiproliferative activities compared to

conjugates with alkyl diester connectors 6, 7, and 9. The length increase of the diamide connector was

associated with a decrease in cytotoxicity against all tested cancer cells, and this eﬀect was stronger in

cisplatin resistant cancer cells A2780 and (D-)A24cisPt8.0.

A diﬀerence was perceived between the ester 15 and amide compounds 17 and 18 with two

di-ruthenium units and aromatic connectors, the ﬁrst one being signiﬁcantly more cytotoxic on all

tested cell lines. Thus, diester compound 15 remained among the most cytotoxic of the series in all cell

lines (IC₅₀values between 0.036 to 0.094 µM). Diamide compounds 17 and 18 were equally cytotoxic in

HEK293 and A2780 cells but were less cytotoxic in the cisplatin resistant A2780cisR cells. Nevertheless,

the cytotoxicity proﬁle of human lung adenocarcinoma A24 and (D-)A24cisPt8.0 cancer cells was

diﬀerent from human ovarian cancer cells A2780 and A2780cisR. No important diﬀerence was observed

between conjugates

important in the case of alkyl 11

6

,

7

, and

9

with alkyl and 15 with aryl diester linkers. The variations were more

13 and aryl 17 18 diamide connected conjugates, especially regarding

–

,

the antiproliferative activity in the human ovarian cancer cells A2780 and A2780cisR.

Interestingly, noticeable diﬀerences in activity could be observed between compounds containing

three di-ruthenium units. While compound 20 (with a linear distribution of the three complexes)

remains among the most cytotoxic of the series (IC₅₀values ranging between 0.033 and 0.074

µM),

the tripodal compound 24 was less cytotoxic (IC₅₀values of 0.167 to 0.277 M). Whereas in conjugate

µ

20 the three di-ruthenium units are connected via ester bonds, they are anchored to the central core

through amide bonds in compound 24. No important diﬀerences in cytotoxicity levels were found in

all cancer cells and in HEK293 cells.

Cytotoxicity is a multifactorial process, and resistance to platinum compounds in the A2870cisR

cell line is attributed amongst others to decreased uptake, increased glutathione and glutathione

S-transferase levels, and increased DNA repair (adduct removal). Uptake may be correlated with

Ctr1 transporter expression, which is expressed at lower levels in cisplatin resistant A2870cisR cells

compared to sensitive cell lines [106–108].

3. Materials and Methods

3.1. Chemistry

The materials and methods corresponding to the chemistry part are presented with full details in

the ‘Materials and methods—Chemistry’ section in Supporting information.

3.2. In Vitro Activity Assessment against T. gondii Tachyzoites and HFF

If not otherwise stated, all tissue culture media were purchased from Gibco-BRL, and biochemical

agents purchased from Sigma-Aldrich. HFF (SCRC-1041.1) were purchased from ATCC, and were

maintained in DMEM medium containing 10% fetal calf serum (FCS, Gibco-BRL, Waltham, MA, USA),

and antibiotics as described [109]. Transgenic T. gondii β-gal (expressing the β-galactosidase gene from

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Escherichia coli) [110] were kindly provided by Prof. David Sibley (Washington University, St. Louis,

MO, USA) and were maintained in HFF as host cells, and were isolated as described before [109].

Compounds were prepared as 1 mM stock solutions in DMSO (dimethyl sulfoxide, Sigma,

St. Louis, MO, USA). For activity assays, HFF monolayers were cultured in 96 well plates by seeding

5

×

10³HFF per well and allowing them to grow to conﬂuence in phenol-red free culture medium

◦

at 37 C/5% CO₂. For infection, T. gondii

β-gal tachyzoites were separated from their host cells as

described [109], parasites were isolated and HFF monolayers were infected with freshly isolated

tachyzoites (1 × 10³per well), with compounds solutions added concomitantly during infection.

For the primary screening, T. gondii β-gal infected HFF were exposed to 0.1 and 1µM of each

compound for a period of 72 h, or the corresponding concentration of DMSO (0.01% and 0.1%,

respectively) as control. For the determination of IC₅₀values against T. gondii -gal, compounds were

M. After 72 h of culture

L PBS

L 5 mM chlorophenol

β

added at 8 concentrations: 0.007, 0.01, 0.03, 0.06, 0.12, 0.25, 0.5, and 1

at 37 C/5% CO₂, the medium was aspirated, and cells were permeabilized by adding 90

µ

◦

µ

(phosphate buﬀered saline) containing 0.05% Triton X-100. After addition of 10

µ

red-β-D-galactopyranoside (CPRG; Roche Diagnostics, Rotkreuz, Switzerland) dissolved in PBS,

the absorption shift was measured at 570 nm wavelength at various time points using an EnSpire^®

multimode plate reader (PerkinElmer, Inc, Waltham, MA, USA). For the primary screening at 0.1 and

1

µM, the activity, measured as the release of chlorophenol red over time, was calculated as percentage

from the respective DMSO control, which represented 100% of T. gondii -gal growth. For the

β

IC₅₀assays, the activity measured as the release of chlorophenol red over time was proportional to

the number of live parasites down to 50 per well as determined in pilot assays. IC₅₀values were

calculated after the logit-log-transformation of relative growth and subsequent regression analysis.

All calculations were performed using the corresponding software tool contained in the Excel software

package (Microsoft, Redmond, WA, USA).

Cytotoxicity assays using uninfected conﬂuent HFF host cells were performed as previously

described [111] by AlamarBlue assay. In brief, conﬂuent HFF monolayers in 96 well-plates were

exposed to 0.1, 1 and 2.5

0.1% and 0.25%) were included. After 72 h of incubation at 37 ^◦C/5% CO₂, the medium was removed,

and plates were washed once with PBS. Resazurin stock solution was diluted 1:200 in PBS and 200

µM of each compound. Non-treated HFF as well as DMSO controls (0.01%,

µL

were added to each well. Plates were read at excitation wavelength 530 nm and emission wavelength

590 nM at the EnSpire^®multimode plate reader (PerkinElmer, Inc). Fluorescence was measured at

diﬀerent timepoints. Relative ﬂuorescence units were calculated from timepoints with linear increase.

3.3. In Vitro Anticancer Activity

Human ovarian carcinoma cells A2780 and the cisPt resistant variant A2780cisR were purchased

from the European Center of Cell Cultures (ECACC, Salisbury, UK). The HEK-293 cell line was kindly

provided by Prof. Mühlemann, University of Bern. The A24 cells and its subline (D-)A24cisPt8.0 were

described previously [105]. The cancer cells A24 and (D-)A24cisPt8.0 [105] (human lung adenocarcinoma

cells wild type A24 and cisPt resistant (D-)A24cisPt8.0 subline), A2780 and A2780cisR (human ovarian

cisplatin sensitive and resistant cancer cells), together with HEK293 (human embryonic kidney)

cells as model for normal cells, were included in the study for the evaluation of the ruthenium

compounds’ selectivity and cross-resistance with cisplatin. The A24 sublines, A2780 and A2780cisR

were cultured in RPMI 1640 medium without riboﬂavin, phenol red and antibiotics, buﬀered with

4.5 mM HEPES (BioConcept, Allschwil, Switzerland), supplemented with 10% (v/v) fetal bovine serum

(FBS) (Thermo Scientiﬁc, Weltham, MA, USA) as the only source of ﬂavins, and with 13.5 mM NaHCO₃.

In order to retain cisplatin resistance in the A2780cisR cell line, 1 µM cisplatin was added to the medium

every 2–3 passages. HEK293 cells were cultured in DMEM/F12 medium, supplemented with 10%

(v/v) FBS. At subcultivation, cell monolayers were rinsed with PBS and exposed for 3 min to StemPro

Accutase Cell Dissociation Reagent (Thermo Scientiﬁc, Weltham, MA, USA) at 36.5 ^◦C. Detached cells

were resuspended in culture medium. Cell densities were determined using Moxi ﬂow cytometer

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according to manufacturer instructions (Orﬂo Technologies, Ketchum, ID, USA). Experiments and

subcultivations were performed using light with wavelengths above 520 nm to prevent photochemical

artifacts. Cytostatic drug response was quantiﬁed as previously reported [105]. Brieﬂy, HEK293, A24,

A2780, A2780cisR, and (D-)A24cisPt8.0 single cell suspensions were freshly prepared from conﬂuent

cell layers using Accutase and diluted in culture medium to a cell density of 2

×

10⁴or 8

×

10⁴cells/mL

(D-)A24cisPt8.0. 150 l of this suspension was combined with 150 l of twice the cisPt or Ru-compound

µ

concentration in a 96-well microtiter plate (TPP, Trasadingen, Switzerland). Test plates were incubated

under standard conditions for 72 h. Cell densities were determined in a Casy I cell analyzer using

Casystat software (OLS OMNI Life Science, Bremen, Germany). Drug concentrations leading to 50%

growth inhibition compared to controls were calculated using GraphPad Software (La Jolla, CA, USA).

Each experiment was done in triplicates.

4. Conclusions

The polynuclear organometallic approach was pioneered by the inorganic trinuclear platinum

compound BBR 3464. The aim of this study was to extend and apply this concept to new conjugates

containing two or three trithiolato-bridged ruthenium(II)-arene units and to assess the biological

activity of the new compounds. We synthesized a library of 16 new compounds containing two and

three di-ruthenium moieties as well as the corresponding 3 trithiolato di-ruthenium(II)-p-cymene

intermediates and evaluated the activities of these compounds against T. gondii tachyzoites and cancer

cells in vitro.

Remarkably, the in vitro activity assessment against T. gondii tachyzoites grown in HFF led to seven

compounds (three with one, three with two, and one with three di-ruthenium units), which displayed

lower IC₅₀values than the standard drug pyrimethamine. However, the IC₅₀values show that the

compounds with two or three trithiolato di-ruthenium(II)arene units do not appear more promising

than the intermediates

hindered aryl diester analogues 15 and 16 with two trithiolato di-ruthenium units are less active against

T. gondii -gal and less toxic for HFF host cells compared to the alkyl diester derivatives and 8–9.

3–5 with only one di-nuclear unit. Of note, the more rigid and sterically

β

6

This insight could be used for further reﬁnement of the structures to improve the activity against

T. gondii. From these results we conclude that the molecular ﬂexibility and volume of the compounds

have an important impact on the measured IC₅₀values against T. gondii, while the global positive

charge of the compounds inﬂuences the biological activity to a lesser extent.

We also determined the cytotoxicity of 14 selected compounds against the cell lines HEK293,

A2780, A24, A2780cisR, and (D-)A24cisPt8.0, which resulted in IC₅₀values ranging from 23 to 650 nM.

Remarkably, all tested compounds were considerably more cytotoxic than cisplatin in A2780cisR,

A24 and de-induced (D-)A24cisPt8.0 subline cancer cells. Compounds 3–9, 11, 15, and 20 presented

IC₅₀values more than two orders of magnitude lower than those measured for cisplatin against

(D-)A24cisPt8.0 cells. Interestingly, irrespective of the nature of the connectors (alkyl or aryl) or of

the number or di-ruthenium units (two or three), the conjugates with ester bonds exhibited similar

antiproliferative proﬁle, and were more cytotoxic than the conjugates in which the di-ruthenium

units were linked via amide bonds. Compounds 3–5 with one and 11 with two di-ruthenium units

showed a moderate selectivity for the tested cancer cells, and they could be exploited to design

new derivatives for future studies. The compounds with two and three di-ruthenium units do not

seem more advantageous compared to the intermediate complexes with only one di-nuclear unit

Nevertheless, the results suggest the potential to circumvent cisplatin cross-resistance.

3–5.

Overall, this study shows that the concept of extended multinuclearity applied to di-nuclear

trithiolato-bridged ruthenium (II)-p-cymene compounds is worth additional assessment.

Pharmaceuticals 2020, 13, 471

19 of 25

Supplementary Materials: The following are available online at http://ww w .mdpi.com /1424-8247 /13 /12 /471 /s1,

Figure S1: General structure of the dicationic and tricationic conjugates., Figure S2: Structure of acetyl ester 25 and

amide 26 derivatives, Figure S3: Dose response curves for compounds

of T. gondii tachyzoites proliferation, Figure S4: 1H-NMR Spectra of

25 ^◦C, Figure S5: 1H-NMR spectra of 10 recorded in DMSO-d6 at 25 ^◦C, Figure S6: 1H-NMR spectra of 11

recorded in DMSO-d6 at 25 ^◦C, Figure S7: 1H-NMR spectra of 15 18 recorded in DMSO-d6 at 25 ^◦C, Figure S8:

1H-NMR spectra of 23 and 24 recorded in DMSO-d6 at 25 ^◦C, Table S1: Primary eﬃcacy/cytotoxicity screening of

compounds in non-infected HFF cultures and T. gondii -gal tachyzoites cultured in HFF, Table S2: IC50 values

M) determined after 72 h exposure of HEK293, A2780, A2780cisR, A24 and (D-)A24cisPt8.0 cells to cisplatin

6,

9

,

10

,

12

25 and 26 recorded in DMSO-d6 at

14

, 13, 15, 16, 24 and 25 as inhibitors

3–5,

6

–

β

(

µ

(cisPt) and selected trithiolato-bridged dinuclear ruthenium(II)-p-cymene compounds. Reference [112] are cited in

the supplementary materials.

Author Contributions: Conceptualization, E.P., J.F., A.H., N.R., and C.K.; methodology, E.P., J.F., A.H., G.B., N.A.,

N.R., and C.K.; software, V.S., T.F., O.D., E.P., G.B., N.A., Y.A., N.R., and M.H. validation, E.P., J.F., A.H., N.R.,

and C.K.; formal analysis, V.S., T.F., E.P., G.B., N.A., N.R., M.H., and C.K.; investigation, V.S., T.F., E.P., J.R., G.B.,

N.A., Y.A., N.R., M.H., and C.K.; resources, J.F., A.H, and J.T.H.; data curation, E.P., O.D., J.F., A.H., G.B., N.A.,

N.R., and C.K., writing—original draft preparation, E.P., J.F., G.B., N.A., N.R., and C.K.; writing—review and

editing, E.P., J.F., O.D., G.B., A.H., N.R., and C.K.; visualization, E.P., J.F., G.B., A.H., N.R., and C.K.; supervision,

E.P., O.D., J.F., A.H., N.R., C.K., and J.T.H.; project administration, J.F., A.H., and J.T.H.; funding acquisition, J.F.,

A.H., and J.T.H. All authors have read and agreed to the published version of the manuscript.

Funding: SNF Grant: Sinergia Project CRSII5_173718 (E.P., O.D., J.F., A.H., G.B., N.A.).

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Conjugates containing two and three trithiolato-bridged dinuclear ruthenium(Ii)-arene units as in vitro antiparasitic and anticancer agents

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