The quest of the best – A SAR study of trithiolato-bridged dinuclear Ruthenium(II)-Arene compounds presenting antiparasitic properties

Laboratoire de Parasitologie, Universite de la Manouba, Institution de la Recherche et de l’Enseignement Superieur Agricoles, Ecole Nationale de Medecine

c

ꢁ

ꢁ ꢁ

Veterinaire de Sidi Thabet, Sidi Thabet, 2020, Tunisia

a r t i c l e i n f o

a b s t r a c t

Article history:

A structure activity relationship (SAR) study of a library of 56 compounds (54 ruthenium and 2 osmium

derivatives) based on the trithiolato-bridged dinuclear ruthenium(II)-arene scaffold (general formula

Received 8 April 2021

Received in revised form

18 May 2021

Accepted 1 June 2021

Available online 8 June 2021

[(h h

⁶-arene)₂Ru₂(m₂-SR)₃]^þ, symmetric and [( ⁶-arene)₂Ru₂(m₂-SR¹)₂(m₂-SR²)]^þ, mixed, respectively) is

reported. The 56 compounds (of which 34 are newly designed drug candidates) were synthesized by

introducing chemical modiﬁcations at the level of bridge thiols, and they were grouped into eight

families according to their structural features. The selected ﬁttings were guided by previous results and

focused on a ﬁne-tuning of the physico-chemical and steric properties. Newly synthesized complexes

were characterized by NMR spectroscopy, mass spectrometry and elemental analysis, and four single-

crystal X-ray structures were obtained.

Keywords:

Trithiolato-bridged dinuclear ruthenium(II)-

Arene compounds

Toxoplasma gondii

The in vitro biological assessment of the compounds was realized by applying a three-step screening

cascade: (i) evaluation of the activity against Toxoplasma gondii RH strain tachyzoites expressing

b-

Structure activity relationship study

Antiparasitic activity

Selectivity

Human foreskin ﬁbroblast viability

Inhibition of immune cell proliferation

galactosidase (T. gondii- -gal) grown in human foreskin ﬁbroblast monolayers (HFF) and assessment of

b

toxicity in non-infected HFF host cells; (ii) dose-response assays using selected compound, and (iii)

studies on the effects in murine splenocytes.

A primary screening was performed at 1 and 0.1

that inhibited parasite proliferation at 1 M by more than 95% and reduced the viability of HFF by less

than 49%. In the secondary screening, dose-response assays showed that the selected compounds

exhibited half maximal inhibitory concentration (IC₅₀) values for T. gondii- -gal between 0.01 M and

0.45 M, with 30 compounds displaying an IC₅₀lower than 0.1 M. When applied to non-infected HFF

monolayers at 2.5 M, 8 compounds caused more than 90% and 31 compounds more than 30% viability

impairment. The tertiary screening included 14 compounds that did not cause HFF viability loss higher

than 50% at 2.5 M. These derivatives were assessed for potential immunosuppressive activities. First,

splenocyte viability was assessed after treatment of cells with concanavalin A (ConA) and lipopolysac-

charide (LPS) with compounds applied at 0.1 and 0.5 M. Subsequently, the 5 compounds exhibiting the

lowest splenocyte toxicity were further evaluated for their potential to inhibit B and T cell proliferation.

Overall, compound 55 [(

⁶-p-MeC₆H₄Prⁱ)₂Ru₂(m₂-SC₆H₄-o-CF₃)₂(m₂-SC₆H₄-p-OH)]Cl exhibited the most

mM, and resulted in the selection of 39 compounds

m

b

m

h

favorable features, and will be investigated as a scaffold for further optimization in terms of anti-parasitic

efﬁcacy and drug-like properties.

1. Introduction

* Corresponding author.

** Corresponding author.

*** Corresponding author.

Despite the fact that parasites inﬂict widespread and important

diseases in animals as well as in humans, drug development against

parasitic diseases remains, with few exceptions, a largely neglected

area. Many currently employed antiparasitic therapies exhibit

ꢀ

E-mail addresses: paunescu_emilia@yahoo.com (E. Paunescu), andrew.

hemphill@vetsuisse.unibe.ch (A. Hemphill), julien.furrer@dcb.unibe.ch (J. Furrer).

1

Equal contributions.

https://doi.org/10.1016/j.ejmech.2021.113610

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

important setbacks, such as adverse side effects and low efﬁcacy,

and the occurrence of resistance is being recognized as a serious

constraint [1]. Consequently, the development of novel compounds

is highly needed.

linked ferrocenyl moieties to bioactive ligands based on thiazoli-

dinone units or atovaquone, and are efﬁcient against Toxoplasma

[54,55]. SAR (structure activity relationship) studies demonstrated

that small modiﬁcations of the bioactive ligand were associated

with important changes in antiparasitic activity and general cyto-

toxicity. For instance, ferrocenyl aminohydroxynaphtoquinone de-

rivatives (Fig. 1, F) presented a slightly lower activity compared to

atovaquone against the atovaquone sensitive T. gondii PLK strain,

but an improved activity against the atovaquone resistant ATO

strain. Only compounds presenting a certain length of the hydro-

phobic chain, and thus an associated lipophilicity, exhibited inter-

esting antiparasitic activity, without exerting cytotoxicity in HFF

(human foreskin ﬁbroblast).

Toxoplasma gondii (T. gondii), a member of the phylum Api-

complexa, is an obligate intracellular protozoan that infects most

mammalian species [2,3] and birds [4]. Toxoplasma has a high

zoonotic potential, and it is estimated that up to 30% of the human

population is infected [5,6]. However, serious disease affects only a

fraction of infected persons. For example, primary infection during

pregnancy can cause congenital toxoplasmosis [7,8], and foetal

infection may lead to severe complications, including spontaneous

miscarriage and malformations such as hydrocephalus, develop-

mental retardation, and ocular toxoplasmosis causing eyesight

impairment and even blindness in later life [9,10]. Likewise, reac-

tivated toxoplasmosis, or acquisition of a primary infection, in

immunocompromised patients will lead to acute toxoplasmosis,

multiple organ failure and possibly death [1 1,12]. Most importantly,

T. gondii is a major economic burden for the food industry, in that it

affects food safety and food security by infecting most farm animal

species used in food production [13].

The most frequently applied treatments for toxoplasmosis rely

mainly on the use of a combination of pyrimethamine and sulfa-

diazine, supplemented with folinic acid to alleviate folic acid deﬁ-

ciency in the host [14]. Additional treatment options include

trimethoprim/sulfamethoxazole, clindamycin, spiramycin and

atovaquone [14]. Nevertheless, these drugs were initially developed

for other diseases [15e18], and they are either of moderate efﬁcacy

and/or are prone to adverse side effects. Additionally, the current

treatments target only tachyzoites, the rapidly replicating stage,

while the latent bradyzoite cyst stage remains unaffected [2]. The

increasing number of reports on treatment failures suggests that

drug resistant strains are emerging [19]. Concluding, there is an

urgent need for new, safe, low-cost and efﬁcacious treatments

against T. gondii.

Organometallic compounds could potentially provide inter-

esting alternatives to current treatment options [20e26]. Following

the pathway opened by cisplatin, important attention was given to

the development of various metalorganic derivatives as prospective

anticancer agents [27e35]. Among other ruthenium-based com-

plexes, trithiolato-bridged dinuclear ruthenium(II)-arene com-

pounds have shown high cytotoxicity against various cancer cell

lines, as well as promising in vivo activity when assessed in tumor

mouse models [36]. Besides anticancer activity, numerous metal-

based compounds also display considerable antiparasitic proper-

ties [23,24,37e40].

Recent studies have shown promising anti-Toxoplasma activities

of various metal-based compounds [41e52]. For example auranoﬁn

(A, Fig. 1), a gold(I)-based complex approved for rheumatoid

arthritis treatment, was shown to have a broad-spectrum antipar-

asitic activity against protozoan parasites [53] and to be effective

against acute toxoplasmosis in vitro and in vivo [41].

A range of hydrolytically stable half-sandwich h

⁶-arene ruth-

enium(II) phosphite compounds [49,56] were previously evaluated

for their in vitro activities against protozoan parasites such as

T. gondii and the closely related Neospora caninum, and the hel-

minth Echinococcus multilocularis. Results on T. gondii and

N. caninum demonstrated that two compounds, namely G1 and G2

(Fig. 1) exhibited a highly selective inhibitory effect on in vitro

proliferation of tachyzoites without harming host cells. These

ﬁndings prompted to further biological activity assessment of a

small library of trithiolato-bridged dinuclear ruthenium(II)-arene

complexes against protozoan parasites including T. gondii [50],

N. caninum [57] and Trypanosoma brucei [58]. Three compounds

H1eH3 (Fig. 1) were highly active against T. gondii with IC₅₀values

(half maximal inhibitory concentrations) in low nanomolar range

(34, 62 and 1.4 nM, respectively). Moreover, the viability of human

foreskin ﬁbroblast cells (HFF used as host cells) was not affected

subsequent to incubation with high doses of complex H1 or H2

(~200

mM), whereas H3 started to compromise HFF survival at

~5 M. In contrast to the in vitro efﬁcacy assessment data, com-

m

pounds H1eH3 were shown to be ineffective in the neosporosis

mouse model at a dosage of 10 mg,kg^ꢀ1$day^ꢀ1) for 5 days [57].

Metallodrugs present a high potential not only as anticancer but

also as antimicrobial, antiviral and antiparasitic agents

[21,24,25,59]. Recent studies in medicinal inorganic chemistry

aimed to extrapolate and adapt strategies that are well-established

in organic drug discovery. This includes for example, the design of

new compounds by employing fragment-based approaches [60,61]

and pharmacophore conjugation strategy [62], the use of combi-

natorial coordination chemistry to access and study larger libraries

of compounds [63,64], as well as the use of high-throughput

screening assays [65,66]. Also, in the perspective of applying

medicinal-chemistry approaches to metal-based bioactive com-

pounds, an increasing number of reports focus of the development

of series of compounds with the identiﬁcation of descriptors and

structure-activity relationships [67e72].

The limitations observed in vivo for compounds H1eH3

[50,57,58] highlighted the necessity to identify thiolato-bridged

dinuclear ruthenium(II)-arene compounds with improved proper-

ties as antiparasitic agents and to understand the structural fea-

tures inﬂuencing their efﬁcacy. The structure of trithiolato

complexes (hydrophobic capping arene ligands, strong thiol

bridges and absence of labile ligands) make them particularly inert

towards hydrolysis, and more generally, ligand substitution. A ﬁne

balance of compounds' polarity/hydrophobicity can be reached by

ﬁnely adjusting the substituents present on the bridge thiols. Based

on H1eH3, a library of 56 thiolato-bridged dinuclear ruthenium(II)-

arene and osmium(II)-arene compounds was designed, comprising

a systematic tuning of the nature, position and number of the

substituents present on the thiol bridge ligands. According to their

respective structural characteristics the compounds were orga-

nized into eight families. For the biological assessment of the 56

Various coordination complexes of ﬁrst row transition metal

ions were shown to be active against the highly virulent T. gondii RH

strain, with low cytotoxicity for non-infected mammalian cells, e.g.,

iron(III) complex B (Fig. 1) [42], and a cobalt(II) complex [43] pre-

senting similar organic ligands. Other compounds active against

Toxoplasma include mononuclear copper(II) complexes C1 and C2

[44], with different naphthyl substituents (Fig. 1).

Coordination complexes of zinc(II) [45] or iron(III) [46] (Fig. 1, D)

with sulfadiazine (drug used in combination with pyrimethamine

for the treatment of toxoplasmosis) [14,15] exhibited better activity

against T. gondii RH tachyzoites in vitro and lower host cell toxicity

compared to sulfadiazine alone.

Compounds E1/E2 [47] and F [48] (Fig. 1) comprise covalently

compounds, a sequence of consecutive screening steps was

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Fig. 1. Structures of various metal complexes presenting anti-Toxoplasma gondii activity.

considered (Fig. 4). In the primary screening, all compounds were

assessed for activity against T. gondii- -gal and non-infected HFF at

0.1 and 1

M. Compounds exbiting ꢁ95% parasite proliferation

inhibition and ꢂ49% impairment of HFF viability at 1 M were then

selected for secondary screening, which comprised dose-

response experiments yielding IC₅₀values for T. gondii- -gal pro-

liferation and an assessment of viability impairment in HFF

monolayers at 2.5 M. Only the compounds that did not cause HFF

viability loss with more than 50% were subjected to a tertiary

screening on their inhibitory potential in murine splenocyte cul-

tures stimulated with lipopolysaccharide (LPS) and concanavalin A

(ConA) to determine their effects on B and T cells viability and

proliferative responses, respectively.

The symmetric trithiolato-bridged dinuclear ruthenium(II)-

arene derivatives were obtained using adapted literature proced-

ures [73e75] in one step as shown in Scheme 1A, starting from the

b

m

ruthenium dimer [Ru(h

⁶-p-MeC₆H₄Prⁱ)Cl₂]₂and excess of the cor-

a

responding thiol (general procedure A in Supporting information).

The mixed cationic compounds were prepared in two steps

adapting previously described protocol [76] following the reaction

sequence presented in Scheme 1B. The dithiolato intermediates

isolated after the reaction of one equivalent of ruthenium dimer

b

m

[Ru(h

⁶-p-MeC₆H₄Prⁱ)Cl₂]₂with two equivalents of a ﬁrst type of

thiol (R1-SH in Scheme 1B, see general procedure B and C in

Supporting information) were further reacted with a second type of

thiol (R2-SH in Scheme 1B) used in excess (general procedure D in

Supporting information). Most of the reactions for obtaining the

mixed trithiolato compounds from the dithiolato compounds were

performed in EtOH. In some cases (e.g., carboxylate analogues 3, 4

and 53) a mixture of CH₂Cl₂/acetone was used as solvent to avoid

esteriﬁcation side reaction favored by the acid produced during the

reaction. For the synthesis of compound 23 the solvent was a

mixture of CH₂Cl₂/MeOH to increase the solubility of the dithiolato

intermediate P4.

2. Results and discussion

2.1. Chemistry

Eight families of compounds based on the trithiolato-bridged

dinuclear ruthenium(II)-arene scaffold were designed by applying

a systematic approach. Various substituents were introduced at

different positions of the thiols' aromatic ring, for tuning their

physico-chemical properties (polarity, hydrophobicity, ability to

participate in H-bonding interactions). Detailed synthesis proced-

ures and analysis of the new compounds are given in the

Supporting information. An overview of the 56 compounds inves-

tigated in this study is presented (Fig. 2).

Compounds 15 and 17 were obtained from the respective hy-

droxy and amine functionalized ruthenium complexes 1 and 2

using reactions presented in Schemes S1 and S2 in Supporting

information.

The new compounds were fully characterized by ¹H and ¹³C

NMR spectroscopy, mass spectrometry and elemental analysis. The

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Fig. 2. Structure of the 56 compounds investigated in this study organized in 8 families based on structural features modiﬁcations.

Scheme 1. Synthesis of the symmetric (A) and mixed (B) trithiolato-bridged dinuclear ruthenium (II)-arene complexes.

representative peaks for the neutral dithiolato-bridged dinuclear

The compounds are readily soluble and stable in DMSO‑d₆

which make them suitable for further biological tests (Figures S19-

S27 in Supporting information).

The crystal structures of the symmetric complexes 13 and 43,

mixed complex 38 and dithiolato intermediate P13 were estab-

lished in the solid state by single-crystal X-ray diffraction (ORTEP

ruthenium(II)-p-cymene (p-cymene: p-MeC₆H₄Prⁱ) intermediates

P1eP13 correspond to [M ꢀ Cl]^þion, due to the loss of a labile

chlorine ligand. For the cationic trithiolato compounds 1e56, the

parent ion peak corresponds to the cation trithiolato di-ruthenium

[M ꢀ Cl]^þion.

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

representations shown in Fig. 3 (A and B, S8 and S9), conﬁrming the

expected structure. Data collection and reﬁnement parameters are

given in Table S5 (Supporting information). Selected bond lengths

and angles are presented in Tables S6, S7, S8 and S9.

[77], the RueSeRu and cent h

⁶-cent(SeSeS)-cent h⁶angles in the

mixed complex 38 show that the central di-ruthenium trithiolate

unit is not symmetric. The presence of the two 4-(triﬂuoromethyl)

thiophenolate ligands forces the dinuclear arene ruthenium unit to

adopt a slightly distorted geometry, the planes of the two p-cymene

ligands (C21eC26 and C31eC36) being tilted.

Compounds 13 and 38 co-crystallize with solvent and H₂O

molecules (13$CH₂Cl₂$H₂O and 38$H₂O, Table S5). These solvent

molecules not only ﬁll the voids in the crystal packing, but also form

a series of H-bonding interactions as presented in Figures S2 and S5

and Table S7 in Supporting information. In both structures, the

ruthenium atoms adopt a pseudo-octahedral geometry with three

sulphur atom ligands and the p-cymene ligand. In the case of the

symmetric trithiolato compound 13, both Ru(II) atoms adopt the

typical piano-stool geometry with the p-cymene ligand being

bound facially while the other three positions are occupied by three

4-aminothiophenolate ligands. The mixed 38 complex presents

In the network of 13 is observed an interplay of H-bonding in-

teractions involving both the chlorine anions and the H₂O mole-

cules trapped in the crystal. A dimeric organization is mediated by

four H-bonding interactions: two chlorine anions bridge two di-

ruthenium units at the level of their respective two amino groups

(Figure S2, Supporting information). The remaining amino group of

each of the symmetric di-ruthenium complexes is involved in H-

bonding interactions with an H₂O molecule. Thus, all three amino

groups of the symmetric trithiolato di-nuclear ruthenium(II)-arene

complex 13 are involved in intermolecular H-bonding interactions,

two of them interact with two chlorine anions and lead to the

formation of a dimer with another di-ruthenium unit, while the

third NH₂group interacts via H-bonds with the H₂O molecule

present. In network, these intermolecular H-bonding interactions

lead to further arrangements e.g., with the formation of parallel

chains of di-ruthenium complexes (Figure S3). In the crystal of 13

two

4-(triﬂuoromethyl)

thiophenolate

and

one

4-

hydroxythiophenolate as bridge thiols. To date, this is only the

second example of an X-ray analysis of a mixed dinuclear trithiolato

ruthenium(II)-arene complex [77]. The Ru₂S₃unit forms a trigonal-

bipyramidal framework; no metal-metal bonds are present, the

corresponding distances being Ru(1)eRu(2) 3.345 and Ru(3)eRu(4)

3.346 for 13 (asymmetric unit contains two independent com-

plexes) and Ru(1)eRu(2) 3.347 Å for 38. In both cations, the values

of RueS bonds as well as the RueSeRu angles (Table S6) are similar

to those found in other symmetric or mixed p-cymene derivatives

previously reported [74,77]. In both structures, the aromatic rings

of the bridge thiols are tilted compared to the plane of the three

sulphur atoms. Similar to the reported mixed complex I (Table S6)

an intermolecular H-p interaction (distance 3.404 Å) between a H-

atom from the p-cymene iso-propyl group and the aromatic ring of

one of the aminothiol ligands was also identiﬁed (Figure S4).

In the crystal packing of 38, a H-bonding interaction between

the chlorine anion and OH group of the 4-hydroxythiophenolate

unit (Figure S5, Supporting information) was acknowledged. The

chlorine anion also interacts via H-bonds with two H₂O molecules

trapped in the network. These cooperative H-bonding interactions

mediated by chlorine anions and H₂O molecules lead to further

dimeric organization (Figure S5) and dictate network packing for

example in antisense chains connected via a zipper of H-bonds

(Figure S7, Supporting information.). For 38 other short intermo-

lecular contacts (2.675 and, respectively, 2.615 Å) were identiﬁed

between the O atom of the phenol and a p-cymene H atom, and a H

atom from the p-cymene methyl substituent of a neighbouring

molecule (Figure S6).

Experimental details regarding the X-ray structure determina-

tion for complexes 43 and P13, as well as the corresponding

structural details are presented in Tables S5, S8 and S9, and

Figures S8 and S9 in the Supporting information.

2.2. Biological assessment

2.2.1. Screening strategy

The 56 compounds considered for this study were organized in

8 families based on their structural features, and were subjected to

a sequential biological screening as summarized in Fig. 4. The

antiparasitic activity (proliferation inhibition) was evaluated using

T. gondii-b-gal grown in HFF host cell monolayers [50], and the

cytotoxic effects were studied in non-infected HFF monolayers. The

potential impact on T and B cell viability and proliferation was

investigated using concanavalin A (ConA) and lipopolysaccharide

(LPS)-stimulated murine splenocyte cultures [51].

The primary screening (Fig. 4) of all 56 compounds against

T. gondii-b-gal tachyzoites and HFF was carried out at 0.1 and 1 mM

concentrations. These two concentrations were chosen for several

reasons, such as: (i) potential solubility issues at the higher con-

centration, (ii) assessments for anti-parasitic or host cell activity

cannot be done by testing only one concentration, (iii) the T. gondii

IC₅₀values of potentially interesting compounds would be in the

range of 0.1 mM, and (iv) the 1 mM concentrations provides ﬁrst

indications on potential cytotoxic effects. Also, testing two con-

centrations initially opens options for selecting compounds for the

Fig. 3. ORTEP representation of compounds 13 (A) and 38 (B) (thermal ellipsoids are

50% equiprobability envelopes, and H atoms are spheres of arbitrary diameter; the

asymmetric unit of 13 contains two independent complexes and two H₂O and two

CH₂Cl₂molecules, and the asymmetric unit of 38 contains one H₂O molecule).

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European Journal of Medicinal Chemistry 222 (2021) 113610

Fig. 4. Schematic representation of the three-step screening cascade used for the assessment of the antiparasitic activity and cytotoxicity of the trithiolato-bridged dinuclear

c

ruthenium(II)-arene compounds. ^ab-Galactosidase parasite proliferation assay. ^bAlamarBlue cell viability assay. BrdU (5-bromo-2⁰-deoxyuridine) cell proliferations assay.

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Fig. 5. Primary screening: results for the 56 compounds assessed for anti-T. gondii activity. Compounds were organized into 8 families. T. gondii infected HFF cultures were treated at

two concentrations 0.1 M (light grey bars) and 1 M (dark grey bars). Results are presented as mean of three replicates standard deviation. Results are presented as percentage of

T. gondii- -gal proliferation relative to control (CTR) parasites treated with 0.01% or 0.1% DMSO (solvent only).

m

b

next screening steps. When applied at 1

m

M, the compounds that

submitted to dose response studies to determine the IC₅₀values,

and potential cytotoxicity in HFF was assessed at 2.5 M. The

inhibited T. gondii- -gal proliferation by at least 95% and did not

b

m

impair HFF viability by more than 49% were subjected to the next

step. In the secondary screening, the selected compounds were

complexes exhibiting HFF cytotoxicity 50% or less were further

subjected to a tertiary screening using murine splenocytes. For this,

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

splenocyte cultures were subjected to treatments with LPS (to

stimulate B cells) or ConA (to stimulate T cells), and the effects of

bridge thiols. The thiol functionalized compound 5, more prone to

oxidation, presented reduced activity against the parasite and less

pronounced HFF toxicity compared to the hydroxy 1 and amino 2

analogues.

the compounds at 0.1 and 0.5

mM concentrations on cellular

viability were determined. These two concentrations were chosen

because (i) most of the selected compounds (including the anti-

Toxoplasma drug pyrimethamine) inhibit T. gondii in this concen-

tration range, (ii) isolated and proliferating spleen cells are likely to

be more susceptible to compound effects than HFF, and (iii) it is

unlikely that higher drug concentrations will be obtained in tested

animals. The ruthenium complexes that impaired T cell viability by

less than 50% and B cell viability by less than 10% were further

evaluated for their capacity to suppress ConA- and LPS-induced T

and B cell proliferation.

2.2.2.2. Family 2. Family 2 includes 7 compounds, for which the

number (two mixed in complexes 8e10 and three in symmetric

complexes 11e14) and the position (para or meta) of the polar

groups OH and NH₂were systematically varied (Table 1, Fig. 2 and

S12). Compounds 11 and 14 were previously reported [74,78] and

tested against T. gondii-b-gal under similar conditions [50].

The primary screening (Figs. 5 and 6, Table 5) shows that com-

pounds bearing two polar groups, namely 8 (hydroxy) and 9

(amino), were more toxic to both T. gondii-b-gal and HFF compared

2.2.2. Primary screening

Detailed results of the primary screening are also provided in

Table 5.

to their analogues with a single polar substituent (1 and 2,

respectively). The two compounds bearing three polar groups, 12

(hydroxy) and 13 (amino), were ineffective against both T. gondii-b-

gal and HFF. These ﬁndings are supported by previous studies in

which no activity was observed for compounds 11 and 14 [74,78].

The signiﬁcantly increased toxicity in T. gondii-b-gal of 8 compared

2.2.2.1. Family 1. Family 1 is comprised of 8 members whose

structures are presented in Table 1, Fig. 2 and S11. The BF^ꢀ₄salt of

complex H3 was previously shown to exhibit encouraging in vitro

to 10 is interesting, as the structures of these compounds only differ

by the presence of a methylene spacer in the bridge thiol bearing

the Bu^tgroup. However, the effects of 8 and 10 on HFF viability were

comparable, suggesting that the increased mobility of the bridge

thiol bearing a Bu^tgroup could be beneﬁcial for antiparasitic

activity.

Overall, the data obtained for family 2 conﬁrm previous ﬁndings

[36] that the antiparasitic activity is inﬂuenced by the nature of the

groups anchored on the bridged thiols, while the position of the

substituents (para versus meta) has a lower inﬂuence.

efﬁcacy against T. gondii-b-gal tachyzoites [50] and it represents a

lead compound for the design of other derivatives of the current

study. For building the ﬁrst series, the hydroxy group of H3 was

replaced by various polar groups: amino (2), thiol (5), and carboxy

(3 and 4), the mixed compounds being synthesized as Cl^ꢀsalts. In

order to assess the inﬂuence of the counterion on the biological

activity, the series was completed with amino and carboxy com-

pounds 6 and 7, isolated as tetraﬂuoroborate (BF^ꢀ₄) salts (Table 1).

The results of the primary screening at concentrations of 0.1 and

1 mM for family 1 are presented in Fig. 5, 6 and Table 5. Hydroxy

functionalized compounds H3 and 1, with BF^ꢀ₄and Cl^ꢀcounterions,

respectively, showed similar inhibition of parasite proliferation.

Nevertheless, 1 did affect more than H3 the viability of HFF for the

same concentration. Amino derivative 6 with a BF^ꢀ₄counter-ion

exhibited enhanced activity against T. gondii and less pronounced

HFF toxicity compared to the corresponding Cl^ꢀsalt 2. All carboxy

derivatives tested, 3, 4 and 7, were toxic neither for T. gondii nor for

HFF. The inﬂuence of the counterion (BF^ꢀ₄vs Cl^ꢀ) appeared less

important than the nature of the polar substituent on one of the

2.2.2.3. Family 3. Family 3 comprises two esters (15 and 18) and

two amides (16 and 17) derivates from hydroxy amino and carboxy

analogues 1, 2 and 3 (Table 2, Fig. 2 and S13). These structural

modiﬁcations act as a protection of the polar groups preventing

metabolic degradation (e.g., oxidation of the OH and NH₂substit-

uent to quinone or quinone-imine) or reaction/interaction with

biomolecules. The proliferation of T. gondii tachyzoites was almost

completely suppressed by 15e18 at 0.1

mM (Fig. 5 and Table 5),

whereas the polar parent analogues 1 and 2 displayed anti-

Table 1

Structure of the compounds composing families 1 and 2.

Compound

R1

R2

X^-

H3^a

CH₂eC₆H₄-4-Bu^t

C₆H₄-4-OH

BF^ꢀ₄

Family 1

1^b

2

3

4

5

CH₂eC₆H₄-4-Bu^t

C₆H₄-4-OH

C₆H₄-4-NH₂

C₆H₄-4-CH₂-CO₂H

C₆H₄-4-CO₂H

C₆H₄-4-SH

Cl^¡

Cl^ꢀ

BF^ꢀ₄

6

7

C₆H₄-4-NH₂

C₆H₄-4-CH₂-CO₂H

Family 2

8^c

9

10

11^d

12

13

14^e

C₆H₄-4-OH

C₆H₄-4-NH₂

C₆H₄-4-OH

C₆H₄-3-OH

C₆H₄-4-NH₂

C₆H₄-3-NH₂

CH₂eC₆H₄-4-Bu^t

C₆H₄-4-Bu^t

C₆H₄-4-OH

C₆H₄-3-OH

Cl^ꢀ

C₆H₄-4-NH₂

C₆H₄-3-NH₂

a

Previously reported in Ref. [50].

previously reported in Ref. [76].

previously reported in Refs. [50,74].

Previously reported in Refs. [50,78].

b

d

e

8

ꢀ

E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Fig. 6. Primary screening: the 56 compounds were assessed with respect to activity against non-infected HFF. Compounds were organized into 8 families, and tested concentrations

were 0.1 M (light grey bars) and 1 M (dark grey bars). Results are presented as mean of three replicates standard deviation. Results are presented as percentage of HFF viability

relative to a control (CTR), namely HFF treated with 0.01% or 0.1% DMSO (solvent only).

m

T. gondii-

b

-gal activity only when applied at 1

m

M, and 3 was

amide substituents (15e18) enhanced signiﬁcantly the potency

against the parasite, and, to a lesser extent, the toxicity to HFF host

cells.

completely inactive. Overall, compounds 15e18 are slightly more

toxic for HFF than the more polar derivatives 1e3. Modifying polar

hydroxy (1), amino (2) or carboxy (3) groups into less polar ester or

9

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Table 2

Structure of the compounds composing families 3 and 4.

Compound

R1

R2

Family 3

15

16

17

18

CH₂eC₆H₄-4-Bu^t

C₆H₄-4-OAc

C₆H₄-4-NHAc

C₆H₄-4-N-piperidine-2,6-dione

C₆H₄-4-CH₂-CO₂Et

Family 4

19

20

21

22^a

23

CH₂eC₆H₄-4-Bu^t

CH₂eC₆H₅

C₆H₄-3-OH

C₆H₄-2-OH

C₆H₄-2-CH₂OH

C₆H₄-4-OH

CH₂eC₆H₄-4-Me

a

Previously reported in Ref. [76] presenting BF^ꢀ₄as counterion.

2.2.2.4. Family 4. Family 4 includes 5 compounds, all containing a

polar hydroxy group on one of the bridge thiols (Table 2, Fig. 2 and

S14). In this series, the inﬂuence of the OH group position (meta and

ortho) in compounds 19 and 20, was compared to that of para-

analogue 1. In derivative 21 the ortho-hydroxy group of 20 was

replaced by a hydroxymethyl substituent, and in compound 23 the

Bu^tsubstituents anchored on two of the bridged thiols in analogue

1 were replaced with less bulky and hydrophobic methyl sub-

stituents in the same para position. Compound 22 with an H atom

as substituent had been previously reported [76] as BF^ꢀ₄salt and

2.2.2.5. Family 5. Family 5 includes 9 members (Table 3, Fig. 2 and

S15). The role of the OH group in compound 1 was also questioned.

In derivative 24 the hydroxy group was removed, whereas in

compounds 25e27 it was replaced by other polar substituents as

ﬂuoro, bromo or triﬂuoromethyl, respectively. Compared to 1, in

compounds 28 and 29 the para-OH group was protected as a

methoxy and triﬂuoromethoxy ether. As the position of the OH

group in 1 (para), 19 (meta) and 20 (ortho) inﬂuenced the antipar-

asitic efﬁcacy, the effect of a methoxy group in the same positions

was also established by comparing compounds 28 (para), 30 (meta)

and 31 (ortho). The electron-rich para-phenoxy ring 1 was replaced

in 32 with a methylene-furan-2-yl substituent.

when tested against T. gondii-b-gal [50] was considered not inter-

esting for further studies.

The primary screening (Figs. 5 and 6 and Table 5) showed that

meta and ortho hydroxy-substituted analogues 19 and 20, were

more efﬁcient against T. gondii compared to the para-substituted

compound 1. A similar but stronger effect was observed for 21,

presenting a methylene spacer between the OH group and the ar-

omatic ring, as well as for 23 bearing less bulky and hydrophobic

Me substituents on two of the bridge thiols (compared to Bu^tin 1).

The position of the polar OH group present on one of the bridge

thiols (1, 19 and 20), but also the volume and lipophilicity of the

substituents present on the other two thiols (1 vs 23) inﬂuence the

parasite proliferation. The structural modiﬁcations in compounds

19e21 and 23 led to a decrease of host cell toxicity.

Compared to 1, the absence of the OH group in 24 increased both

the anti-Toxoplasma efﬁcacy and the viability of HFF (Figs. 5 and 6

and Table 5). Likewise, the replacement of the hydroxy group in 1

with ﬂuoro (25), bromo (26) and triﬂuoromethyl (27) polar sub-

stituents increased the antiparasitic activity and slightly reduced

HFF toxicity. Both OH and F can participate in H-bonding in-

teractions; nevertheless, while the OH group can be oxidized, this

type of metabolization is less prone to take place if the position is

blocked by ﬂuoro, bromo or triﬂuoromethyl substituents.

Compounds 28 and 29 in which the hydroxy group in para po-

sition was protected as a methoxy or triﬂuoromethoxy group were

more efﬁcient against T. gondii and exhibited a lower HFF toxicity

compared to 1. Meta and ortho methoxy-substituted compounds 30

and 31 were similarly active against T. gondii as their respective

hydroxy analogues 19 and 20, but were signiﬁcantly less toxic to

HFF. Replacement of the para-phenoxy (1) with an electron rich

furan ring in 32 also led to selective antiparasitic toxicity.

Table 3

Structure of the compounds composing families 5 and 6.

As previously observed for family 3, the results obtained for

family 5 suggest that high efﬁcacy against the parasite and reduced

inﬂuence of the HFF viability are correlated with the presence of a

polar group on one of the bridge thiols. Jointly, the presence of

polar, chemically reactive groups, that can participate in H-bonding

interactions (e.g., carboxyl) had a negative impact on the anti-

T. gondii activity. Unreactive, polar substituents like ﬂuoro (25),

bromo (26), triﬂuoromethyl (27), methoxy (28, 30, 31) or tri-

ﬂuoromethoxy (29) answered these requirements and appeared to

be better suited.

Compound R1

Family 5

R2

M

24

25

26

27

28

29

30

31

32

CH₂eC₆H₄-4-Bu^tC₆H₅

Ru

CH₂eC₆H₄-4-Bu^tC₆H₄-4-F

CH₂eC₆H₄-4-Bu^tC₆H₄-4-Br

CH₂eC₆H₄-4-Bu^tC₆H₄-4-CF₃

CH₂eC₆H₄-4-Bu^tC₆H₄-4-OCH₃

CH₂eC₆H₄-4-Bu^tC₆H₄-4-OCF₃

CH₂eC₆H₄-4-Bu^tC₆H₄-3-OCH₃

CH₂eC₆H₄-4-Bu^tC₆H₄-2-OCH₃

CH₂eC₆H₄-4-Bu^tCH₂-furan-2-yl Ru

Regardless of the substituent position, all three methoxy de-

Family 6

rivatives 28, 30 and 31 were highly efﬁcient against T. gondii-

b-gal

when applied at 0.1 M. Some differences were observed in the

effect on the HFF viability, for which meta and ortho substitution

appeared to be more favorable.

m

33

34

35

36

37

38

39

40

C₆H₅

C₆H₄-4-OH

Ru

Os

C₆H₄-4-Me

C₆H₄-4-Prⁱ

C₆H₄-4-Bu^t

C₆H₄-4-F

C₆H₄-4-CF₃

2.2.2.6. Family 6. Family 6 is comprised of 8 compounds designed

to assess the role of the methylene spacers in the bridge thiols,

units correlated with the mobility and ﬂexibility of the pendant

CH₂eC₆H₄-4-Bu^tC₆H₄-4-OH

C₆H₄-4-Bu^t

C₆H₄-4-OH

10

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Table 5

Results of the primary efﬁcacy/cytotoxicity screening.

arms in 1 and H3 (Table 3, Fig. 2 and S16). The hydrophobicity of the

substituents was increased in series H < Me < Prⁱ< Bu^tfor com-

pounds 33e36, while derivatives 37 and 38 present polar F and CF₃

substituents. Two di-osmium compounds 39 and 40 were also

synthesized allowing comparison with ruthenium analogues 1 and

36.

Compound

HFF viability (%)

T. gondii b-gal growth

(%)

0.1

mM

1

mM

0.1

60

m

M

4

1

0

m

M

1

H3^a

119

2

81

3

The results of the ﬁrst screening for this family of compounds

are presented in Figs. 5 and 6 and Table 5. Compared to compound

1, the presence of non-polar hydrophobic substituents (Me, Prⁱ, Bu^t)

on two of the bridge thiols in 34e36 increased the anti-Toxoplasma

activity, whereas polar substituents as F and CF₃in 37 and 38 had an

opposite effect. Nevertheless, irrespective of the nature of the

substituents in para position, the di-ruthenium complexes 33e38

displayed lower HFF cytotoxicity than derivative 1. Limited ﬂexi-

bility of the pendant arms and a reduced polarity of the sub-

stituents present on two of the bridge thiols seem to be associated

with improved efﬁcacy against T. gondii-b-gal and a relative

innocuousness towards HFF host cells.

The change of the metal to osmium in compounds 39 and 40

provided contrasting results. Di-osmium compound 40 was clearly

superior to its corresponding di-ruthenium analogue 1 both in

Family 1

1^b,d

2^b,d

3^b,d

4

76

74

91

98

115

116

6

2

4

6

2

46

48

73

93

65

82

107

6

1

9

5

2

66 14

57

114

102

2

0

1

2

7

2

110

99 12

29

0

2

5^d

1

6^d

1

2

7

118

1

0

7

1

9

1

98

0

Family 2

8^c,d

9^d

62

66

73 10

8

6

56

58

62

e

7

8

2

3

2

108

e

1

0

2

4

e

0

1

10^d

11^a

12

2

e

114

97

e

8

104

61

e

105

115

e

5

4

108

85

e

2

13

4

6

5

14^a

terms of efﬁcacy against T. gondii-b-gal and reduced HFF toxicity.

Family 3

However, no important differences were observed between 36 (di-

ruthenium) and 39 (di-osmium), neither with respect to antipara-

sitic activity nor in relation to host cell viability impairment. These

preliminary results suggest that the biological activity could be

altered not only by modifying the substituents present on the

bridge thiols but also by nature of the metal present in the main

framework. Nevertheless, evaluating a larger number of osmium

derivatives, or of structure related di-nuclear compounds based on

other metals e.g., rhodium or iridium would be necessary to

conﬁrm this hypothesis.

15^d

16

74

62

68

50

2

7

4

52

27

58

46

3

1

7

2

5

4

3

0

2

3

2

4

1

0

1

0

1

0

1

17^d

18

Family 4

19^d

20^d

21^d

22^a

23^d

89

80

e

4

3

1

62

68

69

e

2

9

6

5

8

2

e

1

0

1

0

1

e

1

0

110

9

62

4

1

4

Family 5

24^d

25^d

26^d

27^d

28^d

29^d

30^d

31^d

32^d

81

68

89

59

72

69

90

93

76

5

9

2

8

7

2

8

75

61

67

51

62

66

80

65

8

3

0

8

4

7

3

6

3

2

6

0

1

4

1

ꢀ3

ꢀ2

1

0

1

0

2.2.2.7. Family 7. A group of ten derivatives formed Family 7

(Table 4, Fig. 2 and S17). A previous study [50] identiﬁed symmetric

compounds H1 and H2 (Fig. 1) which exhibited interesting anti-

Toxoplasma activity and low toxicity in HFF. These structures were

the starting point for the evaluation of additional structurally

related symmetric compounds. The hydrophobic Me and Bu^t

groups of H1 and H2 were either removed in 41, or replaced with

ꢀ1

4

8

4

2

0

3

ꢀ1

2

0

2

Family 6

Table 4

33^d

34^d

35^d

36^d

37

93

99

104

74

87

89

87

2

3

73

83

101

64

62

74

60

3

2

5

89

3

10

3

113

97

3

8

1

5

1

96

1

0

Structure of the compounds composing families 7 and 8.

0

Compound R1

Family 7 (R1 ¼ R2)

R2

6

3

7

1

3

9

3

6

1

3

2

0

H1^a

H2^a

41

42

43

44

45

46

47

48

49

50

C₆H₄-4-Me

38^d

39^d

40^d

0

C₆H₄-4-Bu^t

C₆H₅

1

2

1

C₆H₄-4-F

C₆H₄-4-CF₃

C₆H₄-3-CF₃

C₆H₄-2-CF₃

C₆H₃-3,5-(CF₃)₂

CH₂eC₆H₄-4-CF₃

CH₂eC₆H₅

Family 7

H1^a

H2^a

41^d

42^d

43^d

44^d

45^d

46^d

47^d

48^d

49

e

3

98

ꢀ1

1

2

3

117

e

0

1

0

1

0

1

e

115

85

77

36

100

95 11

85

104

66

63

0

3

93

69

61

79

68

69

57

93

15

1

2

3

9

1

3

1

3

1

0

1

0

1

2

0

7

1

0

CH₂eC₆H₄-4-Me

0

CH₂eC₆H₄-4-Bu^t

Family 8

3

51^b

52^b

53^b

54

CH₂eC₆H₄-4-CF₃C₆H₄-4-OH

1

CH₂eC₆H₄-4-CF₃C₆H₄-4-NH₂

CH₂eC₆H₄-4-CF₃C₆H₄-4-CH₂-CO₂H

CH₂eC₆H₄-4-CF₃C₆H₄-4-CH₂-CO₂CH₃

CH₂eC₆H₄-2-CF₃C₆H₄-4-OH

4

0

91

1

50^d

62 10

55

Family 8

56

CH₂eC₆H₄-2-CF₃C₆H₄-4-NH₂

51^b,d

112

7

66

4

101

1

0

a

Previously reported in Ref. [50].

Previously reported in Ref. [51].

(continued on next page)

b

11

ꢀ

E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Table 5 (continued)

T. gondii proliferation even at 0.1

mM, but were less toxic to HFF

when applied at 1 M. Hydroxy and amino derivatives 51 and 52

m

Compound

HFF viability (%)

T. gondii b-gal growth

(%)

with CF₃groups, compared to their respective para-Bu^t-substituted

analogues 1 and 2, exhibited similar antiparasitic activity but

slightly lower host cell toxicity. As previously observed for carboxy

substituted compounds 3 and 4, 53 did not display neither anti-

parasitic activity nor HFF toxicity. However, 54 with the carboxy

group protected as methyl ester, presented increased toxicity

0.1

m

M

1

m

M

0.1

59

m

M

7

1

0

m

M

0

52^b,d

53^b

54

115

103

92

1

3

8

58

93

40

93

86

2

6

2

3

102

76

5

102

3

7

0

55^d

56^d

111

114

2

1

0

against both T. gondii-b-gal and host cells; a similar effect was

observed with ethyl ester derivative 18 as compared to the carboxy

compound 3.

a

Compound reported in Ref. [50].

Compound reported in Ref. [51].

Compound reported in Ref. [79].

b

c

The primary screening revealed that the compounds showing

anti-T. gondii activity while remaining non-toxic against HFF are

structurally varied. Free carboxy groups have a detrimental effect as

compounds 3, 4 and 7 (family 1) are admittedly non-toxic to the

host cells but also show no antiproliferative activity on the parasite.

Likewise, the presence of more than one hydroxy and amino sub-

stituents on the bridge thiols (family 2) is not a good option, as

compounds bearing two polar groups (8e10) are active but also

toxic to the host cells and those bearing three polar groups (11e14)

are not active against the parasite. Interestingly, the protection of

the polar groups OH, NH₂or CO₂H as ester or amide (family 3)

brings no beneﬁt, as the HFF viability is signiﬁcantly reduced when

treated with compounds 15e18. If the presence of one hydroxy

group on one of the bridge thiols (family 4) appears to be important

to the anti-parasitic activity, at this point of the study the inﬂuence

of its positon is not easy to deﬁne, even if compound presenting the

OH group in para position (1) appear more toxic on the host cells

and less active on the parasite compared to meta and ortho

substituted analogues 19 and 20.

d

Compounds selected for the secondary screening.

polar ﬂuoro and triﬂuoromethyl substituents, in compounds 42 and

43, respectively. The role of the position (meta in 44, ortho in 45)

and number (46) of the triﬂuoromethyl substituents was also

assessed. Compounds 47e50 differ from the complementary ana-

logues 43, 41, H1 and H2, by the presence of methylene spacers on

the bridge thiols. Compounds 42, 44 and 46 were available in the

laboratory from previous studies and only their purity was veriﬁed

prior to assessing their biological activity (Supporting information).

The primary screening results (Figs. 5 and 6 and Table 5) showed

that triphenyl compound 41 and analogues bearing triﬂuoromethyl

groups 43e47 were highly efﬁcient against T. gondii-

inhibited parasite proliferation almost entirely already at 0.1

However, triﬂuoromethyl substituted analogues 43e47 had a

higher impact on HFF viability compared to 41. The position and the

number of the triﬂuoromethyl groups in 43e47 inﬂuenced HFF

toxicity only marginally. Para-ﬂuoro substituted compound 42

b-gal, and

m

M.

Interestingly, the ether protection of the OH group (family 5,

compounds 28e31) appears to be a better option than the ester

protection (15), while the nature of the protecting group (CH₃or

CF₃) as well as the position of the methoxy substituent impact the

viability of the host cells. The presence of polar substituents F and

expecially CF₃(25 and 26) on only one of the bridge thiols appears

to be associated to increased toxicity on HFF. Removing the meth-

ylene spacer on two of the bridge thiols while maintaining the

polar hydroxy group on the third thiol (family 6) is associalted with

an increased anti-T. gondii activity. One hydroxy group and two

non-polar substituents such as Me, Prⁱand Bu^t(34, 35 and 36) are

better options than three F or CF₃both for the activity against the

parasite and the toxicity on the host cells. Some of the observations

made in the case of mixed compounds seem to apply also to the

symmetric derivatives from family 7. Fluorine as substituent

(compound 42) led to a decreased activity against the parasite. The

presence of methylene linkers on two of the bridge thiols appears

to be associated to a decreased activity on T. gondii (48) or increased

toxicity to HFF (compounds 47, 49 and 50). Compounds bearing

polar CF₃groups (43, 44, 45 and 46) seem a good compromise

between anti-parasitic activity and effect on the host cell viability.

The compounds from family 8 conﬁrm some of the observations

made previously: a free carboxy group on one of the brigde thiols

(53) is to be avoided, as well as the presence of methylene spacers

on two of the bridging thiols (compounds 51 and 52) or the pro-

tection of the carboxy group as an ester (54). Combining two CF₃

substituents on two of the bridge thiols with one polar hydroxy or

amino group on the third thiol appears promising for ensuring both

anti-T. gondii activity and reduced toxicity to the host cells.

suppressed T. gondii-b-gal proliferation only when applied at 1 mM,

while being more toxic for HFF compared to 41. From the com-

pounds with non-polar substituents and methylene spacers on the

pendant thiols, methyl derivative 49 was more efﬁcient in pre-

venting parasite proliferation compared to H- and Bu^t-substituted

analogues 48 and 50, being active already when applied at 0.1 mM,

but also with increased toxicity for HFF.

Summarizing, a more rigid structure in compounds 41e46

(without methylene groups on the pendant arms) favored anti-

parasitic activity, but an increased number of polar triﬂuoromethyl

groups led also to an increase in HFF toxicity. A ﬁne balance of the

polarity, size and number of the substituents is necessary to achieve

a good compromise between antiparasitic activity and host cell

cytotoxicity.

2.2.2.8. Family 8. Family 8 (Table 4, Fig. 2 and S18) contains six

members and was conceived based on lead derivative H3 and

considering the importance of the triﬂuoromethyl substituents in

the compounds presented in family 7 (Table 4, Fig. 2 and S16). The

hydrophobic bulky Bu^tgroups of two of the bridge thiols in H3 were

replaced with polar CF₃groups, while the third thiol presents polar

substituents as hydroxy (51), amino (52), carboxy (53) and ester

(54). Mixed complexes 55 and 56 present two ortho-CF₃substituted

thiols, while the third thiol is substituted with a para-hydroxy (55)

and -amino group (56). As shown in Figs. 4 and 5, the replacement

of one triﬂuoromethyl substituent in 47 with a hydroxy or amino

group in 51 and 52, respectively, signiﬁcantly decreased antipara-

sitic activity (compounds active only when applied at 1 mM), but

had little impact on HFF cytotoxicity. The absence (38) or presence

(51) of methylene spacers in the CF₃substituted thiols did not

substantially alter the anti-Toxoplasma activity and HFF cytotoxicity

2.2.3. Secondary screening

The selection criteria for a compound to enter secondary

screening were: (i) over 95% inhibition of T. gondii-

tion and (ii) not more than 49% viability impairment of HFF when

applied at 1 M. 39 compounds fulﬁlled these criteria and were

b-gal prolifera-

at 1

mM. Similar to symmetric compound 45, hydroxy and amino

substituted derivatives 55 and 56, were highly potent inhibitors of

m

12

ꢀ

E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Table 6

Secondary screening: determination of IC₅₀(mM) values for T. gondii-b-gal and viability of HFF (as %-age in relation to solvent-treated controls) at 2.5 mM for the 39 compounds

selected after the ﬁrst screening. The 14 compounds selected for the tertiary screening (against murine B and T cells) are tagged with^band the corresponding measured values

are shown in bold.

Compound

Pyrimethamine

H3

T. gondii-

0.326

0.079

b

-gal IC₅₀

(m

M)

[LI; LS]^c

[0.288; 0.396]

[0.061; 0.102]

SE^d

0.052

0.060

HFF viability at 2.5

99

70

m

M (%)^e

SD^f

6

2

Family 1

1^a

0.117

0.153

0.181

0.316

0.098

[0.098; 0.139]

[0.127; 0.185]

[0.274; 1.482]

[0.190; 0.526]

[0.066; 0.145]

0.051

0.049

0.954

0.078

0.085

56

51

99

107

72

6

5

2

8

3

2^a

3^a

5^a

6^b

Family 2

8

9

0.115

0.066

0.294

[0.098; 0.134]

[0.046; 0.095]

[0.280; 0.308]

0.044

0.126

0.011

37

19

26

3

1

5

10

Family 3

15

17

0.065

0.014

[0.041; 0.100]

[0.011; 0.032]

0.092

0.032

16

9

5

1

Family 4

19

20

21

23

0.031

0.050

0.038

0.048

[0.024; 0.040]

[0.039; 0.062]

[0.023; 0.060]

[0.031; 0.071]

0.07

0.06

0.11

16

37

4

2

3

2

4

31

Family 5

24

25

26

27

0.052

0.031

0.013

0.043

0.056

0.072

0.078

0.025

0.039

[0.033; 0.083]

[0.021; 0.047]

[0.012; 0.014]

[0.037; 0.145]

[0.040; 0.079]

[0.035;0.145]

[0.062; 0.097]

[0.017; 0.036]

[0.030; 0.052]

0.116

0.119

0.016

0.184

0.080

0.214

0.054

0.088

0.065

1

34

1

36

31

59

54

40

39

0

7

0

7

4

5

1

10

3

28

29^b

30^b

31

32

Family 6

33

0.454

0.072

0.121

0.041

0.344

0.062

0.043

[0.370; 0.550]

[0.043; 0.119]

[0.067; 0.218]

[0.025; 0.065]

[0.302; 0.393]

[0.035; 0.112]

[0.034; 0.053]

0.046

0.114

0.133

0.112

0.031

0.142

0.05

31

57

56

58

70

64

49

12

6

4

6

34^b

35^b

36^b

38^b

39^b

40

5

3

Family 7

41^b

42^b

43

0.072

0.136

0.031

0.015

0.017

0.010

0.016

0.256

0.268

[0.057; 0.092]

[0.093;0.200]

[0.015; 0.078]

[0.010; 0.020]

[0.011; 0.026]

[0.008; 0.013]

[0.014; 0.017]

[0.202; 0.324]

[0.209; 0.345]

0.053

0.099

0.237

0.057

0.105

0.045

0.019

0.052

0.073

86

55

19

26

73

6

2

89

1

7

3

4

16

7

5

4

5

1

44

45^b

46

47

48^b

50

Family 8

51

0.163

0.063

0.071

0.056

[0.008; 0.319]

[0.049; 0.082]

[0.054; 0.095]

[0.037; 0.087]

1.7

9

2

3

52

0.043

0.063

0.099

37

84

77

55^b

56^b

a

Compounds from family 1 which do not correspond to the ﬁrst screening selection criteria, but for which the IC₅₀values and viability of HFF were determined for

comparison purpose.

b

Compounds selected for the third screening on immune cells.

Values at 95% conﬁdence interval (CI); LI is the inferior limit of CI and LS is the superior limit of CI.

Standard error of the regression (SE), represents the average distance at which the measured values fall from the regression line.

Control HFF monolayers treated with 0.25% DMSO exhibited 100% viability.

c

d

e

f

Standard deviation of the mean (six replicate experiments).

chosen for the determination of IC₅₀values against T. gondii and

HFF viability assessments at 2.5 M (Table 6). The IC₅₀values

against T. gondii ranged between 0.01 and 0.454 M, and 30 com-

pounds exhibited an IC₅₀< 0.1 M, which is signiﬁcantly lower than

the value measured for the standard drug pyrimethamine

(0.326 M) [51]. The viability of HFF at 2.5 M compound concen-

applied at 2.5

than 30%.

mM, and 32 compounds caused viability loss higher

m

Compounds from family 1 showed a moderate to low host cell

toxicity and IC₅₀values against T. gondii- -gal ranging from 0.117 to

0.316

M. Interestingly, replacing the Cl^ꢀ(2) with a BF₄^ꢀas coun-

m

b

m

terion (6) decreased HFF toxicity, but also improved antiparasitic

activity. Interstingly, amino functionalized compound 6 appers to

present rather similar activity and toxicity proﬁle with the

tration ranged between 1 and 89% when compared to non-treated

cells. Eight compounds caused over 90% HFF cell death when

13

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

corresponding hydroxy derivative presenting the same BF^ꢀ₄coun-

terion H3.

All compounds assigned to families 2, 3 and 4 strongly impaired

the viability of HFF at 2.5 mM. For compounds belonging to family 2,

groups (46) as well as the simultaneous presence of methylene

spacers and of bulkier substituents in para position (47 and 50)

increased HFF toxicity. Among the four compounds exhibiting low

HFF toxicity, 48 showed the lowest, and 45 the highest activity

the presence of two polar groups that can also take part in H-

bonding interactions (derivatives 8 and 10 with two OH groups, and

9 with two NH₂substituents) led to increased HFF toxicity

compared to the mono-hydroxy and -amino compounds 1 and 2.

Interestingly, for diamino derivative 9 the T. gondii IC₅₀value was

signiﬁcantly lower compared to the IC₅₀values of the dihydroxy

against T. gondii-b-gal, with IC₅₀values of 0.256 and 0.017 mM,

respectively. Replacement of H with polar ﬂuoro substituents (41 vs

42) reduced both the anti-Toxoplasma activity and the viability of

HFF monolayers. Increased ﬂexibility (presence of methylene

spacers on the pendant substituents) and an increased number of

polar substituents that can potentially interact with molecules from

the environment, had a negative impact on HFF. Notably, the IC₅₀

analogues 8 and 10 (0.066 mM vs 0.115 and 0.294 mM), but com-

pound 9 was also more toxic for the host cells. The presence of the

methylene spacer on one of the pendant arms also inﬂuenced the

antiparasitic activity (8 vs 10). In family 3, both ester 15 and amide

values of compounds H1 and H2 (0.034 and 0.062

mM) are very low

against T. gondii- -gal, while the two compounds are non-toxic

b

against the host cells (IC₅₀values on HFF of 800 and > 1000 mM,

17 were highly cytotoxic for HFF at 2.5

mM, and the effect was much

respectively) [50,57].

stronger compared to the toxicity observed for the corresponding

hydroxy and amino analogues 1 and 2. In family 4, shifting the

position of the OH group from para (1) to meta (19) or ortho (20) led

to increased HFF toxicity. A similar effect was observed when

replacing the para Bu^tgroups (1) with less hydrophobic and bulky

In family 8, the hydroxy and amino derivatives 51 and 52 differ

from their analogues 55 and 56 by the position of the tri-

ﬂuoromethyl groups (para vs ortho) and the presence of methylene

spacers on two of the bridging thiols. However, these structural

differences had a strong inﬂuence on HFF toxicity. Both, 51 and 52

Me substituents (23). Interestingly, when applied at 2.5

mM ortho-

strongly affected HFF viability at 2.5 mM. As previously observed,

methylenehydroxy derivative 21 was signiﬁcantly more toxic for

the host cells compared to the ortho-hydroxy derivative 20. All

compounds from family 4 present methylene spacers on two of the

pendant thiol arms.

compounds exhibiting more rigid structures (the absence of the

methylene linkers), as well as the presence of polar groups in po-

sitions that are less prone to intermolecular interactions (ortho vs

para), were more likely to display reduced HFF toxicity. Thus, while

Compounds 29 and 30 from family 5, moderately impaired HFF

55 and 56 were both highly active against T. gondii-

b-gal (IC₅₀

viability when applied at 2.5

presented similar and low IC₅₀values against T. gondii-

0.072 and 0.078 M. In contrast, compounds 24 and 26 almost

completely abolished HFF viability. Derivatives 25, 27, 28, 31 and 32

applied at 2.5 M reduced HFF viability in a 31e40% range, but

T. gondii IC₅₀values were also lower, ranging from 0.013 to

0.056 M.

The position of the methoxy group appeared to be important for

the cytotoxicity against HFF, with para and ortho derivatives 28 and

31 being more toxic compared to the meta-substituted analogue 30.

For the para-substituted compounds 28 and 29, the replacement of

the hydrophobic methyl by the polar and bulkier triﬂuoromethyl

reduced host cell toxicity.

Five of the seven compounds assigned to family 6 only moder-

ately altered the viability of the host cells when tested at 2.5 mM,

while 33 and 40 exhibited increased HFF toxicity. In this series of

hydroxy analogues, a reduced ﬂexibility of the substituents present

on two of the thiols (no methylene spacers) had a positive effect on

host cell viability. 36 presents the lowest and 38 the highest IC₅₀

m

M (by 41 and 46%, respectively) and

values of 0.071 and 0.056 M, respectively), they presented reduced

HFF toxicity at a concentration that was 35 times the T. gondii IC₅₀

value.

m

b-gal of

m

Overall, the presence of one free (non-protected as ester or

amide) OH or NH₂group appeared to be important for antiparasitic

activity, but the presence of two of these groups had detrimental

effects on host cell viability. In addition, host cell toxicity was

inﬂuenced by the position of the OH group. The position and

number of other polar substituents such as CF₃also affected the

viability of the host cells. More rigid structures, lacking the meth-

ylene spacers on the pendant arms are to be favored. During the

m

secondary screening, 14 compounds, when applied at 2.5

mM,

reduced HFF viability by less than 50%. Compounds 6, 29, 30, 34, 35,

36, 38, 39, 41, 42, 45, 48, 55 and 56 were selected for the tertiary

screening to study their impact on splenocyte viability and

proliferation.

2.2.4. Tertiary screening

Previous studies [80,81] have proposed different types of

ruthenium complexes as potential immunosuppressive agents to

treat autoimmune diseases and allergic disorders. However, as this

study aimed at developing ruthenium-based compounds for the

treatment of T. gondii infection, any potential adverse reaction that

particularly suppresses host immunity should be avoided. An ideal

treatment stategy for toxoplasmosis would promote synergy be-

tween host immunity and the action of the applied antiparasitic

drug [82].

value against T. gondii-b-gal (0.041 and 0.344 mM, respectively),

suggesting that more hydrophobic substituents (Bu^tvs CF₃) can

favor antiparasitic activity. Interestingly, compounds 34, 35 and 36

bearing hydrophobic substituents Me, Prⁱand Bu^ton two of the

bridging thiols showed similar effects on the viability of the host

cells (42e44% reduction), but IC₅₀values against T. gondii were not

linearly dependent on the hydrophobicity/size of the substituents

(IC₅₀values of 0.072, 0.121 and 0.041

same peripheric ligands, ruthenium 36 and osmium 39 compounds

showed comparable effects against T. gondii- -gal and HFF.

mM, respectively). For the

The 14 selected compounds were evaluated for their immuno-

suppressive potential. B cells were stimulated with LPS [83,84] and

b

Replacement of the methyl groups on two of the thiols (34) by more

polar and bulkier triﬂuoromethyl groups (38) strongly reduced the

T cells with Con A [85], and the effects of 0.1 and 0.5 mM of each

compound were measured by assessing viability impairment by the

AlamarBlue assay. For the compounds for which treatments at

antiparasitic activity (IC₅₀values 0.072 vs 0.344

From the compounds assigned to family 7, 43, 44, 46, 47 and 50

strongly impaired HFF viability at 2.5 M (74e99%). The presence of

mM, respectively).

0.5 mM did not induce more than 50% of T cell death and less than

m

90% of B cell death, the potential to inhibit the proliferative re-

sponses of B and T cells was further assessed by BrdU (5-bromo-2⁰-

deoxyuridine) incorporation into DNA measured by ELISA

(enzyme-linked immunosorbent assay) [51].

As shown in Fig. 7A and B, 13 out of the 14 tested compounds

exerted dose-dependent effects on both B and T cells in vitro. The

three or six triﬂuoromethyl polar groups in para or meta positions,

which can be involved in intermolecular interactions (H-bonding,

polar) led to a considerable increase in HFF toxicity. Accordingly,

HFF viability was almost completely abolished in the case of com-

pounds 46, 47 and 50, suggesting that an increased number of polar

14

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Fig. 7. Tertiary screening of murine splenocyte cultures with 14 selected ruthenium complexes. The viability of splenocyte cultures stimulated with ConA (T cells, A) and LPS (B cells,

B) was measured by AlamarBlue assay. Cultures were treated with 0.1 M (dark grey bars) and 0.5 M (light grey bars) of each compound. Results are presented as ﬂuorescence

intensity at 590 nm (excitation at 530 nm), bars represent the mean emission of 4 replicates standard deviation. The numbers above the bars indicate percentage in relation to the

control (CTR ¼ cells only stimulated with ConA (A) and cells only stimulated with LPS (B)). CsA (cyclosporin A, 1 M) was used as a known immunosuppressant.

m

only exception was 41, which was highly toxic and completely

impaired T and B cell viability already at 0.1 M. The remaining 13

compounds did not exhibit excessive T cell toxicity at 0.1

bromo-2⁰-deoxyuridine) incorporation into DNA measured by

ELISA (enzyme-linked immunosorbent assay). The results (Fig. 8A

and B and Tables S3 and S4) demonstrate that T cell proliferation

m

mM

(viability ranging from 80 to 128% in relation to untreated cultures).

B Cells appeared to be generally more susceptible, with relative

values ranging from 35 to 100%. Toxicity was increased when

was not notably affected by any of the compounds at 0.1

the same was true for compounds 29, 42 and 55 at 0.5

ever, treatments at 0.5 M with compounds 6 and 35 had a negative

m

M, and

m

M. How-

m

treatments were carried out at 0.5

mM, with relative viability values

impact on T cell proliferative responses (Fig. 8A). B cell proliferation

ranging from 6 to 74% for T cells and from 2 to 22% for B cells.

Twelve compounds clearly exerted increased toxicity in B cells

compared to T cells. In contrast, cyclosporin A (CsA), a known

immunosuppressive drug used as control, had a more pronounced

effect on T cells compared to B cells (23 vs 59% remaining relative

viability).

was more strongly affected (Fig. 8B). Compounds 6, 29 and 35

exerted partial inhibition of B cell proliferation already at 0.1

and almost entirely abolished cell division at 0.5 M. The impair-

ment effect was less pronounced for compounds 42 and 55 at

0.5 M, with residual relative proliferation occurring at 39 and 52%

mM,

m

compared to non-treated controls, respectively. As observed in the

viabilty tests, cyclosporin A had stronger effect on the proliferation

of T cells compared to B cells (21 vs 60%).

Interestingly, for most compounds the toxicity values for HFF

and T/B cells were not comparable. For instance, treatments of HFF

with 55 and 56 at 2.5

effects (84 and 74% remaining viability, respectively, Table 6), while

the application of 0.5 M of the same compounds resulted in severe

m

M resulted in similarly moderate adverse

Compounds 6, 29, 35, 42 and 55 affected the viability and the

proliferation of B lymphocytes to a higher degree than T cells.

However, when tested at a concentration close to its IC₅₀(0.1 mM),

m

viability impairment of B and T cells (residual relative viability of 22

and 11%, and 55 and 27%, respectively, Fig. 7A and B, Tables S1 and

compound 55 neither affected the viability nor the proliferation of

immune cells. In addition, HFF remained largely viable when

S2). Likewise, 41 applied at 2.5

remaining viability, Table 6), but neither T nor B lymphocytes sur-

vived the treatments with 41 at 0.1 M (Fig. 7A and B, Tables S1 and

m

M did not display HFF toxicity (86%

treated with 2.5 mM (35 times the T. gondii IC₅₀value). As a com-

parison, the standard drug pyrimethamine, which is currently used

in the treatment of toxoplasmosis, was assessed similarly by BrdU

m

S2). These differences could be partly explained by the different

metabolic states of HFF and splenocytes. HFF monolayers are

largely conﬂuent, exhibit contact-inhibition and are proliferating

only slowly. ConA and LPS stimulated splenocyte cultures undergo

rapid proliferation and are metabolically much more active.

Nevertheless, these ﬁndings emphasize the necessity to include

various cell-types for toxicity testing, especially considering that all

nucleated cells are subject to infection with T. gondii [3].

ELISA at its T. gondii IC₅₀value (0.326 mM). Residual proliferation

after treatments amounted up to 88% in ConA stimulated cultures

(T cells) and 57% in LPS stimulated cultures (B cells). Based on these

values, compound 55 performs slightly better than pyrimethamine

and should be considered for further development.

The factors responsible for the observed differences in the sus-

ceptibility of B and T cells upon treatments with these compounds

should be investigated. The increased susceptibility of B cells could

be through interference in LPS binding to B cell receptors, inhibition

of early activation events, or modulation of accessory cells such as

macrophages which provide additional essential signals for B cell

Five compounds (6, 29, 35, 42 and 55) were further investigated

for their capacity to inhibit proliferative responses of T and B cells

stimulated with ConA and LPS, respectively, using the BrdU (5-

15

ꢀ

E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Fig. 8. Tertiary screening: effects of compounds 6, 29, 35, 42 and 55 on proliferative responses of murine splenocytes. T cell proliferation (A) was induced by the addition of ConA, B

cell proliferation (B) was induced by adding LPS. Compounds were added at 0.1 M (dark grey bars) and 0.5 M (light grey bars), and BrdU incorporation into DNA was measured by

ELISA assay. Bars represent standard deviation from the mean of three replicates Results are presented as absorption intensity at 450 nm, the numbers above the bars indicate

percentages in relation to the control (CTR ¼ ConA or LPS plus solvent only). CsA (cyclosporin A, 1 M) was used as a known immunosuppressant.

m

activation [86]. Despite presenting similar effects on the viability of

T and B lymphocytes, compounds 35, 42 and 55 exhibited divergent

proliferation inhibition rates. This showed that the induced cellular

events responsible for viability loss are different from those

responsible for cell division inhibition, and/or that compounds 35

(family 6), 42 (family 7) and 55 (family 8) presenting various

structural modiﬁcations do not share the same cellular targets.

Overall, based on its selective antiparasitic activity and moderate

direct effects on B and T cells, compound 55 appear as the most

promising for further studies.

Even if this SAR study on trithiolato diruthenium derivatives did

not provide conclusive rules for simultaneously improving speciﬁc

anti-Toxoplasma activity and reducing general cytotoxicity, some

structural elements that allowed selected compounds to possess

high toxicity against T. gondii and general low toxicity against B and

T cells were identiﬁed. These structural elements can constitute a

starting point in the design of new, efﬁcient and non toxic anti-

parasitic trithiolato dinuclear ruthenium compounds. Screening

alternative counterions should be considered (e.g., BF^ꢀ₄in 6 vs Cl^ꢀin

2). The presence of a polar group (e.g., OH or NH₂, but not CO₂H)

appears to be favorable. Protection of the hydroxy group as ether

could be interesting, but the nature of the protecting group might

also be important e.g., a polar triﬂuoromethoxy (29) instead of

methoxy group (28) should be favored. Reduced ﬂexibility of

groups pending on the bridge thiols by the absence of the methy-

lene spacers (e.g., 35 and 55 vs 1/H3) should be favored. The Bu^t

substituents on two of the bridge thiols (1/H3) could be replaced by

less hydrophobic/bulky Prⁱsubstituents (35) or by polar CF₃groups

(55), taking also into account the position of the substituents.

Replacement of the hydrophobic Me and Bu^tin the symmetric H1

and H2 with polar ﬂuoro groups in 42 also had positive effects.

Additional studies need to be carried out to elucidate which

structural features of these compounds mediate antiparasitic efﬁ-

cacy, and what modiﬁcations could further reduce HFF toxicity, as

well as alleviate viability impairment and antiproliferative effects

on B and T cells. Antiparasitic efﬁcacy is inﬂuenced by a direct

targeting of metabolic functions of the parasite and whether these

functions are essential, but also by the effects exerted on the host

cell. In addition, these compounds undergo various biophysical and

biochemical processes, such as passage through several layers of

membranes prior to reaching their target, and potential metabolic

breakdown once they are located intracellularly, all of which will

impact their stability and efﬁcacy. Previous transmission electron

microscopy (TEM) studies in Toxoplasma, Neospora and in

16

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E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

Trypanosoma demonstrated structural alterations in the parasite

mitochondrion early after initiation of treatments with ruthenium

complexes. In Toxoplasma and Neospora, these alterations included

distinct changes in the mitochondrial matrix, and in the structural

integrity of the parasite cristae, while HFF mitochondria remained

largely unaltered [50,51,57]. In T. brucei, disruption of the mito-

chondrial membrane potential was demonstrated by tetrame-

thylrhodamine ethyl ester assay, alterations in the trypanosome

mitochondrion were evidenced by immunoﬂuorescence employing

an antibody against mitochondrial Hsp70 and Mitotracker labeling,

and the mitochondrial matrix was converted into a translucent

mass lacking any cristae as seen by TEM [58]. Future investigations

should focus on elucidating the mechanism of action and on the

identiﬁcation of respective intracellular targets.

NMR spectroscopy, mass spectrometry and elemental analysis

data) are presented in detail in the Supporting information.

4.2. X-ray structure determination for 13, 38, 43 and P13

Experimental details regarding the X-ray structure determina-

tion for complexes 43 and P13 are presented in the Supporting

information.

Both the crystal of 13 and the crystal of 38 were mounted in air

at ambient conditions. All measurements were made on a RIGAKU

Synergy S area-detector diffractometer [87] using mirror optics

monochromated Cu K

a

radiation (

l

¼ 1.54184 Å) [88].

For 13 the unit cell constants and an orientation matrix for data

collection were obtained from a least-squares reﬁnement of the

setting angles of reﬂections in the range 5.724^ꢃ< 2

q

< 154.274^ꢃ. For

3. Conclusions

13 a total of 4310 frames were collected using u scans, with 0.05 s

exposure time, a rotation angle of 0.5^ꢃper frame, a crystal-detector

distance of 31.0 mm, at T ¼ 173(2) K. For 38 the unit cell constants

and an orientation matrix for data collection were obtained from a

least-squares reﬁnement of the setting angles of reﬂections in the

We report here a SAR study of a library of trithiolato-bridged

dinuclear ruthenium(II)-arene compounds, which was tested for

antiparasitic activity against T. gondii-b-gal tachyzoites, and cyto-

toxicity against HFF host cells and murine B and T cells. Systematic

structural modiﬁcations of the parent compounds H1, H2 and H3

resulted in 56 compounds that were assigned to eight families, with

successive structural variations, designed for increasing the anti-

parasitic activity and reduce the HFF and immune cell toxicity.

The activities of the compounds were assessed following a

sequential three-step screening protocol. A primary screening for

HFF toxicity and antiparasitic activity was undertaken with all 56

range 2.3^ꢃ<

using

q

< 77.1^ꢃ. For 38 a total of 2900 frames were collected

u

scans, with 1.3 and 5.19 s exposure time, a rotation angle of

0.5^ꢃper frame, a crystal-detector distance of 34.0 mm, at T ¼ 173(2)

K.

Data reduction was performed using the CrysAlisPro [87] pro-

gram. The intensities were corrected for Lorentz and polarization

effects, and an absorption correction based on the multi-scan

method using SCALE3 ABSPACK in CrysAlisPro [87] was applied.

Data collection and reﬁnement parameters are given in Table S5.

The structure was solved by direct methods using SHELXT [89],

which revealed the positions of all non-hydrogen atoms of the title

compound. All non-hydrogen atoms were reﬁned anisotropically.

For 13 H-atoms were assigned in geometrically calculated po-

sitions and reﬁned using a riding model where each H-atom was

assigned a ﬁxed isotropic displacement parameter with a value

equal to 1.2Ueq of its parent atom (1.5Ueq for methyl groups). For

13 all H-atoms were placed in geometrically calculated positions

and reﬁned using a riding model where each H-atom was assigned

a ﬁxed isotropic displacement parameter with a value equal to

1.2Ueq of its parent atom (1.5Ueq for the methyl groups, the hy-

droxy group and water).

compounds at 0.1 and 1

mM. 39 compounds, which, at a concen-

tration of 1 M, inhibited parasite proliferation by more than 95%

m

and impaired HFF viability by less than 49%, were chosen for the

next step. This provided ﬁrst insights into the biological effects of

the different structural variations. For instance, the presence of free

carboxy groups or more than one hydroxy and amino substituents

on the bridge thiols is detrimental, while that of polar CF₃groups

appears beneﬁcial. Methylene linkers in the pendant arms of two of

the bridge thiols should be avoided. Combining one polar hydroxy

or amino group on one bridge thiol and two CF₃substituents on the

other two thiols appears to be the best combination for ensuring

both anti-T. gondii activity and reduced toxicity to the host cells.

The secondary screening included dose-response assays to

determine the T. gondii IC₅₀values, and the assessment of HFF

Reﬁnement of the structures was carried out on F²using full-

matrix least-squares procedures, which minimized the function

Sw(F_o²e F²_c)². The weighting scheme was based on counting sta-

tistics and included a factor to down weight the intense reﬂections.

All calculations were performed using the SHELXL-2014/7 [90]

program in OLEX2 [91]. For 38 about 6% of the unit cell volume is

ﬁlled by heavily disordered co-crystallized solvent molecules. Its

electron density was accounted for by the SQUEEZE procedure of

PLATON [92].

toxicity of the compounds at 2.5

mM. The 14 compounds that

impaired HFF viability by less than 50% were subjected to the ter-

tiary screening that focused on the effects on the viability and the

proliferative potential in murine B and T cells. The results revealed

that the presence of one hydroxy group and polar CF₃groups ap-

pears essential, as 9 out of these 14 compounds possess one or both

of these two structural elements.

First the effects on the viability of immune cells were investi-

gated at 0.1 and 0.5

mM, and subsequently the 5 compounds

exhibiting the least detrimental effects against B and T cells were

further evaluated for their potential to inhibit proliferative re-

sponses. The results showed that B cells exhibited a higher sus-

4.3. Biological assessments

4.3.1. General

ceptibility than T cells. Compound 55 [(

h

⁶-p-MeC₆H₄Prⁱ)₂Ru₂(m₂

-

If not stated otherwise, the cell culture media were purchased

from Gibco-BRL (Zurich, Switzerland) and the biochemical reagents

were obtained from Sigma (St. Louis, MO, USA). The compounds

used for in vitro studies were stored as 1 mM stock solutions in

dimethylsulfoxide (DMSO) at ꢀ20 ^ꢃC. The HFF (human foreskin ﬁ-

broblasts, SCRC-1041.1) were purchased from ATCC and were

cultured and maintained as described earlier [93,94]. Tachyzoites of

SC₆H₄-o-CF₃)₂(m₂-SC₆H₄-p-OH)]Cl emerged as a suitable candidate

for further development.

4. Materials and methods

4.1. Chemistry

transgenic T. gondii strain containing a

b-galactosidase reporter

The chemistry related general information, the experimental

procedures used for the synthesis and the puriﬁcation of the

compounds, as well as characterization information (¹H and ¹³C

strain (T. gondii- -gal, with Tg-RH background) were kindly pro-

vided by Prof. David Sibley (Washington University, St. Louis, USA)

and were cultured in HFF, and were maintained and prepared for

b

17

ꢀ

E. Paunescu, G. Boubaker, O. Desiatkina et al.

European Journal of Medicinal Chemistry 222 (2021) 113610

infections as described earlier [93,94].

according to the standards set up by the animal welfare legislation

of the Swiss Veterinary Ofﬁce. The experimental protocol was

approved by the Commission for Animal Experimentation of the

Canton of Bern, Switzerland (Animal license No. BE101/17). Mice

were euthanized using isoﬂurane and CO₂, and spleens were

aseptically removed from euthanized mice. Spleens from female

BALB/c mice were removed and carefully minced by passing

4.3.2. Cytotoxicity in non-infected HFF

The cytotoxic effect was measured by AlamarBlue assay [49].

HFF were seeded into 96 well-plates at a density of 5 ꢄ 10³cells per

well and monolayers were grown to conﬂuency. For the screening,

the medium was removed, and monolayers received 200

medium containing 0.1, 1, or 2.5 M of each compound. Concen-

trations 0.1 and 1 M were tested in triplicate wells, and 2.5 M was

mL of fresh

m

through 40 mm cell strainers. After lysis of red blood cells, spleno-

m

cytes were counted and seeded in polystyrene 96 well ﬂat bottom

assessed in sextuplicate. Non-treated HFF were also included and

solvent controls contained DMSO at the corresponding concen-

trations of 0.01, 0.01 and 0.25%, respectively. Cultures were further

maintained at 37 ^ꢃC/5% CO_2.After 72 h, the medium was removed,

sterile plastic plates at 2 ꢄ 10⁵cells/100

mL/well. The impact of the

selected compounds on splenic T and B cell viability was assessed

by the AlamarBlue assay. Splenocyte cultures in 96 well-plates

were used either unstimulated (negative control), or stimulated

and cells were washed once with 200

m

L phosphate buffered saline

with ConA (5

plus compound. The compounds were tested at 0.1 and 0.5

Cyclosporine A (CsA) applied at 1 M was included as control strong

m

g/mL), LPS (10

m

g/mL), ConA plus compound or LPS

(PBS). Subsequently, 200 L PBS containing resazurin (10 mg/mL

m

mM.

dissolved in PBS) was added per well. Plates were read (excitation

530 nm, emission 590 nm wavelength) using an EnSpire multilabel

reader (2300 EnSpire™ Multilabel Reader, PerkinElmer, Turku,

Finland) immediately (T0) or after 5 h (T5). The cytotoxicity values

were calculated and represented as percentage of the respective

DMSO control, which represented 100% of HFF viability.

m

immunosuppressive compound. After 72 h of culture maintenance

at 37 ^ꢃC/5% CO₂, resazurin (0.1 mg/mL) was added, and the ﬂuo-

rescence intensity was measured immediately (T0), and then after

5 h (T5). Data are presented as mean of emission standard de-

viation (SD) for the indicated numbers. Data comparisons between

groups were examined using a Student's t-test (signiﬁcant when

p < 0.001). To measure the impact of compounds on the prolifer-

ative potential of B and T cells, the 5-bromo-2⁰-deoxyuridine (BrdU)

cell proliferations assay was performed. Spleen cells were exposed

4.3.3. Activity against T. gondii-

b-gal tachyzoites

The transgenic T. gondii- -gal strain cultured in HFF was used to

b

assess tachyzoites proliferation inhibition [49]. HFF were seeded

into 96 well-plates at a density of 5 ꢄ 10⁵cells per well and

monolayers were grown to conﬂuency. Monolayers were infected

to two concentrations (0.1 and 0.5

ence of ConA (5 g/mL) or LPS (10

Controls were splenocyte cultures with ConA or LPS devoided of

drugs, or without treatment. Cyclosporine A (1 M) treatment was

m

M) of compounds in the pres-

m

g/mL) for 72 h at 37 ^ꢃC/5% CO₂.

with 10³T. gondii-

b-gal tachyzoites per well, and drugs (dilutions

from the 1 mM stock solutions) were applied to cultures concom-

itantly with the infection.

m

also included in this assay. Proliferation was assessed with a BrdU

cell proliferation kit (QIA58, Merck Millipore). BrdU label was

added to the cultures 18 h prior the end of the incubation period,

and incorporation of BrdU into DNA was measured by ELISA using

peroxidase-conjugated anti-BrdU monoclonal antibody. Immedi-

ately after stopping the reaction, the absorbance was measured

using dual wavelength of 450/540 nm, in an EnSpire multilabel

reader (PerkinElmer, Waltham). Data are presented as mean SD

for the indicated numbers. Data comparisons between groups were

examined using a Student's t-test (signiﬁcant when p < 0.01).

Initial assessment of drug efﬁcacy was realized by exposing

parasite cultures to 0.1 and 1 mM solutions of each compound in

triplicate at 37 ^ꢃC/5% CO₂over a period of 72 h, including DMSO

solvent controls (at concentrations of 0.01 and 0.1%, respectively).

For IC₅₀determinations, the compounds were added in the

following concentrations: 0.007, 0.015, 0.03, 0.06, 0.12, 0.25, 0.5 and

1 mM (sextuplicate for all), and cultures were maintained for 72 h at

37 ^ꢃC/5% CO_2.As solvent control, a solution of 0.1% DMSO (repre-

senting the highest DMSO concentration in the test) was used.

Subsequently the medium was removed, wells were washed

once with 200

containing 0.05% Triton X-100 per well. After addition of 10

5 mM chlorophenol red- -galactopyranoside dissolved in PBS

(CPRG, Roche Diagnostics, Rotkreuz, Switzerland), the absorption

shift was measured at wavelength 570 nm at various timepoints on

a EnSpire multilabel reader (2300 EnSpire™ Multilabel Reader,

PerkinElmer, Turku, Finland). For the initial screening at 0.1 and

m

L PBS, and cells were permeabilized with 90

m

L PBS

Declaration of competing interest

The authors declare no competing ﬁnancial interest.

Acknowledgments

mL of

b-D

This work was ﬁnancially supported by the Swiss Science Na-

tional Foundation (SNF, Sinergia project CRSII5-173718 and project

no. 310030_184662) and Y.A. was supported by a Swiss Govern-

mental Excellence Fellowship. The X-ray crystal structure deter-

mination service unit of the Department of Chemistry,

Biochemistry and Pharmaceutical Sciences of the University of Bern

is acknowledged for measuring, solving, reﬁning and summarizing

the structures of compounds 13, 38, 43 and P13. The Synergy

diffractometer was partially funded by the SNF within the R'Equip

programme (project no. 206021_177033).

1

m

M, 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

b

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 calcu-

lated after the logit-log transformation of the relative growth (RG,

control ¼ 1) according to the formula ln(RG/[1-RG]) ¼ a$ln(drug

concentration)þb and subsequent regression analysis by the cor-

responding software tool contained in the Excel software package

(Microsoft, Seattle, WA, USA).

Appendix A. Supplementary data

4.3.4. Activity against murine B and T cells stimulated with LPS and

ConA, respectively

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113610.

Spleen cell suspensions were prepared as previously described

[51]. Female BALB/c mice were purchased from Charles River Lab-

oratories (Sulzfeld, Germany) and were maintained in a common

room under controlled temperature and a 14 h dark/10 h light cycle

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The quest of the best – A SAR study of trithiolato-bridged dinuclear Ruthenium(II)-Arene compounds presenting antiparasitic properties

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