Identification of descriptors for structure-activity relationship in ruthenium (II) mixed compounds with antiparasitic activity

Article abstract of DOI:10.1016/j.ejmech.2020.112084

Herein is presented the synthesis, characterization, electrochemical studies, DFT calculations and in vitro evaluation of amoebicidal activity in trophozoites of Entamoeba histolytica of twenty ruthenium (II) mixed compounds with general formulae: [Ru(pdt

Full text of DOI:10.1016/j.ejmech.2020.112084

Journal Pre-proof
Identification of descriptors for structure-activity relationship in ruthenium (II) mixed
compounds with antiparasitic activity
Erika Lorena Cedillo-Gutiérrez, Luis Felipe Hernández-Ayala, Carolina Torres-
Gutiérrez, Miguel Reina, Marcos Flores-Alamo, Julio C. Carrero, Víctor M. Ugalde-
Saldívar, Lena Ruiz-Azuara
PII:
S0223-5234(20)30051-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112084
EJMECH 112084
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 October 2019
Revised Date: 19 January 2020
Accepted Date: 19 January 2020
Please cite this article as: E.L. Cedillo-Gutiérrez, L.F. Hernández-Ayala, C. Torres-Gutiérrez, M. Reina,
M. Flores-Alamo, J.C. Carrero, Ví.M. Ugalde-Saldívar, L. Ruiz-Azuara, Identification of descriptors for
structure-activity relationship in ruthenium (II) mixed compounds with antiparasitic activity, European
Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112084.
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Identification of descriptors for structure-activity relationship in ruthenium (II)
mixed compounds with antiparasitic activity
Erika Lorena Cedillo-Gutiérrez1, Luis Felipe Hernández-Ayala1, Carolina Torres-Gutiérrez1, Miguel
Reina1, Marcos Flores-Alamo2, Julio C. Carrero3, Víctor M. Ugalde-Saldívar*4 and Lena Ruiz-
Azuara*1
1Laboratorio de Química Inorgánica Medicinal, Facultad de Química Universidad Nacional
Autónoma de México, Av. Universidad 3000, Circuito Exterior s/n, CU, P.O. Box 70-360, 04510
México City, México.
2USAI, Facultad de Química Universidad Nacional Autónoma de México, Av. Universidad 3000,
Circuito Exterior s/n, CU, P.O. Box 70-360, 04510 México City, México.
3Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional
Autónoma de México, Universidad 3000, Circuito Exterior s/n, CU, P.O. Box 70-360, 04510
México City, México.
4Departamento de Química Inorgánica y Nuclear, Facultad de Química Universidad Nacional
Autónoma de México, Av. Universidad 3000, Circuito Exterior s/n, CU, P.O. Box 70-360, 04510
México City, México.
Abstract
Herein is presented the synthesis, characterization, electrochemical studies, DFT
calculations and in vitro evaluation of amoebicidal activity in trophozoites of Entamoeba
histolytica of twenty ruthenium (II) mixed compounds with general formulae: [Ru(pdto)(E-
E)]Clx (E-E bidentate, either neutral or negatively charged ligands). For compounds under
study, O-O, N-O and N-N auxiliary donor ligands demonstrate to have a crucial impact on
the electronic properties and that it is possible to modulate the antiparasitic activity. Among
analyzed complexes, only four present a better performance compared to typically used
metronidazole drug (IC50< 6.80 µmol/L) to treat amoebiasis disease. For studied
compounds, structure-activity relationships are strongly determined by either the redox
potential (E1/2) of RuII/RuIII and calculated molar volume (V) of the complexes.
* Corresponding author: lenar701@gmail.com Phone number (0052-55) 56 22 3529
1

Keywords: ruthenium (II) mixed complexes, amebiasis, amoebicidal activity, SAR studies,
DFT calculations.
1. Introduction
Amebiasis is a harmful tropical disease responsible for approximately 100,000 worldwide
annual deaths [1-5]. It is caused by the protozoan parasite Entamoeba histolytica, an
important pathogen with major global impact [6,7] that colonizes the human gut through
contaminated water or food and might cause life-threatening illness such as liver abscesses
and hemorrhagic colitis [7-10]. After malaria (Plasmodium malaria), amebiasis is the
second leading cause of death due to a protozoan parasite [11]. To face this serious public
health care issue, nitroimidazoles derived compounds have been used for the last decades
since they are considered to be the most effective against amebiasis disease [12-14]. In
particular, metronidazole, tinidazole, ornidazole and secnidazole are effective drugs for
treating amebiasis [15]. Nevertheless, irritation of the gastric mucus lining, vomiting,
diarrhea, blood in urine, headache, and with less frequently a central nervous system
toxicity are important side effects [16,17]. In addition, there is an increasing concern in the
development of E. histolytica strains resistance to these drugs [18].
As an alternative to the widely nitroimidazole-type treatments, metal containing
compounds arise as an attractive, versatile and powerful option to mitigate this disease [19-
22]. In fact, medicinal inorganic chemistry is a rapidly growing field with a direct impact
on the diagnosis and therapy of several diseases. In recent years, novel metal-based
anticancer drugs have been developed to reduce toxicity, to increase clinical efficacy and to
broaden the scope of activity [23-26]. The rational design of these metal-centered
molecules allows tuning some important features, such as coordination modes, geometries
2

and redox behavior and thus, enhances the capability to participate in biological processes.
It has been reported that these systems are useful to interact with DNA and other important
biomolecules, to promote reactive oxygen species (ROS) generation, and to induce cellular
apoptosis [27-35]. Additionally, divalent complexes of the first-row transition metals (Mn,
Fe, Co, Ni, Cu and Zn) and Ru have shown excellent amoebicidal activity [36,37]. In
particular, for some RuII chiral heteroleptic complexes with amoebicidal in vitro and in vivo
activity, it has been recently demonstrated that trophozoites exposed to these compounds
die through an apoptotic pathway triggered by ROS production without apparent cytotoxic
effect to human peripheral blood lymphocytes. Furthermore, generated ROS shown a direct
relationship with the redox potential as well as with the IC50 values. As the redox potential
becomes more positive, a decrease in ROS generation and an increase in the IC50 value
have been recorded [37]. Additionally, another advantage of these inorganic metal-based
compounds over nitroimidazoles is their high solubility in water, which would help in their
use for treatment when the parasite has migrated to a vital organ or when the ameba has
become cystic [38,39].
Furthermore, pdto (1,8-bis-2-pyridyl-3,6-dithioctane) is a flexible open-chain tetradentated
ligand with nitrogen and sulfur as donor atoms [40,41]. In addition, several complexes have
been found to be stable showing uni to four-dentate coordination modes [42-44]. Pdto
related systems have been proposed to act as biological materials (interaction with DNA
and cyclodextrins), conductive polymers, and SOD mimetics [44-47]. Besides, octahedral
Ru complexes have been suggested to play an important role in medicinal inorganic
chemistry acting as anticancer, antimicrobial, antifungal and antiparasitic agents [48-51].
3

In the last decade, various heteroleptic compounds of RuII with pdto and different auxiliary
ligands have been synthesized, characterized and tested showing an important in vitro and
in vivo amoebicidal activity. In particular, acetylacetonate, glycinate and ethylenediamine
Ru-pdto complexes show a relationship between biological activity, ROS production and
redox potential [52].
The aim of this work is to find a structure-activity relationship in ruthenium (II) mixed
compounds with antiparasitic activity. To this purpose, twenty complexes of the type
[Ru(pdto)(E-E)]Clx (E-E bidentate, either neutral or negatively charged ligands, Scheme 1)
were synthesized, characterized by diverse experimental techniques (IR, 1H-NMR, UV-vis,
Log P and redox potential, E1/2) and DFT studies and then, evaluated in vitro in
trophozoites of Entamoeba histolytica.
Scheme 1. Molecular structure of [Ru(pdto)(E-E)]Clx complexes.
To assess the relationship between the in vitro antiparasitic activity and the structure of
these complexes, several experimental and computational parameters were taken into
account. The best structure-activity correlation results arise when redox potential
(experimental and calculated) and theoretical molar volume are used, (alone and
combined). These results could serve to estimate the antiparasitic activity of ruthenium (II)
mixed compounds with only a few electronic and molecular descriptors. Furthermore, these
4

results could also promote the use of theoretical calculations to predict biological activity of
different complexes.
2. Results and discussion
2.1.- Chemistry
Scheme 2 presents the three general synthetic routes followed to obtain the complexes
under study. In all the cases, [Ru(pdto)Cl(PPh3)]Cl was the precursor and only slight
changes are observed between different paths. The general formulae of these complexes is
[Ru(pdto)(E−E)]Clx, in which pdto ligand remains unchanged and coordinated to Ru, and
only the secondary ligand varies (E-E). All auxiliary E-E ligands are bidentate and can be
either neutral or negatively charged ligands. The molecular structures of E-E ligands are
also presented in Scheme 2. It is possible to distinguish N-N donors (synthetic path 1; E-E:
1 to 3 and 7 to 16), O-O donors (synthetic path 2.a corresponds to E-E: 4 and 2.b concerns
E-E: 17 to 20) and mixed N-O donors (synthetic path 3.a and 3.b correspond to E-E: 5 and
6, respectively).
To study these complexes, four main groups were established. The first one contains
pristine auxiliary ligands, i.e. without any structural modification (1 to 6: 1-10-
phenanthroline, 2,2'-bipyridine, ethylenediamine, acetylacetonate, glycinate and
methioninate). The second group includes the 1,10-phenanthroline substituted compounds
(7 to 14). For this group, an additional categorization was done: compounds 7 to 10 are
1,10-phenanthroline substituted in position 5, while complexes 11 to 14 are substituted in
position 4. The third group correspond to the systems containing the two substituted 2,2'-
bipyridine ligands (15 and 16) and the last group is related to the salicylaldehydate
compounds (17-20). As it can be seen in Scheme 2, systems 2, 6 to 9, 11 and 15 to 20 are
5

marked with star indicating that they have been synthesized and characterized for the very
first time, meanwhile, the other 8 complexes (1, 3-5, 10, 12-14) have been already been
presented in other studies [37,42]. For compounds analyzed, racemic mixtures were tested.
1.- [Ru(pdto)(1-10-phenanthroline)]Cl2
2.- [Ru(pdto)(2,2´-bipyridine)]Cl2
3.- [Ru(pdto)( thylenediamine)]Cl2
4.- [Ru(pdto)(acetylacetonate)]Cl
5.- [Ru(pdto)(glycinate)]Cl
11.- [Ru(pdto)(4-methyl-1-10-phenanthroline)]Cl2*
12.- [Ru(pdto)(4,7-dimethyl-1-10-phenanthroline)]Cl2
13.- [Ru(pdto)(4,7-diphenyl-1-10-phenanthroline)]Cl2
14.- [Ru(pdto)(3,4,7,8-tetramethyl-1-10-phenanthroline)]Cl2
15.- [Ru(pdto)(4,4'-dimethyl-2,2'-bipyridine)]Cl2*
*
e
6

16.- [Ru(pdto)(5,5'-dimethyl-2,2'-bipyridine)]Cl2*
17.- [Ru(pdto)(5-nitro-salicylaldehydate)]Cl*
18.- [Ru(pdto)(5-chloro-salicylaldehydate)]Cl*
6.- [Ru(pdto)(methioninate)]Cl
7.- [Ru(pdto)(5-nitro-1-10-phenanthroline)]Cl2
8.- [Ru(pdto)(5-chloro-1-10-phenanthroline)]Cl2
9.- [Ru(pdto)(5-methyl-1-10-phenanthroline)]Cl2
10.- [Ru(pdto)(5,6-dimethyl-1,10-phenanthroline)]Cl2
*
*
*
*
19.- [Ru(pdto)(5-methoxy-salicylaldehydate)]Cl
20.- [Ru(pdto)(5-methyl-salicylaldehydate)]Cl
*
*
Scheme 2. The twenty heteroleptic complexes of RuII under study.
2.2.- Spectroscopic and electrochemical characterization
The complete characterization data for the new compounds can be founded in the S1
section of the Supplementary Material. IR spectra analysis show that significant shifts
occur for complexes compared to the free ligands, i.e. pdto (30-50 cm-1) and E-E secondary
ligands (50-100 cm-1), suggesting that they are properly coordinated to Ru atom, as
previously reported [42]. Furthermore, for the systems under examination, λmax transitions
correspond to the metal-ligand charge transfer, around 330-360 nm. In all cases,
compounds present high molar extinction coefficient values as expected for M-L charge
1
transfer (ε from 2402 to 17862 mol L-1cm-1). H-NMR, 13C-NMR, COSY and HSQC
analysis were also done to characterize the complexes. Additionally, conductivity
evaluations are in accordance with expected global charge of the complexes (1:1 or 1:2
electrolyte behavior). Finally, single crystals for complexes 2, 5 and 15 were obtained and
analyzed by X-ray diffraction technique (Section S2: Figures S1-S4 and Tables S1-S5).
Figure 1 shows the voltammogram of [Ru(pdto)(5,5'-dimethyl-2,2'-bipyridine)]PF6
compound acquired with a glassy carbon electrode from open circuit potential to cathodic
-
direction. It is important to signal that, Cl- counter ion was exchanged by PF6 to avoid Cl-
oxidation to Cl2 interference. This same methodology was employed for all analyzed
voltammograms. Two reduction peaks IC, IIC and their respective oxidation waves IA, IIA,
were observed. Electronic transfer Ia is associated with the [RuII(pdto)(L)]/[RuIII(pdto)(L)]
process, wave IIC correspond to [RuII(pdto)(L)]/[RuII(pdto)(L-)] reduction. Half wave
7

potentials values (E1/2) for process I and II are 1.03 and -1.85 V/Fc+-Fc, respectively.
Although, remain compounds present different redox behavior, for this study, only
RuIII/RuII redox values were taken into account. For all the other compounds under study,
half wave potentials are shown in Table 1.
Figure 1. Cyclic voltammogram of 0.001 mol/L [Ru(pdto)(5,5'-dimethyl-2,2'-bipyridine)](PF6) , 0.2
mol/L TBABF4 in MeCN, v= 0.3 V/s.
Table 1 present redox potential values for ruthenium (II) mixed compounds. It has been
already proposed that redox potential could play a critical role in modulating the biological
activity, at least for copper and ruthenium systems [53-55]. For complexes under study, E1/2
for RuIII/RuII redox pair, are in range between 0.33 and 1.15 V/Fc+-Fc values. It is clear that
compounds containing O-O and N-O donors as auxiliary ligands present lower E1/2 values
(E1/2 = 0.33-0.91 V/Fc+-Fc) compared to N-N secondary ligands (E1/2 = 1.03-1.15 V/Fc+-
Fc). This suggests that the former group is easier to oxidize than the latter, which
corresponds to better reducing systems. It is worthy to note that
[Ru(pdto)(ethylendiamine)]Cl2 (E1/2 = 0.85 V/Fc+-Fc) is the only exception and one
8

plausible explanation refers to the very small size and a complete saturated degree of
ethylendiamine ligand. In addition, in Table 1, experimental partition coefficient (P) values
are presented. Although, Log P is a very useful descriptor to classify hydrophilic behavior
of organic molecules, for systems containing metal atoms and in these particular cases, no
trends are observed. Log P behavior is complicated to analyze, and values range from -1.79
to 0.13, but no tendencies were detected. Log P results suggest that for metallodrugs
systems, this parameter might not be the most accurate and useful.
Table 1. Redox potential (E1/2) and partition coefficient values (Log P)
E1/2 RuII/RuIII
(V/ Fc+-Fc)
1.08
Log P
(octanol/water)
-1.25
Compounds
[Ru(pdto)(1-10-phenanthroline)]Cl2
[Ru(pdto)(2,2'-bipyridine)]Cl2
[Ru(pdto)(ethylenediamine)]Cl2
[Ru(pdto)(acetylacetonate)]Cl
[Ru(pdto)(glycinate)]Cl
1.09
0.85
0.44
0.58
-1.05
-1.33
-0.24
-1.32
[Ru(pdto)(methioninate)]Cl
0.55
-1.41
[Ru(pdto)(5-nitro-1-10-phenanthroline)]Cl2
[Ru(pdto)(5-chloro-1-10-phenanthroline)]Cl2
[Ru(pdto)(5-methyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(5,6-dimethyl-1,10-phenanthroline)]Cl2
[Ru(pdto)(4-methyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,7-dimethyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,7-diphenyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(3,4,7,8-tetramethyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,4'-dimethyl-2,2'-bipyridine)]Cl2
[Ru(pdto)(5,5'-dimethyl-2,2'-bipyridine)]Cl2
[Ru(pdto)(5-nitro-salicylaldehydate)]Cl
1.15
1.15
1.02
1.08
1.10
1.13
1.06
1.01
-0.72
-0.85
-1.17
-0.87
-1.39
-0.95
-0.82
-1.16
1.04
1.03
0.33
-1.79
-1.62
0.13
[Ru(pdto)(5-chloro-salicylaldehydate)]Cl
0.45
0.13
[Ru(pdto)(5-methoxy-salicylaldehydate)]Cl
[Ru(pdto)(5-methyl-salicylaldehydate)]Cl
0.69
0.91
-0.10
0.06
2.3.- DFT calculations
DFT geometry optimizations were performed for RuII complexes assuming a distorted
octahedral arrangement with low multiplicity (singlets or doublets) as it has been
previously observed and reported [37, 42]. In Figure S5, geometry optimizations and
Molecular Electrostatic Potential (MEP) maps are presented and in Table S6, bond lengths
9

and angles for these optimized geometries are reported. In general, the values are in
accordance with the X-ray structures.
Table 2 reports some calculated parameters: molar volume (V, cm3/mol), electron affinity
(EA, eV) and RuIII/RuII redox potential (E1/2(calc)). The values of simulated redox potential
are compared to the experimental results (E1/2(exp)) showing a strong correlation (R2=0.93,
Figure S6 in the Supporting Information). This is important to corroborate the
computational employed strategy. It is noteworthy that also electron affinity presents an
important linear correlation when compared to experimentally founded redox potentials
(R2=0.92, Figure S6). The best electron acceptor system is [Ru(pdto)(4,7-diphenyl-1-10-
phenanthroline)]Cl2 and results imply that 1-10-phenanthroline systems and 2,2'-bipyridine
are much better electron acceptor systems compared to oxygen donor ligands (group of
salicylaldehydate compounds). One possible explanation could arise from the well-known
1-10-phenanthroline and 2,2'-bipyridine electron delocalization and planarity properties.
Furthermore, the obtained molar volume values are in accordance with the expected size of
auxiliary ligands. In general, bulkier systems correspond to the substituted 1-10-
phenanthroline systems, followed by the substituted 2,2'-bipyridine and finally the
salicylaldehydate complexes.
10

Table 2. Calculated and experimental physicochemical properties of RuII-pdto complexes.
EA
(eV)
20.42
19.55
15.79
6.87
11.31
10.04
16.93
19.79
19.31
19.49
19.81
19.24
22.46
18.17
19.45
19.47
5.51
E1/2 calc
(V)
E1/2 exp
(V/Fc+-Fc)
1.08
V
Compounds
(cm3/mol)
329.45
1.11
1.00
0.68
0.28
0.41
0.43
1.06
1.12
0.92
0.98
0.89
0.95
0.99
0.76
0.92
0.9
[Ru(pdto)(1-10-phenanthroline)]Cl2
[Ru(pdto)(2,2'-bipyridine)]Cl2
[Ru(pdto)(ethylenediamine)]Cl2
[Ru(pdto)(acetylacetonate)]Cl
[Ru(pdto)(glycinate)]Cl
322.44
275.53
262.57
253.22
351.80
1.09
0.85
0.44
0.58
0.55
1.15
1.15
1.02
1.08
1.10
1.13
1.06
1.01
1.04
1.03
0.33
[Ru(pdto)(methioninate)]Cl
[Ru(pdto)(5-nitro-1-10-phenanthroline)]Cl2
[Ru(pdto)(5-chloro-1-10-phenanthroline)]Cl2
[Ru(pdto)(5-methyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(5,6-dimethyl-1,10-phenanthroline)]Cl2
[Ru(pdto)(4-methyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,7-dimethyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,7-diphenyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(3,4,7,8-tetramethyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,4'-dimethyl-2,2'-bipyridine)]Cl2
[Ru(pdto)(5,5'-dimethyl-2,2'-bipyridine)]Cl2
[Ru(pdto)(5-nitro-salicylaldehydate)]Cl
359.19
342.10
360.58
376.45
347.96
359.38
427.33
403.71
341.24
368.83
332.41
296.62
354.75
309.13
0.47
0.40
0.76
0.87
7.47
10.31
17.04
0.45
0.69
0.91
[Ru(pdto)(5-chloro-salicylaldehydate)]Cl
[Ru(pdto)(5-methoxy-salicylaldehydate)]Cl
[Ru(pdto)(5-methyl-salicylaldehydate)]Cl
2.4.- Antiparasitic activity
The half inhibitory concentration (IC50,
µmol/L) results for the in vitro evaluation of
Entamoeba histolytica parasite and ruthenium (II) mixed compounds are presented in Table
3 and Figure 2. Metronidazole is the most common drug for treating amebiasis and its IC50
is also presented for comparison purposes (IC50= 6.80 µmol/L and Log (1/IC50)= -0.83).
The obtained values present a wide range due to the strong modulation activity of the
auxiliary ligand donor nature. In addition, the size, the global charge, the number of
aromatic rings, the planarity and the electro donating and releasing groups of each complex
play a crucial role in the amoebicidal activity. In this regard, IC50 values varies from 0.06 to
556.00
µmol/L. Comparing the established groups, substituted 2,2'-bipyridine are better
than substituted 1,10-phenanthroline compounds but worse than salicylaldehydate systems.
The best family correspond to the complexes containing pristine ligands. 1,10-
11

phenanthroline and 2,2'-bipyridine are better than their substituted ligands and it is worthy
to note that among studied compounds, the more effective systems compared to
metronidazole drug reference belong to this non substituted ligand group. In fact, 2,2'-
bipyridine, ethylendiamine, acetylacetonate and glycinate compounds present a lower IC50
value compared to metronidazole (IC50 = 3.70, 0.14, 0.06 and 0.12
6.80 mol/L for metronidazole, dark green bars in Figure 2: Log (1/IC50) > -0.83). From all
the analyzed complexes, [Ru(pdto)(acetylacetonate)]Cl is the more effective to in vitro treat
Entamoeba histolytica (IC50 = 0.06 mol/L). This system is characterized for having a
µmol/L, respectively vs
µ
µ
small, charged, and O-O donor auxiliary ligand. In general, [Ru(pdto)(E-E)]2+ are more
hydrophilic systems compared to [Ru(pdto)(E-E)]+, and thus, the cross through the cellular
membrane could be more difficult. This is in accordance with the MEP diagrams (Figure
S5), in which the electron density distribution for [Ru(pdto)(methioninate)]+ ([Ru(pdto)(E-
E)]+) present a more homogenous distribution and less positive values. Some exceptions
can be observed, principally due to the size or the positions of substituents ([Ru(pdto)(2,2'-
bipyridine)]2+, IC50= 3.70
µ
mol/L and [Ru(pdto)(5,6-dimethyl-1,10-phenanthroline)]2+,
IC50= 22 mol/L). Furthermore, 10 complexes present a similar IC50 value compared to
µ
metronidazole (Figure 2, green bars: -0.83 > Log (1/IC50) > -1.50) but metal containing
compounds offer some advantages: high solubility in water, a priori no resistance and no
harmful side-effects to development of E. histolytica, and ROS production, which increase
apoptosis mechanism [36,37,57,58]. Hence, 14 synthesized compounds might be suitable
and considered as candidates to metallo-drugs against amebiasis disease. Finally, in order to
verify the antiparasitic effect of analyzed compounds, a UV-vis experiment measuring their
stability in aqueous solution was performed. Results indicate that compounds are stable at
12

different times (0, 12, 24, 48 and 72 hours) and this might suggest that [Ru(pdto)(E-E)]Clx
systems are responsible for the biological activity (Figure S7).
Table 3. IC50 value tested in trophozoites of Entamoeba histolytica.
IC50 (µmol/L)
Log (1/IC50)
Compounds
Metronidazole
6.80
12.00
3.70
0.14
0.06
-0.83
-1.08
-0.57
0.85
1.22
0.92
[Ru(pdto)(1-10-phenanthroline)]Cl2
[Ru(pdto)(2,2'-bipyridine)]Cl2
[Ru(pdto)(ethylenediamine)]Cl2
[Ru(pdto)(acetylacetonate)]Cl
[Ru(pdto)(glycinate)]Cl
0.12
[Ru(pdto)(methioninate)]Cl
10.00
-1.00
[Ru(pdto)(5-nitro-1-10-phenanthroline)]Cl2
[Ru(pdto)(5-chloro-1-10-phenanthroline)]Cl2
[Ru(pdto)(5-methyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(5,6-dimethyl-1,10-phenanthroline)]Cl2
[Ru(pdto)(4-methyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,7-dimethyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,7-diphenyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(3,4,7,8-tetramethyl-1-10-phenanthroline)]Cl2
[Ru(pdto)(4,4'-dimethyl-2,2'-bipyridine)]Cl2
[Ru(pdto)(5,5'-dimethyl-2,2'-bipyridine)]Cl2
[Ru(pdto)(5-nitro-salicylaldehydate)]Cl
106.00
556.00
422.00
22.00
152.00
48.00
11.00
117.00
17.00
17.50
9.00
-2.02
-2.74
-2.62
-1.34
-2.18
-1.68
-1.04
-2.07
-1.23
-1.24
-0.95
-1.22
-1.11
-1.28
[Ru(pdto)(5-chloro-salicylaldehydate)]Cl
[Ru(pdto)(5-methoxy-salicylaldehydate)]Cl
[Ru(pdto)(5-methyl-salicylaldehydate)]Cl
11.00
13.00
19.20
2.0
log (1/IC50) > -0.83
log (1/IC50) = -0.83
-0.83 > log (1/IC50) > -1.50
log (1/IC50) < -1.50
1.0
0.0
-1.0
-2.0
-3.0
-4.0
-5.0
Figure 2. Log (1/IC50) values for [Ru(pdto)(E-E)]Clx and metronidazole (reference value) tested in
trophozoites of Entamoeba histolytica.
13

2.5.- Physicochemical indexes related to amoebicidal activity
Structure-activity relationships are a very powerful tool to explain and to predict particular
biological behaviors according to some physicochemical or steric parameters. For organic
molecules, there are well-known geometric and electronic factors that are commonly used
to a priori assess the activity. In many cases, this allows to rationally design molecular
systems with ad hoc desired properties and enhanced activity. For compounds containing
metal atoms, these relationships are often very difficult to obtain. In the present work, to
establish a proper approach relating structure and biological activity, several parameters
were taken into account, but only the redox potential and the calculated molar volume were
accurate to describe the observed antiparasitic activity. Figure 3 presents the results for
each group of compounds under study. To rationalize the obtained results, an equation
considering the two parameters (E1/2 and V) was proposed:
Log (1/IC50) = x E1/2 + y Log V
Where x and y are the normalized fractions, i.e. x + y =1, corresponding to the redox
potential and molar volume, respectively. The proposed equation implies that depending on
the group studied, the relative importance of the two parameters varies. For example,
pristine auxiliary ligands are only correlated to the molar volume (R2= 0.9285; Figure 3.a),
and for salicylaldehydate complexes, the redox potential alone correctly fits (R2= 0.9646;
Figure 3.e). For the former, the most active compound is the acetylacetone derivative, and
for the latter, the best amebicide agent is the one with the lowest redox potential value. This
same behavior has been already described for other Ru-pdto complexes and amoebicidal
activity [37]. For all the other families the ratio of the redox potential and the molar volume
oscillates, indicating that both parameters are important and contribute to explain the
antiparasitic activity. For both 1,10-phenanthroline groups (Figures 3.b and 3.c, R2=0.9689
14

and R2=0.9850, respectively), the volume contribution is more important than E1/2 and the
bulkier the complex the more efficient to inhibited Entamoeba histolytica. In this family the
compound that shows the major ability to inhibit the growth of E. hist. is the 4,7-diphenyl-
1,10-phenanthroline. Contrarily, for 2,2’-bipyridine groups (Figure 3.d, R2=0.9912) the
smaller the complex, the more active against Entamoeba histolytica. The ligand with the
best amebicide effect is the unsubstituted bipyridine that is the most oxidizing ligand. Since
the values of simulated and experimental redox potential show a strong correlation, it is
possible to employ both to describe the antiparasitic efficiency (Figure S8). On the other
hand, although, lipophilicity indicator Log P has been reported to be one crucial factor to
determine the activity of organic molecules, for complexes results do not show a clear trend
and additional studies are required to properly evaluate the importance of it. Those results
should be considered for further rationalize designing of ruthenium (II) mixed compounds
with pdto and enhanced antiparasitic activity.
15

3.a
3.b
3.c
3.d
3.e
Figure 3. E1/2 and Log V as descriptors to explain in vitro growth inhibition of Entamoeba
histolytica results.
16

3. Conclusions
Twenty ruthenium (II) mixed compounds of the type [Ru(pdto)(E-E)]Clx have been
synthesized, characterized and in vitro evaluated against E. histolytica. Twelve of them are
reported for the first time (2, 6, 7-9, 11, 15-20). Cyclic voltammetry results indicate that
complexes containing O-O and N-O donors are easier to oxidized than N-N donor ligands.
In general, antiparasitic activity shows that O-O and N-O are better than N-N donor
ligands. Among the complexes under study, only four of them ([Ru(pdto)(2,2'-
bipyridine)]Cl2, [Ru(pdto)(ethylenediamine)]Cl2, [Ru(pdto)(acetylacetonate)]Cl and
[Ru(pdto)(glycinate)]Cl) are better than the reference metronidazole drug to treat
amoebiasis disease (IC50 < 6.80 µmol/L). Moreover, experimental redox potential values
are in good agreement with biological effect. Additionally, DFT electronic and steric
indicators provide useful information to describe the biological activity against E.
histolytica. In this regard, E1/2 and theoretical molar volume are the most important
parameters to explain the effectivity of these compounds to inhibit in vitro E. histolytica
trophozoite parasite. Those results indicate that computational studies could be a powerful
tool to predict the antiparasitic activity of RuII-pdto mixed compounds.
4. Experimental section
4.1.- Materials and methods
The solvents (J. T. Baker) and reagents (Sigma-Aldrich) used did not require further
purification. Elemental analysis was performed on an EAGER 200 analyzer (EAGER 200
CHNS/method). The infrared spectra obtained in a range of 4000-400 cm-1 were acquired in
1
a Nicolet Avatar 320 FT-IR device using KBr pads. The spectra of MNR H and 13C,
COSY and HSQC were acquired in a VARIAN VNMRS 400 MHz equipment, using an
17

internal standard of tetramethylsilane (TMS) and using methanol-D4 as solvent. The
Packard 5484A diode matrix spectrophotometer was used to acquire UV-vis spectra with
methanol as a solvent, in a range of 190 to 8000 nm. The conductivities were measured
with a JENWAY 4330 conductivity meter, the conductivity data were obtained at
concentrations of 1x10-3 mol/L in methanol solutions at 25 ° C.
Diffraction measurements were made on an Oxford Diffraction Gemini-Atlas
diffractometer with a CCD area detector, the source of molybdenum monochromatic
radiation was λ=0.71073 Å at a temperature of 130K. The structures were solved by direct
methods using the SHELXS-97-2 program package. The molecular structure graphs were
generated using the ORTEP3 program for Windows and Mercury 3.10.3.
All electrochemical measurements were made in acetonitrile (HPLC grade) with a CH
Instruments electrochemical Workstation CH760E potentiostat with a conventional three
electrode array. Experimental determinations of the partition coefficient P were made using
the shake flask method in the octanol-water system. The determination of P was carried out
by absorption spectroscopy in the UV-visible region.
4.2.- Synthesis
The 1,8-bis-(2-pyridyl)-3,6-dithioctane (pdto) and the dichlorotris(triphenylphosphine)
ruthenium (II) raw material, as well as the precursor [RuCl2(PPh3)3] were synthesized from
as previously reported [42].
Precursor of the mixed compounds of ruthenium(II): chlorine chloride(1,8-bis-(2-pyridyl)-
3,6-dithioctane)(triphenylphosphine) ruthenium(II), [RuCl(pdto)(PPh3)]Cl. The pdto is
dissolved in methanol and added to a suspension of [RuCl2(PPh3)3] in methanol. The
mixture is heated at reflux for 3 h under constant stirring, the solution is concentrated for
subsequent precipitation using ethyl ether. The precipitate that forms is yellow, filtered and
18

washed with cold ethyl ether, leaving it to dry under vacuum. Its purification is carried out
by means of a chromatographic column made with neutral alumina using CH2Cl2 as solvent
and a mixture CH2Cl2:MeOH (5: 1) as eluent.
Synthesis of mixed coordination compounds of ruthenium(II). The synthesis of the mixed
compounds of ruthenium(II) with pdto and phenanthrolines, bipyridines, ethylenediamine,
acetylacetonate and glycinate, were carried out according to that already reported in
previous works, placing equimolar amounts of the precursor [RuCl(pdto)(PPh3)]Cl and the
respective bidentate ligands, in reflux with appropriate solvents and the time required
according to the type of compound. Purification is carried out by means of a
chromatographic column made with neutral alumina as support and CH2Cl2 as solvent; and
a mixture CH2Cl2:MeOH (5:1) as eluent for all compounds.
The synthesis of compounds containing different monosubstituted salicylaldehydates in
position 5 [RuCl(pdto)(5-R-salal)]Cl (where R = methyl, methoxy, chlorine, nitro) is
presented below. To obtain 1 mmol of the coordination compounds, [RuCl(pdto)(PPh3)]Cl
(precursor) was dissolved in methanol. Subsequently, a certain amount of the bidentate
salicylaldehyde ligand (1: 1 stoichiometric ratio) was weighed, which was dissolved in
methanol and 1 mL of a 1 mol/L aqueous NaOH previously prepared was added; both
solutions were mixed and heated at reflux for 4 h, under constant stirring. The solution was
concentrated to precipitate with ethyl ether, obtaining a fine brick red precipitate. It was
filtered, washed with ether and dried under vacuum. Purification is carried out by means of
a chromatographic column made with neutral alumina as support and CH2Cl2 as solvent.
4.3.- Computational details
DFT calculations were performed with Gaussian 09 package [56], using M06 functional
[57] in conjunction with SDD basis set to describe Ru atom and Los Alamos LanL2DZ for
19

all the other atoms (H, C, N, O and S) [58-60]. This methodology has been already used to
study metal containing systems [61-65]. All structures were confirmed as minima on the
potential energy surface through the vibrational frequency analysis (0 imaginary
frequencies). Molar volume was obtained with a single point calculation based on
optimized structures and Molecular Electrostatic Potential (MEP) were plotted as a guide to
assess charge distribution and global reactivity.
To calculate redox potentials, we followed previous reports that have proven to be useful
for transition metal complexes [66-78], and in which geometries of ferrocene (Cp2Fe) and
ferrocenium ([Cp Fe]+) in the eclipsed conformation (D ) were taken into account and
5h
2
optimized in gas phase. Gas phase and solvation ∆G energies were calculated by the same
level of theory and using SMD continuum solvation model [69] and MeCN to simulate the
same environment of the electrochemical experiments. To obtain gas phase and solvation
free energies of all species, a thermochemical analysis at 298.15 K and 1 atm was
performed following the next cycle:
Redox potential was determined through the free energy changes and according to the one
electron exchange of Nernst equation:
20

Where F is the Faraday constant. The values are doubly referenced first, to the standard
hydrogen electrode that, which in MeCN has a value of 4.6 V [70] and second to a value of
0.68 V for Cp2Fe estimated by the same level of theory to be in agreement with the
experimental measurements.
4.4.- Amoebicidal activity
For the determination of the mean inhibitory concentration (IC50), amibian viability assays
were performed for Entamoeba histolytica trophozoites of the HM1: IMSS strain, where it
was analyzed using the vital exclusion method with the trypan blue marker. 100 µL are
taken every 24 h for 72 h of each of the tubes and 1 µL of marker is added. Samples are
incubated at room temperature for 5 minutes and viable cell counts are performed in a
hematocytometer. For the calculation of the average inhibitory concentration value IC50, a
multivariable analysis is used such as the Stat graph 2010 program.
4.5.- Electrochemical studies
All electrochemical experiments were carried out in solutions 0.001 mol/L of sample and
0.1 mol/L of terabutylammonium hexafluorophosphate (Bu4NPF6) in anhydrous MeCN.
The measurements were performance in a CH Instruments Electrochemical Workstation
CH760E potentiostat. A conventional three electrode array was used: 3 mm diameter
carbon glassy disk as working electrode, platinum wire as counter-electrode and Ag/AgCl
was the reference. According to IUPAC recommendations [71], Voltammograms were
referenced with ferrocene (99.9% purity) in an internal setting. Due to the chloride
oxidation interferes with the RuII/RuIII process, the electrochemical measures were made
with the PF6- salts of the complexes and this do not affect the magnitude of the metallic
center redox potential. The solutions were bubbled with nitrogen prior and between each
experiment.
21

4.6.- Log P determinations
The experimental determination of this parameter is made from a standard solution with a
known concentration (0.05 mmol/L) of the test sample dissolved in water, an aliquot of 1
mL (aqueous phase) is taken, placed in a falcon tube of 10 mL An equivalent volume (1
mL) of 1-octanol grade HPLC (organic phase) was also added to said tube. Both phases are
vigorously stirred with a vortex type apparatus, for 10 minutes constantly and maintaining a
temperature between 24.5 to 25 °C, this to promote the distribution of the compound in
both phases. An emulsion was obtained, which was separated with the help of a centrifuge.
The time used for the separation was 3 minutes at 1600 revolutions per minute, sufficient
time in which a complete separation of both phases was observed.
Acknowledgements
This work was supported by UNAM-PAPIIT IG200616 and UNAM-FQ 50009047. E.C.G
(grant no. 607665), L.F.H.A and M.R. thanks to CONACyT. V.M.U.S thank to UNAM-
DGPA for PASPA grant. The authors thank to UNAM-DGTIC for supercomputer services.
Thank to Adrian Espinoza-Guillén for technical support.
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