Assessment of the nematocidal activity of metallocenyl analogues of monepantel

Article abstract of DOI:10.1039/c6dt03376h

In this study, we present the design, synthesis, characterization and biological evaluation of structurally new ferrocenyl and ruthenocenyl derivatives of the organic anthelmintic monepantel (Zolvix). All seven metallocenyl derivatives prepared (4a/b, 5a/b, 6a/b and 7) were isolated as racemates and characterized by ¹H, ¹³C and ¹⁹F NMR spectroscopies, mass spectrometry, IR spectroscopy and elemental microanalysis. The molecular structures of four compounds (4a/b, 6a and 7) were further confirmed by X-ray crystallography. The biological activities of the organometallic intermediates (4a/b) and organometallic derivatives of monepantel (5a/b, 6a/b and 7) were evaluated in vitro using parasitic nematodes of major importance in livestock, namely Haemonchus contortus and Trichostrongylus colubriformis. Two ferrocenyl compounds (4a and 6a) showed nematocidal activity, while the analogous ruthenocenyl compounds (4b and 6b) were not active at the highest concentration tested (10 μg mL^-1). In order to obtain insight into the difference in activity between ferrocenyl and ruthenocenyl derivatives, the potential of the compounds for reactive oxidative species (ROS) production in live cells was assessed. Interestingly, neither the ferrocenyl nor the ruthenocenyl compounds (4a/b and 6a/b) produced significant ROS in HeLa cells when checked after 22 h, potentially indicating a redox-independent activity of 4a and 6a on the parasites. The selectivity of the compounds on parasites was confirmed by investigating their cytotoxicity profiles. None of these compounds was toxic either to HeLa or MRC-5 cells. Thus, 4a and 6a could be considered as interesting leads for further development of new classes of anti-parasitic agents.

Full text of DOI:10.1039/c6dt03376h

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Assessment of the nematocidal activity of
metallocenyl analogues of monepantel†
Cite this: DOI: 10.1039/c6dt03376h
a
a
b
a,c
Jeannine Hess, Malay Patra, Abdul Jabbar, Vanessa Pierroz,
a
a
c
b
Sandro Konatschnig, Bernhard Spingler, Stefano Ferrari, Robin B. Gasser* and
a
Gilles Gasser*‡
In this study, we present the design, synthesis, characterization and biological evaluation of structurally
®
new ferrocenyl and ruthenocenyl derivatives of the organic anthelmintic monepantel (Zolvix ). All seven
metallocenyl derivatives prepared (4a/b, 5a/b, 6a/b and 7) were isolated as racemates and characterized
1
13
19
by H, C and F NMR spectroscopies, mass spectrometry, IR spectroscopy and elemental microanalysis.
The molecular structures of four compounds (4a/b, 6a and 7) were further confirmed by X-ray crystallo-
graphy. The biological activities of the organometallic intermediates (4a/b) and organometallic deriva-
tives of monepantel (5a/b, 6a/b and 7) were evaluated in vitro using parasitic nematodes of major impor-
tance in livestock, namely Haemonchus contortus and Trichostrongylus colubriformis. Two ferrocenyl
compounds (4a and 6a) showed nematocidal activity, while the analogous ruthenocenyl compounds (4b
and 6b) were not active at the highest concentration tested (10 μg mL− ). In order to obtain insight into
the difference in activity between ferrocenyl and ruthenocenyl derivatives, the potential of the com-
pounds for reactive oxidative species (ROS) production in live cells was assessed. Interestingly, neither the
ferrocenyl nor the ruthenocenyl compounds (4a/b and 6a/b) produced significant ROS in HeLa cells
when checked after 22 h, potentially indicating a redox-independent activity of 4a and 6a on the para-
sites. The selectivity of the compounds on parasites was confirmed by investigating their cytotoxicity
profiles. None of these compounds was toxic either to HeLa or MRC-5 cells. Thus, 4a and 6a could be
considered as interesting leads for further development of new classes of anti-parasitic agents.
1
Received 29th August 2016,
Accepted 3rd October 2016
DOI: 10.1039/c6dt03376h
www.rsc.org/dalton
2
,3
mintics. The excessive use of these drugs has resulted in
the emergence of resistance, and multi-drug resistant
Introduction
5
–8
The impact of parasitic diseases on animals and humans is parasites are now widespread.
Apart from the classical
substantial around the world. Controlling parasites of domesti- groups of anthelmintics, such as the benzimidazoles, imid-
cated animals is a major issue, and usually relies exclusively azothiazoles and macrocyclic lactones, a new synthetic anthel-
on chemotherapy with an arsenal of broad-spectrum anthel- mintic class, the amino-acetonitrile derivatives (AADs, see
Scheme 1), has been discovered, and a promising candidate of
this class, called monepantel (AAD 1566), was recently
commercialized.
Investigations have shown that the safety profile of mone-
pantel relates to its target, a nematode specific nicotinic acetyl-
a
Department of Chemistry, University of Zurich, Winterthurerstrasse 190,
CH-8057 Zurich, Switzerland. E-mail: gilles.gasser@chimie-paristech.fr;
http://www.gassergroup.com
1,9,10
b
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne,
Parkville, Victoria 3010, Australia. E-mail: robinbg@unimelb.edu.au;
http://www.gasserlab.org/; Tel: +61 3 9731 2283
choline receptor (nAChr) subunit, which is absent in host
11–13
mammals.
Although monepantel now represents a new
c
Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190,
class of anthelmintic drug, unfortunately, some years after its
introduction and use in the field, nematodes with reduced
CH-8057 Zurich, Switzerland
1
13
19
†
Electronic supplementary information (ESI) available: H, C and F NMR
spectra (4a/b, 5a/b, 6a/b and 7) (Fig. S1–S16), ORTEP plot of 7 (Fig. S17), anti- sensitivity to monepantel have been detected in several
1
4–18
parasitic activity against C. felis, L. cuprina and R. sanguineus of organometallic countries including Uruguay, New Zealand and Brazil.
precursors and derivatives (4a/b, 5a/b, 6a/b and 7) (Table S1). CCDC
Given this rapid emergence of resistance to monepantel, there
is an urgent need to develop novel and superior control strat-
egies to ensure the sustainability of parasite control. With this
in mind, our group recently started to derivatize monepantel
1
501422–1501425. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c6dt03376h
Present address: Chimie ParisTech, PSL Research University, 11, rue Pierre et
‡
Marie Curie, F-75005 Paris, France.
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Results and discussion
Synthesis and characterization
The ferrocenyl and ruthenocenyl analogues of monepantel
corresponding to Strategy IV were prepared in a two-step
reaction procedure as presented in Scheme 2. In order to
obtain initial insights into their potential as anthelmintic
agents, we focused on the isolation of the organometallic
derivatives as racemates rather than as their enantiomerically
pure forms.
The syntheses of desired organometallic analogues com-
menced with the preparation of a 2-amino-2-hydroxymethyl-
proprionitrile (1) synthon, following a literature procedure by
38
Gauvry et al. The intermediate N-(2-cyano-1-hydroxypropan-2-yl)-
ferroceneamide (4a) was obtained in a moderate yield by react-
Scheme 1 Schematic representation of previously (green) and newly
(
red) designed organometallic derivatives of monepantel (AAD 1566)
using different strategies. OM = ferrocene, ruthenocene or cymantrene;
FG = SCF , F, Cl, Br, I, SCH , CF , OCF , S(O)CF , S(O) CF
ing
1 with activated ferrocencarboxylic acid (2) under
3
3
3
3
3
2
3
.
basic conditions. Moreover, the same reaction allowed the iso-
lation of a di-ferrocenyl analogue of monepantel, 2-ferrocene-
amido-2-cyanopropyl ferroceneoate (5a), with 11% yield.
Compound 4a was then converted to the desired final com-
with various organometallic moieties using different strategies pound N-(2-cyano-1-(5-cyano-2-(trifluoromethyl)phenoxy)propan-
1
9,20
(Scheme 1).
The derivatization of a known organic drug 2-yl)ferroceneamide (6a) using a Williamson ether synthesis
with organometallic moieties has proven to be extremely suc- in the presence of NaH and commercially available 3-fluoro-
2
1–35
cessful in various fields of medicinal chemistry.
4-(trifluoromethyl)benzonitrile. Compound 6a was isolated
Ferroquine, a ferrocenyl analogue of the antimalarial drug in 26% yield. In addition to the di-ferrocenyl analogue
chloroquine, is one of the best examples of such a deriva- 5a, another disubstituted organometallic analogue, namely
tization. Thanks to the presence of a ferrocenyl moiety,
Ferroquine is active against chloroquine-resistant strains of
the malaria parasite, Plasmodium falciparum (P. falciparum),
2
9,36,37
where the original organic drug chloroquine is inactive.
In our initial study, we replaced the aryloxy unit of mone-
pantel with organometallic moieties and modified the benz-
amide part with various functional groups (Scheme 1, Strategy
1
9
I/II). Subsequently, we replaced the chiral C2 spacer of mone-
pantel by a ferrocenyl moiety that comprised planar chirality
with a 1,2-unsymmetric substitution on a cyclopentadienyl
2
0
ring (Scheme 1, Strategy III).
Extending this work to develop organometallic monepantel
derivatives, we report here a new strategy for achieving structu-
rally distinct organometallic-containing monepantel deriva-
tives. First, we kept the aryloxy unit of monepantel un-
perturbed and substituted the benzamide unit with metallo-
cenes, namely ferrocene and ruthenocene (Scheme 1, Strategy
IV). Then, we replaced simultaneously both the aryloxy and the
benzamide units using two metallocenyl fragments (Scheme 1,
Strategy IV). During the course of present study, we first pre-
pared ferrocenyl monepantel derivatives, since the presence of
the ferrocenyl unit might result in additional metal-specific
modes of action, such as the production of ROS under physio-
logical conditions. In order to investigate if these redox reac-
tions contribute to the antiparasitic activity of our novel
organometallic compounds, we designed ruthenocenyl ana-
logous of the ferrocenyl monepantel derivatives. The replace-
ment of the iron(II) by a ruthenium(II) center is expected to
prevent redox mediated ROS production under physiologically
Scheme 2 Reagents and conditions: (i) (a) oxalyl chloride, dry CH
2 2
Cl ,
r.t.; (b) 2-amino-2-hydroxymethylproprionitrile (1), NEt , dry THF, r.t., o.
3
n., 4a (50%), 4b (31%), 5a (11%), 5b (19%); (ii) NaH, 3-fluoro-4-(trifluoro-
methyl)benzonitrile, dry THF, overnight, 0 °C→r.t., 6a (26%), 6b (11%); (iii)
(
ferrocenylmethyl)trimethylammonium iodide, K
2 3
CO , 18-crown-6, dry
3
6,37
relevant conditions.
CH CN, reflux, 144 h (120 h plus overnight), 7 (43%).
3
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N-(1-(ferrocenyloxy)-2-cyanopropan-2-yl)ferroceneamide (7) was dienyl rings of the 4 ferrocene units are in an almost perfect
also prepared by reacting 4a with (ferrocenylmethyl)trimethyl- eclipsed conformation. One linker starting from the quartern-
ammonium iodide under basic conditions. By contrast to ary carbon to the methylene unit next to the cyclopentadienyl
5
a, where one of the ferrocenyl units is linked to C2 spacer ring is disordered in a ratio 3 : 2 (see ESI† for structure).
by an ester functionality, the similar ferrocene unit in 7 is
attached to the C2 spacer by an ether functionality. Moreover,
we synthesized three ruthenocenyl analogues (4b, 5b and 6b),
which are structurally identical to the ferrocenyl analogues 4a,
Biological evaluation
The anthelmintic potentials of our ferrocenyl and ruthenoce-
nyl precursors (4a/b) and final derivatives 5a/b, 6a/b and 7
were first evaluated on two common parasites of small rumi-
5
a and 6a. Despite iso-structural, ferrocene and ruthenocene
have distinct redox properties, therefore comparison of the
biological activity of ferrocenyl and the corresponding ruthe-
nocenyl analogues might shed light on the possible involve-
ment of the redox properties in their activity. The synthetic
sequences to obtain the desired ruthenocenyl analogues of
monepantel are similar to those of ferrocenyl compounds (see
Scheme 2). All novel ferrocenyl and ruthenocenyl analogues of
monepantel described here were isolated as racemic mixtures
nants,
Haemonchus
contortus
(H.
contortus)
and
Trichostrongylus colubriformis (T. colubriformis) using a larval
development assay (LDA) (see Experimental section for
details). The results are summarized in Table 1. The organic
derivatives AAD85, AAD96 and ivermectin were included as
1
controls.
Two of seven metal-based monepantel derivatives tested
displayed moderate activities against H. contortus and
T. colubriformis. The final ferrocenyl derivative of Strategy IV
1
13
19
and characterized by H, C, F NMR spectroscopies, ESI-
mass spectrometry and IR spectroscopy, and their purities
were analyzed by elemental microanalysis.
(
6a) and its corresponding ferrocene precursor (4a) showed
EC60 values in a similar range when tested against H. contortus
.90 μg mL− (15.70 μM) (4a) and 4.70 μg mL (9.77 μM) (6a)
1
−1
4
X-ray crystallography
−1
and T. colubriformis 8.00 μg mL (25.63 μM) (4a) and 7.00 μg
−
1
The molecular structures of four compounds (4a/b, 6a and 7) mL (14.55 μM) (6a). Interestingly, the potencies of both
were further confirmed by X-ray crystallography. Details of the active derivatives (4a, 6a) are approximately twice higher
crystallization procedure were given in the Experimental against H. contortus compared with T. colubriformis. However,
section. The amide unit is coplanar with the cyclopentadienyl the potencies of 4a and 6a are lower when compared with the
ring. The alcohol of compound 4a is part of a cyclic and a organic controls AAD85 and AAD96, which displayed EC
1
00
2
39
−1
−1
linear hydrogen-bridge network. The R
2
(14) cycle is formed values of 0.01 μg mL (0.022 μM/0.021 μM) and 0.032 μg mL
by an alcohol hydrogen, making contact with a symmetry (−x, (0.07 μM/0.068 μM) against H. contortus and T. colubriformis,
1
1
− y, −z)-related carbonyl oxygen; the corresponding alcohol respectively. Importantly, the ruthenocenyl analogues (4b, 5b)
donates back to the original carbonyl oxygen. Additionally, the of the active ferrocenyl derivatives 4a, 5a displayed no activity
same alcohol is an acceptor of a hydrogen bridge from a sym- against H. contortus and T. colubriformis at the highest concen-
metry related amide nitrogen (−x, 1/2 + y, 1/2 − z). The struc- tration evaluated (10 µg mL− ). At this point, it is reasonable to
tures of 4a and 4b are essentially isostructural, with the major speculate that the iron(II) center in 4a and 6a contributes to
difference being that the Fe–C bond lengths in 4a are between the production of toxic ROS which could be the reason for the
1
2
.0293(14) Å and 2.0619(17) Å, whereas the Ru–C bond lengths observed anti-parasitic activity of these compounds. Therefore,
in 4b are between 2.155(3) Å and 2.192(3) Å (see Fig. 1). we evaluated ROS generation using the ferrocene and rutheno-
Compound 6a does not form any “typical” hydrogen bonds, cene-containing derivatives in live cells (Fig. 2). Although an
despite the presence of an amide unit (see Fig. 1).
assessment of ROS generation in the parasitic nematodes
Compound 7 was measured on a synchrotron; it crystallized would have been the ideal, however for technical reasons we
with two molecules in the asymmetric unit. All cyclopenta- used a mammalian cervical cancer cell line (HeLa) as model.
Fig. 1 Molecular structures of 4a, 4b and 6a with atoms shown as thermal ellipsoids (drawn at 50% probability; hydrogen atoms are omitted for
clarity).
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Table 1 Antiparasitic activity against Haemonchus contortus, Trichostrongylus colubriformis and Dirofilaria immitis and cytotoxicity against HeLa
and MRC-5 cells of 4a/b, 5a/b, 6a/b, 7
EC60 value
EC50 value
D. immitis
24 hours
IC50 value
D. immitis
H. contortus
T. colubriformis
48 hours
HeLa
[μM]
MRC-5
[μM]
[μg mL−
1
]
[μM]
[μg mL−1
]
[μM]
[μg mL−1
]
[μM]
[μg mL−1
]
[μM]
Compound
4
5
6
4
5
6
7
a
a
a
b
b
b
4.90
>10.00
4.70
>10.00
>10.00
>10.00
15.70
>19.08
9.77
8.00
>10.00
7.00
>10.00
>10.00
>10.00
>10.00
25.63
>19.08
14.55
>10.00
>10.00
>10.00
>10.00
>10.00
6.60
>32.04
>19.08
>20.78
>27.98
>16.27
12.54
>10.00
>10.00
>10.00
>10.00
6.60
6.60
>10.00
2.20
>32.04
>19.08
>20.78
>27.98
10.74
12.54
>19.60
4.81
>100
>100
>100
>100
>100
>100
>100
n.d.
>100
>100
>100
>100
>100
>100
>100
n.d.
>27.98
>16.27
>18.99
>19.60
>27.98
>16.27
>18.99
>19.60
>10.00
>10.00
2.40
>19.60
5.25
a
a
a
a
0
.01
0.022
0.032
0.07
a
a
a
a
0
.01
0.021
0.032
0.068
n.d.i.
n.d.i.
n.d.i.
n.d.i.
n.d.
n.d.
a
a
a
a
Ivermectine
Cisplatin
0.001
n.d.
0.001
n.d.
0.01
n.d.
0.011
n.d.
1.00–3.00
n.d.
1.14–3.43
n.d.
1.00–3.00
n.d.
1.14–3.43
n.d.
n.d.
9.6 ± 1.2
n.d.
17.6 ± 4.3
a
1
4
−1
EC100 value . n.d.i. = non-disclosable information and n.d. = not determined. EC values are given in μg mL as well as in μM. EC values are
calculated as a mean of 2 series of triplicated dose responses. Experimental errors are not included as they are too low to influence the overall
EC values.
The ROS production in the cells treated with 4a/b and 6a/b
(25 µM for 22 h) was quantified using the fluorescent indicator
2
′,7′-dichlorofluorescein diacetate (H DCFDA). As positive
2
control, tert-butyl hydroperoxide (TBH) was included in the
same assay. Surprisingly, as shown in Fig. 1, the ROS levels in
cells treated with the ferrocenyl compounds (4a, 6a) and the
ruthenocenyl compounds (4b, 6b) did not display a significant
difference. Moreover, the ROS levels of the cells treated with
1
9
compounds are comparable to that of the untreated cells.
The results from ROS assay suggest that the differences in
anti-parasitic activity between the ferrocene- and ruthenocene-
containing organometallic compounds are not related to the
production of ROS in cells. However, it has to be pointed out
that this is for a timepoint only and that increase in ROS pro-
duction can be faster or can take longer. The potency of the
organometallic monepantel derivatives synthesized using
Strategy IV against H. contortus and T. colubriformis is lower
than for the organometallic derivatives from Strategy I/II (best
EC60 of 1.80 μg mL− ), but superior to those from Strategy III
Fig. 2 Level of ROS production in HeLa cells untreated or treated with
4
a, 4b, 6a and 6b after 22 h. THB = tert-butyl hydroperoxide.
1
−
1
19,20
37,40
(best EC60 of 6.60 μg mL ).
Overall, based on knowledge actions.
Therefore, we decided to evaluate these com-
4
1,42
obtained from these structure–activity relationships of organo- pounds further against other parasites.
The activity of our
metallic monepantel derivatives, it can be concluded that it is organometallic-analogues (4a/b, 5a/b, 6a/b and 7) of mone-
preferable to keep the benzamide part of monepantel un- pantel was evaluated on the canine heartworm, Dirofilaria
perturbed, while structural modifications can be made to the immitis (D. immitis). Two ruthenocenyl compounds (5b, 6b)
aryloxy portion for the designing of new derivatives of mone- showed moderate activity against microfilariae of this nema-
pantel to retain anti-parasitic activity against H. contortus and tode species in a 48 h motility assay with EC values of
5
0
1
T. colubriformis.
6.60 μg mL− (10.74 μM (5b), 12.54 μM (6b)). Interestingly, the
Previous studies have shown that organometallic derivatiza- corresponding ferrocenyl derivatives 5a and 6a are inactive
tion modulates the spectrum of activity of a known organic against D. immitis. However, once again, 5b and 6b are less
drug through the addition of metal-specific mode of potent compared to the organic control AAD85, which displays
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an EC50 value of 2.20 μg mL− (4.81 μM). In addition to this, cation. Solvents were used as received or distilled using stan-
1
4
4
3
we further investigated the activity of the ferrocenyl and ruthe- dard procedures. All preparations were carried out using
nocenyl precursors (4a/b) and final derivatives 5a/b, 6a/b and 7 standard Schlenk techniques. Thin layer chromatography
on three arthropods Ctenocephalides felis (cat flea), Lucilia (TLC) was performed using silica gel 60 F-254 (Merck) plates
cuprina (blow fly) and Rhipicephalus sanguineus (brown dog with detection of spots being achieved by exposure to UV light.
tick). Unfortunately, none of the compounds synthesized dis- Column chromatography was performed using Silica gel 60
played potency at the highest concentration tested on C. felis (0.040–0.063 mm mesh, Merck). Eluent mixtures are expressed
−
1
−1
(
100 μg mL ), L. cuprina (32 μg mL ) or R. sanguineus (100 as volume to volume (v/v) ratios. Chlorocarbonyl ferrocene and
−
1
−1
μg mL and 640 μg mL ) (Table S1†).
chlorocarbonyl ruthenocene were synthesized according to lit-
4
4
For development of new anti-parasitic compounds, the erature procedures.
selectivity of the compounds towards parasites is an important
parameter. An ideal anthelmintic should be non-toxic to the
mammalian host, while being efficient in killing the parasites.
Instrumentation and methods
1
13
19
H, C and F NMR spectra were recorded in deuterated sol-
For this purpose, we evaluated the cytotoxicity of the com- vents on Bruker 400 or 500 MHz NMR at room temperature.
pounds using a cervical cancer cell line (HeLa) and a non- The chemical shifts, δ, are reported in ppm (parts per
cancerous human lung fibroblast cell line (MRC-5). None of million). The signals from the residual protons of deuterated
4
5,46
the compounds tested were toxic up to 100 µM (the highest solvent have been used as an internal reference.
The
concentration assayed), indicating selectivity of our com- abbreviations for the peak multiplicities are as follows: s
pounds (4a, 6a) to the nematodes H. contortus, T. colubriformis (singlet), d (doublet), dd (doublet of doublets), t (triplet), q
and D. immitis over mammalian cells.
(quartet), m (multiplet), and br (broad). ESI mass spectrometry
was performed using a Bruker Esquire 6000 spectrometer. In
the assignment of the mass spectra, the most intense peak is
listed. UPLC-ESI-MS was performed on a Waters Acquity UPLC
System coupled to a Bruker HCTTM, using an Acquity UPLC
Conclusion
The extensive use of commercially available broad-spectrum BEH C18 1.7 μm (2.1 × 50 mm) as a reverse phase column with
organic anthelmintics has led to a situation where parasites a flow rate of 0.6 mL min− . The UV absorption was measured
develop resistant to at least one or more available drug classes. at 254 nm. The runs were performed with a linear gradient of
With the view of developing a new class of metal-based anthel- A (acetonitrile (Sigma-Aldrich HPLC grade) and B (distilled
mintic agents, we synthesized and characterized a series of water containing 0.02% TFA and 0.05% HCOOC)): t =
ferrocene- and ruthenocene-containing organometallic ana- 0–0.5 min, 5% A; t = 4.0 min, 100% A; t = 5 min, 100%
logues of the anthelmintic drug monepantel. The biological A. High-resolution mass spectrometry were performed on a
efficacy of all newly synthesized intermediates as well as final Bruker ESQUIRE-LC quadrupole ion trap instrument (Bruker
analogues (4a/b, 5a/b, 6a/b, 7) was assessed in a LDA against Daltonik GmbH, Bremen, Germany), equipped with a com-
H. contortus and T. colubriformis. Two ferrocenyl derivatives 4a bined Hewlett-Packard Atmospheric Pressure Ion (API) source
and 6a showed moderate efficacy with EC values between (Hewlett-Packard Co., Palo Alto, CA, USA). The solutions
1
6
0
1
−1
4
.70–8.00 μg mL− against both nematode species, while the (about 0.1–1 µmol ml
) were continuously introduced
corresponding ruthenocenyl derivatives displayed no activity. through the electrospray interface with a syringe infusion
The ROS production of both types of organometallic deriva- pump (Cole-Parmer 74900-05, Cole-Parmer Instrument
tives (4a/b and 6a/b) was evaluated in an in vitro model using Company, Vernon Hills, Illinois, USA) at a flow rate of
the HeLa cervical cancer cell line. However, no significant 5 µl min− . The MS-conditions were: nebulizer gas (N
1
) 15 psi,
2
−
1
difference in ROS levels after 22 h was observed between the dry gas (N ) 7 min , dry temperature 300 °C, capillary voltage
2
ferrocenyl (4a and 6a) and ruthenocenyl (4b and 6b) ana- 4000 V, end plate 3500 V, capillary exit 100 V, skimmer1 30 V,
logues. This unexpected finding might suggest a redox inde- and trap drive 70. The MS acquisitions were performed at
pendent mode of action for the anti-parasitic activity of the normal resolution (0.6 u at half peak height), under ion charge
ferrocenyl derivatives (4a and 6a) although more investigations control (ICC) conditions (10 000) in the mass range from m/z
will need to be performed to confirm this hypothesis. The 100 to 2000. To get representative mass spectra, 8 scans were
selectivity of the compounds towards parasites was demon- averaged. Infrared spectra were recorded on Perkin-Elmer FTIR
strated by assessment of their cytotoxicity using a cancer spectrometer using KBr pellets. Peak intensities are given as
(HeLa) and a non-cancer (MRC-5) cell line.
broad (b), very strong (vs), strong (s), medium (m) and weak
w). Elemental microanalyses were performed on
(
a
LecoCHNS-932 elemental analyser.
Experimental section
Synthesis
Materials
N-(2-Cyano-1-hydroxypropan-2-yl)ferroceneamide (4a) and
ferroceneoate (5a).
from commercial suppliers and used without further purifi- Chlorocarbonyl ferrocene (0.648 g, 2.608 mmol) and 2-amino-
All chemicals were of reagent grade quality or better, obtained 2-ferroceneamido-2-cyanopropyl
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1
2
-hydroxymethylproprionitrile (0.261 g, 2.608 mmol) were dis- 1720w, 1633s, 1531s, 1455s, 1376s, 1308s, 1130s, 823s.
H
3
solved in dry THF (100 mL). To this orange reaction solution, NMR (500 MHz, DMSO): δ/ppm = 7.51 (s, 1H, NH), 5.64 (t, J =
NEt (453 μL, 3.26 mmol) was added and the mixture was 6.46 Hz, 1H, OH), 5.23–5.22 (m, 2H, C ), 4.73–4.73 (m, 2H,
stirred overnight at room temperature. The solvent was evapor- ), 4.59 (s, 5H, C ), 3.80–3.76 (m, 1H, CH ), 3.53–3.50
ated under reduced pressure. The crude residue was dissolved (m, CH ), 1.54 (s, 3H, CH ). C NMR (125 MHZ, DMSO): δ/
3
5 4
H
C
5
H
4
5
H
5
2
1
3
2
3
in CH
2 × 10 mL). The organic layer was dried over MgSO
the solvent was evaporated under reduced pressure. The crude C H N OH] (17). Elemental Analysis: calcd for C H O N Ru
2 2 2
Cl (30 mL), washed with H O (2 × 10 mL) and brine ppm = 168.1, 120.5, 79.1, 72.4, 72.3, 71.6, 70.4, 70.3, 64.8, 51.9,
+
(
4
, filtered and 21.7. ESI-MS: m/z (%) = 359.1 [M + H] (100), 259.0 [M −
+
4
4
2
15 16 2 2
product was purified by column chromatography on silica with = C, 50.41; H, 4.51; N, 7.84. Found = C, 50.85; H, 4.44; N, 7.41.
hexane : ethyl acetate (4 : 1) as eluent (R
f
(5a) = 0.69, hexane : Data 5b: IR (KBr, cm− ): 3320m, 3104w, 2956w, 2651w, 2322w,
1
ethyl acetate 1 : 1, R (4a) = 0.30, hexane : ethyl acetate 1 : 1) to 2161s, 2053s, 1976s, 1690s, 1656s, 1521s, 1450s, 1373s, 1267s,
f
1
afford 2-ferroceneamido-2-cyanopropyl ferroceneoate (5a) and 1135s, 1035m, 997m, 808s, 759m. H NMR (500 MHz, DMSO):
5 3
N-(2-cyano-1-hydroxypropan-2-yl)ferroceneamide (4a) as orange δ/ppm = 7.93 (s, 1H, NH), 5.27–5.25 (m, 2H, C H ), 5.15–5.13
solids, respectively. Yield: 11% (5a, 0.075 g, 0.143 mmol) and (m, 2H, C H ), 4.85–4.84 (m, 2H, C H ), 4.76–4.75 (m, 2H,
5
3
5
4
−
1
2
5
3
1
9
4
0% (4a, 0.41 g, 1.31 mmol). Data 4a: IR (KBr, cm ): 3467s,
C
5
H
4
), 4.65 (s, 5H, C
5
H
3
), 4.59 (s, 5H, C
5
H
3
), 4.51 (d, J = 10.8,
2
13
412s, 3103w, 2941w, 2862w, 1635s, 1534m, 1454w, 1377w, 2H, CH
2
), 4.27 (d, J = 10.4, 2H, CH
2
), 1.66 (s, 3H, CH
3
).
C
312m, 1267w, 1201w, 1160w, 1099w, 1056m, 1037w, 1023w, NMR (125 MHZ, DMSO): δ/ppm = 168.6, 168.2, 119.2, 78.7,
98w, 911w, 826w, 772w, 710w, 620m, 528w, 499w, 483w, 74.1, 73.3, 72.5, 71.9, 71.6, 71.4, 70.5, 70.3, 64.9, 49.7, 22.1.
1
6
+
64w. H NMR (400 MHz, d -acetone): δ/ppm = 7.12 (s, 1H, ESI-MS: m/z (%) = 639.3 [M + Na] (100). Elemental analysis:
3
NH), 5.01 (t, J = 6.4 Hz, 1H, OH), 4.85–4.84 (m, 2H, C H ), calcd for C H O N Ru = C 50.81; H 3.94; N 4.56. Found = C
5
4
26 24
3
2
2
4
.39–4.38 (m, 2H, C
5
H
4
), 4.24 (s, 5H, C
5 5
H ), 4.0–3.93 (m, 1H, 50.92; H 3.96; N 4.53.
13
CH ), 3.90–3.86 (m, 1H, CH
2
2
), 1.71 (s, 3H, CH
3
). C NMR
N-(2-Cyano-1-(5-cyano-2-(trifluoromethyl)phenoxy)propan-2-
6
(125 MHz, d -acetone): δ/ppm = 170.9, 121.1, 76.1, 71.5, 70.5, yl)ferroceneamide (6a). N-(2-Cyano-1-hydroxypropan-2-yl)ferro-
6
9.5, 69.4, 67.0, 66.9, 53.3, 22.6. ESI-MS: m/z (%) = 351.02 [M + ceneamide (4a, 0.020 g, 0.064 mmol) was dissolved in dry THF
+
+
+
K] (8), 335.04 [M + Na] (100), 312.06 [M] (52). HR ESI-MS: (30 mL). The orange solution was cooled to 0 °C and NaH
m/z (%) = 312.05557, calcd for C H FeN O (M ) m/z (%) = (0.0018 g, 0.074 mmol) was added. After stirring the reaction
+
1
5
16
2 2
3
5
5
1
1
12.05508. Elemental analysis: calcd for C15
16 2 2
H N O Fe = C, mixture for 30 min at 0 °C 3-fluoro-4-(trifluoromethyl)benzo-
7.72; H, 5.17; N, 8.97. Found = C, 57.48; H, 5.13; N, 8.69. Data nitrile (0.012 g, 0.064 mmol) was added. After stirring the reac-
−
1
a: IR (golden gate, cm ): 3368w, 1690m, 1656m, 1520m, tion mixture overnight at room temperature, additional NaH
453w, 1378w, 1288m, 1264w, 1214w, 1164m, 1144m, 1104w, (0.0018 g, 0.074 mmol) and 3-fluoro-4-(trifluoromethyl)benzo-
1
027w, 999w, 918w, 827m, 814m, 770m, 741w. H NMR nitrile (0.012 g, 0.064 mmol) were added to the reaction
6
(500 MHz, d -acetone): δ/ppm = 7.53 (s, 1H, NH), 4.88–4.87 (m, mixture. Another portion of NaH (0.0018 g, 0.074 mmol) was
2
4
C
(
H, C
, CH
s, 5H, C H ), 1.90 (s, 3H, CH ). C NMR (125 MHz, d - ethyl acetate (3 × 10 mL). The combined organic layers were
5
H
4
), 4.77 (d, J = 10.8 Hz, 1H, CH
2
), 4.82–4.50 (m, 3H, added 2 h later. The reaction was quenched with H
2
O (2 mL)
5
H
4
2
), 4.44–4.41 (m, 2H, C ), 4.27 (s, 5H, C
5
H
4
5 5
H
), 4.25 and brine (6 mL) and the aqueous layer was extracted with
1
3
6
5
5
3
acetone): δ/ppm = 171.7, 170.7, 120.21, 75.8, 72.7, 71.7, 71.0, dried over MgSO
4
, filtered and the solvent was evaporated
7
0.8, 70.6, 69.5, 69.4, 66.9, 51.5, 23.1. ESI-MS: m/z (%) = 524.1 under reduced pressure. The crude product was purified by
+
[M] (100). Elemental analysis: calcd for C H N O Fe = C column chromatography on silica with hexane : ethyl acetate
26 24 2 3 2
5
9.58; H 4.62; N 5.34. Found = C 59.51; H 4.52; N 5.25.
f
(6 : 1) as the eluent (R = 0.36, hexane : ethyl acetate 2 : 1) to
N-(2-Cyano-1-hydroxypropan-2-yl)ruthenoceneamide
(4b) give N-(2-cyano-1-(5-cyano-2-(trifluoromethyl)phenoxy)propan-
and 2-cyano-2-(ruthenocenecarboxamido)propyl ruthenocne- 2-yl)ferroceneamide (6a) as an orange solid. Yield: 26%
carboxylate (5b). Chlorocarbonyl ruthenocene (1.67 g, (0.041 g, 0.085 mmol). IR (KBr, cm− ): 3478s, 3414s, 3355s,
1
6
(
.96 mmol) and 2-amino-2-hydroxymethylproprionitrile 2925s, 2851m, 2358m, 2336m, 2240s, 1653s, 1613s, 1574w,
1.05 g, 10.5 mmol) were dissolved in dry THF (50 mL). To this 1527m, 1510w, 1465m, 1415m, 1409w, 1373w, 1309m, 1281m,
colourless reaction solution NEt (6.8 mL, 50 mmol) was 1261w, 1211w, 1180m, 1141m, 1131m, 1037m, 895w, 841w,
3
1
6
added and the mixture was stirred overnight at room tempera- 822w, 632w, 606w, 531w, 503w, 486w. H NMR (400 MHz, d -
ture. The solvent was evaporated under reduced pressure. The acetone): δ/ppm = 7.90 (d, J = 8.4 Hz, 1H, arom.), 7.84 (s, 1H,
crude product was purified by column chromatography on arom.), 7.61 (d, J = 7.6 Hz, 1H, arom.), 7.52 (s, 1H, NH),
silica with hexane : ethyl acetate 7 : 1 → 1 : 7, and the methanol 4.88–4.83 (m, 2H, C
3
3
2
5
H
4
; 1H, CH
2
), 4.67 (d, J = 9.2 Hz, 1H,
as eluent (R (4b) = 0.05, hexane : ethyl acetate 7 : 1, R (5b) = CH ), 4.41–4.40 (m, 2H, C H ), 4.22 (s, 5H, C H ), 1.93 (s, 3H,
f
f
2
5
4
5 5
1
3
6
0
.2, methanol) to afford N-(2-cyano-1-hydroxypropan-2-yl) CH
3
). C NMR (125 MHz, d -acetone): δ/ppm = 170.9, 157.0,
ruthenocenamide (4b) and 2-cyano-2-(ruthenocenecarboxa- 129.3, 129.2, 129.2, 129.1, 126.0, 125.1, 123.2, 123.0, 122.9,
mido)propyl ruthenocnecarboxylate (5b) as pale yellow solids, 119.7, 118.4, 118.0, 75.6, 71.9, 71.7, 71.6, 70.6, 69.6, 69.4, 51.2,
1
9
6
respectively. Yield: 31% (4b, 0.77 g, 2.06 mmol) and 19% (5b, 23.0. F NMR (282.23 MHz, d -acetone): δ/ppm = −63.6.
0
−
1
+
+
.81 g, 1.32 mmol). Data 4b: IR (KBr, cm ): 3248br, 31122s, ESI-MS: m/z (%) = 504.0 [M + Na] (100), 985.1 [2M + Na] (12).
3
056w, 2943w, 2887w, 2641w, 2324w, 2241w, 2050w, 1981w, HR ESI-MS: m/z (%) = 504.05927 calcd for C H F FeN NaO
2
3
18
3
3
2
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+
(
[M + Na] ) m/z (%) = 504.05958. Elemental analysis: calcd for (1-(ferrocenyloxy)-2-cyanopropan-2-yl)ferroceneamide (7) as a
−1
C H N O F Fe = C 57.40; H 3.77; N 8.73. Found = C 57.62; H yellow solid. Yield: 43% (0.021 g, 0.041 mmol). IR (KBr, cm ):
2
3
18 3 2 3
4
.01; N 8.38.
N-(2-Cyano-1-(5-cyano-2-(trifluoromethyl)phenoxy)propan-2-
3469s, 2929w, 2852w, 1637s, 1518s, 1378w, 1339w, 1305w,
1280w, 1101m, 1008m, 825m, 523m, 502m, 485m. H NMR
1
yl)ruthenoceneamide (6b). N-(2-Cyano-1-hydroxypropan-2-yl) (500 MHz, CD CN): δ/ppm = 6.48 (s, 1H, NH), 4.73–4.70 (m,
3
ruthenocenamide (4b, 0.150 g, 0.42 mmol) was dissolved in 2H, C
5
H
4
), 4.46 (s, 2H, RCH
2
OR), 4.39–4.38 (m, 2H, C
5 4
H ),
dry THF (30 mL). The colorless solution was cooled to 0 °C 4.32–4.31 (m, 2H, C
5
H
4
), 4.20 (s, 7H, C
5
H
5
, C ), 4.17 (s, 5H,
5 4
H
3
3
and NaH (0.015 g, 0.63 mmol) was added. After stirring the C H ), 3.80 (d, J = 9.3 Hz, 1H, CH ), 3.70 (d, J = 9.3 Hz, 1H,
5
5
2
1
3
reaction mixture for 30 min at 0 °C 3-fluoro-4-(trifluoromethyl) CH
2
3 3
), 1.68 (s, 3H, CH ). C NMR (125 MHz, CD CN): δ/ppm =
benzonitrile (0.080 g, 0.42 mmol) was added. After stirring the 207.9, 171.2, 121.2, 83.9, 75.6, 73.5, 71.9, 70.7, 70.6, 70.5, 69.6,
+
reaction mixture overnight at room temperature, additional 69.5, 69.5, 69.4, 51.7, 22.9. ESI-MS: m/z (%) = 484.03 [M − CN]
+
+
NaH (0.005 g, 0.21 mmol) and 3-fluoro-4-(trifluoromethyl) (12), 510.04 [M] (100), 533.00 [M + Na] (7). HR ESI-MS: m/z
+
benzonitrile (0.040 g, 0.21 mmol) were added to the reaction (%) = 510.06882, calcd for C26
2 2 2
H26Fe N O ([M + Na] ) m/z (%) =
mixture and the reaction was allowed to stir for another 3 h. 510.06864. Elemental analysis: calcd for C H N O Fe = C
2
6
26
2
2
2
The reaction was quenched with H
and the aqueous layer was extracted with ethyl acetate (3 ×
0 mL). The combined organic layers were dried over Na SO ,
2
O (2 mL) and brine (6 mL) 61.21; H 5.14; N 5.49. Found = C 61.06; H 5.20; N 5.31.
Crystallographic studies
1
2
4
filtered and the solvent was evaporated under reduced Single crystals of 4a and 7 were grown by slow evaporation of
pressure. The crude product was purified by column chromato- acetonitrile solutions of 4a or 7 respectively. Single crystals of
graphy on silica with hexane : ethyl acetate 2 : 1 as the eluent 4b were grown by slow evaporation of an dichloromethane
(
R
f
= 0.60) to give N-(2-cyano-1-(5-cyano-2-(trifluoromethyl) solution of 4b. Single crystals of 6a were grown by slow evapor-
phenoxy)propan-2-yl)ruthenoceneamide (6b) as white solid. ation of an acetone solution of 6a.
−
1
Yield: 11% (0.024 g, 0.046 mmol). IR (KBr, cm ): 3341br,
Crystallographic data of 4a, 4b and 6a were collected at 183
3
1
076br, 2952w, 2234s, 2165w, 1977s, 1630s, 1575s, 1528s, (2) K with Mo Kα radiation (λ = 0.7107 Å) that was graphite-
1
416s, 1285s, 1264s, 1186s, 1132s, 1039s, 816s, 815s, 735s. H monochromated on an Oxford Diffraction CCD Xcalibur
6
3
NMR (500 MHz, d -acetone): δ/ppm = 7.89 (d, J = 8.0 Hz, 1H, system with a Ruby detector. Suitable crystals were covered
arom. H), 7.80 (s, 1H, arom. H), 7.61 (d, J = 8.0 Hz, 1H, arom. with oil (Infineum V8512, formerly known as Paratone N),
H), 7.34 (s, 1H, NH), 5.20–5.19 (m, 2H, C
3
3
5
H
4
), 4.77 (d, J = 9.5 placed on a nylon loop that is mounted in a CrystalCap
Hz, 1H, CH ), 4.71 (s, 2H, C H ), 4.60–4.58 (m, 6H, CH and Magnetic™ (Hampton Research) and immediately transferred
2
5
4
2
1
3
6
Pro
C
5
H
5
3
), 1.87 (s, 3H, CH ). C NMR (125 MHZ, d -acetone): δ/ to the diffractometer. The program suite CrysAlis was used
ppm = 169.6, 169.5, 157.0, 156.9, 129.3, 129.3, 129.2, 129.2, for data collection, multi-scan absorption correction and data
4
7
1
1
7
27.2, 126.1, 125.1, 123.5, 123.3, 123.0, 122.9, 122.8, 120.7, reduction. Crystallographic data of 7 were collected at 100(2)
19.6, 118.4, 118.4, 118.1, 118.0, 79.9, 79.9, 73.2, 73.2, 72.5, K at the PXIII beamline of the SLS synchrotron with a radiation
1
9
2.0, 72.0, 71.2, 71.1, 51.2, 51.1, 22.9, 22.9.
F NMR wavelength of 0.71255 Å. The data was integrated with the XDS
6
48
49
(470.59 MHz, d -acetone): δ/ppm = −63.4. ESI-MS: m/z (%) = software
and further processed with the CCP4
and
50.0 [M + Na] (100). HR ESI-MS: m/z = 528.04751, calcd for POINTLESS software. The data has a low completeness
Ru ([M]) m/z = 528.04733; m/z (%) = 550.02942, because of the one-circle geometry at the beamline and the
calcd for C H F N NaO Ru ([M + Na]) m/z (%) = 550.02928. low symmetry. All structures were solved with direct methods
+
50
5
23 18 3 3 2
C H F N O
2
3
18
3
3
2
5
1
Elemental analysis: calcd for C23
H 3.70; N 7.72. Found = C 50.61; H 3.40; N 7.51.
H
21
F
3
N
3
O
3
Ru(H
2
O) = C 50.73; using SIR97
methods on
and were refined by full-matrix least-squares
2
52
F
with SHELXL-2014.
CCDC entries
N-(1-(Ferrocenyloxy)-2-cyanopropan-2-yl)ferroceneamide (7). 1501422–1501425 contain the X-ray data of compounds 4a, 4b,
N-(2-Cyano-1-hydroxypropan-2-yl)ferroceneamide (4a, 0.03 g, 6a and 7.
0
.096 mmol), (ferrocenylmethyl)trimethylammonium iodide
Bioassay/s to assess anti-parasitic activity
8-crown-6 (0.0076 g, 0.0288 mmol) were dissolved in dry Some parasites were produced in vivo in or on animals.
CN (20 mL) and refluxed (90 °C) for 120 h. Additional Haemonchus contortus and Trichostrongylus colubriformis (stron-
ferrocenylmethyl)trimethylammonium iodide (0.065 g, gylid nematodes) were maintained in sheep, and Dirofilaria
.17 mmol) was added to the reaction and further refluxed immitis (filarial nematodes) in dogs. Rhipicephalus sanguineus
(
0.065 g, 0.17 mmol), K CO (39.8 mg, 0.288 mmol) and
2
3
1
CH
3
(
0
overnight. The orange reaction mixture was allowed to reach (tick) was maintained on dogs. All animal experiments were
room temperature. The solvent was evaporated under reduced approved by the State of Fribourg, Switzerland, and supervised
2
pressure. The crude residue was redissolved in Et O (10 mL) by the Animal Welfare Officer of Novartis Animal Health.
and washed with H O (2 × 5 mL) and brine (2 × 5 mL). The Other parasites, that is, Ctenocephalides felis (flea) and Lucilia
2
organic phase was dried over MgSO , filtered and the solvent cuprina (fly), were produced in vitro and maintained on defibri-
4
was evaporated under reduced pressure. The crude product nated cattle blood. All bioassays were performed by Novartis
was purified by column chromatography on silica using Animal Health employing industry-standard operating
hexane : ethyl acetate 3 : 1 as the eluent (R = 0.29) to afford N- procedures.
f
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Assay to test activity in vitro against Haemonchus contortus
and Trichostrongylus colubriformis
compounds were tested by serial dilution to determine their
minimum effective doses. Ticks were left in contact with the
test compound for 10 min and then incubated at 28 °C and
80% relative humidity for seven days, during which the test
compounds’ effects were monitored. Acaricidal activity was
confirmed based on the pattern of lethality observed.
This method was conducted as described by Kaminsky et al.
1
0
(
2008). In brief, freshly harvested and cleaned nematode
eggs were seeded into a 96-well plate containing the test sub-
stances to be evaluated for anthelmintic activity. Each com-
pound was tested by serial dilution in order to determine its
minimum effective dose. The test compounds were embedded
in an agar-based nutritive medium allowing the full develop-
ment of eggs through to third stage larvae (L3). The plates
were incubated for 6 days at 28 °C and 80% relative humidity.
Egg hatching and ensuing larval development were recorded to
identify a possible nematocidal activity. Efficacy was expressed
as a percentage of reduced egg hatch, reduced development of
L3, or paralysis and death of larvae of all stages.
Contact (tarsal) test. This test was conducted as described
5
6
by Lovis et al. (2013). In brief, the test was performed by pre-
coating wells of a 96-well microliter plate with a serial dilution
of compound, allowing the evaluation of anti-parasitic activity
by contact with ticks. Adult ticks were then distributed to indi-
vidual wells of the plate and incubated at 28 °C and 80% rela-
tive humidity for seven days, during which the test com-
pound’s effect was monitored. Acaricidal activity was con-
firmed upon death of the adult ticks.
Determination of ROS level
Activity in vitro against Dirofilaria immitis
The assay was performed following a procedure published by
our group with slight modification. Briefly, HeLa cells (8000
in 100 µL medium per well) were seeded in a 96 well plate
Microfilariae present in blood from donor dogs chronically
infected with D. immitis were seeded into 96-well microplates.
Individual test compounds were tested by serial dilution to
determine their minimum effective doses. The plates were
incubated for 48 h at 26 °C and 60% relative humidity. The
motility of microfilariae was then recorded to identify any anti-
filarial activity. Efficacy was expressed as the percentage of
reduced motility compared to the control (untreated) and stan-
dards. In these assays, a compound needed to exhibit a nemato-
dicidal efficacy of >60% at a concentration of 32 µg ml−
57
(
black, clear flat bottom from corning). Next day, the media
was aspirated and 200 µM fresh media containing 4a, 4b, 6a or
b (freshly prepared stock solution in DMSO, after dilution
with culture medium final concentration of DMSO ≤0.3%,
5 µM exposure concentration) was added. Cells were then
6
2
incubated at 37 °C incubator for 22 h. The medium was then
removed, washed with 150 µL PBS and then 150 µL solution of
H2DCFDA (final concentration 20 μM) in serum free medium
was added and incubated for 40 min in dark. The fluorescence
generated by intracellular ester cleavage followed by oxidation
of H2DCFDA by intracellular ROS was quantified at 528 nm
emission with 485 nm excitation wavelength in a SpectraMax
M5 microplate reader. For the positive control TBH, 100 µM
concentration and 6 h incubation time was used. After quanti-
fication of the ROS, cell viability on HeLa cells was determined
to quantify the potential decrease in cellular mass due to treat-
ment with different compounds. The media was aspirated and
cells were washed with 150 µL PBS. Afterwards, 100 µL 0.4%
formaldehyde in PBS was added and cells were allowed to fix
for 20 min at room temperature. The formaldehyde solution
was aspirated and cells were washed with 150 µL PBS. 0.02%
crystal violet (CV) solution in PBS was then added (100 µL per
well) and incubated at room temperature for 30 min. The CV
solution was then aspirated, washed with 150 µL of distilled
water and dried overnight. Next day, 180 µL 80% ethanol was
added to each well and the plates were gently shaken on a
rocker for 2–3 h and were read at 570 nm in a SpectraMax
M5 microplate reader. The results expressed as mean and stan-
dard error of six replicates, corrected for the viable cell
population.
1
(32 ppm) to qualify for further testing.
Activity in vitro against Ctenocephalides felis
Oral test. This test was conducted as described by Wade
5
3,54
et al. (1988) and Zakson-Aiken et al. (2001).
In brief, adult
fleas were placed in a suitably formatted microtitration plate,
allowing fleas to access and feed on treated blood via an artifi-
cial feeding system. Each compound was tested by serial
dilution to determine its minimum effective doses. Fleas were
fed on treated blood for 24 h, after which the compound’s
effect was recorded. Insecticidal activity was determined on
the basis of the number of dead fleas recovered from the
feeding system.
Contact test. This test was conducted as described by Wade
5
3,54
et al. (1988) and Zakson-Aiken et al. (2001).
In brief, adult
fleas were distributed into wells of a microplate pre-coated
with a serial dilution of the compounds to be evaluated for
insecticidal activity. The fleas were left in contact with the
compound for 24 h. Insecticidal activity was confirmed upon
death of the adult fleas. In these assays, a compound needed
to exhibit an insecticidal efficacy of >80% at a concentration of
1
1
00 ppm (100 µg ml− ) to qualify for further testing.
Activity in vitro against Rhipicephalus sanguineus (dog tick)
Cell culture
Immersion test. This test was conducted as described by Human cervical carcinoma cells (HeLa) were cultured in
5
5
Lovis et al. (2011). In brief, adult Rhipicephalus sanguineus DMEM (Gibco) supplemented with 5% fetal calf serum (FCS,
were seeded into individual wells of a microtitration plate con- Gibco), 100 U ml− penicillin, 100 μg ml streptomycin at
1
−1
taining the test substances to be evaluated. Individual test 37 °C and 5% CO . The normal human fetal lung fibroblast
2
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MRC-5 cell line was maintained in F-10 medium (Gibco) sup- Dr Jacques Bouvier (Novartis Animal Health, St-Aubin,
−
1
plemented with 10% FCS (Gibco), penicillin (100 U ml ), and Switzerland) and Dr Noëlle Gauvry (Novartis Animal Health,
−
1
streptomycin (100 μg ml ).
Basel, Switzerland) for their help with the biological assays.
Cytotoxicity studies
Cytotoxicity studies were performed on two different cell lines,
namely HeLa, and MRC-5, by a fluorometric cell viability assay
using Resazurin (Promocell GmbH). Briefly, one day before
treatment, cells were seeded in triplicates in 96-well plates at a
density of 4 × 10 cells per well for HeLa and 7 × 10 for MRC-5
in 100 μl growth medium. Upon treating cells with increasing
concentrations of organometallic-monepantel derivatives for
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AADs
Amino-acetonitrile derivatives
C. felis
Ctenocephalides felis
D. immitis
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H. contortus
H2DCFDA
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