Synthesis, bioevaluation and docking studies of some 2-phenyl-1H-benzimidazole derivatives as anthelminthic agents against the nematode Teladorsagia circumcincta

Article abstract of DOI:10.1016/j.ejmech.2020.112554

Gastrointestinal nematode infections are the main diseases in herds of small ruminants. Resistance to the main established drugs has become a worldwide problem. The purpose of this study is to obtain and evaluate the in vitro ovicidal and larvicidal activity of some 2-phenylbenzimidazole derivatives on susceptible and resistant strains of Teladorsagia circumcincta. Compounds were prepared by known procedures from substituted o-phenylenediamines and arylaldehydes or intermediate sodium 1-hydroxyphenylmethanesulfonate derivatives. Egg Hatch Test (EHT), Larval Mortality Test (LMT) and Larval Migration Inhibition Test (LMIT) were used in the initial screening of compounds at 50 μM concentration, and EC₅₀ values were determined for the most potent compounds. Cytotoxicity evaluation of compounds was conducted on human Caco-2 and HepG2 cell lines to calculate their Selectivity Indexes (SI). At 50 μM concentration, nine out of twenty-four compounds displayed more than 98% ovicidal activity on a susceptible strain, and four of them showed more than 86% on one resistant strain. The most potent ovicidal benzimidazole (BZ) 3 showed EC₅₀ = 6.30 μM, for the susceptible strain, while BZ 2 showed the lowest EC₅₀ value of 14.5 μM for the resistant strain. Docking studies of most potent compounds in a modelled Teladorsagia tubulin indicated an inverted orientation for BZ 1 in the colchicine binding site, probably due to its fair interaction with glutamic acid at codon 198, which could justify its inactivity against the resistant strain of T. circumcincta.

Full text of DOI:10.1016/j.ejmech.2020.112554

Journal Pre-proof
Synthesis, bioevaluation and docking studies of some 2-phenyl-1H-benzimidazole
derivatives as anthelminthic agents against the nematode Teladorsagia circumcincta
Nerea Escala, Elora Valderas-García, María Álvarez Bardón, Verónica Castilla
Gómez de Agüero, Ricardo Escarcena, José Luis López-Pérez, Francisco A. Rojo-
Vázquez, Arturo San Feliciano, Rafael Balaña-Fouce, María Martínez-Valladares,
Esther del Olmo
PII:
S0223-5234(20)30526-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112554
EJMECH 112554
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 April 2020
Revised Date: 3 June 2020
Accepted Date: 7 June 2020
Please cite this article as: N. Escala, E. Valderas-García, M.Á. Bardón, V.C. Gómez de Agüero,
R. Escarcena, J.L. López-Pérez, F.A. Rojo-Vázquez, A. San Feliciano, R. Balaña-Fouce, M.
Martínez-Valladares, E.d. Olmo, Synthesis, bioevaluation and docking studies of some 2-phenyl-1H-
benzimidazole derivatives as anthelminthic agents against the nematode Teladorsagia circumcincta,
European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112554.
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Graphical Abstract
2-Phenyl-1H-benzimidazoles as anthelminthic
agents against susceptible and resistant
Teladorsagia circumcincta
BZs 1 (yellow) and 22 (green) as docked in the
colchicine site of modelled T. circumcincta tubulin

1
Synthesis, bioevaluation and docking studies of some 2-phenyl-1H-
benzimidazole derivatives as anthelminthic agents against the nematode
Teladorsagia circumcincta
Nerea Escalaa,#, Elora Valderas-Garcíab,c,#, María Álvarez Bardónb, Verónica Castilla Gómez de
Agüeroc,d, Ricardo Escarcenaa, José Luis López-Péreza,e, Francisco A. Rojo-Vázquezd, Arturo San
Felicianoa,f, Rafael Balaña-Fouce*b, María Martínez-Valladares*c,d, Esther del Olmo*a
# Both authors shared the first position
a
Departamento de Ciencias Farmacéuticas: Química Farmacéutica, Facultad de Farmacia, Universidad de
Salamanca, CIETUS, IBSAL, 37007-Salamanca, Spain.
b Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, 24071-León, Spain.
c Instituto de Ganadería de Montaña, CSIC-Universidad de León, 24346-Grulleros, León, Spain.
d Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de León, 24071-León, Spain
e Facultad de Medicina, Universidad de Panamá, Panamá, R. de Panamá.
f Programa de Pós-graduaçao em Ciências Farmacêuticas, Universidade do Vale do Itajaí, UNIVALI. Itajaí,
SC, Brazil.
* Corresponding authors
E-mail addresses: olmo@usal.es (E. del Olmo, Chemistry), rbalf@unileon.es (R. Balaña-Fouce, Toxicity
studies), mmarva@unileon.es (M. Martínez-Valladares, in vitro helminth assays).
Abstract
Gastrointestinal nematode infections are the main diseases in herds of small ruminants. Resistance
to the main established drugs has become a worldwide problem. The purpose of this study is to
obtain and evaluate the in vitro ovicidal and larvicidal activity of some 2-phenylbenzimidazole
derivatives on susceptible and resistant strains of Teladorsagia circumcincta. Compounds were
prepared by known procedures from substituted o-phenylenediamines and arylaldehydes or
intermediate sodium 1-hydroxyphenylmethanesulfonate derivatives. Egg Hatch Test (EHT), Larval
Mortality Test (LMT) and Larval Migration Inhibition Test (LMIT) were used in the initial
screening of compounds at 50 µM concentration, and EC50 values were determined for the most
potent compounds. Cytotoxicity evaluation of compounds was conducted on human Caco-2 and

2
HepG2 cell lines to calculate their Selectivity Indexes (SI). At 50 µM concentration, nine out of
twenty-four compounds displayed more than 98% ovicidal activity on a susceptible strain, and four
of them showed more than 86% on one resistant strain. The most potent ovicidal benzimidazole
(BZ) 3 showed EC50 = 6.30 µM, for the susceptible strain, while BZ 2 showed the lowest EC50
value of 14.5 µM for the resistant strain. Docking studies of most potent compounds in a modelled
Teladorsagia tubulin indicated an inverted orientation for BZ 1 in the colchicine binding site,
probably due to its fair interaction with glutamic acid at codon 198, which could justify its
inactivity against the resistant strain of T. circumcincta.
Keywords: 2-Phenyl-1H-benzimidazoles, Teladorsagia circumcincta, in vitro assays, cytotoxicity,
tubulin docking studies.
1. Introduction
Nematodes are roundworms belonging to the group of parasitic helminths that affect different host
species, including humans and animals. Soil transmitted helminths (STH) infected more than one
billion people around the world and contributed to 3.45 million disability adjusted life-years
(DALYs) in 2016 [1]. Although these infections are more prevalent in low-income countries, they
also occur in wealthy countries, such as the USA, Australia and some countries of the
Mediterranean basin [2-4]. Moreover, infections produced by gastrointestinal nematodes (GIN)s are
one of the most prevalent parasitic diseases affecting grazing ruminants worldwide. Their
importance is due to the significant economic losses they produce as a result of decreased
production and increased healthcare costs [5-7].
The control of GIN infections is based on the strategic application of anthelmintic drugs, but their
massive and incorrect administration have led to the development of anthelmintic resistance (AR),
especially in ruminants. The emergence of AR has been described in the three classes of broad-
spectrum anthelmintic drugs most commonly used in animals: benzimidazoles such as
fenbendazole, mebendazole and albendazole, among others, macrocyclic lactones (MLs) such as
ivermectin and moxidectin, and imidazothiazoles (IMs) such as levamisole [8-10]. Of these three
classes of anthelmintic drugs, the BZ group that usually interferes tubulin polymerization and
functions in parasites is the most popular and used in the control of GIN infections. Unfortunately,
the current situation of resistance to this family of drugs in animals represents a serious problem for
livestock production, mainly in sheep. This phenomenon is widely distributed, since resistant
populations have been reported all over the world [11-14].

3
In humans, the World Health Organisation (WHO) recommends preventive chemotherapy, as a
public health intervention to at-risk people living in endemic STH areas by mass administration of
BZs, mainly albendazole or mebendazole. In consequence, there are already studies showing the
decrease of BZ efficacy against some STH and the possible development of AR after years of mass
drug administration campaigns [15].
It is well known that the mechanism of action of BZs is targeted to the selective binding of the high
affinity colchicine site of parasite β-tubulin that prevents the formation of microtubules, which
results in the destruction of cell structure and consequently in the death of the parasite [11]; in fact,
competitive inhibition studies in mammalian isolated tubulins show that colchicine and BZ bind at
the same site near the N-terminal domain [16]. Computational studies performed with BZs support
this finding [17]. Three amino acid mutations (F167Y, E198A and F200Y) have been associated
with major causes of anthelmintic drug resistance due to loss of drug affinity by β-tubulin [18],
which seems to indicate that these amino acids would be located at or close to the colchicine
binding site.
The problem of AR is seriously aggravated by the limited development of novel anthelmintic drugs
during the last years. Classical BZ derivatives with nematocidal properties, such as albendazole,
fenbendazole, mebendazole, oxibendazole, parbendazole or luxabendazole display a carbamate
fragment at position C-2. Their activity is due to disruption of functions on the microtubule system
[19]. The carbamate fragment can be substituted by an electron rich fragment such as a phenyl ring
[20]. Here we report on the synthesis and bio-evaluation of some BZ derivatives containing a
substituted phenyl group attached to the C-2 position of the benzimidazole system.
According to Zajíčková et al. [21] an effective approach to obtain new anthelmintics is to exploit
the old ones, synthesizing derivatives and analogues of known drugs with an approved usage.
Interestingly, although many 2-phenylbenzimidazole derivatives were synthesized and tested
against viruses, fungi, and bacteria infecting humans or animals [22-24], and most rarely against
helminths, no evaluation of BZs against Teladorsagia circumcincta could be found. In a previous
study, the south Indian adult earth worm Pheretima posthuma was used as a model for testing the
anthelmintic activity of 2-phenylbenzimidazole derivatives [25]. However, it is important to note
that P. posthuma is not a parasitic worm, does not belong to phylum Nematoda, and therefore it is
not the most suitable model for parasitic infections.
In this context, the goal of the present study is to synthesize several series of BZ derivatives, aiming
to obtain new drugs with improved therapeutic profiles compared to those already marketed, as well
as to evaluate in vitro their anthelmintic activity against Teladorsagia circumcincta, which is a good
experimental model for sheep parasitic nematode infections, as well as one of the most prevalent

4
GIN species in small ruminants worldwide, specifically in template areas [26]. Our research
includes in vitro cytotoxicity tests in order to define candidate molecules for in vivo efficacy and
toxicity assays. With these aims, the anthelmintic activity has been assessed in three life-cycle
stages (eggs and larvae at two levels of growth) of Teladorsagia circumcincta, by means of
assaying the BZs on two isolates, one susceptible and the other resistant to marketed drugs as
albendazole, levamisole and ivermectin. The cytotoxicity evaluation has been carried out on Caco-2
(human colorectal adenocarcinoma) and the HepG2 (hepatocarcinoma) cells
2. Results and Discussion
2.1 Chemistry
Ten BZ derivatives were obtained by a reported two steps procedure. First, the sodium 1-
hydroxyphenylmethanesulfonate derivatives were prepared from the corresponding aldehydes, and
then coupled with substituted o-phenylenediamines in N,N-dimethylformamide (DMF) at 110-120
ºC, to provide the desired BZs in 43 to 87% yield [27]. Eleven BZs were obtained in a single step
by direct condensation of 1,2-phenylenediamines with the corresponding benzaldehyde derivative
in DMF and in the presence of sodium metabisulfite (Na2S2O5) under reflux for 16 to 20 h [28],
(Scheme 1). The wide range of yields observed in these syntheses is similar or even lower than
those reported by other authors. It can be mainly justified by the diverse electronic nature of the
substituents and the influence of their inductive and mesomeric effects on the electrophilia of the
aldehyde carbonyl or on the sulfonate α-carbon, and at a lesser level by the nucleophilia of the
amino groups. Actually, when the substituents involved are p-Cl and the m-NO2 groups at ring B,
the yields are the highest.
Scheme 1. Two procedures for the synthesis of 2-phenylbenzimidazole derivatives
The original articles referenced above comprise proposals for the reaction mechanisms involved in
these BZ syntheses. More recently, Penieres-Castillo et al [29] have published a more complete
study on the subject in which they described different reaction conditions.
BZs 13 and 18 were obtained by reduction of the corresponding nitrobenzimidazoles with H2 under
Pd-C catalysis, while BZ 19 was obtained in 70% yield from the amine 18 by reaction with

5
picolinic acid in the presence of 1,1-carbonyldiimidazole (CDI) as coupling agent. Scheme 2.
Scheme 2. The preparation of BZs 13, 18 and 19
Physicochemical, MS and NMR spectral data for all the BZs are reported in the experimental section.
IR spectra of BZs showed strong or very strong stretching (str) absorption bands of the
benzimidazole moiety near 3315 (N-H), 1613 and 1535 (Ar C-C), 1460 and 1384 (C=N) cm-1
; along
with those associated to the substituents on rings A and B, near 1521 and 1349 cm-1 (NO2 asym and
sym str bands, respectively), 1255 cm-1 (C-O), 731 cm-1 (C-Cl), 679 cm-1 (C-Br) or 1663 cm-1 (HN-
C=O amide I str band). NMR spectra were run either in methanol-d4 or DMSO-d6. 1H-NMR and 13C-
NMR spectra of BZs 1, 2, 3, 23 and 24 resulted simplified due to the symmetry of their structures. As
an example, in the1H-NMR spectrum of BZ 1, five signals were appreciated; one singlet at 3.84 ppm,
corresponding to the methoxy group; two doublets (J = 8.6 Hz) at 7.06 and 7.99 ppm, that correspond
to H-3´+5´and H-2´+6´, respectively, and two multiplets at 7.23 and 7.56 ppm corresponding to H-
13
5+6 and H-4+7, respectively. With respect to the C-NMR spectrum eight signals were observed,
one of a OCH3 at 54.5 ppm, three of aromatic methines at 114.1 (~ double intensity), 122.4 and 128.0
ppm assigned to C-3´+5´and C-4+7, C-5+6 and C-2´+6´, respectively, and four of non-protonated
carbons at 121.7, 138.5, 152.0 and 161.7 ppm, corresponding to C-1´, C-3a+7a, C-2 and C-4´,
respectively. Regarding 1H-NMR spectra of 5-substituted BZs, the signals corresponding to different
protons were assigned taking into account their splitting patterns and chemical shifts. Related to the
benzimidazole fragment, the signal corresponding to proton H-4 resonates in the 7.1 - 7.6 ppm range.
The chemical shift corresponding to H-6 appears at 7.1 – 7.4 ppm, except in the presence of nitrogen
substituents, such as NO2 (fairly downfield, ~ 8.2 ppm), NH2 (fairly upfield, 6.7 ppm) or NHCO
(slightly downfield, 7.5 ppm). The signal for H-7 resonates in the 7.4 - 7.6 ppm interval, though with
13
changes similar to those just above indicated in presence of nitrogen substituents. C-NMR spectra
of 5-substituted BZs, related to the benzimidazole fragment, showed signals of four non protonated
carbons, C-2 (150-157 ppm), C-5 (127-158 ppm), C-3a (131-141 ppm) and C-7a (132-147 ppm).
Related the last two carbons, low and broad signals were observed in some spectra due to poor
relaxation caused by their proximity to nitrogen atoms. Additionally, the signals of three methines C-
4 (96-116 ppm), C-6 (113-124 ppm) and C-7 (112-116 ppm) were appreciated. Chemical shifts of the
signals corresponding to ring B were also appropriately assigned considering the substitution pattern

6
of that ring.
2D-NMR experiments on BZ 19 allowed the assignment of the additional signals corresponding to
1
the pyridine portion. In the H-NMR spectrum, those multiplets at 7.62, 8.23, 8.29 and 8.73 ppm,
corresponding to H-5, H-4, H-3 and H-6, respectively, are correlated with signals at 127.9, 139.1,
123.3 and 149.7 ppm, respectively in the 13C-NMR spectrum.
2.2 Biological assays
2.2.1 Anthelmintic activity and toxicity
All the compounds synthesized according to schemes 1 and 2 were tested against a susceptible strain
of T. circumcinta and their ovicidal (egg hatching inhibition) effects were determined (see table 1).
Those compounds showing ovicidal activity > 98% were also tested against one resistant strain.
Cytotoxicity was assessed on human Caco-2 and HepG2 cells cultures. Compounds listed in Table 1
are organized in first place by the number and type of substituents at position C-5 (C-6) of the
benzimidazole system, and second, by the substituents on the 2-phenyl ring. From the results
included in Table 1, it can be observed that several compounds displaying R1 = H, Me, OMe or Cl, in
combination with R2 = 4-OMe, 4-Cl or 4-Br showed both inhibitory effects, on egg hatching (EHT)
and on larvae motility (LMT) at 50 µM final concentration. Notably, nine BZs displayed their anti-
hatching effect between 98.2 and 100% on the susceptible strain, and five of them showed this effect
between 81.1 and 98.9% on the resistant strain. Additionally, two BZs caused the death of first stage
larvae (L1) at 50 µM. On the other hand, compounds with polar groups such as NO2, NH2 or
picolinamido in R1, did not show a measurable effect on the nematodes, nor on the susceptible or the
resistant strain. Similarly, double substitutions on ring A, as those 5,6-dimethyl (BZ 23) or 5,6-
dichloro (BZ 24), also led to inactivity.
It is also interesting to verify that the type of substituent on C-4’ of the B-phenyl ring conditioned the
anthelmintic potency of active compounds. Curiously, some substituents as -OCH3 and -Cl displayed
higher effects at such 4’-position than if they are attached to the BZ moiety. Thus, with respect to
ring A, the unsubstituted BZs 1, 2 and 3, the 5-CH3 (BZs 4 and 5), the 5-Cl (BZs 8 and 9) and the 5-
OCH3 (BZs 21 and 22) were able to arrest the hatching of wild-type T. circumcinta eggs in more than
98% at the pre-established 50 ꢀM concentration. Among them, the BZs 2, 4, 8, 9 and 22 were also
able to produce a significant hatching inhibition on eggs, between 81.1 and 98,9%, of the resistant
strain of T. circumcincta used in this study. Surprisingly, despite its structural similarity with the
most potent substances in the series, BZ 1 is the only one that loses much of its inhibitory capacity
against the resistant strain.

7
Table 1. Inhibitory effects of BZs on hatching of wild type and resistant eggs of Teladorsagia
circumcinta. Cytotoxicity and selectivity indexes
Cytotoxicity a
CC50, µM
T. circumcincta
susceptible strain
T. circumcincta
resistant strain
Selectivity Indexes b
Compound
Hatch inhib.
% at 50 µM
Ovicidal
EC50, µM
Hatch inhib.
% at 50 µM
Ovicidal
EC50, µM
SIs
Caco-2
SIr
Caco-2
SIs
HepG2
SIr
HepG2
BZ
R1
R2
Caco-2
HepG2
1
2
H
4-OMe
4-Cl
100
99.5
99.8
100
15.78 ± 0.12
9.04 ± 0.23
6.30 ± 0.23
12.21 ± 0.29
15.23 ± 0.78
> 50
18.2
86.4
65.8
81.1
71.8
na
> 50
56.80 ± 8.12
42.63 ± 6.50
39.17 ± 2.46
11.60 ± 3.42
28.06 ± 1.35
9.14 ± 2.32
9.07 ± 2.35
37.38 ± 3.38
22.58 ± 1.69
19.14 ± 2.05
23.28 ± 2.47
67.31 ± 15.31
34.70 ± 4.32
14.37 ± 3.69
12.29 ± 1.09
16.78 ± 9.16
13.38 ± 3.02
> 25
43.08 ± 3.42
51.46 ± 6.27
38.36 ± 2.36
18.75 ± 1.92
27.11 ± 1.69
> 25
3.60
< 1
2.94
nc
2.73
< 1
3.56
nc
H
14.47 ± 1.05
4.72
6.22
0.95
1.84
nc
5.69
6.09
1.53
1.78
nc
3
H
4-Br
< 50
4
5-Me
5-Me
5-Me
5-Me
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-NO2
5-NO2
5-NO2
4-OMe
24.80 ± 0.99
0.47
< 1
nc
0.76
< 1
nc
5
4-Cl
99.5
0,80
1.11
100
< 50
6
2,5-diMe
3-NO2,4-OMe
4-OMe
nc
7
> 50
na
nc
> 25
nc
nc
nc
nc
8
13.74 ± 0.46
6.54 ± 0.40
> 50
89.6
94.0
na
25.76 ± 3.48
36.03 ± 3.02
17.54 ± 0.91
21.73 ± 1.65
30.22 ± 3.35
53.43 ± 16.61
44.91 ± 5.94
22.98 ± 19.57
14.64 ± 0.83
> 12.5
2.72
3.45
nc
1.45
1.08
nc
2.62
2.68
nc
1.39
0.84
nc
9
4-Cl
99.6
1.25
4.00
0.97
1.00
3.16
1.61
4.02
0.88
0.50
17.1
44.1
20.90 ± 0.50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4- NO2
nc
2,5-diMe
3-NO2,4-OMe
3-NH2,4-OMe
4-OMe
> 50
na
nc
nc
nc
nc
nc
> 50
na
nc
nc
nc
nc
nc
> 50
na
nc
nc
nc
nc
nc
> 50
na
nc
nc
nc
nc
nc
4-Cl
> 50
na
nc
nc
nc
nc
nc
2,5-diMe
> 50
na
nc
nc
nc
nc
nc
5-NO2 3-NO2,4-OMe
> 50
na
nc
13.64 ± 4.85
> 50
nc
nc
nc
nc
5-NH2
5-NHpic c
5-OMe
4-OMe
4-OMe
4-OMe
4-Cl
> 50
na
nc
nc
nc
nc
nc
> 50
na
nc
83.17 ± 45.84
21.80 ± 1.47
28.94 ± 1.47
21.69 ±1.61
13.01 ± 1.23
16.68 ± 1.46
> 300
22.76 ± 2.08
19.11 ± 3.68
21.33 ± 1.20
13.48 ± 0.64
31.66 ± 4.09
26.37 ± 2.29
>300
nc
nc
nc
nc
> 50
na
nc
< 50
nc
nc
nc
nc
5-OMe
98.2
99.9
17.4
0.44
100
25.60 ± 1.25
15.34 ± 0.55
> 50
70.6
1.13
1.41
nc
nc
0.83
0.87
nc
nc
5-OMe
4-Br
98.9
na
15.99 ± 0.42
nc
1.36
nc
0.84
nc
5,6-diMe
5,6-diCl
4-OMe
4-OMe
> 50
na
nc
nc
nc
nc
nc
Thiabendazole (TBZ)
100
1.53 ± 0.06
>697
>196
>697
>196
0.43 ± 0.02
a Determined by the Alamar Blue method. b Selectivity index: SI = CC50(Caco-2 or HepG2) / EC50(T.c.), relative to susceptible (SIs)
and resistant (SIr) strains. c picolinamide; na: not appreciable; nc: not calculated. Hatching inhibition values > 80 % and > 90%, the
EC50 values < 10 and < 20 µM, and the SI values > 4 have been bolded to facilitate comparisons.
Egg-hatching dose/response curves of the compounds of interest were performed in at least 6 final
concentrations in order to determine an EC50 value that could be comparable with mammalian
cytotoxicity (in the Supporting Information). Eight out of the nine hits mentioned above showed
similar EC50 values within the 6.30 to 15.78 ꢀM range. The most effective ovicidal BZs were 3 and 9,
which displayed significantly low EC50 values of 6.30 and 6.54 ꢀM, respectively, that is, around 14
times less active than thiabendazole. The EC50 values of the most potent compounds on the resistant
strain ranged between 14.47 and 25.76 ꢀM, which are 9 and 17 times less active than the reference
drug, respectively.
When analysing the effect of this series of 2-phenylbenzimidazole compounds on mammalian cells,
all the compounds were significantly cytotoxic within a range of 9.07 to 83.17 µM. Considering only
those compounds that have a significant effect against T. circumcincta, namely BZs 1 - 5, 8, 9, 21
and 22, their CC50 values were very homogeneous between Caco-2 and HepG2. This determined the

8
SI values calculated for these compounds in the susceptible strain of the parasite to be comprised
between 6.22 (BZ 3) and 0.95 (BZ 4) for Caco-2 cells and 6.22 (BZ 3) and 0.87 (BZ 22) for HepG2
cells. In the case of the resistant strain, the SI values were slightly lower and are comprised between
2.94 (BZ 2) and 0.47 (BZ 4) for the Caco-2 cells and between 3.56 and 0.76 for the same compounds
and HepG2 cells.
In order to study the killing effect of the twenty-four compounds on both susceptible and resistant T.
circumcincta L1, we used the LMT assay with levamisol (LEV) as reference drug for comparison.
Interestingly, three compounds of this series showed activity at this stage of the parasite: BZ 4 with a
larvicidal activity higher than 40%, and BZ 9 and 22, with lesser cytotoxicity which produced 100%
death of susceptible L1 larvae of T. circumcincta. Table 2 includes the larvicidal results showing that
these compounds proved much more potent than LEV, and in the case of BZ 9 also fairly more
selective. Nevertheless, it must be taken into account that SIs,r values in Table 2 are just indicative,
because they were calculated using the cytotoxicity CC50 values on human cells, whereas the EC50
values were determined on a complete and living organism, the nematode larvae. Independently, the
SI is a useful parameter to compare the relative toxicities and selectivities of BZs with those of the
currently used anthelmintic drug LEV. The EC50 values for susceptible L1 were 5.01 and 28.06 µM
for BZs 9 and 22, respectively (see also Supporting Information). The activity of BZ 9 on larvae was
much potent than on Teladorsagia eggs, while the activity of BZ 22 was more potent on
Teladorsagia eggs.
Table 2. Larvicidal effects of active BZs on susceptible and resistant larvae of Teladorsagia
circumcincta.
T. circumcincta L1
susceptible strain
T. circumcincta L1
resistant strain
Selectivity Indexes b
Cytotoxicity a
Mortality
% at 50 µM
Larvicidal
EC50 µM
Mortality
% at 50 µM
Larvicidal
EC 50 µM
Caco-2
CC50 µM
SIs
Caco-2
SIr
Caco-2
BZ
40.9
100
100
0
> 50
nt
100
99.3
0
nd
11.60 ± 3.42
22.58 ± 1.69
21.69 ± 1.61
2912 ± 970
0.95
4.51
0.77
1.63
0.47
1.94
0.75
1.20
4
9
22
5.01 ± 0.04
28.06 ± 2.95
1782 ± 26
11.66 ± 0.21
28.99 ± 3.32
2410 ± 23
LEV
a,b reproduced from Table 1 for comparison with the reference drug levamisol (LEV). SI = CC50(Caco-2) / EC50(L1). s,r: related to L1
of susceptible and resistant strains, respectively. nd: not determined. nt: not tested. EC50 values < 10 and < 30 µM and SI values >4
have been bolded to facilitate comparisons.
Very interestingly, as it can be seen in Table 2, BZs 9 and 22 killed practically all L1 larvae of both
susceptible and resistant strains at 50 µM, with EC50 values in de low-medium µM range.
Furthermore, both BZs were fairly more potent than the reference drug LEV, and BZ 9, the most
active compound, also showed higher SI values than LEV.
The effect of the compounds against the infective form of the parasite, third stage larvae (L3), was
assessed using the LMIT assay. The results showed that they did not produce a significant inhibition

9
of motility, since the highest percentage of inhibition of L3 migration was barely 30% in the case of
BZ 23.
In order to analyze the drugability of the active BZs, a preliminary prediction study was performed
online through the Osiris Data Warrior and preADMET free web services (see Experimental section
4.2.6 for details. Detailed results are shown in table S1 of Supporting Information). To sum up, all the
active BZs fulfil Lipinski’s Rule of Five with MW values in the range of 224.3 to 303.2 amu, clogP
values between 2.8 and 4.1, and the H-bond acceptors and donors (2 - 3 and 1, respectively) under
the established limits. Other properties as their solubility (>1 – 100 µM), total and polar surfaces (171
- 196 and 28 - 38 Å2, respectively), very good intestinal absorption (>92%) and plasma protein
binding (< 90%) ensuring their distribution, as well as their qualification within the 90% cut-off in
the World Drug Index, contributed to configure their Drug Score values in the range 0.40 - 0.69 of
the Osiris algorithm. Most significantly, none of the active BZs was predicted to show any of those
toxicity risks, mutagenic, tumorigenic, reproductive effective or irritant effects, considered in the
Osiris panel.
2.3 Molecular docking studies
In order to justify the differential activity of these compounds against susceptible and resistant strains
of T. circumcincta and further understand the molecular basis of its inhibitory properties, all
compounds described in this study were subjected to molecular docking with the T. circumcincta
tubulin, previously obtained by homology modelling as described in the experimental section 4.2.7.
Taking into account the possibility of tautomerism of the benzimidazole system, which may lead to
mixtures in dynamic equilibrium of asymmetrically substituted compounds, computational
calculations at the DFT level, MN15/6-31+G(d,p), as described in the experimental section 4.2.8
were conducted with the most potent BZs of this study. The results obtained indicate that the
tautomer with the substituent at C-6 (B) is about 0.5 - 1 kcal/mol more stable than the substituent at
C-5 (A) (Scheme 3).
Scheme 3. Tautomeric equilibrium shift and systematic numbering for non-symmetrically
5(6)-monosubstituted BZs.
The activity ranking of the docked compounds of table 1 in the mentioned T. circumcincta tubulin

10
homology model is in agreement with the ovicidal EC50 results. In fact, all BZs that show a value of
EC50 in the µM range (Table 1) appear positioned in the first places of the energetic ranking
according the GlideScore function implemented in GLIDE (see section 4.2.8). Interestingly, the
active conformation of the most efficient compounds corresponds to that of the lowest energy
tautomer. The docking poses of some significant ligands can be visualized in Figure 2 and 2D maps
of interactions in Figure 3 and Supporting Information p. S41.
Figure 2. A: Superimposition of the most potent BZs (4: cyan, 5: yellow, 8: brown 9: red, and
22: green) as docked in the colchicine site of the T. circumcincta tubulin model. B:
Superimposition of docked BZs 1 (yellow) and 22 (green).
Figure 3. 2D interaction maps resulting from the docking studies of several selected compounds
at the colchicine site of T. circumcincta tubulin model. (For other 2D maps, see suppl. info)

11
Looking at the docked molecules of Figure 2 and the 2D-maps of tubulin-BZs interactions of Figure
3, it can be seen that with the exception of BZ 1, the active compounds are arranged with the same
orientation in the site, while other aspects can be deduced from deeper examinations. Experimentally
active ligands have none or only one substituent in the benzene ring of the benzimidazole system,
whereas the presence of two substituents results in a loss of activity. Those with only one substituent
bind tubulin through the lowest energy tautomer; that is, with the substituent located at C-6 according
to the systematic IUPAC numbering. The possibility of interaction of the ligands in the tautomer
substituted at C-5 results unfavourable due to the steric effect exerted by the methyl group of Ala314.
This effect is clearly evident in 5,6-disubstituted BZs, as for example, in the cases of the BZ pairs 4 /
23 or 8 / 24. A decrease in potency is also observed in those BZs with the potent electron attracting
substituent -NO2 located on any benzene ring. This group is placed in an unfavourable hydrophobic
environment and provokes bad contacts with several amino acids of the binding site (Figure 4).
Figure 4. Bad contacts (pink broken lines) of nitro and methoxy groups of BZ 17.
In the colchicine binding site, the active compounds commonly establish a hydrogen bond with the
peptide carbonyl of Val236 through their BZN-H acting as a donor. Additionally, some of the most
potent compounds present ꢀ-stacking interactions between the BZ moiety and the tubulin Phe200
(Figure 5).

12
Figure 5. BZ 4 establishes a hydrogen bond (yellowish broken line) between its 1-BZN-H and
the amide carbonyl of Val236 (left), while BZ 1 establishes the hydrogen bond with Glu198
(right).
Interestingly, as shown in figures 2, 3 and 5, BZ 1 without any substituent on ring A interacts with
tubulin in an inverse arrangement with respect to most compounds of the series, (also BZ 8 in
Supporting Information, p. S41). As a consequence, it establishes its own BZN-H bond with the γ-
carboxyl group of Glu198 instead of the amide carbonyl of Val236. Most of BZs establish a common
H-bond acceptor with Val236 and also display π-stacking interactions with the phenyl group of
Phe200 through its imidazole nucleus (Figure 5). This different model of interaction could account
for the loss of activity of this compound against resistant strains. Indeed, as described in the literature
[18], the mutations associated with BZ-resistant strains for T. circumcincta and Haemonchus
contortus are actually Phe167Tyr, Glu198Ala and Phe200Tyr, and as shown graphically and stated
above, Glu198 and Phe200 are directly involved in tubulin - BZs interactions, which should result
perturbed in some extension by such mutations.
3. Conclusions
In summary, we have identified some 2-phenylbenzimidazoles that exhibit promising nematocidal
activity. Nine compounds displayed more than 98% ovicidal activity on T. circumcincta susceptible
strain at 50 µM, and five of them showed more than 80% ovicidal activity on a resistant strain of T.
circumcincta at the same concentration. BZ 3 was the most potent ovicidal on the susceptible strain,
with an EC50 value of 6.30 µM and SI of 6.22 in relation to Caco-2 cells. BZ 2, showed the lowest
EC50 value of 14.47 µM on the resistant strain and SI of 2.94 on Caco-2 cells. Two BZs produced the
motility inhibition and death of T. circumcincta L1, though none of them showed significant motility
inhibition on the L3 stage. Docking studies of most active BZs on a modelled tubulin of T.
circumcincta indicated a steric orientation of BZs 1 and 8 in their interaction with tubulin, that fairly
differ from the other BZs of this study, and that could justify the lack of effect of BZ 1 against the
resistant strain of T. circumcincta. Globally, these results do not seem very good, mainly because the
infective L3 larvae were resistant to the 24 BZs assayed, however, they constitute a promising
starting point to design and synthesize other compounds with improved tubulin affinity and higher
antiparasitic efficacy. Preliminarily, supported by its activity/selectivity profile and acceptable
ADME/toxicity-risks predictions, BZ 9 could be selected for in vivo efficacy and acute toxicity
evaluation on infected laboratory animals. The combined use of BZ 9 and other approved
anthelminthic agent acting through a different mechanism against the parasite, also looking for
additive or synergic effects could also be considered. However, further and more complete research

13
will be first carried out to obtain better candidates with effects on L3 larvae, with a greater
nematocidal potency and a higher selectivity index.
4. Experimental
4.1. Chemistry
4.1.1. Chemistry General
All commercial chemicals (Aldrich, Alpha, Fischer, SDS) were used as purchased and solvents
(Fischer, SDS, Scharlau) purified by the standard procedures prior to use [30]. Reactions were
monitored by Thin Layer Chromatography (TLC) (Kieselgel 60 F254 precoated plates, E. Merck,
Germany), the spots were detected by exposure to UV light at λ 254 nm, and colorization with 10%
ninhydrin spray, and further heating of the plate. Melting points (Mp) were determined with a Büchi
apparatus in open capillaries and were uncorrected. Separations by flash column chromatography
were performed on Merck 60 silica gel (0.063-0.2 mesh). Infrared spectra were recorded on a FT-IR
spectrometer Perkin Elmer, System BX using solid samples in KBr disks. NMR spectra were
recorded on a Bruker Avance 400 MHz (400 MHz for 1H, 100 MHz for 13C) and Varian Mercury 200
1
13
MHz ((200 MHz for H, 50 MHz for C). The spectra were measured either in methanol-d6 or
DMSO-d6, using tetramethylsilane (TMS) as internal standard, chemical shifts (δ) are given in ppm
and coupling constants (J) in Hertz. High resolution mass spectra (HRMS) were obtained by electron
spray ionisation-mass spectrometry (ESI-MS) technique (5 kV) on a QSTAR XL mass spectrometer.
4.1.2. Synthesis
4.1.2.1. Method A. General procedure for the synthesis of intermediate sodium hydroxy-(4-
methoxyphenyl)methanesulfonate, SMS-1.
4-methoxybenzaldehyde (7.34 mmol) and ethanol (22 mL) were added to a round-bottom flask
equipped with a magnetic stir bar under argon atmosphere; then a 16% solution of Na2S2O5 (5 mL)
was added. The reaction mixture was vigorously stirred for one hour at room temperature, and kept
overnight a 4 ºC in a refrigerator. The resulting precipitate was filtered off under vacuum to obtain a
white solid in quantitative yield.
4.1.2.1.1. Sodium hydroxy-(4-methoxyphenyl)methanesulfonate, SMS-1.
White crystalline solid (100% yield); thermal decomposition of the compound prohibits melting point
1
measurement. IR (KBr), νmax: 3435, 3200, 2980, 1610, 1517, 1250, 1114, 839 cm-1. H NMR

14
(C2D6SO): δ 3.70 (s, 3H), 4.85 (d, J = 5.0 Hz, D2O, 1H), 5.65 (d, J = 5.0 Hz, 1H), 6.77 (d, J = 8.6 Hz,
2H), 7.31 (d, J = 8.6 Hz, 2H). 13C NMR (C2D6SO): δ55.8, 83.7, 114.5 (2C), 128.1 (2C), 133.2, 159.2.
HRMS (ESI+) calcd. for C8H9Na2O5S[M+Na]+: 262.9961; found: 262.9959.
4.1.2.1.2. Sodium hydroxy-(4-chlorophenyl)methanesulfonate, SMS-2.
White crystalline solid (100% yield); thermal decomposition of the compound prohibits melting point
1
measurement. IR (KBr): νmax 3440, 2930, 1660, 1595, 1244, 1136, 832, 621 cm-1. H NMR
(C2D6SO): δ 4.91 (d, J = 5.4 Hz, D2O, 1H), 5.94 (d, J = 5.4 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.41
(d, J = 8.4 Hz, 2H). 13C NMR (C2D6SO): δ 84.2, 126.8 (2C), 129.5 (2C), 131.2, 138.8. HRMS (ESI+)
calcd. for C7H7ClNaO4S [M+Na]+: 266.9465, found: 266.9462.
4.1.2.2. General procedure for the preparation of 2-phenylbenzimidazole derivatives from
intermediates SMS-1 and SMS-2 (compounds 1, 2, 4, 5, 8, 9, 14, 15, 23 and 24).
0.82 mmol of the corresponding intermediate was dissolved in N,N-dimethylformamide (2 mL) and
0.58 mmol of the substituted 1,2-phenylenediamine was added. The mixture was heated at 110-120
ºC for 20–24 h with magnetic stirring. After cooling, the reaction crude was poured on a mixture of
ice/water (150 mL) to give a solid that was filtered off in a Büchner funnel. The resulted solid was
purified by column chromatography on silica gel with solvent gradient (hexane/ethyl acetate from 9:1
to 8:2), and recrystallized from the adequate solvent. Reaction yields ranged within 43 - 87%.
4.1.2.2.1. 2-(4-Methoxyphenyl)-1H-benzimidazole, 1.
Yield: 43 %. Compound crystallized from EtOH to give a yellow solid, Mp: 229-230 ºC IR (KBr):
.
νmax 3460, 2836, 1611, 1436, 1254, 1179, 1035, 836, 803, 744 cm-1. 1H NMR (400 MHz, CD3OD): δ
3.84 (s, 3H), 7.06 (d, J = 8.6 Hz, 2H), 7.23 (m, 2H), 7.56 (m, 2H), 7.99 (d, J = 8.6 Hz, 1H). 13C NMR
(100 MHz, CD3OD): δ 54.5 (OCH3), 114.1 (C-4 + C-7 + C3´ + C-5´), 121.7 (C-1´), 122.4 (C-5 + C-
6), 128.0 (C-2´ + C-6´), 138.5 (C3a + C-7a), 152.0 (C-2), 161.7 (C-4´). HRMS (ESI+) calcd. for
C14H13N2O [M+H]+: 225.1028, found: 225.1027.
4.1.2.2.2. 2-(4-Chlorophenyl)-1H-benzimidazole, 2.
Yield: 65 %. Compound crystallized from EtOH to give a white solid, Mp: 300-301 ºC IR (KBr):
.
νmax 3459, 2920, 1611, 1429, 1272, 1176, 1089, 831, 803, 745 cm-1. 1H NMR (400 MHz, CD3OD): δ
7.27 (m, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.61 (m, 2H), 8.06 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz,
CD3OD): δ 124.2 (2C), 129.3 (4C), 129.6, 130.4 (2C), 137.4 (3C), 152.2. HRMS (ESI+) calcd. for

15
IR
C13H10ClN2 [M+H]+: 229.0533, found: 229.0536.
4.1.2.2.3. 2-(4-Methoxyphenyl)-5-methyl-1H-benzimidazole, 4.
Yield: 59 %. Compound crystallized from EtOH to give a light yellow solid, Mp: 163-164 ºC
.
1
(KBr): νmax 3460, 2921, 1611, 1438, 1255, 1176, 1030, 836, 803, 738 cm-1. H NMR (200 MHz,
CD3OD): δ 2.46 (s, 3H), 3.87 (s, 3H), 7.07 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.2 Hz, 1H), 7.36 (bs,
1H), 7.44 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 8.6 Hz, 2H). 13C NMR (50 MHz, CD3OD): δ 20.3 (CH3),
54.4 (OCH3), 113.6 (C-7), 114.0 (C-4 + C-3´ + C-5´), 122.0 (C-1´), 123.7 (C-6), 127.8 (C-2´ + C-6´),
132.1 (C-5), 137.2 (C-7a), 138.6 (C-3a), 151.8 (C-2), 161.4 (C-4´). HRMS (ESI+) calcd. for
C15H15N2O [M+H]+: 239.1184, found: 239.1180.
4.1.2.2.4. 2-(4-Chlorophenyl)-5-methyl-1H-benzimidazole, 5.
Yield: 87 %. Compound crystallized from EtOH to give a white solid, Mp: 189-190 ºC IR (KBr):
.
1
νmax 3580, 2921, 1630, 1520, 1418, 1090, 965, 834, 801, 731, 676 cm-1. H NMR (200 MHz,
CD3OD): δ 2.46 (s, 3H), 7.10 (dd, J1 = 7.8; J2 = 1.0 Hz, 1H), 7.38 (d, J =1.0 Hz, 1H), 7.47 (d, J = 7.8
13
Hz, 1H), 7.52 (d, J = 9.0 Hz, 2H), 8.02 (d, J = 9.0 Hz, 2H). C NMR (50 MHz, CD3OD): δ 20.3,
113.8, 114.4, 124.4, 127.7 (2C), 128.2, 128.9 (2C), 133.0, 135.8, 137.0, 138.4, 150.3. HRMS (ESI+)
calcd. for C14H12ClN2 [M+H]+: 243.0689, found: 243.0687.
4.1.2.2.5. 5-Chloro-2-(4-methoxyphenyl)-1H-benzimidazole, 8.
Yield: 63 %. Compound crystallized from EtOH to give a white solid, Mp: 175-176 ºC IR (KBr):
.
1
νmax 3429, 2910, 1613, 1494, 1432, 1391, 1257, 1178, 924, 835, 739 cm-1. H NMR (200 MHz,
CD3OD): δ 3.83 (s, 3H), 7.03 (d, J = 8,6 Hz, 2H), 7.17 (dd, J1 = 8.6; J2 = 1,8 Hz, 1H), 7.47 (d, J = 8.6
13
Hz, 1H), 7.50 (d, J = 1,8 Hz, 1H), 7.95 (d, J = 8,6 Hz, 2H). C NMR (50 MHz, CD3OD): δ 54.5
(OCH3), 114.0 (C-7), 114.1 (C-3´ + C-5´), 115.0 (C-4), 121.3 (C-1´), 122.6 (C-6), 127.7 (C-5), 128.0
(C-2´ + C-3´), 137.2 (C-7a), 139.6 (C-3a), 153.3 (C-2), 161.8 (C-4´). HRMS (ESI+) calcd. for
C14H12ClN2O [M+H]+: 259.0638; found: 259.0635.
4.1.2.2.6. 5-Chloro-2-(4-chlorophenyl)-1H-benzimidazole, 9.
Yield: 80 %. Compound crystallized from EtOH to give a white solid, Mp: 229-230 ºC IR (KBr):
.
1
νmax 3510, 2923, 1621, 1582, 1480, 1422, 1302, 1086, 925, 833, 731 cm-1. H NMR (200 MHz,
CD3OD): δ 7.21 (dd, J1 =8.6; J2 = 2.0 Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.49 (d, J = 8.6 Hz, 2H),
13
7.54 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 8.6 Hz, 2H). C NMR (50 MHz, CD3OD): δ 114.4, 115.5,
123.1, 127.9 (2C), 128.2, 128.9 (2C), 136.3 (2C), 137.6, 139.8, 152.1. HRMS (ESI+) calcd. for
C13H9Cl2N2 [M+H]+: 263.0143; found: 263.0143.

16
IR
4.1.2.2.7. 2-(4-Methoxyphenyl)-5-nitro-1H-benzimidazole, 14.
Yield: 75 %. Compound crystallized from MeOH to give a brown-red solid, Mp: 237-239 ºC
.
1
(KBr): νmax 3351, 3085, 1621, 1574, 1509, 1347, 1277, 1012, 881, 817, 737 cm-1. H NMR (200
MHz, C2D6SO): δ 3.84 (s, 3H), 7.14 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 9.0 Hz, 1H), 8.08 (dd, J1 = 9.0;
J2 = 2.4 Hz, 1H), 8.15 (d, J = 8.8 Hz, 2H), 8.40 (d, J =2.4 Hz, 1H). 13C NMR (50 MHz, C2D6SO): δ
55.9 (OCH3), 108.0 (C-4), 111.8 (C-7), 115.0 (C-3´ + C-5´), 118.3 (C-6), 121.8 (C-1´), 129.1 (C-2´ +
C-6´), 140.3 (C-3a), 142.9 (C-5), 143.6 (C-7a), 155.8 (C-2), 161.9 (C-4´). HRMS (ESI+) calcd. for
C14H12N3O3 [M+H]+: 270.0879, found: 270.0884.
4.1.2.2.8. 2-(4-Chlorophenyl)-5-nitro-1H-benzimidazole, 15
Yield: 85 %. Compound crystallized from MeOH to give a yellow solid, Mp: 306-307 ºC IR (KBr):
.
νmax 3441, 2921, 1601, 1498, 1333, 1289, 1060, 837, 735 cm-1. 1H NMR (200 MHz, C2D6SO): δ 7.67
(d, J = 8.6 Hz, 2H), 7.76 (d, J = 9.0 Hz, 1H), 8.13 (dd, J1 = 9.0 Hz; J2= 2.0 Hz, 1H), 8.21 (d, J = 8.6
Hz, 2H), 8.47 (d, J = 2.0 Hz, 1H). 13C NMR (50 MHz, C2D6SO): δ 113.1, 115.0, 118.5, 128.3, 129.0
(2C), 129.6 (2C), 136.1, 138.8, 140.2, 143.2, 155.0. HRMS (ESI+) calcd. for C13H9ClN3O2 [M+H]+:
274.0383, found: 274.0394.
4.1.2.2.9. 2-(4-Methoxyphenyl)-5,6-dimethyl-1H-benzimidazole, 23.
Yield: 60 %. Compound crystallized from EtOH to give a light yellow solid, Mp: 227-229 ºC IR
.
1
(KBr): νmax 3435, 2921, 1612, 1500, 1440, 1384, 1253, 1151, 1031, 923, 836, 795 cm-1. H NMR
(200 MHz, CD3OD): δ 2.14 (s, 6H), 4.07 (s, 3H), 7.02 (d, J = 8.9 Hz, 2H), 7.33 (s, 2H), 7.92 (d, J =
8.9 Hz, 2H). 13C NMR (50 MHz, CD3OD): δ 19.5 (2 x CH3), 54.6 (OCH3), 114.0 (C-4 + C-7), 114.4
(C-3´ + C-5´), 119.7 (C-1´), 128.2 (C-2´ + C-6´), 132.8 (C-5 + C-6), 134.9 (C-3a + C-7a), 150.2 (C-
2), 162.1 (C-4´). HRMS (ESI+) calcd. for C16H17N2O [M+H]+: 253.1341, found: 253.1352.
4.1.2.2.10. 5,6-Dichloro-2-(4-methoxyphenyl)-1H-benzimidazole, 24.
Yield: 71 %. Compound crystallized from EtOH to give a beige solid, Mp: 218-220 ºC IR (KBr):
.
νmax 3321, 2939, 1610, 1573, 1492, 1383, 1261, 1192, 1090, 1028, 969, 832, 735 cm-1.1H NMR (200
13
MHz, CD3OD): δ 3.87 (s, 3H), 7.08 (d, J = 7.4 Hz, 2H), 7.69 (s, 2H), 7.99 (d, J = 7.4 Hz, 2H). C
NMR (50 MHz, CD3OD): δ 54.6 (OCH3), 114.3 (C-3´ + C-5´), 115.4 (C-4 + C-7), 120.9 (C-1´),
125.9 (C-5 + C-6), 128.3 (C-2´ + C-6´), 138.1 (C-3a + C-7a), 154.5 (C-2), 162.2 (C-4´). HRMS
(ESI+) calcd. for C14H11Cl2N2O [M+H]+: 293.0248, found: 293.0238.
4.1.2.3. Method B. General procedure for the preparation of 2-phenylbenzimidazole derivatives

17
(compounds 3, 6, 7, 10, 11, 12, 16, 17, 20, 21 and 22).
A mixture of the corresponding 1,2-phenylenediamine (1.00 mmol), the substituted benzaldehyde
(1.00 mmol) and the Na2S2O5 (1.00 mmol) in dry DMF (4 mL) was heated to reflux for 6-12 h.
Then, the reaction was cooled to room temperature and water (20 mL) was added to provide a solid
that was filtered off in Büchner. The solid was purified by column chromatography on silica gel with
hexane/ethyl acetate (8:2) as elution system, and recrystallized from the adequate solvent in most
cases. Reaction yields ranged within 40 - 73%.
4.1.2.3.1. 2-(4-Bromophenyl)-1H-benzimidazole, 3.
Yield: 40 %. Compound crystallized from EtOH to give an orange solid, Mp: 295-296 ºC IR (KBr):
.
νmax 3460, 2924, 1620, 1430, 1260, 1178, 1070, 835, 802, 670 cm-1. 1H NMR (400 MHz, CD3OD): δ
7.26 (m, 2H), 7.60 (m, 2H), 7.70 (d, J = 8.0 Hz, 2H), 7.99 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz,
CD3OD): δ 124.2 (2C), 126.3, 129.5 (2C), 129.9 (2C), 130.2, 133.4 (2C), 138.9 (2C), 152.3. HRMS
(ESI+) calcd. for C13H10BrN2 [M+H]+: 273.0027, found: 273.0026.
4.1.2.3.2. 2-(2,5-Dimethylphenyl)-5-methyl-1H-benzimidazole, 6.
Yield: 42 %. Compound crystallized from EtOH to give a light yellow solid, Mp: 112-113 ºC IR
.
(KBr): νmax 3430, 2920, 1629, 1447, 1276, 1000, 805 cm-1. 1H NMR (200 MHz, CD3OD): δ 2.33 (s,
3H), 2.43 (s, 3H), 2.45 (s, 3H), 7.07 (dd, J1 = 8.2; J2 = 1.2 Hz, 1H), 7.18 (bs, 2H), 7.38 (m, 2H), 7.46
(d, J = 8.2 Hz, 1H). 13C NMR (50 MHz, CD3OD): δ 18.7 (CH3 at C-2´), 19.5 (CH3 at C-5´), 20.3 (CH3
at C-5), 113.8 (C-7), 114.3 (C-4), 123.8 (C-6), 129.8 (C-1´), 130.0 (C-4´), 130.2 (C-3´), 130.6 (C-6´),
132.3 (C-5), 133.8 (C-2´), 135.3 (C-5´), 136.8 (C-7a), 138.1 (C-3a), 152.4 (C-2). HRMS (ESI+)
calcd. for C16H17N2 [M+H]+: 237.1392; found: 237.1396.
4.1.2.3.3. 5-Methyl-2-(4-methoxy-3-nitrophenyl)-1H-benzimidazole, 7.
Yield: 66 %. Compound crystallized from MeOH to give a yellow solid, Mp: 221-222 ºC IR (KBr):
.
1
νmax 3313, 2936, 1622, 1550, 1521, 1436, 1336, 1267, 1010, 815, 793 cm-1. H NMR (200 MHz,
CD3OD): δ 2.45 (s, 3H), 4.02 (s, 3H), 7.09 (dd, J1 = 8.2; J2 = 1.6 Hz, 1H), 7.35 (d, J2 = 1.6 Hz, 1H),
7.43 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 8.25 (dd, J1 = 8.6; J2 = 2.4 Hz, 1H), 8.49 (d, J = 2.4
13
Hz, 1H). C NMR (50 MHz, CD3OD): δ 20.3 (CH3), 56.0 (OCH3), 112.0 (C-7), 112.2 (C-4), 114.3
(C-5´), 122.3 (C-1´), 122.9 (C-2´), 124.3 (C-6), 131.6 (C-6´), 132.4 (C-5), 136.8 (C-7a), 137.2 (C-
3a), 140.0 (C-3´), 151.2 (C-2), 153.7 (C-4´). HRMS (ESI+) calcd. for C15H14N3O3 [M+H]+: 284.1035,
found: 284.1031.

18
4.1.2.3.4. 5-Chloro-2-(4-nitrophenyl)-1H-benzimidazole, 10.
Yield: 55 %. Compound crystallized from MeOH to give a yellow solid, Mp: 249-250 ºC
.
IR (KBr):
1
νmax 3314, 2900, 1604, 1511, 1413, 1349, 1109, 924, 854, 809, 707 cm-1. H NMR (200 MHz,
CD3OD): δ 7.25 (dd, J1 = 8.6; J2 = 2.0 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H),
8.21 (d, J = 9.0 Hz, 2H), 8.34 (d, J = 9.0 Hz, 2H). 13C NMR (50 MHz, CD3OD): δ 113.9 (C-4), 116.1
(C-7), 123.8 (C-6), 123.9 (C-3´ + C-5´), 127.3 (C-2´ + C-6´), 131.6 (C-5), 131.9 (C-7a), 135.0 (C-1´),
141.3 (C-3a), 148.7 (C-4´), 151.2 (C-2). HRMS (ESI+) calcd. for C13H9ClN3O2: 274.0383; found:
274.0381.
4.1.2.3.5. 5-Chloro-2-(2,5-dimethylphenyl)-1H-benzimidazole, 11.
Yield: 62 %. Compound crystallized from EtOH to give a beige solid, Mp: 192-193 ºC IR (KBr):
.
1
νmax 3435, 2919, 1613, 1584, 1496, 1440, 1375, 1275, 1060, 926, 807 cm-1. H NMR (200 MHz,
CD3OD): δ 2.35 (s, 3H), 2.44 (s, 3H), 7.23 (m, 3H) 7.42 (bs, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.58 (d, J
13
= 2.0 Hz, 1H). C NMR (50 MHz, CD3OD): 18.7, 19.5, 114.3, 115.4, 122.7, 127.8, 129.3, 129.9,
130.5, 130.8, 134.0, 135.5, 136.9, 139.2, 154.1. HRMS (ESI+) calcd. for C15H14ClN2: 257.0846;
found: 257.0845.
4.1.2.3.6. 5-Chloro-2-(4-methoxy-3-nitrophenyl)-1H-benzimidazole, 12.
Yield: 70 %. Compound crystallized from MeOH to give a pale yellow solid, Mp: 239-240 ºC IR
.
(KBr): νmax 3306, 2920, 1626, 1515, 1442, 1343, 1267, 1011, 923, 891, 822, 643 cm-1. 1H NMR (200
MHz, C2D6SO): δ 4.00 (s, 3H), 7.21 (dd, J1= 8.6; J2= 2.4 Hz, 1H), 7.56 (d, J= 9.0 Hz, 1H), 7.60 (d,
J= 8.6 Hz, 1H), 7.63 (d, J= 2.4 Hz, 1H), 8.42 (d, J1= 9.0; J2= 2.2 Hz, 1H), 8.64 (d, J= 2.2 Hz, 1H).
13C NMR (50 MHz, C2D6SO): δ 57.5, 115.2, 115.6, 116.4, 122.6, 123.0, 123.6, 127.0, 132.9, 138.4,
139.6, 140.9, 151.1, 153.8. HRMS (ESI+) calcd. for C14H11ClN3O3: 304.0489; found: 304.0478.
4.1.2.3.7. 2-(2,5-Dimethylphenyl)-5-nitro-1H-benzimidazole, 16.
Yield: 58 %. Compound crystallized from MeOH to give a white solid, Mp: 257-258 ºC IR (KBr):
.
1
νmax 3420, 2923, 1626, 1510, 1469, 1334, 1287, 1065, 921, 884, 737 cm-1. H NMR (200 MHz,
CD3OD): δ 2.33 (s, 3H), 2.45 (s, 3H), 7.19 (d, J = 8.6 Hz, 1H), 7.21 (d, J = 8.6, 1H), 7.42 (s, 1H),
7.62 (d, J = 8.6 Hz, 1H), 8.09 (dd, J1 = 8.6; J2 = 2.0 Hz, 1H), 8.41 (d, J = 2.0 Hz, 1H). 13C NMR (50
MHz, CD3OD): δ 18.9, 19.5, 112.1, 114.0, 117.9, 128.6, 129.9, 131.0 (2C), 134.2, 135.6, 140.1,
141.9, 143.4, 157.2. HRMS (ESI+) calcd. for C15H14N3O2 [M+H]+: 268.1086; found: 268.1080.
4.1.2.3.8. 2-(4-Methoxy-3-nitrophenyl)-5-nitro-1H-benzimidazole, 17.

19
Yield: 43 %. Compound crystallized from MeOH to give a dark yellow solid, Mp: 290-292 ºC
.
IR
1
(KBr): νmax 3433, 3351, 2923, 1621, 1508, 1444, 1347, 1277, 1066, 1012, 881, 817, 736 cm-1. H
NMR (200 MHz, C2D6SO): δ 4.02 (s, 3H), 7.61 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 8.12
(dd, J1 = 9.0; J2 = 2.4 Hz, 1H), 8.44 (d, J = 2.4 Hz, 1H), 8.46 (dd, J1 = 9.0; J2 = 2.4 Hz, 1H), 8.70 (d,
J= 2.4 Hz, 1H). 13C NMR (50 MHz, C2D6SO): δ 57.2, 113.0 113.7, 115.3, 118.2, 121.4, 123.7, 132.9,
137.9, 139.1, 142.8, 145.3, 153.9, 154.0. HRMS (ESI+) calcd. for C14H11N4O5 [M+H]+: 315.0729;
found: 315.0718.
4.1.2.3.9. 5-Methoxy-2-(4-methoxyphenyl)-1H-benzimidazole, 20.
Yield: 46 %. Compound crystallized from EtOH to give a yellow solid, Mp: 150-152 ºC IR (KBr):
.
νmax 3350, 2920, 1623, 1512, 1430, 1335, 1330, 1285, 1220, 1060, 920, 818, 795 cm-1. 1H NMR (400
MHz, CD3OD): δ 3.87 (s, 3H), 3.89 (s, 3H), 7.00 (dd, J1= 9.2; J2 = 2.0 Hz, 1H), 7.12 (d, J = 2.0 Hz,
1H), 7.13 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 9.2 Hz, 1H), 7.99 (d, J =8.4 Hz, 2H). 13C NMR (100 MHz,
CD3OD): δ 54.7 (OCH3), 54.9 (OCH3), 96.2 (C-4), 113.5 (C-6), 114.2 (C-7), 114.6 (C-3´ + C-5´),
118.9 (C-1´), 128.3 (C-2´ + C-6´) 130.4 (C-7a), 136.2 (C-3a), 150.5 (C-2), 157.7 (C-5), 162.5 (C-4´).
HRMS (ESI+) calcd. for C15H15N2O2 [M+H]+: 255.1134, found: 255.1122.
4.1.2.3.10. 2-(4-Chlorophenyl)-5-methoxy-1H-benzimidazole, 21.
Yield: 65 %. Compound crystallized from EtOH to give a yellow solid, Mp: 227-228 ºC IR (KBr):
.
νmax 3350, 2910, 1635, 1595, 1424, 1330, 1271, 1158, 1032, 966, 825, 724 cm-1. 1H NMR (200 MHz,
CD3OD): δ 3.84 (s, 3H), 6.90 (dd, J1 = 8.6; J2 = 2.2Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 7.47 (d, J = 8.6
13
Hz, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.99 (d, J =8.6 Hz, 2H). C NMR (50 MHz, CD3OD): δ 56.1,
97.8, 113.9, 117.0, 128.9 (2C), 129.9, 130.2 (2C), 136.9, 139.0, 142.2, 151.6, 158.4. HRMS (ESI+)
calcd. for C14H12ClN2O [M+H]+: 259.0638, found: 259.0636.
4.1.2.3.11. 2-(4-Bromophenyl)-5-methoxy-1H-benzimidazole, 22.
Yield: 73 %. IR (KBr): νmax 3300, 2910, 1635, 1595, 1424, 1330, 1271, 1158, 1023, 966, 825, 724
cm-1. 1H NMR (400 MHz, CD3OD): δ 3.85 (s, 3H), 6.90 (dd, J1 = 8.8; J2 = 1.6 Hz, 1H), 7.08 (d, J =
13
1.6 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 7.6 Hz, 2H), 7.95 (d, J = 7.6 Hz, 2H). C NMR
(100 MHz, CD3OD): δ 54.7, 96.4, 112.6, 115.6, 123.7, 127.7 (2C), 128.8, 131.8 (2C), 136.0, 138.8,
150.3, 157.1. HRMS (ESI+) calcd. for C14H12BrN2O [M+H]+: 303.0133, found: 303.0128.
4.1.2.4. Procedure for the reduction of nitrobenzimidazoles (compounds 13 and 18).
A solution with 0.32 mmol of the nitrobenzimidazole in MeOH (3 mL) in a two-necked round bottom

20
flask was prepared, then Pd-C catalyst (3 mg) was added. The air was removed, and a balloon with
H2 was attached to one of the necks. The mixture was kept at room temperature and under magnetic
stirring for 2 h. Then, the Pd-C was removed by filtration through celite, and the residue was washed
with MeOH. The solvent was removed in vacuo to provide a dark yellow solid.
4.1.2.4.1. 5-Chloro-2-(3-amino-4-methoxyphenyl)-1H-benzimidazole, 13.
Yield: 91 %. Compound crystallized from MeOH to give a light yellow solid, Mp: 185-186 ºC IR
.
1
(KBr): νmax 3306, 3210, 2918, 1618, 1501, 1453, 1340, 1243, 1026, 927, 802 cm-1. H NMR (200
MHz, CD3OD): δ 3.91 (s, 3H), 6.96 (d, J= 8.2 Hz, 1H), 7.20 (dd, J1= 8.6; J2= 1.6 Hz, 1H), 7.38 (dd,
J1= 8.2; J2= 2.0 Hz, 1H), 7.42 (d, J= 2.0 Hz, 1H), 7.49 (d, J= 8.6 Hz, 1H), 7.52 (d, J= 1.6 Hz, 1H).
13C NMR (50 MHz, CD3OD): δ 54.7, 110.0, 112.7, 113.9, 114.9, 117.0, 121.6, 122.4, 127.6, 137.3,
139.8, 140.0, 149.7, 154.0. HRMS (ESI+) calcd. for C14H13ClN3O: 274,0747, found: 304.0735.
4.1.2.4.2. 5-Amino-2-(4-methoxyphenyl)-1H-benzimidazole, 18.
Yield: 65 %. Compound crystallized from MeOH to give a brown-red solid, Mp: 195-197 ºC IR
.
1
(KBr): νmax 3300, 2910, 1626, 1573, 1355, 1330, 1267, 1011, 923, 820, 795 cm-1. H NMR (200
MHz, CD3OD): δ 3.84 (s, 3H), 6.73 (dd, J1 = 8.6; J2 = 1.8 Hz, 1H), 6.92 (d, J = 1.8 Hz, 1H), 7.03 (d,
13
J = 9.0 Hz, 2H), 7.35 (d, J =8.6 Hz, 1H), 7.94 (d, J = 9.0 Hz, 2H). C NMR (50 MHz, CD3OD): δ
54.4 (OCH3), 99.0 (C-4), 112.9 (C-6), 113.9 (C-3´ + C-5´), 115.4 (C-7), 122.3 (C-1´), 127.4 (C-2´ +
C-6´), 133.9 (C-7a), 138.5 (C-3a), 143.1 (C-5), 150.9 (C-2), 161.0 (C-4´). HRMS (ESI+) calcd. for
C14H14N3O [M+H]+: 240.1137, found: 240.1131.
4.1.2.5. Coupling of BZ 18 with picolinic acid, 19. 79 mg (0.49 mmol) of 1,1-carbonildiimidazole
(CDI) was added to a solution of 50 mg (0.41 mmol) of picolinic acid in dry DMF (2 mL), and the
mixture was maintained at room temperature for 1 h. Then, 88 mg (0.37mmol) of compound BZ 18
was added, and the mixture was kept at room temperature with magnetic stirring for 16 h. The
progress of the reaction was monitored by TLC, and a mixture of n-hexane/ethyl acetate (1:9) was
used as eluent. After completion of the reaction, the solvent was removed under vacuum on a rotary
evaporator, and the solid purified by column chromatography with n-hexane/ethyl acetate (2:8) as
eluent gave 40 mg (67% yield) of an orange solid.
4.1.2.5.1. 2-(4-Methoxyphenyl)- 5-picolinamidil-1H-benzimidazole, 19.
Yield: 70 %. IR (KBr): νmax 3300, 2910, 1626, 1573, 1355, 1330, 1267, 1011, 923, 820, 795 cm-1. 1H

21
NMR (400 MHz, CD3OD): δ 3.88 (s, 3H), 7.09 (d, J = 8.0 Hz, 2H), 7.50 (dd, J1 = 8.6; J2 = 1.8 Hz,
1H), 7.58 (d, J = 8.6 Hz, 1H), 7.62 (m, 1H), 8.03 (d, J = 8.0 Hz, 2H), 8.04 (d, J =1.8 Hz, 1H), 8.23
(m, 1H), 8.29 (m, 1H), 8.73 (m, 1H).13C NMR (100 MHz, CD3OD): δ 55.9 (OCH3), 107.1 (C-4),
115.6 (C-3´ + C-5´), 116.2 (C-7), 117.7 (C-6), 123.1 (C-1´), 123.3 (C-3´´), 127.9 (C-5´´), 129.4 (C-2´
+ C-6´), 134.6 (C-5), 139.1 (C-4´´), 139.2 (C-3a), 140.1 (C-7a), 149.7 (C-6´´), 151.2 (C-2´´), 154.2
(C-2), 163.1 (C-4´), 164.4 (CONH). HRMS (ESI+) calcd. for C20H17N4O2 [M+H]+: 345,1352, found:
345.1340.
4.2 Biological evaluation
4.2.1 Chemicals and drugs
Stock solutions of all the compounds and positive controls were prepared in DMSO (Sigma 276855),
while the final dilutions were made with distilled water in order to maintain a maximum
concentration of 0.5% DMSO per well. To perform the different in vitro tests, thiabendazole (TBZ,
Sigma T8904) or ivermectin (IVM, Sigma I8898) were used as positive controls, while 0.5% DMSO
was used as negative control in all tests. Levamisole (LEV, Sigma 31742) was also used as positive
control but in this case, the stock solution was prepared in distilled water.
4.2.2. Teladorsagia circumcincta
Different in vitro tests were carried out to test the anthelmintic activity of the new compounds in
three stages of the parasite: eggs, first stage larvae (L1) and third stage larvae (L3). All of these tests
were performed using two different strains, one susceptible and one resistant to BZs, LEV and IVM
(the latter kindly provided by Dr. Dave Bartle, Moredun Research Institute, Edinburgh, UK).
4.2.3. Animals
To obtain biological material for further experiments, six 3 months-old Merino lambs were infected
with 25,000 L3 of T. circumcincta at the facilities of the “Instituto de Ganadería de Montaña” (León,
Spain). As indicated above, four of them were infected with a susceptible strain and the other two
with the resistant one. All study protocols were revised and approved by the University of León
Animal Care Committee (León, Spain) to AGL2016-79813-C2-1R/2R, according to current national
and European regulations of animal wellbeing (R.D 53/2013 and EU Directive 2010/63/EU).
4.2.4. In vitro tests
A total of three in vitro experiments were undertaken to determine the activity of the 24 BZ

22
derivatives against different stages of T. circumcincta using a susceptible strain of the parasite. An
initial screening of the compounds was performed using a first discriminant concentration of 50 µM
by the Egg Hatch Test (EHT), and the Larval Migration Inhibition Test (LMIT). The next step was to
calculate the half maximal effective concentration (EC50) in those compounds with activities higher
than 90% at this initial screening. Then, with those compounds, the same initial screening at a
discriminant dose of 50 µM within a resistant strain of the parasite was performed, and as in the
previous case, EC50 was later calculated in those compounds with efficacies higher than 80%. When
the EHT was carried out, some compounds showed larvicidal activity, so a Larval Mortality Test
(LMT) was also performed in parallel. The procedure in this case was the same as in the EHT: an
initial screening at a discriminant dose of 50 µM and the estimation of their EC50.
All compounds were tested at least in duplicate at 3 different days to accurate the results.
Cytotoxicity assays were conducted on human Caco-2 and HepG2 cell lines with the aim to define
the Selectivity Indexes (SI) for those compounds in which the EC50 was determined, and to compare
the SI with those of the reference drug thiabendazole (TBZ).
4.2.4.1. Egg hatching test (EHT)
EHT is based on a modification of a protocol previously described by [31]. Briefly, parasite eggs
were freshly obtained from lamb faeces experimentally infected with T. circumcincta and distributed
in 24-well cell culture plates (Costar®, CLS3738) at a density of 100-150 eggs per well in a volume
of 1 mL. To carry out the initial testing, a 10 mM stock solution was prepared by dissolving each
compound in DMSO, from which working solutions were prepared by further dilutions with distilled
water. Each well included 1 mL of the egg suspension, 10 µL of a stock solution with each
compound and distilled water up to 2 mL to achieve a final concentration in well of 50 µM.
Additionally, in each plate TBZ was included at concentration of 0.1 µg/mL (0.5 µM) as positive
control. After 48 h incubation at 23 ºC, the number of L1 and eggs present per well were counted
using an inverted microscope to determine the percentage of hatched eggs (number of L1 / number of
L1 larvae and eggs
Χ
100). The ovicidal activity was expressed by the percentage of egg hatching
inhibition using the following formula:
% Egg hatching inhibition = [100 – (% egg hatching per well / % egg hatching in control well)]
Χ
100.
The EC50 of the compounds was calculated using 6 concentrations ranging from 50 µM to 1.56 µM.
4.2.4.2. Larval Mortality test (LMT)
This test was carried out only on those compounds in which dead larvae appeared during the EHT

23
reading (BZs 4, 9 and 22), in order to discard a possible activity against this parasite stage.
With the aim to obtain L1, fresh eggs (previously extracted from the faeces) were incubated during
24 h at 23 ºC. Then, larvae were collected in a known volume of water to obtain a density of 100-150
larvae per mL. The LMT was performed in the same way as the EHT but using the L1 instead. In this
case a stock solution of LEV at 10 mg/mL was added as positive control on every plate to reach a
final concentration of 1 mg/mL (4.15 mM) per well. The number of dead and alive L1 present per
well was counted using an inverted microscope to determine the larvicidal activity of the compound
([number of dead L1/number of dead and alive L1]
Χ
100). Apparently motionless and rod-shaped
larvae were considered dead, while those that presented some kind of movement or curvature in their
body were considered alive. The efficacy of the compound was expressed by the percentage of
viability inhibition using the following formula:
% Viability inhibition = (% larvicidal activity per well / % larvicidal activity in control well) 100.
Χ
The EC50 of each compound was calculated using 6 concentrations ranging from 50 µM to 1.56 µM.
4.2.4.3. Larval Migration Inhibition test (LMIT)
LMIT was conducted in following the method described previously by [32]. To obtain L3, faeces
from infected lambs were cultured in aerated and humidified closed boxes twice a week and placed in
a climatic chamber at 25 ºC for 13 days. Briefly, L3 were exsheathed by incubation in 0.5% (v/v)
sodium hypochlorite solution for 10 min at room temperature. After incubation, the larvae suspension
was washed 3 times by adding distilled water and centrifugation for 15 min at 300 g. Then, the
number of larvae was adjusted to a density of 1,000 L3 per mL and aliquots of 100 µL were added to
each well in a 96-well culture plate (VWR, 734-2327) containing 100 µL of a suspension of distilled
water with the compounds. As in the other test, all compounds were tested at a single dose of 50 µM;
and 100 µM final concentration IVM was used as positive control. Each compound was tested in
triplicate on the same plate and at least two technical replicas were performed. After 24 h of
incubation in the dark at 28 ºC, the whole content of each well was transferred to a 96-wells
MultiScreen-Mesh Filter Plate (Sigma-Aldrich, MANMN2010) and left for a further 24 h to allow
the motile L3 to pass through the sieves (20 µm mesh size) for counting. The size of the mesh was 20
µm to prevent the “fall” of the larvae since the cross-diameter of these is slightly larger [33].
The efficacy was expressed by the percentage of larval migration inhibition using the following
formula:
% Larval migration inhibition = [(number of larvae migrating through sieves in negative controls wells
– number of larvae migrating through sieves in treatment wells) / number of larvae migrating through

24
sieves in negative controls wells.] 100
Χ
The EC50 of each compound was calculated using 6 concentrations ranging from 50 µM to 1.56 µM.
4.2.5. Cytotoxicity assay and selectivity indexes
The cytotoxicity evaluation of 2-phenylbenzimidazoles was carried out on two human cell lines: the
colorectal adenocarcinoma Caco-2 (ATCC® HTB-37™) and the hepatocarcinoma HepG2 (ATCC®
HB-8065™). Briefly, 10,000 cells were seeded on 96 well-plates in RPMI 1640 Medium
supplemented with 2.0 g/L sodium bicarbonate (Fisher Scientific®), 1% (w/v) L-glutamine (Sigma-
Aldrich®), and 25 mM HEPES buffer, 10% (v/v) inactivated foetal bovine serum (FBS), and
antibiotic mixture containing 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Cultures were
incubated at 37 ºC in a humidified atmosphere containing 5% CO2. After 24 h, different
concentrations of testing compounds (ranging from 1 to 100 µM) were added for 72ꢀh. After this
time, the viability of the cells was assessed using the Alamar Blue (BioRad) staining method
according to manufacturer's recommendations (Invitrogen).
Cytotoxic concentration 50 (CC50) values were determined by plotting the concentration of each
compound vs. cell viability expressed as the fluorescence emitted by resorufin at 590ꢀnm. Dose-
response curves were fitted using non-linear regression plots, using Sigma Plot programme (10.0
Software, Inc., San José, California; USA).
As a safety parameter of each compound, selectivity index (SI) was calculated as the CC50/ EC50 ratio
of those compounds with a measurable effect on eggs or larvae. Despite the fact that the SI was
calculated using non-congruent magnitudes (cells vs organisms), we considered that it was useful to
compare the relative toxicity of the compounds.
4.2.6. Predictive Drug-likeness and ADME-Toxicity parameters for BZs
Compounds with the best nematocidal activity and weak cytotoxicity were submitted to in silico
pharmacokinetic properties and adverse effects prediction. To predict the druggability of these
compounds, we got the parameters (see supplementary data) related to Lipinski’s rule of five,
absorption rate, drug-likeness and potential toxicity risks provided online by the freely accessible
web-based applications Osiris Data Warrior (http://www.openmolecules.org/datawarrior and
https://www.organic-chemistry.org/prog/peo); and preADMET (https://preadmet.bmdrc.kr/adme/ and
https://preadmet.bmdrc.kr/druglikeness/).

25
4.2.7. Tubulin homology modelling
Homology modelling of T. circumcincta β-tubulin was performed with SWISS-MODEL Workspace
[34]. The sequence of this protein was taken from the one published in the literature [35]. This
protein has a highly conserved region, which includes the most representative amino acids of the
active site. This conserved region was identified after comparison with other β-tubulin sequence
species using GCG software. Residues 1-419 of chain B of the crystallographic structure of Ovis
aries tubulin (PDB ID: 3N2G) were used as a template [36].
The protein structure was validated by the use of Rampage server (http://mordred.bioc.cam.ac.uk/
~rapper/rampage.php) which predict the stereochemical quality and accuracy of the generated model.
Ramachandran plot analysis revealed 92.7% residues of modelled structure in the favoured region,
6.0% residues in the allowed region, and 1.3% residues in the disallowed region.
4.2.8. Molecular docking
Docking calculations were performed using Glide integrated into the Schrödinger Molecular
Modeling Suite (Schrödinger, Inc., USA, 2016-4). T. circumcinta β-tubulin previously obtained by
homology modelling was used as a target to further understand the molecular bases of the inhibitory
properties of these compounds. This target was processed with the Protein Preparation Wizard
available in the modelling package. The process preparation of the protein included assignation of
bond orders, addition and optimization of hydrogens, filling in missing side chains, cap termini
addition, optimization of protonation states of some residues, H-bond network optimization, etc. The
model was refined using OPLS-2005 Force Field. The ligands which appear in table 1 were
processed with the LigPrep module implemented in the same package previous to optimizing the
structure of each ligand; afterwards different tautomers were generated considering the different
protonation states (pH 7.0 ± 2.0) and partial charges using the OPLS-2005 force-field were assigned.
A receptor grid was generated and centred in the colchicine binding site of the β-tubulin. The size of
the box was 25
Χ
25 25 Å, with a van der Waals scaling factor of 1.0 and a partial charge cut-off of
Χ
0.25. Docking was carried out using the Extra Precision (XP) mode of Glide. The final pose was
selected using the energetic parameter GlideScore function. The docking poses of some significant
ligands can be visualized and analysed for the key elements of interaction with the target enzyme
using the Maestro’s Pose Viewer utility in Figures 2 - 5 and supplementary material (section 4).
Author contributions
All authors participate in their respective areas of research and collaborate to the writing of the
manuscript.

26
Acknowledgements
We would like to express our gratitude to Dr. Dave Bartley at Moredun Research Institute for
providing the triple-resistant strain of T. circumcincta. Authors thank Bioinformatic & Molecular
Design Research Centre (BMDRC, Yonsey University, Seoul, Korea) and Idorsia Pharmaceuticals
Ltd. (Allschwil, Switzerland) for complimentary access to preADMET and Osiris Data Warrior
databases, respectively. RE thanks financiation from RICET-USAL Program RD16/0027/0018.
Financial support came from MINECO: RETOS (AGL2016-79813-C2-1R/2R) and Junta de Castilla
y León co-financed by FEDER, UE (LE020P17). EVG was funded by FPU16/03536, VCGA by
Junta de Castilla y León and FSE (LE082-18), MAV by Junta de Castilla y León (LE051-18) and
MMV by the Spanish “Ramon y Cajal” Programme (Ministerio de Economía y Competitividad;
MMV, RYC-2015-18368), respectively.
Appendix A. Supplementary data
13
1H, C NMR and HRMS spectra of compounds BZ 1 to 24. Egg hatching and larval dose-response
curves. Additional results of docking studies. Molecular and ADME properties prediction for most
active compounds.
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