Enhanced Antigiardial Effect of Omeprazole Analog Benzimidazole Compounds

Article abstract of DOI:10.3390/molecules25173979

Giardiasis is a diarrheal disease that is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; however, failures in drug therapy are common, and have adverse effects and increased resistance of the parasite to the drug, generating the need to find new alternative treatments. In this study, we synthesized a series of 2-mercaptobenzimidazoles that are derivatives of omeprazole, and the chemical structures were confirmed through mass, ¹H NMR, and ¹³C NMR techniques. The in vitro efficacy compounds against Giardia, as well as its effect on the inhibition of triosephosphate isomerase (TPI) recombinant, were investigated, the inactivation assays were performed with 0.2 mg/mL of the enzyme incubating for 2 h at 37 ^?C in TE buffer, pH 7.4 with increasing concentrations of the compounds. Among the target compounds, H-BZM2, O₂N-BZM7, and O₂N-BZM9 had greater antigiardial activity (IC₅₀: 36, 14, and 17 μM on trophozoites), and inhibited the TPI enzyme (K₂: 2.3, 3.2, and 2.8 M^?1 s^?1) respectively, loading alterations on the secondary structure, global stability, and tertiary structure of the TPI protein. Finally, we demonstrated that it had low toxicity on Caco-2 and HT29 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.

Full text of DOI:10.3390/molecules25173979

molecules
Article
Enhanced Antigiardial Effect of Omeprazole Analog
Benzimidazole Compounds
Beatriz Hernández-Ochoa 1,*, Saúl Gómez-Manzo 2 , Adrián Sánchez-Carrillo 1,
Jaime Marcial-Quino 3 , Luz María Rocha-Ramírez 4 , Araceli Santos-Segura 1,
Edson Jiovany Ramírez-Nava 2 , Roberto Arreguin-Espinosa 5 , Miguel Cuevas-Cruz 5
,
Alfonso Méndez-Tenorio 6 and Ernesto Calderón-Jaimes 1,
*
1
Laboratorio de Inmunoquímica, Hospital Infantil de México Federico Gómez, Secretaría de Salud,
Ciudad de Mexico 06720, Mexico; ausbir@yahoo.com.mx (A.S.-C.); chelyss68@yahoo.com.mx (A.S.-S.)
Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Secretaría de Salud,
Ciudad de México 04530, Mexico; saulmanzo@ciencias.unam.mx (S.G.-M.);
edsonjiovany@ciencias.unam.mx (E.J.R.-N.)
2
3
4
Consejo Nacional de Ciencia y Tecnolog
Ciudad de Mexico 04530, Mexico; jmarcialq@ciencias.unam.mx
Unidad de Investigación en Enfermedades Infecciosas, Hospital Infantil de México Federico Gómez,
Secretar a de Salud Dr. M rquez No. 162, Col Doctores, Delegaci n Cuauht moc, Ciudad de M xico 06720,
Mexico; luzmrr7@yahoo.com.mx
Departamento de Química de Biomacromoléculas, Instituto de Química,
ía (CONACYT), Instituto Nacional de Pediatría, Secretaría de Salud,
í
á
ó
é
é
5
Universidad Nacional Autónoma de Mexico, Ciudad de Mexico 04510, Mexico;
arrespin@unam.mx (R.A.-E.); miguel.ccqi@yahoo.com.mx (M.C.-C.)
6
Laboratorio de Biotecnología y Bioinformática Genómica, Escuela Nacional de Ciencias Biológicas,
Instituto Politécnico Nacional, Ciudad de México 11340, Mexico; amendezt@ipn.mx
Correspondence: beatrizhb_16@comunidad.unam.mx (B.H.-O.); ecalderj5@yahoo.com.mx (E.C.-J.);
Tel.: +52-5228917 (ext. 2000) (B.H.-O.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Tiziano Tuccinardi
Received: 7 August 2020; Accepted: 28 August 2020; Published: 1 September 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Giardiasis is a diarrheal disease that is highly prevalent in developing countries.
Several drugs are available for the treatment of this parasitosis; however, failures in drug therapy
are common, and have adverse effects and increased resistance of the parasite to the drug,
generating the need to find new alternative treatments. In this study, we synthesized a series
of 2-mercaptobenzimidazoles that are derivatives of omeprazole, and the chemical structures were
confirmed through mass, 1H NMR, and 13C NMR techniques. The in vitro efficacy compounds against
Giardia, as well as its effect on the inhibition of triosephosphate isomerase (TPI) recombinant, were
investigated, the inactivation assays were performed with 0.2 mg/mL of the enzyme incubating for
2 h at 37 ◦C in TE buffer, pH 7.4 with increasing concentrations of the compounds. Among the target
compounds, H-BZM2
,
O2N-BZM7, and O2N-BZM9 had greater antigiardial activity (IC50: 36, 14,
−1) respectively,
and 17
µ
M on trophozoites), and inhibited the TPI enzyme (K2: 2.3, 3.2, and 2.8 M−1
s
loading alterations on the secondary structure, global stability, and tertiary structure of the TPI protein.
Finally, we demonstrated that it had low toxicity on Caco-2 and HT29 cells. This finding makes it an
attractive potential starting point for new antigiardial drugs.
Keywords: omeprazole analogs; giardiasis; inhibition enzyme; giardiacidal compounds
1. Introduction
Parasitic infections such as giardiasis are a human health concern worldwide. Giardia lamblia is
the parasite cause of this human diarrheal disease and is still a major problem in developing countries,
Molecules 2020, 25, 3979; doi:10.3390/molecules25173979
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3979
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mainly affecting the infant population, with high morbidity and mortality indexes due to the effects of
severe diarrhea, invasive infections, malabsorption syndrome, and resultant impairment of growth
and development [1]. This parasitosis is considered the main cause of epidemic enteritis worldwide,
and a re-emerging infectious disease of public health concern [2,3]. Due to the impact of this disease
in regions with a poor socioeconomic status and developing countries, in 2004, the World Health
Organization (WHO) included giardiasis in the group of neglected tropical diseases [4].
Current pharmacological therapies for giardiasis include metronidazole, tinidazole, ornidazole,
and secnidazole; these drugs are derived from the nitroimidazole family, while derivatives of the
benzimidazole group include albendazole and mebendazole [5–8]. All these drugs have different
mechanisms of action, involving distinct targets and downstream metabolic pathways and mechanisms
that affect cellular processes in trophozoites, such as proliferation and conversion into infective cysts.
Although current drug therapy for the treatment of giardiasis is effective, most available drugs
have significant side effects that restrict their use. In addition, the use of metronidazole is seriously
undermined by the development of resistance in the target parasite species [9–11]. Considering the side
effects of the antiprotozoal drugs and the lack of activity of metronidazole against G. lamblia, there is
an urgent need to develop new drugs to treat giardiasis. In this context, the proton pump inhibitors
(PPIs) are extensively used in clinical practice, Omeprazole with other PPIs has been proposed to be
potential drugs for the treatment of several diseases including the giardiasis. Has been demonstrated
that omeprazole shows cytotoxic effects in G. lamblia and concomitantly inactivates the TPI enzyme [12].
The TPI from G. lamblia, is an enzyme of the glycolytic pathway of 247 amino acids, with an α/β
barrel structure, which catalyzes the isomerization between dihydroxyacetone phosphate (DHAP) and
glyceraldehyde-3-phosphate (GAP) [13].
Therefore, we have synthesized compounds with heterocyclic scaffolds bearing different
substituents and have found them to be active against the parasite G. lamblia, and with the
ability to inactivate the TPI. They can be explored as future drug candidates with higher efficacy,
resistance effectiveness, and lesser side effects. Among the heterocycles used are benzimidazole,
and 2-mercaptobenzimidazoles [14,15], which possess extensive pharmacological effects such as
antimicrobial, antihistamine, analgesic, anticonvulsant, and antigiardial activities [16]. In addition,
a recent study revealed the synthesis of a series of 2-[(2yridine-2-ylmethyl)sulfinyl]-1H-benzimidazole
derivatives named BHO1, BHO2, and BHO3 (Figure 1) and their antiprotozoal activity against
G. lamblia was evaluated; two of the evaluated compounds showed antigiardial activity without
cytotoxicity in mammalian culture cells [17]. BHO2 and BHO3 showed the best antigiardial activity;
in addition, it has been reported that one of the pharmacological targets of these compounds is the
triosephosphate isomerase from G. lamblia. Interestingly, BHO2 and BHO3 also have the property
of inactivating the TPI enzyme, and it was confirmed by mass spectrometric techniques that the
benzimidazole moiety of compounds forms a covalent bond with the cysteine residues present in TPI,
which leads to their inactivation.
H C
H C
3
O
3
O
N
N
S
N
S
N
N
H
N
H
CH
3
H C
O
BHO1
3
BHO2
H C
3
H C
3
O
N
S
N
N
H
H C
O
3
F
F
F
BHO3
Figure 1. Chemical structure of 2-[(3yridine-2-ylmethyl)sulfinyl]-1H-benzimidazole derivatives.

Molecules 2020, 25, 3979
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Therefore, in this work, we improve the antigiardial activity of new derivatives of
2-mercaptobenzimidazoles with different substituent groups linked to the benzimidazole ring. To reach
these aims, five compounds named H-BZM1, H-BZM2, H2N-BZM6, O2N-BZM7, and O2N-BZM9,
(Figure 2), containing the scaffold of proton pump inhibitors but with hydrogen, amino, and nitro
substituents groups linked at Position 5 of the benzimidazole ring (R1), were synthesized, purified,
and characterized, we also perform the synthesis of lansoprazole. In addition, we evaluated the
TPI enzyme inactivation ability and the antigiardial capacity of these synthesized compounds.
Finally, we demonstrated that H-BZM2, O2N-BZM7, and O2N-BZM9 displayed greater antigiardial
activity and low toxicity on Caco-2 and HT29 cells concerning commercial drug omeprazole and the
BHO2 compound previously reported. This finding makes an attractive potential starting point for
new antigiardial drugs.
Figure 2. Chemical structure of synthesized 2-mercapto-benzimidazoles, the numbering of the
benzimidazole and pyridine core in blue and red color.
2. Results and Discussion
2.1. Design and Synthesis of Analog Benzimidazoles of Omeprazole
Heterocycle compounds are biologically and structurally important in the fields of chemistry,
biochemistry, and medicine. Often, a simple heterocyclic scaffold is utilized for obtaining new
biologically active compounds with diverse properties. Recently, it has been reported that
2-mercaptobenzimidazole-derived compounds possess antiparasitic properties [18
one of the compounds that has the benzimidazole ring in its structure is omeprazole, which has
been studied for its antigiardial properties [12 14 17 21 22]. Furthermore, it has been observed that
–20]. Interestingly,
,
, , ,
omeprazole and certain analogs exhibit antigiardial activity, because these compounds act as inhibitors
of the glycolytic enzyme triosephosphate isomerase (TPI) of Giardia lamblia by covalent binding of
benzimidazole moiety with cysteine residues [17], suggesting that this enzyme could be proposed
as a potential drug target in the antigiardial activity of these compounds [12]. Based on the above,
and in order of increase, the antigiardial activity of 2-mercaptobenzimidazole-derived compounds,
five analogs (H-BZM1, H-BZM2, H2N-BZM6, O2N-BZM7, and O2N-BZM9) with different substituent
groups linked to the benzimidazole ring was designed and synthesized. It is important to note that the
synthesis of the O2N-BZM7 compound, previously was described in Patent Number 4,791,114 [23];
however, in the present work, a different synthesis method was used to obtain the O2N-BZM7
compound. Besides, the synthesis of the lansoprazole drug was performed to also be evaluated [24–26].
In Position 5 of the benzimidazole core (R1), we placed a hydrogen atom in compounds H-BZM1 and
H-BZM3 (Figure 2), as well as lansoprazole, while an amine group was placed in the H2N-BZM6

Molecules 2020, 25, 3979
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compound. Finally, a nitro group was placed in both the O2N-BZM7 and O2N-BZM9 compounds in
the same position (Position 5 of the benzimidazole core (R1)). The changes performed in the pyridine
ring were as follows: no substituents (R2–R4) for H-BZM1 and O2N-BZM7; H-BZM2 has a pyridine
ring substituted with two methyl groups (R2 and R4), and a methoxy group (R3); and H2N-BZM6
,
and O2N-BZM9 possess a pyridine with substituents of lansoprazole a methyl group (R2) and
trifluoroethoxy group (R3; Figure 2).
Syntheses of compounds were carried out as previously reported by Hernández-Ochoa et al. [17].
Briefly, the synthesis was in two steps, as shown in Scheme 1. First, the precursors were prepared
by nucleophilic substitutions between the 2-mercapto-5-methylbenzimidazole with one of the
◦
substituted 2-chloromethy pyridine and in the presence of potassium hydroxide, at 50 C for 8 h,
which gave the desired the 5-methyl-2-[(4yridine-2-ylmethyl)sulfanyl]-1H-benzimidazole compound.
Then, the precursors were purified by silica gel column chromatography and their chemical structures
confirmed by NMR spectroscopy. Second, oxidation of the thioether group in precursors was done to
obtain the sulfoxide products by using peroxide acid (Scheme 1), in this step, it was important to keep
the reaction at 0 ◦C and to a slow addition of the peroxide acid, which allowed to obtain the desired
final sulfoxides and avoid the formation of the sulfone. Afterward, the final compounds were purified
by silica gel column chromatography and their chemical structures confirmed by NMR spectroscopy
(see Figures S1, S2, S4, S5, S7, S8, S10, S11, S13, and ◦S14 in Supplementary Materials). The compounds
were stored in perfectly closed amber flasks at 4 C, protected from light until later use, to avoid
decomposition. The stock solutions of each of the compounds were prepared immediately before
the experiments.
a
H
N
H
N
R
R
1
N
1
i
Cl
S
SH
N
+
N
N
R
4
R
R
2
4
R
2
3
3
1
R
R
3
2
b
O
O
H
N
H
N
CH
3
R
1
R
1
O
S
N
S
ii
N
N
+
N
R
4
R
4
Cl
R
R
2
3
4
R
R
2
3
3
Scheme 1. Syntheses of compounds. The synthesis was performed through a two-step
method. ) Obtaining intermediaries, prepared by nucleophilic substitutions between the
(
a
2-mercapto-5-methylbenzimidazole with one of the substituted 2-chloromethy pyridine, in an alkaline
medium. (b) Sulfoxide synthesis. Oxidation of thioether group with a peroxide acid.
The compounds were obtained with yields between 70% and 85% as brown solids with similar
melting points. The 1H NMR and 13C NMR spectra of all final synthesized compounds and precursors
are in total agreement with the suggested structures (Figure 2 and Scheme 1). Additional support for
the proposed structures comes from mass spectral data obtained from both low- and high-resolution
mass spectra of the synthesized compounds showing the correct molecular ions, (M+), as suggested by
their molecular formulas or fragments that stem from the molecular ion. Analysis of the molecular ions
and the fragmentation pattern are used in the identification and characterization of these compounds
(Figures S3, S6, S9, S12, and S15 in Supplementary Materials).

Molecules 2020, 25, 3979
5 of 22
2.2. Dose-Dependent Effect of New Compounds on Giardia lamblia
In order to determine the antigiardial effects of each of the synthesized compounds and the
lansoprazole, we used dose-dependent curves to determine the percentage of viable trophozoites from
G. lamblia after incubation at different concentrations of each of the compounds using a trypan blue
dye exclusion assay. The mechanism of action of omeprazole and other PPIs has been studied, and it is
documented that in G. lamblia the efficacy and species-specific action of omeprazole to inhibit TPI is
possible under an acidic or neutral pH [12
,
17]. Based on the above, in the present study, the cytotoxicity
◦
and inactivation assays were carried out at pH 7.4 and 37 C. As seen in Figure 3, all compounds
evaluated here inhibited the viability of G. lamblia trophozoites to different extents. The inhibitory
effects of the synthesized compounds on the viability of the parasite are shown in Figure 3, where a
dose-dependent inhibition was observed. Then, the IC50 values (the concentration of compound that is
required to kill 50% of trophozoites in vitro) were calculated. For the compounds with a hydrogen
atom at Position 5 in the benzimidazole ring (R1), the IC50 values calculated were 676
36 M for H-BZM2, and 45 M for lansoprazole, respectively (Figure 3A and Table 1); H2N-BZM6
with an amino group at the same position inhibited parasite viability and had an IC50 value of 135
(Figure 3B). The maximal inhibitory effects were observed with compounds that have a nitro group in the
benzimidazole ring; IC50 values were 14 M and 17 M for O2N-BZM7 and O2N-BZM9, respectively.
µM for H-BZM1,
µ
µ
µM
µ
µ
It is important to note that H-BZM2, lansoprazole, O2N-BZM7, and O2N-BZM9 showed an increased
cytotoxic effect on G. lamblia trophozoites from 2.6- to 8.5-fold compared with that previously reported
for BHO2 derived from omeprazole, where an IC50 value of 120 µM was determined [17]. These results
suggest that the five compounds are more active against Giardia trophozoites. Based on the above,
we observe that, despite the structural similarity concerning the ring pyridine between compounds
lansoprazole, H2N-BZM6, and O2N-BZM9, they showed differences in their biological activities,
which indicates the importance of the substituents on the benzimidazole ring. According to this,
compounds lansoprazole and O2N-BZM9 exhibited approximately three- to eight-fold higher activity
than H2N-BZM6 against G. lamblia. This increase in the antigiardial effect obtained with the O2N-BZM9
compound is probably due to the presence of the nitro group, which could be involved in the redox
metabolism, affecting the survival of the trophozoites in a similar pathway to the mechanism observed
with metronidazole (MTZ) [13]. Additionally, the experimental results showed that in the compounds
with no substituted pyridine (H-BZM1 and O2N-BZM7), the presence of a nitro group at Position
5 of the benzimidazole nucleus (R1) is essential for increased antigiardial activity, explaining why
compound H-BZM1 is 48-fold less active than O2N-BZM7 in terms of the antigiardial effect.
Figure 3. Antigiardial activity of the five novels synthesized compounds and lansoprazole on G. lamblia
trophozoites’ viability. The trophozoites were incubated in the presence of (
compounds, and lansoprazole; ( H2N-BZM6 O2N-BZM7, and O2N-BZM9 compounds by 48 h at
37 ◦C with increasing concentrations of each of the compounds, and later the viability of the trophozoites
A) H-BZM1, H-BZM2
B
)
,
were determined. The values represent mean
standard errors were lower than 5%.
±
standard deviation from three independent experiments,

Molecules 2020, 25, 3979
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Table 1. In vitro antigiardial activities of compounds, and the cytotoxic activity on Caco-2 and
HT29 cells.
IC50 (µM)
CC50 (µM)
IC50 (µM)
Compounds
Giardia lamblia
Caco-2 (SI)
HT29 (SI)
TPI
H-BZM1
H-BZM2
676
36
45
135
14
17
4012 (6)
3184 (88)
2637 (59)
4006 (29)
640 (45)
663 (39)
-
3918 (5)
1912 (53)
548 (12)
1888 (14)
519 (37)
622 (36)
-
>500
37
66
>500
12
20
Lansoprazole
H2N-BZM6
O2N-BZM7
O2N-BZM9
Omeprazole [17]
300
225
IC50: The concentration of compound that is required to kill 50% of trophozoites in vitro. CC50: Concentration of
the compounds that killed 50% of the cells. The numbers inside the parentheses correspond to the ratio of CC50/IC50
values between mammal cells and trophozoites (selectivity index).
2.3. Cytotoxic Effect of Compounds on Human Intestinal Caco-2 and HT29 Cells
One of the main problems in the development of new drugs is the high toxicity to
mammalian cells. Therefore, we performed an assay to determine the cytotoxic effect of the six
novel synthesized compounds with antigiardial activity on two mammalian cell lines. For this,
we used the cultures of Caco-2 and HT29 cells as a model of the intestinal epithelium. By XTT
(2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl) 2H-tetrazolium-5-carboxamide) assay, we identified the
−
concentration-dependent effects on Caco-2 and HT29 cells’ viability of all compounds, and the CC50
values (the concentration of the compounds that killed 50% of the cells) were determined as shown
in Table 1. The results demonstrate that H-BZM2 and lansoprazole had low toxicity towards Caco-2
and HT29 eukaryotic cells, even at the highest concentrations tested (500
of approximately 20% was seen in Figure 4. It is interesting to note that these same two compounds
H-BZM2 and lansoprazole) have a good antigiardial effect against parasites, with IC50 values of 36
and 45 M for H-BZM2 and lansoprazole, respectively. These results are important because they
indicate that these compounds could be proposed as new antigiardial drugs for further studies.
µM); a cell viability decrease
(
µM
µ
Concerning the O2N-BZM7 and O2N-BZM9 compounds, we observed a negative effect on Caco-2
and HT-29 mammalian cells, lowering the viability to almost 50% when the cells were treated with
500 µM of these compounds. However, at 150 µM, where the viability of the G. lamblia trophozoites
was abolished at 100% (Figure 3B), we observed that at the same concentration Caco-2 and HT29
mammalian cells’ viability was decreased by around 20% (Figure 4C,D).
The selectivity index (SI) demonstrates the differential activity of a pure compound: the greater
the SI value, the more selective it is; thus, it is desirable to have a high SI, giving maximum antigiardial
activity with minimal cell toxicity. Based on the SI, the data shown in Table 1 indicate that the
compounds exhibit a high degree of cytotoxic selectivity on G. lamblia. We propose that H-BZM2
lansoprazole, O2N-BZM7, and O2N-BZM9 be considered for follow-up as antigiardial candidates
with minimal cell toxicity. These results are in concordance with those previously observed in VERO
cells, where the compound BHO2 exhibited CC50 values for mammal cells in the millimolar ranges,
which are 38- to 61-fold higher concerning the IC50 obtained for trophozoites [17].
,

Molecules 2020, 25, 3979
7 of 22
Figure 4. Cytotoxic effect of compounds on Human Intestinal Caco-2 and HT29 cells Dose-response
curves for cell viability of ( ) Caco-2 cells and ( ) HT29 cells treated with H-BZM1 H-BZM2
and H-BZM3. Dose-response curves for cell viability ( ) Caco-2 cells and ( ) HT29 of HT29 cells
treated with H2N-BZM6 O2N-BZM7, and O2N-BZM9. The viability was determined by using an
A
B
,
,
C
D
,
XTT assay. The values represent the mean
standard errors were lower than 5%.
±
standard deviation from three independent experiments,
2.4. In Vitro Screening of Triosephosphate Isomerase Inactivation
As we observed antigiardial activity of all the novel synthesized compounds on G. lamblia
trophozoites, we decided to evaluate in vitro the effect of the compounds in the inactivation TPI
enzyme from G. lamblia.
Previously, it was reported that both omeprazole and certain analogs such as BHO2 have as
a target in nonconservative Cys residue at Position 222 of glycolytic TPI enzyme from G. lamblia,
although this residue is not near the catalytic site [12,17,21]. Hence, it is expected that these novel
synthesized compounds will affect the TPI activity of G. lamblia by derivatization cysteines but in a
more efficient form. We performed an inactivation assay to evaluate the effect of compounds on the
TPI activity using different concentrations of these novel synthesized compounds. Figure 5 shows the
residual activity of the TPI enzyme after incubation for 2 h at 37 ◦C. As the concentration of each of the
compounds was increased, the residual TPI activity decreased for all the compounds. The exposure
of the TPI enzyme to H-BZM2, lansoprazole, O2N-BZM7, and O2N-BZM9 induced the abolition of
enzyme activity in a very similar manner. The TPI enzyme lost 100% of its activity when incubated
with these compounds at 500
µM. In contrast, H-BZM1 and H2N-BZM6 scarcely inactivated the TPI
enzyme, with 25% and 40% reductions, respectively, at the highest concentration (500
µM). From the
plots of residual activity data, we calculated the IC50 values; the efficacy of H-BZM2, lansoprazole,
O2N-BZM7, and O2N-BZM9 was particularly remarkable because the concentrations required to
reduce the activity of the TPI enzyme by 50% were 37
(Figure 5). It is interesting to note that, of the five synthesized compounds, three of them, H-BZM2
O2N-BZM7, and O2N-BZM9, and lansoprazole led to increased inactivation of the TPI enzyme from
µM, 66 µM, 12 µM, and 20 µM, respectively
,

Molecules 2020, 25, 3979
8 of 22
G. lamblia concerning BHO2 and omeprazole, previously reported [17]. As described before, BHO2
affects the activity of the TPI enzyme, with IC50 of 64 M [17], where this compound has a methyl
µ
group Position 5 in the benzimidazole ring (R1). In the results obtained here, we observed that with the
substitution of a hydrogen atom in Position 5 of the benzimidazole ring (R1), we were able to enhance
the inactivation of the TPI enzyme with compound H-BZM2 and the same result was observed with
lansoprazole which also has a hydrogen. However, when a nitro group is placed in that same position,
the compound shows a high ability to inactivate the TPI enzyme since O2N-BZM7 and O2N-BZM9
show IC50 values of 12 µM and 20 µM, respectively, with enhancements of 5.3-fold (O2N-BZM7) and
3.2-fold (O2N-BZM9) observed with respect to BHO2.
Finally, It is interesting to note that compounds H-BZM2, lansoprazole, O2N-BZM7
,
and O2N-BZM9 had a good inhibitory effect on the viability of G. lamblia, and that these compounds
did not affect the viability of human intestinal cells, making them good candidate antigiardial drugs.
Probably, the inhibitory effect on the viability could be due to TPI inactivation in trophozoites, as was
demonstrated recently by López-Velazquez et al. [27], who demonstrated that omeprazole targets and
inhibits recombinant GlTIM (TPI from G. lamblia) in bacterial cells.
Figure 5. Inactivation assays of triosephosphate isomerase (TPI) enzyme from G. lamblia with the
compounds. TPI protein was adjusted at a final concentration of 200 µg/mL and incubated with
increasing concentrations of each of the compounds. After 2 h of incubation at 37 ◦C, the reactions were
stopped by dilution, and the remaining activity was determined as described in Section 3.5 of Materials
and Methods. The figure shows representative experiments performed in triplicate. The values
represent the mean
lower than 5%.
±
standard deviation from three independent experiments, standard errors were
2.5. Second-order Inactivation Constant (k2) of TPI Exposed to Selected Hit Compounds
To compare the reactivity of five compounds and the lansoprazole on the TPI enzyme with
respect to BHO2 and omeprazole, we calculated the rate constants of inactivation. The second-order
inactivation constant k2 (M−1 s−1) represents the rate of formation of the enzyme-inhibitor complex.
First, we determine the pseudo-first-order inactivation constants (k1) after the enzyme was incubated
with different fixed concentrations of H-BZM2, lansoprazole, O2N-BZM7, and O2N-BZM9, and the
initial velocities were determined at different incubation times. Figure 6 shows the behavior of the
four best-synthesized compounds, where a single-exponential decay of time-course inactivation was
observed (from 0 to 120 min). This behavior indicates that the inhibitor-enzyme complex forms rapidly.
The k1 values for each compound were calculated and plotted against their concentrations (Figure 6),
and a linear behavior was obtained and fitted with the linear equation. The calculated k2 values for
H-BZM2 and lansoprazole compounds were 2.3 M−1 s−1 and 1.78 M−1 −1, respectively (Figure 6B,D);
s
these results confirm that a hydrogen atom at Position 5 on the benzimidazole ring (R1) is important in
order to improve the inactivation of the TPI enzyme from G. lamblia. Furthermore, it is interesting
to note that these results are in accordance with the k2 value reported by García-Torres et al. [12] for

Molecules 2020, 25, 3979
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rabeprazole (k2 = 2.66 M−1
s
−1), which also contains a hydrogen atom on the benzimidazole ring
(R1). However, we managed to obtain two compounds that inactivate TPI more powerfully; this was
observed with the O2N-BZM7 and O2N-BZM9 compounds, which showed k2 values of 3.2 M−1s−1
and 2.8 M−1s−1, respectively (Figure 6F,H). These results suggest that, by placing an electroattractor
substituent, such as a nitro group, on the benzimidazole ring (R1), an increase in the inactivation of the
TPI enzyme from G. lamblia can be obtained.
The values of the second-order rate constant for synthesized compounds (H-BZM2, O2N-BZM7,
and O2N-BZM9) and lansoprazole are higher than omeprazole (0.78 M−1 s−1) and BHO2 (1.6 M−1 s−1),
whose second-order rate constant values were previously reported [17]. Our results show a high ability
of the novel synthesized compounds to form enzyme-inhibitor complexes with the TPI enzyme from
G. lamblia. It is interesting to note that the four synthesized compounds showed an increase from
2.3- to 5.6-fold with respect to omeprazole, and an increase from 1.1- to 1.6-fold was obtained with
respect to BHO2. Thus, changes in the benzimidazole ring substantially improved the inactivating
reactivity of compounds against the TPI enzyme from G. lamblia.
Figure 6. Inactivation assays of the TPI enzyme from G. lamblia with H-BZM2, lansoprazole, O2N-BZM7
,
and O2N-BZM9. (A,C,E,G) The pseudo-first-order rate constant values (k1) for each compound

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were calculated by fitting the data to the exponential decay equation A = A0e-k1t, where A0 is the initial
activity value. All experiments were performed in triplicate; the bars represent the standard error.
) The second-order rate constant values of inactivation (k2) were obtained by plotting the k1
(B,D,F,H
values against the concentrations of compounds and adjusted to a linear regression model. All the
experiments were performed in triplicate; standard errors were lower than 5%.
2.6. Structural Studies of TPI Enzyme with the Inhibitory Compound
2.6.1. Circular Dichroism (CD) Assay
To analyze the effect of the synthesized compounds and lansoprazole on the secondary structure
of the TPI protein, circular dichroism (CD) assays were performed in the absence (free of compound)
or presence of the IC50 concentration after incubating the enzyme with each of the compounds.
As seen in Figure 7A, the spectra of the TPI enzyme from G. lamblia in the absence of the compounds
showed minimum absorption peaks at 208 nm and 222 nm, corresponding to β-sheets and α-helices,
as previously noted [17]. However, when the TPI enzyme was incubated with the three synthesized
compounds and lansoprazole, differences were observed in the minimum absorption signals with
respect to the enzyme free of treatment. The compounds that most altered the secondary structure
were O2N-BZM7 and O2N-BZM9 because these compounds showed spectra closer to the blank.
These results are in agreement with those previously obtained in inactivation assays, which revealed
that these two compounds lowered the activity of the enzyme more efficiently. Furthermore, it is
interesting to note that these compounds showed a higher alteration of the secondary structure of
the TPI enzyme from G. lamblia with respect to BHO2, as previously reported [17]. H-BZM2 and
lansoprazole showed less of an alteration in the secondary structure of the TPI enzyme, and the
signals related to the β-sheets were the most altered. These results indicate that the four compounds
affect the secondary structure of the TPI enzyme from G. lamblia, which explains the loss of catalytic
activity and is in concordance with the results of biochemical assays in which it was detected
that the compounds H-BZM2
, O2N-BZM7, O2N-BZM9, and lansoprazole were fastest at forming
enzyme-inhibitor complexes and had a good inhibitory effect on the viability of G. lamblia.
Figure 7. Spectroscopic characterization of TPI from G. lamblia. (
A) Far-UV circular dichroism (CD)
spectra and ( ) thermal stability of the TPI protein. Changes in molar ellipticity were monitored by
B
circular dichroism when the temperature was increased progressively from 35 to 70 ◦C at 1 ◦C/2.5 min.
In both assays, the protein was adjusted to 0.2 mg/mL in 50 mM phosphate buffer (buffer P) and
incubated with any of the four compounds at the IC50 concentration of each inhibitor for 2 h at 37 ◦C
prior to measurement. The experiment is representative of triplicate experiments.

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2.6.2. Thermal Stability Assays
Because the compounds and lansoprazole reduce the catalytic activity and alter the secondary
structure of the TPI enzyme from G. lamblia, we evaluated the effect on the thermal stability of
the TPI enzyme after incubation with the three compounds and the lansoprazole, following changes
™
in the CD signal at 222 nm measuring the unfolded fraction of the α-helices through a temperature
gradient (35–75 ◦C). As seen in Figure 7B, in the denaturation profiles of TPI free of treatment and
incubated with the compounds, we observed that the protein remains in its native structure around
◦
50 C (baseline); above this temperature, the protein was denatured and completely unfolded at
approximately 65 ◦C. From the plot, the Tm value (temperature at which half the
α
-helices remain in
the native state and the other half are in an unfolded state) calculated for the TPI free of treatment was
60.6 ◦C, while for the H-BZM2 and lansoprazole the Tm values determined were 59.9 ◦C and 60.2 ◦C,
respectively (Figure 7B). With respect to O2N-BZM7 and O2N-BZM9, the Tm values determined were
57.5 ◦C and 58.4 ◦C (Figure 7B). The TPI enzyme was most affected in the presence of O2N-BZM7
,
with a loss of 3 ◦C in thermal stability (Tm value of 57.5 ◦C), while the rest of the compounds did not
show significant changes in their Tm values. These data confirm that O2N-BZM7 has a strong effect on
the global stability of the TPI enzyme from G. lamblia. However, it is interesting to note that, although
the compounds caused alterations in catalysis and a loss in the secondary structure of the TPI protein,
they did not affect the overall stability of the protein; Tm values close to that of TPI free of treatment
were determined. We suggest that no effect was observed on the thermal stability of the TPI enzyme
because the compounds were synthesized to react with the Cys of the TPI enzyme; this reaction is local
and would not disrupt the global stability of the protein.
2.6.3. Intrinsic and Extrinsic Fluorescence Assays
Previously, it has been reported that TPI from G. lamblia was affected at the functional and
structural level by the use of sulfhydryl reagents such as 5,50-Dithiobis-2-Nitrobenzoic Acid; DNTB)
and 2-carboxyethyl methanethiosulfonate (MTSCE) [13]. Furthermore, Hernández-Ochoa et al. [17]
reported that the derivatization of Cys residues of TPI from G. lamblia induces a reduction in the
maximum peak fluorescence when using the two novel proton pump inhibitor (PPI) derivatives
BHO2 and BHO3. Based on the above, we performed intrinsic and extrinsic fluorescence assays to
determine the changes in the tertiary structure of TPI protein. The intrinsic fluorescence of the four
tryptophan residues contained in the TPI/monomer was monitored to determine structural changes
in the presence of the four synthesized compounds. We found that the intrinsic fluorescence of the
native TPI protein without inhibitors showed a peak at 330 nm, with a maximum intensity of 161
arbitrary units (a.u.; Figure 8A), while in presence of the compounds there was a reduction in the
maximum peak of fluorescence of 25–75% of the initial signal intensity (Figure 8A). In the presence
of H-BZM2 (40.1 a.u.) and lansoprazole (123 a.u.), the maximum intensity of fluorescence of the TPI
enzyme decreased by 75% and 25%, respectively (Figure 8A), while in the presence of O2N-BZM7
and O2N-BZM9, the maximal fluorescence intensity was 107 and 72.6 a.u, respectively, indicating
a reduction of 33% and 55%, respectively, compared to the TPI enzyme free of compounds. It is
interesting to note that the four synthesized compounds showed a higher reduction in the maximum
fluorescence intensity of the TPI enzyme with respect to the PPI derivative BHO2, where 25% of the
signal was lost. As previously noted, the decrease in fluorescence intensity could be due to the fact that
the four tryptophan residues were exposed to the environment after incubation with the compounds
and induced unfolding of the native three-dimensional (3D) structure in the presence of the four
compounds, which had a negative effect on the activity of the TPI enzyme. Furthermore, these results
are correlated with the loss of catalytic activity and are in concordance with the biochemical assays,
in which the compounds and lansoprazole were faster to form the enzyme-inhibitor complex, showed
alterations on the secondary and 3D structure of the TPI protein, and had an inhibitory effect on the
viability of G. lamblia.

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Another probe to identify alterations in the 3D structure of proteins is the extrinsic fluorescent
molecular probe by ANS assay, which is a molecule that has a high affinity for hydrophobic regions
and exhibits higher ANS fluorescence signals. Based on the above, we measured the fluorescence
emission spectra of ANS in the presence of the four synthesized compounds. As seen in Figure 8B,
all the compounds induced alterations in the 3D structure of the TPI protein, with the extrinsic
fluorescence intensities in the presence of the four inhibitors decreasing with respect to the enzyme
without the inhibitor. The results obtained indicate that the TPI enzyme with ANS used as a control
(without compounds) had a maximal fluorescence emission spectrum of 275 a.u. (corresponding to 100%
fluorescence intensity) at 485 nm (Figure 8B), while the TPI enzyme incubated with the compounds
produced an ANS spectrum with maximal fluorescence at 125 nm, 2.2-fold less than with 100%
fluorescence intensity; this behavior was observed for all the compounds. Thus, the decrease in
fluorescence intensity by more than 100% in the derivatized TPI enzyme is clear evidence that the
enzyme does not expose buried hydrophobic regions after treatment with the compounds. These results
are in agreement with what was obtained with the TIM from the human parasite Encephalitozoon
intestinalis (EiTIM), in which a decrease in the fluorescence intensity was also observed [27].
Finally, it is interesting to note that all the results showed in this work indicate that the TPI of Giardia
lamblia caused alterations in the enzymatic activity, secondary structure, global stability, and tertiary
structure of the protein when it was incubated with the synthesized compounds. The compounds had
a good inhibitory effect on the viability of G. lamblia, and no effect on the viability of human intestinal
cells, making them good candidates to be proposed as antigiardial drugs.
Figure 8. Fluorescence emission spectra of the TPI enzyme from G. lamblia. (A) Intrinsic fluorescence
spectra of the TPI enzyme, and ( ) 8-Anilinonaphthalene-1-sulphonate (ANS) assays in the absence or
B
presence of the four synthesized compounds incubated at the IC50 concentration. Values obtained from
buffer containing ANS without protein (open stars) were subtracted from the recordings with protein.
The experimental conditions for all the experiments are described in Section 3.6.1 of the Materials and
Methods. The data are the mean of at least four independent experiments.
2.7. Molecular Docking Studies
To explore the binding affinities of H-BZM2, lansoprazole, O2N-BZM7, and O2N-BZM9 antigiardial
compounds with the TPI from G. lamblia (PDB entry 4BI7), we performed a molecular blind docking
analysis. In Figure 9, the in silico study revealed two main zones of interaction for all the compounds
(Figure 9A). The first zone was found near the Cys14 residue; in this pocket, 12 amino acids were
identified that interact with the compounds Asn11, Lys13, Cys14, Asn15, Ser44, Gln65, Asn66, Ile93,
Glu98, Arg99, Ile102, and Met103 (the amino acid number corresponds to the TPI sequence from G.
lamblia; Figure 9B); the second zone was near Cys202 residue. In the case of crystal 4B17, Cys202
is mutated by the amino acid alanine, and 12 amino acids were identified that interact with the
compounds Lys159, Met160, Trp162, Lys163, Val201, Ala202, Ala203, Glu204, Ala206, His208, Ile209,

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and Arg210 (Figure 9B). It is interesting to note that all the interactions found for each of the compounds
were different.
Figure 9. Docking prediction between TPI protein (PDB entry 4BI7) from Giardia lamblia with the
compounds. (
from G. lamblia. (
binding pocket (blue) near Cys14 (yellow). (
A
) A general view of the binding affinities of all the antigiardial compounds with the TPI
) First zone of interaction of all the compounds with the amino acids common to the
) Second zone of interaction of all the compounds in the
B
C
TPI structure, showing amino acids common to the binding pocket (blue) near C202A (yellow).
The docking predicted with compounds O2N-BZM7 and O2N-BZM9 (compounds having a
nitro group at Position 5 of the benzimidazole ring (R1)) showed an increase in the formation of
hydrogen bridges with respect to the other compounds. We found that near Cys14, the nitro group of
O2N-BZM7 forms four hydrogen bridges with the amino acids Asn11, Gln65, and Glu98 (Figure 10A);
near Cys202, three hydrogen bridges are formed with amino acids Lys159, Lys 163, and Glu204 amino
acids (Figure 10B). With respect to compound O2N-BZM9, we observed that near the Cys14 residue,
this compound formed three hydrogen bonds with amino acids Cys14, Gln65, and Glu98 (Figure 10C).
In addition to the predicted binding near Cys202, O2N-BZM9 showed interactions with Tpr162 and
Gly205 (Figure 10D).

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Figure 10. Prediction of docking between TPI protein (PDB entry 4BI7) and O2N-BZM7 and O2N-BZM9
:
3D representations of the binding model of O2N-BZM7 in the vicinity of (
A
) Cys14 and (
B) C202A,
and of O2N-BZM9 in the vicinity of ( ) Cys14 and (D) C202A of TPI from G. lamblia. The interactions
C
are shown in red, and Cys14 and C202A are shown in yellow.
Previously, it was reported that the sulfur atom of the benzimidazole ring forms a covalent bond
with the cysteine residues present in the TPI protein from G. lamblia [17], which leads to its loss of
activity; the docking study revealed that the nitro group of compounds O2N-BZM7 and O2N-BZM9
possesses the ability to form up to four hydrogen bonds, which probably causes them to have a greater
inhibitory effect on the TPI enzyme from G. lamblia with respect to H-BZM1, H-BZM2, H2N-BZM6,
and lansoprazole, which do not have the nitro substituent. These results strongly suggest that the nitro
group is essential in the benzimidazole ring (R1) to enhance the inhibitory activity of the compounds,
which was confirmed by the values of k2 shown by O2N-BZM7 and O2N-BZM9 (3.2 M−1 s−1 and
2.8 M−1 −1, respectively).
s
Regarding H-BZM2, which experimentally showed a k2 value of 2.3 M−1 s−1, the docking analysis
showed that two interactions are formed between the benzimidazole ring and the amino acids Glu98
and Cys14 (Figure 11A). On the other hand, in the pocket of C202A, the benzimidazole ring forms two
interactions with the amino acids Lys163 and Glu204 (Figure 11B). Finally, for lansoprazole, the docking
analysis showed that two interactions are formed between the benzimidazole ring and the amino
acids Cys14 and Glu98 (Figure 11C); in the pocket of C202A, the benzimidazole ring forms only one

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interaction with the amino acids Met160 (Figure 11D), and this compound showed a K2 value of
1.78 M−1 s−1
.
Figure 11. Prediction of docking between TPI protein (PDB entry 4BI7) and H-BZM2 and lansoprazole:
3D representations of the binding model of H-BZM2 in the vicinity of ( ) Cys14 and ( ) C202A, and of
lansoprazole in the vicinity of ( ) Cys14 and ( ) C202A of TPI from G. lamblia. The interactions are
A
B
C
D
shown in red, and Cys14 and C202A are shown in yellow.
3. Materials and Methods
3.1. Chemicals
All chemicals used in the synthesis of compounds were purchased from Sigma-Aldrich (St. Louis,
MO, USA) were used as received without any prior purification. Penicillin/streptomycin (5000 U/mL),
Trypsin-EDTA (0.25%), phenol red, and Eagle’s Minimum Essential Medium (EMEM) were purchased
from Gibco, Life Technologies (Paisley, UK). The Caco-2 (ATCC HTB-37; Manassas, VA, USA) and
HT-29 (ATCC HHTB-38) cell lines were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).
Chemicals used for the in vitro culture of G. lamblia were sourced as follows: glucose and L-cysteine
(ACROS organics, Thermo Fisher Scientific, Scoresby, VIC, Australia), ammonium iron (III) citrate,
ascorbic acid (Sigma-Aldrich, Castle Hill, NSW, Australia), potassium dihydrogen orthophosphate
(UNIVAR, Ingleburn, NSW, Australia), bovine bile (Fluka analytical (BD)), and di-potassium hydrogen
orthophosphate (Fronine Laboratories and Supplies, Riverstone, NSW, Australia).
3.2. Chemistry
Melting points were determined in an A9200 digital melting point instrument from Thermo
Scientific; the results are uncorrected. All reactions were monitored by thin-layer chromatography

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(TLC) and carried out using glass plates precoated with silica gel 60 F254 (Merck, Kenilworth, NJ, USA).
1H NMR spectra were determined on Varian 600 MHz AR Premium Compact and 13C NMR (150 MHz)
instruments (Varian-Agilent, Santa Clara, CA, USA). Chemical shifts are reported in ppm DMSO-d6
and CDCl3 as deuterated solvents. Mass spectrometry was obtained from a JEOL JMS-700 spectrometer
by electronic impact (JEOL, Tokyo, Japan).
3.3. General Procedure for the Synthesis of Compounds
Five compounds were designed on the basis of the proton pump inhibitor omeprazole and
lansoprazole, maintaining the 2-(2-pyridylmethylsulfinyl)-1Hbenzimidazole core. H-BZM1, H-BZM2,
and lansoprazole have a hydrogen at Position 5 of the benzimidazole ring (R1); with respect to the
pyridine ring H-BZM1 has no substituents, H-BZM2 and O2N-BZM7 have the same as omeprazole,
H2N-BZM6 and O2N-BZM9 has the same as lansoprazole. Compounds were synthesized in the
Laboratory of Immunochemistry, Hospital Infantil de México Federico Gómez (Ciudad de Mexico,
Mexico). The synthesis of the derivatives was carried out according to the following general
procedure, in two steps: first, the precursors were prepared in a mixture of 2-mercapto-5-benzimidazole
(0.5 g, 3.04 mmoles) and the substituted 2-chloromethyl pyridine (0.55 g, 3.3 mmoles) in 10 mL of
1,2-dimethoxyethane (GLIMA), potassium hydroxide (0.34 g, 6 mmoles) was added, and the mixture was
stirred for 8 h at 50 ◦C to give the desired 2-[(pyridin-2-ylmethyl)sulfanyl]-1H-benzimidazole compound.
The total conversion of primary reagents to 2-[(pyridin-2-ylmethyl)sulfanyl]-1H-benzimidazole was
monitored by TLC assay. Later water was added and the reaction mixture was extracted with CHCl3
(2
×
20 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced
pressure, and the residue was purified using column chromatography on silica gel, with CH2Cl2-hexane
(3:1 v/v) as the eluent, to give a pale yellow solid.
Second, smooth oxidation of the thioether group in precursors was carried out to obtain the final
sulfoxide compounds. The oxidation was done according to the following general procedure: to a
stirring mixture of pyridin-sulfanyl benzimidazole (0.2 g, 0.78 mmoles) in 5 mL of CHCl3 at 0 ◦C was
added 3-chloroperoxybenzoic acid (0.19 g, 1.12 mmoles in 7 mL of CHCl3); the peracid was added
slowly, drop by drop, and the reaction was monitored every 5 min by TLC in order to prevent sulfone
formation. After completion of the reaction, the mixture was treated with a saturated solution of
sodium bicarbonate (20 mL) and extracted with CHCl3 (2
×
20 mL). The organic layer was dried over
anhydrous Na2SO4 and the solvent was removed under reduced pressure. The residue was purified by
means of column chromatography on silica gel using CH2Cl2-hexane (3:1 v/v) as the eluent to produce
the desired product as a pale yellow solid, and their chemical structures were confirmed by NMR
spectroscopy. The analogs labeled H-BZM1, H-BZM2, H2N-BZM6, O2N-BZM7, O2N-BZM9 and the
lansoprazole were stored at 4 ◦C until use. Stock solutions of analogs were prepared by dissolving
them in dimethyl sulfoxide (DMSO).
2-{[(pyridin-2-yl)methyl]sulfanyl}-1H-benzimidazole (H-BZM1), light-yellow solid. Yield 85%, mp.
◦
1
121.3–122.9 C. H NMR (400 MHz, DMSO-d6):
δ 4.52–5.00 (2H, J = 11.5 Hz CH2OS), 7.03–7.11
(1H, dd, J = 7.4, 1.2 Hz, H-6), 7.12–7.13 (1H, dd, J = 8.1, 1.7 Hz, H-30), 7.24 (1H, ddd, J = 8.0, 6.9, 1.3 Hz,
H-50), 7.45 (ddd, J = 7.6, 1.2, 0.5 Hz, H-4)), 8.42 (1H, ddd, J = 5.1, 1.9, 0.5 Hz, H-60) ppm. 13C NMR
(175 MHz, DMSO-d6) δ
: 21.3 (CH3); 61.9 (CH2); 123.2 (C-50); 124.8 (broad, C-6), 125.3 (C-30); 132.8
(broad, C-5); 136.9 (C-40); 149.7 (C-60); 150.7 (C-20); 153.2 (C-2) ppm. Carbons C3a, C7a, C4 and C7 were
not observed due to tautomeric equilibrium. MS calculated for C13H11N3OS: 257.31, found 255.
2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methanesulfinyl]-1H-benzimidazole (H-BZM2), light-brown solid.
Yield 72%, mp. 221.3–222.9 ◦C. 1H NMR (400 MHz, DMSO-d6).
δ 2.07 (3H, s, CH3), 2.14 (3H, s, CH3),
3.54 (3H, s, CH3O), 4.63–4.71 (2H, J = 13.5 Hz, CH2), 7.19 (1H, dd, J = 8.0, 1.3 Hz, H-6), 7.22 (1H, dd,
J = 8.1, 1.7 Hz, H-4)), 7.24 (1H, dd, J = 8.1, 0.5 Hz, H-7), 8.13 (1H, s, H-60) ppm. 13C NMR (100 MHz,
CDCl3)
(C-30), 148.7 (C-20), 149.83 (C-60), 164.5 (C-40); too broad resonances only detected through HMBC:
δ
: 11.64 (CH3), 13.48 (CH3), 21.87 (CH3), 60.01(CH3O), 60.85 (CH2SO), 126.54 (C-50), 127.15

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111.96 (C-4), 119.99 (C-7), 125.09 (C-6), 126.27, 133.1, 134.7, 142.18, 152.5 (C-2) ppm. MS calculated for
C17H19N3O2S: 329.1198, found: 328.
2-{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methanesulfinyl}-1H-benzimidazole (lansoprazole), brown
solid. Yield 75%. Light-yellow solid. Mp. 121.3–122.9 ◦C. The results spectroscopic were accordingly
with those previously published [25]. MS calculated for C16H14F3N3O3S: 369.36, found 367.
2-{[3-methyl-4-(2,2,3-trifluoropropoxy)pyridin-2-yl]methanesulfinyl}-1H-benzimidazol-5-amine (H2N-BZM6),
light-yellow solid. Yield 70%. Mp. 121.3–122.9 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ
: 2.27 (3H, s, CH3),
4.36–4.38 (2H, J = 13.6 Hz, CH2-SO), 4.40 (2H, q, J = 8.7 Hz, CH2-CF3), 6.57–6.59 (1H, d, J = 4.5 Hz
H-50, Py), 6.67–6.69 (1H, dd, J = 8.3, 0.6 Hz, H-6), 6.79 (1H, dd, J = 1.8, 0.6 Hz, H-4), 7.29–7.32 (1H,
,
d, J = 4.5 Hz, H-7), 8.35. (1H, d, J = 5.7 Hz, H-60, Py) ppm. 13C NMR (100 MHz, CDCl3)
δ: 10.77
(CH3), 34.91 (CH2CF3), 64.91 (CH2SO), 65.57 (CH2O), 106.01 (C-50), 111.93 (C-30), 111.7 (C-4), 121.08
(C-7), 121.74 (C-6), 124.32, 141.99, 147.53 (C-60), 157.68 (C-20), 162.50 (C-40), ppm. MS calculated for
C17H17F3N4O2S: 398.40, found 396.
5-nitro-2-[(pyridin-2-yl)methanesulfinyl]-1H-benzimidazole (O2N-BZM7), light-yellow solid. Yield 85%.
Mp. 121.3–122.9 ◦C. The results spectroscopic were accordingly with those previously published [23].
MS calculated for C13H10N4O3S: 302.30, found 300.
2-{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methanesulfinyl}-5-nitro-1H-benzimidazole (O2N-BZM9),
light-yellow solid. Yield 70%. Mp. 121.3–122.9 ◦C. 1H NMR (400 MHz, DMSO-d6)
δ: 2.23 (3Hs, CH3),
4.37, 4.39 (2H, J = 13.6 Hz, CH2-CF3), 4.72, 4.75 (2H, J = 13.6 Hz, CH3SO), 6.71 (1H, d, J = 4.4 Hz, H-50,
Py), 7.38 (1H, dd, J = 8.8, 0.5 Hz, H-6), 7.51 (1H, broad, H-4), 8.22 (1H, d, J = 5.7 Hz, H-60, Py), 8.34 (1H,
dd, J = 2.0, 0.5 Hz) ppm. 13C NMR (175 MHz, DMSO-d6) δ
: 21.3 (CH3); 61.9 (CH2); 123.2 (C-50); 124.8
(broad, C-6), 125.3 (C-30); 132.8 (broad, C-5); 136.9 (C-40); 149.7 (C-60); 150.7 (C-20); 153.2 (C-2) ppm.
Carbons C3a, C7a, C4 and C7 were not observed due to tautomeric equilibrium. MS calculated for
C16H13F3N4O4S: 414.35, found 412.
3.4. In Vitro Assays
3.4.1. Antigiardial Activity
Giardia lamblia WB strain (ATCC number 30957) was grown in a modified TYI-S-33 medium
supplemented with 10% fetal bovine serum. The parasite was cultivated in 15 mL screw-capped
borosilicate glass tubes containing 13 mL medium. The tubes were incubated on a 15◦ horizontal slant
◦
at 37 C. Giardia was harvested from confluent culture by chilling the tubes on ice for 5-10 min to
detach cells, followed by centrifugation at 800× g for 5 min.
The antigiardial activity of the prepared compounds was tested as described. One milligram
of a test compound was dissolved in 10 L of DMSO and topped up with 1 mL growth medium.
The solution was filter-sterilized using 0.22-m syringe filters and the appropriate volumes of the
solutions were taken to prepare the concentration of each compound in 1.5 mL microtubes. For each
preparation, concentrations of 500, 250, 125, 62, and 31 g/mL were prepared in a final volume of 1.5 mL.
Each tube was inoculated with 5
×
104 trophozoites of G. lamblia and then incubated for 48 h at 37 ◦C.
After incubation, 75 L of the treated trophozoites were subcultured individually for another 48 h in a
fresh medium free of compounds. At the end of this period, the viable trophozoites were counted. Each
compound was assayed in duplicate in three independent experiments. In each assay, appropriate
controls were also tested, including a sample without compound and another with DMSO.
The parasites in each tube were counted using a standard hemocytometer at 10
count, trypan blue was employed to distinguish live parasites from dead ones. To permit detachment
of Giardia, the tubes were placed on ice for 10 min and the parasites were then centrifuged at 800 g for
10 min. The supernatant was discarded and 1 mL fresh medium was added to each tube. The final
suspension was prepared by mixing 25 L of the parasite suspension in each tube with 100 L of 0.4%
trypan blue in phosphate-buffered saline (PBS). The 50% inhibitory concentration (IC50) was employed
×
objective. In each
×
µ
µ

Molecules 2020, 25, 3979
18 of 22
as a parameter for biological activity. The IC50 is the concentration of compounds that cuts the number
of parasites to half that in the negative control (growth medium + DMSO + parasites). Lastly, the 50%
inhibitory concentration (IC50) was calculated.
3.4.2. In Vitro Cytotoxicity
The cytotoxicity of compounds was investigated on human cell lines Caco-2 (ATCC HTB-37)
and HT-29 (ATCC HHTB-38), using a Cell Proliferation Kit II (XTT; St Louis, MO, USA) according
to the manufacturer’s instructions. A cell suspension of 105 cells/mL was prepared in Dulbecco’s
modified Eagle’s (DMEM); medium supplemented with 10% inactivated fetal bovine serum and 1%
Penicillin/streptomycin (5000 U/mL), from confluent cultures, and 100 mL portions of the suspension
were added to the wells of 96-well plates. The cells were incubated for 24 h at 37 ◦C in an atmosphere
of 5% CO2, and the medium in each well was then replaced with 150 L fresh medium. Solutions of
the compound were prepared and sterilized as described in Section 3.4.1 of Materials and Methods.
Then, 150 µL of two-fold serial dilutions of each of the compounds, starting at a concentration of 500 M,
in a culture medium at a concentration of 0.3% DMSO, were prepared in the plates. After 48 h of
incubation at 37 ◦C in an atmosphere of 5% CO2, the viability of cells in each well was determined
as follows: The medium in each well was gently replaced with 100 L of fresh medium, 50 µL of XTT
labeling mixture were added per well, and the mixture was incubated for 4 h at 37 ◦C in an atmosphere
of 5% CO2 and the absorbance at 550 nm measured. Each compound was assayed in triplicate via
three independent experiments. The CCI50 values were determined using the nonlinear regression
function of GraphPad Prism version 6.04 for Windows, GraphPad Software, La Jolla, CA, USA,
https://www.graphpad.com (accessed on 23 February 2020).
3.5. In Vitro Screening of TPI Inactivation
3.5.1. Purification of TPI Recombinant Enzyme
The TPI from G. lamblia was expressed and purified [13]. Briefly, bacteria containing the plasmid
for TPI was grown in LB medium supplemented with 0.1 mg/mL of ampicillin and incubated at 37 ◦C.
When the culture reached A600 = 0.8, it was induced using 0.4 mM IPTG and incubated overnight at
30 ◦C with shaking at 180 rpm. After the bacteria were collected by centrifugation (10,000
×
g, 15 min)
-mercaptoethanol,
and 1 mM phenylmethylsulfonyl fluoride). The bacterial suspension was disrupted by sonication
and centrifuged at 9000
g for 1 h at 4 ◦C. Protein purification was performed by immobilized metal
and suspended in 40 mL of lysis buffer, pH 8.0 (50 mM Tris, 50 mM NaCl, 5 mM
β
×
affinity chromatography (IMAC) using a Profinity Ni2+ charged resin (Bio-Rad) previously reported by
Enriquez-Flores et al. [13]. The His-tag sequence was removed from the TIM by incubation with TEVP
at a molar ratio of 30:1 (TPI/TEVP) at room temperature for 17 h in digestion buffer, pH 8.0 (50 mM Tris,
0.5 mM ethylene diamine tetraacetic acid (EDTA), and 1 mM dithiothreitol). The enzyme concentration
was spectrophotometrically determined at 280 nm, and the enzyme was used immediately.
3.5.2. Enzyme Activity Assay
Enzyme activity of TPI was determined spectrophotometrically as described [17] by monitoring
the conversion of glyceraldehyde-3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP). Briefly,
the conversion of GAP to DHAP using a coupling system with enzyme
dehydrogenase (
The standard reaction mixture contained 1 mM GAP, 0.2 mM NADH, and 0.9 units/mL of
α
-glycerol-3-phosphate
◦
α
-GDH) was determinate by measuring NADH oxidation at 340 nm, at 25 C.
α
-GDH
in TE buffer (100 mM triethanolamine, 10 mM EDTA, pH 7.4). The reaction was initiated by adding
5 ng/mL of TPI to the reaction mixture.

Molecules 2020, 25, 3979
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3.5.3. Inactivation of TPI with Compounds
To determine the inactivation of the TPI enzyme from G. lamblia, we performed inactivation assays
of the enzyme in the presence of the synthesized compounds. The compounds’ stock solutions were
freshly prepared using 100% dimethyl sulfoxide (DMSO) as the solvent at 25 mM. The inactivation
◦
assays were performed by incubating 0.2 mg/mL of the TPI enzyme for 2 h at 37 C in TE buffer
with increasing concentrations of the compounds (0 to 500
µM). DMSO was maintained at 5%
during incubation; this concentration did not affect the enzyme activity. After the incubation period,
the enzymatic activity was measured by withdrawing an aliquot of each experimental condition,
as described above. The results were expressed in terms of the percentage of activity versus the
compound’s concentration.
The second-order rate constants of inactivation were calculated by obtaining the pseudo-first-order
rate constant (k1) values at each fixed concentration by fitting them to an exponential decay curve.
Then, the k1 values were replotted against the concentrations of each compound; the second-order rate
constant value of inactivation was obtained from the slope of these plots.
3.6. Spectroscopic Characterization
3.6.1. Circular Dichroism (CD) and Thermal Stability Assays
All spectroscopic assays were carried out at a protein concentration of 0.2 mg/mL. Changes
in the secondary structure of proteins were monitored by circular dichroism (CD) in a Jasco-810
spectropolarimeter containing a thermostated Peltier-controlled cell holder. For CD experiments,
the enzyme was suspended in 50 mM phosphate buffer (buffer P), pH 7.4, and incubated with the
four synthesized compounds by 2 h at 37 ◦C. Spectral scans from 190 to 280 nm at 0.1 nm intervals
were performed at 25 ◦C using a quartz cell with 0.1 cm path length. All CD data were reported as
molar ellipticity. In addition, the thermostability of modified TPI enzyme in the absence or presence of
each of the synthesized compounds was evaluated, recording the loss of CD signal at 222 nm during
a temperature scan ranging from 25 to 75 ◦C and an increase of 1 ◦C/2.5 min. The unfolded fraction
of enzyme and melting temperature (Tm) was determined as reported before. The experiments were
performed in triplicate.
3.6.2. Intrinsic and Extrinsic Fluorescence Assays
Changes in tertiary structure were analyzed following the intrinsic and extrinsic fluorescence of the
TPI enzyme in a Perkin-Elmer LS-55 spectrofluorometer (Perkin-Elmer, Wellesley, MA, USA). The TPI
protein was adjusted at 0.1 mg/mL in 50 mM phosphate buffer (buffer P), pH 7.4n, and incubated
with the four synthesized compounds by 2 h at 37 ◦C. After the TPI protein was incubated with the
synthesized compounds, the samples were excited at 280 nm and we recorded the emission spectra
from 300 to 500 nm with a scan speed of 150 nm/min and using excitation and emission slits of 5 and
4 nm, respectively. Furthermore, we determined the extrinsic fluorescence of the TPI enzyme in the
presence of the four compounds by 8-anilinonaphthalene-1-sulfonic acid (ANS) assay to monitor
changes in the hydrophobic regions of the protein. The protein was incubated in the same conditions
as the intrinsic fluorescence assay. Then, we recorded the fluorescence spectra from 400 to 600 nm in
the presence of 25 mM of ANS dissolved in methanol at 25 ◦C with a scanning rate of 150 nm/min.
The samples were excited to 395 nm using slits of excitation and emission of 10 nm, respectively.
The final spectrum was the average of five scans. Later, each spectrum was subtracted from the spectra
of the blank (buffer P solution containing each of the compounds plus ANS).
3.7. In Silico Analysis of the TPI Crystallographic Structure from G. lamblia
Here, we used the crystal structure of mutant (C202A) TPI from Giardia lamblia, complexed
with 2-phosphoglycolic acid with access code PDB 4BI7 [28]. The atomic coordinates of TPI were
submitted to the PDBsum server (PDBsum-EMBL-EBI) [29] to add the hydrogens to the structure.

Molecules 2020, 25, 3979
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In order to identify all interactions on the TPI protein, blind docking was performed using the
SwissDock Server (http://www.swissdock.ch/docking accessed on 23 June 2020) [30]. The ligand
structures of H-BZM2, lansoprazole, O2N-BZM7, and O2N-BZM9 were energy-minimized UCSF
Chimera software developed by the Resource for Biocomputing, Visualization, and Informatics at the
University of California, San Francisco, with support from NIH P41-GM103311 [31] and later docked
on the crystal structure (PDB: 4B17). SwissDock generates all possible binding modes for each ligand;
the most favorable binding modes at a given pocket are clustered. The predictions file provided Cluster
Rank/Element Full Fitness and estimated binding free energy ∆G.
Chimera software was used to visualize the receptor–ligand interactions for all individual clusters
obtained. For each compound, we analyzed the amino acids that interact with the ligand, the number
of hydrogen bonds formed, the specific atoms involved, and the distance between them.
3.8. Statistical Analysis
The results of the in vitro drug efficacy studies were analyzed using GraphPad Prism. For the XTT
(viability) assay, the mean and standard error of the mean were determined, with each assay completed
in triplicate. The IC50 was calculated using the log (inhibitor) vs. normalized response—variable slope
function in GraphPad Prism.
4. Conclusions
We reported five derivatives of 2-mercaptobenzimidazol named H-BZM1, H-BZM2, H2N-BZM6,
O2N-BZM7, and O2N-BZM9 with different substituent groups linked to the benzimidazole,
which showed antigiardial activity, and without cytotoxic effects on mammalian HT29 and Caco-2 cells.
The antigiardial activity of compounds was improved compared to the omeprazole and lansoprazole.
The most active compounds were H-BZM2, O2N-BZM7, and O2N-BZM9 with greater antigiardial
activity (IC50: 36, 14, and 17
µM on trophozoites), and inhibited the TPI enzyme (K2: 2.3, 3.2 and
2.8 M−1 −1) respectively. The plausible modes of action of compounds involve TPI inhibition,
s
causing alterations in the enzymatic activity, secondary structure, global stability, and tertiary structure
of the protein in the presence of the compounds. Finally, as suggested by docking study that the
nitro group of compounds O2N-BZM7 and O2N-BZM9 (R1) possess the ability to form up to four
hydrogen bonds, which probably causes them to have a greater inhibitory effect on the TPI enzyme from
G. lamblia. The results presented in this study demonstrated that the class of benzimidazoles, related to
PPIs, has the potential to be developed as antigiardial agents. The three analogs presented here were
potent and fast-acting. For the above, the compounds H-BZM2, O2N-BZM7, and O2N-BZM9 are an
attractive potential starting point for new antigiardial drugs.
Supplementary Materials: The following are available online, Figures S1, S2, S4, S5, S7, S8, S10, S11, S13, S14:
Nuclear magnetic resonance spectra of compounds. Figures S3, S6, S9, S12, S15: High-resolution mass spectra
of compounds.
Author Contributions: Conceptualization, B.H.-O. and E.C.-J.; methodology, B.H.-O., L.M.R.-R., S.G.-M., A.S.-C.,
J.M.-Q., A.S.-S., E.J.R.-N., R.A.-E., M.C.-C., A.M.-T., and E.C.-J.; software, B.H.-O., A.M.-T.; validation, B.H.-O.,
L.M.R.-R., S.G.-M., A.S.-C., J.M.-Q., A.S.-S., E.J.R.-N., R.A.-E., M.C.-C., A.M.-T., and E.C.-J.; formal analysis, B.H.-O.,
L.M.R.-R., S.G.-M., A.S.-C., J.M.-Q., A.S.-S., E.J.R.-N., R.A.-E., M.C.-C., A.M.-T., and E.C.-J.; investigation, B.H.-O.,
L.M.R.-R., S.G.-M., A.S.-C., J.M.-Q., A.S.-S., E.J.R.-N., R.A.-E., M.C.-C., A.M.-T., and E.C.-J.; data curation, B.H.-O.,
L.M.R.-R., S.G.-M., A.S.-C., J.M.-Q., A.S.-S., E.J.R.-N., R.A.-E., M.C.-C., A.M.-T., and E.C.-J.; writing—original draft
preparation, B.H.-O., L.M.R.-R., S.G.-M., A.S.-C., J.M.-Q., A.S.-S., E.J.R.-N., R.A.-E., M.C.-C., A.M.-T., and E.C.-J.;
writing—review and editing, B.H.-O., L.M.R.-R., S.G.-M., A.S.-C., J.M.-Q., A.S.-S., E.J.R.-N., R.A.-E., M.C.-C.,
A.M.-T., and E.C.-J.; visualization, B.H.-O., L.M.R.-R., S.G.-M., A.S.-C., J.M.-Q., A.S.-S., E.J.R.-N., R.A.-E., M.C.-C.,
A.M.-T., and E.C.-J.; supervision, B.H.-O., E.C.-J.; project administration, B.H.-O., E.C.-J.; funding acquisition,
B.H.-O., L.M.R.-R., S.G.-M., J.M.-Q., A.M.-T., and E.C.-J. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by Fondos Federales, M
and L.M.R.-R. by HIM/2014/013 SSA 1120, and by the E022 Program, National Institute of Pediatrics, Mexico City,
Mexico (Recursos Fiscales para la Investigaci n). S.G.-M. was supported by INP 038/2019. J.M.-Q. was supported
by INP 024/2017, and by CONACYT grant 259201, Cátedras CONACYT (2184) project number 2057.
éxico. B.H.-O. was funded by HIM/2017/100 SSA 1430
ó

Molecules 2020, 25, 3979
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Acknowledgments: The technical assistance of Javier Gallegos Infante (Instituto de Fisiolog
í
a Celular, UNAM)
with the bibliographic materials is greatly appreciated.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Berkman, D.S.; Lescano, A.G.; Gilman, R.H.; Lopez, S.L.; Black, M.M. Effects of stunting, diarrhoeal disease,
and parasitic infection during infancy on cognition in late childhood: A follow-up study. Lancet 2002, 359,
564–571. [CrossRef]
2.
3.
4.
Lane, S.; Lloyd, D. Current trends in research into the waterborne parasite giardia. Crit. Rev. Microbiol. 2002
28, 123–147. [CrossRef] [PubMed]
Plutzer, J.; Ongerth, J.; Karanis, P. Giardia taxonomy, phylogeny and epidemiology: Facts and open questions.
Int. J. Hyg. Environ. Health 2010, 213, 321–333. [CrossRef] [PubMed]
Savioli, L.; Engels, D.; Daumerie, D.; Jannin, J.; Alvar, J.; Asiedu, K.; Gastellu-Etchegorry, M.; Simarro, P.;
Mariotti, S.P. Response from savioli and colleagues from the Department of Neglected Tropical Diseases,
World Health Organization. PLoS Med. 2006, 3, e283. [CrossRef]
,
5.
6.
Gardner, T.B.; Hill, D.R. Treatment of giardiasis. Clin. Microbiol. Rev. 2001, 14, 114–128. [CrossRef]
Escobedo, A.A.;
Álvarez, G.; Gonzalez, M.E.; Almirall, P.; Cañete, R.; Cimerman, S.; Ruiz, A.; Pérez, R. The
treatment of giardiasis in children: Single-dose tinidazole compared with 3 days of nitazoxanide. Ann. Trop.
Med. Parasitol. 2008, 102, 199–207. [CrossRef]
7.
8.
Kulakova, L.; Galkin, A.; Chen, C.Z.; Southall, N.; Marugan, J.J.; Zheng, W.; Herzberg, O. Discovery of novel
antigiardiasis drug candidates. Antimicrob. Agents Chemother. 2014, 58, 7303–7311. [CrossRef]
Lemée, V.; Zaharia, I.; Nevez, G.; Rabodonirina, M.; Brasseur, P.; Ballet, J.; Favennec, L. Metronidazole and
albendazole susceptibility of 11 clinical isolates of Giardia duodenalis from France. J. Antimicrob. Chemother.
2000, 46, 819–821. [CrossRef]
9.
Upcroft, P.; Upcroft, J.A. Drug targets and mechanisms of resistance in the Anaerobic Protozoa. Clin. Microbiol.
Rev. 2001, 14, 150–164. [CrossRef]
10. Ansell, B.R.; McConville, M.J.; Ma’Ayeh, S.; Dagley, M.J.; Gasser, R.B.; Svärd, S.G.; Jex, A.R. Drug resistance
in Giardia duodenalis. Biotechnol. Adv. 2015, 33, 888–901. [CrossRef]
11. Muller, J.; Hemphill, A.; Müller, N. Physiological aspects of nitro drug resistance in Giardia lamblia. Int. J.
Parasitol. Drugs Drug Resist. 2018, 8, 271–277. [CrossRef] [PubMed]
12. Garc
í
a-Torres, I.; de la Mora, I.; Marcial-Quino, J.; G
ó
mez-Manzo, S.; Vanoye-Carlo, A.; Navarrete-V
ázquez, G.;
Col n-Lozano, B.; Guti
í
é
rrez-Castrell n, P.; Sierra-Palacios, E.; Navarrete-Vá
ó
zquez, G.; et al. Proton pump
inhibitors drastically modify triosephosphate isomerase from Giardia lamblia at functional and structural
levels, providing molecular leads in the design of new antigiardiasic drugs. Biochim. Biophys. Acta Gen. Subj.
2016, 1860, 97–107. [CrossRef]
13. Enriquez-Flores, S.; Rodr
Guti rrez-Castrell n, P.; P rez-Hern
ndez, S.-T.; et al. Determining the molecular mechanism of inactivation by chemical modification of
í
guez-Romero, A.; Hern
ández-Alcántara, G.; Oria-Hernández, J.;
é
ó
é
ández, G.; de la Mora, I.; Castillo-Villanueva, A.; Garc
ía-Torres, I.;
M
é
triosephosphate isomerase from the human parasite Giardia lamblia: A study for antiparasitic drug design.
Proteins Struct. Funct. Bioinform. 2011, 79, 2711–2724. [CrossRef] [PubMed]
14.
15.
Pérez-Villanueva, J.; Santos, R.G.; Hernández-Campos, A.; Giulianotti, M.A.; Castillo, R.; Medina-Franco, J.L.
Towards a systematic characterization of the antiprotozoal activity landscape of benzimidazole derivatives.
Bioorganic Med. Chem. 2010, 18, 7380–7391. [CrossRef]
Pérez-Villanueva, J.; Romo-Mancillas, A.; Hernández-Campos, A.; Yépez-Mulia, L.; Hernández-Luis, F.;
Castillo, R. Antiprotozoal activity of proton-pump inhibitors. Bioorganic Med. Chem. Lett. 2011, 21, 7351–7354.
[CrossRef] [PubMed]
16. Salahuddin; Yar, M.S.; Mazumder, A. Benzimidazoles: A biologically active compounds. Arab. J. Chem. 2017
,
10, S157–S173. [CrossRef]
17. Hern
ández-Ochoa, B.; Navarrete-V
á
zquez, G.; Nava-Zuazo, C.; Castillo-Villanueva, A.; M
é
ndez, S.-T.;
Torres-Arroyo, A.; G
ó
mez-Manzo, S.; Marcial-Quino, J.; Ponce-Macotela, M.; Rufino-Gonz
ález, Y.; et al.
Novel giardicidal compounds bearing proton pump inhibitor scaffold proceeding through triosephosphate
isomerase inactivation. Sci. Rep. 2017, 7, 1–14. [CrossRef]

Molecules 2020, 25, 3979
22 of 22
18. Navarrete-V
Morales, R.; Cort
2-(Trifluoromethyl)benzimidazole derivatives. Bioorganic Med. Chem. Lett. 2001, 11, 187–190. [CrossRef]
19. Valdez, J.; Cedillo, R.; Hern ndez-Campos, A.; Y pez, L.; Hern ndez-Luis, F.; Navarrete-V zquez, G.;
Tapia, A.; Cort s, R.; Hern ndez, M.; Castillo, R. Synthesis and antiparasitic activity of 1H-benzimidazole
derivatives. Bioorganic Med. Chem. Lett. 2002, 12, 2221–2224. [CrossRef]
rez-Villanueva, J.; Hern ndez-Campos, A.; pez-Mulia, L.;
ndez-Lucio, O.; Hern ndez-Luis, F.; Castillo, R. Synthesis and antiprotozoal activity of novel
ázquez, G.; Cedillo, R.; Hern
ández-Campos, A.; Y
é
pez, L.; Hern
ández-Luis, F.; Valdez, J.;
é
s, R.; Hern
ández, M.; Castillo, R. Synthesis and antiparasitic activity of
á
é
á
á
é
á
20.
Pé
á
Y
é
Méndez-Cuesta, C.;
M
é
á
2-{[2-(1H-imidazol-1-yl)ethyl]sulfanyl}-1H-benzimidazole derivatives. Bioorganic Med. Chem. Lett. 2013, 23,
4221–4224. [CrossRef]
21. Reyes-Vivas, H.; de la Mora, I.; Castillo-Villanueva, A.; Y
épez-Mulia, L.; Hernández-Alcántara, G.;
Figueroa-Salazar, R.; Garc a-Torres, I.; G mez-Manzo, S.; M ndez, S.-T.; Vanoye-Carlo, A.; et al. Giardial
í
ó
é
triosephosphate isomerase as possible target of the cytotoxic effect of omeprazole in Giardia lamblia.
Antimicrob. Agents Chemother. 2014, 58, 7072–7082. [CrossRef] [PubMed]
22.
L
ópez-Vel
ázquez, G.; Fern
ández-Lainez, C.; de la Mora, J.I.; de La Portilla, D.C.; Reynoso-Robles, R.;
Gonz lez-Maciel, A.; Ridaura, C.; Garc
á
í
a-Torres, I.; Guti rrez-Castrell n, P.; Olivos-Garc a, A.; et al. On
é
ó
í
the molecular and cellular effects of omeprazole to further support its effectiveness as an antigiardial drug.
Sci. Rep. 2019, 9, 1–14. [CrossRef] [PubMed]
23. Constansa, J.F.; Pinol, A.C.; Corominas, J.P. 2-Benzimidazolylalkylthio (or -sulfinyl or -sulfonyl) Derivatives,
Their Preparation and Their Application as Medicinal Products. U.S. Patent US4791114A, 13 December 1988.
24. Raju, M.N.; Kumar, N.U.; Reddy, B.S.; Anitha, N.; Srinivas, G.; Bhattacharya, A.; Mukkanti, K.; Kolla, N.;
Bandichhor, R. An efficient synthesis of dexlansoprazole employing asymmetric oxidation strategy.
Tetrahedron Lett. 2011, 52, 5464–5466. [CrossRef]
25. Jang, S.-Y.K.; Tai Won, K.; Sungbum, K.; Byung-Ku, C.; Chang-Ju, K.; Cheol Kyung, H.; Tae Hee, S.; Kwee
Hyun, L.; Gwan, S. Process for Preparing (r)-(+)-Lansoprazole and Intermediate Used Therein. Patent
Cooperation Treaty (PCT) WO2010068049A2, 17 June 2010.
26. Liqiang, Y.; Chunzhen, H.; Zhihong, L.; Yuanyuan, H.; Xiangan, M. A Kind of R-Lansoprazole and Preparation
Method Thereof and Purposes. CN106946849A, 14 July 2017.
27. Garc
í
a-Torres, I.; de la Mora, I.; Hern ntara, G.; Molina-Ortiz, D.; Caballero-Salazar, S.;
á
ndez-Alc
á
Olivos-Garcia, A.; Nava, G.; L
ó
pez-Vel zquez, G.; Enríquez-Flores, S. First characterization of a microsporidial
á
triosephosphate isomerase and the biochemical mechanisms of its inactivation to propose a new druggable
target. Sci. Rep. 2018, 8, 8591. [CrossRef] [PubMed]
28. Hern
ández-Alcántara, G.; Torres-Larios, A.; Enr
í
quez-Flores, S.; García-Torres, I.; Castillo-Villanueva, A.;
M
é
ndez, S.-T.; de la Mora, I.; G
ó
mez-Manzo, S.; Torres-Arroyo, A.; L
ó
pez-Velá
zquez, G.; et al. Structural and
functional perturbation of Giardia lamblia triosephosphate isomerase by modification of a non-catalytic,
non-conserved region. PLoS ONE 2013, 8, e69031. [CrossRef]
29. Laskowski, R.A.; Jabłon´ska, J.; Pravda, L.; Varˇeková, R.S.; Thornton, J.M. PDBsum: Structural summaries of
PDB entries. Protein Sci. 2017, 27, 129–134. [CrossRef]
30. Grosdidier, A.; Zoete, V.; Michielin, O. SwissDock, a protein-small molecule docking web service based on
EADock DSS. Nucleic Acids Res. 2011, 39, W270–W277. [CrossRef]
31. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF
Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612.
[CrossRef]
Sample Availability: Samples of the compounds are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).