Synthesis, antiprotozoal activity, and cheminformatic analysis of 2-phenyl-2h-indazole derivatives

Article abstract of DOI:10.3390/molecules26082145

Indazole is an important scaffold in medicinal chemistry. At present, the progress on synthetic methodologies has allowed the preparation of several new indazole derivatives with interesting pharmacological properties. Particularly, the antiprotozoal activity of indazole derivatives have been recently reported. Herein, a series of 22 indazole derivatives was synthesized and studied as antiprotozoals. The 2-phenyl-2H-indazole scaffold was accessed by a one-pot procedure, which includes a combination of ultrasound synthesis under neat conditions as well as Cadogan’s cyclization. Moreover, some compounds were derivatized to have an appropriate set to provide structure-activity relationships (SAR) information. Whereas the antiprotozoal activity of six of these compounds against E. histolytica, G. intestinalis, and T. vaginalis had been previously reported, the activity of the additional 16 compounds was evaluated against these same protozoa. The biological assays revealed structural features that favor the antiprotozoal activity against the three protozoans tested, e.g., electron withdrawing groups at the 2-phenyl ring. It is important to mention that the indazole derivatives possess strong antiprotozoal activity and are also characterized by a continuous SAR.

Full text of DOI:10.3390/molecules26082145

molecules
Article
Synthesis, Antiprotozoal Activity, and Cheminformatic
Analysis of 2-Phenyl-2H-Indazole Derivatives
Karen Rodríguez-Villar 1 , Lilián Yépez-Mulia 2 , Miguel Cortés-Gines 3, Jacobo David Aguilera-Perdomo 3,
Edgar A. Quintana-Salazar 3, Kevin Samael Olascoaga Del Angel 4, Francisco Cortés-Benítez 3
,
Juan Francisco Palacios-Espinosa 3, Olivia Soria-Arteche 3 and Jaime Pérez-Villanueva 3,
*
1
Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana (UAM),
Ciudad de México 04960, Mexico; qkarenrodv@hotmail.com
2
3
Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, UMAE Hospital de Pediatría,
Centro Médico Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México 06720, Mexico;
lilianyepez@yahoo.com
Departamento de Sistemas Biológicos, División de Ciencias Biológicas y de la Salud,
Universidad Autónoma Metropolitana-Xochimilco (UAM-X), Ciudad de México 04960, Mexico;
mcortes@pharmometrica.com.mx (M.C.-G.); jacoboaguilera.96@gmail.com (J.D.A.-P.);
edgarqsl2811@gmail.com (E.A.Q.-S.); jcortesb@correo.xoc.uam.mx (F.C.-B.);
jpalacios@correo.xoc.uam.mx (J.F.P.-E.); soriao@correo.xoc.uam.mx (O.S.-A.)
Doctorado en Biología Experimental, Universidad Autónoma Metropolitana (UAM),
Ciudad de México 04960, Mexico; olaskuaga@gmail.com
4
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
*
Correspondence: jpvillanueva@correo.xoc.uam.mx; Tel.: +525-54-83-72-59; Fax: +525-55-94-79-29
Citation: Rodríguez-Villar, K.;
Yépez-Mulia, L.; Cortés-Gines, M.;
Aguilera-Perdomo, J.D.;
Abstract: Indazole is an important scaffold in medicinal chemistry. At present, the progress on
synthetic methodologies has allowed the preparation of several new indazole derivatives with
interesting pharmacological properties. Particularly, the antiprotozoal activity of indazole derivatives
have been recently reported. Herein, a series of 22 indazole derivatives was synthesized and
studied as antiprotozoals. The 2-phenyl-2H-indazole scaffold was accessed by a one-pot procedure,
which includes a combination of ultrasound synthesis under neat conditions as well as Cadogan’s
cyclization. Moreover, some compounds were derivatized to have an appropriate set to provide
structure-activity relationships (SAR) information. Whereas the antiprotozoal activity of six of
these compounds against E. histolytica, G. intestinalis, and T. vaginalis had been previously reported,
the activity of the additional 16 compounds was evaluated against these same protozoa. The
biological assays revealed structural features that favor the antiprotozoal activity against the three
protozoans tested, e.g., electron withdrawing groups at the 2-phenyl ring. It is important to mention
that the indazole derivatives possess strong antiprotozoal activity and are also characterized by a
continuous SAR.
Quintana-Salazar, E.A.; Olascoaga
Del Angel, K.S.; Cortés-Benítez, F.;
Palacios-Espinosa, J.F.; Soria-Arteche,
O.; Pérez-Villanueva, J. Synthesis,
Antiprotozoal Activity, and
Cheminformatic Analysis of
2-Phenyl-2H-Indazole Derivatives.
Molecules 2021, 26, 2145. https://
doi.org/10.3390/molecules26082145
Academic Editor: Josef Jampilek
Received: 28 February 2021
Accepted: 6 April 2021
Published: 8 April 2021
Keywords: Entamoeba histolytica; Giardia intestinalis; Trichomonas vaginalis; indazole; structure-
activity relationships
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Indazole is an important scaffold in medicinal chemistry. So far, a few drugs containing
the indazole moiety are currently available for clinical use; for instance, the antiemetic
granisetron, the nonsteroidal anti-inflammatory drugs benzydamine and bendazac, the
anticancer agents pazopanib, axitinib, niraparib and entrectinib. From these, niraparib
Copyright:
© 2021 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 (https://
creativecommons.org/licenses/by/
4.0/).
and entrectinib were recently approved in 2017 and 2019 respectively [
the progress on synthetic methodologies has allowed the preparation of several new
indazole derivatives with interesting pharmacological properties [ ]. Particularly, the
], Giardia
1,2]. At present,
3–6
antiprotozoal activity of indazole derivatives against Entamoeba histolytica [
7
intestinalis and Trichomonas vaginalis have been recently reported [8,9].
Molecules 2021, 26, 2145. https://doi.org/10.3390/molecules26082145
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2145
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E. histolytica and G. intestinalis are intestinal protozoa, which causes amebiasis and
giardiasis, respectively. Worldwide 50 million amebiasis cases and 100,000 deaths annually
are estimated [10], whereas 280 million people are affected by G. intestinalis [11]. Both
diseases have high impact in the public health, causing diarrhea, which is a major factor
in morbidity and mortality, affecting mainly infant population [12]. On the other hand,
trichomoniasis, caused by T. vaginalis is the most common non-viral sexually transmitted
disease [13]. According to the World Health Organization, more than 156 million new
cases are estimated annually worldwide [14]. Trichomoniasis can be the cause of cervici-
tis, urethritis, vaginitis, and genital ulceration; additionally, it has been associated with
preterm labor, low-birth weight, sterility, cervical cancer, and a predisposition to HIV
infection [14–16]. Although some chemotherapeutic agents are available to treat amebiasis,
giardiasis and trichomoniasis, e.g., nitroderivatives such as metronidazole [13
,16,17], the
increased drug resistance [13 16 19], and absence of new approved drugs with alternative
,
–
mechanism of action, lead us to search new antiprotozoal compounds, particularly based
on the indazole scaffold. Herein, 22 indazole derivatives were synthesized employing
a practical one-pot process. Whereas six of these compounds have been previously re-
ported to have activity against E. histolytica, G. intestinalis, and T. vaginalis, in this study,
the activity of the additional 16 compounds against these three parasites was evaluated.
Moreover, a SAR analysis and activity landscape studies were performed to highlight
relevant structural features for the biological activity and to identify the SAR nature of the
compounds studied.
2. Results and Discussion
2.1. Synthesis of 2-Phenyl-2H-Indazole Derivatives
The synthetic route for the synthesis of 2-phenyl-2H-indazole derivatives is shown in
Scheme 1. For this purpose, a new practical modification of the Cadogan’s method using
an ultrasound assisted one-pot synthesis was employed. This method uses a combination
of ultrasound synthesis as previously described by Crawford under neat conditions as well
as the Cadogan’s cyclization by refluxing the Schiff base with triethyl phosphite [20,21]. A
comparison of the employed one-pot procedure and a previously reported methodology
for representative examples is displayed in Table 1. Noteworthy, the one-pot method
leads to similar or better yields than our previous report [8]. Moreover, it is important
to mention that our method avoids one purification step, save solvents, and reduce the
work up time. To expand the scope of the method, the number of compounds available
for biological assays, and the diversity of substituents for SAR analysis, derivatives with
chlorine, methoxy, methoxycarbonyl, fluorine and trifluoromethyl groups at the 2- or 3-
positions of the phenyl substituent were also synthesized. It is to be noted that compound
18, having a 2-(methoxycarbonyl)phenyl substituent, was not obtained by employing the
described method, instead, 18a having a 2-cyanophenyl was synthesized as alternative
intermediary to obtain 18; however a slight method modification was also needed to
achieve the compound 18a (vide infra). Since compounds substituted with OH and COOH
are important from the medicinal chemistry point of view due to the fact that it forms key
protein-ligand interactions (e.g., hydrogen bonds and salt bridges) [22], derivatives having
hydroxyl (
7,
14 and 21) and carboxyl (8, 15 and 22) groups were synthesized by hydrolysis
of precursors
3
,
10 17, 4, 11 and 18a. All synthesized compounds were characterized by
,
1H NMR and 13C NMR spectra and the new structures were also characterized by mass
spectrometry. The nuclear magnetic resonance spectra of all compounds can be found in
Figures S1–S23 in the Supplementary Materials.

Molecules 2021, 26, 2145
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◦
Scheme 1. Synthesis of the 2-phenylindazole derivatives. Reagents and conditions: (a
) 1. R1C6H4NH2, ultrasound, 40 C,
◦
neat, 2. P(OEt)3, reflux; (b) BBr3, CH2Cl2, 0 C; (c) 1. NaOH, H2O, reflux, 2. HCl; (d) HCl, MeOH, MW.
Table 1. Chemical yield (%) for 2-phenyl-2H-indazole derivatives synthesized by ultrasound assisted
one-pot procedure, and representative examples of these same compounds synthesized by the
conventional method.
Ultrasound Assisted
One-Pot Procedure
Previously Reported by the
Conventional Method [8]
Compound
R
1
2
H
4-Cl
92
62
57
39
71
43
53
45
51
51
46
55
69
60
52
52
38
50
6
3
4-OCH3
4-COOCH3
4-F
4
5
6
4-CF3
3-Cl
9
-
10
11
12
13
16
17
18a
19
20
3-OCH3
3-COOCH3
3-F
-
-
-
3-CF3
2-Cl
-
-
2-OCH3
2-CN
-
32 1
54
-
2-F
2-CF3
-
26
-
The reaction mixture was ultrasonicated by four cycles of 1 h at 40 ◦C (vide infra).
1

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2.2. Antiprotozoal Activity
Compounds
5
,
6,
9–22 were tested in vitro against E. histolytica, G. intestinalis, and T.
vaginalis. It is important to mention that the activity of compounds
1–4, 7 and 8 against
these three protozoa were previously reported by our group [ ], however, the values were
8
included in Table 2 in order to support the SAR analysis. Against E. histolytica, the first
interesting finding was that all tested compounds were more potent than 1H-indazole
reported by López-Vallejo et al. (IC50 = 0.740 µM) [7]. It is important to note that bio-
logical assays were performed using the same strain, under the same conditions as well
as the same laboratory. The results suggest that 2-phenyl substitution is important for
the antiprotozoal activity, since this modification increased 9-fold the activity. The best
potency was found for 2-phenyl-2H-indazole derivatives substituted with methoxycar-
bonyl (
M). On the other hand, the results for the assays performed against G. intestinalis showed
that the derivatives substituted with 2-chlorophenyl (16), 2-(methoxycarbonyl)phenyl
4, 11 and 18), 4-chlorophenyl (2) and 2-(trifluoromethyl)phenyl (20) (IC50 < 0.050
µ
(
(
18), 2-(trifluoromethyl)phenyl (20) and 2-carboxyphenyl (22) displayed the best activity
IC50 < 0.050 µM). Moreover, 4- and 3-(trifluoromethyl)phenyl derivatives ( and 13 re-
6
spectively) showed favorable effect on the giardicidal activity. It is worth to emphasize
that compounds were slightly more potent against E. histolytica and G. intestinalis than T.
vaginalis. Hence, against T. vaginalis, the best activity (IC50 < 0.070
derivatives substituted with 3-(methoxycarbonyl)phenyl (11), 3-(trifluoromethyl)phenyl
13) and 3-carboxyphenyl (15), as well as the 2-chlorophenyl (16) and 2-carboxyphenyl (22).
µM) was found for the
(
The IC50 values and SAR data displayed in Figure 1, indicate that electron-withdrawing
substituents attached to the 2-phenyl ring are favorable for the antiprotozoal activity
against the three evaluated protozoa.
Figure 1. Principally SAR features derived from the 2-phenyl-2H-indazole derivatives tested.

Molecules 2021, 26, 2145
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Table 2. Antiprotozoal activity of the 2-pheny-2H-indazole derivatives synthesized (IC50 [µM] ± SD).
Compound
R 1
R 2
R 3
E. histolytica
G. intestinalis
T. vaginalis
1 1
2 1
3 1
4 1
5
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
0.080 ± 0.004
0.042 ± 0.003
0.154 ± 0.016
0.022 ± 0.003
0.075 ± 0.007
0.053 ± 0.000
0.074 ± 0.010
0.097 ± 0.006
0.070 ± 0.012
0.078 ± 0.010
0.036 ± 0.000
0.097 ± 0.010
0.061 ± 0.005
0.102 ± 0.003
0.074 ± 0.009
0.088 ± 0.006
0.096 ± 0.003
0.050 ± 0.003
0.092 ± 0.010
0.031 ± 0.011
0.145 ± 0.010
0.074 ± 0.003
0.380 ± 0.146
56.533 ± 18.845
0.113 ± 0.022
0.063 ± 0.003
0.205 ± 0.006
0.063 ± 0.006
0.071 ± 0.007
0.034 ± 0.005
0.119 ± 0.007
0.193 ± 0.012
0.083 ± 0.012
0.056 ± 0.010
0.054 ± 0.003
0.087 ± 0.003
0.050 ± 0.005
0.105 ± 0.000
0.053 ± 0.009
0.031 ± 0.006
0.083 ± 0.003
0.032 ± 0.006
0.071 ± 0.007
0.027 ± 0.011
0.074 ± 0.003
0.023 ± 0.009
1.226 ± 0.125
0.037 ± 0.003
0.118 ± 0.022
0.107 ± 0.003
0.372 ± 0.016
0.107 ± 0.006
0.118 ± 0.020
0.084 ± 0.016
0.157 ± 0.014
0.327 ± 0.018
0.101 ± 0.006
0.120 ± 0.019
0.069 ± 0.008
0.092 ± 0.010
0.069 ± 0.005
0.162 ± 0.000
0.065 ± 0.009
0.066 ± 0.012
0.183 ± 0.006
0.109 ± 0.008
0.108 ± 0.007
0.128 ± 0.014
0.112 ± 0.010
0.067 ± 0.006
0.236 ± 0.016
1.591 ± 0.011
OCH3
COOCH3
F
CF3
OH
COOH
H
6
7 1
8 1
9
Cl
OCH3
COOCH3
F
CF3
OH
COOH
H
10
11
12
13
14
15
16
17
18
19
20
21
H
H
H
H
H
H
H
H
H
H
H
H
H
-
-
H
H
H
H
H
H
-
-
OCH3
COOCH3
F
CF3
OH
COOH
-
22
MTZ 2
ABZ 3
-
IC50 values were taken from Pérez-Villanueva et al. 2017 [8]; 2 Metronidazole; 3 Albendazole.
1
2.3. Cheminformatic Analysis
Compounds
1
–
22 were also analyzed employing cheminformatic tools and compared
23 36] and ChEMBL com-
against antiprotozoal databases of benzimidazole derivatives [
7,
–
pounds [37], which have also reported activities against E. histolytica, G. intestinalis and T.
vaginalis. It is worthwhile to mention that benzimidazole derivatives compose an important
reference database since a high number of compounds were tested under the same method
and conditions. These benzimidazole derivatives have been the reference database for
several cheminformatic and QSAR studies as antiprotozoals [31,34,38–40]; and the data
available has been expanded during the last years. Figure 2 shows a representation of the
property space and the activity distribution of the 22 synthesized indazole derivatives as
compared to benzimidazole derivatives and ChEMBL collection for each analyzed par-
asite [41]. Although, 2-phenyl-2H-indazole derivatives are localized in a very focalized
space as compared to benzimidazole and ChEMBL compounds, their activity values are
similar to the majority of benzimidazoles, but higher than ChEMBL compounds in most
cases, as is visually depicted as violin plots in Figure 2. Although the number and diversity
of indazole derivatives synthesized and tested herein are still low, it is of our interest to
expand the database of indazole derivatives as well as their activity landscape to search for
more potent compounds.

Molecules 2021, 26, 2145
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Figure 2. Property space and activity range for 2-phenyl-2H-indazole derivatives (1–22) synthesized in this work (yellow), a
collection of antiprotozoal 1H-benzimidazole derivatives (blue) and ChEMBL collection of antiprotozoals (gray). The two
principal components taken together account for 75.34, 72.44 and 75.71% of the variance for E. histolytica, G. intestinalis, and
T. vaginalis, respectively.
To visualize the activity landscape of the synthesized indazole derivatives as compared
to the benzimidazole reference database, chemotype-based Structure-Activity Similarity
(SAS) maps were generated (Figure 3) [42]. A general form of the SAS map is presented
in Figure 4. SAS maps are divided in four regions: region I is associated with scaffold
hopping (or side chain hopping), region II denotes smooth SAR and region IV indicates
discontinuous SAR and activity cliffs. The structure similarity threshold was established
by the median similarity of the pairwise comparisons plotted in each map and depends
on the database and the fingerprint employed, whereas the activity similarity threshold
was established on two units in activity difference (100-fold difference in potency). Since
Similarity-Property principle establish that similar compounds have similar properties
(e.g., biological activity), all indazole derivatives fall in such definition [43]. Different SAR
classifications have been previously defined as continuous (smooth), heterogeneous and
discontinuous (rough) SAR [43
disadvantages or opportunities implied on different SAR classification has been widely
discussed [44 45]. Therefore, the 22 indazole derivatives studied are characterized by
,44]. Moreover, the implications in medicinal chemistry,
,
a smooth or continuous SAR (high similarity and low activity differences; region II) as
compared to the benzimidazole derivatives database where a heterogeneous SAR can be
observed [38,39]. Therefore, the antiprotozoal indazole database should be expanded by
including more diverse substituted derivatives at different positions to carry out QSAR
studies and lead optimization.

Molecules 2021, 26, 2145
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Figure 3. Structure-Activity Similarity Maps using Molecular ACCess System keys (MACCS) and Extended Connectivity
Fingerprint (ECFP) divided by thresholds in structure similarity and activity differences in regions I–IV. Each data point
indicates a pairwise comparison of the 22 synthesized 2-phenyl-2H-indazole derivatives (231 datapoints in yellow,
and 111 benzimidazoles tested against E. histolytica (6105 datapoints in blue, ), 159 benzimidazoles tested against G.
intestinalis (12,561 datapoints in blue, ), and 134 benzimidazoles tested against T. vaginalis (8911 datapoints in blue, ).
A–F)
A,B
C
,D
E,F
Figure 4. General form of the structure–activity similarity (SAS) maps showing four major regions.
3. Materials and Methods
3.1. Chemicals and Instruments
All chemicals and starting materials were obtained from Sigma-Aldrich (Sigma-
Aldrich, Toluca, MEX, Mexico). Reactions were monitored by TLC on 0.2 mm percolated
silica gel 60 F254 plates (Merck, Darmstadt, Germany) and visualized by irradiation with a
UV lamp (Cole-Parmer, Vernon Hills, IL, USA). Silica gel 60, 70–230 mesh, was used for
column chromatography (Macherey-Nagel, Düren, Germany). Ultrasound assisted synthe-
sis were carried out employing a Cole-Parmer ultrasonic bath model 8891 (Cole-Parmer,
Vernon Hills, IL, USA), operating at 42 kHz. Melting points were determined in open
capillary tubes with a Büchi M-565 melting point apparatus (Büchi, Flawil, Switzerland)
and are uncorrected. Microwave-assisted reactions were carried out in a monomodal
reactor (Anton-Paar Monowave 300) equipped with a hydraulic pressure sensing device

Molecules 2021, 26, 2145
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1
and an infrared temperature-sensor (Anton-Paar, Graz, Austria). H NMR and 13C NMR
were measured with an Agilent DD2 spectrometer (Agilent, Santa Clara, CA, USA), op-
erating at 600 MHz and 151 MHz for 1H and 13C, respectively. Chemical shifts are given
in parts per million relatives to tetramethylsilane (Me4Si,
δ = 0); J values are given in
Hz. Splitting patterns are expressed as follow: s, singlet; d, doublet; t, triplet; q, quar-
tet; dd, doublet of doublet; td, triplet of doublet; ddd, doublet doublet of doublet; dq,
doublet of quartets; m, multiplet; bs, broad singlet. High resolution Mass spectra were
recorded on a Bruker ESI/APCI-TOF, MicroTOF-II-Focus spectrometer (Bruker, Billerica,
MA, USA) by electrospray ionization (ESI); whereas low resolution Mass spectra were
recorded on a Waters Xevo TQ-MS spectrometer (Waters, Milford, MA, USA) by electron
impact (EI). All compounds were named using the automatic name generator tool from the
ChemDraw Professional 16.0.1.4 software (PerkinElmer, Waltham, MA, USA), according to
IUPAC rules.
3.2. Chemistry
3.2.1. General Procedure for the One-Pot Synthesis of 2-Phenyl-2H-Indazole Derivatives
1–6, 9–13, 16, 17, 19 and 20
A 50 mL heavy wall glass Schlenk reaction tube was charged with 2-nitrobenzaldehyde
(1.654 mmol) and aniline or the substituted aniline (1.654 mmol). The reaction mixture
was ultrasonicated for two hours at 40 ◦C. The condensed water under the tube walls was
removed. Afterwards, triethyl phosphite (5 mmol) was added and heated to 150 ◦C under
nitrogen atmosphere for two hours. The phosphite excess was oxidized with 20 mL of 5%
hydrogen peroxide solution. The product was extracted with ethyl acetate (20 mL
×
3)
and the organic layer was washed with brine (20 mL) and dried with anhydrous sodium
sulfate and finally concentrated under vacuum distillation. The evaporation residue was
purified by using column chromatography with hexane–ethyl acetate 98:2 (compound 19),
95:5 (compounds, 9–13, 16, 17), 90:10 (compounds 1–3, 6, 20), and 80:20 (compounds 4, 5).
2-Phenyl-2H-Indazole (
1
), White solid, 92% yield, m.p.: 81–82 ◦C. The spectroscopic
1
data agree with the previously reported data [8,46]. H NMR (600 MHz, CDCl3) δ 8.41 (d,
J = 0.9 Hz, 1H), 7.91–789 (m, 2H), 7.79 (dd, J = 8.8, 0.9 Hz, 1H), 7.71 (dt, J = 8.5, 1.0 Hz, 1H),
7.54–7.49 (m, 2H), 7.41–7.37 (m, 1H), 7.32 (ddd, J = 8.8, 6.6, 1.0 Hz, 1H), 7.11 (ddd, J = 8.4,
6.6, 0.7 Hz, 1H); 13C NMR (151 MHz, CDCl3)
122.46, 121.00, 120.41, 120.38, 117.95.
δ
149.79, 140.54, 129.56, 127.90, 126.83, 122.77,
◦
2-(4-Chlorophenyl)-2H-Indazole (
spectroscopic agree with the previously reported data [
2
), White solid, 62% yield, m.p.: 143–146 C. The
1
8,
47]. H NMR (600 MHz, CDCl3)
δ
8.37 (d, J = 1.0 Hz, 1H), 7.87–7.83 (m, 2H), 7.77 (dq, J = 8.8, 0.9 Hz, 1H), 7.69 (dt, J = 8.5,
1.0 Hz, 1H), 7.51–7.47 (m, 2H), 7.33 (ddd, J = 8.8, 6.6, 1.1 Hz, 1H), 7.12 (ddd, J = 8.5, 6.6,
0.8 Hz, 1H); 13C NMR (151 MHz, CDCl3)
122.87, 122.17, 120.50, 120.42, 118.07.
δ
150.05, 139.19, 133.72, 129.83, 127.26, 123.03,
◦
2-(4-Methoxyphenyl)-2H-Indazole (
spectroscopic data agree with the previously reported data [
CDCl3) 8.31 (d, J = 0.9 Hz, 1H), 7.83–7.76 (m, 3H), 7.70 (dt, J = 8.4, 1.0 Hz, 1H), 7.32 (ddd,
3
), Beige solid, 57% yield, m.p.: 133–136 C. The
8
,
48]. 1H NMR (600 MHz,
δ
J = 8.7, 6.6, 1.0 Hz, 1H), 7.11 (ddd, J = 8.4, 6.6, 0.8 Hz, 1H), 7.06–7.00 (m, 2H), 3.86 (s, 3H);
13C NMR (151 MHz, CDCl3)
120.38, 117.90, 114.77, 55.73.
δ 159.42, 149.71, 134.25, 126.66, 122.83, 122.55, 122.35, 120.43,
Methyl 4-(2H-Indazol-2-yl)benzoate (
spectroscopic data agree with the previously reported data [
CDCl3)
4
), White solid, 39% yield, m.p.: 186–187 ◦C. The
8
,
48]. 1H NMR (600 MHz,
8.47 (d, J = 0.7 Hz, 1H), 8.22–8.18 (m, 2H), 8.02–7.99 (m, 2H), 7.77 (dd, J = 8.8,
δ
0.8 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.33 (ddd, J = 8.8, 6.6, 1.0 Hz, 1H), 7.14–7.10 (m, 1H),
3.95 (s, 3H); 13C NMR (151 MHz, CDCl3)
123.14, 123.12, 120.60, 120.39, 118.20, 52.46.
δ
166.32, 150.32, 143.77, 131.30, 129.40, 127.58,
◦
2-(4-Fluorophenyl)-2H-Indazole (
5), White solid, 71% yield, m.p.: 110–111 C. The
spectroscopic data agree with the previously reported data [49]. 1H NMR (600 MHz,
CDCl3)
δ 8.34 (d, J = 0.9 Hz, 1H), 7.88–7.84 (m, 2H), 7.78 (dq, J = 8.8, 1.0 Hz, 1H), 7.70 (dt,
J = 8.5, 1.0 Hz, 1H), 7.33 (ddd, J = 8.7, 6.6, 1.0 Hz, 1H), 7.23–7.19 (m, 2H), 7.12 (ddd, J = 8.5,

Molecules 2021, 26, 2145
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6.6, 0.8 Hz, 1H). 13C NMR (151 MHz, CDCl3)
δ
162.05 (d, J = 248.1 Hz), 149.83 (s), 136.88
(d, J = 2.7 Hz), 126.94 (s), 122.84 (s), 122.77 (d, J = 8.6 Hz), 122.59 (s), 120.48 (s), 120.34 (s),
117.89 (s), 116.45 (d, J = 22.8 Hz).
◦2-[4-(Trifluoromethyl)phenyl]-2H-Indazole (
6), Pale pink solid, 43% yield, m.p.: 188–
190 C. The spectroscopic data agree with the previously reported data [47]. 1H NMR
(600 MHz, CDCl3)
(dt, J = 8.5, 1.0 Hz, 1H), 7.34 (ddd, J = 8.8, 6.6, 1.0 Hz, 1H), 7.13 (ddd, J = 8.4, 6.6, 0.5 Hz,
1H). 13C NMR (600 MHz, CDCl3)
150.21, 142.95, 129.75 (q, J = 33.1 Hz), 127.52, 126.86 (q,
δ 8.46 (d, J = 0.9 Hz, 1H), 8.06 (d, J = 8.5 Hz, 2H), 7.81–7.76 (m, 3H), 7.70
δ
J = 3.7 Hz), 123.80 (q, J = 272.0 Hz), 123.08, 123.04, 120.76, 120.47, 120.41, 118.09.
2-(3-Chlorophenyl)-2H-Indazole (
spectroscopic data agree with the previously reported data [48
9
), Light pink solid, 53% yield, m.p.: 109–110 ◦C. The
1
,
50]. H NMR (600 MHz,
CDCl3)
δ
8.39 (d, J = 1.0 Hz, 1H), 7.98 (t, J = 2.1 Hz, 1H), 7.81–7.75 (m, 2H), 7.70 (dt, J = 8.5,
1.0 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.39–7.31 (m, 2H), 7.12 (ddd, J = 8.3, 6.6, 0.8 Hz, 1H);
13C NMR (151 MHz, CDCl3)
120.41, 120.39, 118.77, 117.99.
δ
149.95, 141.45, 135.42, 130.57, 127.88, 127.25, 122.83, 121.24,
◦
2-(3-Methoxyphenyl)-2H-Indazole (10), Beige solid, 45% yield, m.p.: 51–52 C. The
spectroscopic data agree with the previously reported data [50]. 1H NMR (600 MHz,
CDCl3)
δ 8.39 (d, J = 1.0 Hz, 1H), 7.79 (dq, J = 8.6, 0.8 Hz, 1H), 7.69 (dt, J = 8.5, 1.0 Hz, 1H),
7.52 (t, J = 2.2 Hz, 1H), 7.44–7.38 (m, 2H), 7.32 (ddd, J = 8.8, 6.6, 1.1 Hz, 1H), 7.11 (ddd,
J = 8.3, 6.5, 0.8 Hz, 1H), 6.93 (ddd, J = 7.9, 2.5, 1.2 Hz, 1H), 3.90 (s, 3H); 13C NMR (151 MHz,
CDCl3)
δ 160.58, 149.72, 141.68, 130.27, 126.87, 122.71, 122.48, 120.57, 120.39, 117.94, 113.94,
112.94, 106.81, 55.61.
Methyl 3-(2H-Indazol-2-yl)benzoate (11), Beige solid, 51% yield, m.p.: 103–104 ◦C. The
1
spectroscopic data agree with the previously reported data [48]. H NMR (600 MHz, CDCl3)
8.55–8.53 (m, 1H), 8.48 (d, J = 0.9 Hz, 1H), 8.18 (ddd, J = 8.1, 2.3, 1.1 Hz, 1H), 8.08–8.05 (m,
1H), 7.79 (dq, J = 8.8, 0.9 Hz, 1H), 7.71 (dt, J = 8.5, 1.1 Hz, 1H), 7.61 (t, J = 7.9 Hz, 1H), 7.33
(ddd, J = 8.7, 6.6, 1.0 Hz, 1H), 7.12 (ddd, J = 8.5, 6.6, 0.9 Hz, 1H), 3.97 (s, 3H); 13C NMR
(151 MHz, CDCl3)
δ 166.10, 149.98, 140.67, 131.69, 129.79, 128.77, 127.18, 125.21, 122.93,
122.77, 121.51, 120.46, 120.44, 118.00, 52.47.
2-(3-Fluorophenyl)-2H-Indazole (12), White solid, 51% yield, m.p.: 96–98 ◦C. The spec-
1
troscopic data agree with the previously reported data [48]. H NMR (600 MHz, CDCl3)
δ
8.40 (m, J = 0.8 Hz, 1H), 7.79–7.76 (m, 1H), 7.73–7.67 (m, 3H), 7.51–7.46 (m, 1H), 7.33 (ddd, J
= 8.8, 6.6, 0.9 Hz, 1H), 7.11 (m, 2H); 13C NMR (151 MHz, CDCl3)
δ
163.22 (d, J = 247.4 Hz),
141.88 (d, J = 10.0 Hz), 130.88 (d, J = 9.1 Hz), 127.24 (s), 122.87 (s), 122.83 (s), 120.42 (s),
120.41 (s), 118.03 (s), 116.14 (d, J = 3.1 Hz), 114.71 (d, J = 21.2 Hz), 108.70 (d, J = 26.2 Hz).
2-[3-(Trifluoromethyl)phenyl]-2H-Indazole (13), White solid, 46% yield, m.p.: 73–75 ◦C.
1
The spectroscopic data agree with the previously reported data [48]. H NMR (600 MHz,
DMSO-d6)
1.0 Hz, 1H), 7.74 (dq, J = 8.8, 0.9 Hz, 1H), 7.36 (ddd, J = 8.8, 6.5, 1.1 Hz, 1H), 7.14 (ddd,
J = 8.5, 6.5, 0.8 Hz, 1H); 13C NMR (151 MHz, DMSO-d6)
149.64, 140.82, 131.55, 130.88 (q,
δ 9.29 (d, J = 0.9 Hz, 1H), 8.48–8.43 (m, 2H), 7.87–7.81 (m, 2H), 7.80 (dt, J = 8.5,
δ
J = 32.9 Hz), 127.84, 124.69 (q, J = 3.8 Hz), 124.18 (q, J = 272.6 Hz), 123.03, 122.96, 122.72,
121.52, 117.96, 117.14 (q, J = 3.9 Hz).
2-(2-Chlorophenyl)-2H-Indazole (16), White solid, 55% yield, m.p.: 60–61 ◦C. The spec-
1
troscopic data agree with the previously reported data [51]. H NMR (600 MHz, CDCl3)
δ
8.34 (d, J = 1.0 Hz, 1H), 7.80 (dq, J = 8.6, 0.8 Hz, 1H), 7.74 (dt, J = 8.5, 1.0 Hz, 1H), 7.70–7.68
(m, 1H), 7.59–7.56 (m, 1H), 7.45–7.40 (m, 2H), 7.35 (ddd, J = 8.7, 6.6, 1.0 Hz, 1H), 7.14 (ddd,
J = 8.4, 6.6, 0.7 Hz, 1H); 13C NMR (151 MHz, CDCl3)
δ 149.40, 138.60, 130.67, 129.94, 128.97,
128.55, 127.68, 126.92, 125.20, 122.42, 121.99, 120.51, 117.91.
2-(2-Methoxyphenyl)-2H-Indazole (17), Light brown oil, 69% yield. The spectroscopic
1
data agree with the previously reported data [50]. H NMR (600 MHz, CDCl3)
δ
8.51 (d,
J = 1.0 Hz, 1H), 7.87 (dd, J = 7.9, 1.7 Hz, 1H), 7.78 (dq, J = 8.7, 0.9 Hz, 1H), 7.72 (dt, J = 8.4,
1.0 Hz, 1H), 7.39 (ddd, J = 8.2, 7.5, 1.7 Hz, 1H), 7.31 (ddd, J = 8.7, 6.6, 1.1 Hz, 1H), 7.14–7.07
(m, 3H), 3.89 (s, 3H); 13C NMR (151 MHz, CDCl3)
δ
151.89, 148.82, 129.95, 129.25, 126.63,
126.52, 125.51, 122.03, 121.84, 121.20, 120.52, 117.71, 112.36, 56.04.

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2-(2-Fluorophenyl)-2H-Indazole (19), Yellow oil, 54% yield. The spectroscopic data agree
1
with the previously reported data [50
,
52]. H NMR (600 MHz, CDCl3)
δ
8.50 (dd, J = 2.8,
0.9 Hz, 1H), 8.08 (m, 1H), 7.79 (m, 1H), 7.72 (dt, J = 8.5, 0.9 Hz, 1H), 7.41–7.27 (m, 4H), 7.12
(ddd, J = 8.5, 6.6, 0.6 Hz, 1H). 13C NMR (151 MHz, CDCl3)
δ
154.20 (d, J = 250.6 Hz), 129.12
(d, J = 7.9 Hz), 125.10 (d, J = 3.7 Hz), 124.66 (d, J = 10.0 Hz), 122.60 (d, J = 1.3 Hz), 117.00 (d,
J = 20.6 Hz).
2-[2-(Trifluoromethyl)phenyl]-2H-Indazole (20), White solid, 26% yield, m.p.: 51–52 ◦C.
1
The spectroscopic data agree with the previously reported data [51]. H NMR (600 MHz,
CDCl3)
7.66–7.62 (m, J = 9.7, 4.3 Hz, 2H), 7.36 (ddd, J = 8.8, 6.6, 1.0 Hz, 1H), 7.15 (ddd, J = 8.6, 6.6,
0.8 Hz, 1H); 13C NMR (151 MHz, CDCl3)
149.74, 139.11 (q, J = 1.6 Hz), 132.83, 129.67,
δ 8.20 (s, 1H), 7.88–7.84 (m, 1H), 7.79 (dd, J = 8.8, 1.0 Hz, 1H), 7.75–7.70 (m, 2H),
δ
127.27 (q, J = 5.1 Hz), 127.09, 126.42 (q, J = 31.7 Hz), 125.87 (q, J = 1.9 Hz), 123.0275 (q, 273.9),
122.69, 122.24, 120.54, 118.05, 77.16.
3.2.2. General Procedure for O-Demethylation
The methoxy-2-phenylindazole compounds (3, 10 or 17) (4 mmol) were dissolved in
dichloromethane (12 mL) and cooled to 0 ◦C under N2 atmosphere. Afterwards, boron
tribromide (12 mmol, 12 mL of 1 M solution in dichloromethane) was added and the
reaction mixture was stirred at room temperature overnight. The solvent was removed
by evaporation and a saturated sodium bicarbonate solution was added. The resulting
solid was filtered and dried under vacuum. The crude product was purified using a short
column packed with silica gel and ethyl acetate-hexanes (6:4) as a mobile phase.
4-(2H-Indazol-2-yl) phenol (
scopic data agree with the previously reported data [
7
), White solid, 64% yield, m.p.: 179–181 ◦C. The spectro-
1
8]. H NMR (600 MHz, DMSO-d6)
δ
9.84 (s, 1H), 8.90 (d, J = 0.9 Hz, 1H), 7.90–7.83 (m, 2H), 7.74 (dt, J = 8.4, 1.0 Hz, 1H), 7.68 (dq,
J = 8.8, 0.9 Hz, 1H), 7.28 (ddd, J = 8.7, 6.6, 1.1 Hz, 1H), 7.08 (ddd, J = 8.3, 6.6, 0.8 Hz, 1H),
6.97–6.92 (m, 2H); 13C NMR (151 MHz, DMSO-d6)
δ 157.18, 148.56, 132.20, 126.19, 122.33,
121.84, 121.66, 120.87, 120.67, 117.21, 115.90.
◦
1
3-(2H-Indazol-2-yl)phenol (14), Beige solid, 47% yield, m.p.: 131–132 C. H NMR
(600 MHz, DMSO-d6) 9.94 (s, 1H), 9.02 (d, J = 0.9 Hz, 1H), 7.75 (dt, J = 8.5, 1.0 Hz, 1H),
δ
7.70 (dq, J = 8.8, 0.9 Hz, 1H), 7.51–7.48 (m, 2H), 7.36 (t, J = 8.0 Hz, 1H), 7.30 (ddd, J = 8.8, 6.6,
1.1 Hz, 1H), 7.09 (ddd, J = 8.5, 6.5, 0.9 Hz, 1H), 6.84 (ddd, J = 8.1, 2.4, 1.0 Hz, 1H); 13C NMR
(151 MHz, DMSO-d6) δ 158.37, 148.77, 141.02, 130.47, 126.70, 122.35, 122.02, 121.51, 120.90,
117.44, 114.84, 110.67, 107.41. MS: m/z 211.08 ([M + H]+, 100%)
2-(2H-Indazol-2-yl)phenol (21), White solid, 85% yield, m.p.: 114–116 ◦C. The spectro-
1
scopic data agree with the previously reported data [52]. H NMR (600 MHz, CDCl3)
δ
12.09 (s, 1H), 8.47 (d, J = 0.8 Hz, 1H), 7.75–7.71 (m, 2H), 7.58 (dd, J = 8.1, 1.5 Hz, 1H), 7.38
(ddd, J = 8.7, 6.6, 1.0 Hz, 1H), 7.29–7.25 (m, 1H), 7.19–7.15 (m, 2H), 6.97 (ddd, J = 8.2, 7.3,
1.4 Hz, 1H); 13C NMR (151 MHz, CDCl3)
δ 150.48, 147.61, 129.08, 127.69, 124.84, 122.96,
121.32, 120.28, 120.21, 119.63, 119.43, 119.22, 116.82.
3.2.3. General Procedure for the Hydrolysis of Ester Derivatives
The methyl ester compounds (4 or 11, 1.2 mmol) were dissolved in methanol (7.5 mL)
and an aqueous solution of NaOH (3.6 mmol in 3 mL of water) was added. The reaction
mixture was refluxed until the starting material was totally consumed. The mixture was
cooled on ice and acidified to pH 1 with HCl to induce precipitation. The solid was filtered
under vacuum and dried.
4-(2H-Indazol-2-yl)benzoic Acid (
spectroscopic data agree with the previously reported data [
◦
8
), White solid, 96% yield, m.p.: 288–289 C. The
1
8
]. H NMR (600 MHz, DMSO-
d6)
δ
9.21 (s, 1H), 8.26–8.23 (m, 2H), 8.15–8.11 (m, 2H), 7.78 (dt, J = 8.5, 1.0 Hz, 1H), 7.72
(dd, J = 8.8, 0.9 Hz, 2H), 7.33 (ddd, J = 8.8, 6.5, 1.1 Hz, 1H), 7.12 (ddd, J = 8.5, 6.6, 0.8 Hz,
1H); 13C NMR (151 MHz, DMSO-d6)
δ 166.54, 149.30, 142.91, 130.90, 129.74, 127.36, 122.62,
122.52, 122.13, 121.07, 119.94, 117.57.

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◦
3-(2H-Indazol-2-yl)benzoic Acid (15), White solid, 43% yield, m.p.: 215–217 C. 1H NMR
(600 MHz, acetone-d6) 9.01 (s, 1H), 8.78–8.69 (m, 1H), 8.36 (ddd, J = 8.1, 2.3, 1.0 Hz, 1H),
8.12–8.05 (m, 1H), 7.82–7.69 (m, J = 11.5, 9.0, 4.6 Hz, 3H), 7.33 (ddd, J = 8.7, 6.6, 0.9 Hz, 1H),
7.15–7.08 (m, 1H); 13C NMR (151 MHz, acetone-d6)
166.84, 150.61, 141.64, 133.09, 130.81,
δ
δ
129.46, 127.85, 125.33, 123.98, 123.28, 122.20, 122.05, 121.73, 118.63. MS: m/z 239.13 ([M +
H]+, 100%).
3.2.4. Synthesis of Compound 18 and 22
2-(2H-Indazol-2-yl)benzonitrile (18a), Synthesized by a slight variation of the general
method (Section 3.2.1). Briefly, the reaction mixture was ultrasonicated by four cycles of
1 h at 40 ◦C; after each 1h sonication step, the mixture was maintained under vacuum for
30 min at 30 mmHg and 40 ◦C. The cyclization with triethyl phosphite and the reaction
workup were carried out as previously described (vide supra). The crude product was
purified by using column chromatography with hexane–ethyl acetate 90:10. White solid,
32% yield, m.p.: 128–129 ◦C. The spectroscopic data agree with the previously reported
1
data [50]. H NMR (600 MHz, CDCl3)
δ 8.60 (d, J = 0.9 Hz, 1H), 7.97 (dd, J = 8.2, 1.1 Hz,
1H), 7.85 (dd, J = 7.8, 1.4 Hz, 1H), 7.82–7.76 (m, 2H), 7.54 (td, J = 7.7, 1.1 Hz, 1H), 7.36 (ddd,
J = 8.8, 6.6, 1.1 Hz, 1H), 7.15 (ddd, J = 8.5, 6.6, 0.8 Hz, 1H); 13C NMR (151 MHz, CDCl3)
δ
150.22, 142.36, 134.52, 134.04, 128.45, 127.68, 125.98, 123.42, 123.09, 122.84, 120.70, 118.03,
116.55, 106.82.
2-(2H-Indazol-2-yl)benzoic Acid (22), A 50 mL round bottom flask was charged with
2-(2H-indazol-2-yl)benzonitrile (2 mmol), sodium hydroxide (16 mmol) and water (25 mL).
The mixture was heated to reflux for four hours. The cold mixture was treated with
aqueous solution of HCl (10%) till total precipitation, the solid was filtered under vacuum
◦
1
and washed with cold water. White solid, 90% yield, m.p.: 210–211 C. H NMR (600
MHz, DMSO-d6) 12.97 (s, 1H), 8.72 (d, J = 0.9 Hz, 1H), 7.86–7.83 (m, 1H), 7.78 (dt, J = 8.5,
1.0 Hz, 1H), 7.75–7.71 (m, 2H), 7.66 (dd, J = 8.8, 0.9 Hz, 1H), 7.63 (ddd, J = 7.7, 6.3, 2.4 Hz,
1H), 7.33–7.28 (m, 1H), 7.13–7.09 (m, 1H); 13C NMR (151 MHz, DMSO-d6)
167.36, 148.77,
δ
δ
138.88, 131.78, 129.83, 129.53, 128.92, 126.32, 126.26, 124.86, 122.07, 121.69, 120.97, 117.38.
MS (HR-ESI) for C14H11N2O2 [M + H]+, calcd: m/z 239.0815, found: m/z 239.0824.
Methyl 2-(2H-Indazol-2-yl)benzoate (18), A 10 mL microwave vial was charged with
2-(2H-indazol-2-yl)benzoic acid (1.26 mmol) and 5 mL of solution of HCl in MeOH (15%).
The reaction was heated at 120 ◦C under microwave irradiation for 25 min. Then the reac-
tion was neutralized with 10% solution of NaHCO3 and extracted with dichloromethane
(3 × 10 mL). The combined organic layers were dried over Na2SO4 and concentrated
under vacuum. The product was purified by column chromatography employing hexanes–
dichloromethane–ethyl acetate (50:35:15) as mobile phase. White solid, 36% yield, m.p.:
1
71–73 ◦C. The spectroscopic data agree with the previously reported data [48]. H NMR
(600 MHz, CDCl3) δ 8.21 (d, J = 0.9 Hz, 1H), 7.90 (dd, J = 7.7, 1.4 Hz, 1H), 7.76 (dd, J = 8.8,
0.9 Hz, 1H), 7.72 (dt, J = 8.5, 0.8 Hz, 1H), 7.66–7.59 (m, J = 9.2, 7.9, 1.4 Hz, 2H), 7.54 (td,
J = 7.6, 1.4 Hz, 1H), 7.32 (ddd, J = 8.8, 6.6, 1.0 Hz, 1H), 7.12 (ddd, J = 8.4, 6.6, 0.6 Hz, 1H),
3.61 (s, 3H); 13C NMR (151 MHz, CDCl3)
δ 166.81, 149.70, 139.80, 132.07, 130.56, 128.82,
128.33, 126.66, 126.33, 123.81, 122.36, 122.34, 120.41, 117.93, 52.46.
3.3. Antiprotozoal Activity Assays
Trichomonas vaginalis strain GT3, Giardia intestinalis isolate IMSS:0981:1 and Entamoeba
histolytica strain HM1-IMSS were used in all the experiments. Trophozoites of G. intestinalis
were maintained in a TYI-S-33 medium supplemented with 10% calf serum and bovine
bile. E. histolytica and T. vaginalis trophozoites were maintained in TYI-S-33 medium
supplemented with 10% bovine serum. Briefly, 5
×
104 trophozoites of G. intestinalis
◦
or T. vaginalis, or 6
×
103 trophozoites of E. histolytica were incubated for 48 h at 37 C
with different concentrations of the compound to be tested, each added as solutions in
DMSO. As a negative control, parasites received an equivalent amount of DMSO only,
while albendazole (ABZ) and metronidazole (MTZ) were included as positive controls. At

Molecules 2021, 26, 2145
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the end of the treatment period, the cells were washed and subcultured for another 48 h in
fresh medium to which no drug was added. The trophozoites were then counted with a
hemocytometer and the 50% inhibitory concentration (IC50), together with the respective
95% confidence limit was calculated by Probit analysis. Experiments were carried out in
triplicate and repeated at least twice.
3.4. Cheminformatic Studies
3.4.1. Molecular Databases
Compounds with IC50 annotations against E. histolytica, G. intestinalis and T. vagi-
nalis were retrieved from ChEMBL version 27 [37]. The crude databases were refined
by using the following criteria: (a) compounds having structural errors or not readable
were eliminated; (b) duplicated compounds were removed to preserve a single structure
(the lowest activity value was kept); (c) only compounds with values of IC50 < 100 µM
were considered; d) counterions were removed from salts and charges were neutralized;
(e) inorganic compounds were removed. The curated databases contain 968 compounds
against E. histolytica, 1211 against G. intestinalis and 445 against T. vaginalis with pIC50
annotations (-log IC50[mol/L]). Moreover, 180 benzimidazole derivatives with activity
against these same protozoa were taken from previous reports to give 111 derivatives
with IC50 against E. histolytica, 159 compounds against G. intestinalis and 134 molecules
against T. vaginalis [7,23–36]. Compounds were drawing employing ChemDraw Profes-
sional 16.0.1.4 software and exported as simplified molecular-input line-entry system
(SMILES) with their pIC50 annotations. Moreover, indazole derivatives (
1–22) were pro-
cessed employing the same methodology.
3.4.2. Property Space
A set of six properties were calculated for all compounds under study employing RD-
Kit 2019.09.3 implemented in python 3.7.6: Molecular Weight (MW), Rotatable bonds (RBs),
Hydrogen Bond Acceptors (HBAs), Hydrogen Bond Donors (HBDs), Topological Polar
Surface Area (TPSA), and Octanol-Water Partition Coefficient (logP). Principal Component
Analysis (PCA) were carried out employing the OSIRIS DataWarrior software Version
5.2.1 [41]. Violin plots were calculated employing the Seaborn 0.11.1 library implemented
in python 3.7.6.
3.4.3. Structure-Activity Similarity (SAS) Maps
SAS maps were calculated by employing the Activity Landscape Plotter V1 [53].
Molecular ACCess System (MACCS) keys fingerprint (166 bits) and Extended Connectivity
Fingerprint (ECFP) with a diameter four were used [54
plotting molecular similarity in the X-axis and the absolute value of the activity differ-
ences in the Y-axis. The Tanimoto coefficient was used to calculate N(N 1)/2 pairwise
structural similarities (SSi,j) for each pair of molecules i and j included in each scaffold
class (benzimidazole or indazole). Moreover, N(N 1)/2 pairwise activity differences,
determined by the equation | A(T)i,j| = |A(T)i A(T)j|, were calculated for indazole and
,55]. Each map was generated by
−
−
∆
−
benzimidazole datasets; where A(T)i and A(T)j are the activities of the ith and jth molecules
(j > i), in pIC50, tested against the target parasite T [42]. SAS maps were divided in four
regions employing a threshold of two units on activity difference (100-fold in potency)
and the median similarity of the total datapoints plotted in each map (including indazole
and benzimidazole derivatives). Briefly, Regions I and II are associated with scaffold hop-
ping (or side chain hopping) and smooth SAR, respectively, whereas Region IV indicates
discontinuous SAR (Figure 4).
4. Conclusions
A one-pot procedure including a combination of ultrasound synthesis under neat
conditions and a Cadogan’s cyclization was implemented for the synthesis of 2-phenyl-2H-
indazole derivatives. The one-pot method leads to similar or better yields as compared

Molecules 2021, 26, 2145
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to our previous reports. The biological assays revealed that electron withdrawing groups
at the 2-phenyl ring are favorable for antiprotozoal activity. Furthermore, although the
SAR information obtained from indazole derivatives as antiprotozoals is still limited,
cheminformatic analysis for the 22 compounds studied, highlight their potent values and
their smooth SAR nature as compared to reference databases. More studies are needed to
expand the activity landscape of indazole derivatives; however, these results represent a
valuable start-point toward the optimization of indazole derivatives as antiprotozoals.
1
Supplementary Materials: The following are available online, Figures S1–S23 H NMR and 13C
NMR for 2-phenyl-2H-indazole derivatives synthesized (1–22 and 18a).
Author Contributions: Conceptualization, K.R.-V., L.Y.-M., and J.P.-V. methodology, K.R.-V., J.P.-V.,
L.Y.-M., O.S.-A., J.F.P.-E., and F.C.-B.; formal analysis, J.P.-V., and K.S.O.D.A.; investigation, K.R.-
V., J.P.-V., M.C.-G., J.D.A.-P., E.A.Q.-S., and K.S.O.D.A.; data curation, K.S.O.D.A.; writing—J.P.-V.;
writing—review and editing, L.Y.-M., O.S.-A., J.F.P.-E., and F.C.-B.; supervision, J.P.-V., J.F.P.-E., F.C.-B.,
and O.S.-A.; project administration, J.P.-V.; funding acquisition, J.P.-V. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by Consejo Nacional de Ciencia y Tecnología (CONACyT), grant
numbers CB-2015-01-258554 and 1238.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors would like to express their sincere thanks to Amparo Tapia for the
technical support with biological assays as well as Ernesto Sánchez Mendoza and Mónica A. Rincón
for their analytical support. Moreover, the authors acknowledge to DIFACQUIM for providing
the web server to aid the cheminformatic study, OpenMolecules.org for providing the DataWarrior
software and Pharmometrica research center for the support with mass spectra determinations.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1–22 and 18a are available from the authors.
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