Synthesis and biological evaluation of 2H-indazole derivatives: Towards antimicrobial and anti-inflammatory dual agents

Article abstract of DOI:10.3390/molecules22111864

Indazole is considered a very important scaffold in medicinal chemistry. It is commonly found in compounds with diverse biological activities, e.g., antimicrobial and anti-inflammatory agents. Considering that infectious diseases are associated to an inflammatory response, we designed a set of 2H-indazole derivatives by hybridization of cyclic systems commonly found in antimicrobial and anti-inflammatory compounds. The derivatives were synthesized and tested against selected intestinal and vaginal pathogens, including the protozoa Giardia intestinalis, Entamoeba histolytica, and Trichomonas vaginalis; the bacteria Escherichia coli and Salmonella enterica serovar Typhi; and the yeasts Candida albicans and Candida glabrata. Biological evaluations revealed that synthesized compounds have antiprotozoal activity and, in most cases, are more potent than the reference drug metronidazole, e.g., compound 18 is 12.8 times more active than metronidazole against G. intestinalis. Furthermore, two 2, 3-diphenyl-2H-indazole derivatives (18 and 23) showed in vitro growth inhibition against Candida albicans and Candida glabrata. In addition to their antimicrobial activity, the anti-inflammatory potential for selected compounds was evaluated in silico and in vitro against human cyclooxygenase-2 (COX-2). The results showed that compounds 18, 21, 23, and 26 display in vitro inhibitory activity against COX-2, whereas docking calculations suggest a similar binding mode as compared to rofecoxib, the crystallographic reference.

Full text of DOI:10.3390/molecules22111864

molecules
Article
Synthesis and Biological Evaluation of 2H-Indazole
Derivatives: Towards Antimicrobial
and Anti-Inflammatory Dual Agents
Jaime Pérez-Villanueva 1,*, Lilián Yépez-Mulia 2, Ignacio González-Sánchez 3
,
ID
Juan Francisco Palacios-Espinosa 1, Olivia Soria-Arteche 1, Teresita del Rosario Sainz-Espuñes 1,
Marco A. Cerbón 4, Karen Rodríguez-Villar 1, Ana Karina Rodríguez-Vicente 1,
Miguel Cortés-Gines 1, Zeltzin Custodio-Galván 1 and Dante B. Estrada-Castro 1
1
Departamento de Sistemas Biológicos, División de Ciencias Biológicas y de la Salud,
Universidad Autónoma Metropolitana-Xochimilco (UAM-X), Ciudad de México 04960, Mexico;
jpalacios@correo.xoc.uam.mx (J.F.P.-E.); soriao@correo.xoc.uam.mx (O.S.-A.);
trsainz@correo.xoc.uam.mx (T.d.R.S.-E.); qkarenrodv@hotmail.com (K.R.-V.);
karovi24@hotmail.com (A.K.R.-V.); miguel_gines@outlook.com (M.C.-G.);
zeltzin_888@hotmail.com (Z.C.-G.); benzaidest@hotmail.com (D.B.E.-C.)
2
Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias-Pediatría, IMSS,
Ciudad de México 06720, Mexico; lilianyepez@yahoo.com
Catedrático CONACYT Comisionado a Departamento de Sistemas Biológicos, División de Ciencias
3
Biológicas y de la Salud, Universidad Autónoma Metropolitana-Xochimilco (UAM-X),
Ciudad de México 04960, Mexico; ignacio.gonzalez.s@gmail.com
Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México,
4
Ciudad de México 04510, Mexico; mcerbon85@yahoo.com.mx
*
Correspondence. jpvillanueva@correo.xoc.uam.mx; Tel.: +525-5483-7259; Fax: +525-5594-7929
Received: 8 October 2017; Accepted: 27 October 2017; Published: 31 October 2017
Abstract: Indazole is considered a very important scaffold in medicinal chemistry. It is commonly
found in compounds with diverse biological activities, e.g., antimicrobial and anti-inflammatory
agents. Considering that infectious diseases are associated to an inflammatory response, we designed
a set of 2H-indazole derivatives by hybridization of cyclic systems commonly found in antimicrobial
and anti-inflammatory compounds. The derivatives were synthesized and tested against selected
intestinal and vaginal pathogens, including the protozoa Giardia intestinalis, Entamoeba histolytica,
and Trichomonas vaginalis; the bacteria Escherichia coli and Salmonella enterica serovar Typhi; and the
yeasts Candida albicans and Candida glabrata. Biological evaluations revealed that synthesized
compounds have antiprotozoal activity and, in most cases, are more potent than the reference
drug metronidazole, e.g., compound 18 is 12.8 times more active than metronidazole against
G. intestinalis. Furthermore, two 2,3-diphenyl-2H-indazole derivatives (18 and 23) showed in vitro
growth inhibition against Candida albicans and Candida glabrata. In addition to their antimicrobial
activity, the anti-inflammatory potential for selected compounds was evaluated in silico and in vitro
against human cyclooxygenase-2 (COX-2). The results showed that compounds 18, 21, 23, and 26
display in vitro inhibitory activity against COX-2, whereas docking calculations suggest a similar
binding mode as compared to rofecoxib, the crystallographic reference.
Keywords: anticandidal; indazole; Entamoeba histolytica; Giardia intestinalis; rational drug design;
Trichomonas vaginalis
Molecules 2017, 22, 1864; doi:10.3390/molecules22111864
www.mdpi.com/journal/molecules

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1. Introduction
Infectious diseases caused by protozoa, bacteria, and yeasts have a major impact on human
health. Enteric pathogenic protozoa and bacteria are a frequent cause of intestinal disease which, in turn,
is an important cause of morbidity and mortality around the world [
agents of intestinal parasitic diseases are the protozoa Giardia intestinalis and Entamoeba histolytica
which have been estimated to affect 280 million and 50 million people worldwide each year,
respectively [ ]. Furthermore, some bacterial strains have been identified as responsible for severe
intestinal illness. Examples of these are pathogenic strains of Escherichia coli, e.g., enterohemorrhagic
E. coli (EHEC) and enteroaggregative E. coli (EAEC), and Salmonella enterica serovar Typhi [ ].
Intestinal diseases caused by protozoa and bacteria affect persons of all ages, but have a high incidence
in children [ ]. Even though infections associated with each pathogen display particular clinical
1]. Two important etiological
,
2,3
1,4
1,5
symptoms, all of them are causal agents of infectious diarrhea with severe health consequences and
that could lead to death [1,4,5].
On the other hand, Trichomonas vaginalis and Candida albicans are two of the major etiological agents of
vaginitis. According to the World Health Organization (WHO), 276 million new cases of trichomoniasis
have been estimated [
which has been associated with preterm labor, low-birth weight, sterility, cervical cancer, and predisposition
to HIV infection [ ]. In addition, it has been reported that 75% of women have at least one vaginal yeast
infection during their lifespan [ ]. Infections by Candida usually cause swelling, itching, and irritation and
6]. Infection by T. vaginalis can cause severe inflammation of the genital tract,
5–7
8
can turn into a very serious problem for pregnant and immunocompromised women [8,9].
Although some antimicrobial drugs are currently available for treatment of intestinal or vaginal
infections, it has been reported that resistant strains of these microbes to the current therapies
are emerging and that patients’ responses to the available chemotherapeutic agents vary [5,7,8,10].
Therefore, it is important to develop new active molecules to address these current health problems.
The indazole nucleus is a very important heterocyclic framework in medicinal chemistry. This scaffold
is present in a large number of compounds with a wide range of biological activities [11]. Some indazole
derivatives have recently been reported as antiprotozoals, with activity against E. histolytica and
T. vaginalis [12
,13]. Furthermore, indazole derivatives have been synthesized and tested against several
Gram-positive and Gram-negative bacterial strains [11
,
14,15]. Particularly, 3-phenyl-1H-indazole,
and some derivatives, have been identified as DNA gyrase B inhibitors [16]. Although, these reports
give an insight of the potential of indazole derivatives as antiprotozoal and antibacterial agents,
the information available is still limited. Therefore, it is necessary to synthesize new indazole
derivatives to obtain more information about their antimicrobial potential. Considering a multitarget
design approach [17], the derivatives presented in this work were designed from a combination of cyclic
systems found in antiprotozoal [12
,13], antibacterial [11,14–16,18], and anti-inflammatory compounds
(Figure 1) [19 21]. This strategy was chosen because an inflammatory response is commonly found
–
in infectious and parasitic diseases (e.g., amebiosis and trichomonosis). Moreover, previous studies
showed that amebic infections induce host cyclooxygenase-2 (COX-2) and consequently the production
of prostaglandin PGE2. Therefore, it has been suggested that PGE2 could play a major role in
pathogenesis of E. histolytica [22,23]. Eighteen compounds including 2-phenyl-2H-indazole and
2,3-diphenyl-2H-indazole derivatives were synthesized and tested against the protozoa G. intestinalis,
E. histolytica, and T. vaginalis. The more active antiprotozoal compounds were tested against some
bacterial and yeast strains including enterohemorrhagic and enteroaggregative E. coli (strain 933/EHEC
and strain 042/EAEC), S. enterica serovar Typhi, C. albicans, and C. glabrata. Additionally, six selected
compounds were assayed in vitro and in silico as potential anti-inflammatory agents using the COX-2
inhibition assay. To shed light on the potential toxic effects, the cytotoxicity of a selected group of
compounds was assessed on HaCaT (aneuploid immortal keratinocytes) and HeLa (human epitheloid
cervix carcinoma) cells.

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Figure 1. Design of the 2,3-diphenyl-2H-indazole derivatives.
2. Results and Discussion
2.1. Chemical Synthesis
The 2-phenyl-2H-indazole and 2,3-diphenyl-2H-indazole derivatives (
7
–
26) were synthesized
as illustrated in Scheme 1. The commercially-available 2-nitrobenzaldehyde (
with aniline or p-substituted aniline under reflux conditions to afford the corresponding Schiff
bases ( ); only the reaction to afford compound was conducted at room temperature to
achieve better results. Compounds were reduced and cyclocondensed with P(OEt)3 to give
the 2-phenyl-2H-indazole derivatives ( 11) by the Cadogan reaction [24]. Compound 12 was
synthesized by o-demethylation of with boron tribromide [25]. Hydrolysis of 10 with NaOH
afforded the carboxylic acid 13. Compounds 14 and 15 were obtained by S-oxidation of 11 with
sodium metaperiodate [26]. Compounds 16 19 and 22 24 were synthesized by a palladium-catalyzed
1) was heated
2
–
4,
6
5
2–6
7
–
9
–
–
arylation of the corresponding 2-phenyl-2H-indazole derivative with a variety of aryl iodides or
bromides as previously reported [27]. Chemical yields for the palladium catalyzed arylation were
slightly lower as compared to previously reported data by Ohnmacht et al. These results can be
explained since the original reported methodology was scaled up tenfold to achieve the needed
quantity of products for the biological assays. With the same procedure of hydrolysis and S-oxidation
described above, compounds 20 and 25 were obtained from 18 and 23, respectively, whereas 19 and 24
were used as starting material to produce 21 and 26.
Scheme 1. Synthesis of indazole derivatives 7–26.
All synthesized compounds have sharp melting points and were characterized by using
1H-NMR (nuclear magnetic resonance) and 13C-NMR spectra. The data on previously-reported
structures were consistent with literature reports. Eight of the synthetized indazole derivatives
resulted in new structures, which were also characterized by high-resolution mass spectrometry.

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The nuclear magnetic resonance and mass spectra of compounds can be found in Figures S1–S49 in the
Supporting Information.
2.2. Antiprotozoal Activity
The in vitro antiprotozoal assays against E. histolytica, G. intestinalis, and T. vaginalis of the
2-phenyl-2H-indazole and 2,3-diphenyl-2H-indazole derivatives were carried out following the method
previously described [28,29]. 2-Phenyl-2H-indazole derivatives 7–15 were evaluated and the results
are shown in Table 1 as IC50 values. Metronidazole (MTZ) and albendazole (ABZ) were used as
reference drugs. The most active 2-phenyl-2H-indazole derivatives against the three protozoa were
compounds 8 and 10, these compounds containing 4-chlorophenyl and 4-(methoxycarbonyl)phenyl
groups at position 2. Similarly, compound 7, having a phenyl at position 2, has good activity against the
three protozoa, ranking third in activity for T. vaginalis and forth against G. intestinalis and E. histolytica.
Additionally, compound 15, with 4-(methylsulfonyl)phenyl at the same position, can be classified
among the fourth most active 2-phenyl-2H-indazole derivatives, at least for two parasites evaluated
(G. intestinalis and T. vaginalis). Considering these results, 2,3-diphenyl-2H-indazole derivatives 16
, 17,
18 21 22 23, and 26 were selected to be tested for their antiprotozoal activity. These derivatives had
,
,
,
groups H, COOCH3, Cl, and SO2CH3 at positions 2 and 3, substituents that induced the best response
in 2-phenyl-2H-indazole derivatives. In addition, carboxylic acids 20 and 25 were of interest for
comparative purposes with ester derivatives. Comparison of the 2,3-diphenyl-2H-indazole derivatives
(
16
indicated that only compounds 16
parasites; however, the improvement in activity was poor. Compound 16 increased only its potency
two-fold against G. intestinalis and T. vaginalis relative to ; whereas compound 17 also increased its
potency against E. histolytica two-fold when compared with , whereas the activity against G. intestinalis
and T. vaginalis is preserved. A similar two-fold improvement in potency was found for compound 20
,
17
,
18
,
20, and 21) with its parent analogs 2-phenyl-2H-indazole derivatives (
7, 8, 10, 13, and 15)
,
17, and 20, retained or increased the potency against at least two
7
8
,
when compared to 13, against G. intestinalis and E. histolytica and a three-fold improvement against
T. vaginalis.
Table 1. Antiprotozoal activity of 2-pheny-2H-indazole derivatives and 2,3-diphenyl-2H-indazole derivatives.
R2
N
R1
N
R1
N
N
7−15
16−26
IC50 (µM)
G. intestinalis
IC50 (µM)
E. histolytica
IC50 (µM)
T. vaginalis
Compound
R1
R2
7
8
9
H
Cl
–
–
–
–
–
–
–
–
–
H
H
H
H
H
Cl
0.1133 ± 0.0218
0.0634 ± 0.0031
0.2051 ± 0.0063
0.0634 ± 0.0056
0.2185 ± 0.0088
0.1189 ± 0.0067
0.1931 ± 0.0119
0.1678 ± 0.0110
0.0900 ± 0.0234
0.0518 ± 0.0052
0.0607 ± 0.0023
0.0959 ± 0.0022
0.0795 ± 0.0045
0.1242 ± 0.0122
0.1132 ± 0.0070
0.1188 ± 0.0086
0.1209 ± 0.0090
0.1062 ± 0.0081
1.2260 ± 0.1250
0.0370 ± 0.0030
0.0798 ± 0.0036
0.0415 ± 0.0031
0.1538 ± 0.0158
0.0218 ± 0.0028
0.0978 ± 0.0147
0.0737 ± 0.0101
0.0965 ± 0.0059
0.0878 ± 0.0083
0.1359 ± 0.0052
0.3033 ± 0.0105
0.0213 ± 0.0023
0.0502 ± 0.0022
0.0445 ± 0.0045
0.2081 ± 0.0061
0.0394 ± 0.0000
0.0731 ± 0.0086
0.0509 ± 0.0000
0.0459 ± 0.0081
0.3798 ± 0.1461
56.5334 ± 18.8445
0.1184 ± 0.0218
0.1071 ± 0.0031
0.3723 ± 0.0158
0.1070 ± 0.0056
0.2725 ± 0.0147
0.1570 ± 0.0135
0.3274 ± 0.0178
0.3121 ± 0.0110
0.1450 ± 0.0026
0.0573 ± 0.0026
0.1034 ± 0.0023
0.1020 ± 0.0151
0.1113 ± 0.0180
0.2138 ± 0.0101
0.1181 ± 0.0046
0.1431 ± 0.0043
0.2402 ± 0.0067
0.1837 ± 0.0162
0.2360 ± 0.0160
1.5905 ± 0.0113
OCH3
COOCH3
SCH3
OH
COOH
SOCH3
SO2CH3
H
10
11
12
13
14
15
16
17
18
20
21
22
23
25
26
MTZ
ABZ
Cl
COOCH3
COOH
SO2CH3
H
H
H
H
–
COOCH3
COOH
SO2CH3
–
–
–

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On the other hand, the influence of changing Cl, COOCH3, SO2CH3, and COOH substituents from
the phenyl group at position 2 to the phenyl group at position 3, was studied (compounds 22 23 25
and 26). The results showed that activities for compounds 22 23 25, and 26 were equal, or even lower,
in most cases than their respective analogs 17 18 20, and 21 against the three parasites evaluated.
,
,
,
,
,
,
,
The only exception was 26, which improved four-fold its activity against E. histolytica compared
to 21. Although 2,3-diphenyl-2H-indazole derivatives have good antiprotozoal activity, most of the
compounds have equal or even lower activity than its corresponding 2-phenyl-2H-indazole analog.
Nevertheless, all tested compounds behave as potent antiprotozoal agents, in almost all cases better
than metronidazole, the drug of choice. Additionally, most compounds were slightly more potent
against E. histolytica, compared with the other two evaluated parasites. Although, some indazole
derivatives have been reported as active compounds against E. histolytica and T. vaginalis, the activity
against G. intestinalis had not been previously reported for derivatives having an indazole nucleus.
Therefore, 2-phenyl-2H-indazole and 2,3-diphenyl-2H-indazole are promising frameworks for the
design of new antiprotozoal agents.
2.3. Antibacterial and Anticandidal Assays
The susceptibility assays against E. coli 933, E. coli 042, S. enterica serovar Typhi, C. albicans,
and C. glabrata were carried out using the disk diffusion test, in accordance with the procedure outlined by
The Clinical and Laboratory Standards Institute (CLSI) [30]. A selection of compounds based on the results
from the antiprotozoal assays were tested at 5 mg/mL (Table S1), however, they were inactive or poorly
active even at high concentration against the bacterial strains tested. Nevertheless, compounds 18 and 23
showed a notable inhibition zone against C. albicans (inhibition halos of 10 and 13 mm, respectively).
Moreover, these same compounds showed activity against C. glabrata (inhibition halos of 3 and 4 mm,
respectively), which is usually less sensitive to the commercial antimycotics. Based on these observations,
the minimum inhibitory concentration (MIC) against C. albicans and C. glabrata was calculated for
compounds 18 and 23, Table 2. The results showed that both compounds display activity in the low
millimolar range, and are slightly more active against C. albicans as compared to C. glabrata.
Table 2. Antimycotic effect for selected compounds.
Compound
MIC (mM) C. albicans
MIC (mM) C. glabrata
18
23
3.807
3.807
0.045
15.227
15.227
0.079
Ketoconazole
2.4. In Vitro and In Silico Studies on Cyclooxygenase-2
Considering that inflammatory response is associated with parasitic infections and the suggested
role of PGE2, and therefore of the host COX-2 in the pathogenesis of E. histolytica [22], in vitro assays
on human recombinant COX-2 were carried out. Additionally, molecular docking studies using
AutoDock Vina software (TSRI, La Jolla, CA, USA) were performed to aid in the interpretation
of the experimental results [31]. Compounds 18 and 23 were evaluated because of their strong
antiprotozoal activity and their moderate anticandidal effects. Moreover, compounds 21 and 26 were
of interest because of their methylsulfonyl group, which is commonly found in COX-2 inhibitors.
Additionally, compounds 7 and 16 were considered as unsubstituted references. The results for in vitro
assays and docking calculations are shown in Table 3. Compounds
at 10 M, whereas the positive reference celecoxib was used at 1
The results showed in vitro COX-2 inhibition by compounds 18
7
µ
,
16
M, as previously described [32].
21 23, and 26 (36–50%, at 10 M);
, 18, 21, 23, and 26 were tested
µ
,
,
µ
however, they are still weak inhibitors (see Table 3). Nevertheless, these compounds represent
an interesting starting point towards the design of new antiparasitic compounds with an additional
COX-2 inhibitory property. Docking studies suggest a similar binding mode of compounds 18
,

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21
, 23, and 26 against human COX-2 as compared to celecoxib and the crystallographic ligand
rofecoxib [33]. The predicted binding mode of the reference celecoxib and 18 are shown in Figure 2.
Additionally, better docking scores were found for the 2,3-diphenyl-2H-indazole derivatives 16
23, and 26 as compared to the 2H-indazole derivatives (e.g., compound 7).
, 18, 21,
Table 3. Results for the in silico and in vitro evaluations against human COX-2 for selected compounds.
Docking Score
(Lowest Energy Conformation)
Compound
% of Inhibition of COX-2
7 1
16
18
21
23
26
−8.0
−9.7
Inactive
Inactive
−9.5
50.01 ± 9.49
44.45 ± 2.65
36.35 ± 1.7
41.22 ± 5.93
64.92 ± 2.36
−10.1
−10.0
−11.1
−11.7
Celecoxib 2
Compounds 7, 16, 18, 21, 23, and 26 were tested at 10 µM. 2 Reference tested at 1 µM.
1
Figure 2. Predicted binding modes on human COX-2 for celecoxib (panel (a)) and 18 (panel (b)).
Docked compounds are shown in green, whereas the crystallographic reference rofecoxib is in pink.
2.5. Cytotoxicity Assays
Biological assays on HaCaT and HeLa cell lines were conducted to gain insight into the cytotoxic
effects of these derivatives on human cells, as compared with the effects observed on protozoa.
Cellular viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay [34,35]. Compounds 18 and 23 were chosen considering their antiprotozoal and
anticandidal effect, in addition to their COX-2 inhibitory activity. Additionally, compound 16 was
considered as an unsubstituted reference. The results of the cytotoxicity assays are shown in Table 4.
Table 4. Results for cytotoxicity assays in HaCaT and HeLa cell lines.
% Viability (10 µM)
% Viability (10 µM)
IC50 (µM)
IC50 (µM)
HeLa Cells
Compound
HaCaT Cells 1
HeLa Cells
HaCaT cells 2
16
18
23
95.01 ± 2.44
96.25 ± 4.14
97.83 ± 5.19
93.04 ± 4.57
94.14 ± 3.31
93.72 ± 7.48
93.65 ± 17.30
125.00 ± 29.60
-
-
-
-
1 Percent of viability for selected compounds at 10 M. 2 Half maximal inhibitory concentration for a selected compound.
µ
Although the IC50 determinations were limited by the low solubility of the compounds in the cell
culture medium at higher concentrations than 10 M, they did not exhibit important cytotoxic effect at
10 M in either of the cell lines (% viability > 90%). Therefore, the IC50 values are higher than 10
in all cases. Since all tested compounds showed antiprotozoal activity (IC50 values) lower than one
micromolar (high nanomolar range), the results indicated that compounds 16 18, and 23 are selective
µ
µ
µM
,
antiprotozoal compounds. Only compound 16 was soluble enough for IC50 determination on HaCaT
and HeLa cell lines, having values of 93.65 and 125.00 µM, respectively.

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In summary, nine 2-phenyl-2H-indazole derivatives (
derivatives (16
7
–
15) and eleven 2,3-diphenyl-2H-indazole
–
26) were synthesized. Eight compounds resulted in new structures (14, 15, 18–21, 25,
and 26). Biological evaluations revealed that 2-phenyl-2H-indazole and 2,3-diphenyl-2H-indazole
derivatives have giardicidal, amebicidal, and trichomonicidal activity lower than one micromolar and,
in most cases, are more potent than the drug of choice metronidazole. Although the compounds are
mainly inactive against the used bacterial strains, a major finding was that most of the compounds
are selective antiprotozoal agents. In addition, compounds 18 and 23 inhibit in vitro growth of
C. albicans and C. glabrata. Furthermore, compounds 18
, 21, 23, and 26 showed inhibition of COX-2 at
10 M, which adds an interesting property to these 2,3-diphenyl-2H-indazole derivatives, since COX-2
µ
inhibition has been suggested to be beneficial on E. histolytica infections. Assays in HaCaT and HeLa
cells revealed low cytotoxicity in human cells for a selection of these derivatives. These results suggest
that 2-phenyl-2H-indazole and 2,3-diphenyl-2H-indazole are promising scaffolds for the design of new
compounds against intestinal and vaginal pathogens, such as protozoa and yeasts. The mechanisms of
action of indazole derivatives synthesized in this work as antiprotozoal and anticandidal agents are
still unknown and constitutes a further research topic to be addressed in future research.
3. Materials and Methods
3.1. Chemicals and Instruments
All chemicals and starting materials were obtained from Sigma-Aldrich (Toluca, MC, 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. Silica gel 60 (70–230 mesh) was used for column
chromatography. Melting points were determined in open capillary tubes with a Büchi M-565
1
melting point apparatus (Flawil, Switzerland) and are uncorrected. H-NMR and 13C-NMR spectra
were measured with an Agilent DD2 spectrometer (Santa Clara, CA, USA), operating at 600 MHz
1
and 151 MHz for H and 13C, respectively. Chemical shifts are given in parts per million relative
to tetramethylsilane (Me4Si,
δ = 0); J values are given in Hz. Splitting patterns are expressed as
follow: s, singlet; d, doublet; q, quartet; dd, doublet of doublet; t, triplet; m, multiplet; bs, broad
singlet. High-resolution mass spectra were recorded on a Bruker ESI/APCI-TOF, MicroTOF-II-Focus
spectrometer (Billerica, MA, USA) by electrospray ionization (ESI). All compounds were named using
the automatic name generator tool implemented in ChemBioDraw Ultra 13.0 software (PerkinElmer,
Waltham, MA, USA), according IUPAC rules.
3.2. Chemical Synthesis
General procedure for the synthesis of 1-(2-nitrophenyl)-N-phenylmethanimines (2–6). 2-Nitrobenzaldehyde
(5 g, 33.08 mmol) and aniline or the corresponding substituted aniline (33.08 mmol, 1 eq) were
dissolved in ethanol (12–40 mL; the minimum quantity to dissolve the starting materials) and stirred
at reflux temperature for 1–4 h to yield compounds 2–4, 6. Finally, the mixture was cooled to induce
crystallization and the solid formed was separated using vacuum filtration and washed with cold
ethanol. This same reaction was carried out at room temperature to yield compound 5.
General procedure for the synthesis of 2-phenyl-2H-indazole derivatives (7–11). 2-Phenyl-2H-indazole
derivatives were synthesized employing a slight modification of the Cadogan m◦ethod [24].
The corresponding imine 2–6 (20 mmol) was heated in triethyl phosphite (60 mmol) at 150 C (0.5–2 h)
until the starting material was totally consumed. Then, phosphite and phosphate were separated using
vacuum distillation and the residue was purified using column chromatography with hexane–ethyl
acetate (90:10) as a mobile phase to give the respective 2-phenyl-2H-indazole derivatives 7–9 and 11.
A slightly more polar mobile phase was used for the purification of the compound 10, hexane-ethyl
acetate (80:20).

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4-(2H-indazol-2-yl) phenol (12). Compound
9
(4 mmol) was dissolved in dichloromethane (12 mL)
and cooled to 0 ◦C under N2 atmosphere. Then, boron tribromide (12 mL of 1 M solution in
dichloromethane, 12 mmol) was added and the reaction mixture was warmed to room temperature
and stirred overnight. After completion of the reaction, a saturated sodium bicarbonate solution was
added and the solid formed was filtered 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 to give compound 12
General procedure for the synthesis of derivatives 13 20, and 25. The appropriate methyl ester derivative
10 18, and 23, 1.2 mmol) was 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 heated under reflux for five hours.
After completion of the reaction, the mixture was cooled on ice and acidified to pH 1 with HCl to
induce precipitation. The solid was separated using vacuum filtration and dried.
2-(4-(Methylsulfinyl) phenyl)-2H-indazole (14). To a solution of compound 11 (0.8 mmol) in 28 mL
of CH3CN/CH3COOH (1:1), NaIO4 (0.8 mmol) dissolved in 2 mL of H2O/AcOH (4:1) was
added. The reaction mixture was stirred at room temperature for 24 h. Then, the reaction was
neutralized with a saturated solution of sodium bicarbonate and the product was extracted with
dichloromethane (3 × 50 mL). The organic phase was dried with anhydrous sodium sulfate and
concentrated under vacuum. The evaporation residue was purified by column chromatography using
dichloromethane/methanol (98:2) as a mobile phase to give compound 14.
General procedure for the synthesis of derivatives 15
H2O/AcOH (4:1) were added to a solution of the proper indazole derivative 11
,
21, and 26. NaIO4 (5 mmol) dissolved in 5 mL of
19, or 24 (2 mmol) in
,
28 mL of CH3CN/CH3COOH (1:1). The reaction mixture was stirred at reflux temperature for 12 h.
Then, the mixture was neutralized with a saturated solution of sodium bicarbonate and brine solution
was added until complete precipitation. The solid was separated using vacuum filtration and dried.
The crude product was purified by column chromatography using dichloromethane as a mobile phase.
General procedure for the synthesis of 2,3-diphenyl-2H-indazole derivatives 16
16 19 and 22 24 were synthesized by a palladium catalyzed arylation as previously described by
Ohnmacht et al. [27]. It is worth mentioning that the previously-reported methodology was scaled up
to 0.5 g of starting 2-phenyl-2H-indazole. Whereas compounds 16 19 22, and 23, were synthesized
–19 and 22–24. Compounds
–
–
–
,
using the proper 2-phenyl-2H-indazole and the substituted 4-iodobenzene, only compound 24 was
synthesized from 2-phenyl-2H-indazole and 4-bromothioanisole.
1-(2-Nitrophenyl)-N-phenylmethanimine (
1H-NMR (600 MHz, CDCl3)
8.94 (s, 1H), 8.31 (dd, J = 7.8, 1.4 Hz, 1H), 8.07 (dd, J = 8.2, 1.1 Hz, 1H)
7.74 (t, J = 7.6 Hz, 1H), 7.64–7.60 (m, 1H), 7.45–7.40 (m, 2H), 7.31–7.27 (m, 3H)
13C-NMR (151 MHz, CDCl3)
δ 155.84, 151.07, 149.34, 133.58, 131.18, 131.12, 129.75, 129.28, 126.92, 124.54, 121.18.
2
). Yellow solid (93% yield); m.p.: 64.1–64.9 ◦C (lit [24]: 63–64 ◦C);
δ
,
;
N-(4-Chlorophenyl)-1-(2-nitrophenyl) methanimine (
). Dark yellow solid (91% yield); m.p.: 91.2–92.2 ◦C
(lit [36]: 91–92 ◦C). 1H-NMR (600 MHz, CDCl3)
3
δ
8.93 (s, 1H), 8.29 (dd, J = 7.8, 1.5 Hz, 1H)
,
;
8.08 (dd, J = 8.2, 1.2 Hz, 1H), 7.78–7.72 (m, 1H), 7.67–7.61 (m, 1H), 7.41–7.36 (m, 2H), 7.25–7.20 (m, 2H)
13C-NMR (151 MHz, CDCl3) δ 156.24, 149.49, 149.32, 133.64, 132.58, 131.40, 130.87, 129.72, 129.40,
124.61, 122.54.
N-(4-Methoxyphenyl)-1-(2-nitrophenyl) methanimine (
81–82 ◦C); 1H-NMR (600 MHz, CDCl3)
8.97 (s, 1H), 8.32 (dd, J = 7.8, 1.4 Hz, 1H),
4
). Yellow solid (92% yield); m.p.: 79.1–79.9 ◦C (lit [36]:
δ
8.06 (dd, J = 8.2, 1.1 Hz, 1H), 7.75–7.70 (m, 1H), 7.62–7.57 (m, 1H), 7.35–7.29 (m, 2H), 6.98–6.94 (m, 2H)
,
3.85 (s, 3H); 13C-NMR (151 MHz, CDCl3)
122.78, 114.50, 55.53.
δ 159.09, 153.31, 143.88, 133.48, 131.36, 130.81, 129.55, 124.53,
Methyl 4-((2-nitrobenzylidene) amino)benzoate (
(600 MHz, CDCl3) 8.93 (s, 1H), 8.30 (dd, J = 7.7, 1.0 Hz, 1H), 8.10 (d, J = 8.4 Hz, 3H)
7.68–7.63 (m, 1H), 7.30–7.25 (m, 2H), 3.93 (s, 3H); 13C-NMR (151 MHz, CDCl3) δ 166.66, 157.50, 155.14,
5
) Pale yellow solid (73% yield); m.p.: 122.7–124.4 ◦C; 1H-NMR
7.76 (t, J = 7.6 Hz, 1H)
δ
,
,

Molecules 2017, 22, 1864
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149.39, 133.71, 131.66, 130.95, 130.70, 129.84, 128.26, 124.65, 120.93, 52.15; MS (HR-ESI) for C15H12N2O4
[M + H]+, calcd: m/z 285.0870, found: m/z 285.0861.
N-(4-(Methylthio)phenyl)-1-(2-nitrophenyl)methanimine (
6). Burnt orange solid (92% yield); m.p.:
69.3–70.4 ◦C; 1H-NMR (600 MHz, CDCl3)
δ
8.96 (s, 1H), 8.31 (dd, J = 7.8, 1.4 Hz, 1H),
8.07 (dd, J = 8.2, 1.1 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.63–7.58 (m, 1H), 7.33–7.22 (m, 4H), 2.52 (s, 3H);
13C-NMR (151 MHz, CDCl3) δ 154.86, 149.25, 148.06, 137.45, 133.51, 131.07, 129.63, 127.37, 124.53, 121.89,
16.06; MS (HR-ESI) for C14H12N2O2S [M + H]+, calcd: m/z 273.0692, found: m/z 273.0683.
2-Phenyl-2H-indazole (
data matched previously reported data [37]: 1H-NMR (600 MHz, CDCl3)
7.91–7.88 (m, 2H), 7.79 (dd, J = 8.8, 0.9 Hz, 1H), 7.70 (dt, J = 8.5, 1.0 Hz, 1H), 7.54–7.50 (m, 2H)
7
). White solid (64% yield); m.p.: 81.2–81.6 ◦C (lit [24]: 81–82 ◦C); the spectroscopic
8.40 (d, J = 0.9 Hz, 1H)
δ
,
,
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) δ (ppm): 149.78, 140.52, 129.54, 127.88, 126.81, 122.76, 122.44, 120.99, 120.39,
120.37, 117.94.
◦
2-(4-Chlorophenyl)-2H-indazole (8 ;
). White solid (57% yield); m.p.: 143.0–145.5 C (lit [38]: 138–140 ◦C)
the spectroscopic data matched previously reported data [38]: 1H-NMR (600 MHz, CDCl3) δ 8.37
(d, J = 1.0 Hz, 1H), 7.87–7.82 (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) δ 149.89, 139.02, 133.55, 129.67, 127.09, 122.87, 122.71, 122.00, 120.29, 117.90.
2-(4-Methoxyphenyl)-2H-indazole (9 ;
). Beige solid (56 % yield); m.p.: 133.2–135.8 ◦C (lit [39]: 130–131 ◦C)
the spectroscopic data matched previously reported data [40]: 1H-NMR (600 MHz, CDCl3) δ 8.30
(d, J = 0.9 Hz, 1H), 7.82–7.76 (m, 3H), 7.69 (dt, J = 8.4, 1.0 Hz, 1H), 7.31 (ddd, J = 8.7, 6.6, 1.0 Hz, 1H),
7.10 (ddd, J = 8.4, 6.6, 0.8 Hz, 1H), 7.05–6.99 (m, 2H), 3.86 (s, 3H); 13C-NMR (151 MHz, CDCl3)
δ 159.28,
149.58, 134.12, 126.53, 122.70, 122.41, 122.22, 120.30, 120.25, 117.77, 114.63, 55.60.
Methyl 4-(2H-indazol-2-yl) benzoate (10). White solid (52% yield); m.p.: 185.8–186.2 ◦C (lit [41]:
186–187 ◦C); the spectroscopic data matched previously reported data [40]: 1H-NMR (600 MHz,
CDCl3)
δ 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, 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)
120.26, 118.06, 52.33.
δ
166.19, 150.19, 143.64, 131.16, 129.27, 127.45, 123.01, 122.98, 120.47,
◦
2-(4-(Methylthio) phenyl)-2H-indazole (11). Pale yellow solid (61% yield); m.p.: 148.3–149.7 C (lit [38]:
137–139 ◦C); the spectroscopic data matched previously reported data [38]: 1H-NMR (600 MHz, CDCl3)
δ
8.35 (d, J = 0.8 Hz, 1H), 7.84–7.80 (m, 2H), 7.79–7.76 (m, 1H), 7.68 (dt, J = 8.5, 0.9 Hz, 1H), 7.39–7.35 (m, 2H)
,
7.31 (ddd, J = 8.7, 6.6, 1.0 Hz, 1H), 7.10 (ddd, J = 8.4, 6.6, 0.8 Hz, 1H), 2.53 (s, 3H); 13C-NMR (151 MHz,
CDCl3) δ 149.72, 138.63, 137.78, 127.27, 126.82, 122.77, 122.46, 121.26, 120.30, 120.12, 117.84, 15.88.
◦
◦
4-(2H-Indazol-2-yl) phenol (12). Beige solid (64% yield); m.p.: 179–181 C (lit [25]: 193–194 C);
the spectroscopic data matched previously reported data [42]: 1H-NMR (600 MHz, DMSO-d6)
δ
9.85 (s, 1H), 8.91 (d, J = 0.9 Hz, 1H), 7.91–7.84 (m, 2H), 7.75 (dt, J = 8.4, 1.0 Hz, 1H),
7.69 (dq, J = 8.8, 0.9 Hz, 1H), 7.29 (ddd, J = 8.7, 6.6, 1.1 Hz, 1H), 7.08 (ddd, J = 8.3, 6.6, 0.8 Hz, 1H),
6.98–6.92 (m, 2H); 13C-NMR (151 MHz, DMSO-d6) δ 157.09, 148.47, 132.11, 126.10, 122.24, 121.75,
121.57, 120.78, 120.58, 117.12, 115.81.
4-(2H-Indazol-2-yl) benzoic acid (13). White solid (96% yield); m.p.: 288.3–288.5 ◦C (lit [41]: 286–288 ◦C);
1H-NMR (600 MHz, DMSO-d6)
δ 9.23 (s, 1H), 8.29–8.23 (m, 2H), 8.18–8.12 (m, 2H), 7.79 (dt, J = 8.5, 1.0 Hz,
1H), 7.73 (dq, J = 8.8, 0.9 Hz, 2H), 7.35 (ddd, J = 8.8, 6.5, 1.1 Hz, 1H), 7.13 (ddd, J = 8.5, 6.6, 0.8 Hz, 1H);
13C-NMR (151 MHz, DMSO-d6)
δ 166.46, 149.22, 142.83, 130.82, 129.65, 127.28, 122.54, 122.43, 122.04, 120.99,
119.86, 117.48.

Molecules 2017, 22, 1864
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◦
1
2-(4-(Methylsulfinyl) phenyl)-2H-indazole (14). White solid (92% yield); m.p.: 150.1–152.7 C; H-NMR
(600 MHz, CDCl3) 8.47 (d, J = 0.9 Hz, 1H), 8.13–8.07 (m, 2H), 7.83–7.75 (m, 3H),
7.70 (dt, J = 8.5, 1.0 Hz, 1H), 7.34 (ddd, J = 8.8, 6.6, 1.1 Hz, 1H), 7.13 (ddd, J = 8.5, 6.6, 0.8 Hz, 1H), 2.78 (s, 3H)
13C-NMR (151 MHz, CDCl3)
150.14, 145.05, 142.47, 127.45, 125.03, 123.01, 121.49, 120.46, 120.43, 118.01,
44.10; MS (HR-ESI) for C14H12N2OS [M + Na]+, calcd: m/z 279.0562, found: m/z 279.0481.
δ
;
δ
2-(4-(Methylsulfonyl) phenyl)-2H-indazole (15). White solid (68% yield); m.p.: 200.6–201.5 ◦C; 1H-NMR
(600 MHz, CDCl3)
δ 8.50 (d, J = 0.8 Hz, 1H), 8.19–8.05 (m, 4H), 7.76 (m, 1H), 7.70 (m, 1H),
7.35 (ddd, J = 8.8, 6.6, 1.0 Hz, 1H), 7.14 (ddd, J = 8.5, 6.6, 0.7 Hz, 1H), 3.11 (s, 3H); 13C-NMR
(151 MHz, CDCl3) δ 150.43, 144.23, 139.27, 129.18, 127.87, 123.36, 123.18, 120.99, 120.57, 120.54, 118.11,
44.62; MS (HR-ESI) for C14H12N2O2S [M + H]+, calcd: m/z 273.0692, found: m/z 273.0659.
◦
◦
2,3-Diphenyl-2H-indazole (16). White solid (77% yield); mp: 107.4–107.9 C (lit [27]: 102–103 C);
1H-NMR (600 MHz, CDCl3)
7.82–7.79 (m, 1H), 7.73–7.70 (m, 1H), 7.45–7.42 (m, 2H), 7.41–7.34 (m, 9H)
7.14 (ddd, J = 8.4, 6.6, 0.8 Hz, 1H); 13C-NMR (151 MHz, CDCl3)
148.99, 140.24, 135.41, 129.91, 129.69,
128.97, 128.76, 128.30, 128.25, 126.98, 126.02, 122.50, 121.74, 120.52, 117.76.
δ
,
δ
◦
2-(4-Chlorophenyl)-3-phenyl-2H-indazole (17). White solid (45% yield); m.p.: 124.4–125.0 C (lit [43]:
◦
1
126 C); H-NMR (600 MHz, CDCl3)
δ
7.78 (dt, J = 8.8, 0.9 Hz, 1H), 7.68–7.69 (dt, J = 8.5, 0.9 Hz,
149.12,
1H), 7.45–7.32 (m, 10H), 7.14 (ddd, J = 8.4, 6.6, 0.8 Hz, 1H); 13C-NMR (151 MHz, CDCl3)
δ
138.75, 135.47, 134.09, 129.67, 129.63, 129.18, 128.94, 128.55, 127.26, 127.10, 122.73, 121.86, 120.49, 117.72;
MS (HR-ESI) for C19H13ClN2 [M + H]+, calcd: m/z 305.0840, found: m/z 305.0736.
◦
Methyl 4-(3-phenyl-2H-indazol-2-yl) benzoate (18). Pale yellow solid (40% yield); m.p.: 152.4–154.9 C;
1H-NMR (600 MHz, CDCl3)
δ
8.07–8.04 (m, 2H), 7.80 (dt, J = 8.8, 0.8 Hz, 1H), 7.69 (dt, J = 8.6, 1.0 Hz, 1H)
,
7.55–7.52 (m, 2H), 7.44–7.34 (m, 6H), 7.15 (ddd, J = 8.5, 6.6, 0.8 Hz, 1H), 3.93 (s, 3H); 13C-NMR (151 MHz,
CDCl3) δ 166.21, 149.34, 143.76, 135.69, 130.42, 129.70, 129.62, 128.98, 128.66, 127.46, 125.69, 122.89, 122.08,
120.53, 117.81, 52.33; MS (HR-ESI) for C21H16N2O2 [M + H]+, calcd: m/z 329.1285, found: m/z 329.1103.
2-(4-(Methylthio) phenyl)-3-phenyl-2H-indazole (19). Pale yellow solid (71% yield) m.p.: 87.7–89.0 ◦C;
1H-NMR (600 MHz, CDCl3)
δ
7.79 (dt, J = 8.9, 1.0 Hz, 1H), 7.70 (dt, J = 8.6, 1.0 Hz, 1H), 7.43–7.34 (m, 8H)
,
7.24–7.21 (m, 2H), 7.13 (ddd, J = 8.4, 6.6, 0.8 Hz, 1H), 2.49 (s, 3H); 13C-NMR (151 MHz, CDCl3)
δ
148.97,
139.16, 137.23, 135.26, 129.88, 129.68, 128.83, 128.35, 127.00, 126.40, 126.19, 122.50, 121.78, 120.46, 117.68,
15.58; MS (HR-ESI) for C20H16N2S [M + H]+, calcd: m/z 317.1107, found: m/z 317.1108.
◦
1
4-(3-Phenyl-2H-indazol-2-yl) benzoic acid (20). White solid (70% yield); m.p.: 129.2–130.1 C; H-NMR
(600 MHz, DMSO-d6) δ 8.04–7.99 (m, 2H), 7.77 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.59–7.55 (m, 2H),
7.51–7.37 (m, 6H), 7.18 (dd, J = 8.4, 6.6 Hz, 1H); 13C-NMR (151 MHz, DMSO-d6) δ 166.41, 148.44, 143.00,
135.18, 130.45, 130.07, 129.44, 128.95, 128.87, 128.63, 127.18, 125.91, 122.73, 121.30, 120.32, 117.41; MS (HR-ESI)
for C20H14N2O2 [M + H]+, calcd: m/z 315.1128, found: m/z 315.1142.
2-(4-(Methylsulfonyl) phenyl)-3-phenyl-2H-indazole (21). Pale yellow solid (77% yield), m.p.: 101.8–102.7 ◦C
1H-NMR (600 MHz, CDCl3)
7.98–7.94 (m, 2H), 7.78 (dt, J = 8.9, 0.8 Hz, 1H), 7.70–7.66 (m, 3H), 7.48–7.42
(m, 3H), 7.39 (ddd, J = 8.8, 6.5, 1.0 Hz, 1H), 7.38–7.35 (m, 2H), 7.16 (ddd, J = 8.4, 6.5, 0.7 Hz, 1H), 3.08 (s, 3H);
;
δ
13C-NMR (151 MHz, CDCl3)
δ 149.58, 144.52, 139.70, 135.94, 129.70, 129.28, 129.23, 129.00, 128.41, 127.85,
126.45, 123.23, 122.29, 120.58, 117.81, 44.52; MS (HR-ESI) for C20H16N2O2S [M + H]+, calcd: m/z 349.1005,
found: m/z 349.1005.
◦
3-(4-Chlorophenyl)-2-phenyl-2H-indazole (22). White solid (67% yield); m.p.: 141.1–142.8 C (lit [27]:
1
134–135 ◦C); the spectroscopic data matched previously reported data [27
,
44]: H-NMR (600 MHz,
CDCl3)
7.16 (ddd, J = 8.4, 6.5, 0.7 Hz, 1H); 13C-NMR (151 MHz, CDCl3)
129.14, 129.12, 128.48, 128.38, 127.08, 126.01, 122.86, 121.71, 120.11, 117.91.
δ
7.80 (dt, J = 8.8, 0.8 Hz, 1H), 7.67 (dt, J = 8.6, 1.0 Hz, 1H), 7.44–7.35 (m, 8H), 7.30–7.27 (m, 2H),
δ
149.00, 139.98, 134.45, 134.08, 130.84,

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Methyl 4-(2-phenyl-2H-indazol-3-yl) benzoate (23). Pale yellow solid (76% yield): m.p.: 164.5–166.3 ◦C
the spectroscopic data matched previously reported data [45]: 1H-NMR (600 MHz, CDCl3)
8.08–8.04
(m, 2H), 7.84–7.80 (m, 1H), 7.72 (dt, J = 8.5, 0.9 Hz, 1H), 7.45–7.37 (m, 8H), 7.19 (ddd, J = 8.5, 6.5, 0.6 Hz,
1H), 3.93 (s, 3H); 13C-NMR (151 MHz, CDCl3)
166.55, 149.08, 139.99, 134.37, 134.13, 129.97, 129.66, 129.49,
;
δ
δ
129.18, 128.59, 127.14, 126.04, 123.18, 121.90, 120.09, 118.02, 52.29.
3-(4-(Methylthio) phenyl)-2-phenyl-2H-indazole (24). White solid, (36% yield); m.p.: 119.3–121.4 ◦C;
the spectroscopic data matched previously reported data [45]: 1H-NMR (600 MHz, CDCl3)
δ
7.79
(dt, J = 8.8, 0.9 Hz, 1H), 7.70 (dt, J = 8.5, 1.0 Hz, 1H), 7.46–7.43 (m, 2H), 7.42–7.34 (m, 4H), 7.29–7.23 (m, 4H),
7.14 (ddd, J = 8.5, 6.6, 0.8 Hz, 1H), 2.50 (s, 3H); 13C-NMR (151 MHz, CDCl3)
149.01, 140.22, 139.32, 134.94,
129.90, 129.06, 128.29, 126.99, 126.27, 126.16, 126.02, 122.50, 121.66, 120.43, 117.80, 15.26.
δ
4-(2-Phenyl-2H-indazol-3-yl) benzoic acid (25). White solid (87% yield); mp: 296.2–298.2 ◦C
;
1H-NMR
(600 MHz, DMSO-d6) 7.94–7.90 (m, 2H), 7.75 (d, J = 8.7 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H),
δ
7.49–7.42 (m, 5H), 7.38 (ddd, J = 8.7, 6.6, 0.9 Hz, 1H), 7.30–7.26 (m, 2H), 7.16 (ddd, J = 8.4, 6.6, 0.6 Hz, 1H)
;
13C-NMR (151 MHz, DMSO-d6) δ 169.13, 148.11, 140.15, 139.79, 135.10, 129.32, 129.17, 128.98, 128.37, 128.26,
126.77, 125.88, 122.34, 121.02, 120.38, 117.29; MS (HR-ESI) for C20H14N2O2 [M + H]+, calcd: m/z 315.1128,
found: m/z 315.1139.
3-(4-(Methylsulfonyl) phenyl)-2-phenyl-2H-indazole (26).
Pale yellow solid (60% yield), mp:
◦
1
206.9–208.8 C; H-NMR (600 MHz, CDCl3)
δ
7.98–7.94 (m, 2H), 7.84 (dt, J = 8.7, 0.9 Hz, 1H),
7.71 (dt, J = 8.5, 1.0 Hz, 1H), 7.57–7.54 (m, 2H), 7.45–7.39 (m, 6H), 7.22 (ddd, J = 8.5, 6.6, 0.9 Hz, 1H),
3.11 (s, 3H); 13C-NMR (151 MHz, CDCl3)
δ 149.10, 139.83, 139.71, 135.46, 132.95, 130.24, 129.40, 128.90,
127.86, 127.27, 126.06, 123.70, 122.03, 119.64, 118.22, 44.42; MS (HR-ESI) for C20H16N2O2S [M + H]+,
calcd: m/z 349.1005, found: m/z 349.1005.
3.3. Biological Assays
3.3.1. Antiprotozoal Activity Assays
Trichomonas vaginalis strain GT3, Giardia intestinalis isolate IMSS:0981:1, and Entamoeba histolytica
strain HM1-IMSS were used. 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, parasite cultures received an equivalent amount of DMSO only,
while albendazole and metronidazole were included as positive controls. At the end of the treatment
period, the cells were washed and subcultured for another 48 h in a fresh medium to which
no drug was added. The trophozoites were then counted with a haemocytometer 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.
3.3.2. Antibacterial and Anticandidal Assays
Escherichia coli strain EDL933 (EHEC), Escherichia coli strain 042 (EAEC), Salmonella enterica serovar
Typhi, Candida albicans, and Candida glabrata were used. The susceptibility assays were carried out
using the disk diffusion test, in accordance with the outlined by CLSI (M02-A12) [18]. The inoculum
was adjusted to 1.5
Sparks, MD, USA) were used as growth inhibition controls. Ciprofloxacin (10
(25 g) were used as antibiotics for the positive control; whereas ketoconazole (50
×
108 CFU/mL (0.5 McFarland). Sensi-Discs (Becton Dickinson and Company,
g) and ampicillin
g) was used as
µ
µ
µ
antimycotic. Each compound was tested at 5 mg per disc. Petri dishes with the compounds and
the positive control were incubated at 37 ◦C for 24 h. The degree of effectiveness was measured by

Molecules 2017, 22, 1864
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determining the zone of inhibition in millimeters resulting from the compounds. Experiments were
carried out in triplicate and the results are reported as average values.
3.3.3. Cytotoxicity Assays in Human Cells
HeLa (human cervical carcinoma) and HaCaT (immortalized human keratinocytes) cells were
grown in DMEM (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% FBS
(BioWest, Riverside, MO, USA), and maintained in standard culture conditions (37 ◦C, 95% humidity,
and 5% CO2). Cells were allowed to grow to a density of 80% and then were harvested using
sterile PBS/EDTA (pH 7.4) before starting every experiment. Cells were seeded in 96-well plates
(7000 cells/well in 200
in DMSO (J.T. Baker, Phillipsburg, NJ, USA) at different concentrations and diluted in 50
to reach 250 L in the well. The exposure time was 48 h, and then viability was determined by MTT
µL of DMEM). After 24 h the cells were exposed to test compounds dissolved
µL of DMEM,
µ
assay. The absorbance of formazan was determined for each well and its viability was related to the
vehicle (100%). The IC50 was calculated from dose-response curve by non-linear fit using OriginPro
7.0 software (RockWare, Golden, CO, USA).
3.3.4. Cyclooxygenase Assays
The in vitro assays on human recombinant cyclooxygenase-2 were performed using the
COX inhibitor screening assay kit manufactured by Cayman Chemical, catalog number 560131
(Ann Arbor, MI, USA). Assays were carried out by duplicate following the instructions provided
by the manufacturer. Compounds were tested at 10 µM, whereas the reference compound celecoxib
(purchased from Sigma-Aldrich, catalog number PHR1683) was tested at 1 µM because of its high
inhibition at 10 µM.
3.4. Molecular Docking
The crystal structure of human COX-2 was retrieved from the Protein Data Bank (www.rcsb.org;
www.wwpdb.org) [46 47], PDB ID: 5KIR [33]. The protein structure was prepared using Maestro 9.1
,
(Schrödinger, Cambridge, MA, USA) [48]; first, chain B was selected and solvent molecules were removed.
Then, the pdb structure was submitted for minimization using the YASARA web server (YASARA,
Vienna, Austria) [49]. The protein was exported to Autodock Tools 1.5.6 (TSRI, La Jolla, CA, USA) and the
grid coordinates and the pdbqt files were generated [50–52]. Ligands were constructed and minimized
using the universal force field implemented in Maestro 9.1 [53] and exported to Autodock Tools
1.5.6 to generate the pdbqt files. Docking calculations were carried out using Autodock Vina (TSRI,
La Jolla, CA, USA) employing a grid box of 40 × 40 × 40 centered on the co-crystalized ligand binding
site (rofecoxib) and an exhaustiveness value of 500 [31]. The docking protocol was validated by comparison
of docked rofecoxib and the co-crystalized rofecoxib. Molecular graphics and analyses were performed
with the UCSF Chimera package version 1.10.2 (RBVI, San Francisco, CA, USA) [54].
1
Supplementary Materials: The following are available online. Figures S1–S49: H-NMR and 13C-NMR spectra
of compounds
7–26, and HRMS spectra of compounds 14, 15, 17–21, 25, and 26. Table S1. Antibacterial and
antimycotic effect for selected compounds.
Acknowledgments: This work was supported by project UAM-PTC-503 (Apoyo a la Incorporación de Nuevos PTC)
from Secretaría de Educación Pública (SEP). J.P-V and I.G-S also acknowledge the support provided by project
1238 from Consejo Nacional de Ciencia y Tecnologia (Cátedras CONACYT). The authors would like to express
their sincere thanks to Amparo Tapia for the support with biological assays, and to Ernesto Sánchez Mendoza and
Mónica A. Rincón for the analytical support. The authors gratefully acknowledge Schrödinger, LLC, for providing
the academic version of Maestro; YASARA Biosciences GmbH for providing the YASARA Energy Minimization
Server; the Scripps Research Institute for providing Autodock Tools and Autodock Vina; and the Resource for
Biocomputing, Visualization, and Informatics (RBVI) for providing Chimera (Chimera is developed by the RBVI
at the UCSF supported by NIGMS P41-GM103311).
Author Contributions: Jaime Pérez-Villanueva, Olivia Soria-Arteche, and Juan Francisco Palacios-Espinosa
conceived and designed the experiments and wrote the paper; Jaime Pérez-Villanueva, Karen Rodríguez-Villar,
Miguel Cortés-Gines, Zeltzin Custodio-Galván, and Dante B. Estrada-Castro performed the chemical synthesis;

Molecules 2017, 22, 1864
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Lilián Yépez-Mulia performed the antiprotozoal activity assays; Ignacio González-Sánchez and Marco A. Cerbón,
performed the cytotoxicity assays; Ignacio González-Sánchez and Juan Francisco Palacios-Espinosa performed the
COX-2 inhibition assays; Teresita del Rosario Sainz-Espuñes and Ana Karina Rodríguez-Vicente performed the
antibacterial and anticandidal assays; and Jaime Pérez-Villanueva performed the docking studies.
Conflicts of Interest: The authors declare that they have no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2017 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/).