Efficient MW-Assisted synthesis, spectroscopic characterization, x-ray and antioxidant properties of indazole derivatives

Article abstract of DOI:10.3390/molecules21070903

A small series of tetrahydroindazoles was prepared, starting from 2-acetylcyclohexanone and different hydrazines using reflux and a focused microwave reactor. Microwave irradiation (MW) favored the formation of the desired products with improved yields and shortened reaction times. This is a simple and green method for the synthesis of substituted tetrahydroindazole derivatives. The in vitro antioxidant activity was evaluated using the DPPH and ABTS methods. In these assays, 2-(4-fluorophenyl)-3-methyl-4,5,6,7-tetrahydro-2H-indazole (3f) showed moderate DPPH decoloring activity, while 3-methyl-4,5,6,7-tetrahydro-1H-indazole (3a), 3-methyl-2-phenyl-4,5,6,7-tetrahydro-2H-indazole (3b) and 2-(4-fluorophenyl)-3-methyl-4,5,6,7-tetrahydro-2H-indazole (3f) were the most active in the ABTS assay. All compounds were well characterized by IR, ¹H-, ¹³C-NMR and GC-MS spectroscopy and physical data, while the structure of 4-(3-methyl-4,5,6,7-tetrahydro-2H-indazol-2-yl)benzoic acid (3e) was also determined by single crystal X-ray analysis.

Full text of DOI:10.3390/molecules21070903

molecules
Article
Efficient MW-Assisted Synthesis, Spectroscopic
Characterization, X-ray and Antioxidant Properties of
Indazole Derivatives
Efrain Polo 1, Jorge Trilleras 2, Juan Ramos 2, Antonio Galdámez 3, Jairo Quiroga 4 and
Margarita Gutierrez 1,
*
1
Organic Synthesis Laboratory and Biological Activity (LSO-Act-Bio), Institute of Chemistry of
Natural Resources, Universidad de Talca, Casilla 747, Talca 3460000, Chile; epolo@utalca.cl
Grupo/Semillero de Investigación en Compuestos Heterocíclicos, Programa de Química, Facultad de
Ciencias Básicas, Universidad del Atlántico, Km 7 Antigua vía Puerto Colombia,
Barranquilla 081007, Atlántico, Colombia; jorgetrilleras@mail.uniatlantico.edu.co (J.T.);
jotaramos87@gmail.com (J.R.)
Solid State Chemistry Laboratory, Science Faculty, University of Chile, Santiago, 7800003, Chile;
agaldamez@uchile.cl
Heterocyclic Compounds Research Group, Department of Chemistry, Universidad del Valle, Cali A.A. 25360,
Colombia; jairo.quiroga@correounivalle.edu.co
2
3
4
*
Correspondence: mgutierrez@utalca.cl; Tel.: +56-071-220-0448
Academic Editor: Derek J. McPhee
Received: 9 May 2016; Accepted: 5 July 2016; Published: 9 July 2016
Abstract: A small series of tetrahydroindazoles was prepared, starting from 2-acetylcyclohexanone
and different hydrazines using reflux and a focused microwave reactor. Microwave irradiation (MW)
favored the formation of the desired products with improved yields and shortened reaction times.
This is a simple and green method for the synthesis of substituted tetrahydroindazole derivatives.
The in vitro antioxidant activity was evaluated using the DPPH and ABTS methods. In these
assays, 2-(4-fluorophenyl)-3-methyl-4,5,6,7-tetrahydro-2H-indazole (3f) showed moderate DPPH
decoloring activity, while 3-methyl-4,5,6,7-tetrahydro-1H-indazole (3a), 3-methyl-2-phenyl-4,5,6,7-
tetrahydro-2H-indazole (3b) and 2-(4-fluorophenyl)-3-methyl-4,5,6,7-tetrahydro-2H-indazole (3f
)
were the most active in the ABTS assay. All compounds were well characterized by IR, 1H-, 13C-NMR
and GC-MS spectroscopy and physical data, while the structure of 4-(3-methyl-4,5,6,7-tetrahydro-
2H-indazol-2-yl)benzoic acid (3e) was also determined by single crystal X-ray analysis.
Keywords: tetrahydroindazole; antioxidant activity; microwave irradiation; green chemistry
1. Introduction
In the last decade there has been rapid progress in the synthetic organic chemistry field
associated with the search for new organic compound derivatives with desirable properties.
Such compounds are widely used in the pharmaceutical industry. The search for new synthetic
strategies that enable higher performance, less byproducts and shorter reaction times also seeks new
non-conventional energy sources for obtaining products with industrial, pharmaceutical or other
applications. Microwave-assisted synthesis is a branch of green chemistry that has gained much
attention in recent years. Moreover, it is considered pollution-free and eco-friendly and typically
offers high yields, together with simplified processing and handling [1] as compared to conventional
synthesis methods. Its use in organic synthesis has dramatically increased in the last few years,
receiving widespread acceptance and becoming an indispensable tool for obtain new structures and it
is recognized as a valuable tool for easing some of the bottlenecks in the drug discovery process [2–5].
Molecules 2016, 21, 903; doi:10.3390/molecules21070903
www.mdpi.com/journal/molecules

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In this method, reactions occur more rapidly, safely, and with higher chemical yields, often far better
than conventional methods, which require longer reaction times and larger quantities of solvents and
reagents, cause environmental pollution, and contribute to health hazards [6]. Many heterocyclic
compounds have been synthetized using MW irradiation [ ] and Katritzky and Singh reported the
7
applications of MW technology in well-known cyclisation reactions for heterocyclic ring formation
and on other important reactions, such as nucleophilic substitution, hetero-Diels-Alder reactions,
1,3-dipolar cycloaddition [8]. Six-membered heterocycles and their fused derivatives has been reported
among the compounds synthetized via MW irradiation, using cyclocondensation, cycloaddition,
multicomponents and other modular reactions, generating good yield and short reaction time in an
easy and rapid way [9]. Tetrahydroindazole synthesis using MW irradiation has been previously
reported [10–12].
Tetrahydroindazole are chemical compounds of synthetic origin with a five-membered heterocycle,
two nitrogen atoms, and three adjacent carbons. Tetrahydroindazole are potent medicinal scaffolds
and exhibt a full spectrum of biological activities [13–17]. Due to their attractive pharmacological
properties tetrahydroindazole derivatives have attracted the attention of chemists who have
researched ways to obtain the desired properties through different synthetic strategies [18]. The
most common reactions include: (i) reaction of 2-substituted cyclohexanone with a hydrazine; (ii)
ring closure of o-chlorocyclohexanonephenylhydrazone; (iii) reaction of 2-chlorocyclohexenal with
hydrazine hydrate; (iv) addition of diazomethane to cyclohexene; (v) hydrogenation of indazoles
with platinum in acetic acid; and (vi) the hydrolysis of 1-carbamyl-4,5,6,7-tetrahydroindazole [19].
Accordingly, tetrahydroindazole synthesis has long been the subject of interest. At the beginning
of the 20th century, tetrahydroindazole was first prepared by Ainsworth, the reaction of
2-hydroxymethylenecyclohexanone with hydrazine hydrate in methyl alcohol [20]. Since then,
Ainsworth´s tetrahydroindazole synthesis has been adopted as the standard method because of
its convenience and versatility.
Based on the careful analysis of the literature, the present investigation was aimed to focus on the
synthesis of pyrazoles using conventional and non-conventional methods. The tetrahydroindazole
series was synthesized by conventional methods, using different solvents, and MW irradiation methods
with the purpose of comparing purity, yield and reaction time. The isolated and purified compounds
were characterized on the basis of IR, 1H-, 13C-NMR and mass spectral data. The antioxidant activity
was evaluated using the DPPH and ABTS methods. In this context, we present an efficient, clean and
straightforward procedure to prepare a series of tetrahydroindazole derivatives by MW irradiation
under solvent-free conditions, with short reaction times, and using ethanol as a green solvent in
order to obtain the tetrahydroindazole derivatives from crude reaction mixtures without the use of
complicated work-up.
2. Results and Discussions
Six tetrahydroindazole derivatives with different patterns of substitution were synthesized using
a modified Paal-Knorr reaction [21] between 1,3-dicarbonyl compounds and hydrazines (Scheme 1).
This resulted in good yields with almost no secondary products. These coupling reactions were
performed under reflux and MW irradiation, that helped obtain the best results. Reaction conditions,
substituents, yields and reaction times are listed in Table 1.
O
O
H
N
N
R
NH2
2 a-f
N R
3a-f
MW, 2 min
1
R = H-H2O, C6H5, 4-BrC6H4, 4-CNC6H4, 4-CO2HC6H4, 4-FC6H4.
Scheme 1. Synthesis of compounds 3a–f.

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Table 1. Comparative study of conventional vs. microwave method.
Reflux
Acid Acetic
Time Reaction (h)/Yield (%)
Microwave (MW)
300 W/150 ˝
Time Reaction (min)/Yield (%)
Compound
M.p. ˝C
R
DMF
C
3a
3b
3c
3d
3e
3f
H
C6H5
4-BrC6H4
4-CNC6H4
4-CO2HC6H4
4-FC6H4
6/55
5/60
7/47
6/28
8/33
7/61
2/28
2.5/46
3/41
2/22
2/25
2
2
2
2
2
2
90
90
90
37
40
98
Oil
Oil
150–152
178–180
133–135
118–120
3.5/43
With the results obtained it can be established that the state of aggregation of the reagents is not
a limiting factor for the use of microwaves, since in this work, both liquid and solid reagents were
used. Reagent
used to obtain the compounds 3c
1
was used in a liquid state such as the hydrazine to obtain 3a and 3b; the hydrazines
were solids. This feature is consistent with the principles of green
–f
chemistry recommending a decrease in the use of solvents. The compounds 3a and 3b were obtained
as oils (Table 1).
Dimethylformamide and acetic acid were used in conventional method with the purpose of
comparing the influence of solvent on yield and reaction time. Acetic acid is one of the most important
chemical intermediates and frequently used aliphatic carboxylic acids. Acetic and some other acids
and their alkali-metal salts are components of buffers for pH measurements in mixed solvents. DMF
as a solvent with moderately high permittivity and aprotic nature is useful for acid-base studies and
for a wide range of organic reactions. It appears to be a suitable co-solvent since it is aprotic and fully
miscible with water [22].
This comparison in the use of a polar protic solvent (acetic acid) and another aprotic apolar
one (DMF) allowed us to establish that although there is a decrease in reaction times by using acetic
acid, the reaction yield was not favored, as the use of DMF led to an increase in reaction yields for
all compounds. This may be related to the characteristics of aprotic solvents which are capable of
accepting hydrogen bonds, do not have acidic hydrogens and are capable of dissolving salts, which
facilitates the formation of tetrahydroindazoles, in contrast, the use of acetic acid, a protic polar solvent,
is characterized by display hydrogen bonding, have an acidic hydrogen and dissolve salts. Based on
the dielectric constants of the solvents used, it is confirmed that solvents with high dielectric constant
values are favorable for nucleophilic attack on carbonyl groups.
Comparative analyses of percentage yields and total reaction time for all synthesised
tetrahydroindazole derivatives by both conventional and MW-assisted methods were performed to
determine if MW-assisted synthesis of tetrahydroindazole derivatives added any advantage (Table 1).
It was found that yields were improved while total reaction times were reduced drastically. This would
be highly advantageous for drug discovery laboratories where small amounts of different analogues
have to be synthesized in short periods of time, as well as combinatorial synthesis of new libraries of
compounds [23].
Optimal working conditions were determined using microwave for synthesis, varying the
temperature and reaction time as shown in Table 2. For example, for compound 3c, for which it
˝
was found that 150 C and 2 min were the best conditions, keeping the power constant at 300 W.
Compounds 3c were purified by precipitation and were obtained as solids, while compounds
–f
3a and 3b were obtained as oils. The structures and formulas of all the synthesized compounds were
supported by physical and spectral data and found to be in good agreement with the target compounds.
1
Tetrahydroindazoles 3a
–
f
were characterized by H-NMR, 13C-NMR and mass spectroscopy. The
1H-NMR spectra of the tetrahydroindazole derivatives were very similar, and characterized by
the presence of three groups of signals indicating the presence of aromatic protons, protons near
heteroatoms and aliphatic protons, which resonated in the different regions.

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Table 2. Temperature optimization for the synthesis of 3c using MW at 300 W.
Entry
Reaction Temperature ˝C
Reaction Time (min)
Isolated Yield (%)
1
2
3
4
5
80
5
4
4
2
5
-
100
120
150
180
32
53
90
90
The use of MW irradiation for carrying out chemical transformations produces eco-friendly
processes. The basis of this technique in organic synthesis is the empirical observation that
some reactions proceed much faster and with higher yields compared to conventional heating.
MW irradiation facilitates the polarization of the molecules under irradiation. This causes an increase
in the reaction rate, which is reflected in significant reduction in reaction times ranging from hours
to minutes. The use of MW radiation for the synthesis of tetrahydroindazoles has previously been
reported [24] showing increases in yields and significant decrease in reaction times. With the decrease
in the use of solvents, it was possible to obtain more consistent products in line with the principles of
green chemistry.
Compound 3e was isolated as a single-crystal, and Figure 1 shows the molecular structure with
the atom numbering scheme and a summary of cryst˝allographic data for the compound is given in
Table 3. The torsion angle of C13-N2-C4-C12 is 43.7 (5) . The Cremer and Pople puckering parameters
of 6-membered ring (C10/C14/C18-C15) are: Q2 is 0.359 (6) Å,
θ ϕ
is 49.0 (8)˝ and is 148.4 (11)˝.
˝
The torsion angle (C7-C1-C1-O9) at carboxyl substituent group is 9.2 (6) . This group is almost coplanar
with aromatic ring. All relevant structural parameters (bond distances and angles) are as expected
and in acceptable agreement with other organic molecules [25]. Crystallographic data reported in this
paper for compound 3e has been deposited at the Cambridge Crystallographic Data Center (CCDC),
No. 1470060. Copies of the data can be obtained, free of charge, on application to CCDC 12 Union
Road, Cambridge CB2 1EZ, UK (Fax: +44-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
Figure 1. A view of molecular structure showing the displacement ellipsoids are drawn at the 50%
probability level. H-atoms are shown as small spheres of arbitrary radii.

Molecules 2016, 21, 903
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Table 3. Crystal data and details of the structure determination.
Compound
C15H17N2O2, H2O
Crystal shape, color
Crystal size (mm)
Crystal system, Space group
Polyhedron, yellow
0.32 ˆ 0.12 ˆ 0.10
Monoclinic, P21/n (N˝14)
10.704 (2)
a (Å)
10.106 (2)
b (Å)
13.735 (3)
95.35 (3)
1479.3 (5)
c (Å)
β (˝)
V (Å3)
Z
Wavelength, Mo Kα (Å)
T (K)
4
0.71073
297 (2)
F(000)
580
θ-range (˝)
hkl-range
2.50 < θ < 25.0
´12:12, ´12:11, ´16:16
0.086
µ (mm´1
)
Reflections collected/Rint/Rσ
Reflections unique/ parameters
R1, wR2 [F2 > 2σ (F2)]
10,360/0.099/0.0909
2588/ 412
0.0586, 0.0927
1.106
0.15/´0.11
Goodness-of-Fit on F2 (GooF = S)
Residual electron density ∆ρ
/ )
∆ρmin (e Å´3
max
The supramolecular architecture is assembled from the molecules with solvent water molecules via
hydrogen-bonding interactions. The water molecules serve to link molecules forming two-dimensional
array (Figure 2). Each water molecule acts as an acceptor,˝in O-H¨ ¨ ¨ OW hydrogen bond, and as a
donor. The O1W-H2W¨ ¨ ¨ O9 distance is 1.91 Å (angle of 166 ) and O13-H13¨ ¨ ¨ O1W distance is 1.78 Å
˝
(angle of 166 ). The central core constitutes a graph-set descriptor R44p12
q
motif (Figure 2-top) [26]. The
network is reinforced by C-H¨ ¨ ¨ π (pyrazole) interactions [27]. The C7-H7¨ ¨ ¨ Cg1 distance is 2.90 Å and
Cg1[symmetry code: 1/2
´
x, 1/2 + y, 3/2
´
z] represents the centroid of the tetrahydroindazole ring.
These intermolecular interaction form chains run zig-zagging parallel to the [010] plane (Figure 2-top).
Figure 2. A view of partial packing of 3e showing: (top) the central core linking the components
mediated by hydrogen bonds and (bottom) C-H...
π (tetrahydroindazole) interactions. H-atoms are
shown as small spheres of arbitrary radii. Dashed lines represent intermolecular interactions.

Molecules 2016, 21, 903
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In using natural sources as the basis to develop new drugs, industry usually faces the challenge of
low concentrations of selected substances that generally make their commercial exploitation unfeasible.
However, the synthesis of these substances and their derivatives frequently allow the pharmacophore
to be established, and modulation of biological profiles represent an excellent opportunity to explore
the biological actions of these synthetic and semi-synthetic compounds [28].
Antioxidant activity is related to compounds capable of protecting a biological system against
the harmful effects of oxidative stress. Antioxidants have received increased attention in recent years
from nutritionists and medical researchers for their potential activities in the prevention of several
degenerative diseases such as cancer and cardiovascular disorder, as well as aging [29]. On the other
hand, there are few reports on the antioxidant activities of synthetic compounds [30]. Consequently
the synthesis of new active derivatives with potential applications in this area and prepared by
simple chemical procedures are of increasing interest. In order to have a standard antioxidant activity
measurement system to compare with natural antioxidants and to be incorporated into food, synthetic
antioxidants have been developed. Synthetic antioxidants compounds are widely used in food products
to inhibit progress of lipid oxidation [31]. In the United States, the following synthetic antioxidants
have been evaluated by the FDA and are the most widely used: tert-butylhydroquinone (TBHQ),
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate [32]. These
antioxidants are cheaper and easier to process than natural antioxidants. Used in approved quantities,
synthetic antioxidants are safe and effective [33]. Synthetic tetrahydroindazoles and derivatives have
been reported as good antioxidants [34–37], therefore the research and evaluation of tetrahydroindazole
derivatives are a promising area of study for obtained new compounds to use as antioxidant agents.
All compounds obtained were evaluated as antioxidant agents. As shown in Table 4, the DPPH
scavenging activity of the compounds varied greatly. The data showed clearly that compound 3f
has good activity in both assays, with best activity in the capture assay ABTS radical, showing better
IC50 value than ascorbic acid used as a reference (Table 5). Obtained results are concordant with
results previously reported where described compounds having electron with- drawing groups like
chlorine and fluorine, and electron donating groups like methoxy on the phenyl rings attached to
tetrahydroindazole exhibit good antioxidant activity [38].
Table 4. Decoloration of DPPH solution by the synthesized compounds in comparison with ascorbic
acid. Values are the average of triplicate experiments.
Compound/(µg/mL)
100
50
10
IC50
3a
3b
3c
3d
3e
3f
13.51 ˘ 1.5
29.19 ˘ 4.3
31.35 ˘ 2.6
25.89 ˘ 5.3
47.72 ˘ 4.4
85.41 ˘ 12.6
-
8.10 ˘ 0.6
17.84 ˘ 3.4
16.49 ˘ 2.2
13.45 ˘ 2.8
21.32 ˘ 2.1
55.14 ˘ 9.7
-
3.78 ˘ 0.3
11.09 ˘ 1.7
8.92 ˘ 1.0
3.05 ˘ 0.8
7.87 ˘ 1.2
19.46 ˘ 6.7
-
>100
>100
>100
>100
>100
42.35 ˘ 4.3
Ascorbic acid
1 ˘ 0.3
Table 5. ABTS radical scavenging activity of the synthesized compounds measured at 745 nm as
compared to standard ascorbic acid. Values are the average of triplicate experiments.
Compound/(µg/mL)
100
75
50
25
IC50
3a
3b
3c
3d
3e
3f
56.47 ˘ 12.2
52.14 ˘ 10.5
46.88 ˘ 9.7
21.34 ˘ 10.3
49.24 ˘ 15.3
93.12 ˘ 17.5
-
35 ˘ 8.7
37.23 ˘ 11.6
27.38 ˘ 9.9
17.65 ˘ 8.7
31.09 ˘ 9.0
88.37 ˘ 10.3
-
28.62 ˘ 5.3
12.04 ˘ 8.1
19.67 ˘ 7.4
12.10 ˘ 5.5
3.87 ˘ 1.6
83.79 ˘ 8.5
-
0
87.94 ˘ 15.8
92.15 ˘ 18.3
>100
5.08 ˘ 0.8
9.19 ˘ 1.7
5.55 ˘ 2.4
0
>100
>100
53.65 ˘ 3.5
17.54 ˘ 4.8
Ascorbic Acid
-
35 ˘ 2.7

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3. Materials and Methods
3.1. General Information
The experiments were performed in a Discover microwave apparatus (CEM Corporation,
1
Matthews, NC, USA). All the products were characterized by spectral data (IR, MS, H-NMR,
13C-NMR). 1H and 13C-NMR spectra (400.1 MHz for proton and 100.6 MHz for carbon) were recorded
on an AM-400 spectrometer (Bruker, Rheinstetten, Germany), using CDCl3, DMSO-d6 and CD3OD as
solvents. Tetramethylsilane (TMS) was used as an internal standard. Chemical shifts (δ) and J values
are reported in ppm and Hz, respectively, relative to the solvent peak CDCl3 at 7.24 ppm for protons
and 77 ppm for carbon atoms; DMSO-d6 2.5 ppm for protons and 39.7 ppm for carbon atoms, and
CD3OD with 3.35 and 4.78 ppm for protons and 49.3 ppm for carbon. Signals are designated as
follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet; br.s, broad singlet.
IR spectra (KBr pellets, 500–4000 cm´1) were recorded on a NEXUS 670 FT-IR spectrophotometer
(Thermo Nicolet, Madison, WI, USA). GC-MS analyses were performed on a model Trace 1300 GC-MS
instrument (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Rtx-5MS on-column
auto injector and a fused silica capillary column (DB-5, 30 m
ˆ
0.25 mm ID, 0.25 µm film thickness).
Operating conditions were as follows: helium as the carrier gas with a flow rate of 1.5 mL/min; column
˝
˝
˝
temperature 40 C as initial temperature, then increasing at 10 C/min to 280 C; injector temperature,
˝
250 C; volume injected, 1 µL; split ratio, 33.3. MS were recorded in electron ionization (EI) mode, with
energy of 70 eV. The ion source temperature was 200 ˝C; 4.00 min solvent cut time. The compounds
were identified by comparison with the data held in the NIST 11 Library; data are reported as m/z
ratio with the relative abundance of the respective peak. Melting points (uncorrected) were measured
on a Fisher-Johns melting point apparatus. Reaction progress was monitored by means of thin-layer
chromatography using silica gel 60 (Merck, Darmstadt, Germany). All reagents were purchased from
either Merck or Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Final
purification of all products for analysis was carried out by recrystallization.
3.2. Synthesis
The Knorr pyrazole synthesis is the reaction of hydrazine or substituted hydrazine with
1,3-dicarbonyl compounds to provide the pyrazole or pyrazolone ring system. Dimethylformamide
(DMF) and acetic acid (AcOH) were used in the conventional method for the purpose of comparing
the influence of the solvent on yield and reaction time.
Method A (Reflux)
(a)
A mixture of 2-acetylcyclohexanone (1, 1 mmol), and hydrazines 2 (1.0 mmol) in DMF (10 mL)
was heated under reflux for 6–8 h. The precipitated solid product was filtered, washed with
ethanol (EtOH), dried and finally recrystallized from DMF.
(b)
2-Acetylcyclohexanone (1, 1 mmol) and hydrazines 2 (1.0 mmol) in AcOH (10 mL) were stirred
at 80 ˝C for the given times. After completion of the reaction, the mixture was cooled to room
temperature and the precipitate was filtered and purified by recrystallization from EtOH.
Method B (MW)
A mixture of 2-acetylcyclohexanone (
1
, 1.0 mmol) and hidrazines 2 (1.0 mmol) was subjected
to microwave irradiation (Scheme 1). The solid products were isolated by crystallization of
the reaction mixture from EtOH and washed with a mixture of hexane/ethanol (7:3) to get the
corresponding compounds. Hydrazines used to obtain compounds 3c–f was applied as the
corresponding hydrochloride.
´1
δ
3-Methyl-4,5,6,7-tetrahydro-1H-indazole (3a). Yellow oil. Yield 90%, IR (KBr)
ν
/cm 3150 (–CH3), 2919,
1.67–1.62 (m, 4H),
2841 (CH2), 1601, 1440 (C=C arom.), 1319, 845, 822; 1H-NMR (400.1 MHz, CDCl3)

Molecules 2016, 21, 903
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2.11 (s, 3H), 2.30 (t, 2H, J 6.0), 2.53 (t, 2H, J 5.6), 9.68 (s, 1H); 13C-NMR (100.6 MHz, CDCl3)
δ
10.28,
19.94, 22.20, 23.0, 23.41, 112.62, 140.14, 1143.67. GC-MS m/z (rel. int. %): 136 (40), 108 (100), 94 (10).
Preparation of compound 3a and other derivatives with a yield of 77% using cyclic (alkyl) (amino)
carbene (CAAC)-gold(I) catalysts, has been described previously [39], and the use of tungstate sulfuric
acid, under solvent-free conditions has been reported to give a similar yield [40].
´1
3-Methyl-2-phenyl-4,5,6,7-tetrahydro-2H-indazole (3b). Yellow oil. Yield 90%, IR (KBr)
ν
/cm 3316, 3107
1
(–CH3), 2969, 2876 (–CH2–), 1750 (C=N), 1618, 1590, 1504, 1423 (C=C arom.). H-NMR (400.1 MHz,
CDCl3)
δ
1.74–1.63 (m, 4H), 2.01 (s, 3H), 2.28 (t, 2H, J 6.0), 2.54 (t, 2H, J 6.0), 7.25–7.18 (m, 5H); 13C-NMR
10.60, 20.30, 22.78, 23.18, 23.21, 115.27, 124.30, 126.73, 128.73, 134.44, 139.90, 149.51.
(100.6 MHz, CDCl3)
δ
GC-MS m/z (rel. int. %): 212 (100), 197 (34), 184 (78), 118 (32), 77 (82), 51 (20). Compound 3b and other
derivatives have been previously prepared by the condensation reaction of 1,3-diones and hydrazines
under layered zirconium sulfophenyl phosphonate catalysis, under solvent-free conditions [41]. The
˝
use of tungstate sulfuric acid at 80 C under solvent-free conditions has been reported to give a yield
of 80% [40].
´1
ν/cm
2-(4-Bromophenyl)-3-methyl-4,5,6,7-tetrahydro-2H-indazole (3c). Brown solid. Yield 90%, IR (KBr)
3037 (–CH3), 2907, 2846 (–CH2–), 1679 (C=N), 1605, 1496, 1440 (C=C arom.), 1066 (C-Br), 835 (–C6H4-Br).
1H-NMR (400.1 MHz, CDCl3)
1.69 (m, 4H), 2.12 (s, 3H), 2.37 (m, 2H), 2.61 (m, 2H), 7.44–7.23 (m, 3H);
13C-NMR (100.6 MHz, CDCl3)
10.84, 20.41, 22.89, 23.32, 23.79, 115.91, 120.22, 120.22, 123.88, 125.69,
δ
δ
131.98, 134.38, 139.21, 150.21.GC-MS m/z (rel. int. %): 292 (90), 289 (40), 275 (20), 155 (68), 120 (42),
91 (30), 77 (40), 76 (100).
´1
4-(3-Methyl-4,5,6,7-tetrahydro-2H-indazol-2-yl)benzonitrile (3d). White solid. Yield 37%, IR (KBr)
ν
/cm
1
3021 (–CH3), 2926, 2876 (–CH2–), 1654 (C=N), 1617, 1608, 1508, 1418 (C=C aromt). H-NMR (400.1
MHz, CD3OD)
δ
1.77–1.65 (m, 4H), 2.26 (s, 3H), 2.46 (t, 2H, J 5.8), 2.64 (t, 2H, J 5.8), 7.63 (d, 2H, J 8.00),
13.87, 26.19, 26.57, 26.84, 27.65, 113.62, 120.78,
7.82 (d, 2H, J 8.0); 13C-NMR (100.6 MHz, CD3OD)
δ
125.87, 127.89, 137.03, 139.55, 147.23, 155.13. GC-MS m/z (rel. int. %): (50), 222 (24), 209 (48), 143 (32),
102 (100), 77 (12).
4-(3-Methyl-4,5,6,7-tetrahydro-2H-indazol-2-yl)benzoic acid (3e). Orange crystal. Yield 40%, IR (KBr)
ν
/cm´1 3437 (–COOH), 3162 (–CH3), 2934, 2841, 2780 (–CH2–), 1693 (C=N), 1606, 1573, 1517, 1490
(C=C arom.), 1427 (–OH), 1277 (C-O). 1H-NMR (400.1 MHz, DMSO d6)
2.42 (t, 2H, J 5.8), 2.57 (t, 2H, J 5.8), 7.63 (d, 1H, J 8.9), 8.02 (d, 1H, J 8.9), 12.98 (s, 1H); 13C-NMR
(100.6 MHz, DMSO d6) 10.92, 19.87, 22.79, 22.85, 22.93, 116.03, 122.73, 128.15, 130.24, 134.48, 143.34,
δ 1.74–1.68 (m, 4H), 2.26 (s, 3H),
δ
149.70, 166.67. GC-MS m/z (rel. int. %): 256 (100), 239 (10), 228 (10), 139 (10), 123 (50).
´1
/cm
2-(4-Fluorophenyl)-3-methyl-4,5,6,7-tetrahydro-2H-indazole (3f). Brown solid. Yield 98%, IR (KBr)
ν
3030 (–CH3), 2893, 2846 (–CH2–), 1673 (C=N), 1605, 1496, 1440 (C=C arom.), 1060 (C-F), 850 (–C6H4-F).
1H-NMR (400.1 MHz, CDCl3)
1.69 (m, 4H), 2.01 (s, 3H), 2.37 (m, 2H), 2.79 (m, 2H), 7.01–7.16 (m, 4H);
13C-NMR (100.6 MHz, CDCl3)
9.40, 18.46, 20.72, 21.17, 21.42, 116.12, 125.75, 127.14, 129.98, 139.74,
δ
δ
142.59, 160.55. GC-MS m/z (rel. int. %): 230 (70), 214 (40), 217 (20), 155 (68), 120 (40), 91 (30), 77 (30),
76 (100).
3.3. X-ray Crystallography
The X-ray data of 3e was collected at 297 (2) K with a Bruker CCD diffractometer using
MoKα radiation [42]. The structure was solved by direct methods and refined with the full-matrix
least-squares method on F2 with the use of SHELX-97 program package [43]. H-atoms of
water molecules were found in difference Fourier maps and refined freely. Other H-atoms were
placed in calculated positions. The isotopic displacement parameters were calculated as follows:
Uiso(H) = 1.2 Ueq(O) and Uiso(H) = k Ueq(C), where k = 1.5 and 1.2.

Molecules 2016, 21, 903
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3.4. Biological Activity
All compounds were evaluated as antioxidant agents. Each sample was dissolved in
dimethylsulfoxide (DMSO) to make a solution of 1 mg/mL concentration, and then diluted to prepare
the series concentrations for antioxidant assays. In all assays the final concentration of DMSO was
inferior to 5%. Reference chemicals were used for comparison in all assays.
3.4.1. Measurement of DPPH Radical Scavenging Activity
1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a well-known radical which reacts with different
antioxidant compounds where by its deep violet color in methanol solution changes to yellow
(Scheme 2), the hydrogen atom or electron donation ability of the compounds was measured from the
bleaching of the purple colored methanol solution of DPPH.
Scheme 2. Visual change in the coloration in DPPH assay.
The scavenging activities of the compounds were estimated using DPPH as the free radical model
according to the method previously described and adapted [44
,45]. Briefly, an aliquot of 1 mL of the
tested compound (10, 50 and 100 g/mL) and control (2% DMSO final), respectively, were mixed
µ
with 2 mL of a methanolic solution of DPPH (0.5 mg/mL). The mixture was shaken vigorously and
left to stand at room temperature for 5 min, in the absence of light. The mixture was measured
spectrophotometrically at 515 nm. The free radical scavenging activity was calculated as percentage of
DPPH decoloration using the following equation:
%scavenging DPPH free radical “ 100 ˆ p1 ´ AE{ADq
where AE, is the absorbance of the solution after adding the extract and AD is the absorbance of the
blank DPPH solution. Ascorbic acid was used as reference compounds, with IC50 value of 1 µg/mL.
3.4.2. Measurement of ABTS Radical Scavenging Activity
The 2,21-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assay is a
rapid and efficient method, based on the ability of the hydrogen donating antioxidants to scavenge the
long-life radical cation ABTS+. In this method, the preformed radical monocation of ABTS is generated
by oxidation of ABTS with potassium persulfate (a blue chromogen) and is reduced in the presence of
such hydrogen donating antioxidants (Scheme 3).
Scheme 3. Visual change of coloration in ABTS assay.

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The ABTS assay was performed according to a published protocol [46]. The stock solution was
prepared by mixing equal volumes of 7 mM ABTS solution and 2.45 mM potassium persulfate solution
followed by incubation for 12 h at room temperature in the dark to yield a dark-colored solution
containing ABTS
the stock solution by mixing of stock solution to 50% methanol for an initial absorbance of about
0.700 (
0.02) at 745 nm, with temperature control set at 30 ˝C. Free radical scavenging activity was
assessed by mixing 300 L of different compounds (25–250 g/mL in respective solvents) with 3.0 mL
‚+ radicals. A working solution was freshly prepared before each assay by diluting
˘
µ
µ
of ABTS working standard. The decrease in absorbance was measured exactly 1 minute after mixing
the solution, the final absorbance was noted up to 6 min. Data for each assay was recorded in triplicate.
Ascorbic acid, with an IC50 value of 35 µg/mL, was used as positive control. The scavenging activity
was estimated based on the percentage of ABTS radicals scavenged by the following formula:
%scavenging “ rpA0 ´ Asq{A0s ˆ 100
where A0 is absorption of control, As is absorption of tested compound solution.
4. Conclusions
In summary, in the present investigation we have described a simple and green method for
the synthesis of a novel series of substituted tetrahydroindazole derivatives. The structures of
these compounds were confirmed by spectral analysis and their antioxidant activity was evaluated.
The results reveal that most of the synthesized tetrahydroindazoles can be considered as a scaffold
for the development of novel and effective antioxidant agents. The antioxidant activity shown by the
compounds obtained is totally dependent on the concentration and the type of substituent having
the tetrahydroindazole ring. The use of other substituents can lead to improvements in antioxidant
activity reported. Other tests to evaluate antioxidant activity can complement the results obtained and
also provide information related to the mechanism of action displayed by these compounds.
The synthesis of tetrahydroindazole compounds via the microwave-assisted method of reaction is
clean and offers shorter reaction times, with mild reaction conditions, is eco-friendly, produces excellent
yields as compared to conventional methods, and reduces the use of volatile organic compounds.
Finally, it is aligned with the principles of green chemistry. The uses of other conditions were evaluated
to achieve improvements in the yields of derivatives 3d and 3c. Additional work is in progress in order
to obtain different kinds of heterocycles using the same strategy.
Acknowledgments: This research was supported by a FONDECYT project number 1150712, PIEI QUIMBIO,
project, Utalca. E.P. thanks the CONICYT beca No. 63140046, for financial support. J.T., J.R. and J.Q. thanks to
Universidad del Atlántico and Universidad del Valle.
Author Contributions: E.P. Synthesis and analyzed the data J.T. and J.Q. contributed reagents/materials/analysis
tools, J.R. performed the experiments, A.G. carried out the X-ray single crystal diffraction, M.G. Biological activity
and analyzed the data. All authors wrote, read and appoved the final manuscript. Part of this work constitutes
the doctoral thesis of E.P.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2016 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/).