Synthesis of indole-2-carbohydrazides and 2-(indol-2-yl)-1,3,4-oxadiazoles as antioxidants and their acetylcholinesterase inhibition properties

Article abstract of DOI:10.1007/s00706-019-02462-y

Abstract: A range of novel 4,6-dimethoxy-1H-indole-2-carbohydrazides was prepared starting from methyl 4,6-dimethoxy-1H-indole-2-carboxylate which underwent cyclodehydration to generate the corresponding 2-(indol-2-yl)-1,3,4-oxadiazole scaffolds in the presence of N,N-diisopropylethylamine and p-toluenesulfonyl chloride in acetonitrile. All novel compounds were fully characterized by ¹H NMR, ¹³C NMR, FT-IR, and high-resolution mass spectroscopic data. Biological importance of the designated compounds was identified by employing three different antioxidant property determination assays, namely DPPH free radical scavenging, ABTS cationic radical decolarization, and cupric reducing antioxidant capacity (CUPRAC). The anticholinesterase properties were also evaluated by the acetylcholinesterase and butyrylcholinesterase enzyme inhibition assays. According to the results, the indole compounds possessing carbohydrazide functionality were found to be more promising antioxidant targets than the 2-(indol-2-yl)-1,3,4-oxadiazole systems. N?-Benzoyl-4,6-dimethoxy-1H-indole-2-carbohydrazide, a member of the dimethoxyindole-2-carbohydrazide group, demonstrated a better inhibition performance than the standards. Additionally, extremely important results were obtained in the anticholinesterase enzyme inhibition assays in the case of 2-(indol-2-yl)-1,3,4-oxadiazole derivatives. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-019-02462-y

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02462-y
ORIGINAL PAPER
Synthesis of indole‑2‑carbohydrazides
and 2‑(indol‑2‑yl)‑1,3,4‑oxadiazoles as antioxidants and their
acetylcholinesterase inhibition properties
Murat Bingul1 · Mehmet F. Saglam2 · Hakan Kandemir3 · Mehmet Boga4 · Ibrahim F. Sengul2
Received: 11 April 2019 / Revised: 21 May 2019 / Accepted: 12 June 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
A range of novel 4,6-dimethoxy-1H-indole-2-carbohydrazides was prepared starting from methyl 4,6-dimethoxy-1H-indole-
2-carboxylate which underwent cyclodehydration to generate the corresponding 2-(indol-2-yl)-1,3,4-oxadiazole scafolds
in the presence of N,N-diisopropylethylamine and p-toluenesulfonyl chloride in acetonitrile. All novel compounds were
fully characterized by 1H NMR, 13C NMR, FT-IR, and high-resolution mass spectroscopic data. Biological importance of
the designated compounds was identifed by employing three diferent antioxidant property determination assays, namely
DPPH free radical scavenging, ABTS cationic radical decolarization, and cupric reducing antioxidant capacity (CUPRAC).
The anticholinesterase properties were also evaluated by the acetylcholinesterase and butyrylcholinesterase enzyme inhibi-
tion assays. According to the results, the indole compounds possessing carbohydrazide functionality were found to be more
promising antioxidant targets than the 2-(indol-2-yl)-1,3,4-oxadiazole systems. Nʹ-Benzoyl-4,6-dimethoxy-1H-indole-2-
carbohydrazide, a member of the dimethoxyindole-2-carbohydrazide group, demonstrated a better inhibition performance
than the standards. Additionally, extremely important results were obtained in the anticholinesterase enzyme inhibition assays
in the case of 2-(indol-2-yl)-1,3,4-oxadiazole derivatives.
Graphic abstract
Keywords Heterocycles · Bioorganic chemistry · Enzymes inhibition · Indoles
Introduction
Electronic supplementary material The online version of this
The reactive oxygen species (ROS), namely hydroxyl radi-
article (https://doi.org/10.1007/s00706-019-02462-y) contains
cal, superoxide anion, cationic radicals, and nitric oxide,
supplementary material, which is available to authorized users.
have an important role in a variety of diseases such as can-
cer, diabetes, cardiovascular diseases, auto-immune dis-
eases, and ageing [1, 2]. Additionally, the excessive ROS
have also been determined to be the main reason for the
neurological disorders, specifcally Alzheimer’s disease
(AD), Parkinson’s disease (PD), and stroke. Although the
enzymatic systems assist in minimizing the oxidative stress
and protecting the living systems by blocking the exceeding
oxidant species formation, unhealthy physical conditions,
ageing or a stressful environment can decrease or even elimi-
nate the scavenging ability of the endogenous antioxidants
* Ibrahim F. Sengul
fazilsengul@gtu.edu.tr
1
Department of Pharmaceutical Chemistry, Dicle University,
Diyarbakır, Turkey
2
Department of Chemistry, Gebze Technical University,
Kocaeli, Turkey
3
Department of Chemistry, Namık Kemal University,
Tekirdag, Turkey
4
Department of Pharmaceutical Technology, Dicle University,
Diyarbakır, Turkey
Vol.:(0123456789)
1 3

M. Bingul et al.
[3–6]. Therefore, there is an urgent necessity for the devel-
opment of new antioxidant agents in medicinal chemistry.
An intense literature search revealed that the prepara-
tion of a better antioxidant agent has been achieved by the
combination of biologically active heterocyclic compounds.
Among these heterocycles, both indole and oxadiazole are
considered as important scafolds which provide prime com-
pound systems for the development of valuable antioxidant
agents [7–10]. The indole moiety is one of the most widely
spread nitrogen-containing aromatic heterocyclic systems
found in nature. In addition, the indole scafold appears in
biological systems as a part of the amino acid tryptophan
and the neurotransmitter serotonin [11, 12]. The biologi-
cal importance of indole scafold can be explained by its
anticancer [13, 14], antimicrobial [14], anti-infammatory
[15], antimalarial [16], and antitubercular activities [17].
Furthermore, various indole-containing compounds act as
a free radical scavenger in antioxidant studies and they have
also been found in many important drugs [18–20].
the 2-position functionality of the indole as well as comple-
mentary comparison with the previous work [40–42], car-
ried out on its antioxidant properties.
For this purpose, some novel Nʹ-indolyl-4,6-
dimethoxyindole-2-carbohydrazides 5–8 and their cor-
responding cyclisation products 2-(4,6-dimethoxyindol-
2-yl)-1,3,4-oxadiazoles 9–12 have been successfully
prepared. Furthermore, the targeted compounds have been
subjected to three diferent assays, namely DPPH, ABTS,
and CUPRAC, to investigate their antioxidant properties.
Moreover, acetylcholinesterase (ACh) and butyrylcholinest-
erase (BCh) enzyme inhibition properties have also been
evaluated. To the best of our knowledge, our study is the
frst report about the preparation of targeted systems and the
designated biological properties.
Results and discussion
On the other hand, 1,3,4-oxadiazole systems appear in a
large number of medically valuable compounds and there
has been a considerable efort to synthesize these structures.
The 1,3,4-oxadiazole moiety provides valuable bioisosteric
replacement between amide and ester functional groups
and contribute to display important therapeutic potential by
participating in hydrogen bonding interactions with vari-
ous biomolecules [21, 22]. A broad spectrum of biological
activities, including anticancer [23], antimicrobial [24], anti-
convulsant [25], antiepileptic [26], antitubercular [27], and
antiallergic [28] have been determined for the 1,3,4-oxadia-
zole ring systems [29]. Particularly, a number of compounds
containing 1,3,4 or 1,2,4-oxadiazole scafolds have also
been reported to possess antioxidant activity [7–9]. Com-
pounds comprising −NH–NH–C = O− moiety are known
as hydrazide linkers. It is worth noting here that hydrazides
as key intermediate building blocks in the preparation of
1,3,4-oxadiazole compounds which are also important tar-
gets due to their potential biological activities [30–32]. More
specifcally, the antioxidant activities of the hydrazide-based
compounds are well known in the literature [33, 34].
Synthesis
It is well known that the most straightforward method to
synthesize indole-2-carboxylate is the Hemetsberger indole
synthesis. Accordingly, 4,6-dimethoxyindole-2-carboxylate
(3) was prepared by the sequential reaction of commercially
available 2,4-dimethoxybenzaldehyde (1) with methyl azi-
doacetate in the presence of sodium methoxide to form
the corresponding azido ester intermediate 2 followed by
thermal cyclisation in xylene under refux according to the
reported procedure [43]. The indole ester 3 was then con-
densed with hydrazine hydrate in ethanol under refux for
3 h to produce the indole carbohydrazide 4 [44] (Scheme 1).
Indole carbohydrazide 4 served as a key building block in
the preparation of a range of hydrazide bridged derivatives.
For instance, when indole carbohydrazide 4 was reacted
with benzoyl chloride in tetrahydrofuran in the presence of
trimethylamine at room temperature overnight, hydrazide
linked indole 5 was produced as a single product in quantita-
tive yield. Under similar reaction conditions, the treatment
of readily available compound 4 with the m- or p-substituted
benzoyl chloride derivatives aforded the corresponding
benzoyl-4,6-dimethoxy-indole-2-carbohydrazides 6–8 in
excellent yield (Scheme 2). The characteristic peak of all
Considering the importance of indole, indole carbohy-
drazide, and 1,3,4-oxadiazole nucleus, we were interested in
the development of novel indole-2-carbohydrazide, indole-
2-carbohydrazides, and 2-(indol-2-yl)-1,3,4-oxadiazoles, as
well as the evaluation of their antioxidant and anticholinest-
erase properties. The Hemetsberger reaction was chosen as
the synthetic preparation method for the designated indole
compound, since it readily forms derivatives of 2-carbox-
yindoles from aromatic or heterocyclic aldehydes [35–37].
Although the previous studies reported the substitution on
the 3-position of the indole with the oxadiazole structures
and investigated a range of diferent biological activities [38,
39], our study provided new indolyl-oxadiazole systems with
1
these indole-2-carbohydrazides 5–8 in the H NMR spec-
trum was two singlets integrating for 2H at~10 ppm indicat-
ing the presence of the hydrazine NH groups. The 1H NMR
spectrum also showed the characteristic amide NH peaks at
10.27 and 10.36 ppm as a singlet, while indole NH resonated
at 11.53 ppm. The appearance of NH protons at a signif-
cantly downfeld chemical shift indicated a strong hydrogen
bonding between the carbonyl groups and NH protons. The
13C NMR spectrum of the compound 6 also indicated car-
bonyl groups resonance at 166.27 and 161.27 ppm.
1 3

Synthesis of indole-2-carbohydrazides and 2-(indol-2-yl)-1,3,4-oxadiazoles as antioxidants…
Scheme 1
Scheme 2
With the indole-2-carbohydrazides 5–8 in hand, atten-
tion subsequently turned to the preparation of 2-(indol-
2-yl)-1,3,4-oxadiazoles via one-pot dehydrative cyclisation
of carbohydrazides 5–8. Cyclisation of 5 with p-toluene-
sulfonyl chloride (p-TsCl) and N,N-diisopropylethylamine
(DIPEA) in acetonitrile led to the formation of 2-(indol-
2-yl)-1,3,4-oxadiazole (9) in 65% yield. Similarly, cyclisa-
tion of indole-2-carbohydrazides 6–8 under the same reac-
tion conditions gave the corresponding oxadiazoles 10–12 in
good to average yields. The structures of compounds 9–12
were supported by 1H and 13C NMR spectroscopic data. The
1H and 13C NMR spectrum of compound 10 were charac-
appearing at 12.13 ppm. Furthermore, the molecular weight
of the indolyl-1,3,4-oxadiazoles 9–12 was determined by
high-resolution mass spectrometry, which revealed the antic-
ipated molecular ion peaks at 322, 336, 367, 390 (M+1),
respectively.
Biological studies
With the Nʹ-indolyl-4,6-dimethoxyindole-2-carbohydrazides
5–8 and 4,6-dimethoxyindolyl-1,3,4-oxadiazoles 9–12 in
hand, it was of interest to determine the anticholinesterase
activity using acetylcholine and butyrylcholine enzyme inhi-
bition assay. Their antioxidant profles against the free radi-
cals, cationic radicals and reductive efciencies for Cu metal
were also investigated using DPPH, ABTS, and CUPRAC
antioxidant assays. The assays were briefy described in
the method section. The mean and standard deviation (SD)
1
teristic for compounds 11, 12. The H NMR spectrum of
compound 10 showed the disappearance of the amide NH
protons of the starting materials at 10.27 and 10.36 ppm
which clearly confrmed that cyclisation had taken place.
The indole NH peak was also observed to shift downfeld
1 3

M. Bingul et al.
Table 1 The IC50 values of the synthesised compounds 5–12 for
inhibition efciency was identifed in the case of compound
8 and the concentration value was determined to be 8.21 μM
for 50% inhibition. The determined values by compound 5
and 8 were found to be more potent than the standards α-TOC
and BHT. It was also important to highlight that the IC50
values for the designated compounds were very close to the
best standard BHA (5.50 μM). In the case of compounds 6
and 7, better inhibition patterns were also observed with the
lower IC50 concentrations of 10.06 μM and 9.26 μM, respec-
tively. The antioxidant potency of two compounds was better
than the standard α-TOC and almost as good as the standard
BHT. In the case of 2-(indol-2-yl)-1,3,4-oxadiazolyl deriva-
tives 9–12, the compound 9 demonstrated the best inhibi-
tion concentration with the IC50 value of 9.87 μM and was
found to be better than α-TOC and almost as good as the
BHT standard. The compounds 10 and 11 showed identical
inhibition patterns and the IC50 values were determined to be
18.99 μM and 17.96 μM.
DPPH and ABTS activities
Compound
IC50 values/μM
DPPH free Radical
ABTS cation radical
5
337.4 0.25
497.3 0.82
405.0 0.31
285.1 0.30
963.2 0.18
625.4 0.69
332.7 0.87
940.6 0.63
7.88 0.20
7.05 0.62
10.06 0.28
9.26 0.93
8.21 0.43
9.87 0.32
18.99 0.29
17.96 1.19
245.3 1.31
5.50 0.05
20.95 0.41
8.45 0.31
6
7
8
9
10
11
12
BHA
α-TOC
BHT
16.30 0.79
58.86 0.50
Values are means S.D., n=3
Values were given as IC50
Cupric reducing antioxidant capacity (CUPRAC)
values for each compound were calculated from at least three
replicate experiments.
Figure 1 illustrated the absorbance values obtained from
cupric reducing antioxidant capacity assay for the synthe-
sised compounds 5–8 and 9–12 along with the standards
BHT, BHA, and α-TOC. Compounds with a higher absorb-
ance value were identifed to show better reducing capacity.
As in the cationic radical scavenging activity, the reducing
potency of indole-2-carbohydrazides 5–8 was found to be
more promising than the analogues of 2-(indol-2-yl)-1,3,4-
oxadiazoles 9–12. The compounds 5 and 6 were determined
as the most potent derivatives and the absorbance values
at 10 μM and 25 μM concentrations were as good as all
the standards. The concentration-dependent behaviours of
the designated compounds were observed by the increas-
ing absorbance values through the higher concentrations.
A similar antioxidant capacity pattern was observed in the
case of compound 8 and increasing concentration provided
a better absorbance value than the standard α-TOC. On the
other hand, the results suggested that the formation of the
oxadiazole systems attached to the indole moiety reduced
the cupric reducing capacity. The best absorbance values
were obtained in the presence of compound 11 which was
found to show a similar absorbance pattern as that of the
α-TOC standard.
DPPH free radical scavenging assay
Table 1 showed the IC50 values of the indole-2-carbohy-
drazides 5–8 and 2-(indol-2-yl)-1,3,4-oxadiazolyl systems
9–12 and the positive controls, BHT, BHA, and α-TOC for
the DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) radical
scavenging activity. According to the results, the synthesised
compounds, carbohydrazide intermediates and oxadiazoles
have been found less efective compared to the standards
BHA, BHT, and α-TOC. The lower IC50 value has been deter-
mined as the better inhibition activity for the assay. Among
the indole-2-carbohydrazide derivatives 5–8, compound 8
had the best IC50 value and illustrated the best inhibition ef-
ciency. The compound 11, a member of 2-(indol-2-yl)-1,3,4-
oxadiazolyl scafold inhibited 50% of the free radicals forma-
tion at the 332.79 μM concentration which was the highest
concentration value for the oxadiazole systems.
ABTS cation radical decolorization assay
The cationic radical scavenging activities of the synthesised
compounds and controls BHT, BHA, and α-TOC are illus-
trated in Table 1 as IC50 concentration values. The scavenging
abilities of the synthesised compounds were found to be more
potent than the free radical scavenging activities. Among
the synthesised compounds, the concentration values for the
indole-2-carbohydrazide analogues 5–8 were determined to
be greater than the analogues of 2-(indol-2-yl)-1,3,4-oxadi-
azolyl systems 9–12. The compound 5 exhibited the greatest
scavenging activity with the value of 7.05 μM. A similar
Determination of anticholinesterase activity
The acetylcholinesterase (ACh) and butyrylcholinesterase
(BCh) enzyme inhibitory activities were evaluated for the
determination of therapeutic potential of the indole-2-car-
bohydrazide and 2-indolyl-1,3,4-oxadiazole scafolds for the
treatment of AD. The inhibition of AChE and BChE by the
compounds was given as percentage values at 200 µg/cm3
1 3

Synthesis of indole-2-carbohydrazides and 2-(indol-2-yl)-1,3,4-oxadiazoles as antioxidants…
Fig. 1 Absorbance values for
the cupric ion reducing antioxi-
dant capacity (CUPRAC) of the
compounds 5–12 and controls
BHT, BHA, α-Toc. Values are
means SD, n=3, p<0.05,
signifcantly diferent from each
other when compared using
Student’s t test
Cupric reducing anꢀoxidant capacity (CUPRAC)
2.000
1.800
1.600
1.400
1.200
1.000
0.800
0.600
0.400
0.200
0.000
5
6
7
8
9
10
11
12
BHA
TOC
BHT
10 µM
25 µM
50 µM
100 µM
Table 2 Acetylcholinesterase and butyrylcholinesterase enzyme
inhibitory activities of the indole-2-carbohydrazides 5–8 and
2-indolyl-1,3,4-oxadiazoles 9–12a, b
with the value of 90.6% which was the highest percentage
among the compounds and the standard. AChE inhibition
potency of the designated compound was also well enough
to consider for further studies. In the case of compounds
containing the carbohydrazide linkage, the AChE inhibition
activities were also considered quite efective with the val-
ues of more than 50% inhibitions. The compound 7 was the
best candidate for both AChE and BChE inhibition with the
values of 55% and 70%, respectively.
Compounds
AChE inhibition/%
BChE inhibition/%
5
52.90 0.53
51.31 1.45
55.23 0.93
51.12 0.86
88.22 1.02
88.69 1.21
41.31 0.17
67.38 0.98
78.47 0.49
38.37 0.64
36.85 0.20
70.61 1.61
59.51 0.71
86.87 1.54
89.50 0.75
75.68 1.35
90.60 1.56
79.41 0.20
6
7
8
9
10
11
Conclusion
12
Galanthamineb
The methyl 4,6-dimethoxyindole-2-carboxylate, as the
indole backbone for the current work, was prepared by the
Hemetsberger indole synthesis method. The methyl-2-ester
was reacted with the hydrazine hydrate to aford the desired
dimethoxyindole-2-carbohydrazide in excellent yield. The
indole-2-carbohydrazide and benzoyl chloride systems were
successfully bonded with the hydrazide linkage and three
diferent antioxidant (DPPH, ABTS, and CUPRAC) assays
and acetylcholinesterase and butyrylcholinesterase enzyme
inhibitory activities were employed to identify the biologi-
cal capacities of the designated compounds. The obtained
data demonstrated that the dimethoxyindole-2-carbohy-
drazide intermediates 5–8 could be promising structures
than 2-(indol-2-yl)-1,3,4-oxadiazole systems 9–12 for the
investigation of novel potent antioxidant scaffolds. The
ABTS cationic scavenging activity and the cupric reduc-
ing antioxidant capacity (CUPRAC) assays were identifed
as the most sensitive methods due to their higher inhibi-
tion values. The best antioxidant property was obtained
in the case of the compound 5 for both antioxidant assays
and displayed similar inhibition and absorbance values as
that of the standards. Although the DPPH results revealed
that these compounds were found to be not efective for the
a200 µM
bStandard
concentration in Table 2 and compared with galanthamine as
the standard. As expected, the positive control galanthamine
showed very potent inhibition both on AChE and BChE with
the values of 82.37% and 84.06%, respectively. A higher
inhibition percentage demonstrated better anticholinesterase
activity. It was observed that the synthesised compounds
showed quite a promising impact on the acetylcholinesterase
and butyrylcholinesterase enzyme inhibition. The compounds
containing the oxadiazole group were determined to be more
potent than the analogues of carbohydrazide systems. The
most potent inhibition was obtained in the presence of com-
pound 9 with the values of 88.69% and 88.50% in both ACh
and BCh enzymes. Similarly, compound 10 was also a prom-
ising candidate for the ACh and BCh enzyme inhibition with
the values of 88.22% and 86.87%, respectively. Both of the
compounds were determined as better enzyme inhibitors than
the galanthamine standard. The compound 12 was found to
be the most promising compound for the BChE inhibition
1 3

M. Bingul et al.
free radical scavenging ability, the most exciting result was
determined for the anticholinesterase activity assays. All the
synthesised compounds demonstrated a variety of impact
on ACh and BCh enzyme inhibitions and the compounds
9 and 10, containing oxadiazole moiety on the structures,
showed very high activity for the both AChE and BChE
enzyme inhibitions. Moreover, the compound 12 illustrated
the highest potency on the BChE inhibition assay with the
value of more than 90%.
4,6‑Dimethoxy‑N’‑(4‑methylbenzoyl)‑1H‑indole‑2‑carbo‑
hydrazide (6, C19H19N3O4) The carbohydrazide 6 was pre-
pared from 0.25 g indole 4 (1.06 mmol), 0.22 cm3 triethyl-
amine (1.59 mmol), and 0.21 cm3 4-methylbenzoyl chloride
(1.59 mmol) according to GP-1 and the title compound 6
1
was obtained as yellow solid (0.37 g, 100%). H NMR
(500 MHz, DMSO-d6): δ=11.54 (s, 1H, indole NH), 10.37
(s, 1H, NH), 10.28 (s, 1H, NH), 7.85 (d, J= 8.1 Hz, 2H,
aryl H), 7.34 (d, J=8.0 Hz, 2H, aryl H), 7.25 (d, J=1.3 Hz,
indole H), 6.51 (s, 1H, indole H), 6.20 (d, J=1.3 Hz, 1H,
indole H), 3.87 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 2.39 (s,
3H, CH3) ppm; 13C NMR (126 MHz, DMSO-d6): δ=166.2,
161.2 (C=O), 159.0, 154.5, 142.3, 138.7, 130.2, 129.5,
127.9, 127.3, 113.2, 101.8, 92.5, 87.2 (aryl C), 55.7, 55.6
(OCH3), 21.49 (CH3) ppm; IR (KBr): ̄ꢀ =3333, 3282, 2935,
2834, 1646, 1626, 1590, 1548, 1479, 1266, 1219, 1152, 815,
741 cm−1; HRMS: m/z calculated for C19H20N3O4 ([M+H]+)
354.1454, found 354.1455.
Experimental
All reagents and solvents were obtained from commer-
cial sources and appropriately purifed, if necessary. The
deuterated solvent (DMSO-d6) for NMR spectroscopy
was obtained from Merck. The following chemicals were
obtained from Sigma-Aldrich. Measurements IR spectra
were recorded between 4000 and 650 cm−1 using a Perkin
Elmer Spectrum 100 FT-IR spectrometer. 1H and 13C NMR
spectra were recorded in DMSO-d6 solution on a Varian
500 MHz spectrophotometer. High-resolution ESI mass
spectra were recorded by an Orbitrap LTQ XL (Thermo
Scientifc, Waltham, MA, USA) ion trap mass spectrometer
using a nanospray (nano-electrospray) ionization source.
4,6‑Dimethoxy‑Nʹ‑(4‑nitrobenzoyl)‑1H‑indole‑2‑carbohydr‑
azide (7, C18H16N4O6) The carbohydrazide 7 was prepared
from 0.16 cm3 indole 4 (0.68 mmol), 0.15 cm3 triethyl-
amine (1.08 mmol), and 0.19 g 4-nitrobenzoyl chloride
(1.02 mmol) according to GP-1 and the title compound 7 was
obtained as a white solid (1.8 g, 47%). 1H NMR (500 MHz,
DMSO-d6): δ=11.56 (s, 1H, indole NH), 10.81 (s, 1H, NH),
10.43 (s, 1H, NH), 8.39 (d, J = 8.7 Hz, 2H, aryl H), 8.17
(d, J=8.6 Hz, 2H, aryl H), 7.26 (s, 1H, indole H), 6.51 (s,
1H, indole H), 6.20 (s, 1H, indole H), 3.87 (s, 3H, OCH3),
3.78 (s, 3H, OCH3) ppm; 13C NMR (126 MHz, DMSO-d6):
δ=164.9, 161.1 (C=O), 159.1, 154.5, 149.9, 138.7, 138.6,
131.1, 129.5, 127., 124.4, 113.2, 102.1, 92.6, 87.2 (aryl C),
55.7 (OCH3) ppm; IR (KBr): ꢀ̄ = 3400, 3202, 3000, 1637,
1625, 1600, 1522, 1344, 1268, 1212, 1152, 850, 713 cm−1;
HRMS: m/z calculated for C18H16N4O6 ([M+H]+) 385.1148,
found 385.1143.
General procedure 1 (GP‑1): preparation
of indole‑2‑carbohydrazides 5–8
To a suspension of indole in 15 cm3 THF, triethylamine was
added and followed by the addition of acyl chlorides. The
reaction mixture was stirred at room temperature overnight.
The reaction mixture was then poured in to ice water and the
resulting precipitate was fltered, washed with water, and air
dried to yield the carbohydrazide.
Nʹ‑Benzoyl‑4,6‑dimethoxy‑1H‑indole‑2‑carbohydrazide (5,
C18H17N3O4) The carbohydrazide 5 was prepared from 0.25 g
indole 4 (1.06 mmol), 0.22 cm3 triethylamine (1.59 mmol),
and 0.18 cm3 benzoyl chloride (1.59 mmol) according to
GP-1 and the title compound 5 was obtained as yellow solid
(0.36 g, 100%). 1H NMR (500 MHz, DMSO-d6): δ=11.55
(s, 1H, indole NH), 10.46 (s, 1H, NH), 10.32 (s, 1H, NH),
7.98–7.92 (m, 2H, aryl H), 7.47–7.55 (m, 3H, aryl H), 7.26
(d, J=1.7 Hz, 1H, indole H), 6.51 (d, J=1.0 Hz, 1H, indole
H), 6.20 (d, J=1.6 Hz, 1H, indole H), 3.87 (s, 3H, OCH3),
3.77 (s, 3H, OCH3) ppm; 13C NMR (126 MHz, DMSO-d6):
δ=166.3, 161.2 (C=O), 159.0, 154.5, 138.7, 133.0, 132.3,
129.7, 128.9, 127.9, 127.3, 113.2, 101.9, 92.5, 87.2 (aryl C),
55.7, 55.6 (OCH3) ppm; IR (KBr): ̄ꢀ =3325, 3293, 2837,
1644, 1629, 1593, 1527, 1381, 1267, 1216, 1151, 816, 804,
713, 706 cm−1; HRMS: m/z calculated for C18H18N3O4
([M+H]+) 340.1297, found 340.1292.
4,6‑Dimethoxy‑N’‑[3‑(trifluoromethyl)benzoyl]‑1H‑in‑
dole‑2‑carbohydrazide (8, C19H16F3N3O4) The carbohy-
drazide 8 was prepared from 0.18 g indole 1 (0.76 mmol),
0.16 cm3 triethylamine (1.15 mmol), and 0.17 cm3 3-(tri-
fuoromethyl)benzoyl chloride (1.15 mmol) according to
GP-1 and the title compound 8 was obtained as yellow solid
(0.27 g, 87%). 1H NMR (500 MHz, DMSO-d6): δ = 11.56
(s, 1H, indole), 10.75 (s, 1H, NH), 10.42 (s, 1H, NH), 8.33–
8.17 (m, 2H, aryl H), 8.01 (d, J=7.5 Hz, 1H, aryl H), 7.81
(s, 1H), 7.27 (s, 1H, indole H), 6.51 (s, 1H, indole H), 6.20
(s, 1H, indole H), 3.87 (s, 3H, OCH3), 3.77 (s, 3H, OCH3)
ppm; 13C NMR (126 MHz, DMSO-d6): δ = 165.0, 161.1
(C=O), 159.1, 154.5, 138.7, 133.8, 132.0, 130.5, 127.1,
124.5, 113.2, 102.1, 92.5, 87.2 (aryl C), 55.7, 55.6 (OCH3)
ppm; IR (KBr): ̄ꢀ = 3330, 3182, 2967, 1707, 1625, 1540,
1327, 1267, 1227, 1215, 1121, 821, 692 cm−1; HRMS: m/z
1 3

Synthesis of indole-2-carbohydrazides and 2-(indol-2-yl)-1,3,4-oxadiazoles as antioxidants…
calculated for C19H17F3N3O4 ([M+H]+) 408.1171, found
408.1166.
p-TsCl (3.6 mmol) according to GP-2 and the title compound
11 was obtained as a red solid (0.14 g, 37%). Satisfactory
1H and 13C NMR data could not be obtained for this com-
pounds due to the low solubility in common NMR solvents;
IR (KBr): ̄ꢀ = 3216, 2940, 2839, 1710, 1597, 1519, 1461,
1336, 1308, 1153, 1134, 1011, 853, 811, 725 cm−1; HRMS:
m/z calculated for C18H14N4O5 ([M+H]+) 367.1042, found
367.1032.
General procedure 2 (GP‑2): preparation
of 2‑(indolyl)‑1,3,4‑oxadiazoles 9–12
To a mixture of indole-2-carbohydrazide in 25 cm3 acetoni-
trile, DIPEA and p-TsCl were added, successively and the
reaction mixture was then stirred at room temperature for
3–4 h. The resulting reaction mixture was fltered through a
sintered funnel, washed with acetonitrile and water, succes-
sively and air dried to yield the oxadiazole.
2‑(4,6‑Dimethoxy‑1H‑indol‑2‑yl)‑5‑[3‑(trifluoromethyl)‑
phenyl]‑1,3,4‑oxadiazole (12, C19H14F3N3O3) The oxadia-
zole 12 was prepared from 0.15 g indole-2-carbahydrazide
8 (0.37 mmol), 10 cm3 acetonitrile, 0.33 cm3 DIPEA
(1.9 mmol), 0.25 g p-TsCl (1.3 mmol) according to GP-2
and the title compound 12 was obtained as a yellow solid
(71 mg, 49%). 1H NMR (500 MHz, DMSO-d6): δ=12.18
(s, 1H, indole NH), 8.49–8.38 (m, 2H, aryl H), 8.04 (d,
J=7.8 Hz, 1H, aryl H), 7.91 (d, J=7.8 Hz, 1H, aryl H), 7.32
(d, J=1.8 Hz, 1H, indole H), 6.55 (s, 1H, indole H), 6.26 (d,
J=1.7 Hz, 1H, indole H), 3.89 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3) ppm; 13C NMR (126 MHz, DMSO-d6): δ = 162.3,
161.0, 159.7, 154.3, 140.1, 133.7, 131.2, 130.9, 124.8,
123.4, 118.5, 113.7, 104.0, 93.0, 87.2 (aryl C), 55.8, 55.7
(OCH3) ppm; IR (KBr): ̄ꢀ =3203, 2955, 2848, 1605, 1589,
1465, 1337, 1278, 1223, 1153, 1130, 807, 725, 698 cm−1;
HRMS: m/z calculated for C18H18N3O4 ([M+H]+) 390.1066,
found 390.1060.
2‑(4,6‑Dimethoxy‑1H‑indol‑2‑yl)‑5‑phenyl‑1,3,4‑oxadiazole
(9, C18H15N3O3) The oxadiazole 9 was prepared from 0.40 g
indole-2-carbahydrazide 5 (1.2 mmol), 25 cm3 acetonitrile,
1.1 cm3 DIPEA (6.1 mmol), 0.79 g p-TsCl (4.1 mmol)
according to GP-2 and the title compound 9 was obtained
as a pale yellow solid (0.25 g, 65%). 1H NMR (500 MHz,
DMSO-d6): δ=12.16 (s, 1H, indole NH), 8.21–8.03 (m, 2H,
aryl H), 7.73–7.51 (m, 2H, aryl H), 7.22 (s, 1H, indole H),
6.55 (s, 1H, indole H), 6.26 (d, J=1.2 Hz, 1H, indole H),
3.89 (s, 3H, OCH3), 3.81 (s, 3H, OCH3) ppm; 13C NMR
(126 MHz, DMSO-d6): δ = 163.4, 159.7, 159.6, 154.3,
140.0, 132.3, 129.9, 127.0, 123.8, 118.7, 113.7, 103.4,
92.9, 87.2 (aryl C), 55.7 (OCH3) ppm; IR (KBr): ̄ꢀ =3188,
2957, 2841, 1609, 1588, 1556, 1523, 1490, 1463, 1450,
1377, 1308, 1151, 1133, 1009, 823, 791, 684 cm−1; HRMS:
m/z calculated for C18H15N3O3 ([M+H]+) 322.1192, found
322.1186.
DPPH free radical scavenging activity
2‑(4,6‑Dimethoxy‑1H‑indol‑2‑yl)‑5‑(p‑tolyl)‑1,3,4‑oxadia‑
zole (10, C19H17N3O3) The oxadiazole 10 was prepared from
0.30 g indole-2-carbahydrazide 6 (0.85 mmol), 20 cm3
acetonitrile, 0.77 cm3 DIPEA (4.4 mmol), 0.57 g p-TsCl
(3.0 mmol) according to GP-2 and the title compound 10 was
obtained as a white solid (0.21 g, 74%). 1H NMR (500 MHz,
DMSO-d6): δ=12.14 (s, 1H, indole NH), 8.02 (d, J=7.8 Hz,
2H, aryl H), 7.45 (d, J= 7.8 Hz, 2H, aryl H), 7.20 (s, 1H,
indole H), 6.54 (s, 1H, indole H), 6.25 (s, 1H, indole H),
3.89 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 2.43 (s, 3H, CH3)
ppm; 13C NMR (126 MHz, DMSO-d6): δ = 163.4, 159.5,
154.3, 142.5, 139.9, 130.4, 127.0, 121.0, 118.8, 113.7,
103.3, 92.9, 87.2 (aryl C), 55.7 (OCH3), 21.6 (CH3) ppm;
IR (KBr): ̄ꢀ = 3204, 2939, 2842, 1607, 1555, 1523, 1496,
1452, 1376, 1326, 1307, 1283, 1206, 1251, 1132, 818, 795,
726 cm−1; HRMS: m/z calculated C19H17N3O3 ([M+H]+)
336.1348, found 336.1343.
The DPPH (2,2-diphenyl-1-picryl-hydrazyl hydrate) is
an assay which was used to measure the hydrogen atom
(or one electron) donation ability of the scavenging mol-
ecule to the DPPH, resulting in the reduction of DPPH
to DPPH2. DPPH radical scavenging activity assay was
performed according to the method of Blois et al. with
minor modifcations [45].
ABTS cation radical decolorization activity
The ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sul-
fonic acid)) assay was carried out to determine the cationic
radical reducing ability of synthesised compounds. The
procedure described in the literature [46] was followed to
perform the assay.
2‑(4,6‑Dimethoxy‑1H‑indol‑2‑yl)‑5‑(4‑nitrophenyl)‑1,3,4‑
oxadiazole (11, C18H14N4O5) The oxadiazole 11 was pre-
pared from 0.40 g indole-2-carbahydrazide 7 (1.0 mmol),
24 cm3 acetonitrile, 0.94 cm3 DIPEA (5.4 mmol), 0.69 g
Cupric reducing antioxidant capacity (CUPRAC)
The cupric reducing antioxidant capacity (CUPRAC)
method is an antioxidant capacity assay used to identify the
1 3

M. Bingul et al.
20. Metwally MA, Shaaban S, Abdel-Wahab BF, El-Hiti GA (2009)
Curr Org Chem 13:1475
reductive efciencies of the compounds for Cu metal. The
assay was performed according to the procedure given in
the literature [47].
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Anticholinesterase activity determination method
ACh and BCh enzyme inhibitory activities were measured
by the slightly modifed spectrophotometric method devel-
oped by Ellman [48].
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All statistical comparisons were performed by the means
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1 3