Design, synthesis, and antimicrobial activity of certain new indole-1,2,4 triazole conjugates

Article abstract of DOI:10.3390/molecules26082292

The increasing prevalence of microbial infections and the emergence of resistance to the currently available antimicrobial drugs urged the development of potent new chemical entities with eminent pharmacokinetic and/or pharmacodynamic profiles. Thus, a series of new indole-triazole conjugates 6a-u was designed and synthesized to be assessed as new antimicrobial candidates using the diameter of the inhibition zone and minimum inhibitory concentration assays against certain microbial strains. Their in vitro antibacterial evaluation revealed good to moderate activity against most of the tested Gram-negative strains with diameter of the inhibition zone (DIZ) values in the range of 11–15 mm and minimum inhibition concentration (MIC) values around 250 μg/mL. Meanwhile, their in vitro antifungal evaluation demonstrated a potent activity against Candida tropicalis with MIC value as low as 2 μg/mL for most of the tested compounds. Moreover, compound 6f is the most potent congener with an MIC value of 2 μg/mL against Candida albicans.

Full text of DOI:10.3390/molecules26082292

molecules
Article
Design, Synthesis, and Antimicrobial Activity of Certain New
Indole-1,2,4 Triazole Conjugates
Reem I. Al-Wabli 1,*, Mona A. Alsulami 1, Sarah I. Bukhari 2 , Nadine M. S. Moubayed 3, Maha S. Al-Mutairi 1
and Mohamed I. Attia 1,4,
*
1
2
3
4
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia; aa_88_22@hotmail.com (M.A.A.); malmutbiri@ksu.edu.sa (M.S.A.-M.)
Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia; Sbukhari@ksu.edu.sa
Botany and Microbiology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
nmoubayed@ksu.edu.sa
Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research
Division, National Research Centre (ID: 60014618), El Bohooth Street, Dokki, Giza 12622, Egypt
Correspondence: ralwabli@ksu.edu.sa (R.I.A.-W.); mattia@ksu.edu.sa (M.I.A.)
*
Abstract: The increasing prevalence of microbial infections and the emergence of resistance to the
currently available antimicrobial drugs urged the development of potent new chemical entities with
eminent pharmacokinetic and/or pharmacodynamic profiles. Thus, a series of new indole-triazole
conjugates 6a-u was designed and synthesized to be assessed as new antimicrobial candidates using
the diameter of the inhibition zone and minimum inhibitory concentration assays against certain
microbial strains. Their in vitro antibacterial evaluation revealed good to moderate activity against
most of the tested Gram-negative strains with diameter of the inhibition zone (DIZ) values in the range
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Al-Wabli, R.I.; Alsulami,
M.A.; Bukhari, S.I.; Moubayed,
N.M.S.; Al-Mutairi, M.S.; Attia, M.I.
Design, Synthesis, and Antimicrobial
Activity of Certain New Indole-1,2,4
Triazole Conjugates. Molecules 2021,
26, 2292. https://doi.org/10.3390/
molecules26082292
of 11–15 mm and minimum inhibition concentration (MIC) values around 250
their in vitro antifungal evaluation demonstrated a potent activity against Candida tropicalis with MIC
value as low as 2 g/mL for most of the tested compounds. Moreover, compound 6f is the most
µg/mL. Meanwhile,
µ
potent congener with an MIC value of 2 µg/mL against Candida albicans.
Keywords: indole; 1,2,4-triazole-3-thiol; antibacterial; antifungal
Academic Editor: David StC Black
1. Introduction
Received: 17 March 2021
Accepted: 12 April 2021
Published: 15 April 2021
Bacterial infections are the major cause of some high mortality rate diseases such
as typhus, tuberculosis, plague, diphtheria, cholera, dysentery, and pneumonia. On the
other hand, fungi infect many people worldwide every year, yet their contribution to the
global burden of diseases is largely unrecognized, and most of them cause relatively minor
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
infections but kill at least as many people as tuberculosis or malaria [1]. This is due to an
increasing population with reduced immune system or immunocompromised individuals
having AIDS, cancer or those undergoing organ transplantation. Furthermore, microbial
infections are becoming more resistant to antibiotics due to years of their overuse and/or
misuse, which might lead to a potential global health disaster [2]. In addition, the current
hospital-acquired infections are resistant to most of the clinically available antibiotics such
as methicillin and vancomycin. Therefore, the use of these drugs was reserved to treat only
Copyright:
© 2021 by the authors.
the most intractable infections to retard the development of their resistance [3–5]. This
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
makes the design and development of new antimicrobial candidates with novel chemical
structures and/or with different modes of action rather than analogues of the existing ones
are necessary for clinical needs.
Indole ring is one of the most potent pharmacodynamic nucleus and constitutes the
backbone of the neurotransmitter serotonin and the natural hormone melatonin as well as
the essential amino acid tryptophan. The recognition of tryptophan importance in animal
Molecules 2021, 26, 2292. https://doi.org/10.3390/molecules26082292
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2292
2 of 13
and human nutrition served to bring about a renaissance in indole chemistry, and there-
fore, indole derivatives have been synthesized and/or isolated from nature in abundance.
Mitomycin is an example of bioactive indole alkaloids and its analogues are gaining great
attention because of their extensive use in cancer chemotherapy and for its antibacterial
activity [6,7]. Consequently, compounds bearing an indole nucleus have a considerable
importance in the development of diverse bioactive compounds by varying the substituents
or substitution pattern of the indole ring. Such compounds have been reported for various
biological activities including antimicrobial [8], antiviral [9], anticonvulsant [10], analgesic,
anti-inflammatory anti-tubercular [11] and anticancer [12]. Thus, this wide spectrum of
activities of indole-bearing compounds makes them suitable candidates for the develop-
ment of novel antibacterial and antifungal treatments. On the other hand, there has been a
considerable interest in the development of novel heterocyclic compounds with variable
biological activities. Among them, triazoles are a special class of heterocyclic compounds
with a broad spectrum of biological activities such as antibacterial [13,14], antifungal [15,16],
anti-inflammatory [17], antitubercular [18], anticancer [19], antimalarial [20], antiviral [21],
and analgesic [22]. Furthermore, fluconazole and itraconazole [23] are therapeutically
significant antifungal azoles containing 1,2,4-triazole moiety.
Hybridization is an important modality in drug design and development based on a
combination of biologically active moieties to produce a new hybrid compound with an
improved biological activity when compared to the parent compounds. Additionally, this
strategy can result in compounds presenting a modified selectivity profile, different and/or
dual modes of action, and reduced undesired side effects [24]. A literature review revealed
that 1,2,4 triazole-indole hybrid compounds were synthesized and demonstrated excellent
antibacterial and antifungal activity [25]. In addition, it was found that a free N–H in the
indole ring was essential and increased the antibacterial activity to an acceptable extent
against K. pneumoniae, E. coli, and P. aeruginosa [26]. Moreover, the 1,2,4-triazole nucleus
has received attention due to its low toxicity, wide safety, and good pharmacokinetic
characteristics [27], and it was noticed that upon the introduction of the electron-rich sulfur
atom into the triazole ring, the bioactivities of the target compounds greatly improve [28].
This may be attributed to the effect of the sulfur atom in increasing the lipophilicity and
modulating of the electron density in the triazole ring, which increases the transmembrane
diffusion and their interaction with macromolecular targets [29].
On the basis of the aforementioned premises, it was of our interest to design and
synthesize certain indole-based 1,2,4-triazole-3-thiol molecular hybrids 6a-u with different
N4 substituent on the triazole nucleus with increasing order of lipophilicity (cyclohexyl <
phenyl < 4-Cl-phenyl) and to test their antibacterial and antifungal activities.
2. Results and Discussions
2.1. Chemistry
The target compounds 6a-u have been successfully synthesized according to Scheme 1
.
The synthesis was commenced by esterification of the commercially available indole-2-
carboxylic acid (
ester derivative
1
2
) in ethanol in the presence of a catalytic amount of sulfuric acid. The ethyl
was converted to the corresponding acid hydrazide through reaction
3
with hydrazine hydrate in ethanol. Subsequently, the appropriate isothiocyanate, namely
cyclohexyl isothiocyanate, phenyl isothiocyanate, and 4-chlorophenyl isothiocyanate was
allowed to react with compound 3 to yield the respective thiosemicarbazide derivatives
4a-c. Cyclodehydration reaction of the thiosemicarbazide derivatives 4a-c was carried
out in aqueous 10% NaOH to give compounds 5a-c. Thereafter, the target compounds
6a-u were achieved through a nucleophilic substitution reaction in which the appropriate
benzyl halide was subjected to nucleophilic attack by the thiol group of compounds 5a-c
It is noteworthy to mention that the use of polar protic solvents such as ethanol, in a
strong alkaline medium such as sodium hydroxide, favors the formation of both S-alkyl
derivatives as major products and N-alkyl derivatives as minor products. On the other
hand, carrying out the reaction in a polar aprotic solvent such as acetone, in the presence of
.

Molecules 2021, 26, 2292
3 of 13
potassium carbonate favors the formation of S-alkyl derivatives as a sole product [30]. Thus,
the triazole derivatives 5a-c were elaborated to their respective N-alkyl derivatives 6a-u as
sole products through the reaction with the appropriate benzyl halide in dry acetone [31].
The 1H-NMR spectra of compounds 6a-u showed the cyclohexyl protons for compounds
6o-u in the range of
singlets at = 2.28 ppm representing three protons of CH3 group. Singlets integrated
for two protons were noticed in the range of = 4.36-4.70 ppm, which were assigned to
be for the two protons of the benzylic CH2. The aromatic protons were observed in the
region of
= 6.94-8.19 ppm. Additionally, the SH signals disappeared, and the 13C-NMR
spectra of the target compounds 6a-u exhibited signals in the range of = 34.7-37.3 ppm
indicating carbons of the benzylic CH2. The cyclohexyl carbons for compounds 6o-u were
δ = 1.05-4.40 ppm. Meanwhile, compounds 6g, 6n, and 6u showed
δ
δ
δ
δ
observed in the range of
δ = 24.9-56.8 ppm and the aromatic carbons appeared in the range
of δ = 101.8-151.7 ppm.
Scheme 1. Synthesis of the target compounds 6a-u.
2.2. Antimicrobial Evaluation
The in vitro antibacterial potential of the tested compounds 6a-u was estimated against
five standard bacterial strains, namely the Gram-negative Escherichia coli, Pseudomonas
aeruginosa, and Salmonella, and the Gram-positive Staphylococcus aureus and Bacillus subtilis.
Meanwhile, the antifungal potential was estimated against three standard fungal strains,
namely Candida albicans, Candida tropicalis, and Candida glabrata. The results are expressed
as diameter of the inhibition zone (DIZ) and the minimum inhibition concentration (MIC).
Ampicillin (antibacterial) and fluconazole (antifungal) were used as reference drugs, and
the antimicrobial results are presented in Tables 1 and 2.

Molecules 2021, 26, 2292
4 of 13
Table 1. Antibacterial activity of the target compounds 6a-u against E. coli, Pseudomonas aeruginosa, Salmonella ty-
phimurium, Staphylococcus aureus, and Bacillus subtilis.
Pseudomonas
aeruginosa
Salmonella
typhimurium
Staphylococcus
aureus
E. coli
Bacillus subtilis
Compound
No.
DIZ ±
MIC
DIZ ±
MIC
(µg/mL)
DIZ ±
MIC
(µg/mL)
DIZ ±
MIC
(µg/mL)
DIZ ±
MIC
(µg/mL)
SD *
(
µg/mL)
SD *
SD *
SD *
SD *
11 ±
10 ±
10 ±
6a
1000
0
-
1000
250
250
250
250
500
250
500
250
500
250
250
250
250
1000
500
250
250
500
500
1000
500
0
-
1000
1.06
0.00
0.70
11 ±
13 ±
10 ±
10 ±
6b
250
250
250
250
500
250
250
250
1000
250
250
250
500
1000
500
500
250
250
500
1000
500
250
250
250
-
0
-
250
0.70
7.50
0.70
2.12
11 ±
14 ±
11 ±
6c
0
-
9 ± 0.00
9 ± 0.00
9 ± 0.00
250
2.12
8.08
2.88
10 ±
15 ±
11 ±
6d
0
-
250
1.41
8.66
3.46
12 ±
11 ±
6e
0
0
0
-
500
1.41
1.73
11 ±
10 ±
10 ±
6f
-
0
-
-
250
0.70
0.00
0.70
11 ±
10 ±
10 ±
6g
10 ±5.50
250
250
250
500
250
250
250
250
1000
500
250
500
250
500
1000
1000
0
250
0.70
1.15
0.70
12 ±
10 ±
10 ±
6h
9 ± 0.57
9 ± 1.52
1000
250
500
250
500
250
250
-
0
0
0
0
0
0
0
0
0
0
0
0
0
-
1.41
1.00
0.57
12 ±
11 ±
10 ±
6i
-
2.12
1.52
0.70
14 ±
12 ±
11 ±
11 ±
6j
-
3.53
1.32
0.35
1.41
14 ±
11 ±
10 ±
10 ±
6k
-
3.53
0.57
0.57
1.41
14 ±
10 ±
11 ±
6l
9 ± 0.00
-
3.53
1.15
1.00
12 ±
10 ±
10 ±
10 ±
6m
-
2.82
0.57
1.73
0.57
13 ±
12 ±
11 ±
11 ±
6n
-
2.12
1.15
1.00
1.00
11 ±
13 ±
11 ±
6o
0
0
0
0
0
0
0
0
-
0.70
2.51
1.00
11 ±
12 ±
11 ±
6p
-
-
0.00
2.00
2.00
12 ±
12 ±
11 ±
6q
-
-
-
1.06
1.52
1.15
12 ±
13 ±
12 ±
6r
-
0.70
2.64
1.52
10 ±
14 ±
11 ±
6s
-
-
1.41
3.60
1.52
11 ±
13 ±
11 ±
6t
6u
-
-
0.00
3.01
1.73
11 ±
13 ±
12 ±
12 ±
-
250
500
1.41
2.00
1.15
1.00
08 ±
24 ±
14 ±
08 ±
Ampicillin
500
0.57
0.40
0.60
0.28
* The arithmetic mean of the inhibition zone diameters in mean ± standard deviation. (-) not determined.

Molecules 2021, 26, 2292
5 of 13
Table 2. Antifungal activity of the target compounds 6a-u against Candida albicans, Candida tropicalis, and Candida glabrata.
Candida albicans Candida tropicalis Candida glabrata
DIZ ± SD * MIC (µg/mL)
Compound
No.
DIZ ± SD *
MIC (µg/mL)
DIZ ± SD *
MIC (µg/mL)
6a
26 ± 0.28
26 ± 0.11
26 ± 0.28
27 ± 0.00
26 ± 0.28
26 ± 0.28
26 ± 0.11
0
1000
250
250
250
250
2
13 ± 0.57
14 ± 1.52
14 ± 2.08
14 ± 3.46
14 ± 3.21
14 ± 2.00
14 ± 1.15
12 ± 0.57
11 ± 0.57
11 ± 0.57
10 ± 0.00
10 ± 0.00
10 ± 0.57
11 ± 0.57
11 ± 0.57
11 ± 0.57
11 ± 0.57
14 ± 0.57
12 ± 1.52
11 ± 0.57
17 ± 0.28
15 ± 0.11
2
2
10 ± 0.57
10 ± 0.00
10 ± 0.57
10 ± 1.00
11 ± 1.15
10 ± 0.00
10 ± 0.57
1000
250
250
125
250
250
1000
6b
6c
2
6d
4
6e
4
6f
4
6g
250
-
4
6h
2
0
6i
0
-
4
0
-
6j
0
-
4
0
-
6k
0
-
2
0
-
6l
0
-
2
0
-
6m
0
-
2
0
-
6n
0
-
2
0
-
6o
11 ± 0.70
10 ± 0.00
10 ± 0.70
10 ± 0.70
10 ± 0.00
11 ± 0.70
10 ± 0.00
07 ± 0.28
1000
32
32
32
250
250
1000
250
16
4
0
-
-
6p
0
6q
12.5
2
0
-
6r
0
-
6s
2
10 ± 0.57
10 ± 0.57
13 ± 1.00
15 ± 0.57
62.5
32
16
1000
6t
6u
2
2
Fluconazole
1000
* The arithmetic mean of the inhibition zone diameters in mean ± standard deviation. (-) not determined.
Table 1 illustrates the antibacterial activity of the target compounds 6a-u in the DIZ
and MIC assays against the tested bacterial strains. They showed varying degrees of
activities against the tested microorganisms. Most of the investigated compounds 6a-u
manifested good to moderate activity in the DIZ assay toward the tested Gram-negative
bacterial strains with DIZ values in the range of 11-15 mm. Whereas, their MIC value was
around 250
µg/mL which is lower than the MIC values of ampicillin against Escherichia
coli (500 g/mL), Pseudomonas aeruginosa (1000
µ
µ
g/mL), and Salmonella (500 g/mL).
µ
In addition, Gram-positive bacteria were less sensitive toward the tested compounds
6a-u than Gram-negative bacteria. Usually, increasing the lipophilic characteristic of
the synthesized compound increases the antibacterial activity on Gram-positive bacteria.
This can be explained on the bases of the low lipid content of the cell wall of Gram-
positive bacteria, which would permit the activity of more lipophilic compounds [32].
Only compounds 6h-n, bearing the 4-chlorophenyl moiety, exhibited good activity against
the tested standard Staphylococcus aureus as it showed MIC values of 250-1000
Furthermore, compounds 6b-g bearing phenyl moiety on the triazole ring showed MIC
values of 250-500 g/mL against Bacillus subtilis being similar to or better than the MIC
value of the reference drug ampicillin (500 g/mL). In contrast, compounds with N-4-
µg/mL.
µ
µ
chlorophenyl or N-cyclohexyl substituents did not show activity against Bacillus subtilis in
the DIZ assay (Table 1). It was noticed that in general, the unsubstituted benzyl derivatives
6a, 6h, and 6o exhibited lower antibacterial activity against both Gram-negative and

Molecules 2021, 26, 2292
6 of 13
Gram-positive bacteria, which indicates the importance of the para substitution in the
benzyl moiety.
The in vitro antifungal activity of the target compounds 6a-u is presented in Table 2
.
They displayed potent in vitro antifungal activity against Candida tropicalis with an MIC
value as low as 2
a DIZ value around 27 mm against the tested Candida albicans strain. Most of compounds
6a-g exhibited MIC value of 250 g/mL being equal to that of the reference drug flu-
conazole except for compound 6f bearing 3, 4-dichlorbenzyl moiety, which revealed a
potent MIC value of 2 g/mL. Meanwhile, compounds 6o-u with N-cyclohexyl substituent
displayed moderate to good activity against the tested Candida albicans strain, in which
compounds 6p 6q, and 6r are the most active congeners with an MIC value of 32 g/mL.
In addition, compounds 6a-g exhibited MIC values of 125-1000 g/mL against Candida
µg/mL. Moreover, compounds 6a-g bearing N-phenyl substituent showed
µ
µ
,
µ
µ
glabrata in which compound 6d was the most active candidate. Furthermore, compounds 6s
(bearing 2,4-dichlorobenzyl and N-cyclohexyl substituents), 6t (bearing 3,4-dichlorobenzyl
and N-cyclohexyl substituents), and 6u (bearing 4-methylbenzyl and N-cyclohexyl sub-
stituents) manifested good activity toward Candida glabrata with MIC values of 62.5, 32,
and 16 µg/mL, respectively. Unexpectedly, the presence of 4-chlorophenyl moiety on
the triazole ring of compounds 6h-n seems to be unfavorable for their activities against
both Candida albicans and Candida glabrata. In addition, the influence of the para benzyl
substituent in increasing the antifungal activity can be detected from the lower antifungal
activity of the unsubstituted benzyl derivatives 6a and 6b.
The in vitro antifungal activity of the target compounds 6a-u against most of the tested
fungal strains was generally higher than their antibacterial activity. This could be attributed
to the presence of the triazole ring in their scaffold, which is consistent with the presence
of the triazole ring in a number of clinically used antifungal agents [33].
3. Experimental
3.1. General
The melting points were measured using a Gallenkamp melting point device and
are uncorrected. The NMR samples of the synthesized compounds 6a-u were dissolved
in DMSO-d6 and the NMR spectra were recorded using a Bruker NMR spectrometer
(Bruker, Reinstetten, Germany) at 500 MHz for 1H and 125.76 MHz for 13C at the Research
Center, College of Pharmacy, King Saud University, Saudi Arabia. TMS was used as an
internal standard, and chemical shift values were recorded in ppm on the
δ scale. The
1H NMR spectral data are represented as follows: chemical shifts, multiplicity (s, singlet;
d, doublet; t, triplet; m, multiplet) and number of protons. The 13C NMR spectral data
were represented as chemical shifts and type of carbon. Mass spectra were measured on
an Agilent Quadrupole 6120 LC/MS with ESI (electrospray ionization) source (Agilent
Technologies, Palo Alto, CA, USA). Elemental analysis was carried out at the Microanalysis
Laboratory, Cairo University, Cairo, Egypt, using an Elemental C, H, N analyzer Vario EL
III, Germany, and the results agreed favorably with the proposed structures within
±
0.4%
of the theoretical values. Silica gel thin layer chromatography (TLC) plates from Merck
(silica gel precoated aluminium plates with fluorescent indicator at 254 nm) were used for
thin layer chromatography. Visualization was performed by illumination with a UV light
source (254 nm). The microanalysis data, 1HNMR and 13CNMR of compound 6d are also
available in the Supplementary Materials.
3.2. Chemistry
3.2.1. Synthesis of Ethyl 1H-Indole-2-Carboxylate (2)
1H-indole-2-carboxylic acid was subjected to esterification in absolute ethanol and a
catalytic amount of sulfuric acid according to the reported method. The crude ester
2 was
used in the next step without further purification [34,35].

Molecules 2021, 26, 2292
7 of 13
3.2.2. Synthesis of 1H-Indole-2-Carbohydrazide (3)
Compound (0.875 g, 5 mmol) was suspended in absolute ethanol (10 mL), and
2
hydrazine hydrate (2.5 mL, 50 mmol) was added. The reaction mixture was subjected to
reflux for six hours under stirring. The reaction mixture was cooled to ambient temperature.
The precipitated solid was filtered and dried to afford the carbohydrazide 3 [34,36].
3.2.3. General Procedure for the Synthesis of the
Indole-1H-2-yl-4-Substituted-Thiosemicarbazides (4a-c)
A mixture of 1H-indole-2-carbohydrazide (3, 6 mmol) and the appropriate isothio-
cyanate (6 mmol) in absolute ethanol (10 mL) was heated under reflux for five hours,
cooled, the precipitated solid was filtered off and dried to give the thiosemicarbazides
4a-c [34], which were used without further purification [31].
3.2.4. General Procedure for the Synthesis of
5-(1H-Indol-2-yl)-4-Substituted-1,2,4-Triazole-3-Thiols (5a-c)
Aqueous 10% NaOH (3.3 mL) was added to the appropriate thiosemicarbazide deriva-
tive 4a-c (10 mmol). The reaction mixture was heated to reflux for three hours, cooled, and
neutralized with 5N HCl. The precipitated products 5a-c [34] were collected by filtration
and re-crystallized from ethanol [31].
3.2.5. General Procedure for the Synthesis of the Target Compounds 6a-u
The appropriate benzyl halide (1.1 mmol) was added dropwise to a stirred suspension
of the appropriate triazole derivative 5a-c (1 mmol) and anhydrous potassium carbonate
(1.1 mmol) in dry acetone (15 mL). The reaction mixture was heated to reflux under stirring
for two hours. The corresponding alkylated indoles 6a-u were collected by filtration and
recrystallized from ethanol [37].
2-(5-(Benzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-indole (6a) [34]. White powder m.p.
◦
250 C (yield 99%); IR (KBr):
ν
(cm−1) 3163 (NH), 3061 (C-H, aromatic), 2980 (C-H,
aliphatic), 1577, 1570 (C=N), 1494, 1450, 701; 1H NMR (DMSO-d6) ppm: 4.42 (s, 2H, CH2),
5.58 (d, J = 2.1 Hz, 1H, H-3), 6.94-6.96 (m, 1H, Har.), 7.14-7.15 (m, 1H, Har.), 7.27-7.29 (m,
1H, Har.), 7.30-7.31 (m, 2H, Har.), 7.34-7.36 (m, 3H, Har.), 7.39-7.40 (m, 2H, Har.), 7.43-7.44
(m, 1H, Har.), 7.62-7.64 (m, 2H, Har.), 7.67-7.69 (m, 1H, Har.), 12.01 (s, 1H, NH); 13C NMR
(DMSO-d6) ppm: 37.11 (-S-CH2), 101.8, 112.3, 120.2, 121.2, 123.6, 124.3, 127.6, 128.0, 128.4,
128.9, 129.5, 130.6, 131.1, 134.1, 136.9, 137.4, 149.6, 151.7 (Car., CHar., CN); MS m/z (ESI):
383.2 [M + H]+.
2-(5-(4-Nitrobenzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-indole (6b). Yellow powder
m.p. 271 ◦C (yield 90%); IR (KBr):
ν
(cm−1) 3147 (NH), 3061 (C-H, aromatic), 2987 (C-H,
aliphatic), 1597, 1590 (C=N), 1516, 1338 (NO2), 1490, 1309, 800; 1H NMR (DMSO-d6) ppm:
4.55 (s, 2H, CH2), 5.59 (s, 1H, H-3), 6.94-6.96 (m, 1H, Har.), 7.13-7.15 (m, 1H, Har.), 7.35 (d,
J = 7.7 Hz, 1H, Har.), 7.43-7.44 (m, 1H, Har.), 7.48 (d, J = 7.7 Hz, 2H, Har.), 7.64-7.70 (m,
5H, Har.), 8.18 (d, J = 8.4 Hz, 2H, Har.), 11.99 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 35.8
(-S-CH2), 101.9, 112.3, 120.2, 121.2, 123.6, 124.0, 124.2, 127.6, 128.4 130.7, 130.8, 131.2, 134.0,
136.9, 146.0, 147.1, 149.8, 151.3 (Car., CHar.); MS m/z (ESI): 428.1 [M+H]+.
4-((5-(1H-Indol-2-yl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)methyl)benzonitrile (6c). White
◦
powder m.p. 252 C (yield 90%); IR (KBr):
ν
(cm−1) 3273 (NH), 3100 (C-H, aromatic), 2980
(C-H, aliphatic), 2227 (CN), 1595, 1590 (C=N), 1494, 1450, 700; 1H NMR (DMSO-d6) ppm:
4.49 (s, 2H, CH2), 5.59 (d, J = 1.4 Hz, 1H, H-3), 6.94-6.96 (m, 1H, Har.), 7.14 (t, J = 7.7 Hz,
1H, Har.), 7.35 (d, J = 8.4 Hz, 1H, Har.), 7.44 (d, J = 8.4 Hz, 1H, Har.), 7.47 (d, J = 8.4 Hz, 2H,
Har.), 7.59 (d, J = 8.4 Hz, 2H, Har.), 7.64-7.66 (m, 2H, Har.), 7.68-7.70 (m, 1H, Har.), 7.79 (d,
J = 8.4 Hz, 2H, Har.), 11.99 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 36.2 (-S-CH2), 101.9,
110.6, 112.3, 119.2, 120.2, 121.2, 123.6, 124.2, 127.6, 128.4, 130.5, 130.7, 131.2, 132.8, 134.0,
136.9, 143.8, 149.8, 151.4 (Car., CHar., CN); MS m/z (ESI): 405.9 [M-H]−.
2-(5-(4-Chlorobenzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-indole (6d). White powder
m.p. 257 ◦C (yield 90%); IR (KBr):
ν
(cm−1) 3242 (NH), 3100 (C-H, aromatic), 2808 (C-H,

Molecules 2021, 26, 2292
8 of 13
aliphatic), 1600, 1597 (C=N), 1400, 1195, 700; 1H NMR (DMSO-d6) ppm: 4.41 (s, 2H, CH2),
5.58 (d, J = 1.4, 1H, H-3), 6.94-6.96 (m, 1H, Har.), 7.13-7.15 (m, 1H, Har.), 7.35 (d, J = 8.4,
1H, 1H, Har.), 7.37-7.40 (m, 4H, Har.), 7.43-7.45 (m, 3H, Har.), 7.65 (t, J = 7.7 Hz, 2H, Har.),
7.68-7.70 (m, 1H, Har.), 11.98 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 36.0 (-S-CH2), 101.9,
112.3, 120.2, 121.2, 123.6, 124.3, 127.6, 128.4, 128.9, 130.7, 131.2, 131.4, 132.6, 134.1, 136.8,
136.9, 149.7, 151.6 (Car., CHar.); MS m/z (ESI): 415.7 [M-H]−, 417 [(M + 2)-H]−.
2-(5-(2,4-Dichlorobenzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-indole (6e). White pow-
◦
der m.p. 255 C (yield 99%); IR (KBr):
ν
(cm−1) 3250 (NH), 3111 (C-H, aromatic), 2935
(C-H, aliphatic), 1600, 1597 (C=N), 1496, 1398, 800; 1H NMR (DMSO-d6) ppm: 4.45 (s, 2H,
CH2), 5.59 (s, 1H, H-3), 6.94-6.96 (m, 1H, Har.), 7.13-7.15 (m, 1H, Har.), 7.35 (d, J = 7.7 Hz,
1H, Har.), 7.41 (dd, J = 7.7, 2.1 Hz, 1H, Har.), 7.43 (d, J = 7.7 Hz, 1H, Har.), 7.46 (d, J = 7 Hz,
2H, Har.), 7.54 (d, J = 8.4 Hz, 1H, Har.), 7.64-6.66 (m, 3H, Har.), 7.69 (t, J = 7.7 Hz, 1H, Har.),
12.00 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 34.7 (-S-CH2), 101.9, 112.3, 120.2, 121.2, 123.7,
124.2, 127.6, 127.9, 128.4, 129.5, 130.7, 131.2, 133.2, 133.7, 134.0, 134.2, 134.7, 136.9, 149.9,
150.9 (Car., CHar.); MS m/z (ESI): 448.8 [M-H]−, 450.8 [(M + 2)-H]−, 452.9 [(M + 4)-H]−.
2-(5-(3,4-Dichlorobenzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-indole (6f). White pow-
◦
der m.p. 215 C (yield 88%); IR (KBr):
ν
(cm−1) 3207 (NH), 3057 (C-H, aromatic), 2918 (C-H,
aliphatic), 1597, 1566 (C=N), 1496, 1398, 700; 1H NMR (DMSO-d6) ppm: 4.41 (s, 2H, CH2),
5.59 (s, 1H, H-3), 6.95 (t, J = 7.7 Hz, 1H, Har.), 7.13-7.15 (m, 1H, Har.), 7.35-7.38 (m, 2H, Har.),
7.43 (d, J = 8.4 Hz, 1H, Har.), 7.47 (d, J = 7.7 Hz, 2H, Har.), 7.58 (d, J = 8.4 Hz, 2H, Har.),
7.64-7.66 (m, 2H, Har.), 7.68-7.70 (m, 2H, Har.), 12.00 (s, 1H, NH); 13C NMR (DMSO-d6) ppm:
35.41 (-S-CH2), 101.9, 112.3, 120.2, 121.2, 123.6, 124.2, 127.6, 128.4, 129.8, 130.5, 130.7, 131.−0,
131.2, 131.3, 131.6, 134.0, 136.9, 139.3, 149.9, 151.4 (Car., CHar.); MS m/z (ESI): 448.8 [M-H] ,
450.8 [(M + 2)-H]−, 452.5 [(M + 4)-H]−.
2-(5-((4-Methylbenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1H-indole (6g): White powder
m.p. 274 ◦C (yield 95%); IR (KBr):
ν
(cm−1) 3336 (NH), 3111 (C-H, aromatic), 2933 (C-H,
aliphatic), 1647,1630 (C=N), 1436, 1344, 701; 1H NMR (DMSO-d6) ppm: 2.28 (s, 3H, CH3),
4.37 (s, 2H, CH2), 5.57 (s, 1H, H-3), 6.95 (t, J = 7.7 Hz, 1H, Har.), 7.11-7.13 (m, 3H, Har.),
7.22 (d, J = 7.7 Hz, 2H, Har.), 7.35 (d, J = 7.7 Hz, 1H, Har.), 7.40 (d, J = 7 Hz, 2H, Har.), 7.43
(d, J = 8.4 Hz, 1H, Har.), 7.63-7.65 (m, 2H, Har.), 7.67-7.69 (m, 1H, Har.), 12.00 (s, 1H, NH);
13C NMR (DMSO-d6) ppm: 21.1 (CH3), 36.9 (-S-CH2), 101.7, 112.3, 120.2, 121.2, 123.6,124.3,
127.6, 128.4, 129.4, 129.5, 130.6, 131.1, 134.1, 134.3, 136.9, 137.3, 149.6, 151.8 (Car., CHar.); MS
m/z: 394.9 [M-H]−.
2-(5-(Benzylthio)-4-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)-1H-indole (6h): beige powder
m.p. 245 ◦C (yield 70%); IR (KBr):
ν
(cm−1) 3147 (NH), 3089 (C-H, aromatic), 2924 (C-H,
aliphatic), 1595, 1571 (C=N), 1494, 1436, 800, 701; 1H NMR (DMSO-d6) ppm: 4.41 (s, 2H,
CH2), 5.67 (s, 1H, H-3), 6.96 (t, J = 7 Hz, 1H, Har.), 7.15 (t, J = 7 Hz, 1H, Har.), 7.27-7.29 (m,
1H, Har.), 7.31-7.32 (m, 4H, Har.), 7.41-7.44 (m, 2H, Har.), 7.46 (d, J = 8.4 Hz, 2H, Har.), 7.70
(d, J = 8.4 Hz, 2H, Har.), 11.99 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 37.3 (-S-CH2), 102.0,
112.3, 120.2, 121.3, 123.6, 124.1, 127.6, 128.1, 128.9, 129.5, 130.4, 130.7, 133.0, 135.7, 136.9,
137.4, 149.5, 151.7 (Car., CHar.); MS m/z: 414.9 [M-H]−, 416.9 [(M + 2)-H]−.
2-(5-(4-Nitrobenzylthio)-4-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)-1H-indole (6i). Yellow
◦
powder m.p. 217 C (yield 82%); IR (KBr):
ν
(cm−1) 3248 (NH), 3103 (C-H, aromatic), 2924
(C-H, aliphatic), 1598, 1471 (C=N), 1492, 1350 (NO2), 800, 701; 1H NMR (DMSO-d6) ppm:
4.53 (s, 2H, CH2), 5.69 (s, 1H, H-3), 6.96 (t, J = 7.7 Hz, 1H, Har.), 7.15 (t, J = 7.7 Hz, 1H,
Har.), 7.42 (d, J = 8.4 Hz, 2H, Har.), 7.55(d, J = 9.1 Hz, 2H, Har.), 7.66 (d, J = 8.4 Hz, 2H,
Har.), 7.72 (d, J = 8.4 Hz, 2H, Har.), 8.18 (d, J = 8.4 Hz, 2H, Har.), 11.97 (s, 1H, NH); 13
C
NMR (DMSO-d6) ppm: 36.0 (-S-CH2), 102.0, 112.3, 120.2, 121.3, 123.7, 124.0, 127.6, 130.4,
130.7, 130.8, 132.9, 135.8, 136.9, 145.9, 147.2, 149.7, 151.2, 207.1 (Car., CHar.); MS m/z: 462.9
[M+H]+.
4-((4-(4-Chlorophenyl)-5-(1H-indol-2-yl)-4H-1,2,4-triazol-3-ylthio)methyl)benzonitrile (6j):
Pale Yellow powder m.p. 190 ◦C (yield 67%); IR (KBr):
ν
(cm−1) 3354 (NH), 3091 (C-H,
aromatic), 2920 (C-H, aliphatic), 2225 (CN), 1598, 1590 (C=N), 1498, 1454, 800, 701; 1H NMR
(DMSO-d6) ppm: 4.48 (s, 2H, CH2), 5.69 (s, 1H, H-3), 6.96 (t, J = 7.7 Hz, 1H, Har.), 7.15 (t,

Molecules 2021, 26, 2292
9 of 13
J = 7.7 Hz, 1H, Har.), 7.43 (d, J = 8.4 Hz, 2H, Har.), 7.54 (d, J = 8.4 Hz, 2H, Har.), 7.58 (d,
J = 7.7 Hz, 2H, Har.), 7.70 (d, J = 7.70 Hz, 1H, Har.), 7.73 (d, J = 9.1 Hz, 2H, Har.), 7.79 (d,
J = 7.7 Hz, 2H, Har.), 11.97 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 36.0 (-S-CH2), 102.0,
112.3, 120.3, 121.4, 123.8, 124.1, 127.7, 130.4, 130.5, 130.8, 132.9, 133.1, 133.2, 133.7, 135.9,
136.8, 143.9, 149.6, 151.5 (Car., CHar., CN); MS m/z: 439.9 [M-H]−, 442 [(M + 2)-H]−.
2-(5-(4-Chlorobenzylthio)-4-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)-1H-indole (6k): Yellow
◦
powder m.p. 235 C (yield 37%); IR (KBr):
ν
(cm−1) 3178 (NH), 3032 (C-H, aromatic), 2918
(C-H, aliphatic), 1490, 1400 (C=N), 1350, 800, 701; 1H NMR (DMSO-d6) ppm: 4.40 (s, 2H,
CH2), 5.69 (s, 1H, H-3), 6.95-6.97 (m, 1H, Har.), 7.15 (t, J = 7.7 Hz, 1H, Har.), 7.37-7.40 (m, 4H,
Har.), 7.42-7.44 (m, 2H, Har.), 7.52 (d, J = 8.4 Hz, 2H, Har.), 7.72 (d, J = 8.4 Hz, 2H, Har.) 11.99
(s, 1H, NH); 13C NMR (DMSO-d6) ppm: 36.2 (-S-CH2), 102.0, 112.3, 120.2, 121.3, 123.7, 124.1,
127.6, 128.9, 130.4, 130.8, 131.4, 132.6, 132.9, 135.8, 136.7, 136.9, 149.5, 151.5 (Car., CHar.); MS
m/z (ESI): 450.8 [M-H]−, 452.7 [(M + 2)-H]−, 454 [(M + 4)-H]−.
2-(5-(2,4-Dichlorobenzylthio)-4-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)-1H-indole (6l): White
◦
powder m.p. 239 C (yield 79%); IR (KBr):
ν
(cm−1) 3155 (NH), 3091 (C-H, aromatic), 2922
(C-H, aliphatic), 1591, 1580 (C=N), 1490, 1388, 800, 701. 600; 1H NMR (DMSO-d6) ppm: 4.43
(s, 2H, CH2), 5.70 (s, 1H, H-3), 6.96 (t, J = 7.7 Hz, 1H, Har.), 7.14-7.16 (m, 1H, Har.), 7.40-7.42
(dd, J =8.4, 2.1 Hz, 1H, Har.), 7.43 (d, J = 7.7 Hz, 2H, Har.), 7.53-7.57 (m, 3H, Har.), 7.64 (d,
J = 2.1 Hz, 1H, Har.), 7.72-7.73 (m, 2H, Har.), 12.00 (s, 1H, NH); 13C NMR (DMSO-d6) ppm:
34.8 (-S-CH2), 102.13, 112.3, 120.2, 121.3, 123.7, 124.1, 127.6, 127.9, 129.5, 130.4, 130.4, 130.−7,
132.9, 133.2, 133.7, 134.7, 135.8, 137.0, 149.8, 150.8 (Car., CHar.); MS m/z (ESI): 482.8 [M-H] ,
484.8 [(M + 2) + H]+, 486.8 [(M+4)-H]−, 488.7 [(M+6)-H]−.
2-(5-(3,4-Dichlorobenzylthio)-4-(4-chlorophenyl)-4H-1,2,4-triazol-3-yl)-1H-indole (6m): Beige
◦
powder m.p. 229 C (yield 83%); IR (KBr):
ν
(cm−1) 3251 (NH), 3053 (C-H, aromatic), 2922
1
(C-H, aliphatic), 1620, 1597 (C=N), 1496, 1342, 800, 701, 600; H NMR (DMSO-d6) ppm:
4.39 (s, 2H, CH2), 5.70 (s, 1H, H-3), 6.96 (t, J = 7 Hz, 1H, Har.), 7.15 (t, J = 7 Hz, 1H, Har.),
7.36-7.38 (m, 1H, Har.), 7.43 (d, J = 7 Hz, 2H, Har.), 7.55-7.67 (m, 3H, Har.), 7.67 (s, 1H, Har.),
7.72-7.73 (m, 2H, Har.), 12.00 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 35.7 (-S-CH2), 102.1,
112.3, 120.2, 121.3, 123.7, 124.1, 127.6, 129.8, 130.4, 130.5, 130.7, 131.0, 131.3, 131.5, 132.9,
135.8, 137.0, 139.2, 149.7, 151.3 (Car., CHar.,CClar); MS m/z (ESI): 482.8 [M-H]−, 484.8 [(M +
2) + H]+, 486.8 [(M+4)-H]−, 488.7 [(M+6)-H]−.
2-(4-(4-Chlorophenyl)-5-((4-methylbenzyl)thio)-4H-1,2,4-triazol-3-yl)-1H-indole (6n): Yel-
low powder m.p. 295 ◦C (yield 80%); IR (KBr):
ν
(cm−1) 3203 (NH), 3061 (C-H, aromatic),
2918 (C-H, aliphatic), 1685, 1577 (C=N), 1492, 1346, 800, 701; 1H NMR (DMSO-d6) ppm: 2.10
(s, 3H, CH3), 4.36 (s, 2H, CH2), 5.68 (s, 1H, H-3), 6.95-6.97 (m, 1H, Har.), 7.12 (d, J = 7.7 Hz,
2H, Har.), 7.15 (t, J = 7.7 Hz, 1H, Har.), 7.21 (d, J = 8.4 Hz, 2H, Har.), 7.42-7.46 (m, 4H, Har.),
7.71 (d, J = 9.1 Hz, 2H, Har.), 12.00 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 21.18 (CH3), 37.1
(-S-CH2), 101.9, 112.3, 120.2, 121.3, 123.7, 124.1, 127.6, 127.9, 129.5, 130.4, 130.7, 133.0, 134.2,
135.7, 136.9, 137.3, 149.5, 151.7, (Car., CHar.); MS m/z: 428.8 [M-H]+, 430.9 [(M + 2)-H]−
2-(5-(Benzylthio)-4-cyclohexyl-4H-1,2,4-triazol-3-yl)-1H-indole (6o): White powder m.p.
◦
> 300 C (yield 84%); IR (KBr):
ν
(cm−1) 3448 (NH), 3138 (C-H, aromatic), 2939 (C-H,
aliphatic), 1676, 1660 (C=N), 1571, 1460, 701; 1H NMR (DMSO-d6) ppm: 1.05-1.11 (m, 1H,
cyclohexyl), 1.26-1.32 (m, 2H, cyclohexyl), 1.60 (d, J = 13.3 Hz, 1H, cyclohexyl), 1.76 (s, 4H,
cyclohexyl), 1.97-2.03 (m, 2H, cyclohexyl), 4.34-4.38 (m, 1H, cyclohexyl), 4.56 (s, 2H, CH2),
6.75 (s, 1H, H-3), 7.07-7.09 (m, 1H, Har.), 7.19-7.22 (m, 1H, Har.), 7.28-7.30 (m, 1H, Har.),
7.33-7.35 (m, 2H, Har.), 7.43 (d, J = 7 Hz, 2H, Har), 7.47 (d, J = 7.7 Hz, 1H, Har.), 7.65 (d, J = 8.4
Hz, 1H, Har.), 11.83 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 24.9, 25.7, 31.1(cyclohexyl),
37.7 (-S-CH2), 56.7 (cyclohexyl), 103.6, 112.3, 120.2, 121.3, 123.4, 124.2, 127.9, 128.0, 128.9,
129.5, 136.9, 137.4, 149.7, 149.8 (Car., CHar); MS m/z: 386.9 [M-H]−.
2-(5-(4-Nitrobenzylthio)-4-cyclohexyl-4H-1,2,4-triazol-3-yl)-1H-indole (6p): Yellow pow-
◦
der m.p. 255 C (yield 59%); IR (KBr):
ν
(cm−1) 3165 (NH), 3090 (C-H, aromatic), 2931
(C-H, aliphatic), 1618, 1598 (C=N), 1517, 1346 (NO2), 1444, 1436, 701; 1H NMR (DMSO-d6)
ppm: 1.08-1.14 (m, 1H, cyclohexyl), 1.28-1.34 (m, 2H, cyclohexyl), 1.62 (d, J = 13.3 Hz, 1H,
cyclohexyl), 1.78-1.79 (m, 4H, cyclohexyl), 1.97-2.03 (m, 2H, cyclohexyl), 4.35-4.39 (m, 1H,

Molecules 2021, 26, 2292
10 of 13
cyclohexyl), 4.70 (s, 2H, CH2), 6.78 (s, 1H, H-3), 7.07-7.09 (m, 1H, Har.), 7.20-7.22 (m, 1H,
Har.), 7.47 (d, J = 8.4 Hz, 1H, Har.), 7.65 (d, J = 7.7 Hz, 1H, Har.), 7.74 (d, J = 9.1 Hz, 2H, Har),
8.21 (d, J = 8.4 Hz, 2H, Har.), 11.83 (s, 1H, NH); 13C NMR (DMSO-d6) ppm: 24.9, 25.7, 31.1
(cyclohexyl), 36.5 (-S-CH2), 56.7 (cyclohexyl), 103.7, 112.3, 120.2, 121.3, 123.4, 124.0, 124.1,
127.9, 130.8, 136.9, 146.1, 147.2, 149.1, 150.1 (Car., CHar); MS m/z: 431.9 [M-H]−.
4-((4-Cyclohexyl-5-(1H-indol-2-yl)-4H-1,2,4-triazol-3-ylthio)methyl)benzonitrile (6q): White
◦
powder m.p. 235 C (yield 56%); IR (KBr):
ν
(cm−1) 3192 (NH), 3057 (C-H, aromatic), 2970
(C-H, aliphatic), 2225 (CN), 1685, 1606 (C=N), 1436, 701; 1H NMR (DMSO-d6) ppm: 1.10-1.12
(m, 1H, cyclohexyl), 1.29-1.34 (m, 2H, cyclohexyl), 1.62 (d, J = 12.6 Hz, 1H, cyclohexyl), 1.78
(d, J = 10.5 Hz, 4H, cyclohexyl), 1.96-2.02 (m, 2H, cyclohexyl), 4.37-4.38 (m, 1H, cyclohexyl),
4.65 (s, 2H, CH2), 6.78 (s, 1H, H-3), 7.07-7.09 (m, 1H, Har.), 7.20-7.22 (m, 1H, Har.), 7.46 (d,
J = 8.4 Hz, 1H, Har.), 7.65-7.67 (m, 3H, Har.), 7.82 (d, J = 8.4 Hz, 2H, Har), 11.83 (s, 1H, NH);
13C NMR (DMSO-d6) ppm: 24.9, 25.7, 31.1 (cyclohexyl), 36.8 (-S-CH2), 56.7 (cyclohexyl),
103.6, 110.59 112.3, 119.2, 120.3, 121.4, 123.4, 124.1, 127.9, 130.5, 132.8, 136.9, 143.9, 149.1,
150.1 (Car., CHar); MS m/z: 411.9 [M-H]−.
2-(5-(4-Chlorobenzylthio)-4-cyclohexyl-4H-1,2,4-triazol-3-yl)-1H-indole (6r): White powder
m.p. 250 ◦C (yield 85%); IR (KBr):
ν
(cm−1) 3176 (NH), 3055 (C-H, aromatic), 2931 (C-H,
aliphatic), 1589, 1580 (C=N), 1494, 1438, 800, 701; 1H NMR (DMSO-d6) ppm: 1.07-1.13 (m,
1H, cyclohexyl), 1.28-1.34 (m, 2H, cyclohexyl), 1.61 (d, J = 13.3 Hz, 1H, cyclohexyl), 1.78 (d,
J = 10.5 Hz, 4H, cyclohexyl), 1.97-2.03 (m, 2H, cyclohexyl), 4.35-4.39 (m, 1H, cyclohexyl),
4.56 (s, 2H, CH2), 6.79 (s, 1H, H-3), 7.07-7.09 (m, 1H, Har.), 7.20-7.22 (m, 1H, Har.), 7.41 (d,
J = 8.4 Hz, 2H, Har.), 7.47-7.48 (m, 3H, Har.), 7.66 (d, J = 8.4 Hz, 1H, Har), 11.83 (s, 1H, NH);
13C NMR (DMSO-d6) ppm: 24.9, 25.7, 31.1 (cyclohexyl), 36.7 (-S-CH2), 56.7 (cyclohexyl),
103.6, 112.3, 120.2, 121.3, 123.4, 124.2, 127.9, 128.9, 131.4, 132.6, 136.9, 137.0, 149.5, 149.9
(Car., CHar); MS m/z (ESI): 420.9 [M-H]−, 422.9 [(M + 2)-H]−.
2-(5-(2,4-Dichlorobenzylthio)-4-cyclohexyl-4H-1,2,4-triazol-3-yl)-1H-indole (6s): Yellow
◦
powder m.p. 200 C (yield 60%); IR (KBr):
ν
(cm−1) 3288 (NH), 3078 (C-H, aromatic),
1
2927 (C-H, aliphatic), 1570, 1560 (C=N), 1467, 1436, 800, 750, 701; H NMR (DMSO-d6)
ppm: 1.07-1.13 (m, 1H, cyclohexyl), 1.28-1.34 (m, 2H, cyclohexyl), 1.61 (d, J = 12.6 Hz,
1H, cyclohexyl), 1.77 (s, 4H, cyclohexyl), 1.95-2.01 (m, 2H, cyclohexyl), 4.34-4.39 (m, 1H,
cyclohexyl), 4.61 (s, 2H, CH2), 6.79 (s, 1H, H-3), 7.07-7.09 (m, 1H, Har.), 7.20-7.22 (m, 1H,
Har.), 7.42 (dd, J = 8.4, 2.1 Hz, 1H, Har.), 7.47 (d, J = 7.7 Hz, 1H, Har.), 7.55 (d, J = 8.4 Hz,
1H, Har), 7.66-7.68 (m, 1H, Har.), 7.70 (d, J = 2.1 Hz, 1H, Har.), 11.84 (s, 1H, NH); 13C NMR
(DMSO-d6) ppm: 24.9, 25.7, 31.2 (cyclohexyl), 35.5 (-S-CH2), 56.8 (cyclohexyl), 103.6, 112.3,
120.2, 121.3, 123.4, 124.1, 127.9, 129.5, 133.2, 133.8, 134.1, 134.8, 136.9, 148.9, 149.9 (Car.,
CHar); MS m/z (ESI): 454.9 [M-H]−, 456.8 [(M + 2)-H]−, 458.9 [(M + 4)-H]−.
2-(5-(3,4-Dichlorobenzylthio)-4-cyclohexyl-4H-1,2,4-triazol-3-yl)-1H-indole (6t): Yellow
◦
powder m.p. 202 C (yield 53%); IR (KBr):
ν
(cm−1) 3186 (NH), 3086 (C-H, aromatic),
1
2935 (C-H, aliphatic), 1577, 1540 (C=N), 1494, 1436, 800, 760, 701; H NMR (DMSO-d6)
ppm: 1.10-1.14 (m, 1H, cyclohexyl), 1.30-1.35 (m, 2H, cyclohexyl), 1.62 (d, J = 12.6 Hz, 1H,
cyclohexyl), 1.79 (d, J = 11.2 Hz, 4H, cyclohexyl), 1.98-2.02 (m, 2H, cyclohexyl), 4.36-4.40
(t, J = 12.6 Hz, 1H, cyclohexyl), 4.57 (s, 2H, CH2), 6.79 (s, 1H, H-3), 7.07-7.09 (m, 1H, Har.),
7.20-7.22 (m, 1H, Har.), 7.45-7.47 (m, 2H, Har.), 7.61 (d, J = 8.4 Hz, 1H, Har.), 7.66 (d, J = 8.4
Hz, 1H, Har), 7.75 (d, J = 2.1 Hz, 1H, Har.), 11.84 (s, 1H, NH); 13C NMR (DMSO-d6) ppm:
24.9, 25.7, 31.1 (cyclohexyl), 36.09 (-S-CH2), 56.8 (cyclohexyl), 103.6, 112.3, 120.2, 121.4,
123.4, 124.1, 127.9, 129.9, 130.5, 131.0, 131.3, 131.6, 137.0, 139.3, 149.3, 150.0 (Car., CHar); MS
m/z (ESI): 454.9 [M-H]−, 456.8 [(M + 2)-H]−, 458.9 [(M + 4)-H]−.
2-(4-Cyclohexyl-5-((4-methylbenzyl)thio)-4H-1,2,4-triazol-3-yl)-1H-indole (6u): Yellow pow-
◦
der m.p. 190 C (yield 84%); IR (KBr):
ν
(cm−1) 3200 (NH), 3055 (C-H, aromatic), 2922 (CH,
aliphatic), 1637,1640 (C=N), 1494, 1436, 800, 700; 1H NMR (DMSO-d6) ppm: 1.07-1.12 (m,
1H, cyclohexyl), 1.27-1.33 (m, 2H, cyclohexyl), 1.61 (d, J = 12.5 Hz, 1H, cyclohexyl), 1.77
(d, J = 11.2 Hz, 4H, cyclohexyl), 1.98-2.04 (m, 2H, cyclohexyl), 2.28 (s, 3H, CH3), 4.35-4.39
(m, 1H, cyclohexyl), 4.52 (s, 2H, CH2), 6.79 (s, 1H, H-3), 7.07-7.09 (m, 1H, Har.), 7.15 (d,
J = 7.7 Hz, 2H, Har.), 7.19-7.22 (m, 1H, Har.), 7.33 (d, J = 7.7 Hz, 2H, Har.), 7.47 (d, J = 8.4

Molecules 2021, 26, 2292
11 of 13
Hz, 1H, Har), 7.66 (d, J = 8.4 Hz, 1H, Har), 11.84 (s, 1H, NH); 13C NMR (DMSO-d6) ppm:
21.2 (CH3), 24.9, 25.7, 31.1 (cyclohexyl), 37.52 (-S-CH2), 56.7 (cyclohexyl), 103.5, 112.3, 120.2,
121.3, 123.4, 124.3, 128.0, 129.5, 129.6, 134.3, 136.9, 137.3, 149.7, 149.8 (Car., CHar); MS m/z:
400.9 [M-H]−.
3.3. Antimicrobial Evaluation
3.3.1. Materials
Dimethyl sulfoxide (100%) was used to dissolve the standards ampicillin, fluconazole,
and the tested compounds 6a-u to give an initial concentration of 1 mg/mL.
3.3.2. Organisms
All the tested strains bacterial and Candida in this study were provided from King
Khaled University hospital, Microbiology Department. Bacterial strains were divided into
Gram-positive bacteria namely Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC
6633). Escherichia coli (ATCC 25966). Pseudomonas aeruginosa (ATCC 27853) and Salmonella
(LT2) as negative strains. Candida tropicalis (ATCC 66019), Candida glabrata (clinical isolates)
and Candida albicans (ATCC10231) as fungal strains. All these microorganisms were pre-
cultured on nutrient agar (Oxoid) and PDA (potato dextrose agar, Oxoid). Then, 0.5
McFarland turbidity of each microbial suspension was prepared in 5 mL nutrient broth
tubes for the antibacterial assays (Agar well diffusion technique and MIC test).
3.3.3. Agar Well Diffusion Technique
The antimicrobial activity of the tested compounds was determined using the agar
well diffusion method. The prepared microbial suspensions were loaded on the surface
of Mueller Hinton (Oxoid) plates using a sterile cotton swab. The agar surface was perfo-
rated with a sterile cork borer (6mm), and 100 µL of each compound (1000 µg/mL) were
transf◦erred into each well correspondingly. Plates were incubated aerobically for 18-24 h
at 37 C. Diameters of the inhibition zones were measured around each well and were
recorded in mm as average of triplicate trials. DMSO was used as a negative control, while
ampicillin and fluconazole were used as positive controls [38].
3.3.4. MIC Test
The minimum inhibitory concentration of the synthesized compounds in the current
study against the microbial strains was determined using the micro-broth dilution assay
(MIC). The concentrations of the extracts used for MICs ranged from 100% (1mg/mL) to
0.25%. In brief, 100
µL of Mueller–Hinton broth (MH) was first loaded in polystyrene
sterile flat-bottom 96-well plates; then, 100
µL of each compound in study was added, and
two fold dilutions were performed until the 0.25% concentration is reached; finally, 50 ◦
µL
of each of the bacterial suspensions was loaded respectively. Plates were incubated at 37 C
for 18-24 hrs, following incubation plates that were read at 600 nm with an Elisa reader
(Biotech). The first column of the 96-well plate was loaded with only with Mueller Hinton
broth; the bacterial suspension was a positive control and the last column was the negative
control consisting of the medium broth (MH) and the extract correspondingly. The lowest
concentration of compounds where no visible bacterial growth was observed and recorded
after 24 h of incubation was considered as the MIC. The experiments were repeated in
triplicate [39].
4. Conclusions
A series of indole-1,2,4-triazole molecular hybrids 6a-u was synthesized and their
structures were confirmed by different spectroscopic tools. Their in vitro antibacterial
evaluation revealed that they possess good to moderate antibacterial activity against most
of the tested Gram-negative strains with DIZ values in the range of 11-15 mm and MIC
value around 250
µg/mL. Meanwhile, their antifungal evaluation showed higher activity
especially against Candida tropicalis with MIC value as low as 2
µg/mL for most of the

Molecules 2021, 26, 2292
12 of 13
tested compounds. Moreover, compound 6f bearing N-phenyl and 3, 4-dichlorbenzyl
moieties revealed a potent growth inhibition zone of 26 mm in the DIZ assay and MIC
value of 2 µg/mL against Candida albicans. In addition, the antibacterial and the antifungal
activities results imply the significance of the para benzyl substituent.
Supplementary Materials: The microanalysis data are available in Table S1 in the supplementary
1
materials. HNMR and 13CNMR of compound 6d are also available in the supplementary materials.
Author Contributions: M.A.A. synthesized the target compounds 6a-u and carried out their spectro-
scopic identification. M.S.A.-M. contributed in the spectroscopic identification. S.I.B. and N.M.S.M.
performed the in vitro antimicrobial evaluation of the synthesized compounds 6a-u. R.I.A.-W. and
M.I.A. proposed the work and prepared the manuscript for publication. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the Deanship of Scientific Research at King Saud University,
Research Group Project No. RG-1440-140.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or Supplementary Materials.
Acknowledgments: The authors would like to extend their sincere appreciation to the Deanship of
Scientific Research at King Saud University for its funding of this research through the Research
Group Project No. RG-1440-140
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the synthesized compounds are available from the corresponding
authors.
References
1.
2.
Brown, G.D.; Denning, D.W.; Levitz, S.M. Tackling human fungal infections. Science 2012, 336, 647. [CrossRef] [PubMed]
Roberts, R.R.; Hota, B.; Ahmad, L.; Scott, D.; Foster, S.D.; Abbasi, F.; Schabowski, S.; Kampe, L.M.; Gr-Ciavarella, G.; Supino, M.;
et al. Hospital and societal costs of antimicrobial-resistant infections in a chicago teaching hospital: Implications for antibiotic
stewardship. Clin. Infect. Dis. 2009, 49, 1175–1184. [CrossRef] [PubMed]
3.
4.
Zhang, F.F.; Gan, L.L.; Zhou, C.H. Synthesis, antibacterial and antifungal activities of some carbazole derivatives. Bioorganic Med.
Chem. Lett. 2010, 20, 1881–1884. [CrossRef] [PubMed]
Liu, C.; Bayer, A.; Cosgrove, S.E.; Daum, R.S.; Fridkin, S.K.; Gorwitz, R.J.; Kaplan, S.L.; Karchmer, A.W.; Levine, D.P.; Murray,
B.E.; et al. Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant
Staphylococcus aureus infections in adults and children. Clin. Infect. Dis. 2011, 52. [CrossRef] [PubMed]
Kaku, N.; Morinaga, Y.; Takeda, K.; Kosai, K.; Uno, N.; Hasegawa, H.; Miyazaki, T.; Izumikawa, K.; Mukae, H.; Yanagihara, K.
Antimicrobial and immunomodulatory effects of tedizolid against methicillin-resistant Staphylococcus aureus in a murine model
of hematogenous pulmonary infection. Int. J. Med. Microbiol. 2016, 306, 421–428. [CrossRef] [PubMed]
Inman, M.; Moody, C.J. Indole synthesis-something old, something new. Chem. Sci. 2013, 4, 29–41. [CrossRef]
Berger, M.; Gray, J.A.; Roth, B.L. The expanded biology of serotonin. Annu. Rev. Med. 2009, 60, 355–366. [CrossRef]
Sharma, V.; Kumar, P.; Pathaka, D. Biological importance of the indole nucleus in recent years: A comprehensive review. J.
Heterocycl. Chem. 2010, 47, 491–502. [CrossRef]
5.
6.
7.
8.
9.
Zhang, M.Z.; Chen, Q.; Yang, G.F. A review on recent developments of indole-containing antiviral agents. Eur. J. Med. Chem.
2015, 89, 421–441. [CrossRef]
10. Frost, J.M.; Dart, M.J.; Tietje, K.R.; Garrison, T.R.; Grayson, G.K.; Daza, A.V.; El-Kouhen, O.F.; Yao, B.B.; Hsieh, G.C.; Pai, M.; et al.
Indol-3-ylcycloalkyl ketones: Effects of N1 substituted indole side chain variations on CB2 cannabinoid receptor activity. J. Med.
Chem. 2010, 53, 295–315. [CrossRef]
11. Rathod, A.S.; Godipurge, S.S.; Biradar, J.S. Microwave Assisted, Solvent-Free, “Green” Synthesis of Novel Indole Analogs as
Potent Antitubercular and Antimicrobial Agents and Their Molecular Docking Studies. Russ. J. Gen. Chem. 2018, 88, 1238–1246.
[CrossRef]
12. Niemyjska, M.; MacIejewska, D.; Wolska, I.; Truszkowski, P. Synthesis, structural investigations, and anti-cancer activity of new
methyl indole-3-carboxylate derivatives. J. Mol. Struct. 2012, 1026, 30–35. [CrossRef]
13. Varvaresou, A.; Tsantili-Kakoulidou, A.; Siatra-Papastaikoudi, T.; Tiligada, E. Synthesis and biological evaluation of indole con-
taining derivatives of thiosemicarbazide and their cyclic 1,2,4-triazole and 1,3,4-thiadiazole analogs. Arzneimittel-Forschung/Drug
Res. 2000, 50, 48–54. [CrossRef] [PubMed]

Molecules 2021, 26, 2292
13 of 13
14. Pintilie, O.; Profire, L.; Sunel, V.; Popa, M.; Pui, A. Synthesis and Antimicrobial Activity of Some New 1,3,4-Thiadiazole and
1,2,4-Triazole Compounds Having a D,L-Methionine Moiety. Molecules 2007, 12, 103–113. [CrossRef] [PubMed]
15. Zou, X.J.; Lai, L.H.; Jin, G.Y.; Zhang, Z.X. Synthesis, fungicidal activity, and 3D-QSAR of pyridazinone-substituted 1,3,4-
oxadiazoles and 1,3,4-thiadiazoles. J. Agric. Food Chem. 2002, 50, 3757–3760. [CrossRef] [PubMed]
16. Chen, H.; Li, Z.; Han, Y. Synthesis and fungicidal activity against Rhizoctonia solani of 2-alkyl (alkylthio)-5-pyrazolyl-1,3,4-
oxadiazoles (thiadiazoles). J. Agric. Food Chem. 2000, 48, 5312–5315. [CrossRef] [PubMed]
17. Banu, K.M.; Dinakar, A.; Ananthanarayanan, C. Synthesis, characterization, antimicrobial studies and pharmacological screening
of some substituted 1,2,3-triazoles. Indian J. Pharm. Sci. 1999, 61, 202–205.
18. Keri, R.S.; Patil, S.A.; Budagumpi, S.; Nagaraja, B.M. Triazole: A Promising Antitubercular Agent. Chem. Biol. Drug Des. 2015, 86,
410–423. [CrossRef] [PubMed]
19. Duran, A.; Dogan, H.N.; Rollas, S. Synthesis and preliminary anticancer activity of new 1,4-dihydro-3-(3-hydroxy-2-naphthyl)-4-
substituted-5H-1,2,4-triazoline-5-thiones. Il Farm. 2002, 57, 559–564. [CrossRef]
20. Gujjar, R.; Marwaha, A.; El Mazouni, F.; White, J.; White, K.L.; Creason, S.; Shackleford, D.M.; Baldwin, J.; Charman, W.N.;
Buckner, F.S.; et al. Identification of a metabolically stable triazolopyrimidine-based dihydroorotate dehydrogenase inhibitor
with antimalarial activity in mice. J. Med. Chem. 2009, 52, 1864–1872. [CrossRef]
21. Johns, B.A.; Weatherhead, J.G.; Allen, S.H.; Thompson, J.B.; Garvey, E.P.; Foster, S.A.; Jeffrey, J.L.; Miller, W.H. The use of
oxadiazole and triazole substituted naphthyridines as HIV-1 integrase inhibitors. Part 1: Establishing the pharmacophore.
Bioorganic Med. Chem. Lett. 2009, 19, 1802–1806. [CrossRef] [PubMed]
22. Schenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Filippelli, W.; Falcone, G.; Giordano, L.; Vitelli, M.R. 3-Arylsulphonyl-
5-arylamino-1,3,4-thiadiazol-2(3H)ones as anti-inflammatory and analgesic agents. Bioorg. Med. Chem. 2001, 9, 2149–2153.
[CrossRef]
23. Ghodhani, D.R.; Jogel, A.A.; Sanghani, A.M.; Jignasu, P.M. Synthesis and biological screening of 1,2,4-triazole derivatives. Indian
J. Chem. 2015, 54B, 556–564.
24. Viegas-Junior, C.; Danuello, A.; da Silva Bolzani, V.; Barreiro, E.J.; Fraga, C.A.M. Molecular Hybridization: A Useful Tool in the
Design of New Drug Prototypes. Curr. Med. Chem. 2007, 14, 1829–1852. [CrossRef]
25. Shirinzadeh, H.; Süzen, S.; Altanlar, N.; Westwell, A.D. Antimicrobial Activities of New Indole Derivatives Containing 1,2,4-
Triazole, 1,3,4-Thiadiazole and Carbothioamide. Turkish J. Pharm. Sci. 2018, 15, 291–297. [CrossRef]
26. Mane, Y.D.; Sarnikar, Y.P.; Surwase, S.M.; Biradar, D.O.; Gorepatil, P.B.; Shinde, V.S.; Khade, B.C. Design, synthesis, and
antimicrobial activity of novel 5-substituted indole-2-carboxamide derivatives. Res. Chem. Intermed. 2017, 43, 1253–1275.
[CrossRef]
27. Schulze, B.; Schubert, U.S. Beyond click chemistry-supramolecular interactions of 1,2,3-triazoles. Chem. Soc. Rev. 2014, 43,
2522–2571. [CrossRef]
28. Küçükgüzel, G.; Çikla-Süzgün, P. Recent advances bioactive 1,2,4-triazole-3-thiones. Eur. J. Med. Chem. 2015, 97, 830–870.
[CrossRef]
29. Dilmaghani, K.A.; Nasuhi Pur, F.; Nezhad, M.H. Synthesis and antibacterial evaluation of new thione substituted 1,2,4-triazole
schiff bases as novel antimicrobial agents. Iran. J. Pharm. Res. 2015, 14, 693–699. [CrossRef]
30. Metwally, M.A.; Bondock, S.; El-Azap, H.; Kandeel, E.E.M. Thiosemicarbazides: Synthesis and reactions. J. Sulfur Chem. 2011, 32,
489–519. [CrossRef]
31. Palaska, E.; S¸ahin, G.; Kelicen, P.; Durlu, N.T.; Altinok, G. Synthesis and anti-inflammatory activity of 1-acylthiosemicarbazides,
1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazole-3-thiones. Farmaco 2002, 57, 101–107. [CrossRef]
32. Biagi, G.L.; Guerra, M.C.; Barbaro, A.M.; Gamba, M.F. Influence of lipophilic character on the antibacterial activity of
cephalosporins and penicillins. J. Med. Chem. 1970, 13, 511–516. [CrossRef] [PubMed]
33. Asami, T.; Mizutani, M.; Shimada, Y.; Goda, H.; Kitahata, N.; Sekimata, K.; Han, S.Y.; Fujioka, S.; Takatsuto, S.; Sakata, K.; et al.
Triadimefon, a fungicidal triazole-type P450 inhibitor, induces brassinosteroid deficiency-like phenotypes in plants and binds to
DWF4 protein in the brassinosteroid biosynthesis pathway. Biochem. J. 2003, 369, 71–76. [CrossRef] [PubMed]
34. Sarhan, A.E.W.A.O. On the synthesis and reactions of indole-2-carboxylic acid hydrazide. Monatshefte fur Chemie 2001, 132,
753–763. [CrossRef]
35. Coowar, D.; Bouissac, J.; Hanbali, M.; Paschaki, M.; Mohier, E.; Luu, B. Effects of indole fatty alcohols on the differentiation of
neural stem cell derived neurospheres. J. Med. Chem. 2004, 47, 6270–6282. [CrossRef] [PubMed]
36. Al-Wabli, R.; Zakaria, A.; Attia, M. Synthesis, Spectroscopic Characterization and Antimicrobial Potential of Certain New
Isatin-Indole Molecular Hybrids. Molecules 2017, 22, 1958. [CrossRef]
37. Boraei, A.T.A.; El Ashry, E.S.H.; Duerkop, A. Regioselectivity of the alkylation of S-substituted 1,2,4-triazoles with dihaloalkanes.
Chem. Cent. J. 2016, 10, 22. [CrossRef]
38. Vanden Berghe, D.A. Screening methods for antibacterial and antiviral agents from higher plants. Methods Plant. Biochem. 1991
,
47–69.
39. Cos, P.; Vlietinck, A.J.; Berghe, D.V.; Maes, L. Anti-infective potential of natural products: How to develop a stronger in vitro
“proof-of-concept”. J. Ethnopharmacol. 2006, 106, 290–302. [CrossRef]