Design, synthesis, antibacterial and quorum quenching studies of 1,2,5-trisubstituted 1,2,4-triazoles

Article abstract of DOI:10.1007/s13738-020-02093-9

Abstract: In view of discovering novel bioactive molecules, 1-phenyl-1H-2-(1-aryl-5-methyl-1H-1,2,3-triazol-4-yl)-3-(N-aryl-carbamoylmethylthio)-1,2,4-triazoles (8a–n) were designed and synthesized in good yield. Preliminary antibacterial activity was tested against Chromobacterium violaceum and Xanthomonas campestris pv. Campestris (Xcc). Out of 14 derivatives, compound 8g selectively possessed antibacterial activity against C. violaceum. Further derivatives that possessed an electron-withdrawing group and halogen atoms in N-phenylacetamide moiety were moderately active against Xcc (plant pathogen). After observing the reduction of violacein production through plate assay, compounds 8a, 8c, 8h, 8i and 8m were subjected to quantification of quorum sensing inhibition. Compounds with the electron-withdrawing group in N-phenylacetamide moiety showed admirable activity with > 80% inhibition of violacein. Mainly compound 8c which was inactive against the growth of bacteria were identified as excellent QSI which could be a lead compound for further development. Graphic abstract: One of the best approaches to acquire anti-virulence strategies and new direction for the discovery of antibacterial drugs[Figure not available: see fulltext.]

Full text of DOI:10.1007/s13738-020-02093-9

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-02093-9
ORIGINAL PAPER
Design, synthesis, antibacterial and quorum quenching studies
of 1,2,5‑trisubstituted 1,2,4‑triazoles
Reshma Sathyanarayana1 · Sukesh Kumar Bajire2 · Boja Poojary1 · Rajesh P. Shastry2 · Vasantha Kumar3 ·
Revanasiddappa Bistuvalli Chandrashekarappa4
Received: 12 April 2020 / Accepted: 9 October 2020
© Iranian Chemical Society 2020
Abstract
In view of discovering novel bioactive molecules, 1-phenyl-1H-2-(1-aryl-5-methyl-1H-1,2,3-triazol-4-yl)-3-(N-aryl-
carbamoylmethylthio)-1,2,4-triazoles (8a–n) were designed and synthesized in good yield. Preliminary antibacterial activity
was tested against Chromobacterium violaceum and Xanthomonas campestris pv. Campestris (Xcc). Out of 14 derivatives,
compound 8g selectively possessed antibacterial activity against C. violaceum. Further derivatives that possessed an electron-
withdrawing group and halogen atoms in N-phenylacetamide moiety were moderately active against Xcc (plant pathogen).
After observing the reduction of violacein production through plate assay, compounds 8a, 8c, 8h, 8i and 8m were subjected
to quantifcation of quorum sensing inhibition. Compounds with the electron-withdrawing group in N-phenylacetamide
moiety showed admirable activity with>80% inhibition of violacein. Mainly compound 8c which was inactive against the
growth of bacteria were identifed as excellent QSI which could be a lead compound for further development.
Graphic abstract
One of the best approaches to acquire anti-virulence strategies and new direction for the discovery of antibacterial drugs
Keywords 1,2,3-Triazole · 1,2,4-Triazole · Quorum quenching · Molecular docking · ADME · Xcc
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-020-02093-9) contains
supplementary material, which is available to authorized users.
Antibiotic resistance is a common problem worldwide due
to the selection pressure from human applications of anti-
biotics. The pathogenic bacteria use various mechanisms
to get shelter against antimicrobials. Due to this, bacteria
* Boja Poojary
bojapoojary@gmail.com
Extended author information available on the last page of the article
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
attain genotypic and phenotypic resistance to antibiotics and
emerge as superbugs [1]. As a result, antibiotic treatments
remain inefective to the patients [2]. Hence, it’s the foremost
duty of the researchers to discover novel targets with better
mechanisms of action [3, 4]. Among several mechanisms,
quorum sensing (QS) inhibition shows to be an encourag-
ing approach, as these molecules will not prompt resistance
[5]. These pathogenic bacteria communicate by producing
low molecular weight chemical signalling molecules called
autoinducers (AIs) by the process called quorum sensing
[6]. Bacteria use QS signalling pathway to govern virulence
factor and bioflm formation which help in antibiotic resist-
ance. Hence, researchers believe that directing the QS as an
important pathway to acquire anti-virulence with minimal
risk of resistance development. The QS targeted antimi-
crobial drugs enact diferently from traditional antibiotics;
hence this kind of drug is considered as ideal antibacterial
drugs in the near future [7, 8]. The bacterial cell density is
proportional to the concentration of the signalling molecules
in a given environment [9]. QS mechanism is one of the rea-
sons for pathogenicity and survival of bacteria [10] which
should be under consideration for the development of novel
anti-quorum drugs. The inhibition of QS results in control
over bioflm formation, virulence factor expression, biolumi-
nescence, motility and drug-resistant [11]. Henceforth, the
novel research strategy includes the development of novel
synthetic ligands that behave as antagonists of autoinducer.
Numerous works have been carried on synthetic QS modu-
lators [12–14]. Most of the QS modulators imitates natural
products produced by natural antifoulants and certain algae
[15, 16].
production. The most efective quorum sensing inhibitors
reported to date are chloro lactone (CL) (Fig. 1) [21]. As
AHLs posses hydrophilic homoserine lactone ring and
hydrophobic aliphatic amide chain, it can freely acess
cell membrane and associate with receptor protein [22].
Recently Srinivasarao, S et al. [23] worked on QSI and
stated that the presence of at least one methylene function-
ality between triazole nucleus and N-acyl group enhances
the activity and moieties with no methylene group showed
poor QSIs. Figure 3 depicts few synthetic QSI (1–5) [19,
21–24] possessing 1,2,3-triazole, amide and methylene
functionalities.
Xanthomonas campestris pv. campestris (Xcc) is a gram-
negative plant pathogen which causes black rot. This is con-
sidered as one of the trivial and most damaging disease of
cruciferous crops which is present globally. One of the meth-
ods to increase agricultural crop is through usage of organic
fertilizers, agrochemicals and biological controls [25]. Some
of the commercially available agrofungicides are dinicona-
zole, triadimenol, fusilazole, triadimefon, difenoconazole,
oxathiapiprolin and acetazoiamide which has 1,2,4-triazole
as a core moiety, amide linkage and electron-withdrawing
atoms like chlorine and fuorine (Fig. 2).
Few researchers discovered drug against phytopatho-
gens and explained the importance of electron-withdraw-
ing groups attached to the core moiety like 1,2,4-triazole
and quinoline to enhance the activity [26–30]. From these
fndings it can be assumed that the moieties responsible for
potent antibacterial activity is 1,2,3-triazole, 1,2,4-triazole
and electron-withdrawing groups like chlorine. Moreover,
most of the commercially available agrofungicides (6, 7)
and synthesized drug (8, 9) [28, 29] against phytopathogenic
bacteria contain 1,2,4-triazole moieties along with amide
functionality (Fig. 3).
Chromobacterium violaceum is a gram-negative,
aquatic bacterium that rarely behaves as a pathogen caus-
ing skin lesions, extreme virulence, fatal septicaemia
[17]. The gram-negative bacteria communicate by pro-
ducing violet pigment called violacein with the help of
autoinducer like N-acyl homoserine lactone (C6-HSL) to
maintain threshold cell density [18]. The best chemical
approach to obstruct QS is to block signal receptor binding
via non-native small molecules [19]. The quorum sensing
inhibitors (QSIs) will not afect the bacterial growth but
stop the bacterial signalling [20]. A prodigious number
of derivatives of N-acyl-homoserine lactone (AHL) has
been discovered based on their inhibition of violacein
Considering all above facts we are herewith report-
ing novel 1,2,4-triazole analogues to evaluate for their QS
against C. violaceum, antibacterial activity against C. vio-
laceum and Xanthomonas campestris pv. campestris (Xcc).
Drug-receptor interaction can be understood by one of the
routinely used computational studies (molecular docking).
It provides binding orientations of ligands to their targeted
proteins. To understand the efciency of targeted molecules
as anti-quorum inhibitors, it was docked against CviR pro-
tien (PDB ID: 3QP5).
Fig. 1 Structure of autoinducer
(C6-HSL) and QSI (CL)
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Fig. 2 Commercially available agrofungicides
Fig. 3 Synthetic strategy for designing target drug
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yielded carbothioamide (5), which underwent cyclization
in basic condition to obtain pre-fnal 5-(5-methyl-1-(2,4,5-
trichlorophenyl)-1H-1,2,3-triazol-4-yl)-4-phenyl-2,4-dihy-
dro-3H-1,2,4-triazole-3-thione (6). Later on intermediate
6 was treated with diferent N-(substituted phenyl)chloro-
acetamide to yield the target compounds 8a–n. Physical
properties and structural features of target compounds are
descriped in Table S1 (ESI).
Results and discussion
Chemistry and spectral analysis
A new series of 1,2,4-triazole conjugated with 1,2,3-tria-
zole 8a–n was synthesized in good yield following the
synthetic pathway depicted in Scheme 1. At frst trisub-
stituted aniline was converted to azide (1) by treating
with sodium nitrite and sodium azide in 1:1 mixture of
Conc. HCl and water. The intermediate 5-methyl-1-(2,4,5-
trichlorophenyl)-1H-1,2,3-triazole-4-carbohydrazide (4)
was obtained by converting azides (1) to acid derivative
of N-aryl-1,2,3-triazole (2), followed by esterifcation (3)
and converting to corresponding carbohydrazide (4). Fur-
ther intermediate 4 upon treating with phenyl isocyanate
The structures of intermediate and target compounds were
1
confrmed by H NMR, 13C NMR, FT-IR, mass spectrum
(MS) and elemental analyses. The fnal compound taking 8e
into the consideration, the IR spectrum showed absorption
band at 3240, 1683 and 1176 cm−1 which were assigned
to NH, amide C=O and C–F, respectively. The 400 MHz
NMR spectrum showed the characteristic sharp singlet at δ
3.95 ppm for SCH2 proton thus confrming the attachment
Scheme 1 The synthesis of target compounds 8a–n. Reagents and
conditions: a NaNO2/H2O/HCl, NaN3, 0 °C to R.T. 16 h; b Ethylace-
toacetate/EtOH, refuxed, 4 h; c EtOH/HCl, 90 °C, 16 h; d NH2NH2/
EtOH, 90 °C, 16 h; e PhNCS/EtOH, 90 °C, 8h; f 5% NaOH, 100 °C,
3 h; g ClCH2COCl/Et3N/EtOH, 90 °C, 3 h; h K2CO3/CH3COCH3,
R.T. 10 h
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Table 1 Zone of inhibition (mm) of 8a–n against tested microorgan-
of substituted phenyl acetamide to C=S group, singlet at
δ 10.55 ppm was attributed for NH proton and signal at δ
2.53 ppm was assigned for –CH3 group. The chemical shift
values for the aromatic region are in agreement with the
structure. The structure was also supported by 13C NMR,
the desheilded signal resonance at 166.8 ppm confrmed
the presence of amide C=O group. The signal at 36.1 ppm
showed the presence of CH3 carbon. Its mass spectrum dis-
played [M+NH4]+ at m/z 638.00 along with isotopic peaks.
ism at 300 µg/ml
Compound
R
Zone of inhibition (mm)**
C. violaceum
Xcc
8a
NA
7
0.2
Cl
N O
O
8b
8c
21
14 0.5
13 0.6
F
Pharmacological studies
NA
Antibacterial activity and quorum sensing inhibition
against C. violaceum
N O
O
The quorum sensing inhibition (QSI) is directed to inhibi-
tion of bacterial quorum sensing signal/receptor and not
on the growth of bacteria, therefore, we subjected the syn-
thesized moieties 8a–n initially for antibacterial activity
over gram-negative bacteria C. violaceum. The antibacte-
rial activity was evaluated in terms of zone of inhibition
(ZOI) against bacterial strain by disc difusion method, and
results are reported in Table 1. Only compounds 8b, 8f, 8g,
8n showed antibacterial activity against gram-negative C.
violaceum bacteria. The SAR trends revealed that monosub-
stituted N-phenylacetamide with electron-donating groups
and halogen derivative in the para position showed better
activity when compared to halogen in ortho position and
disubstituted N-phenylacetamide moiety. Ciprofoxacin was
used for comparison.
8d
8e
NA
NA
8
7
0.2
0.4
F
F
Cl
8f
20
8
9
0.2
0.5
8g
8h
27
O
NA
12 0.2
Br
The synthesized derivatives 8a–n were subjected to
quorum sensing inhibition (QSI). The autoinducer acyl
HSLs control the production of violet pigment (violacein)
which is used as a reporter system in detecting QS com-
munication between bacteria. Henceforth, hindrance of
QS efcacy in C. violaceum will forbid the production
of violacein. Here, we observed for reduction of viola-
cein production upon QSI activity for the compounds 8a,
8c, 8h, 8i and 8m (Table 2) and violacein production as
well as growth was quantifed (Fig. 4). The SAR trends
revealed that among 14 derivatives, 8a, 8c, 8h, 8i, 8m
which possessed electron-withdrawing group on N-phe-
nylacetamide moiety showed excellent quorum sensing
inhibition activity (> 60% inhibition) at 300 µg/mL. These
moieties which displayed QSI were antibacterial inactive.
Among the fve derivatives which showed QSI, 8c and 8a
exhibited outstanding activity with> 80% QSI which pos-
sessed strong electron-withdrawing nitro and 4-Cl-3-NO2
groups, respectively, attached to N-phenyl acetamide
moiety (Fig. 4) when compared to standard quercetin.
Exchanging 4-Cl-3-NO2 in 8a with a combination of weak
electron-withdrawing group bromo and donating group
methyl led to compound 8h which showed 70% inhibition,
8i
8j
NA
NA
7 0.3
F
11 0.5
Cl
8k
8l
NA
NA
8
8
0.2
0.3
8m
8n
NA
10 0.2
Br
16
10 0.2
18 0.5
Br
Ciprofoxacin (30 μg/disc)
27 0.5
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Table 1 (continued)
NA no activity
whereas monosubstituted halogen derivatives (8m and 8i)
showed < 70% inhibition. The above experiment revealed
that target compounds 8a, 8c, 8h, 8i, 8m had no efect on
the normal growth of C. violaceum but inhibited bacterial
signalling.
**The zone of inhibition was expressed as an average from three
experiments standard deviation
Table 2 Anti-QS activity against tested C. violaceum biosensor
(ATCC 12427)
In silico studies
Compound
Inhibition of quorum
sensing in mm (300 µg/
disc)*
Target protein LuxR‑type protein CviR
8a
12 0.2
–
12 0.4
–
–
–
–
The molecules which play a role as an antagonist of LuxR-
type proteins are likely to be a lead molecule in the evolu-
tion of novel antibacterial therapeutics. The gram-negative
bacteria frequently use acyl-homoserine lactone molecules
(AHLs) as autoinducers. The stable LuxR:AHL complexes
bind DNA to initiate transcription of quorum sensing in a
broad range of pathogens.
8b
8c
8d
8e
8f
8g
8h
10 0.2
C. violaceum strain produces an autoinducer N-hex-
anoyl homoserine lactone (C6-HSL), which is detected via
the LuxR-type protein CviR [31]. The autoinducer C6-HSL
could bind to the CviR protein from C. violaceum to regu-
late the production of violacein. Chlorolactone (CL) com-
pound is one of the potent quorum sensing antagonists
which is successful in competing against C6-HSL for
binding to the autoinducer binding site resulting in closed
conformation incapable to bind DNA [32].
8i
8
–
–
–
8
–
0.1
8j
8k
8l
8m
8n
0.4
Quercetin
10 0.2
*The zone of violacein inhibition was expressed as an average from
three experiments standard deviation
Fig. 4 Anti-quorum sensing
assay (plate assay-insert) and
quantifcation of violacein
inhibition with measurement of
growth under assay condition
by microtiter plate method.
Quantifcation of violacein
produced was expressed in
percentage, more than 60% inhi-
bition of violacein was observed
by respective compounds
(p<0.0001), and the absence of
growth inhibition. The error bar
represents standard deviations
from reproduced results in three
repeated experiments
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Molecular docking
value, greater is the binding efciency of molecules with
CviR protein (PDB ID: 3QP5). The activity trend was rep-
licated in the docking results. The docking results obtained
for binding interaction of the molecules (8a, 8c, 8h, 8i and
8m) with protein are shown in Fig. 5. The docking score of
To obtain a possible correlation of in vitro quorum sens-
ing inhibition, all the newly synthesized molecules were
subjected to in silico docking studies. Larger the negative
Fig. 5 The docking poses of the
compounds with CviR protein
(PDB ID: 3QP5). Lines indicate
the type of interactions: pur-
ple—halogen bond, yellow—
hydrogen bond
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Journal of the Iranian Chemical Society
Table 3 Docking score and interacting amino acid residues for bind-
exhibited halogen bonding to GLU37 by bromo atom of
N-(4-bromo-3-methylphenyl)acetamide group. These moi-
eties which showed good activity also showed excellent
docking score.
ing of the compounds with CviR protien
Compound
Docking score Halogen bond Hydrogen bond
8a
8c
8h
−8.69
−8.17
−8.11
SER155
SER155
ASN77
ASN77
ASN77
SER155
Physiochemical properties
GLU37
8i
−8.06
−8.20
SER155
SER155
–
ASN77
ASN77
ASP97
SER155
TRP84
The important tool for the determination of drug-likeness
property of a bioactive molecule is the Lipinski rule of fve.
Drug-likeness is nothing, but to know whether the synthe-
sized novel bioactive molecule is alike to marketed drugs.
For any molecule to be an efective drug candidate it should
obey Lipinski’s rule of fve [33] which in turn determined by
ADME properties, i.e. absorption, distribution, metabolism
and excretion. According to this, a chemical species should
possess a molecular weight≤500, partition coefcient (log
P) ≤ 5, the number of hydrogen bond donor and accep-
tor ≤ 5 and ≤ 10, respectively. In addition to this, the drug
should have less than or equal to ten rotatable bonds and
polar surface area≤140Å which determine oral bioavailabil-
ity [34]. Other parameters such as #stars (0–5), percentage
human oral absorption (>80% high,<25% low), CNS (−2:
inactive; +2: active), QlogKp (− 8.0–− 10.0), QPlogKhsa
(− 1.5–1.5), #metab (1–8), QPPMDCK (< 25 poor > 500
Great) and QPHERG (>−5) were also determined.
8m
CL (reference) −8.46
the molecules varied from −8.69 to −8.06 kJ/mol (Table 3).
Compared to the chlorolactone (CL) which was taken as
a reference (− 8.46 kJ/mol), the best docking score was
obtained to compound 8a which showed − 8.69 kJ/mol.
And from Fig. 6, It is evident that the ligand 8a is bound
to the same pocket of CviR protein as that of the reference.
This represents that the docking procedure reproduced the
receptor-bound conformation with the ligand molecules.
All the molecules exhibited halogen bonding to SER155
via C2 chlorine atom of the 2,4,5-trichlorophenyl ring,
whereas hydrogen bonding to ASN77 through an oxygen
atom of the amide group. Additionally, the compound 8h
Fig. 6 Surface representation of
LuxR-type protein CviR with
ligand 8a (a) and CL (b)
Table 4 Predicted Lipinski’s
parameter for 8a, 8c, 8h, 8i and
8m
Compounds
Molecular weight Donor HB Acceptor HB logPo/wa
#rotorb Rule of 5
Acceptable range ≤ 500
≤ 5
1
≤ 10
7.5
7.5
6.5
6.5
6.5
(−2.0 to 6.5) ≤ 10
< 5
3
8a
8c
8h
8i
647.98
614.02
660.96
587.03
646.95
6.24
6.23
7.86
6.59
7.05
5
5
4
4
4
1
1
1
1
3
2
2
2
8m
aPredicted octanol/water partition coefcient logp
bPredicted rotatable bonds
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Table 6 Antibacterial
activity against Xanthomonas
campestris pv. campestris
compounds
In this study, the physiochemical properties of 8a, 8c,
8h, 8i and 8m were determined and summarised in Tables 4
and 5. For a drug to be efective orally it should not violate
more than one Lipinski’s rule of fve. It’s worth noting that
though all the compounds violate Lipinski’s rule, it showed
an excellent percentage of human oral absorption. Absorp-
tion is the main criteria for the bioavailability of the drug
sequentially absorption depends on permeability and solu-
bility of the drug [35]. All the compounds obey most of the
parameters expect molecular weight and log Po/w. Though
8a and 8c violate 3 rules it has a good percentage of human
oral absorption=60%. As these compounds possess good
oral absorption (60–100%) violation in any rule will not
afect the bioavailability [36].
Compound
MIC (µg/mL)
8a
300
< 200
< 200
200
400
300
200
< 200
200
< 200
200
300
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
200
200
< 32
8n
Antibacterial activity against Xcc
Ciprofoxacin
Furthermore, the newly synthesized compounds (8a–n) were
also screened for their in vitro growth inhibitory activity
against phytopathogenic bacteria Xanthomonas campestris
pv. campestris. The preliminary screening was carried out
through the disc difusion method and MIC by broth micro-
dilution method. The results of the preliminary antimicrobial
testing of compounds 8a–n (300 μg/mL) and standard anti-
bacterial drug ciprofoxacin are shown in Table 1. As shown
in Table 1, in terms of ZOI, most of the compounds showed
promising antibacterial activity against Xanthomonas camp-
estris pv. campestris at 300 µg/mL. Among all the deriva-
tives, the strong antibacterial activity was observed in 8b
which has fuoro substitution on N-phenylacetamide moiety,
produced growth inhibition zone of 14 mm. The
N-phenylacetamide was selectively active against bacteria
C. violaceum, whereas fve molecules bearing electron-with-
drawing on N-phenylacetamide moiety exhibited fabulous
anti-QS inhibition. Hence, the statement “The QSIs will not
afect the bacterial evolution but stop the bacterial signal-
ling” is proved by this experiment as these moieties (8a,
8c, 8h, 8i, 8m) were antibacterial inactive. The compounds
showing antibacterial activity are not suitable for the anti-
QS activity. Henceforth, the design was successful in dis-
covering novel anti-QS inhibitors. Also, the moieties with
the electron-withdrawing group exhibited moderate activity
against Xcc.
bromo derivatives exhibited optimal activity (8h, 8m,
8n). Except for the 8b, other fuoro derivatives exhibited
weak activity (8d, 8e, 8i). The moiety with the strong elec-
tron-withdrawing nitro group at meta position (8c) showed
excellent activity, whereas 8a with 4-Cl-3-NO2 compara-
tively showed weak activity. However, N-phenylacetamide
moiety with the electron-donating group did not have notice-
able antibacterial activity (8f, 8g, 8k, 8e). The minimum
concentration of antibacterial agent required to inhibit bacte-
rial growth (MIC µg/mL) was determined for entire deriva-
tives. Out of 14 derivatives, compounds 8b, 8c, 8h and 8j
showed MIC value at<200 µg/ml (Table 6).
Experimental section
Materials and methods
The reagents and chemicals used in this series were bought
from Sigma-Aldrich, S.D. Fine and Merck company. All
reagents were analytical grade and were used directly. The
open capillary tube method was used to determine the melt-
ing point of the target compounds. The characterization
1
of desired compounds was done using H NMR (Bruker
Avance III, 400 MHz NMR spectrometer), 13C NMR
(Bruker Avance III, 100 MHz) and IR (ATR). Mass spectra
were recorded on Waters, synapt G2 high detection mass
spectrometry. All the reactions were monitored by TLC, per-
formed on silica plate and observed under UV light. Tetra-
methylsilane was used as a standard in 1H NMR, chemical
shift values (ꢀ) were denoted in ppm and coupling constants
in Hz. Spin multiplicities were stated singlet (s), doublet (d),
triplet (t), quartet (q) and multiplet (m). Elemental analysis
was conducted in CHNS ElementarVarioEL III.
Conclusions
In this study, novel 1-phenyl-1H-2-(1-aryl-5-methyl-1H-
1,2,3-triazol-4-yl)-3-(N-aryl-carbamoylmethylthio)-1,2,4-
triazoles derivatives (8a–n) were designed, synthesized
in good yield and screened for in vitro antibacterial activ-
ity and anti-QS activity. The results revealed that moi-
ety with monosubstitution bearing electron-donating on
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Journal of the Iranian Chemical Society
heated under refux for 16 h. After the completion of the
reaction, the reaction mass was added to ice-cold water. The
precipitate formed was fltered, dried and recrystallized from
ethanol to yield intermediate 4 (4.5 g) as of-white solid.
Yield: 80%; Mp. 184–186 °C; Of-white solid; FT-IR
(ATR, υmax, cm−1): 3309 (N–H), 2966 (C–H), 1625 (C=O),
Synthesis of 1‑azido‑2,4,5‑trichlorobenzene (1)
To a stirred solution of 2,4,5-trichloroaniline (5.0 g,
25.6 mmol) in water (25 mL), added 15% HCl solution
(25 mL). The reaction mixture was then cooled to 0 °C fol-
lowed by dropwise addition of 30% NaNO2 solution (7 mL)
at the same temperature and allowed to stir for 15 min. To the
above reaction mixture, NaN3 (3.3 g, 51.3 mmol) was added a
lot wise at the same temperature and was stirred overnight at
room temperature. The precipitate formed was fltered, washed
thoroughly with water, dried and recrystallized from ethanol
to give intermediate 1 (5.2 g) as of-white solid. Yield: 91%;
Mp. 48–50 °C.
1
1585 (C=C), 854 (C–Cl); H NMR (400 MHz, CDCl3, δ
ppm): 8.28 (s, 1H, NH), 7.69 (s, 1H, Ar–H), 7.50 (s, 1H,
Ar–H), 4.00 (bs, 2H, NH2), 2.48 (s, 3H, CH3); ESI–MS
(m/z): 319.95 [M+H]+; Anal. calcd. for C10H8N5Cl3O: C,
37.47; H, 2.52; N, 21.85%. Found: C, 37.45; H, 2.50; N,
21.83%.
Procedure for synthesis of 2‑(5‑methyl‑1‑(2,4,5‑trich
lorophenyl)‑1H‑1,2,3‑triazole‑4‑carbonyl)‑N‑phenyl‑
hydrazine‑1‑carbothioamide (5)
Procedure for synthesis of 5‑methyl‑1‑(2,4,5‑trichlor
ophenyl)‑1H‑1,2,3‑triazole‑4‑carboxylic acid (2)
Intermediate 1 (5.2 g, 23.3 mmol) in ethanol (50 mL) was
taken in a round bottom fask fxed with a guard tube, added
ethyl acetoacetate (3.0 g, 23.3 mmol). The reaction mixture
was cooled to 0 °C, followed by the lot wise addition of
NaOMe (2.5 g, 46.7 mmol) and refuxed for 4 h. The comple-
tion of the reaction was monitored by thin-layer chromatogra-
phy. After completion of the reaction, the reaction mass was
added to ice-cold water and neutralized with acetic acid. The
precipitate formed was fltered, dried and recrystallized from
EtOAc to yield intermediate 2 (6.0 g) as white solid.
To a solution of intermediate 4 (4.5 g, 17.1 mmol) in EtOH
(50 mL), added phenyl isothiocyanate (1.7 mL, 17.1 mmol)
and heated to refux for 8 h. The progress of the reaction
was monitored by thin-layer chromatography. Excess sol-
vent was removed under high vacuum. Solid formed was
recrystallized from ethanol to yield intermediate 5 (5.0 g)
as white solid.
Yield: 81%; Mp. low melting; Of-white solid; FT-IR
(ATR, υmax, cm−1): 3219 (N–H), 2972 (C–H), 1680 (C=O),
1593 (C=C), 1145 (C=S), 742 (C–Cl); 1H NMR (400 MHz,
CDCl3, δ ppm): 9.77 (bs, 1H, NH), 8.70 (bs, 1H, NH),
8.47 (s, 1H, NH), 7.25–7.74 (m, 7H, Ar–H), 2.47 (s, 3H,
–H3); ESI–MS (m/z): 454.99 [M + H]+; Anal. calcd. for
C17H13Cl3N6OS: C, 44.80; H, 2.88; N, 18.44%. Found: C,
44.78; H, 2.87; N, 18.42%.
Yield: 84%; Mp. 146–148 °C; Off-white solid; FT-IR
(ATR, υmax, cm−1): 3442 (OH), 1685 (C=O), 1577 (C=C), 775
(C–Cl) cm−1; ESI–MS (m/z): 305.95 [M+H]+; Anal. calcd.
for C10H6N3Cl3O2: C, 39.18; H, 1.97; N, 13.71%. Found: C,
39.15; H, 1.95; N, 13.69%.
Procedure for synthesis of ethyl 5‑methyl‑1‑(2,4,5‑tr
Procedure for synthesis of intermediate
5‑(5‑methyl‑1‑(2,4,5‑trichlorophenyl)‑1H‑1,2,3
‑triazol‑4‑yl)‑4‑phenyl‑2,4‑dihydro‑3H‑1,2,4‑tria‑
zole‑3‑thione (6)
ichlorophenyl)‑1H‑1,2,3‑triazole‑4‑carboxylate (3)
To a solution of intermediate 2 (6.0 g, 19.5 mmol) in ethanol, a
catalytic amount of Conc. H2SO4 was added and heated under
refux for 16 h. After completion of the reaction, the excess
solvent was removed under vacuum. The leftover residue was
poured into ice-cold water. The precipitate formed was fltered
and dried to obtain intermediate 3 as white solid (5.9 g).
Yield: 90%; Mp. 128–130 °C; Of-white solid; FT-IR
(ATR, υmax, cm−1): 1732 (C=O), 1577 (N=N), 1448 (C–H),
773 (C–Cl); ESI–MS (m/z): 334.00 [M+H]+; Anal. calcd.
for C12H10N3Cl3O2: C, 43.08; H, 3.01; N, 12.56%. Found:
C, 43.05; H, 3.02; N, 12.54%.
30% NaOH (50 mL) solution was added to intermediate 5
(5.0 g, 11.3 mmol) and heated to refux for 3 h. After the
completion of the reaction, the reaction mass was cooled and
neutralized to pH=7 using dil. HCl. The precipitate formed
was fltered, dried and recrystallized from ethanol to yield
intermediate 6 (4.0 g) as of-white solid. Yield: 81%.
Yield: 81%; Mp. 270–272 °C; Of-white solid; FT-IR
(ATR, υmax, cm−1): 3255 (N–H), 2908 (C–H), 1591 (C=C),
1
1143 (C=S), 763 (C–Cl); H NMR (400 MHz, DMSO-
d6, δ ppm): 14.27 (s, 1H, NH), 8.20–8.24 (m, 1H, Ar–H),
7.35–7.48 (m, 6H, Ar–H), 2.21 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO-d6, δ ppm): 164.4, 139.3, 133.4, 131.0,
130.3, 128.0, 127.6, 127.4, 127.2, 126.9, 126.2, 125.0,
124.8, 124.4, 4.3; ESI–MS (m/z): 437.00 [M+H]+; Anal.
Procedure for synthesis of 5‑methyl‑1‑(2,4,5‑trichlor
ophenyl)‑1H‑1,2,3‑triazole‑4‑carbohydrazide (4)
Hydrazine hydrate 98% (20 mL) was added to a solution of
intermediate 3 (5.9 g, 17.6 mmol) in ethanol (50 mL) and
1 3

Journal of the Iranian Chemical Society
calcd. for C17H11N6Cl3S: C, 46.65; H, 2.53; N, 19.20%.
Found: C, 46.63; H, 2.50; N, 19.15%.
160.5 (J = 210.1 Hz), 144.7, 144.0, 141.9, 137.5, 137.4,
137.3, 130.5, 129.8, 129.3, 129.2, 129.0, 124.7, 122.1, 118.7
(J=8.0 Hz), 117.5 (J=25.2 Hz), 117.1, 32.1, 9.6; ESI–MS
(m/z): 588.03 [M+H]+; Anal. calcd. for C25H17N7Cl3FOS:
C, 50.99; H, 2.91; N, 16.65%. Found: C, 50.95; H, 2.92; N,
16.63%.
Procedure for synthesis of intermediates 7a–n
To a stirred solution of substituted aniline (8.2 mmol) in
ethanol (10 mL), added dry triethylamine (8.2 mmol). The
reaction mixture was cooled to 0 °C, chloroacetyl chloride
(8.2 mmol) was added dropwise and heated under refux for
3 h. The reaction mass was quenched with ice-cold water.
The precipitate formed was fltered, dried and washed with
hexane to yield intermediate 7a–n in good yield. Yield:
70–90%.
2‑((5‑(5‑Methyl‑1‑(2,4,5‑trichlorophenyl)‑1H‑1,2,3‑tria‑
zol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑triazol‑3‑yl)thio)‑N‑(3‑nitrophe‑
nyl)acetamide (8c)
Yield: 75%; Mp. 196–197 °C; Of-white solid; FT-IR (ATR,
υmax, cm−1): 3201 (NH), 2985 (C–H), 1684 (C=O), 1613
(C=N), 1580 (C=C), 1327, 1514 (C–NO2), 764, 815 (C–Cl),
1H NMR (400 MHz, CDCl3, δ ppm): 9.98 (s, 1H, NH),
7.89 (s, 1H, Ar–H), 7.40–7.75 (m, 9H, Ar–H), 3.97 (s, 2H,
–CH2–), 2.52 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3,
δ ppm): 167.2, 144.7, 144.0, 141.9 140.5, 137.6, 137.5,
137.4, 137.1, 130.5, 129.8, 129.4, 129.3, 129.2, 129.0,
124.7, 122.1, 119.1, 117.3, 117.1, 103.4, 32.1, 9.6; ESI–MS
(m/z): 614.02 [M+H]+; Anal. calcd. for C25H17N8Cl3O3S:
C, 48.75; H, 2.78; N, 18.19%. Found: C, 48.70; H, 2.75; N,
18.15%.
Procedure for synthesis of target compound 8a–n
To a mixture of triazole (0.3 g, 0.7 mmol) and substituted
amides (0.7 mmol) in acetone, added K2CO3 (0.7 mmol) and
stirred at room temperature for 5 h. The completion of the
reaction was monitored by thin-layer chromatography. The
reaction mass was fltered, and the fltrate was evaporated
under a high vacuum to yield the desired product which was
recrystallized using ethanol. Yield 70–95%.
N‑(4‑Chloro‑3‑nitrophenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑trich
lorophenyl)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑yl)thio)acetamide (8a)
N‑(3‑Fluoro‑2‑methylphenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑trich‑
lorophenyl)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑yl)thio)acetamide (8d)
Yield: 72%; Mp. 200–202 °C; Brown solid; FT-IR (ATR,
υmax, cm−1): 3132 (NH), 2939 (C–H), 1683 (C=O), 1608
(C=N), 1585 (C=C), 1510, 1327 (C–NO2) 765, 819 (C–Cl);
1H NMR (400 MHz, CDCl3, δ ppm): 10.95 (s, 1H, NH),
8.24 (d, J=4.0 Hz, 1H, Ar–H), 7.71–7.78 (m, 1H, Ar–H),
7.51–7.56 (m, 3H, Ar–H), 7.37–7.44 (m, 3H, Ar–H), 7.16
(s, 2H, Ar–H), 4.02 (s, 2H, –CH2–), 2.49 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3, δ ppm): 167.0, 148.4, 147.9,
137.9, 137.0, 136.4, 133.0, 132.6, 132.5, 132.2, 132.1,
131.8, 131.7, 130.5, 130.5, 130.4, 129.8, 128.8, 128.1,
127.3, 123.9, 121.0, 116.3, 36.3, 9.4; ESI–MS (m/z): 648.98
[M + H]+; Anal. calcd. for C25H16N8Cl4O3S: C, 46.17; H,
2.48; N, 17.23%. Found: C, 46.10; H, 2.45; N, 17.20%.
Yield: 75%; Mp. 160–162 °C; Brown solid; FT-IR (ATR,
υmax, cm−1): 3109 (NH), 2940 (C–H), 1682 (C=O), 1607
(C=N), 1583 (C=C), 766, 820 (C–Cl), 1023 (C-F); 1H NMR
(400 MHz, CDCl3, δ ppm): 9.92 (s, 1H, NH), 7.73–7.79
(m, 2H, Ar–H), 7.53–7.54 (m, 4H, Ar–H), 7.36–7.38 (m,
2H, Ar–H), 7.12–7.18 (m, 1H, Ar–H), 6.81–6.86 (m, 1H,
Ar–H), 4.04 (s, 2H, –CH2–), 2.50 (s, 3H, CH3), 2.28 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 167.0, 161.0
(J = 240.2 Hz), 154.0, 148.4, 137.7 (J = 6.4 Hz), 136.8,
136.3, 133.1, 132.4, 132.2, 131.7, 130.4, 130.3, 129.7,
127.2, 126.8, 126.7, 118.0, 116.9, 116.7, 111.4 (J=22.5 Hz),
35.8, 9.4; ESI–MS (m/z): 602.04 [M+H]+; Anal. calcd. for
C26H19N7Cl3FOS: C, 51.80; H, 3.18; N, 16.26%. Found: C,
51.78; H, 3.15; N, 16.25%.
N‑(4‑Fluorophenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑trichloropheny
l)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑triazol‑3‑yl)thio)
acetamide (8b)
N‑(3‑Chloro‑4‑fuorophenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑trich‑
lorophenyl)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑yl)thio)acetamide (8e)
Yield: 77%; Mp. 202–203 °C; Creamish solid; FT-IR (ATR,
υmax, cm−1): 3120 (N–H), 2940 (C–H), 1682 (C=O), 1607
(C=N), 1583 (C=C), 1021 (C-F), 766, 820 (C–Cl); 1H NMR
(400 MHz, CDCl3, δ ppm): 10.23 (s, 1H, N–H), 7.73 (s,
1H, Ar–H), 7.73–7.74 (m, 2H, Ar–H), 7.44–7.55 (m, 6H,
Ar–H), 7.01–7.02 (m, 2H, Ar–H), 3.97 (s, 2H, –CH2–), 2.52
(s, 3H, CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 167.2,
Yield: 77%; Mp. 209–210 °C; Light cream solid; FT-IR
(ATR, υmax, cm−1): 3240 (NH), 2989 (C–H), 1683 (C=O),
1612 (C=N), 1583 (C=C), 1176 (C-F), 763, 823 (C–Cl); 1H
NMR (400 MHz, CDCl3, δ ppm): 10.55 (s, 1H, NH), 7.85
(d, J = 4.0 Hz, 1H, Ar–H), 7.73 (s, 1H, Ar–H), 7.51–7.56
(m, 4H, Ar–H), 7.45–7.48 (dd, 1H, Ar–H), 7.34–7.38 (m,
1 3

Journal of the Iranian Chemical Society
3H, Ar–H), 3.95 (s, 2H, –CH2–), 2.53 (s, 3H, CH3); 13C
NMR (100 MHz, CDCl3, δ ppm): 166.8, 154.2, 148.4, 137.7,
136.9, 133.1, 132.6, 132.5, 132.3, 131.7, 130.5, 130.4,
129.8, 127.3, 121.4, 119.1, 36.1, 9.5; ESI–MS (m/z): 638.00
[M + NH4]+; Anal. calcd. for C25H16N7Cl4FOS: C, 48.17;
H, 2.59; N, 15.73%. Found: C, 48.15; H, 2.55; N, 15.70%.
ppm): 166.6, 154.1, 148.3, 138.4, 137.4, 136.8, 136.3, 133.1,
132.8, 131.7, 130.5, 130.5, 134.4, 129.7, 122.0, 119.3,
118.9, 36.2, 23.0, 9.5; ESI–MS (m/z): 660.96 [M + H]+;
Anal. calcd. for C26H19N7BrCl3OS: C, 47.04; H, 2.98; N,
14.77%. Found: C, 47.03; H, 2.95; N, 14.72%.
N‑(2‑Fluorophenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑trichloropheny
l)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑triazol‑3‑yl)thio)
acetamide (8i)
2‑((5‑(5‑Methyl‑1‑(2,4,5‑trichlorophenyl)‑1H‑1,2,3‑tria‑
zol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑triazol‑3‑yl)thio)‑N‑(4‑tolyl)
acetamide (8f)
Yield: 65%; Mp. 176–178 °C; Of-white solid; FT-IR (ATR,
υmax, cm−1): 3121 (NH), 2940 (C–H), 1682 (C=O), 1607
(C=N), 1583 (C=C), 766, 820 (C–Cl), 1021 (C–F); 1H NMR
(400 MHz, CDCl3, δ ppm): 10.17 (s, 1H, NH), 8.27–8.30 (m,
1H, Ar–H), 7.72 (s, 1H, Ar–H), 7.51–7.55 (m, 4H, Ar–H),
7.37–7.39 (m, 2H, Ar–H), 7.03–7.13 (m, 3H, Ar–H), 4.07
(s, 2H, –CH2–), 2.16 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3, δ ppm): 166.8, 153.6, 152.9 (J=200.0 Hz), 148.3,
136.9, 136.2, 133.1 (J = 27.6 Hz), 132.4 (J = 3.3 Hz),
131.7, 130.5, 130.4, 130.3, 129.7, 127.3, 126.6, 126.5,
124.6, 124.5, 124.36, 124.39, 122.0, 114.9 (J = 15.6 Hz),
36.0, 9.5; ESI–MS (m/z): 588.02 [M+H]+; Anal. calcd. for
C25H17N7Cl3FOS: C, 50.99; H, 2.91; N, 16.65%. Found: C,
50.95; H, 2.92; N, 16.63%.
Yield: 88%; Mp. 120–122 °C; Light cream solid; FT-IR
(ATR, υmax, cm−1): 3086 (NH), 2989 (C–H), 1687 (C=O),
1
1612 (C=N), 1583 (C=C), 767, 817 (C–Cl); H NMR
(400 MHz, CDCl3, δ ppm): 10.02 (s, 1H, NH), 7.72 (s, 1H,
Ar–H), 7.49–7.54 (m, 6H, Ar–H), 7.36–7.38 (m, 2H, Ar–H),
7.11 (d, 2H, J=8.3 Hz, Ar–H), 3.97 (s, 2H, –CH2–), 2.52 (s,
3H, CH3), 2.30 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3,
δ ppm): 166.4, 154.1, 148.3, 136.8, 136.3, 135.6, 133.8,
133.2, 132.9, 132.5, 132.3, 131.7, 130.5, 130.4, 129.7,
129.3, 127.3, 119.9, 36.1, 20.8, 9.5; ESI–MS (m/z): 584.03
[M + H]+; Anal. calcd. for C26H20N7Cl3OS: C, 53.39; H,
3.45; N, 16.76%. Found: C, 53.30, H, 3.44; N, 16.70%.
N‑(4‑Methoxyphenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑trichlo‑
rophenyl)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑yl)thio)acetamide (8g)
N‑(3‑Chloro‑2‑methylphenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑trich‑
lorophenyl)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑yl)thio)acetamide (8j)
Yield: 75%; Mp. 188–190 °C; Light grey solid; FT-IR (ATR,
υmax, cm−1): 3041 (N–H), 2940 (C–H), 1676 (C=O), 1606
Yield: 65%; Mp. 180–182 °C; Light brown solid; FT-IR
1
(C=N), 1552 (C=C), 1082 (C–O), 767, 829 (C–Cl); H
(ATR, υmax, cm−1): 3112 (NH), 2985 (C–H), 1684 (C=O),
1
NMR (400 MHz, CDCl3, δ ppm): 9.98 (s, 1H, NH), 7.72 (s,
1H, Ar–H), 7.50–7.54 (m, 6H, Ar–H), 7.36–7.38 (m, 2H,
Ar–H), 6.83–6.86 (m, 2H, Ar–H), 3.97 (s, 2H, –CH2–), 3.77
(s, 3H, –OCH3–), 2.51 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3, δ ppm): 166.2, 156.3, 154.1, 148.2, 136.8, 136.3,
133.2, 132.9, 132.5, 132.3, 131.7, 131.4, 130.5, 130.4,
129.7, 127.3, 121.5, 114.0, 55.4, 30.9, 9.5; ESI–MS (m/z):
600.05 [M + H]+; Anal. calcd. for C26H20N7Cl3O2S: C,
51.97; H, 3.35; N, 16.32%. Found: C, 51.95; H, 3.30; N,
16.30%.
1613 (C=N), 1580 (C=C), 764, 815 (C–Cl); H NMR
(400 MHz, CDCl3, δ ppm): 10.23 (s, 1H, NH), 7.89 (s, 1H,
Ar–H), 7.46–7.55 (m, 6H, Ar–H), 6.64–7.29 (m, 3H, Ar–H),
3.68 (s, 2H, –CH2–), 2.56 (s, 3H, CH3), 2.24 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3, δ ppm): 167.2, 144.7, 144.0,
141.9, 141.1, 137.5, 137.4, 137.1, 134.4, 131.7, 129.9, 129.8,
129.3, 129.2, 129.0, 124.8, 124.7, 122.1, 117.7, 117.1, 32.1,
14.6, 9.6; ESI–MS (m/z): 618.01 [M+H]+; Anal. calcd. for
C26H19N7Cl4OS: C, 50.42; H, 3.09; N, 15.83%. Found: C,
50.41; H, 3.08; N, 15.85%.
N‑(4‑Bromo‑3‑methylphenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑tric
hlorophenyl)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑yl)thio)acetamide (8h)
N‑(3,4‑Dimethylphenyl)‑2‑((5‑(5‑methyl‑1‑(2,4,5‑trichlo‑
rophenyl)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑yl)thio)acetamide (8k)
Yield: 72%; Mp. 194–195 °C; Creamish solid; FT-IR (ATR,
υmax, cm−1): 3251 (NH), 2939 (C–H), 1683 (C=O), 1608
(C=N), 1585 (C=C), 765, 819 (C–Cl), 650 (C–Br); 1H NMR
(400 MHz, CDCl3, δ ppm): 10.23 (s. 1H, NH), 7.73 (s, 1H,
Ar–H), 7.50–7.55 (m, 5H, Ar–H), 7.43 (d, 1H, J=8.0 Hz,
Ar–H), 7.32–7.38 (m, 3H, Ar–H), 3.96 (s, 2H, CH2), 2.52 (s,
3H, CH3), 2.37 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3, δ
Yield: 83%; Mp. 132–134 °C; Brown solid; FT-IR (ATR,
υmax, cm−1): 3098 (NH), 2940(C–H), 1681 (C=O), 1609
(C=N), 1583 (C=C), 766, 817 (C–Cl), 1H NMR (400 MHz,
CDCl3, δ ppm): 9.93 (s, 1H, NH), 7.73 (s, 1H, Ar–H),
7.52–7.55 (m, 4H, Ar–H), 7.26–7.39 (m, 4H, Ar–H),
7.05–7.07 (d, 1H, Ar–H), 3.97 (s, 2H, –CH2–), 2.52 (s,
3H, CH3), 2.24 (s, 3H, CH3), 2.17 (s, 3H, CH3); 13C NMR
1 3

Journal of the Iranian Chemical Society
(100 MHz, CDCl3, δ ppm): 167.2, 144.7, 144.0, 141.9,
139.5, 137.5, 137.4, 137.1, 135.8, 132.6, 130.5, 129.9,
129.8, 129.3, 129.2, 129.0, 124.7, 122.1, 120.8, 116.9,
32.1, 19.7, 19.4, 9.6; ESI–MS (m/z): 598.07 [M+H]+; Anal.
calcd. for C27H22N7Cl3OS: C, 54.14; H, 3.70; N, 16.37%.
Found: C, 54.12; H, 3.69, N, 16.35%.
(100 MHz, CDCl3, δ ppm): 167.2, 144.7, 144.0, 1419, 137.5,
137.4, 137.1, 132.0, 130.5, 129.8, 129.3, 129.2, 124.7,
122.1, 121.6, 117.9, 117.1, 32.1, 9.6; ESI–MS (m/z): 647.95
[M+H]+; Anal. calcd. for C25H17 N7BrCl3OS: C, 46.1; H,
2.64; N, 15.09%. Found: C, 46.3; H, 2.66; N, 15.02%.
Biological screening and computational studies
N‑(2‑Ethyl‑6‑methylphenyl)‑2‑(5‑(5‑methyl‑1‑(2,4,5‑trichlo‑
rophenyl)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑ylthio)acetamide (8l)
Bacterial strains
The bacterial cultures used in the study Xanthomonas camp-
estris pv. campestris (Xcc) (5028) were procured National
Collection of Industrial Microorganisms Pune, India. Stock
cultures of bacteria were maintained on nutrient agar (Hime-
dia, Mumbai) slants at 4 °C with periodical subculturing.
The wild-type strain C. violaceum ATCC 12427 (provided
by Robert McLean) used as a biosensor for anti-quorum
sensing studies. C. violaceum was maintained in Luria–Ber-
Yield: 60%; Mp. 118–120 °C; Light brown solid; FT-IR
(ATR, υmax, cm−1): 3211 (NH), 2989 (C–H), 1687 (C=O),
1
1612 (C=N), 1583 (C=C), 767, 817 (C–Cl); H NMR
(400 MHz, CDCl3, δ ppm): 10.23 (s, 1H, NH), 7.89 (s,
1H, Ar–H), 7.50–7.58 (m, 5H, Ar–H), 7.43 (s, 1H, Ar–H),
6.85–7.00 (m, 3H, Ar–H), 3.74 (s, 2H, –CH2–), 2.65–2.71
(q, 2H, –CH2–), 2.56 (s, 3H, CH3), 2.24 (s, 3H, CH3),
1.09–1.06 (t, 3H, J=12.0 Hz, CH3); 13C NMR (100 MHz,
CDCl3, δ ppm): 167.2, 144.7, 144.0, 141.9, 140.9, 137.5,
137.4, 137.1, 136.5, 130.5, 129.8, 129.7, 129.5, 129.3,
129.3, 129.2, 129.0, 128.5, 124.7, 122.6, 122.1, 117.1, 32.1,
24.4, 18.2, 14.0, 9.6; ESI–MS (m/z): 612.08 [M+H]+; Anal.
calcd. for C28H24N7Cl3OS: C, 54.86; H, 3.95; N, 16.00%.
Found: C, 54.85; H, 3.93; N, 16.02%.
◦
tani (LB) broth at 30 C in a rotary shaker incubator for 24 h.
Antibacterial activity
The antibacterial activity of the compounds was screened
preliminarily by the disc difusion method [37]. Briefy, the
synthetic compounds were loaded into the sterile paper discs
and these were placed on the pre-seeded Mueller–Hinton
(Himedia, Mumbai) agar plates. Overnight culture of test
microorganisms in broth (turbidity adjusted to 0.5 McFar-
land unit) was used for the seeding. Ciprofoxacin discs
(6 mm, 30 μg/disc) were used as a positive control for bac-
teria. Then, the bacterial plates were incubated at 37 °C for
24 h and fungal plates for three days at 30 °C temperature.
The diameters of the inhibition zones (in mm) were meas-
ured after the incubation.
N‑(2‑Bromophenyl)‑2‑(5‑(5‑methyl‑1‑(2,4,5‑trichloropheny
l)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑triazol‑3‑ylthio)
acetamide (8m)
Yield: 60%; Mp. 126–128 °C; Creamish solid; FT-IR (ATR,
υmax, cm−1): 3099 (NH), 2939 (C–H), 1683 (C=O), 1608
1
(C=N), 1585 (C=C), 765, 819 (C–Cl), 650 (C–Br); H
NMR (400 MHz, CDCl3, δ ppm): 10.23 (s, 1H, NH), 7.89
(s, 1H, Ar–H), 7.74–7.76 (m, 1H, Ar–H), 7.50–7.55 (m, 5H,
Ar–H), 6.90–7.48 (m, 4H, Ar–H), 3.97 (s, 2H, –CH2–), 2.52
(s, 3H, CH3); 13C NMR (100 MHz, CDCl3, δ ppm): 167.2,
144.7, 144.0, 141.9, 137.5, 137.4, 137.1, 134.9, 132.4,
130.5, 129.8, 129.3, 129.3, 129.0, 128.4, 126.7, 124.7,
122.1, 121.9, 117.1, 113.5, 32.1, 9.6; ESI–MS (m/z): 647.95
[M+H]+; Anal. calcd. for C25H17N7BrCl3OS: C, 46.1; H,
2.64; N, 15.09%. Found: C, 46.3; H, 2.66; N, 15.02%.
Determination of minimum inhibitory concentration (MIC)
MIC of the synthetic compounds against Xanthomonas
campestris pv. Campestris was performed as per the prevoius
method [37]. A serial two-fold dilution of the synthetic com-
pounds was prepared in a 96-well microtiter plate (Tarsons,
U bottom) over the range 1.25 mg/ml–0.019 mg/ml. Each
of the wells was inoculated with 1X108 of the inoculum.
The plates were incubated at 37 °C for 24 h. Appropriate
growth controls and sterility controls were maintained. The
bacterial growth was assessed by measuring the turbidity
after the incubation.
N‑(4‑Bromophenyl)‑2‑(5‑(5‑methyl‑1‑(2,4,5‑trichloropheny
l)‑1H‑1,2,3‑triazol‑4‑yl)‑4‑phenyl‑4H‑1,2,4‑triazol‑3‑ylthio)
acetamide (8n)
Yield: 70%; Mp. 212–214 °C; Of-white solid; FT-IR (ATR,
υmax, cm−1): 3044 (N–H), 2939 (C–H), 1683 (C=O), 1608
(C=N), 1585 (C=C), 765, 819 (C–Cl), 650 (C–Br); 1H NMR
(400 MHz, CDCl3, δ ppm): 10.41 (s, 1H, N–H), 7.73 (s,
1H, Ar–H), 7.43–7.53 (6H, m, Ar–H), 7.36–7.41 (m, 4H,
Ar–H), 3.95 (s, 2H, –CH2–), 2.52 (s, 3H, CH3), 13C NMR
Anti‑quorum sensing activity
The anti-quorum sensing assay was performed as per the
previously reported method with some modifcation [38].
Briefy, the synthetic compounds were loaded into the sterile
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Journal of the Iranian Chemical Society
paper discs and placed into LB agar plates were pre-seeded
with an overnight culture of C. violaceum. The plates were
incubated at 30 °C for 24 h. Quercetin (Sigma Aldrich, USA)
was used as a standard QSI molecule [39]. After the incuba-
tion time, the plates were observed for the presence of clear
zone (inhibition of violacein production) around the discs
suggesting the inhibition of quorum sensing.
Statistical analysis
Statistical signifcance of variance for collected data was
determined by ANOVA. The quantifcation of QSI activ-
ity by microtiter plate assay was analysed by one-way
ANOVA followed by Tukeys test. Statistical analyses were
performed using Graphpad Prism 5.03 software.
Acknowledgements The authors are thankful to Mangalore University
for providing facility, also grateful to IISC Bangalore for providing
spectral data. We also thank Robert McLean, Professor, Department
of Biology, Texas State University, for the gift of Chromobacterium
strains. Also authors thank Department of Biochemistry, Mangalore
University for providing docking facilities.
Quantifcation of anti‑quorum sensing activity
The quorum sensing inhibition by synthetic compounds
was quantifed as per the reported method [40] with few
modifcations. Briefy, the overnight culture of C. violaceum
inoculated to LB medium along with synthetic compounds
in a 10 ml culture tube. The culture tubes were incubated
Compliance with ethical standards
◦
at 30 C for 24 h. Then, 1 ml of culture from each tube
Conflict of interest All the authors declare that they have no confict
was transferred to a microcentrifuge tube and centrifuged at
10000 rpm for 5. Supernatant was separated, cell pellet was
used to extract violacein with an equal volume of DMSO
(Dimethyl sulphoxide). It was vortexed for 1 to 2 min and
centrifuged at 10,000 rpm for 5 min to remove the cells. Two
hundred microlitres of violacein containing supernatants
were transferred to 96-well microplates, and the absorb-
ance was read with a microplate reader at a wavelength of
585 nm. The harvested bacterial cells from individual treat-
ment were resuspended in 1 ml sterile water to check the
cell growth by measuring the optical density at 600 nm. The
results were compared with respective assay control (without
synthetic compound) and standard (quercetin).
of interest.
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Afliations
Reshma Sathyanarayana1 · Sukesh Kumar Bajire2 · Boja Poojary1 · Rajesh P. Shastry2 · Vasantha Kumar3 ·
Revanasiddappa Bistuvalli Chandrashekarappa4
1
2
3
4
Department of Chemistry, Mangalore University,
Mangalagangothri, Karnataka 574199, India
Department of Pharmaceutical Chemistry, NGSM Institute
of Pharmaceutical Sciences Nitte University, Mangaluru,
Karnataka, India
Yenepoya Research Centre, Yenepoya (Deemed to be
University), University Road, Mangalore 575018, India
Department of Chemistry, Sri Dharmasthala
Manjunatheshwara College (Autonomous), Ujire,
Karanataka 574240, India
1 3