Design, synthesis, biological activity, molecular docking and computational studies on novel 1,4-disubstituted-1,2,3-Triazole-Thiosemicarbazone hybrid molecules

Article abstract of DOI:10.1016/j.molstruc.2020.127951

A library of some novel 1,4-disubstituted-1,2,3-triazole-thiosemicarbazone hybrid molecules were designed and synthesized from (4-Prop-2-ynyloxy-benzylidene)-thiosemicarbazone and aryl azides under Cu(I)-catalyzed cycloaddition reaction. All newly synthesized [4-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethoxy)-benzylidene] -thiosemicarbazone hybrid molecules were efficiently characterized by IR, ¹H NMR, ¹³C NMR, HRMS and structure of alkynes 3 & 12 were finally supported by X-ray crystallographic data. Compounds 5c, 5d, 9c, 9d 13c and 13d demonstrated excellent potency results for B. Subtilis and P. Aeruginosa bacterial strains with MIC values 0.0141, 0.0152, 0.0562, 0.0608, 0.0141, 0.0608, 0.0141, 0.0304, 0.0281, 0.0304, 0.0281, 0.0304, respectively as compared to reference drug Ciprofloxacin. Antibacterial activity results were supported by molecular docking and DFT studies.

Full text of DOI:10.1016/j.molstruc.2020.127951

Journal Pre-proof
Design, synthesis, biological activity, molecular docking and computational studies on
novel 1,4-disubstituted-1,2,3-Triazole-Thiosemicarbazone hybrid molecules
Naveen, Ram Kumar Tittal, Vikas D. Ghule, Nikhil Kumar, Lokesh Kumar, Kashmiri
Lal, Ashwini Kumar
PII:
S0022-2860(20)30276-3
DOI:
https://doi.org/10.1016/j.molstruc.2020.127951
MOLSTR 127951
Reference:
To appear in: Journal of Molecular Structure
Received Date: 28 November 2019
Revised Date: 11 February 2020
Accepted Date: 21 February 2020
Please cite this article as: Naveen, R.K. Tittal, V.D. Ghule, N. Kumar, L. Kumar, K. Lal, A. Kumar,
Design, synthesis, biological activity, molecular docking and computational studies on novel 1,4-
disubstituted-1,2,3-Triazole-Thiosemicarbazone hybrid molecules, Journal of Molecular Structure (2020),
doi: https://doi.org/10.1016/j.molstruc.2020.127951.
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© 2020 Published by Elsevier B.V.

Credit Author Statement
Naveen:
Validation,
Formal
analysis,
Investigation,
Molinspiration
Physicochemical Parameters Software Ram Kumar Tittal: Conceptualization,
Methodology, Resources, Data Curation, Visualization, Writing -Original Draft,
Writing- Review & Editing, Supervision Ghule Vikas D.: Software, Data Curation
Nikhil Kumar: Load & solved X-ray Single Crystal data Lokesh Kumar: Formal
analysis, Investigation Kashmiri Lal: Conceptualization, Methodology,
Resources, Data Curation, Ashwani Kumar: Docking study.

Design, Synthesis, Biological Activity, Molecular Docking and Computational Studies
on Novel 1,4-Disubstituted-1,2,3-Triazole-Thiosemicarbazone Hybrid Molecules
Naveen,[a] Ram Kumar Tittal,*[a] Ghule Vikas D.,[a] Nikhil Kumar,[a] Lokesh Kumar,[b] Kashmiri Lal,*[b] and
Ashwini Kumar[c]
aDepartment of Chemistry, National Institute of Technology, Kurukshetra, Haryana 136119, India.
E-mail: rktittaliitd@mail.nitkkr.ac.in
bDepartment of Chemistry, GJUS&T, Hisar, Haryana 125001, India.
cDepartment of Pharmaceutical Sciences, GJUS&T, Hisar, Haryana 125001, India.
S
S
N
O
H
N
O
H
N
N
H
H2N
H2N
CH2N3
H
CuSO4.5H2O
+
sodum ascorbate,
DMF:Water (8:2),
2-3 h.
R
4a-d
R: a=H, b=Cl, c=Br, d=NO2
N
N
N
o, m, p: 3, 8, 12
R
5a-d, 9a-d & 13a-d
Biologically Significant 1,2,3-triazole-thiosemicarbazone hybrid molecules were synthesized
through Cu(I)-catalysed cycloaddition reaction of thiosemicarbazone-linked alkynes and
aromatic azides. The structures of all alkynes and 1,2,3-triazole-thiosemicarbazone hybrid
molecules were characterized by IR, 1H NMR, 13C NMR & HRMS data structure of alkynes
3 &
12 were finally supported by X-ray crystallographic data. However, some alkynes were finally
supported by single-crystal structures. The synthesized 1,2,3-triazole-TSC hybrid molecules were
screened as active antimicrobials and activity results were supported by the molecular docking
and DFT studies.

Design, Synthesis, Biological Activity, Molecular Docking and
Computational Studies on Novel 1,4-Disubstituted-1,2,3-Triazole-
Thiosemicarbazone Hybrid Molecules
Naveen,a Ram Kumar Tittal,*,a Ghule Vikas D.,a Nikhil Kumar,a Lokesh Kumar,b Kashmiri Lal,*,b and Ashwini
Kumarc
aDepartment of Chemistry, National Institute of Technology, Kurukshetra, Haryana 136119, India.
E-mail: rktittaliitd@mail.nitkkr.ac.in
bDepartment of Chemistry, GJUS&T, Hisar, Haryana 125001, India.
cDepartment of Pharmaceutical Sciences, GJUS&T, Hisar, Haryana 125001, India.
Abstract: A library of some novel 1,4-disubstituted-1,2,3-triazole-thiosemicarbazone hybrid molecules
were designed and synthesized from (4-Prop-2-ynyloxy-benzylidene)-thiosemicarbazone and aryl
azides under Cu(I)-catalyzed cycloaddition reaction. All newly synthesized [4-(1-Benzyl-1H-
[1,2,3]triazol-4-ylmethoxy)-benzylidene] -thiosemicarbazone hybrid molecules were efficiently
characterized by IR, 1H NMR, 13C NMR, HRMS and structure of alkynes 3 & 12 were finally supported
by X-ray crystallographic data. Compounds 5c, 5d, 9c, 9d 13c and 13d demonstrated excellent potency
results for B. Subtilis and P. Aeruginosa bacterial strains with MIC values 0.0141, 0.0152, 0.0562,
0.0608, 0.0141, 0.0608, 0.0141, 0.0304, 0.0281, 0.0304, 0.0281, 0.0304, respectively as compared to
reference drug Ciprofloxacin. Antibacterial activity results were supported by molecular docking and
DFT studies.
Keywords: 1,2,3-triazole-thiosemicarbazone • CuAAC • Antibacterial activity • Molecular docking •
DFT study.
Introduction:
In most of the cases, infections in human beings are non-invasive and temporarily silent. According
to WHO, these slumbering infections lead to an active disease which may be cured by medicines.
Sometimes the situation becomes intricate due to multidrug resistance (MDR) to bacterial and fungal
diseases throughout the whole world.[1] This prominence MDR to pathogenic strains delays the proper
remedy to the infections imposed by various strains.[2] Bacterial diseases many times become worse
and even lead to death. In spite of substantial advancement in medicinal chemistry, it remains a
challenge to the medicinal chemist to develop more efficient and safer antibacterial drugs. There is a
desperate need of coherent approach for the development of novel and more potent drugs. The potency
of drug mainly demonstrated by its prime efficacy, lesser virulence, and better selectivity. Now,
medicinal chemists are mainly attracted towards the synthetic tactics of molecular hybridization. It
involves the coupling of several pharmacophoric units via orthodox bonding to form a single paramount
hybrid molecule. The resulted hybrid molecules generally show improved properties as compared to
parent molecules.
Recently, authors have reported the synthesis, molecular docking and DFT studies of biologically
active 1,4-disubstituted-1,2,3-triazolesemicarbazone hybrid molecules with better antibacterial activities
as compared to commonly used antibiotic Ciprofloxacin.[3] In ancient times, elemental sulfur was
known for pharmacophoric properties and was used for insecticides and fungicides. With these
observations, we presumed to investigate, design and synthesize thiosemicarbazone linked triazole
hybrid molecules. Thiosemicarbazone unit is a sulfur analogy of semicarbazone which may possess
better biological activities. Thiosemicarbazide commonly abbreviated as TSC, also known as hydrazine
carbothioamide, is a sulphur-containing thiocarbonyl organic compound that shares C=N-NH-(C=S)-
NH moiety and known as thiosemicarbazone (TSC). The TSC and its derivatives usually prepared by
the condensation reaction between aldehydes or ketones with thiosemicarbazide under Schiff’s base
condition.[4] Because of better chemical reactivity, physiological properties, versatile biological and
pharmacological activities, TSC and its derivatives have unparalleled applications in the synthetic and

medicinal fields. The broad antimicrobial spectrum of thiosemicarbazone has made a suitable place in
the pharmaceutical industry. Also, TSC is an excellent chelating ligand which remains a versatile
synthon for the synthesis of important therapeutic compounds. Antiviral properties of
thiosemicarbazone were first reported by Hamre et al in 1950.[5] In fact, thiosemicarbazone was the
first representative to be used as an active molecule for the development of an antiviral drug.
Thiosemicarbazone linked with isatin unit reportedly found active against smallpox or cowpox.[6]
Sidwell and co-workers found purine-6-carboxyaldehyde linked with thiosemicarbazone effective
against the herpes virus.[7] Brockman and co-workers have reported thiosemicarbazone as active
against herpes simplex virus.[8] Klayman et al. tested the antimalarial activity of thiosemicarbazone
linked with 2-acetyl pyridine.[9] Dobek lab found the activity of thiosemicarbazone substituted-2-acetyl
pyridine activity against mycobacteria.[10] Hybrid compounds containing TSC moiety or metal
complexes of TSC have been reported in the literature to possess more promising biological activities
like
antiviral,[11]
antitumor,[12]
antibacterial,[13]
antitubercular,[14]
antidiabetic,[15]
antimicrobials,[16] anticonvulsant,[17] anticancer,[18] antimalarial,[19] anti-inflammatory and
antiameobic activity, etc.[20]
Similarly, 5-member nitrogen-containing heterocycles like 1,2,3-triazoles are the most affluent
constitutional ingredient to various drugs and biomaterials. This is due to stability towards acidic and
basic hydrolysis even under extreme redox conditions.[21] Because of polar nature, triazoles behave as
a hydrogen acceptor, hydrogen donor, can show dipole-dipole interaction and increases solubility in an
aqueous medium. Thus, show several types of interactions with the target bioactive conjugates and
protect it from metabolic degradation under extreme reaction conditions, chemical environments, and
reagents. The 1,3-dipolar cycloaddition reaction between terminal alkyne and azide by Huisgen method
is the most prevalent and robust strategy for the synthesis of 1,4-disubstituted-1,2,3-triazole but leads to
the formation of an almost equivalent amount of 1,4 and 1,5-isomers. Recently, the modified Huisgen
Cu(I)-catalyzed cycloaddition reaction between an azide and terminal alkyne under CuAAC reaction
condition by Medal,[22] and Sharpless[23] has emerged as a premier example of click reaction. The
term click reactions are versatile, highly selective, compatible with a wide variety of functional groups,
efficient and accelerate the transformation of reactant into only one isomeric product. The removal of
homogeneous Cu-catalyst from the reaction mixture always remains a big issue. Various types of
procedures have emerged in literature for the regioselective synthesis of 1,4-disubstituted-1,2,3-
triazole.[24] The ease in formation, high yield and stability of 1,4-disubstituted-1,2,3-triazoles are
currently used as building blocks in fascinating biological activities like anticancer,[25-27]
antioxidant,[28] antitubular,[29] antimalarial,[30] anti-HIV,[31] antimicrobial,[32-34] and anti-
inflammatory etc.[35]
Considering medical importance of both TSC & 1,4-disubstituted-1,2,3-triazole units in the light of
our attention for synthesizing pharmacologically active compounds, it was envisioned to synthesize a
family of 1,2,3-triazole-thiosemicarbazone hybrids (Figure 1) via molecular hybridization approach
with a view to assess their antimicrobial properties against the human pathogens like E. coli, B. subtilis,
S.
aureus,
P.
Aeruginosa, & S. Enteric.
Figure 1. Designed 1,2,3-triazole-thiosemicarbazone hybrid molecule.
Results and Discussion

A series of novel 1,2,3-triazole-thiosemicarbazone hybrid molecules were synthesized in three steps
using the reported procedures with slight modifications as described in Scheme 1.[3] The first step
commenced with propargylation of commercially available o, m & p-hydroxybenzaldehyde by refluxing
R1
H
R1
H
O
H
H
O
(iii)
(ii)
(i)
O
N
N
CH2N3
O
O
N
HO
o, m, p: 1, 6, 10
3, 8, 12
2, 7, 11
R2
R2
conditions:
4
5, 9, 13
(i) Propargyl bromide, K2CO3, Acetone, refulx, 8h
(ii) NH2NHCSNH2.HCl, ethanol, reflux
(iii) 4a-d, CuSO4.5H2O, sodum ascorbate, DMF:Water (8:2), 2-3 h.
R1: NNHCSNH2, R2: a=H, b=Cl, c=Br, d=NO2
Scheme 1. Synthesis of TSC-triazole hybrid molecules (5a-d, 9a-d, 13a-d).
with propargyl bromide in acetone using anhydrous K2CO3. In the second step, the prepared o,m,p-
propargyloxy benzaldehyde was subjected for condensation reaction with thiosemicarbazide in dry
C2H5OH for 4 hrs. The so obtained thiosemicarbazone linked alkynes (TSC-alkyne) were recrystallized
with alcohol in 90-94% yields. The formation of TSC-alkynes (3, 8, 12) was efficiently characterized by
1
IR, H NMR, and 13C NMR spectroscopic data. For reference, the Infra-Red spectroscopic data of
compound 12 showed two major stretching peaks at νmax 3282 & 1606 cm-1 due to terminal alkyne ≡CH
and −C=N−, respectively for the formation of thiosemicarbazone of propargyl benzaldehyde. Also, the
1H NMR spectrum of 12 showed a triplet peak at δ 3.60 ppm equivalent to one hydrogen and another
peak at δ 11.34 ppm equivalent to another one hydrogen due to ≡CH(alkyne) group and H−C=N− group,
respectively to confirm the formation of TSC-alkynes. The HRMS spectrum of alkyne 12 showed a
major peak at m/z 234.077 corresponds to [M+H]+. Finally, the structures of TSC-alkynes 3 and 12
were supported by the x-ray diffraction data.[36] The crystal structure diagram of alkynes 3 and 12
shown in Figure 2 and 3. The crystal data and crystal structure refinement parameters of 3 and 12 are
given in Table SI-1 (S.I. file).
Figure 2. Crystal structure of compound 3. Asymmetric unit contains two such molecular units.

Figure 3. ORTEP diagram of 12 with 50% probability (H atoms are removed for clarity).
The starting material for the click reaction, i.e. TSC-alkyne (3, 8 & 12) were subjected to react with
in situ generated organic azides in the presence of CuSO4.5H2O in DMF as a solvent to afford different
1,4-disubstituted-1,2,3-triazole-TSC hybrids (5a-d, 9a-d & 13a-d). The results with reaction details are
summarized in Table 1. The structures of all the synthesized triazole hybrids were efficiently
characterized with the help of IR, 1H NMR, 13C NMR and HRMS data. For instance, the appearance of
a band at 3151 cm-1 in the IR spectrum of compound 13b showed the formation of the triazole ring. The
1H NMR spectrum of compound 13b showed singlet peak at δ 7.93 ppm equivalent to one hydrogen due
13
to -CH of triazole ring to confirm the formation of 1,2,3-triazole-TSC. The C NMR spectrum of 13b
showed peaks at δ 143.29 and 125.25 ppm, which were assigned to C-4 and C-5 carbon atoms of the
triazole rings. Two peaks appeared at δ 61.64 and 52.53 ppm were due to the presence of two methylene
carbons attached to N-1 and C-4 of the triazole ring, respectively. The HRMS spectrum of 13b showed
the m/z peak at 401.1084 corresponds to [M+H]+. All other compounds also exhibited a similar pattern
in their spectra and the characterization details are presented in S.I. file.
Table 1. Synthesis of terminal alkyne (3, 8 & 12) and 1,2,3-triazole-linked-thiosemicarbazone (5a-d,
9a-d, 13a-d)a.
S. No.
1
2
3
4
5
6
7
8
Compounds
R2
--
Time (h)
Yield (%)b
4
92
85
86
90
92
90
90
91
90
89
94
88
85
90
92
3
5a
5b
5c
5d
8
9a
9b
9c
9d
12
13a
13b
13c
13d
H
3
2.5
2.5
2
4
3
2.5
2.5
2
Cl
Br
NO2
--
H
Cl
Br
NO2
--
9
10
11
12
13
14
15
4
H
2.5
2.5
2
Cl
Br
NO2
2
aBenzyl azide 4a-d & alkynes 3, 8, & 12 (1 mmol); CuSO4.5H2O (10 mol%) and Na-asc. (20 mol%), rt,
2-3 h. bYield refers to pure compound obtained after recrystallization.
A library of newly synthesized TSC-1,2,3-triazole hybrid molecules was screened in vitro for
antibacterial activity against gram-positive bacteria: Staphylococcus Aureus (MTCC 3160),

Staphylococcus Enteric and Bacillus Subtilis (MTCC441) and gram-negative bacteria: Escherichia Coli
(MTCC 16521) and Pseudomonas Aeruginosa (MTCC 424) by a standard serial dilution method. A
stock solution of 100 µg/ml concentration in DMSO solvent was prepared. A commonly used antibiotic
drug Ciprofloxacin was selected as a reference drug. The stock solution of compounds and reference
were diluted to 50 to 6.25 µg/mL concentration. Now each concentration lot was inoculated with 100
µg/mL suspension of respective bacteria in sterile saline and incubated at 37 °C for 24 hrs. To observe
the effect of solvent on bacterial growth, a blank test was also performed at the experimental condition
with the nutrient medium. The minimum inhibitory concentration (MIC) of the triazole was recorded in
µmol/mL, as shown in Table 2.
Table 2. In vitro antibacterial data of compounds 3, 5a-d, 8, 9a-d, 12, 13a-d (MIC in µmol/mL).
Comp.
3
5a
5b
5c
5d
8
9a
9b
9c
9d
12
13a
13b
13c
13d
R
---
H
Cl
A
B
C
D
E
0.1073
0.0683
0.0312
0.0141
0.0152
0.1073
0.0683
0.0624
0.0562
0.0608
0.1073
0.0683
0.0624
0.0141
0.0608
0.1465
0.1073
0.1366
0.0624
0.0562
0.0608
0.1073
0.0683
0.0312
0.0281
0.0304
0.1073
0.0683
0.0312
0.0562
0.0304
0.0366
0.2145
0.0683
0.0312
0.0281
0.1217
0.2146
0.0683
0.0624
0.0562
0.0304
0.2146
0.0683
0.0312
0.0281
0.0608
0.0732
0.1073
0.1366
0.0624
0.0141
0.0304
0.1073
0.0683
0.0312
0.0281
0.0304
0.1073
0.1366
0.0156
0.0281
0.0304
0.0732
0.2146
0.0683
0.0624
0.0562
0.1217
0.2146
0.0683
0.1248
0.0281
0.0608
0.2146
0.1366
0.0624
0.1124
0.0304
0.0732
Br
NO2
----
H
Cl
Br
NO2
----
H
Cl
Br
NO2
Ciprofloxacin
A: B. Subtilis; B: S. Aureus; C: E. Coli; D: P. Aeruginosa; E: S. Enteric.
The antibacterial screening results of alkynes and triazole hybrids (3, 5a-d, 8, 9a-d, 12 & 13a-d)
were compared with each other and reference drug. Wherein, it was observed that the incorporation of
triazole units on to the TSC-alkynes have increased the antibacterial potency of almost all 1,4-
disubstituted-1,2,3-triazole-TSC hybrid molecules for all bacterial strains except few. Also, it was
observed that on comparing antibacterial activity results of TSC-alkynes and triazole-TSC hybrid
molecules with commonly used reference drug Ciprofloxacin, compounds 5c, 5d, 9c, 9d 13c & 13d
demonstrated excellent potency results for B. Subtilis and P. Aeruginosa bacterial strains with MIC
values 0.0141, 0.0152, 0.0562, 0.0608, 0.0141, 0.0608, 0.0141, 0.0304, 0.0281, 0.0304, 0.0281, 0.0304,
respectively. However, among other bacterial strains, S. Aureus showed excellent potency for 9c, 9d, &
13d; E. Coli for 5c, 9c, 9d, 13c & 13d and S. Enteric showed excellent activity for 5c, 9c, 9d & 13d.
Similarly, for compound 5c best potency results were observed for B. Subtilis, E. Coli and P.
Aeruginosa with MIC 0.01405, 0.02809 & 0.01405 µmol/mL as compared to Ciprofloxacin with MIC
0.14648, 0.07324 & 0.07324 µmol/mL, respectively. Likewise, compound 9c showed the better potency
for both S. Aureus, and S. Enteric with MIC 0.0281 µmol/mL and compound 13c showed best results
for B. Subtilis and E. Coli with MIC 0.01405 & 0.02809 µmol/mL, respectively as compared to drug
Ciprofloxacin (MIC 0.14648 & 0.07324 µmol/mL).
Also, the antibacterial activities of recently reported 1,2,3-triazole-semicarbazone hybrid molecules
(Figure 4a) were compared with these newly synthesized 1,2,3-triazole-TSC molecules (Figure 4b) for
some tested strains of bacteria, i.e. E. Coli, B. Subtilis, S. Aureus, P. Aeruginosa & S. Enteric as listed in
Table 3. It was observed and revealed that TSC-derivatives are more potent or active as compared to

Figure 4. a: Semicarbazone hybrid & b: Thiosemicarbazone hybrids.
Table 3. Comparison of antimicrobial activity of SC & TSC hybrids.
E. Coli
TSC
B. Subtilis
TSC
Alkyne/R
SC
SC
o-Alkyne
H
Cl
Br
NO2
p-Alkyne
H
Cl
Br
0.11520
0.07440
0.06747
0.06024
0.13123
0.23041
0.07440
0.13495
0.12048
0.06562
0.21459
0.06831
0.03121
0.02809
0.12166
0.21459
0.06831
0.03121
0.02809
0.06083
0.11520
0.07440
0.06747
0.06024
0.06562
0.11520
0.07440
0.06747
0.12048
0.06562
0.10730
0.06831
0.03121
0.01405
0.01521
0.10730
0.06831
0.06242
0.01405
0.06083
NO2
S. Aureus
P. Auroginosa
Alkyne/R
SC
TSC
SC
TSC
o-Alkyne
H
Cl
Br
NO2
p-Alkyne
H
Cl
Br
0.11520
0.03720
0.06747
0.06024
0.06562
0.05760
0.03720
0.03374
0.12048
0.03281
0.10730
0.13661
0.06242
0.05618
0.06083
0.10730
0.06831
0.03121
0.05618
0.03041
0.11520
0.07440
0.06747
0.06024
0.06562
0.11520
0.07440
0.06747
0.12048
0.06562
0.10730
0.13661
0.06242
0.01405
0.03041
0.10730
0.13661
0.01561
0.02809
0.03041
NO2
o-/P-Alk: ortho/para-alkyne; SC: Semicarbazone; TSC: Thiosemicarbazone
the corresponding semicarbazone analogues except for 5a and 13a with unsubstituted benzyl (R=H),
which showed little discouraging results for S. Aureus & P. Auroginosa bacterial strains. Also, TSC-lin-
-ked ortho-alkynes for E. Coli and para-alkyne for S. Aureus showed less potency then corresponding
semicarbazone linked alkynes.
The structure-activity relationship (SAR) results showed that all triazole derivatives exhibited better
efficacy and synergistic effect in the biological activity of TSC-triazole hybrids than the parent
pharmacophoric units i.e. either TSC or 1,2,3-triazole. The Table SI-1 (S.I. file) revealed that the
substitution of benzyl moiety by electron-withdrawing groups (Br and NO2) like 5c, 5d, 9c, 9d, 13c, and
13d exhibited broad-spectrum activity against studied bacterial strains. It was also observed that the
compounds substituted with Br- on benzyl moiety (5c, 9c and 13c) showed more promising antibacterial
activity than other compounds.

The physicochemical properties of 1,2,3-triazole-TSC hybrids were calculated by using Lipinski’s
rule of five, which acts as a filter for drug likeliness and to deduce their pharmacokinetic parameters. It
includes absorption, distribution, metabolism and excretion from the human body. The value of milogP
(octanol-water partition coefficient) of all synthesized compounds is less than 5.0 (2.1 to 4.09)
indicating their high affinity and excellent permeability across the lipid bilayers of biological
membranes. Also, the molecular weight less than 500 (366.45 to 445.35) showed easy diffusion,
transportation and absorption. The sum of -OH and -NH groups showed that the hydrogen bond donor is
less than 5.0 (3 for all) and hydrogen bond acceptor are less than 10 (i.e. 7-10). Therefore, the no. of
violation to the Lipinski’s rule of five is zero, confirming their easy binding to receptors. Also, the
numbers of rotational bonds are less than 10 (i.e. 5-9), thus confirming their good flexibility. The value
of total polar surface area (TPSA) was less than 160Å2 (59.65 Å2 to 136.19 Å2) which presented it to
be a good predictor of transport properties like intestinal absorption, blood barrier penetration, etc. The
mathematical equation of %ABS is equal to 109-(0.345 x TPSA).[37] All the presented alkyne-TSC and
triazole-TSC hybrid molecules exhibited good absorption (%ABS = 62.01 to 88.42). According to
Lipinski’s rule of five, any molecule violating more than one of these rule decreases the activity and
selectivity of a drug-like compound to make unlikely orally-active for human beings. It is clear from the
above discussion that parameters of all the tested compounds were within the limits of Lipinski’s rule of
five with violation number equal to zero and have good drug likeliness property. The bioactivity score
of all the synthesized compounds summarized in table SI-2 (S.I. file) by calculating the activity score
of GPCR (protein-coupled receptor) ligand, ICM (ion channel modulator), KI (kinase inhibitor), NRL
(nuclear receptor ligand), PI (protease inhibitor), and EI (enzyme inhibitor). The bioactivity score of all
the compounds was >-0.5, which revealed that the synthesized compounds are active.
The reason for the higher activity of 1,2,3-triazole-TSC hybrid molecules as compared to TSC-
alkynes could also be explained by the docking studies, the results of study represented in Figure 5 & 6.
Figure 5. Interactions of alkyne 12 with active sites of DNA gyrase.

Figure 6. Interactions of a triazole 13d with the active site residues of DNA gyrase.
Figure 7. Cartoon diagram of DNA gyrase with co-crystallized ligand, alkyne 12, and triazole 13d.
Both molecules were docked in the active site of topoisomerase II of E. Coli (PDB ID: 1KZN).[38-40]
This showed interactions of alkyne and triazole with the active sites of DNA gyrase. The two hydrogens
of the terminal amino group were involved in hydrogen bonding (Figure 5) with oxygen atoms of
Glu50 and Gly77. Amide-π interaction was observed between the aromatic ring of alkyne 12 and the
peptide bond between Asn46 and Ala47. A one carbon-hydrogen bonding was seen between carbon
attached to oxygen in alkyne and the carbonyl oxygen of Val43. In triazole 13d, the NO2 group has
created a web of hydrogen bonding with His95, Ala96, Val120 and Ser121 (Figure 6). The terminal –

NH2 group exhibited hydrogen bonding with Val71 and Thr165 while second NH group involved in
hydrogen bonding with Asp73. The π-orbitals of the aromatic ring were involved in the amide-π
stacking interactions with the peptide bond between Asn46 and Ala47. The hydrogen bonding was
represented by the intense green colour dotted lines and the amide-π stacking represented by the pink
dotted lines. In addition, the binding affinity of triazole was -8.2 kcal/mol in comparison to that of
alkyne -6.6 kcal/mol. All these facts support the higher activity of triazole as compared to the alkyne.
The cartoon diagram of DNA gyrase along with co-crystallized ligand, alkyne, and triazole is shown in
Figure 7.
All the molecules included in the study were optimized at the B3LYP/6-311G(d,p) level using the
Gaussian09 program package.[41] With the help of frontier molecular orbitals (FMOs) of any chemical
compounds, one can determine important parameters such as chemical reactivity needed for drug
design.[42-44] A molecule with a high value of highest occupied molecular orbitals (HOMOs) can
donate electrons to the least unoccupied molecular orbitals (LUMOs). The value of HOMO and LUMO
determines the electronic transitions and the reactivity of molecules. These values also help to predict
the frontier orbital energies (FMOs), chemical potential (ꢀ), chemical hardness (η), and electrophilicity
index (ω) as presented in Table 4. The values of ꢀ, η, and ω can be obtained by using Koopmans’
theorem and Parr approximation,[45] as described in the SI (see, S. I. file). We have observed that 5a-5d
derivatives exhibited higher bandgap as compared to 9a-9d and 13a-13d derivatives. Replacement of
Cl- of 5b with –Br in 5c didn’t show any change in the bandgap.
Table 4. Calculated FMOs, energy gap, chemical potential, chemical hardness, and electrophilicity
index in eV at the B3LYP/6-311G(d,p) level of theory.
EHOMO
(eV)
ELUMO-HOMO
(eV)
3.91
Compd.
E
LUMO (eV)
ꢀ (eV)
η (eV)
ω (eV)
-5.60
-5.64
-5.91
-5.91
-6.01
-5.90
-5.62
-5.68
-5.67
-5.73
-5.71
-5.58
-5.75
-5.75
-5.86
-1.69
-1.59
-1.72
-1.71
-2.73
-1.84
-1.93
-1.60
-1.59
-2.78
-1.80
-1.64
-1.91
-1.90
-2.85
-3.64
-3.62
-3.81
-3.81
-4.37
-3.87
-3.77
-3.64
-3.63
-4.26
-3.75
-3.61
-3.83
-3.82
-4.35
1.96
2.03
2.10
2.10
1.64
2.03
1.84
2.04
2.04
1.48
1.96
1.97
1.92
1.92
1.50
3.39
3.23
3.47
3.46
5.84
3.69
3.87
3.24
3.24
6.14
3.60
3.31
3.81
3.80
6.30
3
5a
5b
5c
5d
8
9a
9b
9c
9d
12
13a
13b
13c
13d
4.05
4.19
4.19
3.27
4.05
3.69
4.08
4.08
2.95
3.91
3.94
3.84
3.84
3.01
However, the introduction of –NO2 group in 5d significantly reduced the bandgap and revealed more
mobility of electrons. A similar trend was observed in meta and para derivatives. The predicted
hardness values of –NO2 derivatives of 5d, 9d, and 13d were 1.64 eV, 1.48 eV, and 1.50 eV,
respectively that showed higher reactivity. On the other hand, the corresponding electrophilicity index
of these molecules revealed their fewer stabilization energies. The HOMO and LUMO of 13a-d along
with molecular electrostatic potential (MESP) maps were calculated as illustrated in Figure 8. In a
comparison study of bandgap energy (∆ELUMO-HOMO) of TSC-triazoles (5a-d & 13a-d) with recently
reported semicarbazone derivatives as shown in table 5 revealed that 1,2,3- triazole-TSC hybrids
molecules are soft with less bandgap energy. Thus, 1,2,3-triazole-TSC hybrid molecules with lower
bandgap energy were more reactive as compared to the 1,2,3-triazole-SC hybrid molecules. It was

revealed that 1,2,3-triazole-thiosemicarbazone hybrid molecules are more reactive and are better
antimicrobial substitutes to current drug Ciprofloxacin.
13a (HOMO)
13a (LUMO)
13b (LUMO)
13b (HOMO)
13c (LUMO)
13c (HOMO)
13d (HOMO)
13d (LUMO)

13b
13a
13c
13d
Figure 8. HOMO and LUMO distribution at the ground state of 13a-d molecules and Molecular
electrostatic potential (MESP) analysis of 13a-d molecules.
Table 5. Comparison of bandgap energy (ELUMO-HOMO) of 1,2,3-triazole-TSC hybrid and 1,2,3-triazole-
SC hybrid molecules.
TSC
SC
Compd.
EHOMO
(eV)
ELUMO-HOMO
(eV)
EHOMO
(eV)
ELUMO-HOMO
(eV)
4.37
ELUMO (eV)
ELUMO (eV)
-5.60
-5.64
-5.91
-5.91
-6.01
-5.71
-5.58
-5.75
-5.75
-5.86
-1.69
-1.59
-1.72
-1.71
-2.73
-1.80
-1.64
-1.91
-1.90
-2.85
3.91
4.05
4.19
4.19
3.27
3.91
3.94
3.84
-5.91
-5.82
-5.99
-5.99
-6.53
-5.69
-5.82
-6.22
-5.87
-6.31
-1.54
-1.56
-1.69
-1.68
-2.71
-1.06
-1.31
-1.7
3
5a
5b
5c
5d
12
13a
13b
13c
13d
4.26
4.3
4.31
3.82
4.63
4.51
4.52
4.3
3.84
3.01
-1.57
-3.04
3.27
Conclusions
In precise, we have designed and synthesized a library of 1,4-disubstituted-1,2,3-triazole-
thiosemicarbazone hybrid molecules via Cu(I)-catalyzed click reaction of thiosemicarbazone-linked
alkynes with aromatic azides. Newly synthesized compounds were fully characterized by IR, 1H NMR,
13C NMR, HRMS and structures of alkynes 3 & 12 were finally supported by x-ray crystallographic
data. Also, a synergistic effect in biological activity was exhibited by the incorporation of
thiosemicarbazide unit with 1,2,3 triazoles. Compounds 5, 9, 13 (c & d) with MIC values 0.0141,

0.0152, 0.0562, 0.0608, 0.0141, 0.0608, 0.0141, 0.0304, 0.0281, 0.0304, 0.0281, 0.0304, respectively
for B. Subtilis and P. Aeruginosa bacterial strains exhibited excellent potency as compared to classically
used drug Ciprofloxacin. The FMOs revealed that incorporation of triazole moiety on to semicarbazone
linked alkyne has improved the pharmacological activities of the resulted hybrid molecules.
Antibacterial potency results of Triazole-TSC hybrid molecules were supported by computational
docking and DFT studies.
Experimental Section
General Procedure for the synthesis of a terminal alkyne (2)
To a solution of o-/m-/p-substituted hydroxybenzaldehyde (1.0 mmol) in 20 mL acetone, anhydrous
potassium carbonate (1.5 mmol) was added and the resulting suspension was refluxed for 30 min.
Propargyl bromide (80% in toluene, 1.5 mmol) was added to it slowly and the reaction mixture was
refluxed for 7-8 hrs. After completion of the reaction as monitored by TLC, the solvent was evaporated
by a rotatory evaporator and the resulting solid residue was washed with ice-cold water then
recrystallized from chloroform: hexane (7:3) to get pure terminal alkyne 2.
General procedure for the synthesis of thiosemicarbazone linked alkyne (3, 8 & 12)
Thiosemicarbazide hydrochloride (1 mmol dissolved in 10 mL water) was added to a stirred solution of
ortho-propargyl-benzaldehyde (1 mmol) taken in ethanol at 70 °C. The reaction mixture was allowed to
stir for 4 hours at this temperature. The progress and completion of the reaction were monitored by
taking TLC at a frequent interval of time. After completion, the reaction mixture was cooled and light
brown colour precipitates have appeared in the reaction mixture. Then, 20 mL of ice cool water was
added to the reaction mixture. When the number of precipitates increased, filtered and dried the
precipitates. Then, the precipitate was washed with ice-cold water followed by washing with ethanol to
remove unreacted starting materials to produce the pure compound in 90-94% yield.
General procedure for the synthesis of thiosemicarbazone linked triazoles (5a-d, 9a-d & 13a-d)
A mixture of substituted benzyl bromide 4a-d (1.0 mmol), sodium azide (3.0 mmol), alkyne 3, 8 & 12
(1.0 mmol) in DMF/water (8:2), copper sulphate pentahydrate (10 mol%) and sodium ascorbate (20
mol%) was stirred for 2 to 3 hrs at room temperature. The progress and completion of the reaction was
monitor by TLC at a frequent interval of time. After completion, the reaction mixture was diluted with
ice-cold water (30 mL). The solid residues were filtered, washed with aqueous ammonium chloride:
ammonia solution (9:1 v/v) followed by washing with water and recrystallized with ethyl acetate to get
the desired 1,2,3-triazole derivatives (5a-d, 9a-d & 13a-d).
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
We thank Dr A. Bhankhar, Department of Bio & Nano Technology, Hisar, GJUS&T, Haryana (India)
for rendering Pharmacological facilities.
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Highlights:
1) Newly designed 1,4-disubstituted-1,2,3-triazole-thiosemicarbazone hybrid molecules
were synthesized in excellent yield.
1
2) All triazole hybrid molecules were fully characterized by IR, H NMR, 13C NMR,
HRMS and alkynes 3 & 12 were supported by single-crystal structures.
3) Compounds 5, 9, 13 (c & d) exhibited excellent potency for B. Subtilis and P.
Aeruginosa bacterial strains as compared to reference drug Ciprofloxacin.
4) Thiosemicarbazone hybrid molecules with lower bandgap energy were soft and more
reactive than corresponding semicarbazone hybrid molecules and alkynes.
5) The antibacterial activity results were supported by the molecular docking and DFT
studies.
Thank You,
Yours truly,
(Ram Kumar Tittal)

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
The authors declare no conflict of interest.