Novel isoniazid embedded triazole derivatives: Synthesis, antitubercular and antimicrobial activity evaluation

Article abstract of DOI:10.1016/j.bmcl.2020.127434

In the present study, a series of new isoniazid embedded triazole derivatives have been synthesized. These compounds were evaluated for their in vitro antitubercular and antimicrobial activities. Among the screened compounds, six have exhibited potent antitubercular activity against Mycobacterium tuberculosis H37Rv strain with MIC value 0.78 μg/mL, whereas, three compounds have displayed activity with MIC value ranging from 1.56 to 3.125 μg/mL. The cytotoxicity of the active compounds was studied against RAW 264.7 cell line by MTT assay and no toxicity was observed even at 25 μg/mL concentration. The five compounds have displayed good antimicrobial activities. Molecular docking have been performed against mycobacterial InhA enzyme to gain an insight into the plausible mechanism of action which could pave the way for our endeavor to identify potent antitubercular candidates. We believe that further optimization of these molecules may lead to potent antitubercular agents.

Full text of DOI:10.1016/j.bmcl.2020.127434

Journal Pre-proofs
Novel isoniazid embedded triazole derivatives: synthesis, antitubercular and
antimicrobial activity evaluation
Pravin S. Patil, Sanghratna L. Kasare, Nitin B. Haval, Vijay M. Khedkar,
Prashant P. Dixit, Estharla Madhu Rekha, Dharmarajan Sriram, Kishan P.
Haval
PII:
DOI:
Reference:
S0960-894X(20)30545-X
https://doi.org/10.1016/j.bmcl.2020.127434
BMCL 127434
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
4 April 2020
17 June 2020
21 July 2020
Please cite this article as: Patil, P.S., Kasare, S.L., Haval, N.B., Khedkar, V.M., Dixit, P.P., Rekha, E.M., Sriram,
D., Haval, K.P., Novel isoniazid embedded triazole derivatives: synthesis, antitubercular and antimicrobial
activity evaluation, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127434
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Novel isoniazid embedded triazole derivatives: synthesis,
antitubercular and antimicrobial activity evaluation
Pravin S. Patil,a Sanghratna L. Kasare,a Nitin B. Haval,a Vijay M. Khedkar,b Prashant P. Dixit,c Estharla
Madhu Rekha,d Dharmarajan Sriram,d Kishan P. Haval*a
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University SubCampus, Osmanabad 413501 (MS) India
bDepartment of Pharmaceutical Chemistry, School of Pharmacy, Vishwakarma University, Pune 411048 (MS) India
cDepartment of Microbiology, Dr. Babasaheb Ambedkar Marathwada University SubCampus, Osmanabad 413501 (MS) India
dDepartment of Pharmacy, Birla Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal,
R. R. District, Hyderabad 500078, India
Email: havalkp@gmail.com
GRAPHICAL ABSTRACT
ABSTRACT
In the present study, a series of sixteen new isoniazid embedded triazole derivatives have
been synthesized. These compounds were evaluated for their in vitro antitubercular and
antimicrobial activities. Among the screened compounds, six have exhibited potent
antitubercular activity against Mycobacterium tuberculosis H37Rv strain with MIC value
0.78 μg/mL, Whereas, three compounds have displayed activity with MIC value ranging
from 1.56 to 3.125 μg/mL. The cytotoxicity of the active compounds was studied against
RAW 264.7 cell line by MTT assay and no toxicity was observed even at 25 μg/mL
concentration. The five compounds have displayed good antimicrobial activities. Molecular
docking have been performed against mycobacterial InhA enzyme to gain an insight into the
plausible mechanism of action which could pave the way for our endeavor to identify potent
antitubercular candidates. We believe that further optimization of these molecules may lead
to potent antitubercular agents.
Keywords: Isoniazid, 1,2,3-Triazole, Antitubercular Activity, Antimicrobial Activity,
Cytotoxicity, Molecular Docking, Click Chemistry.
1

Tuberculosis (TB) is the leading cause of death from a single infectious agent (ranking
above HIV/AIDS). It is a chronic disease that gets transmitted through air and caused by the
bacillus Mycobacterium tuberculosis. It typically affects the lungs (pulmonary TB) but can
also affect other sites (extrapulmonary TB).1 As per Global Tuberculosis Report-2019,
tuberculosis (TB) is a communicable disease and one of the top ten causes of death
worldwide.2 The current efforts in the drug development process are insufficient to
completely eradicate the TB epidemic.3 There are two main health concerns around the
tuberculosis: drug-resistant tuberculosis and co-infection with HIV. It is observed that interest
in tuberculosis research is rising, and the control of its spread has become one of the main
health priorities in the world, with the United States, the United Kingdom, and India, leading
the research in this area.4 As constrained treatment options for multi-drug resistant (MDR-
TB) and extensively drug resistant (XDR-TB) are available, TB researchers have the
challenge of synthesizing new anti-TB drugs with novel modes of action.5, 6
Isoniazid (INH) has been used as first-line anti-TB drug combined with Rifampicin
(RIF), Ethambutol (EMB) and Pyrazinamide (PZA) to treat TB infection for more than 60
years, and it still remains one of the most effective anti-TB drugs. Its mechanism of action
includes multiple effects on lipids, glycolysis, biomembranes, proteins and nucleic acid
synthesis.7 Unfortunately, it has several adverse effects particularly, psychiatric, hepatitis,
peripheral neuropathy, GI Intolerance, allergic reactions and drug interactions.8 The bacterial
strains resistant to isoniazid are becoming common which mainly due to the long-term widely
use even abuse, so there is an urgent need to develop novel isoniazid derivatives.9
To overcome the drug resistance and adverse effects, combination of isoniazid with other
active moiety is frequently applied. Numerous efforts have been undertaken to develop
isoniazid hybrids as new anti-TB agents (Fig. 1).10-15 1,2,3-Triazole derivatives inhibit the
growth of bacteria by blocking lipid biosynthesis and/or additional mechanisms which is one
of the most attractive strategies for developing effective anti-TB agents (development of cell
wall biosynthesis inhibitors). Both isoniazid and triazole act by similar mechanism, i.e.
inhibition of cell wall synthesis and both are very potent anti-TB agents, thus these two
entities conjugated covalently into one single molecule may offer a new lead with potent anti-
tubercular activity.16-21 Recently, we have reported phthalimide bearing 1,2,3-triazole
derivatives possessing antitubercular activity.22 Encouraged by the previous studies23-28 and
in continuation of our efforts towards the synthesis of new anti-tuberculosis agents, herein,
we designed and synthesized a series of novel isoniazid embedded 1,4-disubstituted 1,2,3-
triazole analogues starting from 5-nitrosalicylaldehyde.
2

Fig. 1. (a) Representative isoniazid and 1,2,3-triazole analogues having antitubercular
activity; (b) Our approach of isoniazid embedded triazole derivatives as antitubercular agents.
For the synthesis of the title compounds, firstly 2-hydroxy-5-nitrobenzaldehyde (1) was
treated with propargyl bromide in presence of K2CO3 in DMF at room temperature to obtain
aldehyde (2).29 The condensation of aldehyde (2) and isonicotinohydrazide was carried out in
diisopropylethylammonium acetate (DIPEAc) to obtain the alkyne (3) (Scheme 1).30, 31 The
click reaction of alkyne (3) and substituted azidobenzenes (4a-p) in presence of CuSO4.5H2O
and sodium ascorbate was performed to furnish corresponding triazole derivatives (5a-p) in
good yields as depicted in Scheme 2.
3

Scheme 1. Reaction conditions: (i) Propargyl bromide, K2CO3, DMF, rt, 6h (90%); (ii)
Isonicotinohydrazide, DIPEAc, rt, 1h (92%).
Scheme 2. Reaction conditions: (i) CuSO4, Sodium ascorbate, DMF, rt (80-92%)
1
The newly synthesized compounds were characterized by their H NMR, 13CNMR and
1
HRMS spectral data analysis. In H NMR spectrum of compound 5o, two singlets at  2.33
and 5.45 ppm are observed due to the -CH3 attached to aromatic ring and –O-CH2 attached to
triazolyl ring, respectively. The characteristic peak at  12.02 is due to amide N-H. The two
characteristics peaks in 13CNMR spectrum of compound 5o at  18.67 and 62.75 ppm
confirms the presence of -CH3 and -OCH2 carbons, respectively. The HRMS spectrum further
strengthen the structure assigned to compound 5o, as it displays [M+H]+ ion peak at m/z
503.1427 for the molecular formula C23H18N8O6.
The antitubercular screening was performed by preparing inoculum from fresh LJ
medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone, 0.5% glycerol,
supplemented oleic acid, albumin, dextrose, and catalase [OADC]), adjusted to OD590 1.0,
and diluted 1:20; 100 µl was used as inoculum. Each drug stock solution was thawed and
diluted in 7H9-S at four-fold the final highest concentration tested. The serial two-fold
dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100 µl
7H9-S. A growth control containing no antibiotic and a sterile control were also prepared on
each plate. Sterile water was added to all perimeter wells to avoid evaporation during the
4

incubation. The plate was covered, sealed in plastic bags and incubated at 37oC in normal
atmosphere. After 7 days incubation, 30 µl of alamar blue solution was added to each well,
and the plate was re-incubated overnight. A change in colour from blue (oxidised state) to
pink (reduced) indicated the growth of bacteria, and the MIC was defined as the lowest
concentration of drug that prevented this change in colour.32, 33 The compounds 5b, 5d, 5i,
5m, 5o and 5p have shown excellent antitubercular activity with MIC value 0.78 μg/mL.
While, compounds 5f, 5j and 5n have displayed noticeable antitubercular activity with MIC
value 1.56, 1.56 and 3.125 μg/mL, respectively (Table 1).
The in vitro cytotoxicity of the antitubercular active analogues with lower MIC value
were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
against growth inhibition of RAW 264.7 cells at 25 μg/mL concentration.34 The cell lines
were maintained at 37oC in a humidified 5% CO2 incubator (Thermo scientific). Detached the
adhered cells and followed by centrifugation to get cell pellet. Fresh media was added to the
pellet to make a cell count using haemocytometer and plate 100 μL of media with cells
ranging from 5,000-6,000 per well in a 96-well plate. The plate was incubated overnight in
CO2 incubator for the cells to adhere and regain its shape. After 24h, cells were treated with
the test compounds at 25 μg/mL diluted using the media to deduce the percentage inhibition
on normal cells. The cells were incubated for 48h to assay the effect of the test compounds on
different cell lines. Zero hour reading was noted down with untreated cells and also control
with 1% DMSO to subtract further from the 48h reading. After 48h incubation, cells were
treated by MTT (4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide) dissolved in
PBS (5 mg/ml) and incubated for 3-4h at 37oC. The formazan crystals thus formed were
dissolved in 100 μL of DMSO and the viability was measured at 540nm on a multimode
reader (Spectra max). The values were further calculated for percentage inhibition, which in
turn helps us to know the cytotoxicity of the test compounds. The results obtained are
summarized in Table 1.
Table 1. Antimycobacterial activity of isoniazid embedded triazole derivatives (5a-p).
MIC against
Mtb H37Rv
strain (μg/mL)
% Inhibition
Cytotoxicity at
25 μg/mL
Yield
(%)
Entry
Triazole derivatives
5

83
85
82
92
25
NA
5a
5b
5c
5d
0.78
12.5
0.78
25.69
19.04
22.71
84
>25
NA
5e
82
86
1.56
>25
24.18
5f
NA
5g
6

84
81
83
82
86
12.5
0.78
1.56
12.5
12.5
20.12
23.86
24.16
19.27
23.18
5h
5i
5j
5k
5l
80
82
0.78
27.72
17.65
5m
5n
3.125
7

90
84
0.78
0.78
23.88
26.95
5o
5p
Isoniazid
0.1
0.2
Rifampicin
Ciprofloxacin
Ethambutol
1.56
3.125
Newly synthesized isoniazid embedded triazole derivatives (5a-p) were evaluated for
their in vitro antimicrobial activities against both Gram positive and negative bacteria by agar
well diffusion assay.35 Tetracycline and Fluconazole were used as reference standard for
antibacterial and antifungal activities, respectively. The antibacterial pathogens DMSO was
used as negative control. The compounds were dissolved in DMSO and concentration used
for this assay was 1 mg/mL. Inoculums of each bacterial and fungal pathogen was developed
by inoculating pathogens in nutrient broth and keeping them for 24 h at 370C. The bacterial
suspension was diluted to adjust the turbidity to the 0.5 McFarland standards by using sterile
saline. 200 µL diluted suspension of each pathogen was inoculated on sterile Mueller Hinton
agar plates. Wells were prepared in agar and wells were filled with 100 µL of the samples.
Incubation of all experimental Petri plates was allowed at 37oC for 24h. After incubation, the
plates were keenly observed and results were recorded. Zones were measured and recorded
by using scale in millimeter (mm). The compounds 5e, 5f, 5g, 5h and 5j have exhibited good
antimicrobial activities against both antibacterial and antifungal pathogens. The presence of –
OCH3 and –Cl substituents on these derivatives have played vital role for their activities. The
compound 5i, 5k and 5l have displayed moderate antimicrobial activities (Table 2).
Table 2. Antimicrobial activity of isoniazid embedded triazole derivatives (5a-p).
Pathogens→
Antibacterial
E.
Antifungal
A. S.
S.
B.
B.
E.
S.
P.
S.
S.
Entry ↓
aureus cerus subtilis aerogenes coli typhi aeruginosa boydii abony niger cerevisiae
8

--
--
--
--
13
--
15
13
--
16
--
10
10
12
--
5a
--
--
--
--
12
--
--
--
--
--
14
12
--
5b
--
--
10
--
--
--
--
--
5c
--
--
--
--
--
--
--
--
5d
10
12
12
15
--
12
11
12
10
--
06
09
12
11
12
11
11
10
--
14
10
12
13
06
10
10
08
12
12
11
10
09
12
10
11
10
12
11
10
10
10
11
--
12
--
14
07
13
12
--
13
--
11
10
10
09
13
09
--
121
12
10
10
--
5e
5f
08
08
10
10
13
12
--
08
10
08
12
5g
5h
5i
13
--
12
--
14
12
--
--
5j
--
--
--
--
--
--
5k
13
--
13
--
08
--
--
5l
--
--
--
--
--
--
5m
--
--
--
--
--
--
--
--
--
5n
5o
--
--
12
10
29
NA
09
10
28
NA
13
10
NA
30
--
--
--
--
--
--
--
--
--
--
--
--
--
5p
22
NA
32
NA
18
NA
24
NA
28
NA
29
NA
27
NA
NA
30
Tetracycline
Fluconazole
(--): Not active; NA: Not applicable; Diameter of zone of inhibition is given in millimeter (mm).
The MIC values were determined for the compounds having good antimicrobial
activities and performed by following the method and guidelines of Clinical and Laboratory
Standard Institute (CLSI). The results are summarized in Table 3 and expressed in µg/mL.
Table 3. MIC values of most potent isoniazid embedded triazole derivatives
Pathogens
5e
5f
5g
5h
5j
Tetracycline
S. typhi
ATCC9207
S. aureus
ATCC 6538
E. aerogenes
ATCC13048
340
180
150
170
200
130
230 140 270
450 220 310
370 260 190
20
20
15
The significant anti-tubercular activities exhibited by isoniazid embedded triazole
derivatives motivated us to elucidate the plausible target and mechanism of their
antimycobacterial action. Molecular docking has emerged has a reliable tool complementary
to the in vivo and in vitro biological study to identify the potential biological target and gain
an insight into the ligand–receptor interactions for the bioactive molecules, especially in the
absence of available resources to perform enzyme-based assays.36, 37 With this objective
molecular docking study was carried out for these bioactive molecules against a crucial
9

mycobacterial target enoyl ACP reductase (InhA) using the Glide (Grid-Based Ligand
Docking With Energetics) program of the Schrödinger molecular modeling suite
(Schrödinger, LLC, New York, NY, 2018).38-41
Molecular docking study revealed that all the bioactive isoniazid embedded triazole
derivatives (5b, 5d, 5f, 5i, 5j, 5m, 5n, 5o and 5p) could recognize the binding site of InhA
and got deeply embedded in it engaging in a network of bonded and non-bonded interactions.
They have produced an average docking score (Glide Score) of -8.031 with an average
binding energy (Glide energy) of -50.051 kcal/mol signifying a promising binding affinity
towards the target. Furthermore, to gain a more detailed insight into the most significant
interactions guiding the ligand-receptor binding, a per-residue interaction analysis was
performed between these molecules and the active site residues (Table 4).
Table 4. Per-residue interaction analysis data for isoniazid embedded triazole derivatives (5b,
5d, 5f, 5i, 5j, 5m, 5n, 5o and 5p) with mycobacterial enoyl ACP reductase (InhA)
Per-Residues Interactions
Coulombic (kcal/mol)
Glide
Glide Interaction
Entry &
MIC
(µg/mL)
H-
Pi-Pi
Score
Energy
(kcal/mol)
Van der Waals (kcal/mol)
bonding Stacking
(Å)
(Å)
Leu218 (-2.342), Leu217 (-2.205), Ile215 (-3.224),
Gln214(-2.334), Leu207 (-2.171), Ile202 (-2.326), Met199 Asp261 (-2.463), Glu220 (-2.275),
(-3.256), Ala198 (-3.314), Thr196 (-2.227), Ile194 (- Glu219 (-2.932), Gln214 (-2.261),
3.113), Pro193 (-2.249), Gly192 (-2.301), Ala191 (-2.418), Glu210 (-2.277), Glu209 (-2.351),
-
8.21
7
Lys165 (-2.118), Met161 (-2.002), Tyr158 (-3.198),
Ala157 (-2.361), Pro156 (-2.229), Met155 (-2.471),
Phe149(-3.195), Asp148 (-2.124), Met147 (-2.222),
Met199 (-2.360), Thr196 (-2.447),
Arg195 (-2.253), Ile194 (-2.415),
Lys165 (-2.342), Pro156 (-2.118),
5b
(0.78)
Thr196
(2.17)
Phe149
(2.143)
-50.551
Gly104 (-2.144), Met103 (-2.254), Met98 (-2.245), Phe97 Asp150 (-2.167), Asp148 (-2.127),
(-3.235), Gly96 (-2.298), Ser94 (-2.225), Ile21 (-2.125), Met147 (-2.103), Gly96 (-2.184),
Ser20 (-2.113), Ile16 (-2.080)
Ser20 (-2.361)
Leu218 (-2.448), Leu217 (-2.476), Ile215 (-3.348), Gln214
(-2.626), Leu207 (-2.472), Ile202 (-2.619), Met199 (-
3.443), Ala198 (-3.114), Thr196 (-2.235), Ile194 (-3.151), Glu219 (-3.041), Gln214 (-2.352),
Pro193 (-2.557), Gly192 (-2.472), Ala191 (-2.523), Lys165 Glu210 (-2.458), Glu209 (-2.447),
Asp261 (-2.553), Glu220 (-2.382),
Ile194
(2.16),
Thr196
(2.27),
Glu104
(2.68),
Ser94
-
8.37
2
(-2.039), Met161 (-2.011), Tyr158 (-3.171), Ala157 (-
-51.71 2.462), Pro156 (-2.215), Met155 (-2.513), Phe149 (-3.499), Arg195 (-2.277), Ile194 (-2.426),
Asp148 (-2.135), Met147 (-2.278), Gly104 (-2.195), Lys165 (-3.444), Pro156 (-2.208),
Met199 (-2.339), Thr196 (-2.556),
5d
(0.78)
Phe149
(2.23)
Met103 (-2.391), Met98 (-2.342), Phe97 (-3.320), Gly96 (- Asp150 (-2.333), Asp148 (-2.193),
2.554), Ser94 (-2.438), Ile21 (-2.023), Ser20 (-2.081), Ile16 Met147 (-2.122), Gly96 (-2.484),
(2.75)
(-2.211)
Ser20 (-2.636)
Leu218 (-2.134), Leu217 (-1.976), Ile215 (-2.067), Gln214
(-1.926), Leu207 (-1.773), Ile202 (-1.619), Met199 (-
2.156), Ala198 (-2.114), Thr196 (-2.135), Ile194 (-2.112),
Pro193 (-2.106), Gly192 (-1.727), Ala191 (-2.317), Lys165
(-2.043), Met161 (-1.607), Tyr158 (-1.736), Ala157 (-
-48.322 1.457), Pro156 (-1.525), Met155 (-1.678), Phe149 (-2.144),
Asp148 (-2.011), Met147 (-2.111), Gly104 (-1.597),
Met103 (-1.238), Met98 (-2.014), Phe97 (-1.435), Gly96 (-
1.891), Ser94 (-1.725), Ile21 (-1.845), Ser20 (-2.088), Ile16
(-1.821)
Asp261 (-1.693), Glu220 (-
1.585), Glu219 (-2.041), Gln214
(-1.564), Glu210 (-1.778),
Glu209 (-1.791), Met199 (-
1.999), Thr196 (-1.847), Arg195
(-1.893), Ile194 (-2.415), Lys165
(-1.641), Pro156 (-1.758),
Asp150 (-1.666), Asp148 (-
1.027), Met147 (-1.223), Gly96 (-
1.442), Ser20 (-1.361)
Tyr158
(2.589),
Phe149
(2.518)
-
7.83
6
5f
(1.56)
Ile194
(2.39)
Leu218 (-2.599), Leu217 (-2.576), Ile215 (-3.547),
Gln214 (-2.823), Leu207 (-2.673), Ile202 (-2.841),
Met199 (-3.656), Ala198 (-2.449), Thr196 (-2.343),
Ile194 (-3.263), Pro193 (-2.839), Gly192 (-2.479),
Ala191 (-2.561), Lys165 (-2.045), Met161 (-2.096),
Asp261 (-2.693), Glu220 (-
2.585), Glu219 (-3.241), Gln214
(-2.464), Glu210 (-2.278),
Glu209 (-2.223), Met199 (-2.47),
Thr196 (-2.847), Arg195 (-
Ile194
(2.15),
Thr196
(2.02)
Tyr158
(2.111),
Phe149
(2.113)
-
8.39
5
5i
(0.78)
-52.373
10

Tyr158 (-2.71), Ala157 (-2.525), Pro156 (-2.425),
Met155 (-2.584), Phe149 (-3.636), Asp148 (-2.193),
Met147 (-2.333), Gly104 (-2.597), Met103 (-2.43),
Met98 (-2.445), Phe97 (-3.435), Gly96 (-2.898), Ser94
(-2.725), Ile21 (-2.044) , Ser20 (-2.383), Ile16 (-2.271)
2.393), Ile194 (-2.455), Lys165 (-
3.51), Pro156 (-2.228), Asp150 (-
2.867), Asp148 (-2.427), Met147
(-2.443), Gly96 (-2.841), Ser20 (-
2.361)
Leu218 (-2.112), Leu217 (-1.996), Ile215 (-2.043),
Gln214 (-1.986), Leu207 (-1.893), Ile202 (-1.763),
Met199 (-2.341), Ala198 (-2.313), Thr196 (-2.119),
Ile194 (-2.046), Pro193 (-2.115), Gly192 (-1.880),
Ala191 (-2.636), Lys165 (-1.943), Met161 (-1.867),
Tyr158 (-1.973), Ala157 (-1.949), Pro156 (-1.925),
Met155 (-1.973), Phe149 (-1.988), Asp148 (-2.087),
Met147 (-2.054), Gly104 (-1.997), Met103 (-1.896),
Met98 (-2.041), Phe97 (-2.543), Gly96 (-1.896), Ser94
(-1.626), Ile21(-1.975), Ser20 (-1.883), Ile16 (-1.991)
Asp261 (-1.893), Glu220 (-
1.943), Glu219 (-2.343), Gln214
(-1.864), Glu210 (-1.843),
Glu209 (-1.891), Met199 (-
2.142), Thr196 (-1.829), Arg195
(-1.996), Ile194 (-2.051), Lys165
(-1.849), Pro156 (-1.608),
Asp150 (-1.867), Asp148 (-
1.031), Met147 (-1.443), Gly96 (-
1.841), Ser20 (-1.561)
Tyr158
(2.176),
Phe149
(2.343)
-
7.92
5
5j
(1.56)
Ile194
(2.33)
-49.524
-51.704
-45.67
Leu218 (-2.241), Leu217 (-2.116), Ile215 (-3.154),
Gln214 (-2.192), Leu207 (-2.073), Ile202 (-2.248),
Met199 (-2.973), Ala198 (-2.925), Thr196 (-2.167),
Ile194 (-3.207), Pro193 (-2.339), Gly192 (-2.216),
Ala191 (-2.441), Lys165 (-2.113), Met161 (-2.095),
Tyr158 (-2.949), Ala157 (-2.367), Pro156 (-2.235),
Met155 (-2.404), Phe149 (-2.919), Asp148 (-2.087),
Met147 (-2.215), Gly104 (-2.112), Met103 (-2.145),
Met98 (-2.014), Phe97 (-3.225), Gly96 (-2.198), Ser94
(-2.232), Ile21 (-2.214), Ser20 (-2.083), Ile16 (-2.121)
Asp261 (-2.169), Glu220 (-
2.134), Glu219 (-2.274), Gln214
(-1.216), Glu210 (-2.243),
Glu209 (-2.191), Met199 (-
2.247), Thr196 (-2.534), Arg195
(-2.193), Ile194 (-2.234), Lys165
(-2.489), Pro156 (-2.308),
Asp150 (-2.261), Asp148 (-
2.023), Met147 (-2.001), Gly96
(-2.441), Ser20 (-2.238)
Ile194
(2.52),
Thr196
(2.07)
-
8.22
5
5m
(0.78)
Phe149
(2.286)
Leu218 (-1.952), Leu217 (-1.706), Ile215 (-2.021),
Gln214 (-1.192), Leu207 (-1.071), Ile202 (-1.486),
Met199 (-1.887), Ala198 (-2.114), Thr196 (-2.131),
Ile194 (-1.936), Pro193 (-1.898), Gly192 (-1.721),
Ala191 (-2.302), Lys165 (-2.141), Met161 (-1.067),
Tyr158 (-1.276), Ala157 (-1.162), Pro156 (-1.125),
Met155 (-1.271), Phe149 (-1.206), Asp148 (-2.009),
Met147 (-2.111), Gly104 (-1.197), Met103 (-1.135),
Met98 (-2.011), Phe97 (-1.413), Gly96 (-1.189), Ser94
(-1.241), Ile21 (-1.291), Ser20 (-1.083), Ile16 (-1.211)
Asp261 (-1.564), Glu220 (-
1.413), Glu219 (-1.101), Gln214
(-1.143), Glu210 (-1.324),
Glu209 (-1.230), Met199 (-
1.342), Thr196 (-1.590), Arg195
(-1.165), Ile194 (-2.039), Lys165
(-1.675), Pro156 (-1.208),
Asp150 (-1.186), Asp148 (-
1.111), Met147 (-1.003), Gly96
(-1.341), Ser20 (-1.131)
Tyr158
(2.373),
Phe149
(2.424)
-
7.12
9
5n
(3.125)
Ile194
(2.30)
Leu218 (-2.522), Leu217 (-2.207), Ile215 (-3.025),
Gln214 (-2.128), Leu207 (-2.423), Ile202 (-2.146),
Met199 (-3.053), Ala198 (-2.314), Thr196 (-2.235),
Ile194 (-2.486), Pro193 (-2.265), Gly192 (-2.211),
Ala191 (-2.388), Lys165 (-1.999), Met161 (-2.177),
Tyr158 (-3.225), Ala157 (-3.062), Pro156 (-2.612),
Met155 (-2.332), Phe149 (-2.245), Asp148 (-2.075),
Met147 (-2.133), Gly104 (-2.119), Met103 (-2.143),
Met98 (-2.020), Phe97 (-3.113), Gly96 (-2.143), Ser94
(-1.725), Ile21 (-2.421), Ser20 (-2.003), Ile16 (-2.023)
Asp261 (-2.233), Glu220 (-
2.313), Glu219 (-3.012), Gln214
(-1.964), Glu210 (-2.488),
Glu209 (-1.291), Met199 (-
2.231), Thr196 (-2.147), Arg195
(-1.144), Ile194 (-2.455), Lys165
(-2.675), Pro156 (-1.605),
Asp150 (-2.186), Asp148 (-
2.126), Met147 (-2.113), Gly96 (-
2.184), Ser20 (-2.236)
Tyr158
(2.30),
Lys165
(2.44)
-
8.12
6
5o
(0.78)
Phe149
(2.396)
-50.349
Leu218 (-2.455), Leu217 (-2.736), Ile215 (-2.927),
Gln214 (-2.669), Leu207 (-2.679), Ile202 (-2.486),
Met199 (-2.967), Ala198 (-3.364), Thr196 (-2.632),
Ile194 (-2.646), Pro193 (-2.439), Gly192 (-2.301),
Ala191 (-2.234), Lys165 (-1.924), Met161 (-2.154),
Tyr158 (-2.377), Ala157 (-2.285), Pro156 (-2.389),
Met155 (-2.271), Phe149 (-2.168), Asp148 (-2.228),
Met147 (-2.136), Gly104 (-2.137), Met103 (-2.106),
Met98 (-2.172), Phe97 (-3.435), Gly96 (-2.391), Ser94
(-1.698), Ile21 (-2.293), Ser20 (-1.982), Ile16 (-1.992)
Asp261 (-2.221), Glu220 (-
2.165), Glu219 (-2.935), Gln214
(-1.872), Glu210 (-2.274),
Glu209 (-1.929), Met199 (-
2.210), Thr196 (-2.129), Arg195
(-1.192), Ile194 (-2.143), Lys165
(-2.006), Pro156 (-1.518),
Asp150 (-2.280), Asp148 (-
1.027), Met147 (-1.988), Gly96 (-
1.849), Ser20 (-1.966)
Lys165
(2.24),
Gly96
(2.44)
-
8.05
6
5p
(0.78)
Tyr158
(2.572)
-50.259
The lowest energy docked conformation of 5i showed that the compound could
snuggly fit into the active site of mycobacterial enoyl ACP reductase (InhA) with an excellent
binding affinity (Glide score of -8.395 and Glide binding energy -52.373 kcal/mol) at the
coordinates close to the native ligand engaging in a series of steric and electrostatic
interactions (Fig. 2).
11

Figure 2. Binding mode of 5i into the active site of mycobacterial enoyl ACP reductase
(InhA). (On the right side: green lines indicate π-π stacking interactions, while the pink lines
signify the hydrogen bonding interactions)
The per-residue interaction analysis showed that the enzyme-inhibitor complex is
stabilized by a network of favorable van der Waals interactions (steric) observed with active
site residues wherein the 1-(2-methoxy phenyl)-1H-1,2,3-triazol-4-yl component interacted
closely with Leu218 (-2.599 kcal/mol), Leu217 (-2.576 kcal/mol), Ile215 (-3.547 kcal/mol),
Gln214 (-2.823 kcal/mol), Leu207 (-2.673 kcal/mol), Ile202 (-2.841 kcal/mol), Met199 (-
3.656 kcal/mol), Pro193 (-2.839 kcal/mol), Tyr158 (-2.71 kcal/mol), Ala157 (-2.525
kcal/mol), Pro156 (-2.425 kcal/mol), Met155 (-2.584 kcal/mol), Gly104 (-2.597 kcal/mol),
Met103 (-2.43 kcal/mol) and Ser20 (-2.383 kcal/mol) residues, while the 5-nitro-2-(prop-2-
yn-1-yloxy)benzylidene) section interacted similarly with Ile194 (-3.263 kcal/mol), Gly192 (-
2.479 kcal/mol), Lys165 (-2.045 kcal/mol), Phe149 (-3.636 kcal/mol), Asp148 (-2.193
kcal/mol), Met147 (-2.333 kcal/mol), and Ser94 (-2.725 kcal/mol) residues. Furthermore, the
isonicotinohydrazide component of the molecule also has displayed significant van der Waals
interactions with Ala198 (-2.449 kcal/mol), Thr196 (-2.343 kcal/mol), Ala191 (-2.561
kcal/mol), Met161 (-2.096 kcal/mol), Met98 (-2.445 kcal/mol), Phe97 (-3.435 kcal/mol),
Gly96 (-2.898 kcal/mol), Ile21 (-2.044 kcal/mol) and Ile16 (-2.271 kcal/mol) residues. The
enhanced binding affinity of 5i has been also attributed to very significant coulombic
(electrostatic) interactions observed with Asp261 (-2.693 kcal/mol), Glu220 (-2.585
kcal/mol), Glu219 (-3.241 kcal/mol), Gln214 (-2.464 kcal/mol), Glu210 (-2.278 kcal/mol),
Glu209 (-2.223 kcal/mol), Met199 (-2.47 kcal/mol), Thr196 (-2.847 kcal/mol), Arg195 (-
2.393 kcal/mol), Ile194 (-2.455 kcal/mol), Lys165 (-3.51 kcal/mol), Pro156 (-2.228
kcal/mol), Asp150 (-2.867 kcal/mol), Asp148 (-2.427 kcal/mol), Met147 (-2.443 kcal/mol),
12

Gly96 (-2.841 kcal/mol) and Ser20 (-2.361 kcal/mol) residues. While these non-bonded
interactions i.e. steric and electrostatic with active site residues of InhA were the major
driving force for the mechanical interlocking of 5i, the higher binding affinity is also
complemented by a very close hydrogen bonding interactions observed firstly with Ile194
through prop-2-yn-1-yloxy linker in the molecule and secondly with carbonyl (-C=O)
function through the Thr196 residue having bond distances of 2.15Å and 2.02Å, respectively.
Furthermore, the compound 5i was seen to be anchored to InhA through two pi-pi(π-π)
stacking interactions as well, firstly with Tyr158 (2.111Å) and second observed through
Phe149 (2.113Å). Such type of hydrogen bonding and pi-pi stacking interactions serve as an
anchor to direct the orientation of inhibitor into the 3D space of enzyme’s active site and
further facilitate the non-bonded interactions as well contributing to the stability of the
enzyme-inhibitor complex. The other active molecules 5b, 5d, 5f, 5j, 5m, 5n, 5o and 5p (Fig.
S1-S8) were also observed to be deeply embedded in the active site of InhA engaging in a
network of bonded and non-bonded interactions. Overall, the binding affinity data along with
the per-residue ligand interaction analysis suggests that a balanced network of non-bonded
interactions (steric and electrostatic) complemented by hydrogen bonding and pi-pi stacking
interactions contribute to the anchoring of these molecules to mycobacterial InhA. The
quantitative insights derived herein are now being fruitfully utilized to carry out site specific
mutation around the core nucleus to generate tighter binding and potent mycobacterial InhA
inhibitors.
In conclusion, a series of novel isoniazid embedded triazole derivatives (5a-p) have
been synthesized via click reaction in good to excellent yields. The newly synthesized
compounds were evaluated for their in vitro antimycobacterial and antimicrobial activities.
We have identified six compounds as potent antitubercular agents (MIC value 0.78 μg/mL)
against M. tuberculosis H37Rv (Mtb). However, the three compounds have exhibited
moderate antitubercular activity in the range of MIC value 1.56-3.12 μg/mL. These
compounds have displayed low cytotoxicity profile by MTT assay against RAW 264.7 cell
line. Among the compounds screened for their in vitro antimicrobial activities, five
compound have shown good activities against both antibacterial and antifungal pathogens.
Furthermore, in silico binding affinity data against mycobacterial InhA could provide a
valuable insight into the plausible mechanism of antitubercular action for these molecules.
The promising results obtained from this study qualifies these new isoniazid embedded
triazole derivatives as a pertinent starting point for structure-based optimization as InhA
specific antitubercular candidates through iterative design and synthesis approach. The new
13

entities with dual antitubercular and antimicrobial properties are desperately needed, we
believe that the analogues reported in this work will help global efforts for identification of
potential lead molecules for further development.
Acknowledgments
Authors are thankful to Birla Institute of Technology and Science-Pilani, Hyderabad
Campus for providing screening data of synthesized compounds. We are also thanks to
Schrödinger Inc. for providing Small-Molecule Drug Discovery Suite (2018) to perform the
molecular docking studies.
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41. Molecular docking study: The Glide (Grid-Based Ligand Docking With Energetics)
program (Schrödinger, LLC, New York, NY, 2018) was used to perform the molecular
docking study for which the 3D crystal structure of mycobacterial enoyl-ACP reductase
(InhA) complexed with its inhibitor (PDB code: 4TZK) was retrieved from the RCSB’s
Protein Data Bank (http://www.rcsb.org/pdb/) and refined using the Protein Preparation
Wizard to correct the experimental errors which involve elimination of all
crystallographically observed water molecules (since they are not known to be conserved
in the ligand interaction), adding the missing hydrogen atoms and side chains,
assignment of all atom force field (OPSL-2005) charges and atom types, creating the
disulfide bonds, identification of atom/residue overlaps and finally energy minimization
of the cleaned structure until the average RMSD of nonhydrogen atoms converged to 0.3
Å. Next, the shape and properties of the active site of the enzyme for docking was
defined using the receptor grid generation panel to include residues within a 10.0 Å
16

radius of the crystallized ligand for which a grid box of 10X10X10Å dimensions around
the centroid of the native ligand was generated. The 2D structures of the ligands to be
docked were sketched with the build panel in Maestro and converted using the ligand
preparation panel to energy minimized 3D structures. This ligand optimization procedure
involves assignment of correct protonation states and atom types to the molecule,
ascribing partial atomic charges (OPLS-2005 force field) and then final energy
minimization (until their average RMSD reached 0.001Å) to generate single low energy
3D structure for docking. With this setup, flexible docking was executed with extra
precision (i.e., with GlideXP) scoring function to identify the modes of binding for these
bioactive molecule and gauze their binding affinities towards InhA. The docking poses
generated as output file analyzed for the most significant thermodynamic interactions
with the active site residues through the Pose Viewer utility of Maestro.
42. Procedure for the synthesis of 5-nitro-2-(prop-2-yn-1-yloxy)benzaldehyde (2): A
mixture of 2-hydroxy-5-nitrobenzaldehyde (1) (1 mmol), propargyl bromide (80 wt% in
toluene) (1.5 mmol) and K2CO3 (1.5 mmol) was stirred in DMF at room temperature for
6h. The progress of the reaction was monitored by TLC. After completion of reaction,
the reaction mixture was poured on crushed ice. The solid obtained was filtered, washed
with water and crystallized from ethanol. Yield: 90%; M. P.: 90-92oC.
43. Procedure
for
the
synthesis
of
(E)-N'-(5-nitro-2-(prop-2-yn-1-
yloxy)benzylidene)isonicotinohydrazide (3): A mixture of 5-nitro-2-(prop-2-yn-1-
yloxy)benzaldehyde (2) (1 mmol) and isonicotinohydrazide (1 mmol) was dissolved in
diisopropylethylammonium aceate (DIPEAc) (10 ml) and stirred at room temperature for
1h. Then, the reaction mixture was poured on cold water. The solid obtained was filtered
and washed with cold water. The products obtained were crystallized from ethanol.
(E)-N'-(5-Nitro-2-(prop-2-yn-1-yloxy)benzylidene)isonicotinohydrazide (3): Yield:
92%; M. P. : 205-207oC; 1H NMR (400 MHz, DMSO-d6)  = 3.14 (t, J = 4 Hz, 1H), 4.93
(d, J = 4 Hz, 2H), 7.21 (d, J = 8 Hz, 1H), 7.80-7.84 (m, 3H), 8.21 (dd, J = 4 & 8 Hz, 1H),
13
8.70 (bs, 1H), 8.77-8.78 (m, 2H), 12.17 (s, 1H); C NMR (100 MHz, CDCl3 + DMSO-
d6)  = 56.76, 76.75, 78.04, 112.78, 121.44, 121.47, 123.41, 126.09, 139.85, 141.49,
142.17, 149.82, 159.74, 161.71.
44. General procedure for the synthesis of (E)-N'-(5-nitro-2-((1-phenyl-1H-1,2,3-triazol-
4-yl)methoxy)benzylidene)isonicotinohydrazide derivatives (5a-p): The mixture of
(E)-N'-(5-nitro-2-(prop-2-yn-1-yloxy)benzylidene)isonicotinohydrazide (3) (1 mmol) and
substituted azidobenzenes (4a-p) (1 mmol) were stirred in presence of CuSO4.5H2O (20
17

mol%) and sodium ascorbate (20 mol%) in DMF at room temperature. The progress of
the reactions was monitored by TLC. After completion of reactions (6-8h), the reaction
mixtures were poured in ice cold water. The products obtained were filtered, washed
with water and crystallized from ethanol.
(E)-N'-(2-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5a): Yield: 83%; M. P.: 238-240oC; 1H NMR (400 MHz, DMSO-
d6)  = 5.42 (s, 2H), 7.35-7.38 (m, 1H), 7.47-7.50 (m, 2H), 7.72-7.73 (m, 3H), 7.81-7.84
(m, 3H), 8.24 (dd, J = 4 & 8 Hz, 1H), 8.72-8.79 (m, 3H), 12.08 (s, 1H).
(E)-N'-(5-Nitro-2-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)isonicotino
1
hydrazide (5b): Yield: 85%; M. P.: 210-212oC; H NMR (400 MHz, DMSO-d6)  =
5.45 (s, 2H), 7.41-7.54 (m, 3H), 7.82-7.85 (m, 2H), 7.88-7.94 (m, 5H), 8.26 (dd, J = 4 &
8 Hz, 1H), 8.76-8.81 (m, 3H), 12.15 (s, 1H).
(E)-N'-(2-((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5c): Yield: 82%; M. P.: 225-227oC; 1H NMR (400 MHz, DMSO-
d6)  = 5.41 (s, 2H), 7.33-7.44 (m, 1H), 7.63-7.68 (m, 2H), 7.74-7.79 (m, 3H), 7.81-7.85
(m, 3H), 8.20-8.27 (m, 1H), 8.73-8.81 (m, 3H), 12.08 (s, 1H).
(E)-N'-(5-Nitro-2-((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)
1
isonicotinohydrazide (5d): Yield: 92%; M. P.: 254-256oC; H NMR (400 MHz,
DMSO-d6)  = 5.49 (s, 2H), 7.46 (d, J = 8 Hz, 1H), 7.79-7.84 (m, 2H), 7.85 (bs, 3H),
8.25 (d, J = 8 Hz, 2H), 8.36 (d, J = 8 Hz, 1H), 8.75-8.78 (m, 3H), 9.14 (s, 1H), 12.16 (s,
1H).
(E)-N'-(2-((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5e): Yield: 84%; M. P.: 242-244oC; 1H NMR (400 MHz, DMSO-
d6)  = 3.85 (s, 3H), 5.41 (s, 2H), 7.06 (t, J = 8 Hz, 1H), 7.12 (d, J = 8 Hz, 1H), 7.362-
7.43 (m, 2H), 7.64-7.77 (m, 5H), 8.23 (dd, J = 4 & 8 Hz, 1H), 8.41 (s, 1H), 8.74 (s, 1H),
8.81 (d, J = 2 Hz, 1H), 12.11 (s, 1H); 13C NMR (100 MHz, CDCl3 + DMSO-d6)  =
55.10, 61.63, 111.61, 112.01, 120.14, 121.25, 122.69, 124.19, 124.63, 125.85, 129.70,
131.90, 139.36, 140.41, 140.76, 142.14, 150.08, 160.15, 161.43; HRMS (ESI)+ calcd. for
C23H19N7O5 [M+H]+: 474.1481 and found 474.1525.
(E)-N'-(2-((1-(2-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5f): Yield: 82%; M. P.: 216-218oC; 1H NMR (400 MHz, DMSO-
d6)  = 5.40 (s, 2H), 7.02-7.10 (m, 1H), 7.25-7.35 (m, 1H), 7.39-7.48 (m, 2H), 7.541-7.58
(m, 2H), 7.62-7.67 (m, 2H), 7.77 (bs, 1H), 8.21 (dd, J = 4 & 8 Hz, 1H), 8.31-8.41 (m,
18

1H), 8.74-8.76 (m, 1H), 8.80-8.85 (m, 1H), 12.05 (s, 1H); 13C NMR (100 MHz, CDCl3 +
DMSO-d6)  = 61.48, 112.12, 120.04, 122.58, 124.11, 125.49, 125.79, 126.85, 127.19,
127.50, 129.68, 129.85, 130.35, 133.36, 140.70, 140.84, 142.02, 150.02, 159.98, 161.33
HRMS (ESI)+ calcd. for C22H16ClN7O4 [M+H]+: 478.0986 and found 478.1023.
(E)-N'-(2-((1-(2-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5g): Yield: 86%; M. P.: 126-128oC; 1H NMR (400 MHz, DMSO-
d6)  = 3.85 (s, 3H), 5.41 (s, 2H), 7.05-7.13 (m, 2H), 7.34-7.44 (m, 2H), 7.63-7.66 (m,
1H), 7.68-7.70 (m, 3H), 7.80 (bs, 1H), 8.24 (dd, J = 4 & 8 Hz, 1H), 8.40 (d, J = 8 Hz,
1H), 8.73 (d, J = 4 Hz, 1H), 8.80-8.83 (m, 1H), 12.11 (s, 1H); 13C NMR (100 MHz,
CDCl3 + DMSO-d6)  = 55.00, 60.27, 111.54, 114.78, 120.02, 121.07, 121.54, 122.58,
122.63, 123.35, 123.75, 124.09, 125.39, 129.59, 136.83, 139.35, 141.95, 144.90, 150.00,
160.05, 161.30.
(E)-N'-(2-((1-(3-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5h): Yield: 84%; M. P.: 220-222oC; 1H NMR (400 MHz, DMSO-
d6)  = 5.43 (s, 2H), 7.35-7.40 (m, 2H), 7.46-7.50 (m, 1H), 7.73 (bs, 3H), 7.79-7.84 (m,
13
2H), 7.93 (bs, 1H), 8.24 (d, J = 8 Hz, 1H), 8.74-8.81 (m, 3H), 12.08 (s, 1H); C NMR
(100 MHz, CDCl3 + DMSO-d6)  = 61.81, 112.07, 114.98, 117.55, 119.39, 121.79,
122.71, 125.97, 127.90, 130.26, 134.19, 136.68, 140.85, 141.39, 142.28, 142.64, 148.83,
151.58, 160.06, 161.53.
(E)-N'-(2-((1-(3-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5i): Yield: 81%; M. P.: 232-234oC; 1H NMR (400 MHz, DMSO-
d6)  = 3.83 (s, 3H), 5.45 (s, 2H), 6.94-6.98 (m, 1H), 7.41-7.42 (m, 2H), 7.46-7.48 (m,
2H), 7.8 (bs, 2H), 7.95 (d, J = 4 Hz, 1H), 8.26 (dd, J = 4 & 8 Hz, 1H), 8.75 (s, 2H), 8.85
(s, 2H), 12.16 (s, 1H); 13C NMR (100 MHz, CDCl3 + DMSO-d6)  = 54.24, 61.23,
104.63, 110.76, 111.84, 112.98, 119.88, 120.58, 121.71, 122.01, 125.53, 129.41, 134.42,
136.21, 140.20, 141.34, 141.63, 141.69, 150.86, 158.98, 159.69.
(E)-N'-(5-Nitro-2-((1-(o-tolyl)-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)isonicotino
hydrazide (5j): Yield: 83%; M. P.: 228-230oC; 1H NMR (400 MHz, DMSO-d6)  = 2.16
(s, 3H), 5.44 (s, 2H), 7.30-7.40 (m, 4H), 7.66 (bs, 3H), 7.75 (d, J = 8 Hz, 2H), 7.22-7.25
(m, 1H), 8.67 (bs, 1H), 8.77 (s, 1H), 8.81 (d, J = 4 Hz, 1H), 12.07 (s, 1H); 13C NMR (100
MHz, CDCl3 + DMSO-d6)  = 16.83, 61.68, 111.99, 120.66, 120.70, 121.22, 122.69,
124.81, 125.74, 125.91, 128.99, 130.51, 132.28, 135.09, 139.35, 140.72, 140.86, 142.05,
19

149.15, 159.98, 161.28; HRMS (ESI)+ calcd. for C23H19N7O4 [M+H]+: 458.1532 and
found 458.1581.
(E)-N'-(2-((1-(2-Fluorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5k): Yield: 82%; M. P.: 220-222oC; 1H NMR (400 MHz, DMSO-
d6)  = 5.44 (s, 2H), 7.31-7.38 (m, 3H), 7.43-7.49 (m, 1H), 7.68 (d, J = 8 Hz, 1H), 7.75-
7.90 (m, 3H), 8.22 (dd, J = 4 & 8 Hz, 1H), 8.46 (d, J = 4 Hz, 1H), 8.67 (bs, 1H), 8.73 (d,
J = 8 Hz, 1H), 8.79-8.81 (m, 1H), 12.09 (s, 1H); 13C NMR (100 MHz, CDCl3 + DMSO-
d6)  = 61.40, 112.03, 116.05, 116.25, 121.26, 122.65, 124.42, 124.46, 124.76, 125.83,
129.98, 130.05, 140.74, 141.39, 142.12, 143.96, 149.14, 151.32, 160.01, 161.37.
(E)-N'-(5-Nitro-2-((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)
isonicotinohydrazide (5l): Yield: 86%; M. P.: 230-232oC; 1H NMR (400 MHz, DMSO-
d6)  = 2.38 (s, 3H), 5.41 (s, 2H), 7.21 (d, J = 8 Hz, 1H), 7.32-7.38 (m, 2H), 7.55 (d, J =
8 Hz, 1H), 7.59-7.64 (m, 3H), 7.76-7.78 (m, 2H), 8.21 (d, J = 8 Hz, 1H), 8.54 (d, J = 4
Hz, 1H), 8.75 (s, 1H), 8.80 (d, J = 4 Hz, 1H), 12.04 (s, 1H).
(E)-N'-(2-((1-Mesityl-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)isonicotino
1
hydrazide (5m): Yield: 80%; M. P.: 222-224oC; H NMR (400 MHz, DMSO-d6)  =
1.87 (s, 6H), 2.29 (s, 3H), 5.46 (s, 2H), 6.93-6.98 (m, 2H), 7.34-7.37 (m, 1H), 7.69 (s,
1H), 7.73-7.77 (m, 2H), 8.06-8.08 (m, 1H), 8.21 (dd, J = 4 & 8 Hz, 1H), 8.68 (bs, 1H),
13
8.77-8.81 (m, 2H), 12.09 (s, 1H); C NMR (100 MHz, CDCl3 + DMSO-d6)  = 16.50,
20.29, 62.18, 112.30, 121.74, 123.13, 125.14, 125.99, 128.32, 132.36, 134.00, 139.39,
139.73, 139.74, 141.08, 141.25, 142.56, 149.43, 160.22, 161.78
(E)-N'-(2-((1-(2,4-Dimethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitro
1
benzylidene)isonicotinohydrazide (5n): Yield: 82%; M. P.: 248-250oC; H NMR (400
MHz, DMSO-d6)  = 2.12 (s, 3H), 2.35 (s, 3H), 5.46 (s, 2H), 7.12 (d, J = 8 Hz, 1H),
7.17-7.22 (m, 2H), 7.45 (d, J = 8 Hz, 1H), 7.80 (bs, 1H), 7.47 (s, 3H), 8.25 (dd, J = 4 & 8
13
Hz, 1H), 8.34 (s, 1H), 8.74-8.79 (m, 2H), 12.15 (s, 1H); C NMR (100 MHz, CDCl3 +
DMSO-d6)  = 16.37, 19.65, 61.35, 112.06, 113.05, 120.64, 122.28, 124.34, 124.82,
125.52, 126.14, 130.67, 131.56, 132.41, 135.19, 138.56, 140.45, 141.68, 144.01, 148.12,
159.77, 160.94; HRMS (ESI)+ calcd. for C24H21N7O4 [M+H]+: 472.1689 and found
472.1721.
(E)-N'-(2-((1-(2-Methyl-5-nitrophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitro
1
benzylidene)isonicotinohydrazide (5o): Yield: 90%; M. P.: 250-252oC; H NMR (400
MHz, DMSO-d6)  = 2.33 (s, 3H), 5.45 (s, 2H), 7.33 (d, J = 8 Hz, 1H), 7.58-7.61 (m,
20

3H), 7.77 (bs, 1H), 7.79 (s, 1H), 8.21-8.25 (m, 3H), 8.48 (s, 1H), 8.77-8.78 (m, 2H),
12.02 (s, 1H); 13C NMR (100 MHz, CDCl3 + DMSO-d6)  = 18.67, 62.75, 112.97,
121.20, 122.88, 123.79, 124.47, 126.04, 126.91, 132.88, 136.61, 140.41, 141.52, 141.90,
142.69, 143.04, 143.64, 146.41, 150.36, 161.01, 162.55; HRMS (ESI)+ calcd. for
C23H18N8O6 [M+H]+: 503.1383 and found 503.1427.
(E)-N'-(2-((1-(4-Ethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)-5-nitrobenzylidene)
isonicotinohydrazide (5p): Yield: 84%; M. P.: 234-236oC; 1H NMR (400 MHz, DMSO-
d6)  = 1.20 (t, J = 8 Hz, 3H), 2.665 (q, J = 8 Hz, 2H), 5.40 (s, 2H), 7.30 (d, J = 8 Hz,
2H), 7.34 (s, 1H), 7.63 (s, 3H), 7.64 (d, J = 8 Hz, 2H), 7.76 (bs, 1H), 8.23 (dd, J = 4 & 8
Hz, 1H), 8.52 (s, 1H), 8.75 (s, 1H), 8.81 (d, J = 2 Hz, 1H), 12.04 (s, 1H); 13C NMR (100
MHz, CDCl3 + DMSO-d6)  = 15.16, 26.63, 62.42, 113.29, 117.35, 120.02, 123.39,
126.38, 128.84, 134.59, 136.59, 138.12, 139.60, 141.54, 142.38, 145.08, 147.39, 149.88,
159.26, 163.31; HRMS (ESI)+ calcd. for C24H21N7O4 [M+H]+: 472.1689 and found
472.1735.
Supplementary Material
1
Supplementary material to this article containing H NMR, 13C NMR and HRMS
spectrums of new compounds are available for the authorized users.
21

Highlights
 Novel isoniazid embedded triazole derivatives have been synthesized.
 Exhibited potent antitubercular and antimicrobial activities.
 Nontoxic against RAW 264.7 cell line.
22