Synthesis of novel 1,2,3-triazole derivatives of isoniazid and their in vitro and in vivo antimycobacterial activity evaluation

Article abstract of DOI:10.1016/j.ejmech.2014.05.005

We report herein the synthesis and antimycobacterial activity of 1,2,3-triazole derivatives of isoniazid. Most of the compounds exhibited potent activity against Mycobacterium tuberculosis H37Rv strain with MIC₉₉ values ranging from 0.195 to 1.

Full text of DOI:10.1016/j.ejmech.2014.05.005

Accepted Manuscript
Synthesis of novel 1,2,3-triazole derivatives of isoniazid and their in vitro and in vivo
antimycobacterial activity evaluation
Deepak Kumar, Beena, Garima Khare, Saqib Kidwai, Anil K. Tyagi, Ramandeep
Singh, Diwan S. Rawat
PII:
S0223-5234(14)00418-8
10.1016/j.ejmech.2014.05.005
EJMECH 6960
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 December 2013
Revised Date: 1 April 2014
Accepted Date: 1 May 2014
Please cite this article as: D. Kumar, Beena, G. Khare, S. Kidwai, A.K. Tyagi, R. Singh, D.S Rawat,
Synthesis of novel 1,2,3-triazole derivatives of isoniazid and their in vitro and in vivo antimycobacterial
activity evaluation, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.005.
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Highlights (for review)
ACCEPTED MANUSCRIPT
Highlights
 In vitro antituberculosis activity of novel 1,2,3-triazole derivatives of isoniazid was
reported against M. tuberculosis H37Rv.
 Compounds exhibited potent activity with MIC ranging from 0.195 to 1.56 μM in
vitro.
 One compound when tested in vivo, showed significant activity.
 Compounds were nontoxic against THP-1 cell line even at 50 μM concentration.

*Revised Manuscript
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Synthesis of novel 1,2,3-triazole derivatives of isoniazid and their in vitro
and in vivo antimycobacterial activity evaluation
Deepak Kumara, Beenaa, Garima Khareb, Saqib Kidwaic, Anil K. Tyagib, Ramandeep Singhc,
Diwan S Rawata*
Graphical abstract
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Synthesis of novel 1,2,3-triazole derivatives of isoniazid and their in vitro
and in vivo antimycobacterial activity evaluation
Deepak Kumara, Beenaa, Garima Khareb, Saqib Kidwaic, Anil K. Tyagib, Ramandeep Singhc,
Diwan S Rawata*
a Department of Chemistry, University of Delhi, Delhi-110007, India
bDepartment of Biochemistry, University of Delhi, South Campus, New Delhi-110021, India
cTranslational Health Science and Technology Institute, Vaccine and Infectious Disease
Research Centre, Gurgaon, 122016, India
Abstract
We report herein the synthesis and antimycobacterial activity of 1,2,3-triazole derivatives of
isoniazid. Most of the compounds exhibited potent activity against Mycobacterium
tuberculosis H37Rv strain with MIC99 values ranging from 0.195 to 1.56 μM in vitro. One
compound showed better in vitro activity than the reference, whereas five compounds were
equally potent to the reference compound isoniazid. The cytotoxicity of these compounds
was studied against THP-1 cell line and no toxicity was observed even at 50 μM
concentration. The compound with most potent in vitro activity was evaluated for in vivo in
murine model of tuberculosis and significantly reduced bacillary load in both lungs and
spleen at 10 weeks post-treatment. However this clearance effect was more pronounced in the
case of spleen. Molecular docking and molecular dynamics simulations have been performed
using two targets 2IDZ 1 (wild type Enoyl-acyl-carrier-protein reductase) and 4DQU 2
(mutant type Enoyl-acyl-carrier-protein reductase) to study the binding orientation and
stability of the compound 47. Docking studies proved compound 47 fit well into the binding
pocket of both the targets. Molecular dynamic simulations concluded that the highest active
compound 47 in complex with 4DQU was more stable when compared to the 2IDZ. We
believe that further optimization of these molecules may lead to potent anti-tubercular agents.
Key Words: antimycobacterial, isoniazid, MDR-TB, TDR-TB, LL-3858, in vivo.
*Corresponding author:
E. Mail: dsrawat@chemistry.du.ac.in
Phone No: 91-11-27667465
Fax No: 91-11-27667501
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1. Introduction
Tuberculosis (TB), one of the leading infectious diseases, still remains a major global
health problem [1]. The number of people infected with TB and the deaths reported every
year is enormously high. In 2010, WHO reported 9.4 million new TB cases and 1.7 million
deaths due to tuberculosis [2]. In terms of number of TB cases, India alone is responsible for
one-fifth of the global TB incidences [3]. It is anticipated that by 2020, one billion people
may be newly infected and over 30 million may die due to TB. The situation becomes more
severe to people with human immunodeficiency virus (HIV) infection [4,5]. TB in most
instances is a curable disease and more than 90% of people with drug-susceptible TB can be
cured within six months using combinations of first-line drugs. However, the emergence of
multi drug resistant (MDR) and extensively drug resistant (XDR) tuberculosis has worsen the
situation and its treatment is more challenging [1,6]. It requires the use of second-line drugs
that are more costly, cause more severe side effects, and must be taken up for two years [7].
Recently, a more dangerous and completely incurable form of tuberculosis, totally drug
resistance tuberculosis (TDR) has also been reported in Italy, Iran and more recently in India
[8-12]. TDR-TB strain has been shown to be resistant to all the first line, second line and
third line anti-TB drugs. Therefore, there is an urgent need to develop novel anti-TB agents,
which are synthetically feasible, have fewer side effects and shorter duration of treatment.
Although, several new compounds are currently in different stages of clinical trials [13-17],
only one new drug bedaquiline has been recently approved by FDA for its use in drug
resistant tuberculosis [18-20].
Isonicotinic acid hydrazide (Isoniazid or INH), a first-line anti-TB drug is one of the
most effective agents used for the treatment of Mycobacterium tuberculosis infection since
1952 [21,22]. It is a pro-drug and gets activated by a catalase-peroxidase enzyme known as
KatG in mycobacteria. The activated form reacts non-enzymatically with coenzymes, NAD+
and NADP+ to form isonicotinoyl-NAD(P) complex [23-25], which binds with the enoyl-acyl
carrier protein (ACP) reductase InhA. The enzyme, InhA helps in the synthesis of mycolic
acid. Thus isoniazid inhibits the synthesis of mycolic acid, required for the mycobacterial cell
wall. Unfortunately, bacterial strains resistant to INH are becoming common and with the
emergence of multi-drug resistant tuberculosis especially among persons with HIV infection,
even combination therapy is also no longer successful. Last few years have witnessed the
synthesis of a number of lipophilic analogues of isoniazid (INH) and some of them exhibited
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good in vitro antimycobacterial activity [26-33]. It has been suggested that the increased
lipophilicity of INH derivatives may facilitate diffusion through bio-membranes, therefore
enhancing the antimycobacterial activity. Moreover, by functionalising the hydrazine group
of isoniazid and retaining its activity can avoid the toxicity and other severe problems related
to the inactivation of isoniazid by the enzyme N-acetyltransferase-2.
LL-3858 is an isoniazid derivative and is developed by Lupin Limited. It has shown
bactericidal activity against both drug sensitive and multidrug resistant TB [34]. Phase I
clinical trial studies of LL-3858 have shown the potential of treating pulmonary tuberculosis
patients effectively [35]. The mechanism of action of LL-3858 is not yet known. Currently
this compound is in the initial stage of phase II clinical trial for the treatment of tuberculosis
[36].
N
N
F3C
H
O
N
NH2
N
O
NH
N
N
Isoniazid
LL-3858 (Lupin Ltd)
The 1,2,3-triazoles are known for several biological activities such as antibacterial,
antiviral, anti-inflammatory, analgesic, anticancer, anticonvulsant and anti-platelet [37-46].
Triazoles also have shown very promising anti-TB activity and antifungal activity via
inhibiting the cell wall synthesis [47,48]. Recently, the concept of hybrid molecules has been
the most interesting topic in medicinal chemistry, where two or more pharmacophores are
linked covalently resulting into one molecule [49-51]. The two units of final molecule may
act on different targets to exert dual drug action or one part can counterbalance the side
effects caused by another part. As discussed both isoniazid and triazoles act by similar
mechanism i.e. inhibition of cell wall synthesis and both are very potent anti-TB agents, thus
keeping these things in our mind and in continuation of our efforts towards the synthesis of
novel biological important molecules [52-64], we proposed to link isoniazid and triazole
entities together and synthesized novel 1,2,3-triazoles derivatives of isoniazid. These two
scaffolds were joined covalently into one single derivative in anticipation that the hybrid may
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show potent anti-TB activity. All the synthesized compounds were evaluated for their in vitro
antituberculosis activity against M. tuberculosis H37Rv.
2. Chemistry
For the synthesis of the title compounds, firstly different benzaldehydes having
triazole moiety (6-22, 34-38) were synthesized as outlined in scheme 1 and 2. The 4-
hydroxybenzaldehyde (1) was treated with 1,2-dibromoethane or 1,3-dibromopropane to get
the compounds 2 and 3 as depicted in scheme 1. The bromo group of compounds 2 and 3 was
then converted into azido group (4 and 5) by reaction with sodium azide in the presence of
K2CO3 as base and dimethylformamide as solvent. The cycloaddition of azide group and
terminal alkyne via click reaction gave different triazole based compounds (6-22).
Additionally, benzaldehydes having isomeric triazole linkage (34-38) were also synthesized
by different route as shown in scheme 2. The 4-hydroxybenzaldehyde (1) was treated with
propargyl bromide to obtain compound 23 [65]. Also the alkyl halides 29-33, were converted
to alkyl azides (34-38) in presence of K2CO3 using dimethylformamide as solvent. The
cycloaddition of these alkyl azides (34-38) and terminal alkyne (23) via click reaction gave
triazole based compounds (34-38). These compounds (6-22 and 34-38) were then condensed
with isoniazid to get the desired compounds (39-55 and 56-60) in good yields as depicted in
scheme 3.
13
All the new compounds were characterized by their IR, C NMR and mass spectral
data analysis. The formation of compound 39 was confirmed by the IR spectrum, which
1
showed peaks at 3222 and 1675 cm-1 due to NH and C=O groups, respectively. In the H
NMR of compound 39, a triplet at δ 0.88 was assigned to the terminal methyl protons of n-
butyl group. The sextet and quintet at δ 1.57 and 2.26 respectively were assigned to the two
internal methylene protons of n-butyl group. The CH2 protons of n-butyl group directly
attached to the triazole ring appeared as triplet at δ 2.55. The two triplets at δ 4.01 and 4.47
were assigned to the protons of CH2 protons attached to oxygen and nitrogen atoms in the
ethylene linker, respectively. Peak at δ 8.38 was assigned to the N=CH proton. Proton of
triazole ring was observed at δ 7.87. All aromatic protons appeared in the region of δ 6.99 to
8.76. The NH proton of the amide group appeared at δ 11.93. The 13C NMR spectra showed
peaks at δ 13.62, due to the terminal methyl carbon of n-butyl group, whereas peaks at δ
22.27, 27.08 and 29.45 were assigned to the methylene carbons of n-butyl group. The
methylene carbons, NCH2 and OCH2 of the ethylene linker appeared at δ 46.26, 64.70,
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respectively. The carbon of N=CH linkage appeared at δ 148.86. The most downfield peak at
δ 161.42 was assigned to the carbonyl carbon of amide linkage. The remaining nine peaks at
114.87, 121.52, 121.94, 126.72, 128.93, 140.61, 146.77, 150.32 and 160.18 were due to the
rest of the carbons of phenyl, pyridine and triazole nucleus. Further structure of compound 39
was confirmed by the mass spectral analysis which showed peaks at 393.4422 and 415.3933
due to (M + H)+ and (M + Na)+, respectively.
<Insert Scheme 1 here>
<Insert Scheme 2 here>
<Insert Scheme 3 here>
3. Biological Activity
3.1. In vitro anti-tuberculosis assay
A stock culture of M. tb H37Rv (ATCC 27294) was grown to Abs600nm of 0.2 in
Middlebrook 7H9 broth (Difco) supplemented with 0.05% Tween 80, 0.2% glycerol and
albumin/NaCl/glucose (ADS) complex. The culture was diluted 1:1000 in 7H9-based
medium before aliquoting 50 L into each well of a 96-well plate. Compounds were
dissolved in DMSO (Sigma) to make stock solutions of 50 mM. Compounds (100 µL
solution) were added to the first row of the 96-well plate at a final concentration 100 µM. 2-
fold serial dilutions were made and 5 dilutions of each compound (50 M-0.195 M) were
tested for anti-mycobacterial activity. The compounds were diluted 1:1 by addition of 50 L
of 1:1000 diluted cultures. Row 6 and 12 of the 96-well plates were no compounds control.
The plates were incubated at 37 C and the MIC99 values were read macroscopically using an
inverted plate reader after 14 days. MIC99 is defined as the minimum inhibitory concentration
of the compound required for 99% inhibition of bacterial growth. Each measurement was
repeated thrice.
3.2. In vivo anti-tuberculosis assay
The most active compound 47 from the series was selected for in vivo antituberculosis
activity evaluation. For activity evaluation, pathogen-free Balb/c mice of either sex (25-30 g)
were procured from the Division of Laboratory Animals, Central Drug Research Institute,
Lucknow, India. The animals were maintained in a BSLIII animal facility at University of
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Delhi South Campus, New Delhi and routinely cared according to the guidelines of CPCSEA
(Committee for the Purpose of Control and Supervision of Experiments on Animals), India.
All the experimental protocols included in this study were reviewed and approved by the
Institutional Animal Ethics Committee (Ref No. 1/IAEC/AKT/Biochem/UDSC/14.10.2011).
Mice were challenged with M. tb. H37Rv bacilli by respiratory route in inhalation
chamber (Glascol Inc., USA) pre-calibrated to deliver approximately 1000 bacilli per animal
in the lung by using frozen stocks of M. tb. H37Rv with their CFU pre-determined. Mice
were euthanized on the day after infection to determine the number of CFU implanted in the
lungs. Following two weeks of infection, mice were divided into different groups − untreated,
DMSO, isoniazid (25 mg/kg), rifampicin (10 mg/kg), compound 47 (25 mg/kg, 50 mg/kg,
100 mg/kg) and therapy was initiated.
All drugs were administered once daily, five days per week, in a maximum volume of
0.175 mL by oral gavage. Before initiating the chemotherapy, the infection in the animals
was verified by euthanizing a group of animals (N = 5) at 2 weeks post infection followed by
pathological observation and enumeration of the bacillary load in lungs and spleen. Mice
(N = 5 each group) were euthanized by CO2 asphyxiation after three weeks, six weeks and
ten weeks time points post therapy and monitored for gross pathological observations and
bacillary load. For bacterial enumeration, mice were dissected and lungs and spleens were
aseptically removed and homogenized in saline. Appropriate dilutions of the homogenates
were plated in duplicates onto MB7H11 agar and the plates were incubated at 37 C for 3–4
weeks followed by enumeration of colonies. The results were expressed as log10 CFU per
organ.
At 3 weeks time point, a bacillary load of 6.38 and 4.19 log10 CFU was measured in
the lungs and spleens of untreated animals, respectively. The disease continued to persist and
at 6 weeks post infection also, a bacillary load of 5.91 and 3.68 log10 was recorded in the
lungs and spleens of untreated animals, respectively. At 10 weeks post infection, the disease
further progressed with a bacillary load of 6.04 and 4.02 log10 in the lungs and spleens of
untreated animals, respectively.
3.3. In Silico Studies
Selected proteins 2IDZ and 4DQU was obtained from the Protein Data Bank
(www.rcsb.org) and further modified using Schrodinger’s Glide docking protocol. For Glide
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v5.8 calculations, both the complexes were imported to Maestro v9.3. Using Protein
Preparation Wizard (PPW), included biological unit and assigned bond orders, created zero
order bonds to metals, created disulfide bonds, converted any selenomethionines to
methionines, deleted water molecules beyond 5 Å from hetero groups, generated metal
binding states, added missing hydrogens, filled any missing side chains and loops and capped
the termini making use of the prime module integrated within protein preparation wizard
(PPW). Under review and modify tab of PPW, apart from Aricept, all the co-crystallized
ligands were identified and generated states. Under the refine tab of PPW, optimization of the
H-bond network was carried out to fix the overlapping hydrogens and the most likely
positions of hydroxyl and thiol hydrogen atoms, protonation states and tautomers of ‘His’
residues, and Chi ‘flip’ assignments for ‘Asn’, ‘Gln’ and ‘His’ residues were selected by the
protein assignment script shipped by Schrodinger. The pH range was set to 7.0 and the
protein was minimized by applying OPLS 2005 force field. Restrained minimizations were
performed until the average root mean square deviation (RMSD) of the non-hydrogen atoms
converged to 0.3Å. Location and surface area of active site/subsites of the targets were
predicted using Sitemap module integrated within Schrödinger software. Compound 47 was
also refined to its minimized state. Proteins and compound 47 refined was used for XP
docking, which is a flexible ligand docking protocol.
Desmond v3.1 Package was used to study the thermodynamic stability of the ligand-
receptor complex. Predefined TIP4P water model was used to simulate water molecules using
OPLS_2005 force field. Orthorhombic periodic boundary conditions were set up to specify
the shape and size of the repeating unit. In order to neutralize the system electrically, Na+
ions were added to balance the system charge and were placed randomly in the solvated
system. After building the solvated system containing protein in complex with the ligand, the
system was minimized and relaxed using default protocol integrated within Desmond.
Molecular dynamic simulations were carried out with the periodic boundary conditions in the
NPT ensemble. The temperature and pressure were kept at 300 K and 1 atmospheric pressure
using Nose-Hoover temperature coupling and isotropic scaling, and the operation was
followed by running the 10 ns NPT production simulation and saving the configurations thus
obtained with 5ps intervals with a time step of 4.8 ps. All the MD runs were performed on a
Dell workstation with 8 processors, each of which is Intel(R) Xeon(R) CPU E5607@2.27
GHz, with 16 GB DDR RAM. Desmond v3.1 integrated within Schrodinger was compiled
and run under Linux CentOS 6.1 operating system.
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4. Results and Discussion
All the isoniazid-triazoles conjugates (39-60) were tested for their in vitro anti-
mycobacterial activity against M. tuberculosis H37Rv. Isoniazid was used as a reference
drug. Table 1 and 2 shows the minimum concentration for all the derivatives that inhibited
the growth of 99% of the input cells (MIC99). Results show that most of the compounds
exhibited excellent activity with MIC99 values ranging from 0.39 to 1.56 μM. Seven
compounds 43-46, 49, 58 and 59 were found to be potent as standard compound isoniazid
(MIC99 = 0.39 μM). Compound 47 was the most active derivative (MIC99 = 0.195-0.39 μM),
being about two fold more effective than isoniazid. The lipophilicity (ClogP values) of the
isoniazid-triazole conjugates was also calculated and it was remarkably higher than the
isoniazid which is very important in terms of permeability through biomembranes.
<Insert Table 1 here>
<Insert Table 2 here>
After 3 weeks of treatment with isoniazid, CFU in the lungs of infected animals was
reduced by 1.22 log10 while after 6 weeks it showed a further CFU reduction in the bacillary
load by 2.59 log10 and a furthermore reduction of 3.38 log10 at 10 weeks when compared with
the animals in the untreated group (Fig. 1A). In spleens also, isoniazid treatment resulted in a
sharp reduction of bacillary load with a 2.66 log10 reduction at 3 weeks when compared with
the animals in the untreated group (Fig. 1B). At 6 weeks and 10 weeks time point, no bacilli
were recovered from the spleens. Whereas, after 3 weeks of treatment with rifampicin, the
CFU in the lungs of infected animals was reduced by 1.93 log10 while at 6 weeks it showed a
further CFU reduction in the bacillary load by 2.8 log10 when compared with the animals in
the DMSO group. After 10 weeks of rifampicin administration, no bacilli were recovered
from the lungs of the infected animals (Fig. 1A). In spleens also, rifampicin treatment
resulted in a marked reduction in the bacillary load with a 1.76 log10 and 3.31 log10 reduction
at 3 weeks and 6 weeks, respectively when compared with the animals in the DMSO group.
At 10 weeks time point, no bacilli were recovered from the spleens (Fig. 1B).
The evaluation of the efficacy of compound 47 was carried out at 25 mg/kg, 50 mg/kg
and 100 mg/kg concentrations. Compound 47, when administered at a low concentration of
25 mg/kg demonstrated no effect on the multiplication of bacilli in lungs at 3 weeks and 6
weeks time point as was evident from a comparable bacillary load in the lungs of compound
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47 treated animals and DMSO treated animals. An extension of the time period up to 10
weeks also did not make any further improvement in the bacillary burden present in the lungs
of compound 47 treated animals in comparison to DMSO treated animals at this
concentration (Fig. 1A). An increase in the concentration of compound 47 to 50 mg/kg also
showed no significant improvement in the control of pulmonary bacillary load at all the time
points evaluated. Even when compound 47 was given at a higher dose of 100 mg/kg for 3
weeks, there was no significant reduction in the bacillary load in the lungs when compared
with the DMSO treated animals, however, extending the time of therapy to 6 weeks and 10
weeks displayed a 0.82 log10 CFU and 0.91 log10 CFU reduction in the bacillary load in the
lungs of the animals when compared with the DMSO treated animals (Fig. 1A).
<Insert Fig. 1 here>
<Insert Fig. 2 here>
The efficacy of compound 47 was also examined in terms of its ability to reduce the
splenic bacillary load (Fig. 1B). The observations were also made in order to assess the role
of compound 47 in curtailing the hematogenous spread of the bacilli. It was observed that at
25 mg/Kg concentration of compound 47, the compound was unable to exert any influence
on the replication of splenic bacilli of the animals treated with the compound when compared
with the DMSO treated animals at 3 weeks and 6 weeks time points. Extension of the time
period up to 10 weeks exhibited improvement in the efficacy of the compound and splenic
bacillary load was reduced by 1.5 log10 CFU in comparison to the bacillary load in DMSO
treated animals. However, when the concentration of compound 47 was increased to 50
mg/kg, a substantial therapeutic effect was observed which was evident by a significant
reduction in the splenic bacillary load. At this concentration of compound 47, at the end of 3
weeks of chemotherapy, a reduction of 1.08 log10 CFU was observed in the splenic bacillary
load when compared with the DMSO treated animals. The extension of chemotherapy up to 6
weeks resulted in a significantly higher reduction (2.15 log10 CFU) in the bacillary load. On
further extension of therapy period to 10 weeks, a very significant reduction in the bacillary
load (3.31 log10 CFU) in the spleens was observed when compared with the DMSO treated
animals (Fig. 1B). The efficacy of compound 47 was even more pronounced at a
concentration of 100 mg/kg. When animals were administered 100 mg/kg compound 47 for 3
weeks, the splenic bacillary load exhibited a reduction of 1.46 log10 when compared with that
of the DMSO treated animals. Further continuation of the chemotherapy up to 6 weeks and
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10 weeks resulted in a more prominent reduction in the bacillary load by 2.58 log10 in the
spleens of the animals treated with compound 47 when compared with the DMSO treated
animals (Fig. 1B).
All the compounds were further examined for toxicity in THP-1 cell line. The
compounds were found to be non-toxic upto a concentration of 50 μM the highest
concentration tested (Table 1). It was not possible to test toxicity at higher concentrations due
to solubility limitations.
Furthermore, compound 47 was docked into both wild type InhA (2IDZ 1) and mutant
type InhA (4DQU 2) for identifying the binding orientation and stability of the compound 47
[66, 67]. Docking was performed using Glide module present in the Maestro 6.5 3 [68].
Binding pockets of both the proteins are almost similar, but Asp 148 of 2IDZ is changed to
Gly 148 in 4DQU (Fig. 3). Docking scores of -7.14 and -9.48 were obtained for compound 47
against 2IDZ and 4DQU proteins. Compound 47 fit well into the binding pockets of both the
targets. Stability in the binding pockets is due to the presence of hydrophobic amino acids in
the pocket. The 2IDZ protein contains Ile 95, Val 65, Leu 63, Ile 122, Phe 97, Ile 16, Ile 21,
Met 147, Phe 149, Met 155, Ala 191, Tyr 158, Pro 193 and Trp 222 in the binding pocket,
and 4DQU contains Ile 16, Phe 41, Ile 122, Leu 63, Val 65, Ile 95, Phe 97, Met 147, Pro 193
and Ile 194.
Acquired complexes were redirected to molecular dynamic simulations of 10 ns.
Predefined TIP4P water model [69] was used to simulate water molecules using OPLS_2005
force field [70,71]. RMSD (Root mean square deviation) of 2IDZ complex with compound
47 was in between 1.5 to 3.5 Å throughout the simulation (Fig. 4). There was fluctuation
between the 2.5 to 4.5 ns and after 5 ns it got stabilized. Interesting results were observed in
the case of 4DQU complex with compound 47. This complex was more stable when
compared to the initial one. RMSD of this complex was in between 1.5 to 2 Å. RMSF (Root
mean square fluctuation) was also analyzed for the both the complexes and there was no
much difference in the fluctuations of amino acids during the run (Fig. 5). Fluctuations of the
amino acids in complexes were coinciding throughout the simulation showing meagre
difference. Superimposed RMSD and RMSF results confirmed the stability of the resistant
target when compared with the wild type. These theoretical studies may be useful to give
insights into the activity of compound 47 against resistant targets.
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<Insert Fig. 3 here>
<Insert Fig. 4 here>
<Insert Fig. 5 here>
5. Conclusions
A series of 22 isoniazid-triazole conjugates were synthesized and evaluated for their
in vitro and in vivo antitubercular activity. Most of the compounds exhibited potent activity
and one compound exhibited better activity than isoniazid in vitro. When tested for in vivo
activity this compound exhibited mild activity both in case of lung as well as spleen. The
influence of the compound on the replication of the pathogen was far superior in the case of
spleen as compared to the influence on the lung. We do not have clear explanation for this
observation at present however it might suggest that the bio-availability of these compounds
could be the possible reason for the better activity profile in the case of spleen. It is not
unlikely that this difference could stem from the fact that the required concentration of the
compound could be more easily available in the blood stream as compared to pulmonary
tissue thereby leading to a more pronounced effect on the control of bacillary load in spleens
as the bacteria residing in spleens results from the hematogenous spread. However,
translation of this speculation into real evidence would require further experiments. In terms
of toxicity, all the compounds were found to be nontoxic upto 50 μM concentration against
THP-1 cell line. Docking and molecular dynamics simulations results indicated that
compound 47 showed important amino acids interactions with both wild type and mutant
type proteins, and displayed significant stability in the binding pocket, which may be
considered as a positive sign for compound 47 in terms of activity against INH-resistant
strains of M. tuberculosis.
From the activity and toxicity results, it can be concluded that these compounds
exhibited potent activity in in vitro but unfortunately mild activity in in vivo and are safe in
terms of toxicity profile and hence can be used as lead molecule for further optimisation. The
major problem faced during performing the experiments was the solubility of these
compounds therefore efforts are being made to improve the solubility of these compounds so
that the real therapeutic potential of the compounds can be evaluated.
6. Experimental Protocols
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All the chemicals were purchased from Sigma-Aldrich. Solvents used for the
chemical synthesis were acquired from commercial sources, were of analytical grade and
used without further purification. Thin layer chromatography (Merck Kiesel 60 F254, 0.2 mm
thickness) was used to monitor the progress of the reactions and the compounds were purified
by silica gel (60-120 mesh) column chromatography. Melting points were recorded on EZ-
Melt automated melting point apparatus, Stanford Research Systems and are uncorrected. IR
spectra were recorded on Perkin-elmer FT-IR spectrophotometer using KBr pellets or as film
1
in chloroform and the values were expressed in cm-1. H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded on Jeol ECX spectrospin instrument using CDCl3 or
DMSO-d6 as solvent and TMS as internal reference. The chemical shift values were
expressed on δ scale and the coupling constant (J) in Hz. Mass data were recorded in Jeol-
Accu TOF JMS-T100LC mass spectrometer.
6.1. Typical procedure for the synthesis of 4-(2-bromoethoxy)benzaldehyde (2) and related
compound (3): A mixture of 4-hydroxybenzaldehyde (1, 5.0 g, 0.041 mol), 1,2-
dibromoethane (23.0 g, 0.122 mol) and K2CO3 (16.9 g, 0.122 mol) in dry DMF (80 mL) was
o
stirred at 35-40 C for 8-10 h. After completion of the reaction, cold water was added to the
reaction mixture and the compound was extracted with CHCl3 (3 x 50 mL). The organic layer
was washed with cold water to remove DMF. The organic phase was dried over Na2SO4 and
the excess solvent was removed under reduced pressure. The crude product was purified by
column chromatography using EtOAc:hexane as eluent to obtain compound 2.
6.2. Typical procedure for the synthesis of 4-(2-azidoethoxy)benzaldehyde (4) and related
compound (5): To a stirred solution of compound 2 (6.0 g, 0.026 mol) and K2CO3 (10.8 g,
0.078 mol) in DMF (50 mL), NaN3 (5.1 g, 0.078 mol) was added slowly. The reaction
mixture was stirred at 50 °C for 4-5 h. After cooling down to room temperature, water was
added to dissolve excess NaN3 and the solution was extracted with CHCl3 (3 x 50 mL). The
combined organic layer was washed several times with water to remove DMF and then dried
over Na2SO4. Excess solvent was distilled off under reduced pressure to get compound 4 in
quantitative yield.
6.3. Typical procedure for the synthesis of 4-(2-(4-butyl-1H-1,2,3-triazol-1-yl)ethoxy)
benzaldehyde (6) and related compounds (7-22): 4-(2-Azidoethoxy)benzaldehyde (4, 500
mg, 2.6 mmol) and 1-butyne (170 mg, 3.1 mmol) were dissolved in 20 mL of t-BuOH at
room temperature. To this, a solution of CuSO4.5H2O (130 mg, 0.52 mmol) and sodium
13

ACCEPTED MANUSCRIPT
ascorbate (207 mg, 1.0 mmol) in 20 mL of water was added. The reaction mixture was stirred
at 35-40 °C for 4-5 h. After completion, the reaction mixture was extracted with EtOAc (3 x
40 mL). The organic layer was dried over Na2SO4 and the excess solvent was removed under
reduced pressure. Crude product was purified by column chromatography to get the desired
compound 6.
6.3.1. 4-(2-(4-Butyl-1H-1,2,3-triazol-1-yl)ethoxy)benzaldehyde (6). Yield 85%; IR (film, cm-1):
1
2957, 2937, 2871, 2860, 2741, 1690, 1602, 1580, 1312, 1254, 1217, 1162, 1045; H NMR
(400 MHz, CDCl3): δ 0.92 (t, J = 7.3 Hz, 3H, CH2CH2CH2CH3), 1.37 (sextet, J = 7.3 Hz, 2H,
CH2CH2CH2CH3), 1.64 (quintet, J = 7.3 Hz, 2H, CH2CH2CH2CH3), 2.71 (t, J = 7.3 Hz, 2H,
CH2CH2CH2CH3), 4.45 (t, J = 5.8 Hz, 2H, NCH2), 4.76 (t, J = 5.1 Hz, 2H, OCH2), 6.97 (d,
J = 8.8 Hz, 2H, ArH), 7.44 (s, 1H), 7.83 (d, J = 8.8 Hz, 2H, ArH), 9.89 (s, 1H, CHO).
6.3.2. 4-(2-(4-(1-Hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzaldehyde (7). Yield
1
78%; IR (film, cm-1): 3394, 2929, 2854, 1680, 1601, 1254, 1161, 1053, 907; H NMR (400
MHz, CDCl3): δ 1.18 (s, 1H), 1.26-1.32 (m, 1H), 1.47-1.49 (m, 2H), 1.54-1.56 (m, 1H), 1.66-
1.69 (m, 2H), 1.82 (s, 2H), 1.89-1.91 (m, 2H), 2.32 (brs, 1H, OH), 4.39 (t, J = 5.1 Hz, 2H,
NCH2), 4.71 (t, J = 5.1 Hz, 2H, OCH2), 6.90 (d, J = 8.0 Hz, 2H, ArH), 7.59 (s, 1H), 7.76 (d,
J = 8.8 Hz, 2H, ArH), 9.81 (s, 1H, CHO).
6.3.3. 4-(2-(4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzaldehyde (8). Yield 74%;
IR (film, cm-1): 3364, 2925, 2853, 1677, 1600, 1578, 1508, 1311, 1252, 1162, 1042; 1H NMR
(400 MHz, CDCl3): δ 2.37 (brs, 1H, CH2OH), 4.46 (t, J = 5.1 Hz, 2H, NCH2), 4.81 (t, J = 5.1
Hz, 4H), 6.98 (d, J = 8.8 Hz, 2H, ArH), 7.32 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H, ArH), 9.89 (s,
1H, CHO).
6.3.4. (1-(2-(4-Formylphenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl propionate (9). Yield
1
86%; H NMR (400 MHz, CDCl3): δ 1.12 (t, J = 7.3 Hz, 3H, COCH2CH3), 2.34 (q, J = 7.3
Hz, 2H, COCH2CH3), 4.46 (t, J = 5.1 Hz, 2H, NCH2), 4.81 (t, J = 5.1 Hz, 2H, OCH2), 5.22 (s,
2H, CH2OCO), 6.98 (d, J = 8.8 Hz, 2H, ArH), 7.80 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H, ArH),
9.89 (s, 1H, CHO).
1
6.3.5. 4-(2-(4-(Pyridin-2-yl)-1H-1,2,3-triazol-1-yl)ethoxy)benzaldehyde (10). Yield 65%; H
NMR (400 MHz, CDCl3): δ 4.51 (t, J = 5.1 Hz, 2H, NCH2), 4.88 (t, J = 5.1 Hz, 2H, OCH2),
7.01 (d, J = 8.8 Hz, 2H, ArH), 7.23-7.25 (m, 1H, ArH), 7.76-7.81 (m, 1H, ArH), 7.83 (d, J =
14

ACCEPTED MANUSCRIPT
8.8 Hz, 2H, ArH), 8.18 (d, J = 7.3 Hz, 1H, ArH), 8.35 (s, 1H), 8.59 (d, J = 5.1 Hz, 1H, ArH),
9.88 (s, 1H, CHO).
6.3.6. 4-(2-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzaldehyde (12). Yield 70%;
1H NMR (400 MHz, CDCl3): δ 4.45 (t, J = 5.1 Hz, 2H, NCH2), 4.80 (t, J = 5.1 Hz, 2H,
OCH2), 5.22 (s, 2H, CH2OPh), 6.94-6.99 (m, 5H, ArH), 7.26-7.30 (m, 2H, ArH), 7.79 (s,
1H), 7.82 (d, J = 9.8 Hz, 2H, ArH), 9.88 (s, 1H, CHO).
6.3.7. 4-(2-(4-(p-Tolyloxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzaldehyde (13). Yield
78%; 1H NMR (400 MHz, CDCl3): δ 2.28 (s, 3H, CH3), 4.45 (t, J = 5.1 Hz, 2H, NCH2), 4.80
(t, J = 5.1 Hz, 2H, OCH2), 5.19 (s, 2H, CH2OPh), 6.87 (d, J = 8.8 Hz, 2H, ArH), 6.95 (d, J =
8.8 Hz, 2H, ArH), 7.07 (d, J = 8.8 Hz, 2H, ArH), 7.78 (s, 1H), 7.82 (d, J = 8.8 Hz, 2H, ArH),
9.88 (s, 1H, CHO).
6.3.8. 4-(2-(4-((4-Chlorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzaldehyde (14).
1
Yield 75%; H NMR (400 MHz, CDCl3): δ 4.45 (t, J = 5.1 Hz, 2H, NCH2), 4.81 (t, J = 5.1
Hz, 2H, OCH2), 5.19 (s, 2H, CH2OPh), 6.90 (d, J = 9.5 Hz, 2H, ArH), 6.94 (d, J = 8.8 Hz,
2H, ArH), 7.22 (d, J = 8.8 Hz, 2H, ArH), 7.79 (s, 1H), 7.82 (d, J = 8.8 Hz, 2H, ArH), 9.88 (s,
1H, CHO).
6.3.9. 4-(3-(4-Propyl-1H-1,2,3-triazol-1-yl)propoxy)benzaldehyde (15). Yield 85%; IR (film,
cm-1): 3137, 3076, 2960, 2933, 2873, 1688, 1601, 1578, 1510, 1313, 1257, 1161, 1049; 1H
NMR (400 MHz, CDCl3): δ 0.94 (t, J = 7.3 Hz, 3H, CH2CH2CH3), 1.66 (sextet, J = 7.3 Hz,
2H, CH2CH2CH3), 2.43 (quintet, J = 5.9 Hz, 2H, OCH2CH2CH2N), 2.68 (t, J = 7.3 Hz, 2H,
CH2CH2CH3), 4.05 (t, J = 5.9 Hz, 2H, NCH2), 4.56 (t, J = 6.6 Hz, 2H, OCH2), 6.98 (d, J =
8.8 Hz, 2H, ArH), 7.27 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H, ArH), 9.89 (s, 1H, CHO).
6.3.10. 4-(3-(4-Butyl-1H-1,2,3-triazol-1-yl)propoxy)benzaldehyde (16). Yield 80%; IR (film,
cm-1): 2950, 1685, 1603, 1251, 1041, 806; 1H NMR (400 MHz, CDCl3): δ 0.91 (t, J = 7.3 Hz,
3H, CH2CH2CH2CH3), 1.35 (sextet, J = 7.3 Hz, 2H, CH2CH2CH2CH3), 1,62 (quintet, J = 7.3
Hz, 2H, CH2CH2CH2CH3), 2.43 (quintet, J = 6.6 Hz, 2H, OCH2CH2CH2N), 2.43 (t, J = 6.6
Hz, 2H, CH2CH2CH2CH3), 4.03 (t, J = 5.9 Hz, 2H, NCH2), 4.56 (t, J = 6.6 Hz, 2H, OCH2),
6.98 (d, J = 8.8 Hz, 2H, ArH), 7.27 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H, ArH), 9.89 (s, 1H, CHO).
6.3.11. 4-(3-(4-(1-Hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)propoxy)benzaldehyde (17).
Yield 75%; IR (film, cm-1): 3394, 2933, 2857, 1686, 1600, 1577, 1509, 1256, 1218, 1160,
15

ACCEPTED MANUSCRIPT
1047; 1H NMR (400 MHz, CDCl3): δ 1.31-1.36 (m, 1H), 1.50-1.55 (m, 2H), 1.59-1.64 (m,
1H), 1.67-1.78 (m, 2H), 1.82-1.86 (m, 2H), 1.92-1.95 (m, 2H), 2.04 (s, 1H, OH), 2.45
(quintet, J = 5.9 Hz, 2H, OCH2CH2CH2N), 4.07 (t, J = 5.1 Hz, 2H, NCH2), 4.57 (t, J = 6.6
Hz, 2H, OCH2), 6.98 (d, J = 8.8 Hz, 2H, ArH), 7.45 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H, ArH),
9.88 (s, 1H, CHO).
6.3.12. 4-(3-(4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)propoxy)benzaldehyde (18). Yield
77%; IR (film, cm-1): 3370, 2926, 2853, 1686, 1601, 1577, 1509, 1313, 1258, 1218, 1161,
1045; 1H NMR (400 MHz, CDCl3): δ 2.10 (s, 1H, OH), 2.45 (quintet, J = 6.6 Hz, 2H,
OCH2CH2CH2N), 4.07 (t, J = 5.9 Hz, 2H, NCH2), 4.60 (t, J = 6.6 Hz, 2H, OCH2), 4.78 (s,
2H, CH2OH), 6.98 (d, J = 8.8 Hz, 2H, ArH), 7.56 (s, 1H), 7.83 (d, J = 8.8 Hz, 2H, ArH), 9.89
(s, 1H, CHO).
6.3.13. (1-(3-(4-Formylphenoxy)propyl)-1H-1,2,3-triazol-4-yl)methyl propionate (19). Yield
79%; 1H NMR (400 MHz, CDCl3): δ 1.12 (t, J = 5.8 Hz, 3H, COCH2CH3), 2.34 (quintet, J =
7.3 Hz, 2H, OCH2CH2CH2N), 2.45 (q, J = 5.8 Hz, 2H, OCH2CH3), 4.07 (t, J = 5.8 Hz, 2H,
NCH2), 4.59 (t, J = 6.6 Hz, 2H, OCH2), 5.21 (s, 2H, CH2OCO), 6.98 (d, J = 8.8 Hz, 2H,
ArH), 7.61 (s, 1H), 7.84 (d, J = 8.0 Hz, 2H, ArH), 9.89 (s, 1H, CHO).
6.3.14. 4-(3-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)propoxy)benzaldehyde (20). Yield
74%; 1H NMR (400 MHz, CDCl3): δ 2.45 (quintet, J = 5.6 Hz, 2H, OCH2CH2CH2N), 4.06 (t,
J = 5.1 Hz, 2H, NCH2), 4.60 (t, J = 6.6 Hz, 2H, OCH2), 5.21 (s, 2H, CH2OPh), 6.96 (d, J = 8
Hz, 5H, ArH), 7.29 (d, J = 7.3 Hz, 2H, ArH), 7.62 (s, 1H), 7.82 (d, J = 8.8 Hz, 2H, ArH),
9.88 (s, 1H, CHO).
6.3.15. 4-(3-(4-(p-Tolyloxymethyl)-1H-1,2,3-triazol-1-yl)propoxy)benzaldehyde (21). Yield
1
76%; H NMR (400 MHz, CDCl3): δ 2.28 (s, 3H, CH3), 2.45 (quintet, J = 6.6 Hz, 2H,
OCH2CH2CH2N), 4.06 (t, J = 5.9 Hz, 2H, NCH2), 4.60 (t, J = 6.6 Hz, 2H, OCH2), 5.17 (s,
2H, CH2OPh), 6.85 (d, J = 8.8 Hz, 2H, ArH), 6.96 (d, J = 8.8 Hz, 2H, ArH), 7.07 (d, J = 8.8
Hz, 2H, ArH), 7.61 (s, 1H), 7.82 (d, J = 8.8 Hz, 2H, ArH), 9.88 (s, 1H, CHO).
6.3.16. 4-(3-(4-((4-Chlorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy)benzaldehyde (22).
1
Yield 72%; H NMR (400 MHz, CDCl3): δ 2.46 (quintet, J = 5.9 Hz, 2H, OCH2CH2CH2N),
4.05 (t, J = 5.9 Hz, 2H, NCH2), 4.61 (t, J = 6.6 Hz, 2H, OCH2), 5.17 (s, 2H, CH2OPh), 6.89
(d, J = 9.5 Hz, 2H, ArH), 6.95 (d, J = 8.8 Hz, 2H, ArH), 7.22 (d, J = 8.8 Hz, 2H, ArH), 7.61
(s, 1H), 7.83 (d, J = 8.8 Hz, 2H, ArH), 9.89 (s, 1H, CHO).
16

ACCEPTED MANUSCRIPT
6.4. Procedure for synthesis of 4-(prop-2-ynyloxy)benzaldehyde (23): To a mixture of para-
hydroxybenzaldehyde (1, 5.0 g, 0.041 mol) and K2CO3 (16.9 g, 0.122 mol) in dry DMF (25
mL), a solution of propargyl bromide (5.2 g, 0.049 mol) in DMF (50 mL) was added
dropwise. The reaction mixture was stirred at room temperature for 10-12 h. Progress of the
reaction was monitored by thin layer chromatography. After completion, the reaction mixture
was poured into ice cold water and extracted with chloroform (3 x 50 mL). The combined
organic layer was washed with cold water to remove DMF and dried over anhydrous Na2SO4.
Excess solvent was removed under reduced pressure and crude product thus obtained was
purified over silica gel column using EtOAc/Hexane as an eluent to afford compound 23.
6.5. Typical procedure for the synthesis of 1-azidopropane (29) and related compounds (30-33):
Sodium azide (792 mg, 0.012 mol) was added to a solution of 1-bromopropane (24, 500 mg,
0.004 mol) and K2CO3 (1.68 g, 0.012 mol) in dry DMF (15 mL). The mixture was stirred for
o
2-3 h at 50 C. After cooling down to room temperature, water was added to dissolve excess
NaN3 and compound was extracted with CHCl3 (3 x 50 mL). The organic layer was washed
with water. The resulting organic layer was dried over Na2SO4 and solvent was removed
under vacuum to get compound 29.
6.6. Typical procedure for the synthesis of 4-((1-propyl-1H-1,2,3-triazol-4-yl)
methoxy)benzaldehyde (34) and related compounds (35-38): 4-(Prop-2-ynyloxy)benzaldehyde
(23, 500 mg, 3.12 mmol) and 1-azidopropane (29, 265 mg, 3.12 mmol) were dissolved in 20
mL of t-BuOH at room temperature. To this, a solution of CuSO4.5H2O (156 mg, 0.62 mmol)
and sodium ascorbate (248 mg, 1.24 mmol) in 20 ml of water was added. The reaction
mixture was stirred at 35-40 °C for 4-5 h. After completion, the reaction mixture was
extracted with EtOAc (3 x 40 mL). Organic layer was dried over Na2SO4 and the excess
solvent was removed under reduced pressure. Crude product was purified by column
chromatography to get the pure compound 34.
1
6.6.1. 4-((1-Propyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (34). Yield 80%; H NMR
(400 MHz, CDCl3): δ 0.97 (t, J = 7.3 Hz, 3H, NCH2CH2CH3), 1.96 (sextet, J = 7.3 Hz, 2H,
NCH2CH2CH3), 4.34 (t, J = 7.3 Hz, 2H, NCH2), 5.30 (s, 2H, OCH2), 7.11 (d, J = 8.8 Hz, 2H,
ArH), 7.63 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H, ArH), 9.89 (s, 1H, CHO).
6.6.2. 4-((1-iso-Propyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (35). Yield 82%; IR
1
(film, cm-1): 2982, 2928, 2852, 1689, 1600, 1578, 1508, 1311, 1247, 1215, 1161, 1110; H
17

ACCEPTED MANUSCRIPT
NMR (400 MHz, CDCl3): δ 1.60 (d, J = 6.6 Hz, 6H, CH(CH3)2), 4.85 (septet, J = 6.6 Hz, 1H,
CH(CH3)2), 5.29 (s, 2H, OCH2), 7.12 (d, J = 8.8 Hz, 2H, ArH), 7.67 (s, 1H), 7.84 (d, J = 8.8
Hz, 2H, ArH), 9.89 (s, 1H, CHO).
1
6.6.3. 4-((1-Butyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (36). Yield 85%; H NMR
(400 MHz, CDCl3): δ 0.95 (t, J = 7.3 Hz, 3H, NCH2CH2CH2CH3), 1.36 (sextet, J = 7.3 Hz,
2H, NCH2CH2CH2CH3), 1.90 (quintet, J = 7.3 Hz, 2H, NCH2CH2CH2CH3), 4.37 (t, J = 7.3
Hz, 2H, NCH2), 5.30 (s, 2H, OCH2), 7.11 (d, J = 8.8 Hz, 2H, ArH), 7.63 (s, 1H), 7.84 (d, J =
8.8 Hz, 2H, ArH), 9.89 (s, 1H, CHO).
1
6.6.4. 4-((1-Pentyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (37). Yield 75%; H NMR
(400 MHz, CDCl3): δ 0.89 (t, J = 7.3 Hz, 3H, CH2CH2CH2CH2CH3), 1.32-1.35 (m, 4H), 1.92
(quintet, J = 7.3 Hz, 2H, NCH2CH2CH2CH2CH3), 4.36 (t, J = 7.3 Hz, 2H, NCH2), 5.30 (s,
2H, OCH2), 7.11 (d, J = 8.8 Hz, 2H, ArH), 7.62 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H, ArH), 9.89
(s, 1H, CHO).
1
6.6.5. 4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (38). Yield 90%; H NMR
(400 MHz, CDCl3): δ 5.19 (s, 2H, OCH2), 5.47 (s, 2H, NCH2Ph), 7.01 (d, J = 8.8 Hz, 2H,
ArH), 7.19-7.22 (m, 2H, ArH), 7.29-7.33 (m, 3H, ArH), 7.48 (s, 1H), 7.75 (d, J = 8.8 Hz, 2H,
ArH), 9.81 (s, 1H, CHO).
6.7. Typical procedure for the synthesis of (E)-N'-(4-(2-(4-butyl-1H-1,2,3-triazol-1-yl)
ethoxy)benzylidene)isonicotinohydrazide (39) and related compounds (40-60): A mixture of
isoniazid (200 mg, 1.45 mmol) and 4-(2-(4-butyl-1H-1,2,3-triazol-1-yl)ethoxy)benzaldehyde
(6, 360 mg, 1.31 mmol) in EtOH/H2O was stirred at room temperature for 4-6 h. After
completion of the reaction, separated solid was filtered and washed with cold EtOH to obtain
the pure compound 39.
6.7.1. (E)-N'-(4-(2-(4-Butyl-1H-1,2,3-triazol-1-yl)ethoxy)benzylidene)isonicotinohydrazide
(39): Yield 80%; mp 187-190 oC; IR (KBr, cm-1): 3222, 3046, 2955, 2875, 1675, 1608, 1559,
1
1511, 1286, 1259, 1174, 1048, 947, 825; H NMR (400 MHz, DMSO-d6): δ 0.88 (t, J = 7.3
Hz, 3H, CH2CH2CH2CH3), 1.57 (sextet, J = 7.3 Hz, 2H, CH2CH2CH2CH3), 2.26 (quintet, J =
6.5 Hz, 2H, CH2CH2CH2CH3), 2.55 (t, J = 7.3 Hz, 2H, CH2CH2CH2CH3), 4.01 (t, J = 6.5 Hz,
2H, NCH2), 4.47 (t, J = 6.5 Hz, 2H, OCH2) 6.99 (d, J = 8.7 Hz, 2H, ArH), 7.67 (d, J = 8.7
Hz, 2H, ArH), 7.79-7.80 (m, 2H, ArH), 7.87 (s, 1H), 8.38 (s, 1H, N=CH), 8.76 (dd, J = 1.4,
13
4.3 Hz, 2H, ArH), 11.93 (s, 1H, CONH); C NMR (100 MHz, DMSO-d6): δ 13.62, 22.27,
18

ACCEPTED MANUSCRIPT
27.08, 29.45, 46.26, 64.70, 114.87, 121.52, 121.94, 126.72, 128.93, 140.61, 146.77, 148.86,
150.32, 160.18, 161.42; ESI-HRMS (m/z) calculated for C21H24N6O2: 392.1961, found:
393.4422 (M + H)+, 415.3933 (M + Na)+; Anal. calcd. for C21H24N6O2: C, 64.27; H, 6.16; N,
21.41; O, 8.15, found: C, 64.33; H, 6.23; N, 21.57; O, 8.28.
6.7.2.
(E)-N'-(4-(2-(4-(1-Hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzylidene)
o
isonicotinohydrazide (40). Yield 55%; mp 205-207 C; IR (KBr, cm-1): 3294, 3107, 2939,
1668, 1610, 1536, 1508, 1423, 1311, 1279, 1251, 1169, 1040, 971, 840; 1H NMR (400 MHz,
DMSO-d6): δ 1.23-1.27 (m, 1H), 1.37-1.40 (m, 2H), 1.48 (brs, 1H), 1.62-1.69 (m, 4H), 1.81-
1.84 (m, 2H), 4.45 (t, J = 5.1 Hz, 2H, NCH2), 4.73 (t, J = 5.1 Hz, 2H, OCH2), 4.85 (s, 1H,
OH), 7.01 (d, J = 8.7 Hz, 2H, ArH), 7.67 (d, J = 8.7 Hz, 2H, ArH), 7.79-7.80 (m, 2H, ArH),
7.94 (s, 1H), 8.38 (s, 1H, N=CH), 8.77 (brs, 2H, ArH), 11.94 (s, 1H, CONH); 13C NMR (100
MHz, DMSO-d6): δ 21.67, 25.25, 37.83, 48.83, 66.42, 67.99, 115.0, 121.51, 127.08, 128.95,
140.57, 148.73, 150.32, 155.89, 159.69, 161.45; ESI-HRMS (m/z) calculated for C23H26N6O3:
434.2066, found: 435.2711 (M + H)+; Anal. calcd. for C23H26N6O3: C, 63.58; H, 6.03; N,
19.34; O, 11.05, found: C, 63.63; H, 6.15; N, 19.50; O, 11.17.
6.7.3.
(E)-N'-(4-(2-(4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzylidene)
o
isonicotinohydrazide (41). Yield 50%; mp 215-217 C; IR (KBr, cm-1): 3400, 3197, 3144,
2962, 2852, 1654, 1609, 1574, 1574, 1514, 1367, 1303, 1254, 1174, 1058, 1017, 918, 830; 1H
NMR (400 MHz, DMSO-d6): δ 4.45 (t, J = 5.1 Hz, 2H, NCH2), 4.51 (d, J = 5.1 Hz, 2H,
OCH2), 4.76 (t, J = 5.1 Hz, 2H, CH2OH), 5.18 (t, J = 5.1 Hz, 1H, OH), 7.00 (d, J = 8.7 Hz,
2H, ArH), 7.67 (d, J = 8.7 Hz, 2H, ArH), 7.79-7.80 (m, 2H), 8.04 (s, 1H), 8.38 (s, 1H,
N=CH), 8.75-8.77 (m, 2H, ArH), 11.94 (s, 1H, CONH); ESI-HRMS (m/z) calculated for
C18H18N6O3: 366.1440, found: 367.2901 (M + H)+; Anal. calcd. for C18H18N6O3: C, 59.01; H,
4.95; N, 22.94; O, 13.10, found: C, 59.11; H, 5.05; N, 22.87; O, 13.18.
6.7.4.
(E)-(1-(2-(4-((2-Isonicotinoylhydrazono)methyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-
o
yl)methylpropionate (42). Yield 62%; mp 180-181 C; IR (KBr, cm-1): 3233, 3132, 3077,
1743, 1678, 1611, 1553, 1513, 1351, 1288, 1244, 1168, 1052, 840, 823; 1H NMR (400 MHz,
DMSO-d6): δ 1.00 (t, J = 7.6 Hz, 3H, COCH2CH3), 2.30 (q, J = 8.0 Hz, 2H, COCH2CH3),
4.45 (t, J = 5.1 Hz, 2H, NCH2), 4.78 (t, J = 5.1 Hz, 2H, OCH2), 5.11 (s, 2H, CH2OCO), 7.00
(d, J = 8.7 Hz, 2H, ArH), 7.66 (d, J = 8.7 Hz, 2H, ArH), 7.79 (dd, J = 2.2, 4.3 Hz, 2H, ArH),
8.22 (s, 1H), 8.38 (s, 1H, N=CH), 8.75-8.77 (m, 2H, ArH), 11.94 (s, 1H, CONH); ESI-HRMS
(m/z) calculated for C21H22N6O4: 422.1703, found: 423.4100 (M + H)+; Anal. calcd. for
19

ACCEPTED MANUSCRIPT
C21H22N6O4: C, 59.71; H, 5.25; N, 19.89; O, 15.15, found: C, 59.83; H, 5.31; N, 19.88; O,
15.20.
6.7.5.
(E)-N'-(4-(2-(4-(Pyridin-2-yl)-1H-1,2,3-triazol-1-yl)ethoxy)benzylidene)isonicotino
o
hydrazide (43). Yield 55%; mp 212-214 C; IR (KBr, cm-1): 3305, 3130, 2940, 1661, 1605,
1
1543, 1510, 1477, 1381, 1264, 1169, 1045, 1030, 828; H NMR (400 MHz, DMSO-d6): δ
4.53 (t, J = 5.1 Hz, 2H, NCH2), 4.86 (t, J = 5.1 Hz, 2H, OCH2), 7.03 (d, J = 8.7 Hz, 2H,
ArH), 7.31-7.34 (m, 1H, ArH), 7.66 (d, J = 8.7 Hz, 2H, ArH), 7.78-7.79 (m, 2H, ArH), 7.85-
7.90 (m, 1H, ArH), 8.01-8.03 (m, 1H, ArH), 8.37 (s, 1H), 8.58 (m, 1H, ArH), 8.68 (s, 1H,
N=CH), 8.75-8.76 (m, 2H, ArH), 11.93 (s, 1H, CONH); ESI-HRMS (m/z) calculated for
C22H19N7O2: 413.1600, found: 414.4650 (M + H)+, 436.4455 (M + Na)+; Anal. calcd. for
C22H19N7O2: C, 63.91; H, 4.63; N, 23.72; O, 7.74, found: C, 64.11; H, 4.76; N, 23.88; O,
7.83.
6.7.6. (E)-N'-(4-(2-(4-Phenyl-1H-1,2,3-triazol-1-yl)ethoxy)benzylidene)isonicotinohydrazide
(44). Yield 75%; mp 235-237 oC; IR (KBr, cm-1): 3307, 3091, 2941, 1662, 1606, 1542, 1510,
1405, 1381, 1317, 1264, 1169, 1047, 1029, 828; 1H NMR (400 MHz, DMSO-d6): δ 4.51 (t, J
= 5.1 Hz, 2H, NCH2), 4.82 (t, J = 5.1 Hz, 2H, OCH2), 7.04 (d, J = 8.7 Hz, 2H, ArH), 7.30-
7.33 (m, 1H, ArH), 7.41-7.45 (m, 2H, ArH), 7.67 (d, J = 8.7 Hz, 2H, ArH), 7.78 (d, J = 5.8
Hz, 2H, ArH), 7.84 (d, J = 7.3 Hz, 2H, ArH), 8.37 (s, 1H), 8.65 (s, 1H, N=CH), 8.75 (d, J =
13
5.8 Hz, 2H, ArH), 11.93 (s, 1H, CONH); C NMR (100 MHz, DMSO-d6): δ 49.29, 66.33,
115.10, 121.61, 122.05, 125.25, 127.16, 127.99, 129.02, 130.76, 140.66, 146.49, 148.85,
150.39, 159.75, 161.58; ESI-MS (m/z) calculated for C23H20N6O2: 412.16, found: 413.22 (M
+ H)+; Anal. calcd. for C23H20N6O2: C, 66.98; H, 4.89; N, 20.38; O, 7.76, found: C, 67.12; H,
4.98; N, 20.44; O, 7.89.
6.7.7. (E)-N'-(4-(2-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzylidene)isonicotino
o
hydrazide (45). Yield 80%; mp 199-201 C; IR (KBr, cm-1): 3278, 3044, 2936, 1665, 1607,
1
1543, 1511, 1497, 1287, 1247, 1172, 1054, 970, 822, ; H NMR (400 MHz, DMSO-d6): δ
4.46 (t, J = 5.1 Hz, 2H, NCH2), 4.80 (t, J = 5.1 Hz, 2H, OCH2), 5.18 (s, 2H, CH2OPh), 6.93
(t, J = 7.3 Hz, 1H, ArH), 6.99-7.03 (m, 4H, ArH), 7.28 (t, J = 8.0 Hz, 2H, ArH), 7.67 (d, J =
8.7 Hz, 2H, ArH), 7.79-7.81 (m, 2H, ArH) 8.31 (s, 1H), 8.39 (s, 1H, N=CH), 8.76-8.77 (m,
13
2H, ArH), 11.97 (s, 1H, CONH); C NMR (100 MHz, DMSO-d6): δ 49.06, 60.92, 66.32,
114.67, 115.0, 120.84, 121.56, 125.17, 127.13, 128.96, 129.53, 140.60, 142.83, 148.76,
150.33, 158.04, 159.62, 161.50; ESI-HRMS (m/z) calculated for C24H22N6O3: 442.1753,
20

ACCEPTED MANUSCRIPT
found: 443.2369 (M + H)+, 465.1719 (M + Na)+; Anal. calcd. for C24H22N6O3: C, 65.15; H,
5.01; N, 18.99; O, 10.85, found: C, 65.23; H, 5.17; N, 19.11; O, 10.99.
6.7.8.
(E)-N'-(4-(2-(4-(p-Tolyloxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzylidene)
o
isonicotino hydrazide (46). Yield 75%; mp 174-176 C; IR (KBr, cm-1): 3222, 3050, 2963,
1
1654, 1608, 1575, 1552, 1509, 1456, 1383, 1301, 1253, 1230, 1178, 1051, 842, 813; H
NMR (400 MHz, DMSO-d6): δ 2.21 (s, 3H, CH3), 4.46 (t, J = 5.1 Hz, 2H, NCH2), 4.79 (t, J =
5.1 Hz, 2H, OCH2), 5.08 (s, 2H, CH2OPh), 6.90 (d, J = 8.7 Hz, 2H, ArH), 7.0 (d, J = 8.7 Hz,
2H, ArH), 7.07 (d, J = 8.7 Hz, 2H, ArH), 7.67 (d, J = 8.7 Hz, 2H, ArH), 7.80-7.81 (m, 2H,
ArH), 8.29 (s, 1H), 8.39 (s, 1H, N=CH), 8.77 (brs, 2H, ArH), 11.96 (s, 1H, CONH); 13C
NMR (100 MHz, DMSO-d6): 20.08, 49.03, 61.01, 66.32, 114.54, 115.01, 121.52, 125.06,
127.13, 128.94, 129.50, 129.84, 140.60, 142.95, 148.75, 150.32, 155.92, 159.62, 161.47;
ESI-HRMS (m/z) calculated for C25H24N6O3: 456.1910, found: 457.3754 (M + H)+, 479.3582
(M + Na)+; Anal. calcd. for C25H24N6O3: C, 65.78; H, 5.30; N, 18.41; O, 10.51, found C,
65.86; H, 5.41; N, 18.53; O, 10.41.
6.7.9. (E)-N'-(4-(2-(4-((4-Chlorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)benzylidene)
o
isonicotinohydrazide (47). Yield 82%; mp 190-192 C; IR (KBr, cm-1): 3301, 3127, 3072,
2932, 1666, 1608, 1542, 1510, 1489, 1285, 1249, 1208, 1050, 911, 839; 1H NMR (400 MHz,
DMSO-d6): δ 4.46 (t, J = 5.1 Hz, 2H, NCH2), 4.80 (t, J = 5.1 Hz, 2H, OCH2), 5.14 (s, 2H,
CH2OPh), 7.0 (d, J = 8.7 Hz, 2H, ArH), 7.05 (dd, J = 2.2, 6.5 Hz, 2H, ArH), 7.31 (dd, J = 2.2,
6.5 Hz, 2H, ArH), 7.67 (d, J = 8.7 Hz, 2H, ArH), 7.80 (d, J = 5.8 Hz, 2H, ArH), 8.31 (s, 1H),
13
8.38 (s, 1H, N=CH), 8.76 (d, J = 5.8 Hz, 2H, ArH), 11.96 (s, 1H, CONH); C NMR (100
MHz, DMSO-d6): δ 49.09, 61.31, 66.31, 114.97, 116.48, 121.56, 124.61, 125.32, 127.14,
128.96, 129.25, 140.59, 142.49, 148.76, 150.32, 156.84, 159.60, 161.50; ESI-HRMS (m/z)
calculated for C24H21ClN6O3: 476.1364, found: 477.1584 (M + H)+, 499.1008 (M + Na)+;
Anal. calcd. for C24H21ClN6O3: C, 60.44; H, 4.44; Cl, 7.43; N, 17.62; O, 10.06, found: C,
60.56; H, 4.51; Cl, 7.59; N, 17.82; O, 10.23.
6.7.10.
(E)-N'-(4-(3-(4-Propyl-1H-1,2,3-triazol-1yl)propoxy)benzylidene)isonicotino
o
hydrazide (48). Yield 85%; mp 188-190 C; IR (KBr, cm-1): 3222, 3046, 2956, 2933, 1675,
1609, 1560, 1511, 1286, 1258, 1174, 1144, 1048, 947, 825; 1H NMR (400 MHz, DMSO-d6):
δ 0.88 (t, J = 7.3 Hz, 3H, CH2CH2CH3), 1.57 (sextet, J = 7.3 Hz, 2H, CH2CH2CH3), 2.26
(quintet, J = 6.5 Hz, 2H, OCH2CH2CH2N), 2.56 (t, J = 7.3 Hz, 2H, CH2CH2CH3), 4.01 (t, J =
5.8 Hz, 2H, OCH2), 4.48 (t, J = 6.5 Hz, 2H, NCH2), 7.00 (d, J = 8.7 Hz, 2H, ArH), 7.68 (d, J
21

ACCEPTED MANUSCRIPT
= 8.7 Hz, 2H, ArH), 7.80-7.81 (m, 2H, ArH), 7.88 (s, 1H), 8.39 (s, 1H, N=CH), 8.76-8.77 (m,
13
2H, ArH), 11.95 (s, 1H, CONH); C NMR (100 MHz, DMSO-d6): δ 13.61, 22.26, 27.08,
29.44, 46.24, 64.69, 114.86, 121.51, 121.93, 126.70, 128.91, 140.59, 146.75, 148.82, 150.31,
160.16, 161.39; ESI-HRMS (m/z) calculated for C21H24N6O2: 392.1961, found: 393.4258 (M
+ H)+, 415.3584 (M + Na)+; Anal. calcd. for C21H24N6O2: C, 64.27; H, 6.16; N, 21.41; O,
8.15, found: C, 64.39; H, 6.23; N, 21.62; O, 8.28.
6.7.11. (E)-N'-(4-(3-(4-Butyl-1H-1,2,3-triazol-1-yl)propoxy)benzylidene)isonicotinohydrazide
(49). Yield 82%; mp 191-192 oC; IR (KBr, cm-1): 3294, 3129, 2932, 1663, 1609, 1535, 1511,
1306, 1278, 1255, 1172, 1055, 962, 841, 817; 1H NMR (400 MHz, DMSO-d6): δ 0.87 (t, J =
7.3 Hz, 3H, CH2CH2CH2CH3), 1.29 (sextet, J = 7.3 Hz, 2H, CH2CH2CH2CH3), 1.54 (quintet,
J = 7.3 Hz, 2H, CH2CH2CH2CH3), 2.27 (quintet, J = 6.5 Hz, 2H, OCH2CH2CH2N), 2.58 (t, J
= 7.3 Hz, 2H, CH2CH2CH2CH3), 4.01 (t, J = 6.5 Hz, 2H, OCH2), 4.47 (t, J = 6.5 Hz, 2H,
NCH2), 7.00 (d, J = 8.7 Hz, 2H, ArH), 7.68 (d, J = 8.7 Hz, 2H, ArH), 7.79-7.81 (m, 2H,
ArH), 7.87 (s, 1H), 8.39 (s, 1H, N=CH), 8.76-8.77 (m, 2H, ArH), 11.93 (s, 1H, CONH); 13C
NMR (100 MHz, DMSO-d6): δ 13.71, 21.68, 24.71, 29.46, 31.13, 46.26, 64.69, 114.86,
121.53, 121.89, 126.73, 128.93, 140.60, 146.92, 148.84, 150.32, 160.18, 161.41; ESI-HRMS
(m/z) calculated for C22H26N6O2: 406.2117, found: 407.1853 (M + H)+, 429.0985 (M + Na)+;
Anal. calcd. for C22H26N6O2: C, 65.01; H, 6.45; N, 20.68; O, 7.87, found: C, 65.14; H, 6.61;
N, 20.75; O, 7.90.
6.7.12.
(E)-N'-(4-(3-(4-(1-Hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)propoxy)benzylidene)
o
isonicotinohydrazide (50). Yield 62%; mp 185-187 C; IR (KBr, cm-1): 3449, 3199, 3199,
1
2930, 2854, 1669, 1602, 1516, 1302, 1257, 1237, 1172, 1065, 1049, 1002, 833; H NMR
(400 MHz, DMSO-d6): δ 1.23-1.26 (m, 1H), 1.38 (brs, 2H), 1.46 (brs, 1H), 1.62-1.68 (m,
4H), 1.82-1.87 (m, 2H), 2.26-2.29 (m, 2H), 3.97-4.03 (m, 2H), 4.47-4.51 (m, 2H), 4.84 (brs,
1H, OH), 7.0 (d, J = 8.0 Hz, 2H, ArH), 7.68 (d, J = 8.0 Hz, 2H, ArH), 7.80 (d, J = 5.1 Hz,
2H, ArH), 7.92 (s, 1H), 8.39 (s, 1H, N=CH), 8.76 (brs, 2H, ArH), 11.95 (s, 1H, CONH); 13C
NMR (100 MHz, DMSO-d6): δ 21.74, 25.29, 29.50, 37.85, 46.32, 64.78, 67.99, 114.90,
121.18, 121.55, 126.74, 128.97, 140.63, 148.89, 150.34, 155.74, 160.21, 161.46; ESI-MS
(m/z) calculated for C24H28N6O3: 448.22, found: 449.27 (M + H)+; Anal. calcd. for
C24H28N6O3: C, 64.27; H, 6.29; N, 18.74; O, 10.70, found: C, 64.35; H, 6.31; N, 18.91; O,
10.66.
22

ACCEPTED MANUSCRIPT
6.7.13.
(E)-N'-(4-(3-(4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl)propoxy)benzylidene)
o
isonicotinohydrazide (51). Yield 58%; mp 225-228 C; IR (KBr, cm-1): 3265, 3144, 3083,
1
2953, 2914, 1666, 1605, 1557, 1516, 1407, 1367, 1287, 1258, 1175, 1007, 956, 827; H
NMR (400 MHz, DMSO-d6): δ 2.28 (quintet, J = 5.8 Hz, 2H, OCH2CH2CH2N), 4.01 (t, J =
5.8 Hz, 2H, OCH2), 4.49-4.53 (m, 4H) 5.17 (brs, 1H, OH), 7.0 (d, J = 8.7 Hz, 2H, ArH), 7.67
(d, J = 8.7 Hz, 2H, ArH), 7.80 (d, J = 5.8 Hz, 2H, ArH), 8.01 (s, 1H), 8.39 (s, 1H, N=CH),
8.76 (brs, 2H, ArH), 11.94 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6): δ 29.47, 46.32,
55.06, 64.68, 114.88, 121.50, 122.82, 126.73, 128.92, 140.61, 148.03, 148.86, 150.31,
160.17, 161.40; ESI-HRMS (m/z) calculated for C19H20N6O3: 380.1597, found: 381.5581 (M
+ H)+, 403.5226 (M + Na)+; Anal. calcd. for C19H20N6O3: C, 59.99; H, 5.30; N, 22.09; O,
12.62, found: C, 60.15; H, 5.51; N, 22.24; O, 12.77.
6.7.14. (E)-(1-(3-(4-((2-Isonicotinoylhydrazono)methyl)phenoxy)propyl)-1H-1,2,3-triazol-4-
o
yl)methylpropionate (52). Yield 66%; mp 162-163 C; IR (KBr, cm-1): 3286, 3139, 2938,
1735, 1665, 1604, 1542, 1512, 1349, 1289, 1255, 1171, 1054, 951, 836; 1H NMR (400 MHz,
DMSO-d6): δ 1.00 (t, J = 7.6 Hz, 3H, COCH2CH3), 2.28-2.33 (m, 4H), 4.03 (t, J = 5.8 Hz,
2H, OCH2), 4.54 (t, J = 6.5 Hz, 2H, NCH2), 5.11 (s, 2H, CH2OCO), 6.99 (d, J = 8.7 Hz, 2H,
ArH), 7.68 (d, J = 8.7 Hz, 2H, ArH), 7.79-7.81 (m, 2H, ArH), 8.19 (s, 1H), 8.39 (s, 1H,
N=CH), 8.76-8.77 (m, 2H, ArH), 11.94 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6): δ
8.89, 26.68, 29.40, 46.63, 57.10, 64.73, 114.83, 121.52, 124.83, 126.74, 128.91, 140.59,
142.02, 148.86, 150.30, 160.16, 161.43, 173.39; ESI-HRMS (m/z) calculated for C22H24N6O4:
436.1859, found: 437.3393 (M + H)+, 459.2805 (M + Na)+; Anal. calcd. for C22H24N6O4: C,
60.54; H, 5.54; N, 19.25; O, 14.66, found: C, 60.71; H, 5.66; N, 19.19; O, 14.73.
6.7.15.
(E)-N'-(4-(3-(4-(Phenoxymethyl)-1H-1,2,3-triazol-1-yl)propoxy)benzylidene)
o
isonicotino hydrazide (53). Yield 80%; mp 186-188 C; IR (KBr, cm-1): 3286, 3095, 2962,
2873, 1661, 1611, 1541, 1510, 1375, 1264, 1284, 1171, 1037, 841, 822; 1H NMR (400 MHz,
DMSO-d6): δ 2.27 (quintet, J = 6.5 Hz, 2H, OCH2CH2CH2N), 4.0 (t, J = 5.8 Hz, 2H, OCH2),
4.52 (t, J = 6.5 Hz, 2H, NCH2), 5.09 (s, 2H, CH2OPh), 6.90 (t, J = 7.3 Hz, 1H, ArH), 6.97 (t,
J = 8.7 Hz, 4H, ArH), 7.23-7.27 (m, 2H, ArH), 7.65 (d, J = 8.7 Hz, 2H, ArH), 7.78 (d, J = 5.8
Hz, 2H, ArH), 8.24 (s, 1H), 8.36 (s, 1H, N=CH), 8.73-8.74 (m, 2H, ArH), 11.92 (s, 1H,
CONH); 13C NMR (100 MHz, DMSO-d6): δ 29.38, 46.59, 60.99, 64.72, 114.64, 114.85,
120.80, 121.48, 124.61, 126.71, 128.89, 129.48, 140.59, 142.75, 148.83, 150.29, 158.02,
23

ACCEPTED MANUSCRIPT
160.13, 161.38; Anal. calcd. for C25H24N6O3: C, 65.78; H, 5.30; N, 18.41; O, 10.51, found: C,
65.66; H, 5.55; N, 18.33; O, 10.47.
6.7.16.
(E)-N'-(4-(3-(4-(p-Tolyloxymethyl)-1H-1,2,3-triazol-1-yl)propoxy)benzylidene)
o
isonicotinohydrazide (54). Yield 72%; mp 199-200 C; IR (KBr, cm-1): 3291, 3128, 3095,
2926, 2874, 1662, 1611, 1541, 1509, 1265, 1234, 1170, 1021, 841, 822; 1H NMR (400 MHz,
DMSO-d6): δ 2.21 (s, 3H, CH3), 2.30 (quintet, J = 5.8 Hz, 2H, OCH2CH2CH2N), 4.02 (t, J =
5.8 Hz, 2H, OCH2), 4.55 (t, J = 5.8 Hz, 2H, NCH2), 5.07 (s, 2H, CH2OPh), 6.90 (d, J = 8.7
Hz, 2H, ArH), 6.99 (d, J = 8.7 Hz, 2H, ArH), 7.07 (d, J = 8.0 Hz, 2H, ArH), 7.67 (d, J = 8.7
Hz, 2H, ArH), 7.80 (d, J = 5.1 Hz, 2H, ArH), 8.26 (s, 1H), 8.39 (s, 1H, N=CH), 8.76 (brs, 2H,
ArH), 11.95 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6): δ 20.08, 29.40, 46.61, 61.07,
64.72, 114.51, 114.87, 121.53, 124.58, 126.72, 128.92, 129.48, 129.83, 140.65, 142.90,
148.84, 150.32, 155.91, 160.16, 161.41; ESI-HRMS (m/z) calculated for C26H26N6O3:
470.2066, found: 471.2482 (M + H)+, 493.1452 (M + Na)+; Anal. calcd. for C26H26N6O3: C,
66.37; H, 5.57; N, 17.86; O, 10.20, found: C, 66.48; H, 5.49; N, 18.01; O, 10.32.
6.7.17.
(E)-N'-(4-(3-(4-((4-Chlorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy)
o
benzylidene)isonicotinohydrazide (55). Yield 82%; mp 182-184 C; IR (KBr, cm-1): 3291,
1
3095, 2928, 2874, 1661, 1611, 1540, 1510, 1492, 1283, 1264, 1236, 1172, 1018, 825; H
NMR (400 MHz, DMSO-d6): δ 2.30 (quintet, J = 6.5 Hz, 2H, OCH2CH2CH2N), 4.02 (t, J =
5.8 Hz, 2H, OCH2), 4.55 (t, J = 6.5 Hz, 2H, NCH2), 5.13 (s, 2H, CH2OPh), 6.98 (d, J = 8.7
Hz, 2H, ArH), 7.05 (d, J = 8.7 Hz, 2H, ArH), 7.31 (d, J = 8.7 Hz, 2H, ArH), 7.67 (d, J = 8.7
Hz, 2H, ArH), 7.81 (d, J = 5.8 Hz, 2H, ArH), 8.28 (s, 1H), 8.39 (s, 1H, N=CH), 8.77 (d, J =
13
5.8 Hz, 2H, ArH), 11.96 (s, 1H, CONH); C NMR (100 MHz, DMSO-d6): δ 29.37, 46.61,
61.39, 64.70, 114.83, 116.48, 121.48, 124.53, 124.74, 126.71, 128.88, 129.22, 140.58, 142.39,
148.81, 150.28, 156.84, 160.12, 161.37; ESI-HRMS (m/z) calculated for C25H23ClN6O3:
490.1520, found: 491.3194 (M + H)+, 513.2664 (M + Na)+; Anal. calcd. for C25H23ClN6O3:
C, 61.16; H, 4.72; Cl, 7.22; N, 17.12; O, 9.78, found: C, 61.28; H, 4.66; Cl, 7.07; N, 17.27; O,
9.89.
6.7.18. (E)-N'-(4-((1-Propyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)isonicotinohydrazide
(56). Yield 68%; mp 195-197 oC; IR (KBr, cm-1): 3258, 3157, 2935, 1656, 1607, 1542, 1510,
1
1289, 1253, 1172, 1053, 1007, 844; H NMR (400 MHz, DMSO-d6): δ 0.82 (t, J = 7.3 Hz,
3H, CH2CH2CH3), 1.81 (sextet, J = 7.3 Hz, 2H, CH2CH2CH3), 4.31 (t, J = 7.3 Hz, 2H,
CH2CH2CH3), 5.19 (s, 2H, OCH2), 7.12 (d, J = 8.7 Hz, 2H, ArH), 7.68 (d, J = 8.7 Hz, 2H,
24

ACCEPTED MANUSCRIPT
ArH), 7.79 (d, J = 8.7 Hz, 2H, ArH), 8.23 (s, 1H, ArH), 8.39 (s, 1H, N=CH), 8.76 (d, J = 5.8
Hz, 2H, ArH), 11.93 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6): δ 10.78, 23.14, 50.96,
61.28, 115.13, 121.49, 124.51, 126.85, 128.89, 140.58, 142.29, 148.84, 150.29, 159.84,
161.39; Anal. calcd. for C19H20N6O2: C, 62.62; H, 5.53; N, 23.06; O, 8.78, found: C, 62.77;
H, 5.46; N, 23.20; O, 8.84.
6.7.19.
(E)-N'-(4-((1-Iso-propyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)isonicotino
o
hydrazide (57). Yield 74%; mp 186-187 C; IR (KBr, cm-1): 3151, 2982, 2852, 1664, 1598,
1513, 1308, 1240, 1176, 1061, 1031, 1005, 824; 1H NMR (400 MHz, DMSO-d6): δ 1.48 (d, J
= 6.5 Hz, 6H, CH(CH3)2), 4.81 (septet, J = 6.5 Hz, 1H, CH(CH3)2), 5.18 (s, 2H, OCH2), 7.13
(d, J = 8.7 Hz, 2H, ArH), 7.69 (d, 2H, J = 8.7 Hz, ArH), 7.80 (d, J = 5.8 Hz, 2H, ArH), 8.31
(s, 1H, ArH), 8.40 (s, 1H, N=CH), 8.76 (d, J = 5.8 Hz, 2H, ArH), 11.94 (s, 1H, CONH); ESI-
MS (m/z) calculated for C19H20N6O2: 364.16, found: 365.21 (M + H)+; Anal. calcd. for
C19H20N6O2: C, 62.62; H, 5.53; N, 23.06; O, 8.78, found: C, 62.86; H, 5.66; N, 23.19; O,
8.69.
6.7.20. (E)-N'-(4-((1-Butyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)isonicotinohydrazide
(58). Yield 65%; mp 205-207 oC; IR (KBr, cm-1): 3250, 3159, 3047, 2960, 2935, 2873, 1661,
1
1607, 1544, 1511, 1467, 1288, 1252, 1174, 1005, 844; H NMR (400 MHz, DMSO-d6): δ
0.87 (t, J = 7.3 Hz, 3H, CH2CH2CH2CH3), 1.23 (sextet, J = 7.3 Hz, 2H, CH2CH2CH2CH3),
1.79 (quintet, J = 7.3 Hz, 2H, CH2CH2CH2CH3), 4.35 (t, J = 7.3 Hz, 2H, CH2CH2CH2CH3),
5.19 (s, 2H, OCH2), 7.13 (d, J = 8.7 Hz, 2H, ArH), 7.69 (d, J = 8.7 Hz, 2H, ArH), 7.79-7.81
(m, 2H, ArH), 8.24 (s, 1H), 8.40 (s, 1H, N=CH), 8.76-8.77 (m, 2H, ArH), 11.94 (s, 1H,
CONH); 13C NMR (100 MHz, DMSO-d6): δ 13.28, 19.07, 31.71, 49.10, 61.28, 115.14,
121.51, 124.52, 126.86, 128.90, 140.58, 142.30, 148.84, 150.31, 159.85, 161.40; ESI-HRMS
(m/z) calculated for C20H22N6O2: 378.1804, found: 379.1872 (M + H)+, 401.1347 (M + Na)+;
Anal. calcd. for C20H22N6O2: C, 63.48; H, 5.86; N, 22.21; O, 8.46, found: C, 63.63; H, 5.91;
N, 22.34; O, 8.55.
6.7.21. (E)-N'-(4-((1-Pentyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)isonicotinohydrazide
(59). Yield 64%; mp 208-210 oC; IR (KBr, cm-1): 3248, 3157, 2958, 1660, 1607, 1542, 1511,
1
1290, 1254, 1174, 1054, 1006, 845; H NMR (400 MHz, DMSO-d6): δ 0.83 (t, J = 7.3 Hz,
3H, CH2CH2CH2CH2CH3), 1.16-1.21 (m, 2H), 1.23-1.31 (m, 2H), 1.79 (quintet, J = 7.3 Hz,
2H, CH2CH2CH2CH2CH3), 4.34 (t, J = 7.3 Hz, 2H, CH2CH2CH2CH2CH3), 5.19 (s, 2H,
OCH2), 7.12 (d, J = 8.7 Hz, 2H, ArH), 7.68 (d, J = 8.7 Hz, 2H, ArH), 7.79-7.81 (m, 2H,
25

ACCEPTED MANUSCRIPT
ArH), 8.24 (s, 1H, ArH), 8.39 (s, 1H, N=CH), 8.75-8.77 (m, 2H, ArH), 11.94 (s, 1H, CONH);
13C NMR (100 MHz, DMSO-d6): δ 13.77, 21.50, 28.00, 29.39, 49.36, 61.28, 115.14, 121.50,
124.51, 126.86, 128.89, 140.58, 142.29, 148.83, 150.31, 159.84, 161.39; ESI-MS (m/z)
calculated for C21H24N6O2: 392.19, found: 393.25 (M + H)+; Anal. calcd. for C21H24N6O2: C,
64.27; H, 6.16; N, 21.41; O, 8.15, found: C, 64.35; H, 6.32; N, 21.65; O, 8.28.
6.7.22. (E)-N'-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)isonicotinohydrazide
(60). Yield 84%; mp 205-208 oC; IR (KBr, cm-1): 3270, 3032, 2958, 1655, 1607, 1547, 1513,
1
1454, 1408, 1290, 1252, 1175, 1051, 812; H NMR (400 MHz, DMSO-d6): δ 5.19 (s, 2H,
OCH2), 5.60 (s, 2H, NCH2Ph), 7.11 (d, J = 8.7 Hz, 2H, ArH), 7.29-7.33 (m, 3H, ArH), 7.34-
7.38 (m, 2H, ArH), 8.79-8.80 (m, 2H, ArH), 7.68 (d, J = 8.7 Hz, 2H, ArH), 8.29 (s, 1H,
13
ArH), 8.39 (s, 1H, N=CH), 8.75-8.77 (m, 2H, ArH), 11.94 (s, 1H, CONH); C NMR (100
MHz, DMSO-d6): δ 52.88, 61.22, 115.16, 121.53, 124.85, 126.89, 127.99, 128.20, 128.81,
128.92, 136.0, 140.60, 142.73, 148.86, 150.32, 159.84, 161.43; ESI-HRMS (m/z) calculated
for C23H20N6O2: 412.1648, found: 413.4110 (M + H)+, 435.3596 (M + Na)+; Anal. calcd. for
C23H20N6O2: C, 66.98; H, 4.89; N, 20.38; O, 7.76, found: C, 66.83; H, 4.81; N, 20.47; O,
7.88.
Acknowledgements
D.S.R. thanks Council of Scientific and Industrial Research (CSIR) [No. 02(0049)/12/EMR-
II] New Delhi, India for financial support. A.K.T. is thankful to the Department of
Biotechnology, Ministry of Science and Technology, Government of India for financial
support. D.K. and Beena are thankful to CSIR for the award of junior and senior research
fellowship. Authors are also thankful to CIF-USIC, University of Delhi, Delhi for NMR
spectral data and RSIC, CDRI, Lucknow for mass data.
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