Design, synthesis and 3D-QSAR studies of new diphenylamine containing 1,2,4-triazoles as potential antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2014.07.051

A new series of new diphenylamine containing 1,2,4-triazoles were synthesized from 4-arylideneamino-5-[2-(2,6-dichlorophenylamino) benzyl]-2H-1,2,4-triazole-3(4H)-thiones 3a-f. The synthesized compounds were screened for in-vitro antimycobacterial and antibacterial activities. The synthesized compounds 4a, 4e and 4d have shown potential activity against Mycobacteriumtuberculosis H₃₇Rv strain with MIC of 0.2, 1.6 and 3.125 μM respectively. To investigate the SAR of diphenylamine containing 1,2,4-triazole derivatives in more details, CoMFA (q²-0.432, r ²-0.902) and CoMSIA (q²-0.511, r²-0.953) models on M. tuberculosis H₃₇Rv were established. The generated 3D-QSAR models are externally validated and have shown significant statistical results, and these models can be used for further rational design of novel diphenylamine containing 1,2,4-triazoles as potent antitubercular agents.

Full text of DOI:10.1016/j.ejmech.2014.07.051

European Journal of Medicinal Chemistry 84 (2014) 516e529
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and 3D-QSAR studies of new diphenylamine
containing 1,2,4-triazoles as potential antitubercular agents
,
*
K. Mohan Krishna a, Bharathkumar Inturi a, Gurubasavaraj V. Pujar a
,
Madhusudan N. Purohit a, G.S. Vijaykumar b
a Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS University, Mysore 570015, Karnataka, India
b Department of Microbiology, JSS Medical College, JSS University, Mysore 570015, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of new diphenylamine containing 1,2,4-triazoles were synthesized from 4-arylideneamino-
5-[2-(2,6-dichlorophenylamino) benzyl]-2H-1,2,4-triazole-3(4H)-thiones 3aef. The synthesized com-
pounds were screened for in-vitro antimycobacterial and antibacterial activities. The synthesized com-
pounds 4a, 4e and 4d have shown potential activity against Mycobacterium tuberculosis H37Rv strain with
Received 5 September 2013
Received in revised form
15 July 2014
Accepted 15 July 2014
Available online 16 July 2014
MIC of 0.2, 1.6 and 3.125 mM respectively. To investigate the SAR of diphenylamine containing 1,2,4-
triazole derivatives in more details, CoMFA (q2-0.432, r2-0.902) and CoMSIA (q2-0.511, r2-0.953)
models on M. tuberculosis H37Rv were established. The generated 3D-QSAR models are externally vali-
dated and have shown significant statistical results, and these models can be used for further rational
design of novel diphenylamine containing 1,2,4-triazoles as potent antitubercular agents.
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
1,2,4-Triazoles
Mycobacterium tuberculosis
MABA assay
3D-QSAR
CoMFA
CoMSIA
1. Introduction
global medical need [3]. The long treatment regimen, which is
necessary due to the presence of a non-replicating persistent
Tuberculosis is the leading cause of mortality from a single in-
fectious agent and is responsible for more than three million deaths
worldwide every year [1]. Despite half a century of antitubercular
chemotherapy, there are still 8e10 million new cases of active
tuberculosis each year and nearly two billion individuals are
believed to harbor latent tuberculosis. In 2011, there were an esti-
mated 8.7 million new cases of TB (13% co-infected with HIV) and
1.4 million people died from TB [2]. The synergy between tuber-
culosis and the AIDS epidemic as well as the surge of multidrug-
resistant isolates of Mycobacterium tuberculosis has reaffirmed
tuberculosis as a primary public health threat. The current threat in
TB treatment lies in the emergence of strains resistant to two of the
best antitubercular drugs, isoniazid (INH) and rifampicin (RIF).
In the past decade there has been substantial progress in the
development of new antitubercular drugs. The typical challenges of
drug development are augmented in Tuberculosis by the
complexity of disease, the requirement for multi-drug regimens,
the relative lack of Tuberculosis drug development for the past
several decades and inadequate resources despite the urgency of
tuberculosis phenotype (NRP-TB), results in poor patient compli-
ance, causes undesired side effects, and makes a significant
contribution to the emergence of drug resistant Tuberculosis
strains [4e6]. In the new drug development for Tuberculosis, it is
important that new drugs inhibit targets different from those of
currently used drugs. In addition, development of new agents that
can shorten the lengthy TB therapy requires the design of drugs
that can inhibit targets involved in persistence or dormancy.
The M. tuberculosis enoyl acyl carrier protein (ACP) reductase
(InhA) is an excellent target for antitubercular drug Isoniazid (INH)
and ethionamide (ETH). Enoyl CoA reductase of M. tuberculosis
catalyzes the final step in the fatty acid elongation pathway and is a
target for the activated form of isoniazid (INH), a front-line anti-TB
drug. Importantly, the majority of INH-resistant isolates result from
mutations in KatG, the enzyme that activates INH, rather than from
mutations in the ultimate drug target(s). Consequently, it is hy-
pothesized that inhibitors of InhA, which circumvent the need for
KatG activation, will form an effective basis for tackling MDR-TB.
Triclosan, a diphenyl ether derivative inhibits InhA without re-
quires activation of KatG with a Ki value of 1 nM and which has an
MIC99 for MTB of 2e3 mg/mL [7]. Diclofenac sodium and its acid
hydrazides have been reported for their antitubercular potential
* Corresponding author.
[8,9]. LL-3858 from Lupin laboratories has demonstrated significant
E-mail addresses: gbrpujar@gmail.com, gvpujar@jssuni.edu.in (G.V. Pujar).
http://dx.doi.org/10.1016/j.ejmech.2014.07.051
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
517
in-vitro and in-vivo activity against sensitive and resistant strains of
M. tuberculosis and is currently in Phase I clinical trials [10]. Het-
erocyclic derivatives like oxazolidinones, nitroimidazoles [11],
quinolones [12] and pyrroles [13] were found to possess significant
antitubercular activities and are in various stages of clinical trials.
Mannich base derivatives [14], thiazolidinones [15] and triazoles
fused with 6-membered rings [16e19] were reported to possess
significant antitubercular activity. From the above observations and
in continuation of our studies on novel 1,2,4-triazoles [20], it was
felt worthwhile to synthesize new diphenylamine containing 1,2,4-
triazole derivatives which mimic the structural features of LL3858
and Triclosan (Fig. 1) and evaluate them for antitubercular and
antibacterial potential.
Quantitative structureeactivity relationship (QSAR) techniques
are used to correlate physicochemical descriptors from a set of
related compounds to their known molecular activity or molecular
property values. 3D-QSAR models are useful methods of ligand-
based drug design when the molecular targets for the compounds
being investigated are either unknown or have not been structur-
ally resolved. The descriptors used to develop QSAR models can
range from molecular descriptors for lipophilicity (cLogP), steric
bulk (molar refractivity, volume), and electrostatics (polar surface
area, Coulombic charges, and dipole moments) to 3-dimensional
descriptors that involve alignment of the compounds, and calcu-
lating steric and electrostatic values using charged probe atoms at
grid lattice points (Comparative Molecular Field Analysis, CoMFA)
or 3D-Comparative Molecular Similarity Indices Analysis (CoMSIA)
[21]. Several 3D-QSAR models were developed during the study to
understand the relationship between structure and activity. De-
scriptors including cLogP, molar refractivity (CMR), polar surface
area (PSA), CoMFA and CoMSIA variables were investigated for their
ability to predict and correctly rank whole cell MIC activity using
the Partial Least Squares (PLS) method.
dichlorophenyl) amino] benzyl]-4-amino-5-mercapto (4H)-1,2,4-
triazole (2) was conventionally synthesized by Reid and Heindel
method [22]. This synthetic methodology involves synthesis of
dithiocarbazinate salt by treating diclofenac acid hydrazide [20] (1)
with carbon disulfide and alcoholic potassium hydroxide solution.
This salt was on treatment with hydrazine hydrate to give 1,2,4-
triazole (2). The compound 2 was then condensed with different
aldehydes in presence of glacial acetic acid to yield corresponding
Schiff bases 3aef. The compounds 4ael were synthesized by a
multicomponent Mannich reaction involving Schiff bases (3aef),
formaldehyde and appropriate secondary amine. The reaction
proceeds via the formation of immonium salts which subsequently
attack N2-of triazole gives rise to regioselective Mannich base. This
reaction is highly regioselective and furnishes only N-Mannich base
derivatives. The triazolothiazolidinones 5aef were synthesized
from the corresponding Schiff bases 3aef with thioglycolic acid in
presence of dry benzene. This ring closure involves addition reac-
tion by the loss of imine linkage. The triazoloquinazolinones 6aef
were synthesized from corresponding Schiff bases 3aef and an-
thranilic acid by fusion. The physical characterization data of the
synthesized compounds are given in Table 1. The structures of all
the synthesized compounds were characterized on the basis of
their elemental analysis, IR, 1H NMR, 13C NMR, and LC-MS data.
The IR spectrum of the compound 3a exhibited absorption
bands at 1604 cmꢀ1 for C]N stretching and C]S stretching at
1270 cmꢀ1. The 1H NMR spectrum of the compound 3a exhibited a
singlet at 13.88 ppm corresponding to NH adjacent to C]S of 1,2,4-
triazole nucleus and singlet at 10.00 ppm corresponding to N]CH
of imine linkage indicate the formation of Schiff base. The IR
spectrum of compound 4a exhibited absorption band at 1161 cmꢀ1
corresponding to CeO group and other characteristic absorption
bands were observed at 1276 cmꢀ1 for C]S, at 2951 cmꢀ1 for CHe
and at 3325 cmꢀ1 for NH-group. In the 1H NMR spectrum of com-
pound 4a the aromatic protons resonated as multiplet between 6.9
and 7.9 ppm. The singlet at 10.37 ppm was assigned to N]CH
proton. The absence of NH singlet at 13.88 ppm and presence of
methylene protons at 2.82 ppm and 3.62 ppm corresponds to
CH2NCH2 and CH2OCH2 of morpholine ring confirms the formation
of the compound 4a. The IR spectrum of compound 5a has shown
2. Chemistry
Synthesis of the intermediate and target compounds was
accomplished according to the steps depicted in the scheme of
synthesis (Scheme 1). The starting compound 3-[2-[(20, 60-
Fig. 1. Design strategy for diphenyl amino containing 1,2,4-triazole and their clubbed heterocycles.

518
K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
Scheme 1. Reagent and reaction condition: i) CS2, Alcoholic KOH, reflux, 6 h ii) NH2NH2 H2O 99% reflux, 6 h iii) R-CHO, CH3COOH, reflux, 4e6 h; iv) Morpholine/Piperdine, HCHO,
C2H5OH, reflux, 3e4 h; v) HSCH2COOH, Benzene, reflux, 6e8 h; vi) Anthranilic acid, reflux, 2e4 h.
absorption band at 653 cmꢀ1 corresponds to CeS of thiazolidinone
ring and other characteristic absorption bands were observed at
1294 cmꢀ1 for C]S, at 2931 cmꢀ1 for CHe and 1631 cmꢀ1 for C]N
group. In the 1H NMR spectrum of compound 5a, the singlet at
5.66 ppm was assigned to thiomethylene protons; the singlet at
3.31 ppm was assigned to a proton at C5 of thiazolidinone ring. The
aromatic protons appeared between 6.92 and 7.54 ppm confirms
the formation of compound 5a. The IR spectrum of compound 6a
has shown peak at 1594 cmꢀ1 corresponding to C]O of quinazo-
linone ring and other characteristic absorption bands were ob-
tained at 3317 cmꢀ1 for NH, 3059 cmꢀ1 for CHe and 1530 cmꢀ1 for
C]N group. In the 1H NMR spectra of compound 6a has shown the
absence of NH singlet at 13.88 ppm indicating the formation of
quinazolinone ring.
the standard Microplate Alamar Blue Assay (MABA) and agar
dilution method. The antitubercular potential of synthesized
compounds was tested against M. tuberculosis H37Rv at different
concentrations ranging from 50
results (Table 2) that most of the synthesized compounds displayed
antitubercular activity ranging from 25 to 0.2 against
M. tuberculosis 37Rv strain. Among the compounds tested,
mM to 0.2 mM. It is evident from
mM
H
Mannich bases 4ael have shown more significant antitubercular
activity than the triazolothiazolidinones 5aef and tri-
azoloquinazolinones 6aef. The Mannich bases 4a, 4e and 4d have
shown potential activity at 0.2 mM, 1.6 mM and 3.125 mM respec-
tively. Compound 4a has antitubercular activity comparable to the
standard drug Isoniazid and possess benzylidene amino group at
4th position and morpholino methyl group at 2nd position of 1,2,4-
triazole ring system. The cytotoxicity of the most active compounds
3. Results and discussion
(4a, 4b, 4c, 4d, 4e, 4g, 4j) with and less than 6.25 mM were assessed
by Microculture Tetrazolium Assay (MTT) against Vero (African
Green monkey kidney epithelial cells) and HepG2 (hepatic carci-
noma cells) cell lines. From the results (Table 2), it is evident
that the tested compounds are not cytotoxic. Hence, the antitu-
bercular activity of the synthesized compounds was not due to
cytotoxicity.
3.1. Biological activity
3.1.1. Antitubercular activity
The synthesized compounds were screened for their antitu-
bercular efficacy against M. tuberculosis H37Rv (ATCC-27294) using

K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
519
Table 1
Physical data of synthesized compounds.
Compound code
R
R1
Molecular formula
C22H17Cl2N5S
Mol. wt
Analysis (%) found (calculated)
C
H
N
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
Phenyl
e
454
484
514
444
460
455
553
583
613
543
559
554
551
581
611
541
557
552
528
559
589
518
535
529
539
569
599
529
545
540
58.21 (58.15)
56.98 (57.03)
56.12 (56.03)
53.92 (54.06)
52.22 (52.17)
55.31 (55.39)
58.54 (58.59)
56.97 (57.63)
56.80 (56.77)
55.22 (55.25)
53.69 (53.66)
56.27 (56.32)
60.12 (60.08)
59.97 (59.89)
59.02 (58.92)
57.58 (57.67)
56.14 (56.01)
57.75 (58.69)
54.59 (54.54)
53.74 (53.76)
52.99 (53.06)
50.96 (50.97)
49.48 (49.44)
52.20 (52.17)
64.54 (64.57)
63.19 (63.28)
62.19 (62.11)
61.18 (61.26)
59.48 (59.45)
62.25 (62.23)
3.75 (3.77)
3.92 (3.95)
4.13 (4.11)
3.41 (3.40)
3.26 (3.28)
3.56 (3.54)
4.75 (4.73)
4.86 (4.84)
4.94 (4.93)
4.47 (4.45)
4.30 (4.32)
4.55 (4.54)
5.10 (5.12)
5.18 (5.20)
5.25 (5.27)
4.47 (4.48)
4.68 (4.70)
4.94 (4.93)
3.64 (3.62)
3.78 (3.79)
3.92 (3.94)
3.30 (3.31)
3.23 (3.21)
3.41 (3.43)
3.76 (3.74)
3.90 (3.89)
4.01 (4.04)
3.42 (3.43)
3.35 (3.33)
3.56 (3.54)
15.37 (15.41)
14.43 (14.46)
13.58 (13.61)
15.79 (15.76)
15.20 (15.21)
18.42 (18.46)
15.15 (15.18)
14.37 (14.40)
13.67 (13.70)
15.43 (15.46)
15.06 (15.02)
17.65 (17.68)
15.27 (15.24)
14.41 (14.45)
13.72 (13.74)
15.49 (15.52)
15.11 (15.07)
17.76 (17.75)
13.21 (13.25)
12.58 (12.54)
11.88 (11.90)
13.53 (13.51)
13.07 (13.10)
15.91 (15.87)
15.56 (15.58)
14.73 (14.76)
14.07 (14.02)
15.84 (15.88)
15.39 (15.41)
15.19 (18.14)
4-methoxy phenyl
3,4-dimethoxy phenyl
2-furyl
2-thiphenyl
4-pyridyl
e
C
C
C
C
C
C
C
C
C
23H19Cl2N5OS
24H21Cl2N5O2S
20H15Cl2N5OS
20H15Cl2N5S2
21H16Cl2N6S
27H26Cl2N6OS
28H28Cl2N6O2S
29H30Cl2N6O3S
25H24Cl2N6O2S
e
e
e
e
Phenyl
Morpholino
Morpholino
Morpholino
Morpholino
Morpholino
Morpholino
Piperidino
Piperidino
Piperidino
Piperidino
Piperidino
Piperidino
e
e
e
e
e
e
e
e
e
e
e
e
4-methoxy phenyl
3,4-dimethoxy phenyl
2-furyl
2-thiphenyl
4-pyridyl
C25H24Cl2N6OS2
26H25Cl2N7OS
C28H28Cl2N6S
C
Phenyl
4-methoxy phenyl
3,4-dimethoxy phenyl
2-furyl
2-thiphenyl
4-pyridyl
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
29H30Cl2N6OS
30H32Cl2N6O2S
26H26Cl2N6OS
26H26Cl2N6S2
27H27Cl2N7S
24H19Cl2N5OS2
25H21Cl2N5O2S2
26H23Cl2N5O3S2
22H17Cl2N5O2S2
22H17Cl2N5OS3
23H18Cl2N6OS2
29H20Cl2N6O
30H22Cl2N6O2
31H24Cl2N6O3
27H18Cl2N6O2
27H18Cl2N6 OS
28H19Cl2N7O2
Phenyl
4-methoxy phenyl
3,4-dimethoxy phenyl
2-furyl
2-thiphenyl
4-pyridyl
Phenyl
4-methoxy phenyl
3,4-dimethoxy phenyl
2-furyl
2-thiphenyl
4-pyridyl
Table 2
Minimum inhibitory concentrations (MIC) of in-vitro antitubercular activity and antibacterial activity.
Compound code
Minimum inhibitory concentration (
m
M)
Cytotoxicity (mM)
M. tuberculosis H37Rv
S. aureas
B. substilus
E. coli
P. aeruginosa
Vero
HepG2
MABA method
Agar microdilution method
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
0.2
6.25
6.25
3.125
1.6
12.5
6.25
12.5
12.5
6.25
12.5
12.5
50
50
50
25
25
12.5
12.5
25
25
25
0.2
6.25
6.25
12.5
12.5
6.25
12.5
12.5
12.5
12.5
25
12.5
12.5
12.5
12.5
100
12.5
12.5
25
6.25
12.5
12.5
6.25
6.25
12.5
6.25
25
12.5
6.25
12.5
12.5
12.5
25
12.5
25
25
12.5
25
>300
>300
>300
>300
>300
nd
>300
nd
nd
>300
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
>300
>300
282
>300
250
nd
>300
nd
nd
262
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
6.25
6.25
3.125
3.125
12.5
6.25
12.5
6.25
6.25
12.5
12.5
nd
nd
nd
nd
nd
6.25
12.5
nd
nd
nd
12.5
12.5
12.5
12.5
25
12.5
25
25
12.5
25
12.5
100
100
12.5
12.5
12.5
12.5
25
25
12.5
12.5
25
25
12.5
25
12.5
12.5
100
100
12.5
12.5
50
12.5
100
50
12.5
12.5
100
e
100
100
12.5
12.5
50
12.5
100
25
12.5
100
12.5
12.5
12.5
100
e
25
12.5
12.5
100
e
12.5
12.5
25
12.5
25
<0.2
e
12.5
nd
<0.2
e
nd
nd
nd
nd
nd
nd
nd
nd
INH
Ampicillin
e
<6.25
<6.25
<6.25
<6.25
nd: not determined.
3.1.2. In-vitro antibacterial activity
Pseudomonas aeruginosa (ATCC-27853) by cup plate method.
Ampicillin was used as standard and DMSO was used as solvent
control. The Minimum Inhibitory Concentration (MIC) of the syn-
thesized compounds was determined by twofold serial dilution
The synthesized compounds were evaluated for their in-vitro
antibacterial activity against Bacillus subtilus (ATCC 6633), Staphy-
lococcus aureus (ATCC-25923), Escherichia coli (ATCC-25922), and

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K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
Table 3
technique. The minimum inhibitory concentration data of the
synthesized compounds are given in Table 2. The Mannich bases 4a,
PLS statistical results of CoMFA models.
4d, 4e and 4f have shown potential activity at 6.25 mM against all
Model description
CoMFA CoMFA-ClogP CoMFA-PSA CoMFA-CMR
the bacterial strains. The compounds 5a, 5d and 5e of tri-
azolothiazolidinones and 6a, 6d and 6e have shown potential ac-
q2
r2
SE
N.C
F
0.372
0.584
0.260
1
0.39
0.877
0.260
4
0.432
0.902
0.140
4
0.441
0.778
0.203
3
tivity at 12.5
mM against tested bacterial strains. Rest of the
synthesized compounds has shown moderate to low antibacterial
activity against tested bacterial strains.
22.418 23.073
29.754
16.365
Fraction Steric
0.603
0.397
0.435
0.550
0.015
0.427
0.549
e
0.454
0.397
e
Electrostatic
ClogP
PSA
3.2. Structure activity relationship
e
e
e
e
0.025
e
e
CMR
e
0.149
The initial SAR of compounds 4ael reveals that Mannich bases
with morpholine group have shown significant antitubercular ac-
tivity compared to the piperdine derivatives. Compounds 4b, 4c, 5b,
5c, 6b and 6c with substituted phenyl ring at 4th position of 1,2,4-
triazoles have shown reduced activity when compared to unsub-
stituted derivative 4a. Triazolothiazolidinones (5aef) have shown
weak antitubercular activity when compared to corresponding
Mannich base derivatives, Lack of the imine linkage in 5ae5f
compounds might be accounted for their diminished antituber-
cular activity of triazolothiazolidinones. Compounds 6a and 6e of
the fused triazoloquinazolinones derivatives have shown signifi-
The bold values indicate the best models.
Table 4
PLS statistical results of CoMSIA models.
Model description
CoMSIA CoMSIA-ClogP CoMSIA-PSA CoMSIA-CMR
q2
r2
SE
N.C
F
0.454
0.667
0.239
1
0.419
0.753
0.207
2
0.511
0.953
0.101
5
0.492
0.763
0.202
2
32.005 22.843
48.497
24.142
Fraction Steric
0.030
0.353
0.298
0.102
0.150
0.053
0.377
0.020
0.065
0.326
0.174
0.052
0.302
-
0.100
0.100
0.140
0.042
0.343
e
cant antitubercular activity at 12.5 mM concentration.
Electrostatic
Hydrophobic 0.153
Donor
Acceptor
Clop
0.145
0.318
3.2.1. 3D-QSAR model
All structures of diphenylamine containing 1,2,4-trizole de-
rivatives were built and aligned onto the common substructure N-
(2,6-dichlorophenyl)-2-methylbenzenamine nucleus with the
potent compound 4a as the template. The structure alignment is
illustrated Fig. 2. To investigate the SARs of diphenylamine con-
taining 1,2,4 trizole derivatives, the in-vitro antitubercular activity
data were used to build the 3D-QSAR models. Twenty four (4ael,
5aef and 6aef) compounds were employed for 3D-QSAR study and
their inverse logarithms of Minimum Inhibitory Concentration
(pMIC) values were calculated based on the original MIC data. The
detailed operational procedures were followed as per the literature
and are given in experimental section [23e26]. To develop the 3D-
QSAR models, descriptors like cLogP, molar refractivity (CMR), polar
surface area (PSA) were used along with CoMFA and CoMSIA de-
scriptors. Several 3D-QSAR models were developed during the
study to obtain model with high predictivity. Descriptors like cLogP,
CMR, PSA, and 3D-CoMFA and CoMSIA variables were investigated
for their ability to predict and correctly rank whole cell MIC activity
using the method of PLS (Tables 3 and 4). The cross-validation
correlation coefficient (q2) and correlation coefficient (r2) are
e
PSA
CMR
e
e
e
0.082
-
e
e
0.072
The bold values indicate the best models.
considered as two important parameters to evaluate the qualities of
PLS analysis to indicate the predictive capacity and the self-
consistence. A q2 value over 0.3 is considered as significant for
the chance of significant correlation being <95% [23]. From the data
presented in Tables 3 and 4, it is evident that the addition of polar
surface area (PSA) to CoMFA model resulted in a significant model
with q2 (0.432) and r2 (0.902) values. A similar result was obtained
when polar surface area (PSA) was added to CoMSIA study resulted
in higher q2 and r2 values (0.511 and 0.953 respectively). The
standard errors of estimate of CoMFA and CoMSIA models were also
reasonably low amounting to 0.140 and 0.101 respectively. How-
ever, the additions of cLogP and molor refractivity (CMR) to CoMFA
and CoMSIA models have negligible effect on the internal
validation.
Fig. 2. (A) Structure of the template compound 4a, common substructure is in bold, (B) 3D-QSAR structure alignment and superimposition of 24 compounds using compound 4a as
the template.

K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
521
Table 5
Experimental and predicted activities of the target compounds for CoMFA and CoMSIA models against Mycobacterium tuberculosis H37Rv.
Compound code
CoMFA
CoMSIA
pMIC(experimental)
pMIC(predicted)
Residuals
pMIC(experimental)
pMIC(predicted)
Residuals
Training set
4b
4c
4d
4e
4f
4g
4h
4i
5.204
5.204
5.505
5.796
4.903
5.204
4.903
4.903
5.204
4.903
4.301
4.301
4.602
4.903
4.903
4.602
4.602
4.602
5.181
5.089
5.605
5.497
5.122
5.119
4.956
4.864
5.379
4.897
4.343
4.216
4.641
4.918
4.743
4.723
4.667
4.579
0.023
0.115
5.204
5.204
5.505
5.795
4.903
5.204
4.903
4.903
5.204
4.903
4.301
4.301
4.602
4.903
4.903
4.602
4.602
4.602
5.269
5.208
5.483
5.614
5.029
5.26
4.983
4.922
5.197
4.743
4.270
4.282
4.588
4.980
4.842
4.732
4.533
4.600
0.065
0.004
ꢀ0.022
ꢀ0.181
0.126
0.059
0.080
0.019
ꢀ0.007
ꢀ0.159
ꢀ0.030
ꢀ0.018
ꢀ0.013
0.077
ꢀ0.1
0.299
ꢀ0.219
0.085
ꢀ0.053
0.039
4j
4l
ꢀ0.175
0.006
5a
5b
5e
5f
6a
6b
6d
6f
Test set
4a
4k
5c
5d
6c
ꢀ0.042
0.085
ꢀ0.039
ꢀ0.015
0.16
ꢀ0.121
ꢀ0.065
0.023
ꢀ0.061
0.130
ꢀ0.069
ꢀ0.001
6.699
4.903
4.301
4.602
4.602
4.903
5.344
5.271
4.633
4.734
4.59
1.355
ꢀ0.368
ꢀ0.332
ꢀ0.132
0.012
6.699
4.903
4.301
4.602
4.602
4.903
5.549
5.329
4.517
4.503
4.628
4.620
ꢀ1.149
0.426
0.216
ꢀ0.099
0.026
6e
4.679
0.224
ꢀ0.282
Table 6
Results of validation.
active and remaining other groups of the molecule are away from
the desirable streic region which are close to yellow contour. Sub-
stitution of the phenyl ring by thiophene in 4e and furan in 4d
perfectly aligned in the desirable steric region account for potential
activity. Rests of the molecules are found to be less active as they
are away from the desirable steric region.
The contour map of the electrostatic field of CoMFA model is
shown in Fig. 3(B). The red contours represent regions that lead to
the enhancement of activity with electron rich groups where as
blue regions represent desirability of electropositive charge groups.
There are large red contours over the triazole and morpholine ring
and two small red contours near to the triazole (C]S) and imine
nitrogen are favorable to electronegative charge groups. This could
explain the relatively higher activity of compound 4ael than
others. Another blue contour near to the phenyl ring also seems to
favor the electropositive substitutions.
Validation parameter
CoMFA
CoMSIA
q2
0.432
0.902
0.511
0.953
r2 > 0.6
[(r2 ꢀ r20)/r2] < 0.1
0.85 ꢁ k ꢂ 1.15
r2m > 0.5
ꢀ0.067
ꢀ0.003
1.029
1.033
0.745
0.942
Further, external validation was carried out for CoMFA and
CoMSIA models based on Golbraikh and Roy and are satisfying the
external validation parameters. The results of external validation
(Table 6) suggest that the generated CoMFA and CoMSIA models
have good predictive abilities. The derived 3D-QSAR models pro-
vide the tool to guide the design and synthesis of novel and more
potent diphenylamine based antitubercular agents.
3.2.1.1.2. CoMSIA contour analysis. The CoMSIA contour maps
derived using steric, electrostatic, hydrophobic, hydrogen bond
donor and hydrogen bond acceptor fields are represented in Fig. 4.
CoMSIA steric and electrostatic contours in Fig. 4(D) and (E) are
similar to those of CoMFA. In case of CoMSIA, there is a large yellow
contour overlapping the benzene ring, reveals that the bulkier
substituents are not preferred in this region.
The hydrogen bond donor contour map of CoMSIA model is
displayed in Fig. 4(A). In the CoMSIA, hydrogen bond donor field
favored regions are represented by cyan contours and unfavorable
regions by purple contours. A big cyan contour on the nitrogen
atom at 4th position of triazole moiety favors hydrogen bonding
and a purple contour on the aryl substitution of the thiazolidione
compounds 5aef is unfavorable. This could account for reduced
activity of compounds 5aef. The hydrogen bond acceptor contour
map of CoMSIA model is displayed in Fig. 4(B). In the hydrogen
acceptor field, magenta contour favors the regions of hydrogen
bond acceptor substituent's on ligands and the red ones represents
areas where such substituents on compounds may be disfavored. A
big magenta contour around morpholine ring and a small magenta
3.2.1.1. CoMFA and CoMSIA contour analysis. To view the field effect
on the target property, CoMFA and CoMSIA contour maps were
generated. The contour maps may identify the important regions
where any changes in the steric, electrostatic, hydrophobic,
hydrogen bond donor, hydrogen bond acceptor and polar surface
area fields may affect the biological activity. The visualization of the
results of the CoMFA and CoMSIA models were performed using the
StDev* Coeff mapping option contoured by contribution. All the
contours represented the default 80 and 20% level contributions for
favored and disfavored regions. 3D-QSAR contour maps of CoMFA
and CoMSIA models are depicted in Figs. 3 and 4. Compound 4a
with higher pMIC value was chosen to superimpose into the con-
tour maps to illustrate the SAR information in a better manner.
3.2.1.1.1. CoMFA contour analysis. The CoMFA contour map of
steric field is shown in Fig. 3(A). The green contours are indicative
of favorable regions for sterically bulkier groups and the yellow
contours are indicative of regions that are sterically less favorable.
The green contour covers phenyl and morpholine groups as shown
in Fig. 3(A), which could explain why compound 4a is the most

522
K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
Fig. 3. CoMFA contour maps of compound 4a. (A) CoMFA steric contours: Green contours indicate regions where steric interaction is favored. Yellow contours are areas where the
steric interaction is disfavored. (B) CoMFA electrostatic contours: The blue region represents the area where an electropositive group is favorable and the red region refers to the area
where an electronegative group is favorable. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. CoMSIA contour maps based on compound 4a. (A) CoMSIA hydrogen bond donor contours: cyan and purple contours represent favorable and unfavorable hydrogen bond
donor regions, respectively (B) CoMSIA hydrogen bond acceptor contours: magenta and red contours represent favorable and unfavorable hydrogen bond acceptor regions,
respectively (C) CoMSIA hydrophobic contour maps: yellow contours represent regions where hydrophobic groups increase activity, while gray contours highlight regions that
would favors hydrophilic groups. (D) CoMSIA steric contour maps: green contour favors steric or bulky group and yellow contour denotes disfavored region. (E) CoMSIA electrostatic
contour maps: blue contour indicates electropositive charge and red contour electronegative charge. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
contour around the diphenyl amine substitutions indicate the re-
gions important for hydrogen bond acceptor. From these contour
maps we could conclude that the morpholino substitution is
essential for the activity; however, compound 4b and 4c were less
active despite having morpholino functional group which could
attribute to the presence of methoxy group in the unfavorable
region.
Hydrophobic contour maps are shown in Fig. 4(C). The yellow
region and gray region indicates the area of hydrophobic and hy-
drophilic properties are preferred respectively. A yellow contours
are at the 4th and 5th carbons of phenyl ring, and small yellow
contour is near to the thione group at the 2nd position of triazole
ring could indicates the necessity of the hydrophobic groups attach
to the benzene and triazole ring favors for the activity (4ael).

K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
523
4. Experimental
nucleus), 9.85 (s, 1H, N]CH), 7.64e6.69 (m, 11H, AreH), 6.19 (s,
1H, AreNH), 4.25 (s, 2H, AreCH2), 3.80 (s, 3H, eOCH3 group at C4);
4.1. Material and methods
13C NMR (DMSO-d6,
d ppm): 162.10 (C]S), 159.83 (C]N), 148.93
(C5 of triazole), 148.12e112.7 (aromatic carbons), 57.09 (eOCH3),
The melting points of the synthesized compounds were deter-
mined in open glass capillaries and were uncorrected. IR spectra
were recorded on Shimadzu FT-IR 8400-S spectrophotometer by
KBr pellet technique. Elemental analysis was performed and found
values were within 0.4% of theoretical values unless otherwise
noted. 1H NMR and 13C NMR spectra were recorded on AMX-400
NMR spectrophotometer at 400 MHz using DMSO-d6 as the sol-
vent and tetramethylsilane (TMS) as internal standard. The chem-
27.74 (AreCH2); LC-MS m/z: 483.06 (Mþ), 485.06 (Mþ2).
4.3.3. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(3,4-
dimethoxybenzylideneamino)-1H-1,2,4-triazole-5(4H)-thione (3c)
Yield: 75%, M.p. 212e214 ꢃC, IR ( cmꢀ1, KBr): 3344 (NH), 2970
n
(CeH), 1604 (C]N), 1272 (C]S), 1138 (CeO); 1H NMR (DMSO-d6,
ppm): 13.81 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
d
9.72 (s, 1H, N]CH), 7.53e6.78 (m, 10H, AreH), 6.22 (s, 1H, AreNH),
4.21 (s, 2H, AreCH2), 3.84 (s, 3H, eOCH3 group at C4), 3.78 (s, 3H,
ical shifts are expressed in
d ppm. The splitting patterns were
designated as follows; s: singlet; d: doublet; q: quartet; m: multi-
plet. LC-MS were recorded using Shimadzu LC-MS-2010A instru-
ment by ESI.
eOCH3 group at C3); 13C NMR (DMSO-d6,
d ppm): 162.24 (C]S),
159.11 (C]N), 148.64 (C5 of triazole), 147.21e113.09 (aromatic car-
bons), 56.13 (eOCH3), 27.95 (AreCH2); LC-MS m/z: 513.08 (Mþ),
515.08 (Mþ2).
4.2. Synthesis of 3-[2-[(20,60-dichlorophenyl) amino] benzyl]-4-
amino-5-mercapto(4H)-1,2,4-triazole (2)
4.3.4. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(furan-2-
ylmethyleneamino)-1H-1,2,4-triazole-5(4H)-thione (3d)
Diclofenac acid hydrazide 1 (10 mmol) was treated with carbon
disulfide (10 mmol) in the presence of alcoholic potassium hy-
droxide solution (15 mmol). The reaction mixture was refluxed for
6 h, cooled and the separated solid was collected by filtration, dried
to get potassium salt of dithiocarbazinate was used directly without
purification for the next step. Dithiocarbazinate salt (10 mmol) and
hydrazine hydrate (20 mmol) was refluxed for 6 h and profuse
evolution of hydrogen sulfide was observed. The reaction mixture
was cooled and diluted with ice cold water. The reaction mixture
was then acidified with dilute HCl and the separated solid was
collected by filtration, washed with water and recrystallized from
absolute ethanol to give compound 2.
Yield: 79%, M.p. 231e233 ꢃC, IR ( cmꢀ1, KBr): 3340 (NH), 2931
n
(CeH), 1608 (C]N), 1276 (C]S), 1101 (CeO); 1H NMR (DMSO-d6,
ppm): 13.87 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
10.13 (s, 1H, N]CH), 7.95e6.89 (m, 10H, AreH), 6.12 (s, 1H, AreNH),
4.17 (s, 2H, AreCH2); 13C NMR (DMSO-d6,
ppm): 163.18 (C]S),
d
d
161,12 (C]N), 148.79 (C5 of triazole), 144.12e111.63 (aromatic car-
bons), 27.96 (AreCH2); LC-MS m/z: 443.04 (Mþ), 445.04 (Mþ2).
4.3.5. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(thiophen-2-
ylmethyleneamino)-1H-1,2,4-triazole-5(4H)-thione (3e)
Yield: 82%, M.p. 250e252 ꢃC, IR ( cmꢀ1, KBr): 3336 (NH), 2943
n
(CeH), 1597 (C]N), 1274 (C]S), 717 (CeS); 1H NMR (DMSO-d6,
ppm): 13.82 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
10.19 (s, 1H, N]CH), 7.82e6.93 (m,10H, AreH), 6.25 (s, 1H, AreNH),
4.15 (s, 2H, AreCH2); 13C NMR (DMSO-d6,
ppm): 163.24 (C]S),
Yield: 72%, M.p. 188e190 ꢃC, IR (
n
cmꢀ1, KBr): 750 (CeCl), 3215
ppm): 13.58
d
(NH), 1597 (C]N), 1296 (C]S); 1H NMR (DMSO-d6,
d
(s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus), 7.53e6.81 (m,
7H, AreH), 6.27 (s, 1H, AreNH), 5.65 (s, 2H, NeNH2), 4.11 (s, 2H,
d
161.26 (C]N), 149.13 (C5 of triazole), 145.22e119.95 (aromatic
carbons), 28.26 (AreCH2); LC-MS m/z: 459.01 (Mþ), 461.01 (Mþ2).
AreCH2); 13C NMR (DMSO-d6,
d ppm):162.4 (C]S), 148.6 (C3 of
triazole), 134.8e114.6 (aromatic carbons), 27.99 (AreCH2); LC-MS
m/z: 365.03 (Mþ), 367.02 (Mþ þ2).
4.3.6. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(pyridin-4-
ylmethyleneamino)-1H-1,2,4-triazole-5(4H)-thione (3f)
4.3. General procedure for synthesis of 4-arylideneamino-5-(2-
(2,6-dichlorophenylamino) benzyl)-2H-1,2,4-triazole-3(4H)-thiones
(3aef)
Yield: 82%, M.p. 250e252 ꢃC, IR ( cmꢀ1, KBr): 3336 (NH), 2937
n
(CeH), 1600 (C]N), 1288 (C]S), 1269 (CeN); 1H NMR (DMSO-d6,
ppm): 13.45 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
10.25 (s,1H, N]CH), 7.68e6.91 (m,11H, AreH), 6.23 (s,1H, AreNH),
4.18 (s, 2H, AreCH2); 13C NMR (DMSO-d6,
ppm): 162.79 (C]S),
d
To the solution of 1,2,4-triazole 2 (10 mmol) in glacial acetic acid
(15 mL) an equimolar amount of appropriate aromatic aldehyde
was added. The reaction mixture was refluxed on an oil bath for
4e6 h. The reaction mixture was concentrated, cooled and the solid
formed was filtered, dried and recrystallized from ethanol.
d
161.26 (C]N), 149.21 (C5 of triazole), 144.67e117.24 (aromatic
carbons), 28.01 (AreCH2); LC-MS m/z: 454.36 (Mþ), 456.36 (Mþ2).
4.4. General procedure for synthesis of 4-(arylideneamino)-2-
(substituted-aminomethyl)-5-(2 (2,6-dichlorophenylamino)
benzyl)-2H-1,2,4-triazole-3(4H)-thiones (4ael)
4.3.1. 4(E)-4-(Benzylideneamino)-3-(2-(2,6-dichlorophenylamino)
benzyl)-1H-1,2,4-triazole-5(4H)-thione (3a)
Yield: 84%, M.p. 215e217 ꢃC, IR (
n
cmꢀ1, KBr): 3336 (NH), 1604
ppm): 13.88 (s, 1H, NH
The Schiff bases 3aef (5 mmol) were dissolved in ethanol. Then
formaldehyde (37%, 1.5 mL) and appropriate secondary amines
(5 mmol) in ethanol were added to this solution. The reaction
mixture was refluxed for 3e4 h and kept overnight at room tem-
perature. The separated solid was collected by filtration, dried and
recrystallized from ethanol.
(C]N), 1270 (C]S); 1H NMR (DMSO-d6,
d
adjacent to C]S of 1,2,4-triazole nucleus), 10.00 (s, 1H, N]CH),
7.88e6.77 (m, 12H, AreH), 6.22 (s, 1H, AreNH), 4.29 (s, 2H,
AreCH2); 13C NMR (DMSO-d6,
d ppm): 163.01 (C]S), 160.23 (C]
N), 149.32(C5 of triazole), 136.7e113.8 (aromatic carbons), 28.04
(AreCH2); LC-MS m/z: 453.06 (Mþ), 455.06 (Mþ2).
4.4.1. (E)-4-(Benzylideneamino)-3-(2-(2,6-dichlorophenylamino)
4.3.2. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(4-methoxy-
benzyl)-1-(morpholinomethyl)-1H-1,2,4-triazole-5(4H)-thione (4a)
benzylideneamino)-1H-1,2,4-triazole-5(4H)-thione (3b)
Yield: 85%, M.p. 161e163 ꢃC, IR ( cmꢀ1, KBr): 3325 (NH), 2951
n
Yield: 81%, M.p. 234e236 ꢃC, IR (
n
cmꢀ1, KBr): 3331 (NH),
(CeH), 1598 (C]N), 1350 (CeN), 1276 (C]S), 1161 (CeO); 1H NMR
(DMSO-d6, ppm): 10.37 (s, 1H, N]CH), 7.92e6.91 (m, 12H, AreH),
6.9 (s, 1H, AreNH), 5.10 (s, 2H, NeCH2eN), 4.34 (s, 2H, AreCH2),
2931(CeH), 1604 (C]N), 1257 (C]S), (CeO) 1153; 1H NMR (DMSO-
d6, ppm): 13.46 (s, 1H, NH adjacent to C]S in 1,2,4-triazole
d
d

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K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
3.62 (t, 4H, CH2OCH2 of morpholine ring), 2.82 (t, 4H, CH2NCH2 of
morpholine ring); 13C NMR (DMSO-d6,
ppm): 163.17 (C]S),
161.65 (C]N), 149.52 (C5 of triazole), 137.14e112.96 (aromatic
carbons), 69.10 (NeCeN), 66.89 (C2 and C6 of morpholine ring),
50.91 (C3 and C5 of morpholine ring), 28.05 (AreCH2); LC-MS m/z:
552.13 (Mþ), 554.12 (Mþ2).
4.4.6. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-1-(morpholino-
methyl)-4-(pyridin-4-ylmethyleneamino)-1H-1,2,4-triazole-5(4H)-
thione (4f)
d
Yield: 75%, M.p. 166e168 ꢃC, IR ( cmꢀ1, KBr): 3336.96 (NH),
n
2951 (CeH), 1577 (C]N), 1279 (C]S), 1114 (CeO); 1H NMR (DMSO-
d6, ppm): 10.54 (s, 1H, N]CH), 8.4e7.12 (m,10H, AreH), 6.9 (s,1H,
d
AreNH), 5.11 (s, 2H, NeCH2eN), 4.35 (s, 2H, AreCH2), 3.61 (t, 4H,
CH2OCH2 of morpholine ring), 2.85 (t, 4H, CH2NCH2 of morpholine
4.4.2. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(4-
methoxybenzylideneamino)-1-(morpholinomethyl)-1H-1,2,4-
triazole-5(4H)-thione (4b)
ring); 13C NMR (DMSO-d6,
d ppm): 162.35 (C]S), 160.54 (C]N),
149.23 (C5 of triazole), 144.41e118.3 (aromatic carbons), 68.99
(NeCeN), 66.72 (C2 and C6 of morpholine ring), 50.17 (C3 and C5 of
morpholine ring), 28.22 (AreCH2). LC-MS m/z: 553.12 (Mþ), 555.12
(Mþ2).
Yield: 85%, M.p. 161e163 ꢃC, IR ( cmꢀ1, KBr): 3329 (NH), 2955
n
(CeH), 1608 (C]N), 1286 (CeOeC), 1271 (C]S); 1H NMR (DMSO-
d6, ppm): 10.13 (s, 1H, N]CH), 7.68e6.76 (m, 11H, AreH), 6.47 (s,
d
1H, AreNH), 5.15 (s, 2H, NeCH2eN), 4.29 (s, 2H, AreCH2), 3.89 (s,
4.4.7. (E)-4-(Benzylideneamino)-3-(2-(2,6-dichlorophenylamino)
benzyl)-1-(piperidin-1-ylmethyl)-1H-1,2,4-triazole-5(4H)-thione
(4g)
3H, OCH3), 3.74 (t, 4H, CH2OCH2 of morpholine ring), 2.91 (t, 4H,
CH2NCH2 of morpholine ring); 13C NMR (DMSO-d6,
d ppm): 163.57
(C]S), 160.63 (C]N), 148.82 (C5 of triazole), 136.57e113.84 (aro-
matic carbons), 69.25 (NeCeN), 67.06 (C2 and C6 of morpholine
ring), 56.12 (eOCH3), 50.65 (C3 and C5 of morpholine ring), 28.12
(AreCH2). LC-MS m/z: 582.14 (Mþ), 584.14 (Mþ2).
Yield: 81%, M.p. 146e148 ꢃC, IR (
n
cmꢀ1, KBr): 3317 (NH), 2931
ppm): 10.40
(CeH), 1577 (C]N), 1276 (C]S); 1H NMR (DMSO-d6,
d
(s, 1H, N]CH), 7.9e6.85 (m, 12H, AreH), 6.49 (s, 1H, AreNH), 5.11
(s, 2H, NeCH2eN), 4.31 (s, 2H, AreCH2), 2.78 (t, 4H, CH2NCH2 of
piperidine ring), 1.52e1.55 (m, 6H, (CH2)3 of piperidiene ring); 13C
4.4.3. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(3,4-
dimethoxybenzylideneamino)-1-(morpholinomethyl)-1H-1,2,4-
triazole-5(4H)-thione (4c)
NMR (DMSO-d6, d d ppm): 163.14 (C]
ppm): 13C NMR (DMSO-d6,
S), 161.24 (C]N), 149.71 (C5 of triazole), 137.61e110.31 (aromatic
carbons), 68.12 (NeCeN), 52.13 (C3 and C5 of piperidine ring), 28.69
(AreCH2), 25.97 (C4 of piperidine ring), 25.62 (C2 and C6 of piper-
idine ring). LC-MS m/z: 550.12 (Mþ), 554.12 (Mþ2).
Yield: 76%, M.p. 158e160 ꢃC, IR ( cmꢀ1, KBr): 3345 (NH), 2914
n
(CeH), 1600 (C]N), 1273 (C]S), 1238 (CeN); 1H NMR (DMSO-d6,
ppm): 10.05 (s, 1H, N]CH), 7.53e6.65 (m, 10H, AreH), 6.49 (s, 1H,
d
AreNH), 5.10 (s, 2H, NeCH2eN), 4.32 (s, 2H, AreCH2), 3.97 (s, 3H,
eOCH3 group at C4), 3.95 (s, 3H, eOCH3 group at C3); 3.67 (t, 4H,
CH2OCH2 of morpholine ring), 2.82 (t, 4H, CH2NCH2 of morpholine
4.4.8. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(4-methoxy-
benzylideneamino)-1-(piperidin-1-ylmethyl)-1H-1,2,4-triazole-
5(4H)-thione (4h)
ring); 13C NMR (DMSO-d6,
d
ppm): 162.56 (C]S), 159.87 (C]N),
Yield: 74%, M.p. 149e151 ꢃC, IR ( cmꢀ1, KBr): 3329 (NH), 2955
n
149.21 (C5 of triazole), 137.49e111.92 (aromatic carbons), 68.65
(NeCeN), 66.74 (C2 and C6 of morpholine ring), 56.79 (eOCH3),
50.91 (C3 and C5 of morpholine ring), 28.24 (AreCH2). LC-MS m/z:
612.15 (Mþ), 614.15 (Mþ2).
(CeH), 1608 (C]N), 1269 (C]S), 1186.21(CeOeC); 1H NMR (DMSO-
d6, ppm): 10.12 (s, 1H, N]CH), 7.81e6.95 (m, 11H, AreH), 6.72 (s,
d
1H, AreNH), 5.13 (s, 2H, NeCH2eN), 4.45 (s, 2H, AreCH2), 3.92 (s,
3H, OCH3), 2.83 (t, 4H, CH2NCH2 of piperidine ring), 1.46e1.53 (m,
6H, (CH2)3 of piperidiene ring); 13C NMR (DMSO-d6,
d ppm): 164.48
(C]S), 161.98 (C]N), 149.97 (C5 of triazole), 137.49e112.09 (aro-
matic carbons), 68.96 (NeCeN), 56.74 (eOCH3), 52.86 (C3 and C5 of
piperidine ring), 28.11(AreCH2), 25.48 (C4 of piperidine ring), 25.13
(C2 and C6 of piperidine ring). LC-MS m/z: 580.16 (Mþ), 580.16
(Mþ2).
4.4.4. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(furan-2-
ylmethyleneamino)-1-(morpholinomethyl)-1H-1,2,4-triazole-
5(4H)-thione (4d)
Yield: 80%, M.p. 146e148 ꢃC, IR ( cmꢀ1, KBr): 3335 (NH), 2935
n
(CeH), 1604 (C]N), 1196 (CeOeC), 1272 (C]S); 1H NMR (DMSO-
d6, ppm): 10.37 (s, 1H, N]CH), 7.92e6.76 (m, 10H, AreH), 6.9 (s,
d
4.4.9. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-4-(3,4-dimethoxy-
benzylideneamino)-1-(piperidin-1-ylmethyl)-1H-1,2,4-triazole-5(4H)-
thione (4i)
1H, AreNH), 5.10 (s, 2H, NeCH2eN), 4.34 (s, 2H, AreCH2), 3.62 (t,
4H, CH2OCH2 of morpholine ring), 2.82 (t, 4H, CH2NCH2 of mor-
pholine ring); 13C NMR (DMSO-d6,
d ppm): 164.16 (C]S), 161.93
Yield: 72%, M.p. 155e157 ꢃC, IR ( cmꢀ1, KBr): 3314 (NH), 2931
n
(C]N), 149.02 (C5 of triazole), 147.53e115.76 (aromatic carbons),
68.97 (NeCeN), 66.23 (C2 and C6 of morpholine ring), 50.92 (C3 and
C5 of morpholine ring), 28.59 (AreCH2). LC-MS m/z: 542.11 (Mþ),
544.11 (Mþ2).
(CeH), 1604 (C]N), 1423 (CeNeC) 1273 (C]S), 1161.21 (CeO); 1H
NMR (DMSO-d6, ppm): 10.05 (s, 1H, N]CH), 7.52e6.60 (m, 10H,
d
AreH), 6.64 (s, 1H, AreNH), 5.11 (s, 2H, NeCH2eN), 4.36 (s, 2H,
AreCH2), 3.92 (s, 3H, eOCH3 group at C4), 3.87 (s, 3H, eOCH3 group
at C3), 2.78 (t, 4H, CH2NCH2 of piperidine ring), 1.41e1.56 (m, 6H,
4.4.5. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-1-(morpholino-
methyl)-4-(thiophen-2-ylmethyleneamino)-1H-1,2,4-triazole-
5(4H)-thione (4e)
(CH2)3 of piperidiene ring); 13C NMR (DMSO-d6,
d ppm): 163.74
(C]S), 161.50 (C]N), 150.09 (C5 of triazole), 136.46e111.14 (aro-
matic carbons), 68.45 (NeCeN), 56.14 (eOCH3), 53.02 (C3 and C5 of
piperidine ring), 28.94 (AreCH2), 25.31 (C4 of piperidine ring),
24.87 (C2 and C6 of piperidine ring). LC-MS m/z: 610.17 (Mþ), 612.17
(Mþ2).
Yield: 84%, M.p. 174e176 ꢃC, IR ( cmꢀ1, KBr): 3344 (NH), 2941
n
(CeH), 1597 (C]N), 1161 (CeOeC), 1265 (C]S), 707 (CeS); 1H NMR
(DMSO-d6, ppm): 10.68 (s, 1H, N]CH), 7.62e6.88 (m, 10H, AreH),
d
6.59 (s, 1H, AreNH), 5.08 (s, 2H, NeCH2eN), 4.32 (s, 2H, AreCH2),
3.64 (t, 4H, CH2OCH2 of morpholine ring), 2.79 (t, 4H, CH2NCH2 of
4.4.10. 4-((Furan-2-yl)methyleneamino)-5-(2-(2,6-dichlorophenyl-
amino)benzyl)-2-((piperidin-1-yl)methyl)-2H-1,2,4-triazole-3(4H)-
thione (4j)
morpholine ring); 13C NMR (DMSO-d6,
d ppm): 162.43 (C]S),
161.33 (C]N), 148.73 (C5 of triazole), 146.83e116.92 (aromatic
carbons), 68.04 (NeCeN), 66.92 (C2 and C6 of morpholine ring),
51.15 (C3 and C5 of morpholine ring), 28.49 (AreCH2). LC-MS m/z:
558.08 (Mþ), 560.08 (Mþ2).
Yield: 79%, M.p. 141e143 ꢃC, IR (
n
cmꢀ1, KBr): 3325 (NH), 2928
ppm): 10.49
(s, 1H, N]CH), 7.95e6.56 (m, 10H, AreH), 6.84 (s, 1H, AreNH), 5.21
(CeH), 1593 (C]N), 1259 (C]S); 1H NMR (DMSO-d6,
d

K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
525
(s, 2H, NeCH2eN), 4.29 (s, 2H, AreCH2), 2.5 (t, 4H, CH2NCH2 of
piperidine ring), 1.46e1.49 (m, 6H, (CH2)3 of piperidiene ring); 13C
7.62e6.75 (m, 11H, AreH), 6.67 (s, 1H, AreNH), 5.59 (s, 2H, SeCH2),
4.15 (s, 2H, AreCH2), 3.92 (s, 3H, OCH3), 3.28 (s, 1H, C5 of thiazoli-
NMR (DMSO-d6,
d
ppm): 162.94 (C]S), 161.14 (C]N), 149.43 (C5 of
dinone ring); 13C NMR (DMSO-d6,
d ppm): 171.03 (C]O of thia-
triazole), 147.63e117.32 (aromatic carbons), 68.12 (NeCeN), 53.23
(C3 and C5 of piperidine ring), 28.97 (AreCH2), 25.76 (C4 of piper-
idine ring), 24.92 (C2 and C6 of piperidine ring). LC-MS m/z: 540.13
(Mþ), 542.13 (Mþ2).
zolidinone ring), 162.26 (C]S), 154.03 (C]N), 146.18 (C5 of
triazole), 135.89e113.12 (aromatic carbons), 57.61 (OCH3), 56.48 (C2
of thiazolidinone ring), 33.09 (C4 of thiazolidinone ring), 27.87
(AreCH2). LC-MS m/z: 557.05 (Mþ), 559.05 (Mþ2).
4.4.11. (E)-3-(2-(2,6-Dichlorophenylamino)benzyl)-1-(piperidin-1-
ylmethyl)-4-(thiophen-2-ylmethyleneamino)-1H-1,2,4-triazole-
5(4H)-thione (4k)
4.5.3. 3-(3-(2-(2,6-Dichlorophenylamino)benzyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl)-2-(3,4-dimethoxyphenyl) thiazolidin-4-one
(5c)
Yield: 82%, M.p. 149e151 ꢃC, IR (
n
cmꢀ1, KBr): 3338 (NH), 2939
Yield: 73%, M.p. 151e153 ꢃC, IR ( cmꢀ1, KBr): 3329 (NH), 2974
n
(CeH), 1597 (C]N), 1423.51 (CeN), 1273 (C]S); 1H NMR (DMSO-
d6, ppm): 10.71 (s, 1H, N]CH), 7.61e6.63 (m, 10H, AreH), 6.47 (s,
(CeH), 1613 (C]N), 1277 (C]S), 1156 (CeO); 1H NMR (DMSO-d6,
ppm): 13.24 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
d
d
1H, AreNH), 5.09 (s, 2H, NeCH2eN), 4.32 (s, 2H, AreCH2), 2.7 (t, 4H,
CH2NCH2 of piperidine ring), 1.34e1.55 (m, 6H, (CH2)3 of piperidine
7.67e6.64 (m, 10H, AreH), 6.54 (s, 1H, AreNH), 5.66 (s, 2H, SeCH2),
4.11 (s, 2H, AreCH2), 3.89 (s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 3.24
ring); 13C NMR (DMSO-d6,
d
ppm): 163.12 (C]S), 155.65 (C]N),
(s, 1H, C5 of thiazolidinone ring); 13C NMR (DMSO-d6,
d ppm):
149.57 (C5 of triazole), 144.73e114.23 (aromatic carbons), 68.08
(NeCeN), 53.86 (C3 and C5 of piperidine ring), 28.45 (AreCH2),
25.13 (C4 of piperidine ring), 24.84 (C2 and C6 of piperidine ring).
LC-MS m/z: 556.1 (Mþ), 558.12 (Mþ2).
170.27 (C]O of thiazolidinone ring), 162.83 (C]S), 158.24 (C]N),
148.32 (C5 of triazole), 137.75e110.89 (aromatic carbons), 56.78
(OCH3), 56.09 (C2 of thiazolidinone ring), 31.45 (C4 of thiazolidinone
ring), 28.25 (AreCH2). LC-MS m/z: 587.06 (Mþ), 589.06 (Mþ2).
4.4.12. 4-((Pyridin-4-yl)methyleneamino)-5-(2-(2,6-dichloro-
phenylamino)benzyl)-2-((piperidin-1-yl)methyl)-2H-1,2,4-triazole-
3(4H)-thione (4l)
4.5.4. 3-(3-(2-(2,6-Dichlorophenylamino)benzyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl)-2-(furan-2-yl)thiazolidin-4-one (5d)
Yield: 84%, M.p. 144e146 ꢃC, IR ( cmꢀ1, KBr): 3317 (NH), 2927
n
Yield: 79%, M.p. 146e147 ꢃC, IR (
n
cmꢀ1, KBr): 3338 (NH), 2939
(CeH), 1574 (C]N), 1418 (CeN), 1267 (C]S); 1H NMR (DMSO-d6,
ppm): 13.69 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
(CeH), 1597 (C]N), 1423.51 (CeN), 1273 (C]S); 1H NMR (DMSO-
d6, ppm): 10.71 (s, 1H, N]CH), 7.8e6.63 (m, 11H, AreH), 6.52 (s,
d
d
7.85e6.97 (m, 10H, AreH), 6.95 (s, 1H, AreNH), 5.78 (s, 2H, SeCH2),
1H, AreNH), 5.10 (s, 2H, NeCH2eN), 4.34 (s, 2H, AreCH2), 2.73 (t,
4H, CH2NCH2 of piperidine ring), 1.43e1.58 (m, 6H, (CH2)3 of
4.25 (s, 2H, AreCH2), 3.12 (s, 1H, C5 of thiazolidinone ring); 13C NMR
(DMSO-d6,
d ppm): 171.34 (C]O of thiazolidinone ring), 161.58
piperidine ring); 13C NMR (DMSO-d6,
d
ppm): 164.10 (C]S), 156.02
(C]S), 158.46 (C]N), 149.69 (C5 of triazole), 144.12e116.53 (aro-
matic carbons), 57.69 (C2 of thiazolidinone ring), 32.11 (C4 of thia-
zolidinone ring), 28.26 (AreCH2). LC-MS m/z: 517.02 (Mþ), 519.02
(Mþ2).
(C]N), 150.02 (C5 of triazole), 145.56e115.32 (aromatic carbons),
67.98 (NeCeN), 55.05 (C3 and C5 of piperidine ring), 27.90
(AreCH2), 26.85 (C4 of piperidine ring), 25.05 (C2 and C6 of piper-
idine ring). LC-MS m/z: 551.14 (Mþ), 553.14 (Mþ2).
4.5.5. 3-(3-(2-(2,6-Dichlorophenylamino)benzyl)-5-thioxo-1H-
4.5. General procedure for synthesis of 3-(3-(2-(2,6-dichlorophenyl-
amino)benzyl)-5-mercapto-4H-1,2,4-triazol-4-yl)-2-arylthiazolidin-
4-ones (5aef)
1,2,4-triazol-4(5H)-yl)-2-(thiophen-2-yl)thiazolidin-4-one (5e)
Yield: 80%, M.p. 129e131 ꢃC, IR ( cmꢀ1, KBr): 3326 (NH), 2974
n
(CeH), 1613 (C]N), 1263 (C]S), 677 (CeS); 1H NMR (DMSO-d6,
ppm): 12.97 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
d
A mixture of Schiff base 3aef (10 mmol) and thioglycolic acid
(10 mmol) in 30 mL dry benzene was refluxed on a water bath for
6e8 h. The excess benzene was distilled off under reduced pres-
sure. The reaction mixture was neutralized with cold dilute sodium
bicarbonate solution. The product formed was filtered, dried and
recrystallized from absolute ethanol.
7.64e6.76 (m, 10H, AreH), 6.64 (s, 1H, AreNH), 5.53 (s, 2H, SeCH2),
4.32 (s, 2H, AreCH2), 3.35 (s,1H, C5 of thiazolidinone ring); 13C NMR
(DMSO-d6,
d ppm): 172.26 (C]O of thiazolidinone ring), 163.39
(C]S), 154.01 (C]N), 150.18 (C5 of triazole), 143.32e113.37 (aro-
matic carbons), 55.08 (C2 of thiazolidinone ring), 31.17 (C4 of thia-
zolidinone ring), 28.28 (AreCH2). LC-MS m/z: 533 (Mþ), 535 (Mþ2).
4.5.1. 3-(3-(2-(2,6-Dichlorophenylamino)benzyl)-5-thioxo-1H-
4.5.6. 3-(3-(2-(2,6-Dichlorophenylamino)benzyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl)-2-phenylthiazolidin-4-one (5a)
1,2,4-triazol-4(5H)-yl)-2-(pyridin-4-yl)thiazolidin-4-one (5f)
Yield: 85%, M.p. 141e143 ꢃC, IR (
n
cmꢀ1, KBr): 3367 (NH), 2931
Yield: 77%, M.p. 124e126 ꢃC, IR ( cmꢀ1, KBr): 3319 (NH), 2975
n
(CeH), 1594 (C]N), 1273 (C]S), 653 (CeS); 1H NMR (DMSO-d6,
ppm): 13.59 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
(CeH), 1604 (C]N), 1280 (C]S); 1H NMR (DMSO-d6,
d ppm): 13.09
(s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus), 7.87e6.67 (m,
11H, AreH), 6.65 (s, 1H, AreNH), 5.71 (s, 2H, SeCH2), 4.30 (s, 2H,
AreCH2), 3.38 (s, 1H, C5 of thiazolidinone ring); 13C NMR (DMSO-
d
7.54e6.92 (m, 12H, AreH), 6.85 (s, 1H, AreNH), 5.66 (s, 2H, SeCH2),
4.11 (s, 2H, AreCH2), 3.31 (s, 1H, C5 of thiazolidinone ring); 13C NMR
(DMSO-d6,
d
ppm): 170.31 (C]O of thiazolidinone ring), 164.56
d6, d ppm): 172.45 (C]O of thiazolidinone ring), 162.97 (C]S),
(C]S), 160.98 (C]N), 148.54 (C5 of triazole), 136.69e111.24 (aro-
matic carbons), 55.7 (C2 of thiazolidinone ring), 32.72 (C4 of thia-
zolidinone ring), 28.42 (AreCH2). LC-MS m/z: 527.04 (Mþ), 527.04
(Mþ2).
156.64 (C]N), 148.21(C5 of triazole), 144.19e110.48 (aromatic car-
bons), 56.09 (C2 of thiazolidinone ring), 32.10 (C4 of thiazolidinone
ring), 28.01 (AreCH2). LC-MS m/z: 528.04 (Mþ), 530.04 (Mþ2).
4.6. General procedure for synthesis of 2-(2-(2,6-dichllorphenyl-
amino)benzyl)-3-(arylideneamino)-[1,2,4]triazolo [5,1-b]
quinazolin-9(3H)-ones (6aef)
4.5.2. 3-(3-(2-(2,6-Dichlorophenylamino)benzyl)-5-thioxo-1H-
1,2,4-triazol-4(5H)-yl)-2-(4-methoxyphenyl) thiazolidin-4-one (5b)
Yield: 79%, M.p. 136e138 ꢃC, IR ( cmꢀ1, KBr): 3312 (NH), 2945
n
(CeH), 1580 (C]N), 1265 (C]S), 659 (CeS); 1H NMR (DMSO-d6,
ppm): 13.24 (s, 1H, NH adjacent to C]S in 1,2,4-triazole nucleus),
The appropriate Schiff base 3aef (10 mmol) and anthranilic acid
(10 mmol) were taken in a glass mortar and mixed well. The
d

526
K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
mixture was then transferred into a wide mouthed round bottom
flask and heated on an oil bath for 2e4 h at 180 ꢃC. The reaction
mixture was cooled and mixed with water and filtered. The solid
obtained was washed with water and 10% NaHCO3 solution, dried
and recrystallized from dichloromethane.
(C3 of triazole), 159.92 (C]O of quinazoline), 157.24 (C]N), 149.02
(C5 of triazole), 144.12e109.97 (aromatic carbons), 28.95 (AreCH2).
LC-MS m/z: 539.1 (Mþ), 541.1 (Mþ2).
5. Biological activity
4.6.1. (E)-3-(Benzylideneamino)-2-(2-(2,6-dichlorophenylamino)
5.1. Antitubercular activity
benzyl)-[1,2,4]triazolo[5,1-b]quinazolin-9(3H)-one (6a)
Yield: 71%, M.p. 145e147 ꢃC, IR (
n
cmꢀ1, KBr): 3317 (NH), 3059
5.1.1. Microplate Almar Blue assay
(CeH), 1594 (C]O), 1530 (C]N); 1H NMR (DMSO-d6,
(s, 1H, N]CH), 7.72e6.68 (m, 16H, AreH), 6.46 (s, 1H, AreNH), 4.36
(s, 2H, AreCH2); 13C NMR (DMSO-d6,
ppm): 172.84 (C3 of tri-
d
ppm): 10.37
All the synthesized compounds were evaluated for their efficacy
against M. tuberculosis 37Rv (ATCC-27294) at concentration
H
d
ranging from 50 M to 0.2 mM using twofold dilutions in the initial
m
azole), 161.28 (C]O of quinazoline), 153.62 (C]N), 149.94 (C5 of
triazole), 137.02e112.76 (aromatic carbons), 28.72 (AreCH2). LC-MS
m/z: 538.11 (Mþ), 540.11 (Mþ2).
screen. Log phase culture of M. tuberculosis H37Rv was diluted so as
to give final OD550 nm of 0.05 in Middle brook 7H9 broth medium. In
96 well white plates 190
The dimethyl sulfoxide solution of test compound was dispensed to
96 well plates so as to make final test concentration 25 M (5
test compound was dispensed in 10 L of DMSO). Then the plate
was incubated at 37 ꢃC/5% CO2 for 5 days. On 5th day 25
L of
mL of culture was dispensed in each well.
4.6.2. (E)-2-(2-(2,6-Dichlorophenylamino)benzyl)-3-(4-methoxy-
m
mM
benzylideneamino)-[1,2,4]triazolo[5,1-b]quinazolin-9(3H)-one (6b)
m
Yield: 74%, M.p.139e141 ꢃC, IR (
n
cmꢀ1, KBr): 3329 (NH), 3057
m
(CeH), 2956 (CeH),1576 (C]O), 1562 (C]N); 1H NMR (DMSO-d6,
ppm): 10.07(s, 1H, N]CH), 7.67e6.7 (m, 15H, AreH), 6.63 (s, 1H,
AreNH), 4.18 (s, 2H, AreCH2), 3.93 (s, 3H, OCH3); 13C NMR (DMSO-
d6, ppm): 170.03 (C3 of triazole), 160.82 (C]O of quinazoline),
freshly prepared 1:1 mixture of Almar Blue reagent and 10% tween
80 was added to each well of plate. The plate was again incubated
for 24 h at 37 ꢃC/5% CO2 incubator. The fluorescence was read on
BMG polar star with excitation frequency at 544 nm and emission
frequency at 590 nm. The compounds, which were found active
(>90% inhibition as compared with control) at this concentration
were further tested by agar dilution method in concentrations
d
d
153.29 (C]N), 148.13 (C5 of triazole), 132.93e110.25 (aromatic
carbons), 57.78 (OCH3), 28.21 (AreCH2). LC-MS m/z: 568.12 (Mþ),
570.12 (Mþ2).
starting from 50 to 0.2 mM.
4.6.3. (E)-2-(2-(2,6-Dichlorophenylamino)benzyl)-3-(3,4-dimethoxy-
benzylideneamino)-[1,2,4]triazolo[5,1-b]quinazolin-9(3H)-one (6c)
5.1.2. .Agar dilution method
Yield: 70%, M.p. 130e132 ꢃC, IR (
n
cmꢀ1, KBr): 3324 (NH), 3015
Drug susceptibility and determination of MIC of the test com-
pounds against M. tuberculosis H37Rv was performed by agar
microdilution method [27,28], where twofold dilutions of each test
compound were added into Middlebrook7H10 agar supplemented
with OADC and organism. A culture of M. tuberculosis H37Rv
growing on L-J medium was harvested in 0.85% saline with 0.05%
(CeH), 2978 (CeH), 1593 (C]O), 1567 (C]N); 1H NMR (DMSO-d6,
ppm): 10.21 (s, 1H, N]CH), 7.84e6.67 (m, 14H, AreH), 6.35 (s, 1H,
AreNH), 4.18 (s, 2H, AreCH2), 3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3);
13C NMR (DMSO-d6,
ppm): 172.43 (C3 of triazole), 161.83 (C]O of
d
d
quinazoline), 158.87 (C]N), 147.65 (C5 of triazole), 136.45e116.24
(aromatic carbons), 56.74 (OCH3), 28.11 (AreCH2). LC-MS m/z:
598.13 (Mþ), 600.13 (Mþ2).
Tween-80. A solution of 1 mM concentration of compounds was
prepared in DMSO. This suspension was added to (in tubes) 7H10
middle brook's medium (containing 1.7 mL medium and 0.2 mL
OADC supplement) at different concentration of compound keep-
ing the volume constant i.e. 0.1 mL. Medium was allowed to cool
keeping the tubes in slanting position. These tubes were then
incubated at 37 ꢃC for 24 h followed by streaking of M. tuberculosis
4.6.4. 3-((Furan-2-yl)methyleneamino)-2-(2-(2,6-dichlorophenyl-
amino)benzyl)-[1,2,4]triazolo[5,1-b]quinazolin-9(3H)-one (6d)
Yield: 77%, M.p. 161e163 ꢃC, IR ( cmꢀ1, KBr): 3319 (NH), 3011
n
(CeH), 2997 (CeH), 1569 (C]O), 1545 (C]N); 1H NMR (DMSO-d6,
ppm): 10.10 (s, 1H, N]CH), 7.69e6.76 (m, 14H, AreH), 6.41 (s, 1H,
AreNH), 4.21 (s, 2H, AreCH2); 13C NMR (DMSO-d6,
ppm): 169.95
d
H
37Rv (5 ꢄ104 bacilli per tube). These tubes were then incubated at
d
37 ꢃC. Growth of bacilli was seen after 30 days of incubation. Tubes
having the compounds were compared with control tubes where
medium alone was incubated with H37Rv. The concentration at
which complete inhibition of colonies occurred was taken as active
concentration of test compound.
(C3 of triazole), 160.82 (C]O of quinazoline), 156.63 (C]N), 148.12
(C5 of triazole), 147.18e117.24 (aromatic carbons), 27.44 (AreCH2).
LC-MS m/z: 528.09 (Mþ), 530.09 (Mþ2).
4.6.5. 3-((Thiophen-2-yl)methyleneamino)-2-(2-(2,6-dichloro-
phenylamino)benzyl)-[1,2,4]triazolo[5,1-b]quinazolin-9(3H)-one
(6e)
5.2. In-vitro antibacterial activity
Yield: 73%, M.p. 156e158 ꢃC, IR (
n
cmꢀ1, KBr): 3332 (NH), 3052
The Minimum Inhibitory Concentration (MIC) of the synthe-
sized compounds was determined by twofold serial dilution tech-
nique. The study involved a series of six assay tubes for each test
compound against four bacterial strains. The entire test was done in
duplicate. To the first assay tube, 1.8 mL of seeded broth and 0.2 mL
(CeH), 2985 (CeH),1599 (C]O), 1523 (C]N); 1H NMR (DMSO-d6,
ppm) : 10.56 (s, 1H, N]CH),7.73e6.64 (m, 14H, AreH), 6.45 (s, 1H,
AreNH), 4.29 (s, 2H, AreCH2); 13C NMR (DMSO-d6,
ppm): 171.14
d
d
(C3 of triazole), 159.99 (C]O of quinazoline), 155.21 (C]N), 148.27
(C5 of triazole), 145.57e113.64 (aromatic carbons), 28.35 (AreCH2).
LC-MS m/z: 544.06 (Mþ), 546.06 (Mþ2).
of the test compound (1 mM) were added and mixed thoroughly
and the twofold serial dilution was done up to the sixth tube
containing 1 mL of the seeded broth. The additions of the drug
solution and serial dilution were done under strict aseptic condi-
tions. Solvent control, negative control (growth control) and drug
control were maintained. The assay tubes were incubated for 24 h
at 37 ꢃC. The lowest concentration which apparently caused com-
plete inhibition of growth of microorganisms was considered as the
minimum inhibitory concentration (MIC).
4.6.6. (E)-2-(2-(2,6-Dichlorophenylamino)benzyl)-3-(pyridin-4-
ylmethyleneamino)-[1,2,4]triazolo[5,1-b]quinazolin-9(3H)-one (6f)
Yield: 71%, M.p. 157e159 ꢃC, IR ( cmꢀ1, KBr): 3297 (NH), 3010
n
(CeH), 2988 (CeH),1579 (C]O), 1522 (C]N); 1H NMR (DMSO-d6,
ppm): 10.10 (s, 1H, N]CH), 7.59e6.64 (m, 15H, AreH), 6.45 (s, 1H,
AreNH), 4.26 (s, 2H, AreCH2); 13C NMR (DMSO-d6,
ppm): 171.83
d
d

K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
527
5.3. Cytotoxicity screening
along with their calculated molecular descriptors and biological
activity data are given Table 7. MIC activity originally determined in
The cytotoxicity of the best active compounds was assessed by
Microculture Tetrazolium Assay (MTT) [29e32] against Vero and
HepG2 cells. The stock solutions of the test compounds were
diluted in a 96 deep well plate aseptically with MEM (without FBS)
micromolar values (mM/mL) and then converted to a pMIC value by
taking Log (1/MIC). The plot of predicted pMIC against experi-
mental pMIC is represented in Fig. 5. The pMIC values were used as
the dependent variable in all PLS models subsequently developed.
to achieve concentrations 300, 250, 200,150, 100 and 50 mM. The 96
well plates containing the cells were taken and kept inverted on
5.4.4. CoMFA and CoMSIA studies
filter paper to remove the supernatant media and washed gently
CoMFA calculation was carried out using default values. The
steric (Lennard-Jones) and electrostatic (Coulomb) fields were
calculated at each intersection on the regularly spaced grid. 3D
cubic lattice with grid spacing of 2 Å and extending to 4 Å beyond
the aligned molecules in all directions was used. Steric and elec-
trostatic fields energies were calculated using a sp [3] carbon as the
steric probe atom and þ1 charge (default probe atom in SYSYL) for
the electrostatic probe with the cutoff energy of 30 kcal/mol.
CoMSIA similarity index descriptors were derived using the same
lattice box used in CoMFA calculations. The standard settings were
used in CoMSIA to calculate five different fields like steric, elec-
trostatic, hydrophobic, acceptor and donor. The default value of 0.3
was used as the attenuation factor (R). The statistical evaluation for
the CoMSIA analyses were performed in the same way as described
for CoMFA.
with PBS and decanted. 100
perimeter wells. Then 100
m
L of sterile water were added to outer
m
L of each test compound dilutions were
added to the wells. DMSO was used as control and the plates were
incubated in incubator (5% CO2) at 37 ꢃC 72 h and 24 h for Vero cells
and HepG2 respectively. After the incubation, plates were inverted
on filter paper to remove the supernatant media followed by PBS
washing. To this, 50 mL of MTT solution was added to each well in
dark place and incubated for 3 h. After the incubation, the MTT
solution was removed from the well by inverting gently on filter
paper and 50 mL of DMSO was added to each well and kept in dark
place for 1e2 h. Then the optical density readings of the plates were
taken using Elisa reader at 540 nm.
Determination of safety profile (CC50
)
Optical Density of Test
Opatical Density of Control
% Cell Viability ¼
ꢄ 100
(1)
(2)
5.4.5. Partial least squares (PLS) analysis
The CoMFA or CoMSIA descriptors were used as independent
variables and pMIC values as dependant variables in PLS regression
analysis. SAMPLS leave-one-out (LOO) Cross-validated PLS was
performed to determine the optimal number of components to be
used in the subsequent final analysis. Column filtering value was set
2.0 kcal/mol by omitting those lattice points those energy variation
is below this threshold to improve the signal-to-noise ratio. Finally,
no-cross-validated analysis was performed and employed to
analyze the result of the CoMFA and CoMSIA models. The predictive
ability of the models is expressed by conventional correlation co-
efficient r2 value, which is analogous to cross-validated r2 (q2). The
cross-validated coefficient q2 (or r2) was evaluated by following
equations:
% Cell Inhibition ¼ 100 ꢀ % Cell Viability
CC50 was calculated by extrapolating a graph with % cell inhi-
bition on Y-axis against concentration of test compound on X-axis.
5.4. 3D-QSAR model on M. tuberculosis H37Rv
5.4.1. Data set
In this study, in-vitro antitubercular activity data against
M. tuberculosis H37Rv were selected to construct the 3D-QSAR
models, Tables 1 and 2 listed their structures and biological data
respectively. For CoMFA and CoMSIA analysis, 24 compounds with
determinate MIC values of M. tuberculosis H37Rv were employed
and their MIC values were converted to pMIC values according to
the following equation:
Table 7
Physicochemical properties and activity of training set & test set compounds.
Compound
MIC
pMIC
ClogP
CMR
PSA
pMIC ¼ ꢀlogðpMICÞ
(3)
Training set
4b
4c
4d
4e
6.250
5.204
5.204
5.505
5.796
4.903
5.204
4.903
4.903
5.204
4.903
4.301
4.301
4.602
4.903
4.903
4.602
4.602
4.602
6.99
6.73
6.29
6.95
5.80
8.51
8.20
7.94
7.51
7.02
6.13
6.05
5.48
4.64
4.64
5.70
4.76
4.51
16.35
16.97
14.90
15.55
15.53
16.05
16.66
17.28
15.22
15.84
14.28
14.28
14.25
14.06
14.06
16
85.03
6.250
3.125
1.60
12.50
6.250
12.50
12.50
6.250
12.50
50
50
25
12.5
12.5
25
96.859
85.905
95.513
94.80
5.4.2. Structure optimization and alignment
The CoMFA and CoMSIA studies were performed using SYBYL X
2.1 (Certara L.P.) molecular modeling software on windows-7 pro-
fessional operating system. The molecules were drawn using
ChemSketch. Energy minimizations were performed using Tripos
force field and conjugate gradient method with a convergence
criterion of 0.05 kcal/mol (Å). Charges were calculated by the
Gasteiger-hücker method. Subsequently, all structures were
aligned on the common substructure N-(2,6-dichlorophenyl)-2-
methylbenzenamine nucleus (Fig. 2(A) depicted in bold) with
potent compound 4a was chosen as a template molecule.
4f
4g
4h
4i
4j
4l
5a
5b
5e
5f
6a
54.77
68.695
80.529
69.576
78.407
154.075
173.251
177.095
178.444
61.287
74.958
76.934
84.709
6b
6d
6f
5.4.3. Training and test set preparation
25
25
14.55
15.17
The study began with an initial set of 24 diphenylamine con-
taining 1,2,4-triazoles compounds with activity against
M. tuberculosis. The remaining 6 compounds were selected as test
set used in external validation. The test set compounds were
selected such that their activity and physical properties (MW and
cLogP) broadly reflective of the training set characteristics. The
training set and test set 1,2,4-triazole compounds used in this study
Training set
4a
4k
5c
5d
6c
6e
0.20
12.50
50
25
25
6.699
4.903
4.301
4.602
4.602
4.903
7.30
8.16
5.79
4.65
5.44
5.34
15.74
15.86
15.51
13.60
16.62
15.20
71.25
79.132
165.113
138.489
86.548
86.868
12.5

528
K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
Fig. 5. Calculated pMIC versus experimental pMIC values for the 18 training set molecules obtained by PLS analysis using CoMFA and CoMSIA models.
hꢀ
ꢁ.
i
ꢀ
ꢁ
ꢁ
2
P
r2 ꢀ r02 r2 < 0:1
Ypred ꢀ Yactual
y
q2 ¼ 1 ꢀ
(4)
ꢀ
2
P
Ypred ꢀ Ymean
0 : 85 ꢁ k ꢁ 1 : 15
y
where k is the slope of regression lines through the origin.
k is obtained according to the following formula:
where
P
Ypred ¼ predicted activity;
yyi
yi2
k ¼
(7)
P
Yactual ¼ experimental activity;
Ymean ¼ the best estimate of the mean.
where
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
y ¼ Experimental activity of test set of compounds
yi ¼ Predicted activity of test set of compounds
R20 is calculated as follows
PRESS
SEE; SEP ¼
(5)
(6)
n ꢀ c ꢀ 1
where
n ¼ number of compounds;
c ¼ number of components;
À
Á
P
P
2
yi ꢀ yrio
R20 ¼ 1 ꢀ
(8)
ꢀ
ꢁ
2
yi ꢀ yi
ꢀ
ꢁ
2
PRESS ¼ Ypred ꢀ Yactual
where
yrio ¼ kyi
5.4.6. External validation of the 3D-QSAR models
According to Roy and Roy [34e36] for a model with good
external predictability, a difference between r2 and r02 values needs
to be studied. An additional statistic for external validation r2m was
introduced by the following equation.
The predictive abilities of the CoMFA and CoMSIA models were
determined from a test set of compounds not included in the model
generation. The experimental, predicted and their residual values
of the 18 training set inhibitors and six test set inhibitors are given
in Table 5. For a QSAR model, internal validation of leave-one-out
cross-validated q2 is commonly used. But Golbraikh and Tropsha
[33] reported that the high value of q2 was necessary and important
but not the sufficient condition for a model to have a high predic-
tive power. To estimate the predictive ability of the QSAR model,
squared correlation coefficient values between the observed and
predicted values of the test set compounds with intercept (r2) and
without intercept (r20) were calculated. According to Golbraikh and
Tropsha, models are considered acceptable if they satisfy all of the
following conditions:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢂ
ꢂ
rm2 ¼ 1 ꢀ
r ꢀ r0
(9)
2
2
ꢂ
ꢂ
A value of r2m greater than 0.5 may be taken as an indicator of
good external predictability.
6. Conclusion
In summary, twenty four 1,2,4-triazole derivatives comprising of
diphenyl amine moiety were synthesized and evaluated for in-vitro
antitubercular activity against M. tuberculosis H37Rv. The Mannich
bases 4a, 4d and 4e were found to be the most potent molecules
with MIC value in the range of 0.2 mMe3.125 mM. The cytotoxicity
analysis of the most active compounds have carried out by MTT
assay for Vero and HepG2 cell lines, none of the tested compounds
q2 > 0:5
r2 > 0:6

K. Mohan Krishna et al. / European Journal of Medicinal Chemistry 84 (2014) 516e529
529
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