Synthesis and pharmacological evaluation of novel 4-isopropylthiazole-4- phenyl-1,2,4-triazole derivatives as potential antimicrobial and antitubercular agents

Article abstract of DOI:10.1007/s00044-012-0092-0

A series of novel 4-isopropylthiazol-4-phenyl-1,2,4-triazol derivatives, N′-(substituted benzylidene)-2-(5-(4-isopropylthiazol-2-yl)-4-phenyl-4H-1, 2,4-triazol-3-ylthio)acetohydrazides 4a-e, 4-isopropylthiazol-2-yl-4′- phenyl-4′H-1′, 2′,4′-triazol-3′-ylthio (substituted methyl benzylidene) acetohydrazides 5a-f, 3-(4-isopropylthiazol-2- yl)-4-phenyl-5-(5-substituted-1,3,4-oxadiazol-2-ylthio)-4H-1,2,4-triazole derivatives 6a-f and N-acetyl-5′-(4-isopropylthiazol-2-yl)-4′- phenyl-4′H-1,2,4-triazol-3′-ylthio) acetohydrazide 7 were synthesized and characterized by spectroscopy, elemental, and mass spectral analysis. These compounds were evaluated for their preliminary in vitro antibacterial, antifungal, and antitubercular activity against Mycobacterium tuberculosis (M. tuberculosis) H37Rv strain by broth dilution assay method. All the compounds exhibited moderate to significant antibacterial and antifungal activities. Results of the antitubercular screening against Mycobacterium tuberculosis H37Rv showed that compounds 4c and 6c exhibited good antitubercular activity when compared with first line drug isoniazid.

Full text of DOI:10.1007/s00044-012-0092-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:938–948
DOI 10.1007/s00044-012-0092-0
ORIGINAL RESEARCH
Synthesis and pharmacological evaluation of novel
4-isopropylthiazole-4-phenyl-1,2,4-triazole derivatives
as potential antimicrobial and antitubercular agents
•
G. V. Suresh Kumar Y. Rajendra Prasad
•
S. M. Chandrashekar
Received: 26 October 2011 / Accepted: 14 May 2012 / Published online: 26 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of novel 4-isopropylthiazol-4-phenyl-1,
2,4-triazol derivatives, N0-(substituted benzylidene)-2-(5-(4-
isopropylthiazol-2-yl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-
acetohydrazides 4a–e, 4-isopropylthiazol-2-yl-40-phenyl-40H-10,
20,40-triazol-30-ylthio (substituted methyl benzylidene) acet-
ohydrazides 5a–f, 3-(4-isopropylthiazol-2-yl)-4-phenyl-
5-(5-substituted-1,3,4-oxadiazol-2-ylthio)-4H-1,2,4-triazole
derivatives 6a–f and N-acetyl-50-(4-isopropylthiazol-2-yl)-40-
phenyl-40H-1,2,4-triazol-30-ylthio) acetohydrazide 7 were
synthesized and characterized by spectroscopy, elemental,
and mass spectral analysis. These compounds were eval-
uated for their preliminary in vitro antibacterial, anti-
fungal, and antitubercular activity against Mycobacterium
tuberculosis (M. tuberculosis) H37Rv strain by broth
dilution assay method. All the compounds exhibited
moderate to significant antibacterial and antifungal
activities. Results of the antitubercular screening against
Mycobacterium tuberculosis H37Rv showed that com-
pounds 4c and 6c exhibited good antitubercular activity
when compared with first line drug isoniazid.
Keywords 4-Isopropylthiazole ꢀ 1,2,4-Triazole ꢀ
Cytotoxicity ꢀ Antimicrobial ꢀ Antitubercular activity
Introduction
Tuberculosis (TB), a contagious infection caused by
Mycobacterium tuberculosis (MTB), still remains the
leading cause of the worldwide death among the infectious
disease (Espinal, 2003; Mitchison, 1993). The synergy
between tuberculosis and the AIDS epidemic as well as the
surge of multidrug-resistant isolates of MTB has reaffirmed
tuberculosis as a primary public health threat (Young and
Cole, 1993; Bloom and Murray, 1992).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). The current TB treatment comprises of
3–4 drugs for a period of 6–9 months. Novel drugs are
urgently required which can shorten this long-treatment
period and target multidrug-resistant strains of TB.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0092-0) contains supplementary
material, which is available to authorized users.
G. V. Suresh Kumar (&)
Department of Medicinal Chemistry, St Johns Pharmacy
College, 6, 2nd Stage Vijaynagar, R.P.C Layout, Bangalore 560
040, Karnataka, India
A key target for antimycobacterial chemotherapy is cell
wall biosynthesis. The cell envelope of MTB comprises
four classes of polymer (peptidoglycan, arabinogalactan,
mycolic acids, and lipoarabinomanan). This complex li-
poglycan calyx on cell surface provides a significant
physical barrier to intracellular-acting drugs (Kuo et al.,
2003). Azole derivatives have demonstrated interesting
antitubercular activity in addition to its antimicrobial
activity. It is established that these compounds reach target
by transmembrane diffusion because of its lipophilic
property and target the sterol demethylase, a mixed
e-mail: gvsureshkumar@yahoo.com
Y. Rajendra Prasad
AU College of Pharmaceutical Sciences, Andhra University,
Visakhapatnam 530 003, Andhra Pradesh, India
e-mail: dryrp_au@rediffmail.com
S. M. Chandrashekar
Poornaprajna Institute of Scientific Research (PPISR),
Poornaprajnapura, Post: Bidalur, Ta: Devanahalli, Near
Woodrich Resort, Bangalore 562 110, Karnataka, India
e-mail: Chandra_jan25@yahoo.co.in
123

Med Chem Res (2013) 22:938–948
939
function oxidase involved in sterol synthesis in eukaryotic
organism (Walczak et al., 2004; Babaoglu et al., 2003).
Thiazole and its derivatives are reported to be physio-
logically and pharmacologically active heterocycle and
utilized in treatment of several diseases like epilepsy,
diabetes, antifertility, and sedative-hypnotic or anesthetic
action. Isopropyl thiazole is an important pharmacophore
and privileged structure in medicinal chemistry encom-
passing a diverse range of biological activities including
antibacterial and antitubercular activity (Khalaf et al.,
2004).
research on thiazoles, (Shiradkar et al., 2007; Mallikarjun
et al., 2007; Mallikarjuna et al., 2009; Suresh Kumar et al.,
2010a) it was contemplated to synthesize a series of novel
4-isopropylthiazol-4-phenyl-1,2,4-triazol derivatives to
study their cytotoxicity, antimicrobial (bacterial and fun-
gal), and antitubercular activity against H37Rv strain.
Chemistry
The general synthetic strategy employed to obtain the title
compounds with excellent yields is depicted in Scheme 1 and
their physical properties are depicted in Table 1. The key
intermediate in the present study 5-(4-isopropylthiazol-2-yl)-
4-phenyl-4H-1,2,4-triazole-3-thiol 1 was prepared as per the
literature (Suresh Kumar et al., 2010a). Acetylation of com-
pound 1 by refluxing with ethyl bromoacetate in the presence
of sodium hydroxide and absolute ethanol yielded compound
2. The important intermediate 50-(4-isopropylthiazol-2-yl)-4-
phenyl-40H-1,2,4-triazol-3-ylthio) acetohydrazide 3 was
1,2,4-Triazoles represent an overwhelming and rapid
developing field in modern heterocyclic chemistry. For
instance, some biheterocyclic compounds, consisting of
clubbed 1,2,4-triazole and thiazoles or 1,3,4 oxadiazole
rings are reported as novel azole class of antimycobacte-
rials, which are proved to be highly active both in vitro and
in vivo experiments (Demirbas et al., 2004; Bayrak et al.,
2009; Turan-Zitouni et al., 2005; Rida et al., 1986). So,
after extensive literature search and in continuation of our
N
N
N
SH
N
S
1
i
N
N
O
N
S
N
O
S
2
ii
H
R
N
N
N
N
N
H
N
N
N
N
N
H
N
N
N
S
R
N
S
NH2
N
N
O
iii
S
iv
S
O
N
S
O
S
5a-f
4a-e
i
v
3
H
N
N
O
N
N
N
N
N
R
NH
N
N
N
S
S
N
COCH3
S
O
S
6a-f
7
i. ethyl bromoacetate, NaOH, ethanol, reflux 4h ii. hydrazine hydrate, ethanol, reflux 4h iii. aryl aldehyde, ethanol, glacial acetic acid, reflux 2h
iv. acetophenone, ethanol,glacial acetic acid reflux 2h v. aromatic acid, POCl3, reflux 6h
Scheme 1 Synthesis of compounds 2, 3, 4a–4e, 5a–5f, 6a–6f and 7
123

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Med Chem Res (2013) 22:938–948
Table 1 Analytical and physico-chemical data of synthesized compounds CHCl3:CH3OH (9:1)
Compound
R
Molecular
formula
M.W
Solvent system
M.p (°C)a/
crystallization
solvent
Yield % Analysis of C, H ,N found (Calc.)b
(%)
C
H
N
2
–
C18H20N4O2S2
388
374
522
–
98–100 (Ethanol)
85
55.65 (55.62) 5.19 (5.16) 14.42 (14.43)
51.32 (51.38) 4.84 (4.85) 22.44 (22.47)
59.86 (59.88) 5.22 (5.23) 13.43 (13.46)
58.11 (58.16) 4.47 (4.49) 14.12 (14.13)
60.36 (60.35) 4.85 (4.82) 14.66 (14.61)
59.15 (55.16) 4.96 (4.97) 13.80 (13.85)
56.90 (55.92) 4.38 (4.37) 16.59 (16.54)
57.67 (57.65) 4.65 (4.67) 16.14 (16.11)
61.76 (61.75) 5.38 (5.39) 13.85 (13.86)
61.08 (61.09) 5.13 (5.11) 14.25 (14.21)
61.20 (61.23) 5.34 (5.39) 17.13 (16.11)
51.89 (51.84) 4.17 (4.15) 15.13 (15.17)
56.40 (56.44) 4.54 (4.51) 16.44 (16.43)
59.98 (59.90) 4.38 (4.37) 18.25 (18.22)
49.93 (49.97) 3.96 (3.97) 19.41 (19.46)
55.81 (55.79) 3.87 (3.81) 16.98 (16.95)
54.64 (55.66) 3.79 (3.83) 19.39 (19.33)
60.74 (60.72) 4.67 (4.66) 17.71 (17.65)
57.97 (57.89) 4.23 (4.22) 17.63 (17.64)
51.90 (51.89) 4.84 (4.82) 20.18 (20.15)
3
–
C
16H18N6OS2
CHCl3:CH3OH (9:1)
CHCl3:CH3OH (9:1)
CHCl3:CH3OH (9:2)
CHCl3:CH3OH (9:1)
CHCl3:CH3OH (9:2)
CHCl3:CH3OH (9:1)
218–220 (Ethanol) 89
193–195 (Ethanol) 82
148–150 (Ethanol) 92
203–205 (Ethanol) 86
160–162 (Ethanol) 93
223–225 (Ethanol) 84
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
7
3,4-OCH3
4-Cl
C22H26N6O3S2
C
C
23H21ClN6OS2 496
4-OH
23H22N6O2S2
478
507
506
521
506
492
3-OCH3, 4-Cl C24H24N6O3S2
3-NO2
4-NO2
4-OCH3
4-OH
C23H21N7O3S2
C24H23N7O3S2
C25H26N6O2S2
CHCl3:C2H5OH (9:3) 187–189 (Ethanol) 84
CHCl3:C2H5OH (9:2) 175–178 (Ethanol) 79
CHCl3:C2H5OH (9:3) 171–173 (Ethanol) 81
C
24H24N6O2S2
4-CH3
4-Br
C25H26N6OS2
490.16 CHCl3:C2H5OH (9:2) 162–165 (Ethanol) 81
24H23BrN6OS2 554.06 CHCl3:C2H5OH (9:3) 154–157 (Ethanol) 74
24H23ClN6OS2 510.11 CHCl3:C2H5OH (9:1) 163–165 (Ethanol) 68
C
C
4-Cl
C6H5
C23H20N6OS2
460
CHCl3:CH3OH (9:1)
CHCl3:CH3OH (9:2)
117–120 (Ethanol) 69
190–193 (Ethanol) 74
183–185 (Ethanol) 72
192–194 (Ethanol) 68
182–185 (Ethanol) 73
171–174 (Ethanol) 69
198–200 (Ethanol) 67
CH2Cl
C6H4Cl
C6H4NO2
C6H4CH3
C6H4OH
–
C18H17ClN6OS2 432
C23H19ClN6OS2 475
CHCl3:CH3OH (9:1)
C23H19N7O3S2
C24H22N6OS2
C23H20N6O2S2
505.1
CHCl3:CH3OH (9:2)
474.13 CHCl3:CH3OH (9:1)
476.11 CHCl3:CH3OH (9:2)
C
18H20N6O2S2
416
CHCl3:CH3OH (9:1)
M.W Molecular weight of the compounds
a
Melting point range of the compounds
b
Elemental analysis of C, H, and N were with in 0.4 % of theoretical value
obtained on treating compound 2 with hydrazine hydrate in
the presence of absolute ethanol.
treating compound 3 with acetic anhydride in presence of
ethanol.
The treatment of acetohydrazide derivative 3 with
substituted aromatic aldehydes affords schiff bases 4a–e.
The compounds 4a–e having arylidene hydrazide struc-
ture may exist as E/Z geometrical isomers about –C=N
double bond and as cis/trans amide conformers. Accord-
ing to the literature (Galic et al., 2001), the compounds
containing imine bond are present in higher percentage in
dimethyl-d6 sulfoxide solution in the form of geometrical
E isomer about –C=N double bond. The Z isomer can be
stabilized in less polar solvents by an intramolecular
hydrogen bond. In the present study, the spectral data
were obtained in dimethyl-d6 sulfoxide solution, hence no
signal belonging to Z isomer for compounds 4a–e was
observed.
Biological activity
The standard strains were procured from the American
Type Culture Collection (ATCC) and Gene Bank, Institute
of Microbial Technology, Chandigarh, India. The anti-
bacterial activity of the synthesized compounds (3, 4a–e,
5a–f, 6a–f, and 7) was performed by broth dilution method
(Eweiss et al., 1986) against the following standard bac-
terial strains: Staphylococcus aureus (ATCC 11632),
S. faecalis (ATCC 14506), Bacillus subtilis (ATCC
60511), Klebsiella pneumoniae (ATCC 10031), and
Escherichia and Pseudomonas aeruginosa (ATCC 10145);
and antifungal activity against yeasts: Saccharomyces
cerevisiae (ATCC 9763, Sc) and Candida tropicalis
(ATCC 1369, CT); and mold: Aspergillus niger (ATCC
6275). MIC of compounds was determined against MTB
H37Rv strain by broth dilution assay method. The cyto-
toxicity of the synthesized compounds was evaluated for
their cytotoxic potential using A549 (lung adenocarci-
noma) cell lines in the presence of fetal bovine serum.
The treatment of compound 3 with substituted ace-
tophenones in the presence of catalytic amount of glacial
acetic acid affords acetohydrazides 5a–f. Compound 3 on
reflux with appropriate aromatic acids yielded a series of
3-(4-isopropylthiazol-2-yl)-4-phenyl-5-((5-substituted-1,3,
4-oxadiazol-2-yl)methylthio)-4H-1,2,4-triazoles 6a–f.
Compound 7, N-acetyl-5-(4-isopropylthiazol-2-yl)-4-phenyl-
4H-1,2,4-triazol-3-ylthio) acetohydrazide was synthesized on
123

Med Chem Res (2013) 22:938–948
Results and discussion
Chemistry
941
Structurally, the modifications engendering these desir-
able drug properties (enhanced penetration and decreased
resistance) are optimally made at N2 of the hydrazide
framework (Fig. 1). Such modifications block the resulting
molecule against the action of N-arylaminoacetyl trans-
ferases (NATs). These enzymes are found in both myco-
bacteria and their mammalian hosts, and they deactivate
hydrazides by means of reaction at N2. NATs have been
implicated in the development of resistance, particularly
among those patients known as the ‘‘fast acetylators,’’ for
whom there is an inherent problem of chronic underdosing
of INH (isonicotonic hydrazide) under genetic control.
Improving serum drug concentrations should narrow this
range (Nuermberger and Grosset, 2004; Kinzig-Schippers
et al., 2005; Balcells et al., 2006), and structurally blocking
hydrazide toward the actions of NATs at N2 may thus
combat the rise of resistance. In line with the above dis-
cussion, our rationale has been to prepare various Schiff
bases (4a–e, 5a–f) with enhanced lipophilicity and to
examine their efficacy against Gram-positive, Gram-nega-
tive bacteria, yeasts, molds, and MTB H37Rv by inducting
halogens, which are known for inductive, conjugative,
steric and/or electronic effects that can be directly involved
in the biological activity (i.e., antimicrobial).
The –SH proton of compound 1 is acidic enough and
substitution reaction could be achieved on this group in the
presence of a base (Klimesova et al., 2004; Pomarnacka
and Kornicka 2001). In the present study, compound 2, an
acetic acid ester derivative of compound 1, was obtained in
good yield. 1H NMR spectrum of compound 2 depicts
additional signals derived from ester group at 1.33 (–OCH2
CH3), 3.91 (–SCH2), and 4.12 (–OCH2CH3) ppm inte-
grating for three protons, two protons, and two protons,
respectively. The signals of 13C NMR spectrum of the
compound 2 belonging to the same groups were recorded at
13.24, 32.5, and 63.24 ppm, respectively.
The 1H NMR spectrum of compound 3 displayed signals
derived from hydrazide structure appeared at 4.52 and 9.35
(–NHNH2) ppm integrating for two protons and one pro-
ton, respectively (controlled by changing D2O). The 1H and
13C NMR spectra of compounds 4a–e displayed additional
signals due to the aromatic ring derived from aldehyde
moiety at aromatic region, while the signal belonging to
–NH2 group of hydrazide structure was absent. The signals
belonging to –SCH2, –N=CH, and –NH groups were
observed between 3.32 and 3.72, 8.23 and 8.71, and 11.05
and 11.71 ppm, respectively.
1H NMR of compound 5a showed a signal at d 2.32 due
to methyl group and peaks between d 7.2 and d 7.5
accountable for aromatic benzyl group. Further, mass
spectrum of compound 5a showed a molecular ion peak
m/z 520.09 which confirmed its molecular weight. The
structure of compound 6a was confirmed by lack of reso-
nances corresponding to NH and NH2 in the 1H NMR
spectrum and appearance of peaks around 144.11 and
164.32 due to C2 and C5 of oxadiazole in 13C NMR spectra.
Further, molecular ion base peak at m/z 460.11 confirmed
structure of compound 6a.
The antimicrobial activity of acetohydrazide derivatives
4a–e, 5a–f divulged that compound 4a comprising 3,4-di
methoxy and 5b comprising 4-methoxy substitution (elec-
tron withdrawing) showed improved antimicrobial activity
against tested bacterial and fungal species. This excellent
inhibition of compound 4a is attributed to participation of
the free electron pairs on the oxygen by resonance and
increased electron density in the aromatic system. Com-
pounds 4c possessing 4-OH on phenyl ring exhibited
moderate antimicrobial activity and excellent antitubercu-
lar activity against MTB H37Rv at MIC 8 mg/mL.
In drug design, halogen atoms are used to improve
penetration through lipid membranes and tissues. They
may also present a significant reactivity depending on the
structure of the molecule. In this work, the introduction of a
para-halogen substituent in the N-phenyl ring of 4b (4-Cl),
5f (4-Cl), or 5e (4-Br) presented a negative effect as they
reduced the antitubercular activity (Table 2). Probably, the
halogen reactivity at R position compromised the original
interactions of the non-substituted compounds with the
bacterial target. The SAR analysis also pointed the volume
of the halogen as a feasible restrictive factor since bromine,
the largest halogen, is more deleterious to the antituber-
cular activity than chlorine. Based on these data, we may
infer that substituent (R) is a steric and/or restricted posi-
tion that should be carefully considered in the future design
of antitubercular targets.
Biological activity
The results of antimicrobial testing of synthesized com-
pounds (3, 4a–e, 5a–f, 6a–f, and 7) against selected Gram-
positive, Gram negative bacteria, yeasts, molds, and MTB
H37Rv are illustrated in Tables 2 and 3, respectively.
Several recent experiments indicate that the incorpora-
tion of hydrophobic moieties into the framework of acet-
ohydrazide enhance penetration of the drug into the tissues
of the mammalian host and into the waxy cell wall of the
bacterium. This strategy of drug design has been proposed
as a vehicle for controlled study of the growth cycle of the
pathogen as well as a means of augmenting fundamental
drug activity.
Evaluating the antimicrobial activity of the synthesized 2-
(4-isopropylthiazol-2-yl)-5-substituted-1,3,4-oxadiazole
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Med Chem Res (2013) 22:938–948
Table 2 Antimicrobial activity Table 2 Antimicrobial activity of synthesized compounds 2, 3, 4a–e, 5a–f, 6a–f and 7 expressed as MIC (lg/mL)
expressed as MIC (lg/mL)
Compounds
Gram-positive organismsa
Gram-negative organismsb
Fungic
Sa
Sf
Bs
Kp
Ec
Pa
Sc
Ct
An
2
125
16
125
62.5
31.25
8
125
8
125
8
62.5
8
62.5
62.5
16
125
31.25
31.25
31.25
62.5
62.5
31.25
31.25
125
8
62.5
31.25
16
3
31.25
12
4a
8
16
16
31.25
62.5
62.5
16
4b
62.5
125
125
125
31.25
31.25
62.5
125
16
31.25
16
31.25
125
16
62.5
16
31.25
125
8
16
31.25
31.25
31.25
125
125
125
8
4c
162.5
16
4d
125
8
4e
31.25
31.25
31.25
31.25
62.5
125
31.25
125
62.5
62.5
31.25
62.5
125
31.25
31.25
4
16
31.25
62.5
125
16
62.5
62.5
62.5
16
62.5
125
62.5
31.25
62.5
31.25
31.25
16
5a
31.25
250
31.25
16
5b
5c
5d
125
62.5
16
62.5
125
31.25
31.25
16
125
125
31.25
16
31.25
31.25
125
31.25
31.25
125
62.5
125
31.25
8
5e
31.25
62.5
62.5
31.25
62.5
31.25
62.5
125
31.25
B1
5f
31.25
31.25
8
31.25
31.25
16
6a
125
16
6b
62.5
62.5
62.5
125
31.25
16
125
125
31.25
31.25
31.25
8
6c
8
8
62.5
62.5
31.25
62.5
62.5
B1
62.5
31.25
62.5
31.25
125
B5
6d
31.25
62.5
31.25
125
B5
31.25
31.25
62.5
125
62.5
125
31.25
62.5
B1
6e
6f
7
Ciprofloxacin
Norfloxacin
Flucanozole
a
B5
–
–
–
B5
B5
B1
B1
B1
B5
–
–
–
–
–
–
–
–
–
B1
B1
B1
The screening organisms. Gram-positive bacteria: Staphylococcus aureus (ATCC 11632, Sa), Streptococcus faecalis (ATCC 14506, Sf), and
Bacillus subtilis (ATCC 60511, Bs)
b
The screening organisms. Gram-negative bacteria: Klebsiella penumoniae (ATCC 10031, Kp), Escherichia coli (ATCC 10536, Ec), and
Pseudomonas aeruginosa (ATCC 10145, Pa)
c
The screening organisms. Yeasts: Saccharomyces cerevisiae (ATCC 9763, Sc) and Candida tropicalis (ATCC 1369, Ct), mold: Aspergillus
niger (ATCC 6275, An)
derivatives 6a–f revealed that compounds were more effec-
tive against Gram-positive bacteria at MIC 4–31.25 mg/mL.
Compounds 3, 4c, 5c, and 6c were tested for their cytotoxic
potential using A549 (lung adenocarcinoma) cell lines in the
presenceoffetalbovineserum.AsshowninFig. 2, compound
4c showed maximum cytotoxicity at a concentration of
250 lM. The other compounds 3, 5c, and 6c showed appre-
ciable cytotoxicity of about 50 % of the vehicle control at a
concentration of 250 lM.
derivatives (3, 4a–e, 5a–f, 6a–f, and 7) and their in vitro
evaluation of antimicrobial (bacterial and fungal) and
antitubercular activity against H37Rv strain. Antimicrobial
study revealed that compounds 4a, 4c, 5c, and 6c demon-
strated significant activity against tested Gram-positive and
Gram-negative bacteria and fungal species. The in vitro
antituberculosis screening of these series showed that all
compounds were active, in particular compounds 4c and 6c
exhibited excellent antitubercular activity at MIC 8 and
4 lg/mL, respectively, when compared with first line drug
Isoniazid. The promising in vitro antimicrobial activity and
low-toxicity profile of clubbed isopropylthiazole class of
compounds make them certain promising molecules for
further lead optimization in the development of novel an-
timycobacterial agents.
Conclusion
In conclusion, this work demonstrates the synthesis of a
series of novel 4-isopropylthiazol-4-phenyl-1,2,4-triazol
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Med Chem Res (2013) 22:938–948
943
250.0 µM
200.0 µM
150.0 µM
100.0 µM
50.0 µM
Table 3 Primary antitubercular activity of synthesized compounds 2,
3, 4a–e, 5a–f, 6a–f, and 7
4
3
2
1
0
Compound
MIC values (lg/mL)
of MTB H37Rv
2
125
16
3
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
5f
8
125
8
31.25
62.5
62.5
8
16
Treatment
62.5
31.25
31.25
62.5
16
Fig. 2 Cytoxic activity of compounds 3, 4c, 5c, and 6c tested in
A549 cells by MTT assay. The bars reflect the viable cells in each
treatment. Cells represents, cells alone without any treatment, DMSO
denote the vehicle control. The experiment was done in duplicate with
triplicate readings of each experiment
6a
6b
6c
6d
6e
6f
4
125
62.5
62.5
62.5
0.25
solvents in varying proportions and spots were observed
using iodine as visualizing agent.
7
Synthesis of 50-(4-isopropylthiazol-2-yl)-ethyl-2-(40-phenyl-
40H-10,20,40-triazol-30-ylthio)acetate 2
Isoniazid
Compound 2 was synthesized by refluxing an equimolar
mixture of compound 1 (0.01 mol), NaOH, and ethyl
bromoacetate for 4 h. The reaction mixture was cooled and
crystalline mass obtained was recrystallised.
IR (KBr) m max, cm-1: 3,068 (Ar C–H str), 1,730
(C=O), 1,189 (O–C2H5), 765 (C–S).
N1
N1
N
N
N
H
N
H
N
O
N
S
NH2
S
NH2
O
N2
N2
N
Compound-3
Isoniazid
1H NMR (DMSO-d6, 300 MHz) d: 7.2–7.5 (m, 5H,
Ar–H), 7.32 (s, 1H, thiazole-C5), 4.12 (m, 2H, O-CH2),
3.91 (s, 2H, S-CH2), 3.26 (m, 1H, isopropyl), 1.33 (s, 3H,
terminal CH3), 1.23 (d, 6H, terminal CH3), 0.97 (d, 6H,
terminal 2CH3) ppm.
Fig. 1 Structures of isoniazid and synthesized compound 3 showing
the position N2
Experimental
Chemical protocols
13C NMR (DMSO-d6, 300 MHz) d: 166.11 (acetyl C=O),
163.98 (thiazole C4), 153.29 (thiazole-C2), 149.21 (C5 of tri-
azole), 145.32 (C3 of triazole), 123.09 (thiazole-C5), 113.43–
138.08 (Ar–C), 63.24 (CH2), 33.12 (tertiary-1C-isopropyl),
32.54 (S–CH2), 21.24 (terminal 2CH3-isopropyl), 13.24
(acetyl CH3) ppm.
Melting points were determined in open capillary tubes in a
Thomas Hoover melting point apparatus and are uncor-
rected. Infrared spectra were recorded on Shimadzu FT-IR
1
157, H NMR and 13C NMR spectra were recorded (in
CDCl3/DMSO-d6) on a Bruker spectrometer at 300/
400 MHz using TMS as an internal standard. Mass spectra
(EI) on (AMD-604) mass spectrometer operating at 70 eV.
Elemental analysis was performed on Thermo Finnigan
Flash (EA 1112 CHNS Analyzer).
MS (%) 388.10 (M ? 100.0 %), 389.11 (M ? 1,
19.8 %), 390.10 (9.4 %), 390.11 (2.6 %).
Synthesis of 50-(4-isopropyl thiazol-2-yl)-4-phenyl-4H-
1,2,4-triazol-3-ylthio) aceto hydrazide 3
Thin layer chromatography (TLC) was performed
throughout the reaction on Merck silica gel GF254 alu-
minum sheets using mixture of different polar and nonpolar
The mixture of Compound 2 (0.1 mol) an ester and 2.5
equivalents of hydrazine hydrate in 30 mL ethanol was
123

944
Med Chem Res (2013) 22:938–948
refluxed for 4 h. Cooled to room temperature, the precip-
itate obtained was filtered and purified by recrystallization.
IR (KBr) m max, cm-1: 3,423 (NH str of amide), 3,328
(NH2 str), 3,080 (Ar H str), 2,921 (Ali C–H str), 1,450 Ar
(C=C), 1,419 (C=C), 760 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 9.35 (s, 1H, NH of
hydrazine hydrate), 7.40 (s, 1H, thiazole-C5), 7.3–7.6 (m,
5H, Ar–H), 4.52 (d, 2H, NH2 of hydrazine hydrate), 3.93
(s, 2H, S–CH2), 3.61 (m, 1H, isopropyl), 0.95 (d, 6H, ter-
minal CH3) ppm.
(m, 9H, Ar–H), 3.62 (s, 2H, S–CH2), 3.43 (m, 1H, iso-
propyl), 0.98 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 165.91 (C=O),
163.98 (thiazole C4), 163.29 (thiazole-C2), 114.19 (thia-
zole-C5), 149.13 (C5 of triazole), 145.34 (C3 of triazole),
143.41 (N=CH), 111.54–138.28 (Ar–C), 43.28 (S–CH2),
33.31 (tertiary-1C-isopropyl), 21.30 (terminal 2CH3-iso-
propyl) ppm.
MS (%) 496.80 (M ? 1), 343.1, 315.0.
13C NMR (DMSO-d6, 300 MHz) d: 165.9 (C=O),
161.98 (thiazole C4), 160.29 (thiazole-C2), 114.09 (thia-
zole-C5), 149.21 (C5 of triazole), 145.40 (C3 of triazole),
113.21–138.08 (Ar–C), 43.41 (S–CH2), 33.21 (tertiary-1C-
isopropyl), 21.28 (terminal 2CH3-isopropyl) ppm.
MS (%) 375.20 (M ? 1 100.0 %), 348.30, 271.3.
50-(4-isopropylthiazol-2-yl)-40-phenyl-40H-10,20,40-triazol-
30-ylthio-(400-hydroxybenzylidene) acetohydrazide 4c IR
(KBr) m max, cm-1: 3,438 (OH str), 3,204 (NH str), 3,074
(Ar C–H str), 2,863 (Ali C–H str), 1,667 (C=N str) imines,
1,601 (C=C), 1,186 (C–O), 768 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 11.68 (s, 1H, NH),
8.29 (s, 1H, N=CH), 7.32 (s, 1H, thiazole-C5), 7.2–7.5 (m,
9H, Ar–H), 5.02 (s, 1H, OH), 3.65 (m, 1H, isopropyl), 3.32
(s, 2H, S-CH2), 1.02 (d, 6H, terminal 2CH3)ppm.
13C NMR (DMSO-d6, 300 MHz) d: 165.34 (C=O),
163.98 (thiazole C4), 163.29 (thiazole-C2), 114.12 (thia-
zole-C5), 149.31 (C5 of triazole), 145.40 (C3 of triazole),
143.32 (N=CH), 112.11–137.02 (Ar–C), 43.03 (S–CH2),
33.23 (tertiary-1C-isopropyl), 21.16 (terminal 2CH3-iso-
propyl) ppm.
General method for the synthesis of 50-(4-isopropylthiazol-
2-yl)-40-phenyl-40H-10,20,40-triazol-30-ylthio) (substituted
benzylidene)acetohydrazide 4a–e
Aceto hydrazide 3 (0.05 mol) and the substituted aryl
aldehyde (0.05 mol) in 20 ml of ethanol along with cata-
lytic amount of glacial acetic acid (1 mL) was refluxed for
2 h in water bath at 90 °C; the mixture was cooled to room
temperature, the separated solid was filtered, washed with
alcohol dried, and recrystallized.
MS (%) 477.13 (M ? 100.0 %), 478.13 (M ? 1,
29.5 %), 479.13 (10.4 %), 479.14 (3.3 %).
50-(4-isopropylthiazol-2-yl)-40-phenyl-4H-10,20,40-triazol-
30-yl thio-(300,400-dimethoxy benzylidene) acetohydrazide
4a IR (KBr) m max, cm-1: 3,200 (NH str), 3,104 (Ar C–H
str), 1,671 (C=N str), 1,655 (C=N str) imines, 1,601 (C=C),
1,265 (Ar–OCH3), 763 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 11.05 (s, 1H, NH),
8.23 (s, 1H, N=CH), 7.32 (s, 1H, thiazole-C5), 7.2–7.5 (m,
8H, Ar–H), 3.81 (m, 6H, OCH3), 3.72 (s, 2H, S–CH2), 3.62
(m, 1H, isopropyl), 0.95 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 166.3 (C=O),
163.98 (thiazole C4), 161.29 (thiazole-C2), 149.11 (C5 of
triazole), 143.92 (N=CH), 114.11 (thiazole-C5), 111.23–
138.08 (Ar–C), 145.32 (C3 of triazole), 56.23 (2 OCH3),
43.21 (S–CH2), 33.16 (tertiary-1C-isopropyl), 21.21 (ter-
minal 2CH3-isopropyl) ppm.
50-(4-isopropylthiazol-2-yl)-40-phenyl-40H-10,20,40-triazol-
30-ylthio-(300-methoxy-400-hydroxy benzylidene) acetohyd-
razide 4d IR (KBr) m max, cm-1: 3,431 (OH str), 3,294
(NH str), 3,089 (Ar C–H str), 1,676 (C=N str) imines, 1,241
(Ar OCH3), 739 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 8.23 (s, 1H, N=CH),
11.71 (s, 1H, NH), 7.42 (s, 1H, thiazole-C5), 7.2–7.5 (m,
8H, Ar–H), 5.02 (s, 1H, S-OH), 4.46 (s, 1H, OH), 3.83 (s,
3H, OCH3), 3.68 (m, 1H, isopropyl), 3.36 (s, 2H, S–CH2),
0.94 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 165.90 (C=O),
163.98 (thiazole C4), 163.41 (thiazole-C2), 149.22 (C5 of
triazole), 145.26 (C3 of triazole), 143.44 (N=CH), 114.09
(thiazole-C5), 113.15–136.11 (Ar–C), 56.04 (OCH3), 43.65
(S–CH2), 33.15 (tertiary-1C-isopropyl), 21.28 (terminal
2CH3-isopropyl) ppm.
MS (%) 523.3 (M ? 2), 329.2, 255.2.
MS (%) 507.14 (M ? 100.0 %), 508.14 (M ? 1,
30.6 %), 509.14 (10.6 %), 509.15 (3.6 %).
50-(4-isopropylthiazol-2-yl)-4-phenyl-40H-10,20,40-triazol-
30-yl thio-(400-chloro benzylidene) acetohydrazide 4b IR
(KBr) m max, cm-1: 3,413 (NH str), 3,084 (Ar CH str),
2888 (Ali C–H str), 1,673 (C=N str) imines, 763 (C–S), 698
(C–Cl).
1H NMR (DMSO-d6, 300 MHz) d: 11.71 (s, 1H, NH),
8.26 (s, 1H, N=CH), 7.31 (s, 1H, thiazole-C5), 7.3–7.7
50-(40-isopropylthiazol-2-yl)-40-phenyl-40H-10,20,40-triazol-
30-ylthio-(300-nitrobenzylidene) acetohydrazide 4e IR
(KBr) m max, cm-1: 3,328 (NH str), 3,182 (Ar C–H str),
2,850 (Ali C–H str), 1,566 (C–NO2), 1,186 (C–O), 740
(C–S).
123

Med Chem Res (2013) 22:938–948
945
1H NMR (DMSO-d6, 300 MHz) d: 8.36 (s, 1H, N=CH),
11.70 (s, 1H, NH), 7.34 (s, 1H, thiazole-C5), 7.2–7.5 (m,
9H, Ar–H), 3.63 (m, 1H, isopropyl), 3.34 (s, 2H, S–CH2),
0.94 (d, 6H, terminal CH3) ppm.
43.92 (S–CH2), 33.16 (tertiary-1C-isopropyl), 22.24 (CH3),
21.24 (terminal 2CH3 -isopropyl) ppm.
MS (%) 505.16 (M ? 100.0 %), 506.16 (M ? 1,
31.6 %), 507.16 (10.5 %).
13C NMR (DMSO-d6, 300 MHz) d: 165.97 (C=O),
163.96 (thiazole C4), 163.33 (thiazole-C2), 149.25 (C5 of
triazole), 145.32 (C3 of triazole), 143.66 (N=CH), 114.21
(thiazole-C5), 114.11–132.38 (Ar–C), 43.17 (S–CH2),
33.22 (tertiary-1C-isopropyl), 21.34 (terminal 2CH3-iso-
propyl) ppm.
50-(4-Isopropylthiazol-2-yl)-4-phenyl-40H-10,20,40-triazol-30-
ylthio-(400-hydroxy methylbenzylidene) acetohydrazide 5c
IR (KBr) m max, cm-1: 3,417 (OH str), 3,075 (Ar C–H str),
2,926 (C–H str of CH3), 1,668 (C=N str) imines, 690 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 8.09 (s, 1H, NH),
7.37 (s, 1H, thiazole-C5), 7.2–7.5 (m, 9H, Ar–H), 5.07 (s,
1H, OH), 3.64 (m, 1H, isopropyl), 3.32 (s, 2H, S-CH2),
2.13 (s, 3H, CH3), 0.93 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 168.49 (C=O),
164.08 (thiazole C4), 163.39 (thiazole-C2), 149.25 (C5 of
triazole), 145.32 (C3 of triazole), 143.43 (N=CH), 114.31
(thiazole-C5), 115.31–135.08 (Ar–C), 43.19 (S-CH2), 33.36
(tertiary-1C-isopropyl), 22.11 (CH3), 21.26 (terminal 2CH3
-isopropyl) ppm.
MS (%) 506.12 (M ? 100.0 %), 507.12 (M ? 1,
29.9 %), 508.12 (10.7 %), 508.13 (3.3 %).
General method for the synthesis of 50-(4-isopropylthiazol-
2-yl)-40-phenyl-40H-10, 20,40-triazol-30-ylthio) (substituted
methyl benzylidene)acetohydrazide derivatives 5a–f
A mixture of compound 3 aceto hydrazide (0.01 mol) and
appropriate acetophenone (0.01 mol) in ethanol (30 mL)
and few drops of glacial acetic acid was refluxed for 2 h.
The solid that separated out on cooling was filtered and
recrystallised.
MS (%) 491.14 (M ? 100.0 %), 492.15 (M ? 1,
27.4 %), 493.14 (9.1 %).
5-(4-Isopropylthiazol-2-yl)-4-phenyl-4H-1,2,4-triazol-3-
50-(4-Isopropylthiazol-2-yl)-40-phenyl-40H-10,20,40-triazol-
30-ylthio-(400-nitromethylbenzylidene) acetohydrazide 5a
IR (KBr) m max, cm-1: 3,453 (NH str), 3,088 (Ar C–H str),
2,964 (C–H str of CH3), 1,687 (C=N str) imines, 1,560
(C–NO2).
1H NMR (DMSO-d6, 300 MHz) d: 8.23 (s, 1H, NH),
7.35 (s, 1H, thiazole-C5), 7.2–7.5 (m, 9H, Ar–H), 4.27 (m,
1H, isopropyl), 3.32 (s, 2H, S–CH2), 2.32 (m, 3H, CH3),
0.97 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 168.93 (C=O),
163.98 (thiazole C4), 163.29 (thiazole-C2), 149.30 (C5 of
triazole), 145.43 (C3 of triazole), 143.22 (N=C), 114.12
(thiazole-C5), 111.54–138.03 (Ar–C), 43.91 (S–CH2),
33.12 (tertiary-1C-isopropyl), 22.21 (CH3), 21.24 (terminal
2CH3-isopropyl) ppm.
ylthio)-N’-(1-p-tolylethylidene)acetohydrazide
5d IR
(KBr) m max, cm-1: 3,125.38 (Ar C–H str), 2,933.3 (C–H
str of CH3), 2,841.18 (Ali C–H str), 1,662.72 (C=N str)
imines, 694.55 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 8.09 (s, 1H, NH),
7.37 (s, 1H, thiazole-C5), 7.2–7.5 (m, 9H, Ar–H), 3.64 (m,
1H, isopropyl), 3.32 (s, 2H, S–CH2), 2.13 (s, 3H, CH3),
2.53 (s, 3H, CH3), 0.93 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 168.49 (C=O),
164.08 (thiazole C4), 163.39 (thiazole-C2), 149.25 (C5 of
triazole), 145.32 (C3 of triazole), 143.43 (N=CH), 114.31
(thiazole-C5), 115.31–135.08 (Ar–C), 43.19 (S–CH2),
33.36 (tertiary-1C-isopropyl), 22.11 (CH3), 24.56 (CH3),
21.26 (terminal 2CH3 -isopropyl) ppm.
MS (%) 490.16 (M ? 100.0 %), 491.16 (M ? 1,
30.9 %), 493.16 (2.6 %).
MS (%) 522.13 (M ? 2), 377.09, 343.1,
50-(4-Isopropylthiazol-2-yl)-40-phenyl-40H-10,20,40-triazol-
30-yl thio-(400-methoxy methylbenzylidene) acetohydrazide
5b IR (KBr) m max, cm-1: 3,433 (NH str), 3,080 (Ar C–H
str), 2,959 (C–H str of CH3), 2,828 (CH3–O), 1,670 (C=N
str) imines.
1H NMR (DMSO-d6, 300 MHz) d: 8.01 (s, 1H, NH),
7.2–7.5 (m, 9H, Ar–H), 7.37 (s, 1H, thiazole-C5), 3.74 (s,
3H, OCH3), 3.63 (m, 1H, isopropyl), 3.34 (s, 2H, S–CH2),
2.12 (s, 3H, CH3), 0.92 (d, 6H, terminal CH3)ppm.
13C NMR (DMSO-d6, 300 MHz) d: 168.9 (C=O),
163.96 (thiazole C4), 163.21 (thiazole-C2), 114.09 (thia-
zole-C5), 149.33 (C5 of triazole), 145.32 (C3 of triazole),
143.23 (N=CH), 112.11–138.33 (Ar–C), 55.73 (OCH3),
1-(4-Bromophenyl)ethylidene)-2-(5-(4-isopropylthiazol-2-
yl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)acetohydrazide 5e IR
(KBr) m max, cm-1: 3,134 (Ar C–H str), 2,922 (C–H str of
CH3), 2,888 (Ali C–H str), 1,654 (C=N str) imines, 674 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 8.09 (s, 1H, NH),
7.37 (s, 1H, thiazole-C5), 7.2–7.5 (m, 9H, Ar–H), 3.64 (m,
1H, isopropyl), 3.32 (s, 2H, S–CH2), 2.13 (s, 3H, CH3),
0.93 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 168.49 (C=O),
164.08 (thiazole C4), 163.39 (thiazole-C2), 149.25 (C5 of
triazole), 145.32 (C3 of triazole), 143.43 (N=CH), 114.31
(thiazole-C5), 115.31–135.08 (Ar–C), 43.19 (S–CH2),
123

946
Med Chem Res (2013) 22:938–948
6b IR (KBr) m max, cm-1: 2,963 (Ar C–H str), 1,711
(C=N of oxadiazole), 1,626 (C=C), 690 (C–Cl).
1H NMR (DMSO-d6, 300 MHz) d: 7.35 (s, 1H, thia-
zole-C5), 7.2-7.7 (m, 9H, Ar–H), 4.12 (s, 2H, S-CH2), 3.67
(m, 1H, isopropyl), 0.98 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 163.98 (thiazole
C4), 163.29 (thiazole-C2), 114.09 (thiazole-C5), 164.32 (C5
of oxadiazole), 149.21 (C5 of triazole), 145.32 (C3 of tri-
azole), 144.11 (C2 of oxadiazole), 113.1–139.02 (Ar–C),
33.12 (tertiary-1C-isopropyl), 45.11 (CH2), 21.24 (terminal
2CH3-isopropyl) ppm.
33.36 (tertiary-1C-isopropyl), 24.56 (CH3), 21.26 (terminal
2CH3 -isopropyl) ppm.
MS (%) 556.05 (M ? 100.0 %), 554.06 (M ? 1,
94.0 %), 555.06 (26.2 %), 557.06 (24.2 %), 558.05 (9.2 %).
1-(4-Chlorophenyl)ethylidene)-2-(5-(4-isopropylthiazol-2-
yl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)acetohydrazide 5f IR
(KBr) m max, cm-1: 3,133 (Ar C–H str), 2,955 (C–H str of
CH3), 1,649 (C=N str) imines, 645 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 7.85 (s, 1H, NH),
7.66 (s, 1H, thiazole-C5), 7.2–7.8 (m, 9H, Ar–H), 3.55 (m,
1H, isopropyl), 3.32 (s, 2H, S-CH2), 2.44 (s, 3H, CH3),
0.97 (d, 6H, terminal CH3) ppm.
MS (%) 432.06 (M ? 100.0 %), 434.06 (M ? 1,
42.0 %), 433.06 (23.3 %), 435.06 (8.6 %).
13C NMR (DMSO-d6, 300 MHz) d: 162.49 (C=O),
163.55 (thiazole C4), 162.11 (thiazole-C2), 145.35 (C5 of
triazole), 142.32 (C3 of triazole), 141.41 (N=CH), 113.66
(thiazole-C5), 115.31–135.08 (Ar–C), 42.19 (S–CH2),
32.11 (tertiary-1C-isopropyl), 22.33 (CH3), 21.23 (terminal
2CH3-isopropyl) ppm.
3-(4-Isopropyl thiazol-2-yl)-40-phenyl-50-((500-chloro phe-
nyl-100,300,400-oxadiazol-200-yl) methylthio) -40H-10,20,40-tri-
azole 6c IR (KBr) m max, cm-1: 3,162 (Ar C–H str),
1,653 (C=N of oxadiazole), 1,258 (Ar–C–O), 1,079 (C–O
of oxadiazole), 760 (C–S), 674.02 (C–Cl).
1H NMR (DMSO-d6, 300 MHz) d: 7.41 (s, 1H, thia-
zole-C5), 7.2–7.5 (m, 9H, Ar–H), 3.63 (m, 1H, isopropyl),
0.97 (d, 6H, terminal CH3) ppm.
MS (%) 510.11 (M ? 100.0 %), 512.10 (M ? 1,
41.1 %), 511.11 (27.9 %), 513.11 (11.0 %), 512.11 (4.5 %).
13C NMR (DMSO-d6, 300 MHz) d: 164.32 (C5 of
oxadiazole), 163.98 (thiazole C4), 163.29 (thiazole-C2),
149.21 (C5 of triazole), 145.32 (C3 of triazole), 144.11 (C2
of oxadiazole), 116.11–132.08 (Ar–C), 114.09 (thiazole-
C5), 33.12 (tertiary-1C-isopropyl), 21.24 (terminal 2CH3-
isopropyl) ppm.
General method for the synthesis of 3-(4-isopropylthiazol-
2-yl)-40-phenyl-50-((500-substituted-100,300,400-oxadiazol-200-yl)-
methylthio)-40H-10,20,40-triazol 6a–f
A mixture of compound 3 aceto hydrazide (0.001 mol) and
appropriate aromatic acid (0.001 mol) in phosphorus oxy-
chloride was refluxed for 6 h. The reaction mixture was
slowly poured over crushed ice and kept overnight. The solid
thus separated out was filtered, treated with dilute sodium bi
carbonate, washed with water, and recrystallized.
MS (%) 494.08 (M ? 100.0 %), 496.07 (M ? 1,
41.1 %), 495.08 (25.1 %), 497.08 (8.2 %).
3-(4-Isopropylthiazol-2-yl)-5-((5-(4-nitrophenyl)-1,3,4-ox-
adiazol-2-yl)methylthio)-4-phenyl-4H-1,2,4-triazole
6d
IR (KBr) m max, cm-1: 3,162 (Ar C–H str), 1,653 (C=N of
oxadiazole), 1,263 (Ar–C–O), 1,055 (C–O of oxadiazole),
760 (C–S).
3-(4-Isopropyl thiazol-2-yl)-4-phenyl-5-((500-phenyl-1,3,4-
oxadiazol-2-yl) methylthio)-4H-1,2,4-triazole 6a IR
(KBr) m max, cm-1: 3,053 (Ar C–H str), 2,980 (Ar C–H
str), 1,688 (C=N of oxadiazole), 1,078 (C–O of oxadiaz-
ole), 745 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 7.35 (s, 1H, thia-
zole-C5), 7.2–7.5 (m, 10H, Ar–H), 3.65 (m, 1H, isopropyl),
0.96 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 164.32 (C5 of
oxadiazole), 163.98 (thiazole C4), 163.29 (thiazole-C2),
149.21 (C5 of triazole), 145.32 (C3 of triazole), 144.11 (C2
of oxadiazole), 114.09 (thiazole-C5), 111.11–138.08 (Ar–
C), 33.12 (tertiary-1C-isopropyl), 21.24 (terminal 2CH3-
isopropyl) ppm.
1H NMR (DMSO-d6, 300 MHz) d: 7.33 (s, 1H, thia-
zole-C5), 7.3–7.6 (m, 9H, Ar–H), 3.67 (m, 1H, isopropyl),
0.93 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 164.32 (C5 of
oxadiazole), 163.98 (thiazole C4), 163.29 (thiazole-C2),
149.21 (C5 of triazole), 145.34 (C3 of triazole), 144.21 (C2
of oxadiazole), 116.11–132.08 (Ar–C), 114.89 (thiazole-
C5), 33.52 (tertiary-1C-isopropyl), 21.24 (terminal 2CH3-
isopropyl) ppm.
MS ( %) 505.10 (M ? 100.0 %), 506.10 (M ? 1,
29.2 %), 508.10 (2.4 %), 507.10 (1.7 %).
MS (%) 460.11 (M ? 100.0 %), 461.12 (M ? 1,
25.1 %), 462.12 (3.6 %), 463.11 (2.5 %).
3-(4-Isopropylthiazol-2-yl)-4-phenyl-5-((5-p-tolyl-1,3,4-oxa-
diazol-2-yl)methylthio)-4H-1,2,4-triazole 6e IR (KBr) m
max, cm-1: 3,162 (Ar C–H str), 1,673 (C=N of oxadiaz-
ole), 1,057 (C–O of oxadiazole), 767 (C–S).
3-((500-(Chloromethyl)-100,300,400-oxadiazol-200-yl)methylthio)-
50-(4-isopropylthiazol-2-yl)-40-phenyl-40H-10,20,40-triazole
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Med Chem Res (2013) 22:938–948
947
1H NMR (DMSO-d6, 300 MHz) d: 7.34 (s, 1H, thia-
zole-C5), 7.3–7.6 (m, 9H, Ar–H), 3.67 (m, 1H, isopropyl),
2.35 (s, 3H, CH3), 0.93 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 164.32 (C5 of
oxadiazole), 163.98 (thiazole C4), 163.29 (thiazole-C2),
149.55 (C5 of triazole), 145.33 (C3 of triazole), 144.61 (C2
of oxadiazole), 116.11–132.38 (Ar–C), 114.39 (thiazole-
C5), 33.82 (tertiary-1C-isopropyl), 24.44 (CH3), 21.24
(terminal 2CH3-isopropyl) ppm.
Biological protocol
Antimicrobial activity
The antimicrobial susceptibility testing was performed in
vitro by broth microdilution method (Hassan et al., 1983;
Khalil et al., 1993). The MIC determination of the syn-
thesized compounds was carried out in side-by-side com-
parison with ciprofloxacin and norfloxacin against Gram-
positive bacteria (S. aureus, S. faecalis, B. subtilis) and
Gram-negative bacteria (K. penumoniae, E. coli. P. aeru-
ginosa). The antifungal activity was assayed against yeasts
(C. tropicalis, S. cerevisiae) and molds (A. niger). The
minimal inhibitory concentrations (MIC, lg/mL) were
defined as the lowest concentrations of compound that
completely inhibited the growth of each strain. Test com-
pounds (10 mg) were dissolved in dimethylsulfoxide
(DMSO, 1 mL) then diluted in culture medium (Mueller–
Hinton Broth for bacteria and Sabouraud Liquid Medium
for fungi) further progressive dilutions to obtain final
concentrations of 1, 2, 4, 8, 16, 31.25, 62.5, 125, 250, and
500 lg/mL. DMSO never exceeded 1 % v/v. The tubes
were inoculated with 105 cfu mL-1 (colony forming unit/
mL) and incubated at 37 °C for 24 h. The growth control
consisting of media (positive control) and media with
DMSO (negative control) at the same dilutions as used in
the experiments were employed.
MS (%) 474.13 (M ? 100.0 %), 475.13 (M ? 1, 29.8 %),
476.13 (10.3 %), 476.14 (3.3 %).
4-(5-((5-(4-Isopropylthiazol-2-yl)-4-phenyl-4H-1,2,4-triazol-
3-ylthio)methyl)-1,3,4-oxadiazol-2-yl)phenol 6f IR (KBr) m
max, cm-1: 3,162 (Ar C–H str), 1,644 (C=N of oxadiaz-
ole), 1,233 (Ar–C–O), 1,055 (C–O of oxadiazole), 760.58
(C–S).
1H NMR (DMSO-d6, 300 MHz) d: 7.34 (s, 1H, thia-
zole-C5), 7.3–7.6 (m, 9H, Ar–H), 3.67 (m, 1H, isopropyl),
5.23 (s, 1H, OH), 0.95 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 164.32 (C5 of
oxadiazole), 163.98 (thiazole C4), 163.29 (thiazole-C2),
149.21 (C5 of triazole), 145.32 (C3 of triazole), 144.11 (C2
of oxadiazole), 116.11–132.08 (Ar–C), 114.59 (thiazole-
C5), 33.62 (tertiary-1C-isopropyl), 21.24 (terminal 2CH3-
isopropyl) ppm.
MS (%) 476.11 (M ? 100.0 %), 477.11 (M ? 1,
28.8 %), 478.10 (9.1 %), 478.12 (3.0 %).
Anti tubercular activity
The preliminary antitubercular screening for test com-
pounds were obtained for MTB H37Rv, the MIC of each
drug was determined by broth dilution assay (Goto et al.,
1981) and is defined as the lowest concentration of drug,
which inhibits B99 % of bacterial population present at the
beginning of the assay. A frozen culture in Middlebrook
7H9 broth supplemented with 10 % albumin-dextrose-
catalase and 0.2 % glycerol was thawed and diluted in
broth to 105 cfu mL-1 (colony forming unit/mL) dilutions.
Each test compound was dissolved in DMSO and then
diluted in broth twice at the desired concentration. The
final concentration of DMSO in the assay medium was
1.3 %. Each U-tube was then inoculated with 0.05 mL of
standardized culture and then incubated at 37 °C for
21 days. The growth in the U-tubes was compared with
visibility against positive control (without drug), negative
control (without drug and inoculum), and with standard
isoniazid.
Synthesis of N-acetyl-50-(4-isopropylthiazol-2-yl)-40-
phenyl-40H-10,20,40-triazol-(30-ylthio) acetohydrazide 7
Compound 3 aceto hydrazide (0.003 mol) was warmed
with acetic anhydride for 2 h and then the mixture was
allowed to attain room temperature. The deposited solid
was filtered, washed, and recrystallized from ethanol.
IR (KBr) m max, cm-1: 3,265 (NH str), 1,815 (C–O),
1,685 (C=O), 770 (C–S).
1H NMR (DMSO-d6, 300 MHz) d: 10.21 (s, 1H, NH of
hydrazine hydrate), 8.24 (1H, NH2 of hydrazine hydrate),
7.35 (s, 1H, thiazole-C5), 7.2–7.5 (m, 5H, Ar–H), 3.82 (s,
2H, S–CH2), 3.63 (m, 1H, isopropyl), 2.12 (s, 3H, CH3),
0.95 (d, 6H, terminal CH3) ppm.
13C NMR (DMSO-d6, 300 MHz) d: 171.54 (C=O),
169.44 (CH3), 164.32 (C=O), 163.98 (thiazole C4), 163.29
(C2 of thiazole-), 149.21 (C5 of triazole), 145.32 (C3 of
triazole), 114.09 (C5 of thiazole), 114.1–136.2 (Ar–C),
40.91 (S–CH2), 33.12 (tertiary-1C-isopropyl), 21.24 (ter-
minal 2CH3-isopropyl) ppm.
MTT assay for cell viability
MS (%) 416.11 (M ? 100.0 %), 417.11 (M ? 1,
23.4 %), 418.12 (1.8 %).
Toxicity of compounds 3, 4c, 5c, and 6c in A549 cell lines
in the presence of 10 and 0.2 % FBS, respectively, was
123

948
Med Chem Res (2013) 22:938–948
Kinzig-Schippers M, Tomalik-Scharte D, Jetter A, Scheidel B, Jakob
V, Rodamer M, Cascorbi I, Doroshyenko O, Sorgel F, Fuhr U
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determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphe-
nyltetrazolium bromide reduction assay (Mosmann, 1983).
The compounds were dissolved in DMSO at 10 mM con-
centration and stored at -20 °C. The dilutions were made
in culture medium before treatment.
Acknowledgments We thank management of St. Johns Pharmacy
College, Bangalore for providing necessary facilities. We are grateful
to Dr. K.G. Bhat, Maratha Mandal’s Dental College, Hospital and
Research Centre, Belgaum, India for providing the facilities to
determine the antibacterial and antitubercular activities. We also wish
to thank IISC, Bangalore, India for providing IR, NMR, mass spectra,
and elemental analysis data.
Mallikarjun BP, Suresh Kumar GV, Sastry BS, Nagaraj, Manohara
KP (2007) Synthesis and anticonvulsant activity of some potent
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isopropylthiazole hydrazide analogs and some derived clubbed
triazole, oxadiazole ring systems–a novel class of potential
antibacterial, antifungal and antitubercular agents. Eur J Med
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