New pyrazole derivatives containing 1,2,4-triazoles and benzoxazoles as potent antimicrobial and analgesic agents

Article abstract of DOI:10.1016/j.ejmech.2012.12.057

Azole class of compounds are well known for their excellent therapeutic properties. Present paper describes about the synthesis of three series of new 1,2,4-triazole and benzoxazole derivatives containing substituted pyrazole moiety (11a-d, 12a-d and 13a-d). The newly synthesized compounds were characterized by spectral studies and also by C, H, N analyses. All the synthesized compounds were screened for their analgesic activity by the tail flick method. The antimicrobial activity of the new derivatives was also performed by Minimum Inhibitory Concentration (MIC) by the serial dilution method. The results revealed that the compound 11c having 2,5-dichlorothiophene substituent on pyrazole moiety and a triazole ring showed significant analgesic and antimicrobial activity.

Full text of DOI:10.1016/j.ejmech.2012.12.057

European Journal of Medicinal Chemistry 62 (2013) 410e415
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New pyrazole derivatives containing 1,2,4-triazoles
and benzoxazoles as potent antimicrobial and analgesic agents
,
c
d
e
A.M. Vijesh a, Arun M. Isloor b , Prashanth Shetty , S. Sundershan , Hoong Kun Fun
*
a Department of Chemistry, Gitam School of Technology, Gitam University, Nagadenahalli, Doddabellapur 561 203, Bengaluru Rural District,
Karnataka, India
b Medicinal Chemistry Laboratory, Department of Chemistry, National Institute of Technology Karnataka, Surathkal, Mangalore 575 025, India
c Department of Pharmacology, Nitte Gulabi Shetty Memorial College of Pharmacology, Deralakatte, Mangalore 574 160, India
d Department of Microbiology, Veterinary College, UAS-Hebbal, Bangalore 24, India
e Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Azole class of compounds are well known for their excellent therapeutic properties. Present paper de-
scribes about the synthesis of three series of new 1,2,4-triazole and benzoxazole derivatives containing
substituted pyrazole moiety (11aed, 12aed and 13aed). The newly synthesized compounds were
characterized by spectral studies and also by C, H, N analyses. All the synthesized compounds were
screened for their analgesic activity by the tail flick method. The antimicrobial activity of the new de-
rivatives was also performed by Minimum Inhibitory Concentration (MIC) by the serial dilution method.
The results revealed that the compound 11c having 2,5-dichlorothiophene substituent on pyrazole
moiety and a triazole ring showed significant analgesic and antimicrobial activity.
Received 3 August 2012
Received in revised form
21 December 2012
Accepted 25 December 2012
Available online 10 January 2013
Keywords:
1,2,4-Triazole
Benzoxazole
Pyrazole
Ó 2013 Elsevier Masson SAS. All rights reserved.
Analgesic study
Antimicrobial study
1. Introduction
a long time. It is well known from the literature that Schiff bases
derived from 1,2,4-triazoles displayed excellent biological prop-
The treatment of infectious diseases still remained as an impor-
tant and challenging problem due to emerging infectious diseases
and increasing number of multi-drug resistant microbial pathogens.
In spite of the large number of antibiotics and chemotherapeutics,
the emergence of old and new antibiotic resistant bacterial strains
constitutes a substantial need for the new class of potent anti-
microbial agents. Heterocyclic compounds play an important role in
designing a new class of structural entities of medicinal importance
with new mechanisms of action. These heterocyclic compounds
are well known to possess diverse pharmacological properties,
viz. antimicrobial, analgesic, anti-inflammatory, anticancer, anti-
convulsant and antimalarial.
The chemistry of the carbonenitrogen double bond plays a vital
role in the progress of science. Compounds with the structure of e
C¼Ne (azomethine group) are known as Schiff bases, which are
usually synthesized by the condensation of primary amines and
active carbonyl groups. The chemistry of Schiff bases derived from
1,2,4-triazole analogues has been an interesting field of study for
erties. In particular, they show antibacterial, antifungal [1], anti-
tubercular [2], antioxidant [3], anticancer [4], antimalarials,
anticonvulsant, anti-inflammatory [5,6] and pesticidal properties.
They are important molecules in the medicinal and pharmaceutical
fields and it has been suggested that the azomethine linkage might
be responsible for the biological activities displayed by Schiff bases.
Therefore it was planned to synthesize hybrids of substituted 1,2,4-
triazoles. Hydrazone derivatives have shown different biological
properties [7], such as anti-inflammatory, analgesic [8], anti-
convulsant, antituberculous, and antimicrobial activity [9]. Hydra-
zones are important compounds for drug design, as possible ligands
for metal complexes, organocatalysis and also for the syntheses of
heterocyclic compounds. Derivatives of benzoxazole have gained
much importance because of its wide applications in medicinal
sector. They are associated with diverse pharmacological activities
such as analgesic, antituberculosis [10], antibacterial [11], antifungal
[12], anti-inflammatory, antioxidant and anthelmintic activity [13].
Further, pyrazole derivatives have shown significant biological
activities, such as antimicrobial [1], analgesic [14], anti-
inflammatory [15] and anticancer [16]. This gave a great impetus
to the search for potential pharmacologically active drugs carrying
* Corresponding author. Tel.: þ91 824 2474000; fax: þ91 824 2474033.
E-mail address: isloor@yahoo.com (A.M. Isloor).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.057

A.M. Vijesh et al. / European Journal of Medicinal Chemistry 62 (2013) 410e415
411
N
O
pyrazole substituents. The search for novel antimicrobial and
S
analgesic agents devoid of side effects continues to be an active area
of research in medicinal chemistry. Although new and more
expensive drugs have been developed, their cost is beyond the
common man’s reach. As a consequence, these trends have
emphasized the pressing need for new, more effective, cheaper and
safe antimicrobial agents.
X
N
O
NH2NH2.H2O
OC2H5
ClCH2COOC2H5
X
N
O
6
O
S
NH
NH
SH
H2N
X
O
Cl
X
N
4a,b
5a,b
O
NH2
Cl
7
Many azole classes of compounds, mainly 1,2,4-triazoles and
their derivatives constitute an important class of organic com-
pounds with analgesic, anti-inflammatory and antimicrobial ac-
tivities [17,18]. In addition, hydrazone derivatives also exhibit
potent antimicrobial, analgesic and anti-inflammatory activities
[17]. Small modifications in the structure of the targets will alter
their biological character as well as their physiochemical prop-
erties. Keeping in view of this and in continuation of our search for
biologically potent molecules [18e22], we hereby report the syn-
thesis and biological evaluation of some new Schiff base analogous
of 1,2,4-triazoles and benzoxazoles carrying substituted pyrazole
moiety with the hope enhancing the biological activity.
Where X= H, Cl
Scheme 2. Synthetic route for the benzoxazolehydrazides.
3. Results and discussion
All the final derivatives 11aed, 12aed and 13aed were charac-
terized after crystallization from appropriate solvents. IR spectrum
of Schiff Base 11a showed absorption at 3120 cmꢀ1 which is due to
the NH stretching. The band appeared at 1620 cmꢀ1 is due to C¼N
stretching confirmed the structure of the compound. The 1H NMR
spectrum of 11a showed a singlet at
protons. Two doublets at
7.38 (J ¼ 4.8 Hz) and
are due to aromatic protons of p-thioanisyl moiety. A singlet at
d
2.50 which is due to SCH3
d
d
7.65 (J ¼ 4.8 Hz)
2. Chemistry
d
8.21 is due to pyrazole-5H proton. Two doublets at
(J ¼ 6.7 Hz) and
the pyridine ring. eN¼CH protons appeared as a singlet at
Two singlets appeared in 13.81 and d 14.38 are due to triazole SH
d
7.86
d
8.73 (J ¼ 6.7 Hz) are due to aromatic protons of
Intermediate triazoles 3aec were synthesized from corre-
sponding acid hydrazides (1aec) through multi-step reactions
(Scheme 1) as per the reported literature [23,24]. The hydrazides
were converted to potassium salt (2aec) by reacting with carbon
disulphide and potassium hydroxide in absolute ethanol. On
reacting with hydrazine hydrate, the salt undergoes ring closure to
yield the triazoles, 3aec.
d
9.52.
d
and pyrazole-NH protons respectively further confirmed the
structure. The mass spectrum of 11a showed a molecular ion peak
at m/z ¼ 394 (m þ 1), which is in agreement with the molecular
formula C18H15N7S2.
IR spectrum of hydrazone 12a showed absorption at 3215 cmꢀ1
which is due to the NH stretching. Both the C¼O and C¼N
stretching frequencies merged together and resulted in a broad
band in 1652. The 1H NMR spectrum of 12a showed a singlet at
2-(1,3-Benzoxazol-2-ylsulfanyl)-1-(hydrazinyloxy)ethanone
6
was synthesized from 1,3- benzoxazole-2-thiol (5a) by following
the reported literature [13]. The anomalous reaction of hydrazine
hydrate with ethyl[(5,7-dichloro-1,3-benzoxazol-2-yl)sulfanyl]ac-
etate (5b) resulted in the formation of compound 7 [13]. The re-
action sequence has been presented in Scheme 2.
d
4.12 is due to CH2 protons. Similarly multiplets at
due to aromatic protons. A singlet at 7.84 is due to pyrazole-5H
proton. The eN¼CHe protons appeared as a singlet at 8.18. Two
singlets appeared in 11.37 and 13.39 are due to eCOeNHe and
d 7.27e7.80 are
d
d
Synthetic route of 3-substituted-1H-pyrazole-4-carbaldehydes
(10aed) has been reported in our previous work [25]. Substituted
pyrazole aldehydes (10aed) were synthesized by the Vilsmayer
Haack reaction of se/micarbazones (9aed), which in turn obtained
by the reaction of semicarbazide hydrochloride with various
substituted acetophenones in ethanol media [25]. The targeted
Schiff bases (11aed) were obtained in good yield by refluxing
equimolar amount of 3-aryl-1-H-pyrazole-4-carbaldehyde (10aed)
with 4-amino-5-aryl-4H-1,2,4-triazole-3-thiol (3aec) in the pres-
ence of catalytic amount of sulphuric acid using ethanol as solvent.
New hydrazone derivatives (12aed and 13aed) were synthesized
by refluxing equimolar amount of 3-aryl-1-H-pyrazole-4-
carbaldehyde (10aed) with 2-(1,3-benzoxazol-2-ylsulfanyl) aceto-
hydrazide (6)/5,7-dichloro-2-hydrazinyl-1,3-benzoxazole (7) in
ethanol media, in the presence of catalytic amount of acetic acid.
The reaction pathway has been summarized in Scheme 3.
d
d
pyrazole-NH protons respectively. The mass spectrum of 12a
showed a molecular ion peak at m/z ¼ 447 (M þ 1), which is in
agreement with the molecular formula C19H13Cl2N5O2S2. Similarly
the spectral values for all the compounds and C, H, N analyses are
given in the experimental part and the characterization is provided
in Table 1.
4. Pharmacology
4.1. Antimicrobial studies
All the newly synthesized compounds 11a-d, 12a-d and 13a-d
were screened for their antimicrobial activity. Antimicrobial study
was assessed by Minimum Inhibitory Concentration (MIC) by the
serial dilution method [26]. For this, Staphylococcus aureus, Bacillus
subtilis, Escherichia coli and Pseudomonas aeruginosa microorgan-
isms were used to investigate the antibacterial activity. Antifungal
activity was carried out against Candida albicans. Ceftriaxone and
Fluconazole were used as standard drugs for antibacterial and
antifungal activities respectively. Several colonies of S. aureus,
B. subtilis, E. coli, P. aeruginosa and Pseudomonas albicans were
picked off a fresh isolation plate and inoculated in corresponding
tubes containing 5 mL of trypticase soya broth. The broth was
incubated for 6 h at 37 ꢁC until there was visible growth. The MC
Farland No.5 standard was prepared by adding 0.05 mL of 1% w/v
BaCl2.2H2O in Phosphate Buffered saline (PBS) to 9.95 mL of 1% v/v
H2SO4 in PBS. The growth of all the four cultures was adjusted to Mc
O
N
N
O
NH2-NH2.H2O
EtOH
CS2
NH SK
NH
SH
Ar
NH
NH2
Ar
N
NH2
3a-c
Ar
EtOH, KOH
S
1a-c
2a-c
O
N
N
Ar =
,
,
Scheme 1. Synthetic route for the 1,2,4-triazoles.

412
A.M. Vijesh et al. / European Journal of Medicinal Chemistry 62 (2013) 410e415
N
N
N
Ar
N
SH
3a-c
Ar1
N
N
NH2
N
Ar
N
SH
11a-d
N
H
O
N
Ar1
S
O
O
O
O
N
S
Ar1
NH
NH2
6
NHNH2
O
N
N
N
NH
O
12a-d
N
H
(ii)
(i)
Ar
Ar
8a-d
N
Cl
N
H
Cl
9a-d
O
N
10a-d
O
NH
NH2
Ar1
NH
N
Cl
N
7
Cl
N
13a-d
N
H
Where, (i) Semicarbazide. HCl, Sodium Acetate, Ethanol (ii) DMF, POCl3
Ar1 = 2,4-Dichlorophenyl, 4-Thioanisyl, 2,5-Dichlorothiophene, Biphenyl
Ar = 1-Naphthyloxymethyl, Isonicotinyl, 6-Methylnicotinyl
Scheme 3. Synthetic route for the compounds 11aed, 12aed and 13aed.
Farland No.5 turbidity standard using sterile PBS. This gives
a 108 cfu/mL suspension. The working inoculums of aforemen-
tioned four different microorganisms containing 105 cfu/mL sus-
pension was prepared by diluting the 108 cfu/mL suspension, 103
times in trypticase soya broth.
P. aeruginosa and P. albicans suspension was added to the first,
second, third, fourth and fifth rows of tubes respectively. Along
with the test samples and Ceftriaxone/Fluconazole (standard), the
inoculums control (without antimicrobial compound) and broth
control (without antimicrobial compound and inoculum) were
maintained. All the test sample and control tubes were then incu-
bated for 16 h at 37 ꢁC.
4.1.1. Preparation of antimicrobial suspension (1 mg/mL)
Dissolved 10 mg of each compound in 10 mL of dimethyl
formamide to get 1 mg/mL concentration.
4.1.3. Interpretation
After incubation, the tubes showing no visible growth were
considered to be representing the MIC. The details of the results are
furnished in Table 2. Inoculums control showed visible growth,
whereas the broth control showed no growth.
4.1.2. Preparation of dilutions
In all, for each of the 12 antimicrobial compounds and standard
antimicrobial i. e Ceftriaxone for antibacterial and Fluconazole for
antifungal, 24 tubes of 5 mL capacity were arranged in five rows
with each row containing 6 tubes. Then 1.9 mL of trypticase soya
broth was added in the first tube in each row and then 1 mL in the
Among the screened samples, 11c and 11d emerged as potent
antimicrobial agents. 11c showed excellent activity against S. aureus
compared to the standard. 11c showed similar activity against
B. subtilis and P. aeruginosa as compared to standard drug Ceftri-
axone. 11c contains 2,5-dichlorothiophene substituent on a pyr-
azole ring which accounts for the enhanced activity. 11d showed
similar activity as that of standard drugs against S. aureus, B. subtilis
and P. aeruginosa. 11d having 2,4-dichlorophenyl moiety on pyr-
azole ring and the antibacterial activity is due to the presence
of this group. 11b showed similar activity as that of standard,
against P. aeruginosa and 13c had shown similar activity against
S. aureus compared to Ceftriaxone. Compound 11b has 2,4-
dichlorophenyl moiety and 13c has 2,5-dichlorothiophene
remaining tubes. Now,100 mL of antimicrobial suspension dissolved
in dimethyl formamide was added to the first tube in each row
and then after mixing the content, 1 mL was serially transferred
from these tubes to the second tube in each of the rows. Then the
contents of the second tube of each of the rows were mixed
and transferred to the third tube in each of the rows. This serial
dilution was repeated till the sixth tube in each of the rows. This
provided antimicrobial concentrations of 50, 25, 12.5, 6.25, 3.125,
1.6125 mg/mL in the first to sixth tube respectively in each row.
Finally, 1 mL of 105 cfu/mL of S. aureus, B. subtilis, E. coli,
Table 1
Characterization data of the compounds 11aed, 12aed and 13aed.
Compounds
Ar1
Ar
Molecular formula (Mol. wt.)
Yield (%)
M.p. (ꢁC)
11b
11c
11d
12a
12b
12c
12d
13a
13b
13c
13d
2,4-Dichlorophenyl
2,5-Dichlorothiophene
2,4-Dichlorophenyl
2,4-Dichlorophenyl
4-SCH3-C6H4
2,5-Dichlorothiophene
Biphenyl
2,4-Dichlorophenyl
4-SCH3-C6H4
1-Naphthyloxymethyl
1-Naphthyloxymethyl
6-Methylnicotinyl
C23H16Cl2N6OS (495)
C21H14Cl2N6OS2 (501)
C18H13Cl2N7S (430)
C19H17Cl2N5O2S (446)
C20H17N5O2S2 (423)
C17H11Cl2N5O2S2 (452)
C25H19N5O2S (453)
C17H9Cl4N5O (441)
C18H13Cl2N5OS (418)
C15H7Cl4N5OS (447)
C23H15Cl2N5O (448)
77
78
76
90
86
85
88
82
85
81
83
196e198
158e160
156e158
150e152
168e170
184e186
196e198
288e290
244e246
284e286
264e266
e
e
e
e
e
e
e
e
2,5-Dichlorothiophene
Biphenyl

A.M. Vijesh et al. / European Journal of Medicinal Chemistry 62 (2013) 410e415
413
Table 2
2 mg/kg by i.p. route. Remaining groups received test drug at a dose
level of 200 mg/kg and was given by i.p. route. Each of the prepa-
ration was given in such a manner so that the fluid intake was same
in all cases. They were placed into individual restraining cages
leaving the tail hanging out freely. Before testing, the animals were
allowed to adapt to the cages for 30 min. The lower 5 cm portion of
the tail was marked. This part of the tail was immersed in a cup of
freshly filled water at 55 ꢂ 2 ꢁC. Within a few seconds, the rat reacts
by withdrawing the tail. The reaction time was recorded using
a stopwatch. After each determination, the tail was carefully dried.
The reaction time was determined before and periodically after oral
administration of the test substance (after 0, 30, 60 and 120 min).
The cut off time of the immersion was 10 s. Percentage analgesic
activity shown by the tested compounds is presented in Table 3.
Out of the newly synthesized compounds, 11c showed significant
activity at 30 min and highly significant activity at 60 and 120 min. As
regards the relationships between the structure of the heterocyclic
scaffold and the detected analgesic properties, it showed varied bio-
logicalactivity. Probably in this casethe nature of theheterocyclicring
as well as the presence of different substituents causes a certain
change of activity. Among the three series, 11aed which contains
triazole moiety showed good activity compared to the other two
series 12aed and 13aed. In the first series 11aed, compound
11c is showing significant analgesic activity and have 2,5-
dichlorothiophene moiety. From the obtained results, it is clear that
the major role for analgesic activity is played by the substituent pre-
sentonpyrazoleandtheheterocyclicringsbondedtopyrazolemoiety.
Antimicrobial activity data of the compounds 11aed, 12aed and 13aed in MIC
(mg/mL).
Comp. No.
Antibacterial activity
Antifungal
activity
S. aureus
B.subtilis
E.coli
P.aeruginosa C. albicans
11a
11b
11c
11d
12a
12b
12c
12d
13a
13b
13c
13d
Standard
Inoculum
control
3.1250
6.2500
1.6125
3.1250
12.5000
6.2500
6.2500
12.5000
12.5000
6.2500
3.1250
12.5000
3.1250
xxxa
3.1250
3.1250
1.6125
1.6125
6.2500
12.500
3.1250
12.5000
6.2500
12.5000
6.2500
25.0000
1.6125
xxxa
3.1250
3.1250
3.125
3.1250
3.125
6.2500
3.1250
25.0000
12.5000
12.5000
3.1250
12.5000
1.6125
xxxa
6.2500
1.6125
1.6125
1.6125
6.2500
6.2500
6.2500
12.5000
3.1250
12.5000
3.1250
12.5000
1.6125
xxxa
12.5000
12.5000
6.25000
6.25000
12.5000
12.5000
25.0000
25.0000
12.5000
25.0000
12.5000
25.0000
6.2500
xxxa
Broth control No growth No growth No growth No growth
No growth
a
Indicates Growth in all concentrations.
substituent respectively, which is responsible for the enhanced
activity of the compounds. The remaining compounds showed
moderately good activity against all of the four tested bacterial
strains compared to the standard, Ceftriaxone.
Antifungal activity data revealed that compounds 11c and 11d
had shown similar activity against the fungal stain C. albicans com-
pared to the standard drug Fluconazole. Other derivatives were less
active compared to standard. Among the three series, 11aed which
contains triazole moiety showed good antimicrobial activity com-
pared to the other two series 12aed and 13aed, which contains
benzoxazole substituent.
5. Conclusion
Three series of new Schiff bases and hydrazones of substituted
pyrazole were synthesized in reasonably good yields. The newly
synthesized compounds were characterized by 1H NMR, 13C NMR,
mass spectrometry, IR studies and elemental analyses. All the
newly synthesized compounds 11a-d, 12a-d and 13a-d were
screened for analgesic activity by tail flick method and the results
showed moderately good activity compared to the standard drug
Pentazocin. Among the synthesized compounds, 11c showed sig-
nificant analgesic activity and have 2,5-dichlorothiophene substit-
uent on pyrazole ring, which accounts for the enhanced analgesic
activity. The antimicrobial activity of the new derivatives was also
performed and the results revealed that among the screened
samples, 11c and 11d emerged as potent antimicrobial agents. 11c
and 11d contains 2,5-dichlorothiophene, 2,4-dichlorophenyl sub-
stituent respectively on pyrazole ring. Among the synthesized
compounds, 11aed which contains triazole moiety showed good
analgesic and antimicrobial activity compared to 12aed and 13aed,
which contains benzoxazole substituent.
As regards the relationships between the structure of the het-
erocyclic scaffold and the detected antimicrobial/analgesic prop-
erties, it showed varied biological activity. Probably in this case the
nature of the heterocyclic ring as well as the presence of different
substituents caused a certain change of activity. From the anti-
microbial and analgesic screening results, one can conclude that,
a combination of two different heterocyclic systems namely pyr-
azole and 1,2,4-triazoles has enhanced the pharmacological effect
and hence they are ideally suited for further modifications to obtain
more efficacious antibacterial/analgesic compounds.
4.2. Analgesic studies: tail flick method
This method was described by Asongalem et al. [27]. Young male
Wistar strain albino rats (170e210 g body weight) were used for the
study. Fourteen groups having six animals in each group were
taken. Group one served as vehicle control, 0.5% CMC was given
and group two served as standard received Pentazocin at a dose
Table 3
Analgesic activity (tail flick method) of compounds 11a-d, 12a-d and 13a-d.
Compound
% Inhibition ꢂ SEM (n ¼ 6)
0 min
30 min
60 min
120 min
Control
Pentazocin
(Standard)
11a
11b
11c
11d
12a
12b
12c
1.9 ꢂ 0.12
1.9 ꢂ 0.12
4.20 ꢂ 0.12
2.17 ꢂ 0.06c
4.18 ꢂ 0.13
2.18 ꢂ 0.06c
4.20 ꢂ 0.10
2.22 ꢂ 0.07c
2.62 ꢂ 0.28
2.85 ꢂ 0.33
2.67 ꢂ 0.41
2.93 ꢂ 0.30
2.27 ꢂ 0.43
3.05 ꢂ 0.33
2.55 ꢂ 0.42
3.13 ꢂ 0.37
2.75 ꢂ 0.31
3.25 ꢂ 0.38
2.98 ꢂ 0.35
2.43 ꢂ 0.24
2.77 ꢂ 0.21a
2.93 ꢂ 0.32
2.52 ꢂ 0.31b
3.50 ꢂ 0.39
3.38 ꢂ 0.36
3.15 ꢂ 0.37
2.82 ꢂ 0.43a
3.27 ꢂ 0.39
2.92 ꢂ 0.30
3.42 ꢂ 0.36
3.03 ꢂ 0.36
2.65 ꢂ 0.26a
2.83 ꢂ 0.22a
2.25 ꢂ 0.11
2.28 ꢂ 0.20c
4.15 ꢂ 0.32
3.45 ꢂ 0.23
3.53 ꢂ 0.22
2.93 ꢂ 0.44
3.43 ꢂ 0.41
3.05 ꢂ 0.30
3.57 ꢂ 0.38
3.07 ꢂ 0.36
2.77 ꢂ 0.26
2.90 ꢂ 0.22
3.08 ꢂ 0.33
2.20 ꢂ 0.13c
2.98 ꢂ 0.38
3.62 ꢂ 0.55
3.83 ꢂ 0.21
3.06 ꢂ 0.44
3.63 ꢂ 0.39
3.20 ꢂ 0.31
3.63 ꢂ 0.37
3.10 ꢂ 0.35
2.95 ꢂ 0.28a
12d
13a
13b
13c
13d
6. Experimental
Statistical analysis was done by one way analysis of variance (ANOVA) followed by
Dunnet’s test,
pentazocin.
n
¼
6. Values were compared with respect to standard drug
6.1. Chemistry
a
P < 0.05 (Significant from the control).
P < 0.01 (Significant from the control).
P < 0.001 (Significant from the control).
b
c
Melting points were determined by open capillary method and
were uncorrected. The IR spectra (in KBr pellets) were recorded on

414
A.M. Vijesh et al. / European Journal of Medicinal Chemistry 62 (2013) 410e415
a JASCO FT/IR-4100 spectrophotometer. 1H NMR spectra were recor-
ded (DMSO-d6) on a Bruker (400 MHz) using TMS as internal stan-
6.3. General procedure for the synthesis of hydrazones 12a-d and
13a-d
dard. Chemical shift values are given in
d (ppm) scales. The mass
spectrawere recorded ona JEOL JMS-D 300 spectrometeroperating at
70 eV. Elemental analyses were performed on a Flash EA 1112 series
CHNSeO Analyzer. The completion of the reaction was checked by
thin layer chromatography (TLC) on silica gel coated aluminium
sheets (silica gel 60 F254) obtained from Merck. Commercial grade
solvents and reagents were used without further purification.
An equimolar mixture of 3-aryl-1H-pyrazole-4-carbaldehyde,
10a-d (0.1 mol) and corresponding benzoxazolehydrazides, 6 or 7,
(0.1 mol) were dissolved in warm ethanol (20 mL) containing
glacial acetic acid (0.5 mL). The reaction mixture was refluxed for
8 h and then kept at room temperature overnight. The solids
precipitated were filtered, washed with chilled ethanol, dried and
recrystallized from hot ethanol to afford compounds 12a-d and
13a-d.
6.2. General procedure for the synthesis of 4-{[(E)-{4-[aryl]-1H-
pyrazol-3-yl}methylidene] amino}-5-(substituted)-4H-1,2,4-
triazole-3-thiol (11a-d)
6.3.1. 2-(1,3-Benzoxazol-2-ylsulfanyl)-N0-{(E)-[3-(2,4-
dichlorophenyl)-1H-pyrazol-4-yl] methylidene}acetohydrazide
(12a)
An equimolar mixture of 3-aryl-1H-pyrazole-4-carbaldehyde,
10a-d (0.1 mol) and 4-amino-5-substituted-4H-1,2,4-triazole-3-
thiol, 3a-d (0.1 mol) were dissolved in warm ethanol (20 mL). A
catalytic amount of sulphuric acid was added to the reaction mix-
ture and refluxed for 8 h, then kept at room temperature overnight.
The resultant solid was filtered, washed with chilled ethanol, dried
and recrystallized from ethanoledioxane (1:2) mixture to afford
compounds 3aed.
IR (KBr, ymax cmꢀ1): 3215 (NeH-str), 2971 (CeH-str), 1652 (C¼O,
C¼N); 1H NMR (DMSO-d6):
d 4.12 (s, 2H, eCH2eCO), 7.27e7.80
(m, 8H, AreH), 7.84 (s, 1H, pyrazole-5H), 8.18 (s, 1H, N¼CH), 11.37
(s, 1H, COeNH), 13.39 (s, 1H, pyrazole-NH); MS: m/z ¼ 447 (M þ 1).
Anal. calcd. for C19H17Cl2N5O2S : C, 51.13; H, 2.94; N,15.69. Found: C,
51.09; H, 2.91; N, 15.66%.
6.3.2. 2-(1,3-Benzoxazol-2-ylsulfanyl)-N0-[(E)-{3-[4-
(methylsulfanyl)phenyl]-1H-pyrazol-4-yl}methylidene]
acetohydrazide (12b)
6.2.1. 4-{[(E)-{4-[4-(Methylsulfanyl)phenyl]-1H-pyrazol-3-yl}
methylidene]amino}-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol
(11a)
IR (KBr, ymax cmꢀ1): 3154 (NeH-str), 2916 (CeH-str), 1659
IR (KBr, ymax cmꢀ1): 3120 (NeH-str), 2914 (CeH-str), 1620(C¼N),
(C¼O), 1610 (C¼N); 1H NMR (DMSO-d6):
d 2.45 (s, 3H, SCH3), 4.17
1542 (C¼C); 1H NMR (DMSO-d6):
d
2.50 (s, 3H, SCH3), 7.38 (d, 2H,
(s, 2H, eCH2eCO), 7.26e7.62 (m, 8H, AreH), 8.07 (s, 1H, pyrazole-
5H), 8.20 (s, 1H, N¼CH), 11.42 (s, 1H, COeNH), 13.39 (s, 1H,
pyrazole-NH); MS: m/z ¼ 424 (M þ 1). Anal. calcd. for C20H17N5O2S2
: C, 56.72; H, 4.05; N, 16.54. Found: C, 56.69; H, 4.03; N, 16.51%.
J ¼ 4.8 Hz, p-thioanisyl), 7.65 (d, 2H, J ¼ 4.8 Hz, p-thioanisyl), 7.86
(d, 2H, J ¼ 6.7 Hz, 4-pyridyl), 8.21 (s, 1H, pyrazole-5H), 8.73 (d, 2H,
J ¼ 6.7 Hz, 4-pyridyl), 9.52 (s, 1H, N¼CH), 13.81 (s, 1H, SH), 14.38
(s, 1H, pyrazole-NH); MS: m/z ¼ 394 (M þ 1). Anal. calcd. for
C18H15N7S2 : C, 59.94; H, 3.84; N, 24.92. Found: C, 59.91; H, 3.80; N,
24.92%.
6.3.3. 2-(1,3-Benzoxazol-2-ylsulfanyl)-N0-{(E)-[3-(2,5-
dichlorothiophen-3-yl)-1H-pyrazol-4-yl]methylidene}
acetohydrazide (12c)
6.2.2. 4-({(E)-[4-(2,4-Dichlorophenyl)-1H-pyrazol-3-yl]
methylidene}amino)-5-[(naphthalen-1-yloxy)methyl]-4H-1,2,4-
triazole-3-thiol (11b)
IR (KBr, ymax cmꢀ1): 3163 (NeH-str), 2945 (CeH-str), 1654 (C¼O,
C¼N); 1H NMR (DMSO-d6):
d 4.18 (s, 2H, eCH2eCO), 7.25e7.60
(m, 5H, AreH), 8.14 (s, 1H, pyrazole-5H), 8.31 (s, 1H, N¼CH), 11.46
(s, 1H, COeNH), 13.38 (s, 1H, pyrazole-NH); MS: m/z ¼ 453 (M þ 1).
Anal. calcd. for C17H11Cl2N5O2S2 : C, 45.14; H, 2.45; N, 15.48. Found:
C, 45.12; H, 2.43; N, 15.44%.
IR (KBr, ymax cmꢀ1): 3118 (NeH-str), 2917 (CeH-str), 1622 (C¼N),
1596 (C¼C); 1H NMR (DMSO-d6):
d 5.34 (s, 2H, OCH2), 7.09e7.86
(m, 10H, AreH), 8.27 (s, 1H, pyrazole-5H), 8.68 (s, 1H, N¼CH),
13.62 (s, 1H, SH), 14.21 (s, 1H, pyrazole-NH); 13C NMR:
d 141.81,
134.28, 133.86, 133.39, 127.00, 126.29, 125.82, 125.31; MS:
m/z ¼ 496 (M þ 1). Anal. calcd. for C23H16Cl2N6OS : C, 55.76; H, 3.26;
N, 16.96. Found: C, 55.73; H, 3.24; N, 16.93%.
6.3.4. 2-(1,3-Benzoxazol-2-ylsulfanyl)-N0-[(E)-(3- biphenyl -1H-
pyrazol-4-yl)methylidene] acetohydrazide (12d)
IR (KBr, ymax cmꢀ1): 3179 (NeH-str), 2966 (CeH-str), 1656 (C¼O,
C¼N); 1H NMR (DMSO-d6):
d 4.46 (s, 2H, eCH2eCO), 7.21e7.68
6.2.3. 4-({(E)-[4-(2,5-Dichlorothiophen-3-yl)-1H-pyrazol-3-yl]
methylidene}amino)-5-[(naphthalene-1-yloxy)methyl]-4H-1,2,4-
triazole-3-thiol (11c)
(m, 13H, AreH), 8.07 (s, 1H, pyrazole-5H), 8.25 (s, 1H, N¼CH),
11.42 (s, 1H, COeNH), 13.32 (s, 1H, pyrazole-NH); MS: m/z ¼ 454
(M þ 1). Anal. calcd. for C25H19N5O2S : C, 66.21; H, 4.22; N, 15.44.
Found: C, 66.17; H, 4.19; N, 15.41%.
IR (KBr, ymax cmꢀ1): 3368 (NeH-str), 2915(CeH-str),1652 (C¼N),
1571 (C¼C); 1H NMR (DMSO-d6):
d 5.33 (s, 2H, OCH2), 7.11e8.01
(m, 8H, AreH), 8.41 (s, 1H, pyrazole-5H), 9.76 (s, 1H, N¼CH), 13.76
(s, 1H, SH), 14.05 (s, 1H, pyrazole-NH); MS: m/z ¼ 502 (M þ 1). Anal.
calcd. for C21H14Cl2N6OS2: C, 50.30; H, 2.81; N, 16.76. Found: C,
50.27; H, 2.79; N, 16.73%.
6.3.5. 5,7-Dichloro-2-[(2E)-2-{[3-(2,4-dichlorophenyl)-1H-pyrazol-
4-yl]methylidene} hydrazinyl]-1,3-benzoxazole (13a)
IR (KBr, ymax cmꢀ1): 3132 (NeH-str), 2902 (CeH-str), 1660 (C¼N),
1551 (C¼C); 1H NMR (DMSO-d6):
d 7.21e7.76 (m, 5H, AreH), 7.93
(s,1H, pyrazole-5H), 8.19 (s,1H, N¼CH),12.05 (s,1H, NH),13.46 (s,1H,
pyrazole-NH); MS: m/z ¼ 442 (M þ 1). Anal. calcd. for C17H9Cl4N5O :
C, 46.29; H, 2.06; N, 15.88. Found: C, 46.25; H, 2.03; N, 15.84%.
6.2.4. 4-({(E)-[4-(2,4-Dichlorophenyl)-1H-pyrazol-3-yl]
methylidene}amino)-5-(6-methylpyridin-3-yl)-4H-1,2,4-triazole-3-
thiol (11d)
IR (KBr, ymax cmꢀ1): 3240 (NeH-str), 2913 (CeH-str),1626(C¼N),
6.3.6. 5,7-Dichloro-2-[(2E)-2-({3-[4-(methylsulfanyl)phenyl]-1H-
pyrazol-4-yl}methylidene) hydrazinyl]-1,3-benzoxazole (13b)
IR (KBr, ymax cmꢀ1): 3126 (NeH-str), 2909 (CeH-str), 1657
1552 (C¼C); 1H NMR (DMSO-d6):
d 2.49 (s, 3H, CH3), 7.36e7.92
(m, 5H, AreH), 8.43 (s, 1H, pyrazole-5H), 8.75 (s, 1H, pyridine-
2H), 9.49 (s, 1H, N¼CH), 13.83 (s, 1H, SH), 14.13 (s, 1H, pyrazole-
NH); MS: m/z ¼ 431 (M þ 1). Anal. calcd. for C18H13Cl2N7S : C,
50.24; H, 3.05; N, 22.78. Found: C, 50.21 H, 3.01; N, 22.74%.
(C¼N), 1550 (C¼C); 1H NMR (DMSO-d6):
d 2.46 (s, 3H, SCH3), 7.25e
7.57 (m, 6H, AreH), 8.08 (s, 1H, pyrazole-5H), 8.19 (s, 1H, N¼CH),
12.12 (s, 1H, NH), 13.41 (s, 1H, pyrazole-NH); MS: m/z ¼ 419 (M þ 1).

A.M. Vijesh et al. / European Journal of Medicinal Chemistry 62 (2013) 410e415
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6.3.7. 5,7-Dichloro-2-[(2E)-2-{[3-(2,5-dichlorothiophen-3-yl)-1H-
pyrazol-4-yl]methylidene} hydrazinyl]-1,3-benzoxazole (13c)
IR (KBr, ymax cmꢀ1): 3130 (NeHestr), 2912 (CeH-str), 1621
(C¼N), 1583 (C¼C); 1H NMR (DMSO-d6):
d 7.24e7.37 (m, 3H, AreH),
8.05 (s, 1H, pyrazole-5H), 8.24 (s, 1H, N¼CH), 12.14 (s, 1H, NH), 13.51
(s, 1H, pyrazole-NH); MS: m/z ¼ 448 (M þ 1). Anal. calcd. for
C15H7Cl4N5OS : C, 40.29; H, 1.58; N, 15.66. Found: C, 40.26; H, 1.56;
N, 15.63%.
6.3.8. 2-[(2E)-2-{[3-(Biphenyl-4-yl)-1H-pyrazol-4-yl]methylidene}
hydrazinyl]-5,7-dichloro-1,3-benzoxazole (13d)
IR (KBr, ymax cmꢀ1): 3110 (NeH-str), 2905 (CeH-str), 1659 (C¼N),
1548 (C¼C); 1H NMR (DMSO-d6):
d 7.24e8.02 (m, 11H, AreH), 8.07
(s,1H, pyrazole-5H), 8.27 (s,1H, N¼CH),12.15 (s,1H, NH),13.49 (s,1H,
pyrazole-NH); 13C NMR (DEPT, DMSO-d6,100 MHz):
d 140.68,129.53,
129.22, 128.26, 127.46, 120.93, 115.67; MS: m/z ¼ 449 (M þ 1). Anal.
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H, 3.35; N, 15.60%.
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Acknowledgements
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[20] A.M. Vijesh, A.M. Isloor, S. Telkar, S.K. Peethambar, S. Rai, N. Isloor, Eur. J. Med.
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M. Thiageeswaran, H.K. Fun, Eur. J. Med. Chem. 45 (2010) 2695e2699.
[22] A.M. Vijesh, A.M. Isloor, V. Prabhu, S. Ahmad, S. Malladi, Eur. J. Med. Chem. 45
(2010) 5460e5464.
The authors extend their appreciation to The Deanship of
Scientific Research at King Saud University for the funding the
work through the research group project No. RGP-VPP-207.
Appendix A. Supplementary data
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N.A. Isloor, Eur. J. Med. Chem. 46 (2011) 5591e5597.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.057.
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