Synthesis, pharmacological activities and molecular docking studies of pyrazolyltriazoles as anti-bacterial and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2017.08.042

A series of novel pyrazolyl alcohols (5a-h), pyrazolyl azides (6a-h), and pyrazolyltriazoles (8a-h, 10a-p and 12a-l) were prepared and evaluated for their bioactivity (anti-bacterial and anti-inflammatory) profile. The compound 5c displayed the potent anti-bacterial activity against Micrococcus luteus (MIC 3.9 and MBC 7.81 μg/mL). In vitro anti-inflammatory activity data denoted that compound 8b is effective among the tested compounds against IL-6 (IC₅₀ 6.23 μM). Docking analysis of compounds 5f, 8a-b, 8e-f and 8h displayed high binding energies for the compounds 8a-b and 8h towards TNF-α dimer (2AZ5 protein) and IL-6 (1ALU protein). In vivo anti-inflammatory activity of compounds 8b and 8h with respect to LPS induced mice model indicated that compound 8h showed significant reduction in TNF-α.

Full text of DOI:10.1016/j.bmc.2017.08.042

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, pharmacological activities and molecular docking studies of
pyrazolyltriazoles as anti-bacterial and anti-inflammatory agents
Cherupally Dayakar a, Buddana Sudheer Kumar b, Galande Sneha b, Gudem Sagarika c, Koneru Meghana c,
Sistla Ramakrishna c, , Reddy Shetty Prakasham b, , Bhimapaka China Raju a,
⇑
⇑
⇑
a Natural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Medicinal Chemistry and Biotechnology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
c Pharmacology and Toxicology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel pyrazolyl alcohols (5a-h), pyrazolyl azides (6a-h), and pyrazolyltriazoles (8a-h, 10a-p
and 12a-l) were prepared and evaluated for their bioactivity (anti-bacterial and anti-inflammatory) pro-
file. The compound 5c displayed the potent anti-bacterial activity against Micrococcus luteus (MIC 3.9 and
MBC 7.81 mg/mL). In vitro anti-inflammatory activity data denoted that compound 8b is effective among
Received 28 March 2017
Revised 2 August 2017
Accepted 25 August 2017
Available online xxxx
the tested compounds against IL-6 (IC50 6.23
displayed high binding energies for the compounds 8a-b and 8h towards TNF-
IL-6 (1ALU protein). In vivo anti-inflammatory activity of compounds 8b and 8h with respect to LPS
induced mice model indicated that compound 8h showed significant reduction in TNF-
l
M). Docking analysis of compounds 5f, 8a-b, 8e-f and 8h
a
dimer (2AZ5 protein) and
Keywords:
1H-Pyrazole-4-carbaldehydes
Pyrazolyl-1H-1,2,3-triazolyl alcohols
Pyrazolyl-1H-1,2,3-triazoles
Pyrazolyl-1H-1,2,3-triazolylcarboxylates
Anti-bacterial activity
a
.
Ó 2017 Elsevier Ltd. All rights reserved.
Anti-inflammatory activity
1. Introduction
term use of NSAIDs leads to the side effects such as bleeding, gas-
trointestinal ulceration and kidney damage. Celecoxib (Fig. 1),
Valdecoxib and Rofecoxib are the potent and gastrointestinal safe
anti-inflammatory drugs. Recently, pyrazole and triazole linked
derivatives were reported as potential PDE4, inhibitors.6
As part of our ongoing work on synthesis, biological activities of
triazoles7–10 and pyrazoles,11–14 authors found that both these
molecules are effective bioactive compounds. Literature search
against pyrazolyltriazoles revealed no information on synthesis
and biological activities. In view of the above, the present manu-
script describes the preparation of pyrazole based pyrazolyl alco-
hols, pyrazolyltriazoles and evaluation of their anti-microbial as
Pyrazoles and its derivatives are important nitrogen containing
heterocyclic compounds.1 Pyrazoles constitute the core structures
of natural products and pyrazole based well known drugs are avail-
able in the market such as Celebrex, Viagra and Acomplia.2 On the
other hand 1,2,3-triazoles have emerged as an important hetero-
cyclic compounds, have been displayed various biological activities
and widely used as pharmaceuticals.3 Therefore, the development
of synthetic methods for the preparation of pyrazoles and triazoles
are continuous to be an active area of research.
Inflammation is a process with the tumor necrosis factor (TNF-
a
) and interleukin-6 (IL-6) play a major role in pathogenesis.4
well as anti-inflammatory activities against TNF-a and IL-6
Inflammation is a condition of body immunity, swelling, redness,
heat, ache and pain associated with damaged tissue or organ. Fur-
ther, it is associated with diseases including allergy, arthritis,
atherosclerosis and auto-immune diseases. The major pharmaco-
logical action of nonsteroidal anti-inflammatory drugs (NSAIDs)
is the enzymatic inhibition of cyclooxygenase (COX) mediated pro-
duction of pro-inflammatory prostaglandins and thromboxanes.5
NSAIDs are used for the treatment of acute inflammation. The long
(in vitro) and LPS induced mice (in vivo). The structure activity rela-
tionships and molecular modeling studies are also discussed.
2. Results and discussion
2.1. Chemistry
The preparation of target compounds pyrazolyl-1H-1,2,3-triazolyl
alcohols 8a-h, pyrazolyl-1H-1,2,3-triazoles 10a-p and pyrazolyl-
1H-1,2,3-triazolyl carboxylates 12a-l were depicted in Schemes
1–3. Condensation of acetophenones 1a-h with henylhydrazine
⇑
Corresponding author.
E-mail address: chinaraju@iict.res.in (B. China Raju).
http://dx.doi.org/10.1016/j.bmc.2017.08.042
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. Structures of pyrazole (1-2) and triazole (3-4) drugs; present work on pyrazole based heterocyclic compounds (8a-h, 10a-p and 12a-l).
Scheme 1. Synthesis of substituted 1H-1,2,3-triazolyl alcohols 8a-h.
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3
Scheme 2. Synthesis of substituted 1H-1,2,3-triazoles 10a-p.
Scheme 3. Synthesis of substituted 1H-1,2,3-triazole-4,5-dicarboxylates 12a-l.
hydrochlorides 2a-h in the presence of acetic acid at room temper-
ature provided the corresponding phenylhydrazones 3a-h
(Scheme 1). Vilsmeier-Haack reaction of 3a-h in presence of DMF
with POCl3 afforded pyrazole carboxaldehydes 4a-h.15 NaBH4
reduction of carboxaldehydes 4a-h provided the corresponding
alcohols 5a-h. Alcohols 5a-h were converted to corresponding
azides 6a-h in the presence of DPPA and DBU (Scheme 1).16
Click chemistry has been explored a new approach for the
preparation of various heterocyclic molecules that can accelerate
the drug discovery process. Thus prepared azides 6a-h were sub-
jected to cycloaddition with propargyl alcohol 7, phenyl acetylenes
9a-k and dimethyl/diethyl acetylenedicarboxylate 11a-b to
prepare series of target compounds (Schemes 1–3). Initially, we
have carried out the reaction of azides 6a-h with propargyl alcohol
7 in the presence of CuSO4ꢀ5H2O/sodium ascorbate in aqueous
alcohol medium. This provided the corresponding pyrazolyl-1H-
1,2,3-triazolyl alcohols 8a-h (Scheme 1). The cycloaddition of
azides 6a-f and phenyl acetylenes 9a-k under optimized condi-
tions provided pyrazolyl-1H-1,2,3-triazoles 10a-p (Scheme 2).
Similarly, cycloaddition of azides 6a-f with dimethyl/diethyl
acetylenedicarboxylate 11a-b provided the corresponding pyra-
zolyl-1H-1,2,3-triazolyl carboxylates 12a-l (Scheme 3). Thus pre-
pared compounds 8a-h, 10a-p and 12a-l are new and well
characterized by spectral data (Supplementary information).
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C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
2.2. Biology
measuring the MIC and MBC against M. luteus, S. typhi and S.
paratyphi (Table 1). Between 5c and 8f, compound 5c observed to
be most potent against M. leteus and the observed MIC value is
3.9 and 7.81 mg/ml whereas standard (streptomycin) showed
6.25 mg/ml (for MIC and MBC) (Table 1).
The intermediate compounds pyrazolyl alcohols 5a-h, pyrazoly-
lazides 6a-h and target compounds pyrazolyl-1H-1,2,3-triazolyl
alcohols 8a-h, pyrazolyl-1H-1,2,3-triazoles 10a-p, pyrazolyl-1H-
1,2,3-triazolylcarboxylates 12a-l were screened for their anti-
microbial and in vitro anti-inflammatory activities. Based on the
in vitro anti-inflammatory data, the molecular modeling studies
were performed for compounds 5f, 8a-b, 8e-f and 8h while for
in vivo anti-inflammatory activity, compounds 8b and 8h selected
based on activity profile data and experiments were carried out
using LPS induced mice.
2.2.2. In vitro anti-inflammatory activity
The in vitro anti-inflammatory activities of compounds 5a-h,
6a-h, 8a-h, 10a-p and 12a-l were tested by using protein (egg
albumin) denaturation method18 along with the standard drug,
diclofenac sodium (Table 2) while LPS-induced TNF-
a and IL-6
(based on enzyme-linked immunosorbent assay (ELISA)19 was car-
ried out for those compounds which revealed better activity profile
and compared with rolipram (Table 3). Analysis of the structure
activity relationship of compounds 5a-h denoted IC50 in the range
of 54.21–105.45 mg/ml with respect to in vitro protein denaturation
profile. The compound with chlorine substitution (5c, IC50
54.21 mg/ml) at N-aryl displayed equipotency with respect to the
standard compound (IC50 53.70 mg/ml; Table-2). However, com-
pound 5a, 5b (chlorine substitution at C-aryl) and 5h (dimethyl
substitution at N-aryl) displayed moderate activity, while 5d-e
(methyl substitution) and 5g (dichloro) reduced the activity.
Methoxy substituted derivative (5f) drastically reduced the activ-
ity. Addition of triazole moiety to pyrazolyl derivative (8a-h)
denoted IC50 in the range of 58.00 –128.89 mg/ml. Among these,
the compound 8e showed similar trend as that of 5a-b and 5h.
Comparing the activity profile of 5f and 8f (both have the methoxy
substitution) with triazole moiety (8f) improved the activity
(Table 2).
2.2.1. In vitro anti-microbial activity
The in vitro anti-microbial activity, minimum inhibitory con-
centration (MIC) and minimum bactericidal concentration (MBC)
of all the synthesized compounds (5a-h, 6a-h, 8a-h, 10a-p and
12a-l) were analyzed and the data presented in Table 1. The
in vitro anti-bacterial activity of compounds and internal standard,
streptomycin, was tested against two Gram-positive organisms
(Micrococcus luteus and Staphylococcus aureus) and two Gram-neg-
ative organisms (Salmonella typhi and Salmonella paratyphi) by agar
well plate method.17 The in vitro anti-fungal activity of compounds
(5a-h, 6a-h, 8a-h, 10a-p and 12a-l) was tested by agar plate
method against two fungal strains (Aspergillus niger and Aspergillus
fumigates) with fluconazole as standard. The MIC and MBC studies
were performed by using tube dilution.
The data presented in Table 1 describes that the chloro pyra-
zolyl alcohol 5c and pyrazolyl triazole alcohol 8f displayed better
anti-bacterial activity (26.5 and 26 mm zone of inhibition) against
M. luteus compared to standard (22.5 mm); while, none of the syn-
thesized compounds showed anti-bacterial activity against the
other Gram positive bacterium, S. aureus. With respect to Gram
negative bacteria, though some of the synthesized compounds
revealed anti-bacterial activity however, which is less than stan-
dard (Table 1). This data denoted that the synthesized compounds
role in growth inhibition of bacterial strains differ with chemical
nature of the compounds. Critical evaluation of Table 1 data further
indicated that the compound 5c and 8f are effective as anti-bacte-
rial agents in comparison with other synthesized compounds.
Hence, these compounds (5c and 8f) were further studied for eval-
uation of concentration dependent anti-bacterial activity profile by
In vitro anti-inflammatory activity based on TNF-
a and IL-6
revealed that pyrazolyltriazole alcohol compounds showed better
activity compared to pyrazole alcohols (Table 3, Fig. 2). Among
pyrazole alcohols, compound with methoxy substitution (5f)
showed activity, while the same compound denoted weak activity
at protein denaturation (Table 2); while analysis of activity at TNF-
a
and IL-6, compound 5f denoted fourfold improved activity with
IL-6 (Table 3). In case of pyrazolyltriazole alcohols, the compounds
8a-b, 8e-f and 8h showed activity in TNF- and IL-6. IL-6 based
activity is much better to that of TNF- , while standard compound
showed similar activity profile with TNF- and IL-6 (Table 3),
a
a
a
whereas egg albumin based in vitro anti-inflammatory activity
was noticed to 8e and 8f (Table 2).
Table 1
Anti-bacterial activity of the compounds 5a-h and 8a-h.
Zone of inhibition in mm (MIC, MBC [mg/ml])
Compound*
Gram positive Bacteria
Gram negative Bacteria
M. luteus
S. aureus
S. typhi
S. paratyphi
5a
5b
–
–
–
–
11.5 0.71
–
14 0.00
12 0.00
5c
5d
26.5 0.71 (3.9, 7.81)
16.5 0.71
–
–
21.5 0.71 (62.5, 125)
19 0.00
18 0.00 (31.25, 62.5)
14 0.00
5e
5f
5g
5h
8a
8b
–
–
–
–
–
–
–
–
–
–
–
–
17 1.41
–
13.5 0.71
14.5 0.71
–
12.5 0.71
11.5 0.71
11.5 0.71
12 0.00
12.5 0.71
15 0.00
–
8c
8d
11.5 0.71
–
–
–
19.5 0.71
15 0.00
16.5 0.71
14 0.00
8e
8f
8g
8h
–
–
–
–
–
19 0.00
11.5 0.71
19 0.00 (62.5, 62.5)
17 0.00
26 0.00 (125, 125)
–
–
16 0.00 (62.5, 125)
14 1.41
15.5 0.71
27 1.41 (6.25, 6.25)
–
Streptomycin
22.5 0.71 (6.25, 6.25)
20.3 0.57
30 0.00 (6.25, 6.25)
*
Compounds 6a-h, 10a-p and 12a-l were not shown activity; hence they have not incorporated.
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C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 2
A)
Anti-inflammatory activity of the compounds 5a-h and 8a-h based on protein
80
70
60
50
40
30
20
10
0
denaturation method.
Egg Albumin
Compound*
% inhibition
IC50 (mg/ml)
50 (mg/ml)
100 (mg/ml)
5f
5a
5b
5c
5d
5e
5f
5g
5h
8a
8b
8c
8d
8e
8f
8g
8h
75.86 0.17
67.24 0.44
76.72 0.89
60.34 0.43
40.52 0.72
15.52 0.85
60.34 0.58
66.38 0.22
10.34 0.89
49.14 0.47
55.17 0.78
21.55 0.64
66.38 0.95
68.97 0.44
50.86 0.25
46.55 0.77
66.38 0.06
87.93 0.87
83.62 0.46
92.24 0.78
81.90 0.83
68.10 0.33
47.41 0.59
80.17 0.87
83.62 0.53
38.79 0.21
73.28 0.79
78.45 0.77
38.79 0.98
83.62 0.67
86.21 0.34
76.72 0.51
77.59 0.87
93.10 0.12
56.86 0.76
59.79 0.34
54.21 0.87
61.05 0.65
73.42 0.54
105.45 0.85
62.37 0.43
59.79 0.12
128.89 0.74
68.24 0.32
63.74 0.84
128.89 0.75
59.79 0.39
58.00 0.71
65.17 0.82
64.44 0.55
53.70 0.24
8b
8h
0
5
10
15
20
25
Conc. (μM)
B)
100
90
80
70
60
50
40
30
20
10
0
5f
Diclofenac sodium
8b
8h
*
Compounds 6a-h, 10a-p and 12a-l were not shown activity; hence they have
not incorporated.
0
5
10
15
20
25
2.2.3. Docking studies on TNF-a and IL-6
Docking studies were performed to those compounds (5f, 8a-b,
8e-f and 8h) showed the anti-inflammatory activity at in vitro anal-
ysis (Table 4; Figs. 3 & 4) along with rolipram as standard towards
Conc. (μM)
Fig. 2. The active compounds (5f, 8b and 8h) inhibit PMA/LPS-induced production
of IL-6 and TNF-
a
in U937cells. U937 cells (5 ꢂ 104 cells/ml) were pre-incubated for
protein target 2AZ5 (TNF-
a
dimer) and 1ALU (IL-6).20 Among the
30 min in the presence of the indicated concentrations of active compounds, and
tested compounds, compound 8b showed the highest (ꢁ8.27)
then they were treated with 100 ng/ml LPS for 24 h (A, B). The concentration of TNF-
and IL-6 in the culture supernatants was measured using an ELISA assay. Data
shown are typical of three independent experiments with similar results.
binding energy while this value was lowest (ꢁ6.64) for 5f against
a
TNF-a, and rolipram showed binding energy of ꢁ8.86 K.cal/mol.
In case of IL-6, the compounds 8a (ꢁ6.71) and 8f (ꢁ5.55) showed
the highest and lowest binding energy values respectively, and
the rolipram showed ꢁ6.55 (Table 4). Evaluation of interactive nat-
ure (hydrogen bonding) between protein and synthesized com-
pounds suggested that compounds 5f (Fig. 3 A), 8a (Fig. 3 B) and
8f (Fig. 3 E) interacted with protein by single hydrogen bonding
while 8b (Fig. 3 C), 8e (Fig. 3 D) and 8h (Fig. 3 F) showed two inter-
compounds 8e-f (Fig. 4 D-E) and 8h (Fig. 4 F) depicted single
interaction.
2.2.4. In vivo anti-inflammatory activity by LPS challenged mouse
model
actions each with target protein [2AZ5 (TNF-a dimer)] while with
respect to IL-6 target protein (1ALU), the compounds 5f (Fig. 4 A)
and 8a-b (Fig. 4 B-C) denoted two interactions whereas the
LPS has been implicated as an important pathogenic factor for
the induction of sepsis, which is characterized by an inflammatory
cytokine storm. Hence, in vivo anti-inflammatory activity of
Table 3
Anti-inflammatory activity (TNF-
a
and IL-6) of the compounds 5a-h and 8a-h.
TNF-
Compound
a
IL-6
% Inhibition (10 mM)
IC50 (mM)
% Inhibition (10 mM)
IC50 (mM)
5a
5b
5c
5d
5e
5f
5g
5h
8a
8b
8c
8d
8e
8f
8g
8h
Rolipram
1.44 0.60
3.32 0.04
2.61 0.92
0.74 0.22
ND
22.95 1.67
4.92 0.84
8.35 1.30
15.70 1.10
19.24 3.94
12.73 4.68
14.75 2.35
31.19 3.51
40.88 0.10
9.90 2.78
41.73 5.61
59.13 1.76
ND
ND
ND
ND
ND
35.53 2.34
ND
19.57 0.23
24.11 6.56
21.65 1.48
20.87 3.36
23.77 6.95
68.47 1.64
18.33 5.23
46.36 1.80
52.99 4.22
76.94 0.01
38.82 0.86
42.04 1.33
50.40 3.52
46.73 7.34
39.57 9.06
87.00 8.36
53.53 3.05
ND
ND
ND
ND
ND
8.24 1.48
ND
ND
ND
35.74 2.45
30.92 1.50
ND
8.12 0.69
6.23 0.23
ND
ND
ND
21.39 1.89
17.18 1.01
ND
17.40 0.92
1.12 0.03
8.70 0.51
9.50 1.03
ND
7.72 0.82
1.17 0.49
ND: Not detected; results summarized in the table, for both % of cytokines inhibition presented as the mean SEM from three different experiments.
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Table 4
Dock score values for the compounds 5f, 8a-b, 8e-f and 8h.
Compound
TNF-a
IL-6
Binding energy (XP glide score) (K.cal/mol)
Interactions
Binding energy (XP glide score) (K.cal/mol)
Interactions
5f
8a
8b
8e
8f
8h
ꢁ6.64
ꢁ7.51
ꢁ8.27
ꢁ7.40
ꢁ6.98
ꢁ7.19
ꢁ8.86
ꢁ8.32
Leu120
Tyr151
ꢁ5.57
ꢁ6.71
ꢁ6.44
ꢁ5.79
ꢁ5.55
ꢁ6.50
ꢁ6.55
ꢁ9.97
Lys66, Lys86
Met67, Ser176
Met67, Ser176
Lys86
Lys86
Leu64
Tyr151, Gln61
Tyr151, Tyr151
Tyr151
Tyr151, Gln61
Tyr151, Ser60
Tyr151, Gly121
Rolipram
Indomethacin
Lys66, Ala68
Arg182, Arg179
B) 8a
A) 5f
C) 8b
F) 8h
D) 8e
E) 8f
G) Rolipram
Fig. 3. Docking pose of compounds 5f, 8a-b, 8e-f and 8h with protein 2AZ5 (TNF-a dimer).
compounds 8b and 8h were evaluated on the basis of LPS induced
(Fig. 5A) and IL-6 (Fig. 5B) levels compared to the vehicle control
group of animals. In the treatment groups, mice were pre-treated
with the test compounds 8b and 8h at a dose of 100 mg/kg for a
cytokine expression in Balb/c mice (Fig. 5).21,22 The challenge with
LPS in mice showed significant (P < 0.001) increase in TNF-
a
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7
A) 5f
B) 8a
C) 8b
E) 8f
E) 8h
D) 8e
G) Rolipram
Fig. 4. Docking pose of compounds 5f, 8a-b, 8e-f and 8h with protein 1ALU (IL-6).
(A)
(B)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
###
**
###
***
***
***
Fig. 5. Effect of test compounds on LPS induced serum inflammatory cytokine secretions. ELISA of tumor necrosis factor-alpha (TNF-a) (A) and interleukin-6 (IL-6) (B). The
results were expressed as mean S.E.M. of 8 animals in each group. Control, vehicle control mice; LPS control, LPS control mice; LPS + Dex, LPS + Dexamethasone (2 mg/kg)
treated mice; 8b + LPS, LPS + 8b (100 mg/kg) treated mice; 8h + LPS, LPS + 8h (100 mg/kg) treated mice. ### p < 0.001 vs. Control; ** p < 0.01 vs. LPS Control.
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C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
period of 3 consecutive days followed by LPS challenge (10 mg/kg).
Results indicated that the test compound 8h had shown significant
4.1.1. General procedure for the preparation of (E)-1-phenyl-2-(1-
phenylethylidene)-hydrazines (3a-h)
(P < 0.01) inhibition in the TNF-
a
levels when compared to the
Acetic acid (0.4 mmol) was added to a stirred solution of ace-
tophenone 1a (8.3 mmol) and phenyl hydrazine hydrochloride 2a
(9.9 mmol) in methanol. The contents were stirred at room tem-
perature for 1 h. After completion of the reaction (TLC), the solvent
was removed under reduced pressure and yellow colored solid 3a
was obtained. The other phenylhydrazones 3b-h were prepared
from corresponding acetophenones 1b-h and phenyl hydrazine
hydrochlorides 2b-h under similar conditions.
LPS control animals, whereas the levels of IL-6 were not signifi-
cantly (P < 0.05) altered (Fig. 5). Hence, compound 8h may be con-
sidered as a good therapeutic agent to treat the inflammatory
disease.
According to I. M. Garrelds et al23 there is time dependent pro-
duction of cytokines (TNF-
leukemia) cells mentioning that the production of TNF-
a
and IL-6) by U937 (Human monocytic
is maxi-
a
mum after 6 h of incubation with gradual decrease at 24 h. It signi-
fies the importance of time points for the analysis of cytokines in
4.1.1.1. (E)-1-phenyl-2-(1-phenylethylidene)hydrazine (3a). Ref. 24.
in vitro models. The independent regulation of the TNF-
a and
IL-6 induction during differentiation of U937 cells in in vitro and
their synthesis in in vivo plays a prominent role when treated with
compounds to suppress the formation of different inflammatory
mediators.
4.1.1.2. (E)-1-(1-(4-chlorophenyl)ethylidene)-2-phenylhydrazine (3b).
Ref. 24.
4.1.1.3. (E)-1-(4-chlorophenyl)-2-(1-phenylethylidene)hydrazine (3c).
Ref. 24.
Comparative analysis of bioinformatics data and experimental
data (in vitro and in vivo anti-inflammatory) on TNF-
IL-6 proteins revealed that TNF- showed more binding energies
compared to IL-6 for the compounds 5f, 8a-b, 8e-f and 8h. Among
binding energies of TNF- , the compound 8b showed highest bind-
a dimer and
a
4.1.1.4. (E)-1-phenyl-2-(1-p-tolylethylidene)hydrazine (3d). Ref. 24.
a
4.1.1.5. (E)-1-(4-chlorophenyl)-2-(1-phenylethylidene)hydrazine (3e).
ing energy (ꢁ8.27), while in vitro experimental data indicated 8f
compound displayed effective activity (IC50 17.18 mM). With
respect to IL-6 docking data, compounds 8a-b and 8h denoted
almost similar binding energies (ꢁ6.44 to ꢁ6.71), whereas,
in vitro data suggested 8b shown better activity (IC50 6.23 mM);
while in vivo analysis indicated that compound 8h has significant
Ref. 24.
4.1.1.6.
(3f). Ref. 25.
(E)-1-(1-(4-methoxyphenyl)ethylidene)-2-phenylhydrazine
4.1.1.7. (E)-1-(1-(2,4-dichlorophenyl)ethylidene)-2-phenylhydrazine
(3g). Ref. 25.
activity against TNF-
TNF- inhibition in the in vivo studies may be due to inhibition
of the NF- B activation which in turn regulated the expression of
TNF-
a (P < 0.01). This high selectivity towards
a
j
4.1.2. General procedure for the preparation of 1,3-diphenyl-1H-pyrazole-
4-carbaldehydes (4a-h)
a.
Phenylhydrazone (3a, 4.7 mmol) in ethanol was added to a stir-
red cold solution of Vilsmeier reagent, which is prepared by the
addition of DMF (1.1 mL) to POCl3 (2.2 mL) at 0 °C. The contents
were brought to room temperature and the stirring was continued
at 50–60 °C for 5 h. The reaction mixture was poured into ice cold
water and extracted with ethyl acetate (2 ꢂ 10 mL). The organic
layer was separated, washed with brine, dried over Na2SO4 and sol-
vent was removed under reduced pressure. The obtained residue
was purified by column chromatography (silicagel 60:120; ethyl
acetate/hexane 8:92) provided 1,3-diphenyl-1H-pyrazole-4-car-
baldehyde 4a. The 1H-pyrazole-4-carbaldehydes 4b-h were syn-
thesized from corresponding phenylhydrazones 3b-h under
similar conditions.
3. Conclusion
Series of pyrazolyl alcohols (5a-h), pyrazolyl azides (6a-h) and
pyrazolyltriazoles (8a-h, 10a-p and 12a-l) were prepared and
screened for their anti-microbial and anti-inflammatory (in vitro
and in vivo) activities. The compound 5c showed potential anti-
bacterial activity against M. luteus while, the compounds 5f, 8b
and 8h demonstrated potent in vitro anti-inflammatory activity,
whereas the compound 8h was found to be effective in vivo LPS
induced sepsis model in mice.
4. Experimental
4.1.2.1. 1,3-diphenyl-1H-pyrazole-4-carbaldehyde (4a). Ref. 24.
4.1. Chemistry
4.1.2.2.
3-(4-chlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
(4b). Ref. 24.
All chemicals and reagents were purchased from Aldrich
(Sigma-Aldrich, USA), AVRA Chemicals Pvt. Ltd (Hyderabad, India)
and were used without further purification. Reactions were moni-
tored by thin layer chromatography (TLC) on pre-coated silica gel
60 F254 (mesh); spots were visualized under UV light. Column chro-
matography has been carried out by using Merck silica gel (60–120
mesh). Melting points were determined in open glass capillary
tubes on a Stuart melting point apparatus and are uncorrected.
IR spectrum was recorded with a Thermo Nicollet Nexus 670 FT
spectrometer. 1H NMR and 13C NMR spectra were recorded on Bru-
ker Avance 300, 400 and 500 MHz spectrometers. Chemical shifts
(d) are quoted in parts per million and are referenced to tetram-
ethylsilane (TMS) as internal standard. ESI-MS obtained on quarto
micro spectrometer. HRMS were carried out on Agilent 6510, Q-
TOFLC/MS instrument ESI-Technique.
4.1.2.3.
1-(4-chlorophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde
(4c). Ref. 24.
4.1.2.4. 1-phenyl-3-p-tolyl-1H-pyrazole-4-carbaldehyde (4d). Ref.
24.
4.1.2.5. 3-phenyl-1-p-tolyl-1H-pyrazole-4-carbaldehyde (4e). Ref. 25.
4.1.2.6. 3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
(4f). Ref. 15.
4.1.2.7. 3-(3,4-dichlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
(4g). Ref. 26.
Please cite this article in press as: Dayakar C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.08.042

C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
4.1.2.8. 1-(3,4-Dimethylphenyl)-3-phenyl-1H-pyrazole-4-carbalde-
hyde (4h). Yield: 86%; white solid; m.p.: 119–121 °C; 1H NMR
(CDCl3): d 10.03 (s, 1H), 8.48 (s, 1H), 7.83–7.80 (m, 2H), 7.58 (d,
J = 2.1 Hz, 1H), 7.51–7.45 (m, 4H), 7.23 (d, J = 8.2 Hz, 1H), 2.33 (s,
3H), 2.30 (s, 3H); 13C NMR (CDCl3): d 185.12, 154.52, 138.18,
136.89, 136.62, 131.41, 130.79, 130.51, 129.13, 128.92, 128.67,
122.19, 120.93, 116.93, 19.89, 19.34; ESI-MS: m/z, 277 [M+H]+;
ESI-HRMS: m/z calcd for C18H16N2O [M+H]+ 277.1335, found
277.1329.
was removed under reduced pressure. The residue was purified
by column chromatography using silica gel (ethyl acetate/hexane
2:98) provided 4-(azidomethyl)-1,3-diphenyl-1H-pyrazole 6a as
colorless liquid. The azide compounds 6b-h were prepared from
corresponding pyrazolyl alcohols 5b-h under similar conditions.
4.1.4.1. 4-(Azidomethyl)-1,3-diphenyl-1H-pyrazole (6a). Ref. 29.
4.1.4.2.
4-(azidomethyl)-3-(4-chlorophenyl)-1-phenyl-1H-pyrazole
(6b). Ref. 29.
4.1.3. General procedure for the synthesis of (1,3-diphenyl-1H-pyrazol-
4-yl)-methanols (5a-h)
4.1.4.3.
4-(Azidomethyl)-1-(4-chlorophenyl)-3-phenyl-1H-pyrazole
Sodium borohydride (1.2 mmol) was added to a stirred solution
of pyrazole aldehyde 4a (1.0 mmol) in methanol at 0 °C. The reac-
tion mixture was stirred for 1 h at room temperature. After com-
pletion of the reaction (TLC), the solvent was removed under
reduced pressure; cold water was added and extracted with ethyl
acetate (2 ꢂ 10 mL). The organic layer was washed with brine,
dried over Na2SO4 and solvent was removed under reduced pres-
sure provided (1,3-diphenyl-1H-pyrazol-4-yl)methanol 5a. The
1H-pyrazolylmethanols 5b-h were prepared from the correspond-
ing pyrazole aldehydes 4b-h under similar conditions.
(6c). Ref. 29.
4.1.4.4. 4-(Azidomethyl)-1-phenyl-3-p-tolyl-1H-pyrazole (6d). Ref.
29.
4.1.4.5. 4-(Azidomethyl)-3-phenyl-1-p-tolyl-1H-pyrazole (6e). Ref.
29.
4.1.4.6. 4-(Azidomethyl)-3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole
(6f). Ref. 29.
4.1.3.1. (1,3-Diphenyl-1H-pyrazol-4-yl)methanol (5a). Ref. 27.
4.1.4.7. 4-(Azidomethyl)-3-(2,4-dichlorophenyl)-1-phenyl-1H-pyra-
zole (6g). Yield: 88%; colorless liquid; 1H NMR (CDCl3): d 8.04 (s,
1H), 7.72–7.70 (m, 2H), 7.51 (d, J = 2.1 Hz, 1H), 7.47–7.43 (m,
3H), 7.34–7.28 (m, 2H), 4.58 (s, 2H); 13C NMR (CDCl3): d 152.09,
138.24, 132.16, 129.54, 128.75, 128.48, 127.87, 127.83, 120.12,
115.35, 45.23; ESI-MS: m/z, 344 [M+H]+.
4.1.3.2.
(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methanol
(5b). Ref. 28.
4.1.3.3. (1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)methanol (5c).
Ref. 28.
4.1.4.8. 4-(Azidomethyl)-1-(3,4-dimethylphenyl)-3-phenyl-1H-pyra-
zole (6h). Yield: 86%; colorless liquid; 1H NMR (CDCl3): d 7.94 (s,
1H), 7.78–7.75 (m, 2H), 7.54 (d, J = 1.9 Hz, 1H), 7.46–7.36 (m,
4H), 7.17 (d, J = 8.2 Hz, 1H), 4.38 (s, 2H), 2.31 (s, 3H), 2.26 (s,
3H); 13C NMR (CDCl3): d 151.42, 137.86, 137.64, 135.20, 132.49,
130.30, 128.63, 128.15, 128.02, 127.81, 120.36, 116.33, 114.36,
45.24, 19.85, 19.22; ESI-MS: m/z, 304 [M+H]+; ESI-HRMS: m/z calcd
for C18H17N5Na 326.1376, found 326.1370.
4.1.3.4. (1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)methanol (5d). Ref. 27.
4.1.3.5. (3-phenyl-1-p-tolyl-1H-pyrazol-4-yl)methanol (5e). Ref. 27.
4.1.3.6. (3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methanol
(5f). Ref. 27.
4.1.3.7. (3-(2,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methanol
(5g). Yield: 98%; white solid; m.p.: 116–118 °C; 1H NMR (CDCl3):
d 8.04 (s, 1H), 7.73–7.69 (m, 2H), 7.51 (d, J = 2.1 Hz, 1H), 7.47–
7.43 (m, 3H), 7.34–7.28 (m, 2H), 4.57 (s, 2H); 13C NMR (CDCl3): d
148.64, 139.74, 135.11, 134.42, 133.00, 130.68, 129.54, 129.45,
127.16, 126.86, 126.73, 122.62, 119.10, 56.09; ESI-MS: m/z, 319
[M+H]+; ESI-HRMS: m/z calcd for C16H12Cl2N2O [M+H]+ 319.0399,
found 319.0410.
4.1.5. General procedure for the preparation of 1H-1,2,3-triazolyl
methanols (8a-h)
Propargyl alcohol 7 (1.0 mmol) was added to a stirred solution
of pyrazole azide 6a (1.0 mmol) in tert-butanol and H2O (1:1) at
room temperature, followed by CuSO4ꢀ5H2O (0.1 mmol) and
sodium ascorbate (0.05 mmol). The reaction mixture was stirred
for 6–12 h at room temperature. After completion of the reaction
(TLC), the reaction mixture was extracted with ethyl acetate
(2 ꢂ 5 mL). The organic extract was washed with H2O and dried
over anhydrous Na2SO4. The residue was purified by column
chromatography using silica gel (ethyl acetate/hexane) furnished
(1-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,3-triazol-4-yl)
methanol 8a. The 1H-1,2,3-triazolyl methanols 8b-h were synthe-
sized from pyrazole azides 6a-h under similar conditions.
4.1.3.8. (1-(3,4-Dimethylphenyl)-3-phenyl-1H-pyrazol-4-yl)methanol
(5h). Yield: 96%; white solid; m.p.: 138–140 °C; 1H NMR (CDCl3): d
7.95 (s, 1H), 7.85 (d, J = 7.4 Hz, 2H), 7.55 (s, 1H), 7.48–7.35 (m, 4H),
7.19 (d, J = 8.1 Hz, 1H), 4.74 (s, 2H), 2.33 (s, 3H), 2.29 (s, 3H); 13C
NMR (CDCl3): d 151.18, 137.92, 135.03, 133.07, 130.38, 128.66,
128.03, 127.82, 120.46, 120.42, 116.34, 56.07, 19.99, 19.34; ESI-
MS: m/z, 279 [M+H]+; ESI-HRMS: m/z calcd for C18H18N2O [M
+H]+ 279.1492, found 279.1494.
4.1.5.1. (1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,3-triazol-
4-yl)methanol (8a). Yield: 96%; white solid; m.p.: 126–128 °C; IR
mmax (cmꢁ1): 3451, 2924, 1592, 1500, 1325, 1216, 947, 758; 1H
NMR (CDCl3): d 7.98 (s, 1H), 7.76–7.69 (m, 2H), 7.64–7.58 (m,
2H), 7.51–7.37 (m, 6H), 7.32 (d, J = 7.4 Hz, 1H), 5.60 (s, 2H), 4.72
(s, 2H); 13C NMR (CDCl3): d 151.65, 148.00, 139.50, 132.03,
129.53, 128.95, 128.66, 128.57, 127.93, 127.02, 121.50, 119.14,
114.47, 56.35, 44.75; ESI-MS: m/z, 332 [M+H]+; ESI-HRMS: m/z
calcd for C19H17N5O [M+H]+ 332.1505, found 332.1508.
4.1.4. General procedure for the preparation of 4-(azidomethyl)-1,3-
diphenyl-1H-pyrazole (6a-h)
DBU (1.2 mmol) and diphenylphosphoryl azide (1.5 mmol) was
added sequentially to a stirred solution of (1,3-diphenyl-1H-pyra-
zol-4-yl)methanol 5a (1.0 mmol) in dry toluene (10 mL) at room
temperature. The reaction mixture was stirred for 3 h at room tem-
perature. After completion of the reaction (TLC), the reaction mix-
ture was quenched with aqueous NH4Cl solution (8 mL) and
extracted with ethyl acetate (2 ꢂ 10 mL). The organic layer was
separated, washed with brine, dried over Na2SO4 and the solvent
4.1.5.2. (1-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)-
1H-1,2,3-triazol-4-yl)methanol (8b). Yield: 96%; white solid; m.p.:
Please cite this article in press as: Dayakar C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.08.042

10
C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
156–158 °C; IR mmax (cmꢁ1): 3447, 2924, 1595, 1500, 1327, 1217,
949, 754; 1H NMR (CDCl3+DMSO-d6): d 8.19 (s, 1H), 7.75 (d,
J = 7.8 Hz, 2H), 7.64 (d, J = 8.3 Hz, 3H), 7.55–7.40 (m, 4H), 7.33 (t,
J = 7.4 Hz, 1H), 5.61 (s, 2H), 4.70 (s, 2H); 13C NMR (CDCl3 +
DMSO-d6): d 149.62, 148.44, 138.86, 133.70, 130.21, 128.94,
128.63, 128.38, 126.44, 121.16, 118.43, 114.30, 55.62, 43.92;
ESI-MS: m/z, 366 [M+H]+; ESI-HRMS: m/z calcd for C19H16ClN5O
[M+H]+ 366.1116, found 366.1126.
7.40 (td, J = 6.1, 1.8 Hz, 3H), 7.19 (d, J = 8.1 Hz, 1H), 5.61 (s, 2H),
4.74 (s, 2H), 2.32 (s, 3H), 2.29 (s, 3H); 13C NMR (CDCl3): d 151.35,
147.91, 138.05, 137.53, 135.64, 132.20, 130.46, 128.94, 128.56,
128.52, 127.97, 121.41, 120.50, 116.49, 114.04, 56.54, 44.81,
19.95, 19.36; ESI-MS: m/z, 360 [M+H]+; ESI-HRMS: m/z calcd for
C
21H21N5O [M+H]+ 360.1818, found 360.1821.
4.1.6. General procedure for the preparation of 1H-1,2,3-triazoles
(10a-p)
4.1.5.3. (1-((1-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)methyl)-
1H-1,2,3-triazol-4-yl)methanol (8c). Yield: 92%; white solid; m.p.:
154–156 °C; IR mmax (cmꢁ1): 3373, 2862, 1589, 1494, 1392, 1202,
818, 701; 1H NMR (CDCl3): d 7.95 (s, 1H), 7.68–7.65 (m, 2H),
7.62–7.58 (m, 2H), 7.49–7.40 (m, 6H), 5.60 (s, 2H), 4.74 (s, 2H);
Phenyl acetylene 9a (1.0 mmol) was added to a stirred solution
of pyrazole azide 6a (1.0 mmol) in tert-butanol and H2O (1:1) at
room temperature, followed by CuSO4ꢀ5H2O (0.1 mmol) and
sodium ascorbate (0.05 mmol). The reaction mixture was stirred
for 6–12 h at room temperature. After completion of the reaction
(TLC), the reaction mixture was extracted with ethyl acetate
(2 ꢂ 5 mL). The organic extract was washed with H2O and dried
over anhydrous Na2SO4. The residue was purified by column
chromatography using silica gel (ethyl acetate/hexane) furnished
1-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-4-phenyl-1H-1,2,3-tria-
zole 10a. The 1H-1,2,3-triazole compounds 10b-p were synthe-
sized from pyrazole azides 6a-f under similar conditions.
13C NMR (CDCl3):
d 151.92, 147.97, 138.07, 132.52, 131.85,
129.63, 129.00, 128.82, 128.40, 127.96, 121.47, 120.24, 115.00,
56.50, 44.73; ESI-MS: m/z, 366 [M+H]+; ESI-HRMS: m/z calcd for
C
19H16ClN5O [M+H]+ 366.1116, found 366.1121.
4.1.5.4. (1-((1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl)-1H-1,2,3-
triazol-4-yl)methanol (8d). Yield: 89%; white solid; m.p.: 140–
142 °C; IR mmax (cmꢁ1): 3412, 2919, 1516, 1447, 1320, 1213, 813,
766; 1H NMR (CDCl3): d 7.97 (s, 1H), 7.71 (d, J = 7.7 Hz, 2H),
7.53–7.39 (m, 5H), 7.33–7.25 (m, 3H), 5.61 (s, 2H), 4.74 (s, 2H),
2.40 (s, 3H); 13C NMR (CDCl3): d 151.73, 139.59, 138.59, 129.65,
129.52, 129.18, 128.47, 127.81, 126.94, 121.46, 120.43, 119.13,
114.38, 56.55, 44.82, 21.34; ESI-MS: m/z, 346 [M+H]+; ESI-HRMS:
m/z calcd for C20H19N5O [M+H]+ 346.1662, found 346.1665.
4.1.6.1.
1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-4-phenyl-1H-
1,2,3-triazole (10a). Yield: 85%; white solid; m.p.: 156–158 °C; IR
mmax (cmꢁ1): 3083, 2925, 1597, 1502, 1455, 1221, 1067, 769, 692;
1H NMR (CDCl3): d 8.02 (s, 1H), 7.78–7.73 (m, 4H), 7.67–7.64 (m,
2H), 7.63 (s, 1H), 7.51–7.37 (m, 7H), 7.34–7.29 (m, 2H), 5.69 (s,
2H); 13C NMR (CDCl3): d 151.70, 148.03, 139.58, 132.15, 130.47,
129.55, 129.00, 128.85, 128.70, 128.49, 128.24, 127.98, 127.02,
125.71, 119.32, 119.15, 114.69, 44.92; ESI-MS: m/z, 400 [M+H]+;
ESI-HRMS: m/z calcd for C24H19N5Na [M+H]+ 400.1520, found
400.1532.
4.1.5.5. (1-((3-Phenyl-1-(p-tolyl)-1H-pyrazol-4-yl)methyl)-1H-1,2,3-
triazol-4-yl)methanol (8e). Yield: 85%; white solid; m.p.: 166–
168 °C; IR mmax (cmꢁ1): 3447, 2922, 1593, 1499, 1327, 1216, 822,
758; 1H NMR (CDCl3): d 7.94 (s, 1H), 7.63–7.57 (m, 4H), 7.50–
7.38 (m, 4H), 7.25 (d, J = 8.9 Hz, 2H), 5.60 (s, 2H), 4.73 (d,
J = 4.5 Hz, 2H), 2.38 (s, 3H); 13C NMR (CDCl3): d 151.41, 147.94,
137.32, 136.95, 132.16, 130.04, 128.94, 128.59, 128.49, 127.95,
121.42, 119.13, 114.17, 56.50, 44.79, 20.99; ESI-MS: m/z, 346
[M+H]+; ESI-HRMS: m/z calcd for C20H19N5O [M+H]+ 346.1662,
found 346.1664.
4.1.6.2. 1-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)-4-
phenyl-1H-1,2,3-triazole (10b). Yield: 92%; white solid; m.p.: 162–
164 °C; IR mmax (cmꢁ1): 3082, 1598, 1501, 1223, 1071, 833, 758,
688; 1H NMR (CDCl3): d 8.05 (s, 1H), 7.84–7.71 (m, 4H), 7.69 (s,
1H), 7.62 (d, J = 8.5 Hz, 2H), 7.52–7.31 (m, 8H), 5.68 (s, 2H); 13C
NMR (CDCl3): d 150.38, 148.04, 139.36, 134.63, 130.52, 130.27,
129.51, 129.09, 128.80, 128.62, 128.23, 127.10, 125.63, 119.20,
119.07, 114.53, 44.76; ESI-MS: m/z, 412 [M+H]+; ESI-HRMS: m/z
calcd for C24H18N5Cl [M+H]+ 412.1323, found 412.1333.
4.1.5.6.
(1-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazol-4-yl)methanol (8f). Yield: 95%; white
solid; m.p.: 128–130 °C; IR mmax (cmꢁ1): 3427, 2962, 1531, 1461,
1338, 1248, 839, 794; 1H NMR (CDCl3) d 7.97 (s, 1H), 7.71 (d,
J = 7.7 Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.45 (t, J = 7.9 Hz, 3H),
7.32–7.27 (m, 1H), 6.98 (d, J = 8.7 Hz, 2H), 5.57 (s, 2H), 4.75 (s,
2H), 3.84 (s, 3H); 13C NMR (CDCl3): d 159.96, 151.55, 139.57,
129.52, 129.19, 128.53, 126.90, 124.53, 119.09, 114.40, 114.10,
56.51, 55.38, 44.95; ESI-MS: m/z, 362 [M+H]+; ESI-HRMS: m/z calcd
for C20H19N5O2Na 384.1431, found 384.1449.
4.1.6.3. 1-((1-(4-Chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)methyl)-4-
phenyl-1H-1,2,3-triazole (10c). Yield: 90%; white solid; m.p.: 136–
138 °C; IR mmax (cmꢁ1): 3082, 2930, 1593, 1486, 1221, 1070, 770,
694; 1H NMR (CDCl3): d 8.00 (s, 1H), 7.82 (t, J = 2.0 Hz, 1H), 7.78
(dd, J = 5.2, 3.3 Hz, 2H), 7.66–7.64 (m, 2H), 7.63 (s, 1H), 7.60
(ddd, J = 8.2, 2.1, 0.8 Hz, 1H), 7.51–7.47 (m, 2H), 7.46–7.36 (m,
4H), 7.34–7.27 (m, 2H), 5.68 (s, 2H); 13C NMR (CDCl3): d 151.65,
146.92, 139.48, 133.91, 132.04, 129.51, 128.95, 128.67, 128.44,
127.90, 127.01, 126.88, 119.29, 119.08, 114.43, 44.89; ESI-MS:
m/z, 412 [M+H]+; ESI-HRMS: m/z calcd for C24H18N5Cl [M+H]+
412.1323, found 412.1332.
4.1.5.7.
(1-((3-(2,4-Dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazol-4-yl)methanol (8g). Yield: 95%; white
solid; m.p.: 128–130 °C; IR mmax (cmꢁ1): 3311, 2924, 1596, 1502,
1346, 1228, 825, 751; 1H NMR (CDCl3): d 7.95 (s, 1H), 7.69–7.65
(m, 2H), 7.62–7.58 (m, 2H), 7.50–7.41 (m, 5H), 5.60 (s, 2H), 4.74
(s, 2H); 13C NMR (CDCl3): d 148.73, 139.34, 135.76, 134.33,
132.97, 129.76, 129.66, 129.49, 127.60, 127.48, 127.18, 121.66,
119.19, 116.63, 56.21, 44.75; ESI-MS: m/z, 400 [M+H]+; ESI-HRMS:
m/z calcd for C19H15Cl2N5O [M+H]+ 400.0726, found 400.0731.
4.1.6.4. 4-Phenyl-1-((1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl)-
1H-1,2,3-triazole (10d). Yield: 80%; white solid; m.p.: 142–144 °C;
IR mmax (cmꢁ1): 3082, 2923, 1601, 1454, 1252, 1173, 1021, 829,
755, 688; 1H NMR (CDCl3): d 8.00 (s, 1H), 7.80–7.70 (m, 4H), 7.63
(s, 1H), 7.54 (d, J = 8.1 Hz, 2H), 7.46 (t, J = 8.0 Hz, 2H), 7.39 (t,
J = 7.6 Hz, 2H), 7.30 (dd, J = 17.3, 7.7 Hz, 4H), 5.67 (s, 2H), 2.41 (s,
3H); 13C NMR (CDCl3): d 151.75, 147.99, 139.61, 138.62, 130.50,
129.69, 129.54, 129.24, 128.85, 128.42, 128.22, 127.83, 126.93,
125.71, 119.33, 119.11, 114.54, 44.96, 21.36; ESI-MS: m/z, 392
[M+H]+; ESI-HRMS: m/z calcd for C25H21N5 [M+H]+ 392.1870, found
392.1880.
4.1.5.8.
(1-((1-(3,4-Dimethylphenyl)-3-phenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazol-4-yl)methanol (8h). Yield: 81%; white
solid; m.p.: 160–162 °C; IR mmax (cmꢁ1): 3451, 2917, 1509, 1448,
1364, 1214, 865, 767; 1H NMR (CDCl3): d 7.94 (s, 1H), 7.61 (dd,
J = 5.2, 3.2 Hz, 2H), 7.52 (d, J = 1.9 Hz, 1H), 7.47–7.44 (m, 2H),
Please cite this article in press as: Dayakar C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.08.042

C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
4.1.6.5. 4-Phenyl-1-((3-phenyl-1-(p-tolyl)-1H-pyrazol-4-yl)methyl)-
1H-1,2,3-triazole (10e). Yield: 82%; white solid; m.p.: 145–147 °C;
IR mmax (cmꢁ1): 3087, 2998, 1605, 1453, 1295, 1047, 752, 695; 1H
NMR (CDCl3): d 7.98 (s, 1H), 7.81–7.73 (m, 2H), 7.67–7.61 (m,
4H), 7.60 (s, 1H), 7.51–7.25 (m, 8H), 5.68 (s, 2H), 2.39 (s, 3H); 13C
NMR (CDCl3): d 151.44, 148.02, 137.39, 136.91, 132.26, 130.52,
130.02, 128.94, 128.81, 128.59, 128.38, 128.18, 127.97, 125.71,
119.23, 119.12, 114.38, 44.92, 20.95; ESI-MS: m/z, 392 [M+H]+;
139.54, 132.14, 129.47, 128.91, 128.58, 128.37, 127.94, 126.89,
126.60, 119.08, 118.61, 117.77, 114.92, 112.41, 44.78, 40.44; ESI-
MS: m/z, 421 [M+H]+; ESI-HRMS: m/z calcd for C26H24N6 [M+H]+
421.2135, found 421.2153.
4.1.6.11.
1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-4-(4-fluo-
rophenyl)-1H-1,2,3-triazole (10k). Yield: 89%; white solid; m.p.:
156–158 °C; IR mmax (cmꢁ1): 3095, 2925, 1598, 1497, 1224, 1069,
839, 751, 692; 1H NMR (CDCl3): d 8.02 (s, 1H), 7.73 (dd, J = 8.0,
3.2 Hz, 4H), 7.65 (d, J = 7.0 Hz, 2H), 7.59 (s, 1H), 7.53–7.37 (m,
5H), 7.31 (t, J = 7.4 Hz, 1H), 7.07 (t, J = 8.6 Hz, 2H), 5.67 (s, 2H);
ESI-HRMS: m/z calcd for
392.1883.
C
25H21N5 [M+H]+ 392.1870, found
4.1.6.6. 1-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methyl)-
4-phenyl-1H-1,2,3-triazole (10f). Yield: 81%; white solid; m.p.:
130–132 °C; IR mmax (cmꢁ1): 3001, 2958, 1731, 1461, 1224, 1060,
752, 686; 1H NMR (CDCl3): d 8.03 (s, 1H), 7.77 (dd, J = 13.9,
7.8 Hz, 4H), 7.66 (s, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.52–7.29 (m,
6H), 7.01 (d, J = 8.6 Hz, 2H), 5.68 (s, 2H), 3.87 (s, 3H); 13C NMR
(CDCl3): d 163.67, 161.70, 151.69, 139.55, 132.13, 129.56, 129.01,
128.72, 128.53, 127.97, 127.47, 127.41, 127.05, 126.70, 119.13,
115.91, 115.74, 114.58, 44.92; ESI-MS: m/z, 408 [M+H]+; ESI-
13C NMR (CDCl3):
d 163.60, 161.63, 151.63, 139.48, 132.05,
129.49, 128.93, 128.65, 128.44, 127.89, 127.40, 127.33, 126.98,
119.06, 115.84, 115.67, 114.50, 44.85; ESI-MS: m/z, 396 [M+H]+;
ESI-HRMS: m/z calcd for C24H18N5F [M+H]+ 396.1619, found
396.1641.
4.1.6.12.
4-(4-Chlorophenyl)-1-((1,3-diphenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazole (10l). Yield: 85%; white solid; m.p.:
164–166 °C; IR mmax (cmꢁ1): 3083, 2926, 1501, 1453, 1220, 1095,
834, 751, 693; 1H NMR (CDCl3): d 8.03 (s, 1H), 7.73 (d, J = 7.7 Hz,
2H), 7.69 (d, J = 8.5 Hz, 2H), 7.67–7.63 (m, 2H), 7.60 (s, 1H), 7.50–
7.40 (m, 5H), 7.37–7.30 (m, 3H), 5.67 (s, 2H); 13C NMR (CDCl3): d
151.61, 146.87, 139.44, 133.87, 132.00, 129.48, 128.95, 128.65,
128.44, 127.87, 126.98, 126.85, 119.30, 119.04, 114.40, 44.84;
ESI-MS: m/z, 412 [M+H]+; ESI-HRMS: m/z calcd for C24H18N5Cl [M
+H]+ 412.1323, found 412.1341.
HRMS: m/z calcd for
C
25H21N5O [M+H]+ 408.1818, found:
408.1827.
4.1.6.7.
1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-4-(2-(trifluo-
romethyl)phenyl)-1H-1,2,3-triazole (10g). Yield: 95%; white solid;
m.p.: 76–78 °C; IR mmax (cmꢁ1): 2922, 1709, 1440, 1347, 1162,
818, 662, 548; 1H NMR (CDCl3): d 8.01 (s, 1H), 7.93 (d, J = 7.8 Hz,
1H), 7.75–7.71 (m, 3H), 7.68 (d, J = 0.7 Hz, 1H), 7.66–7.59 (m,
3H), 7.50–7.40 (m, 6H), 7.29–7.33 (m, 1H), 5.71 (s, 2H); 13C NMR
(CDCl3): d 151.74, 145.27, 139.45, 132.56, 132.34, 132.08, 131.96,
129.56, 129.02, 128.79, 128.49, 127.91, 127.13, 126.41, 125.56,
123.29 (d, J = 273.37 Hz), 121.57 (q, J = 5.44 Hz), 120.29, 119.11,
114.03, 45.09; ESI-MS: m/z, 446 [M+H]+; ESI-HRMS: m/z calcd for
4.1.6.13.
4-(4-Bromophenyl)-1-((1,3-diphenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazole (10m). Yield: 93%; white solid; m.p.:
170–172 °C; IR mmax (cmꢁ1): 3108, 3080, 2926, 1596, 1501, 1453,
1219, 1069, 829, 752, 691; 1H NMR (CDCl3): d 8.05 (s, 1H), 7.75
(d, J = 7.6 Hz, 2H), 7.72–7.58 (m, 6H), 7.56–7.43 (m, 6H), 7.34 (t,
J = 7.4 Hz, 1H), 5.69 (s, 2H); 13C NMR (CDCl3): d 151.70, 146.98,
139.53, 132.09, 131.98, 129.56, 129.43, 129.01, 128.73, 128.51,
127.95, 127.21, 127.07, 122.11, 119.39, 119.13, 114.47, 44.94;
ESI-MS: m/z, 456 [M+H]+; ESI-HRMS: m/z calcd for C24H18N5Br
[M+H]+ 456.0818, found 456.0846.
C
25H18N5F3Na [M+H]+ 468.1389, found 468.1406.
4.1.6.8. 4-(2,4-Bis(trifluoromethyl)phenyl)-1-((1,3-diphenyl-1H-pyra-
zol-4-yl)methyl)-1H-1,2,3-triazole (10h). Yield: 92%; white solid; m.
p.: 134–136 °C; IR mmax (cmꢁ1): 3116, 2943, 1504, 1278, 1145,
1085, 1145,751, 683; 1H NMR (CDCl3): d 8.21 (s, 2H), 8.08 (s,
1H), 7.82–7.71 (m, 4H), 7.64 (dd, J = 7.9, 1.4 Hz, 2H), 7.52–7.42
(m, 5H), 7.33 (t, J = 7.4 Hz, 1H), 5.73 (s, 2H); 13C NMR (CDCl3): d
151.61, 144.45, 139.54, 132.07, 131.96, 131.64, 129.47, 129.37,
128.89, 128.61, 128.28, 128.24, 127.94, 127.03 (d, J = 29.97 Hz),
126.91, 126.06 (q, J = 5.44 Hz), 124.01 (d, J = 273.37 Hz), 122.64
(q, J = 5.44 Hz), 119.09, 114.70, 44.99; ESI-MS: m/z, 514 [M+H]+;
ESI-HRMS: m/z calcd for C26H17N5F6 [M+H]+ 514.1451, found
514.1460.
4.1.6.14. 1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-4-(p-tolyl)-1H-
1,2,3-triazole (10n). Yield: 89%; white solid; m.p.: 186–188 °C; IR
mmax (cmꢁ1): 3098, 2924, 1596, 1501, 1452, 1218, 823, 752, 693;
1H NMR (CDCl3): d 8.02 (s, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.66 (d,
J = 8.0 Hz, 4H), 7.59 (s, 1H), 7.51–7.41 (m, 5H), 7.30 (dd, J = 14.0,
6.6 Hz, 1H), 7.20 (d, J = 8.0 Hz, 2H), 5.68 (s, 2H), 2.36 (s, 3H); 13C
NMR (CDCl3): d 151.63, 148.05, 139.52, 138.03, 132.09, 129.48,
129.46, 128.93, 128.62, 128.40, 127.92, 127.57, 126.94, 125.55,
119.08, 118.89, 114.69, 44.82, 21.25; ESI-MS: m/z, 392 [M+H]+;
4.1.6.9. 4-(1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,3-tria-
zol-4-yl)aniline (10i). Yield: 78%; white solid; m.p.: 168–170 °C;
IR mmax (cmꢁ1): 3380, 3319, 2922, 1604, 1501, 1454, 1272, 1069,
830, 752, 695; 1H NMR (CDCl3): d 8.00 (s, 1H), 7.76–7.72 (m, 2H),
7.68–7.63 (m, 2H), 7.59–7.54 (m, 2H), 7.50 (s, 1H), 7.49–7.40 (m,
5H), 7.31 (t, J = 7.4 Hz, 1H), 6.69 (t, J = 5.5 Hz, 2H), 5.66 (s, 2H),
3.72 (s, 2H); 13C NMR (CDCl3): d 151.64, 146.51, 139.59, 132.17,
129.49, 128.92, 128.60, 128.37, 127.96, 126.91, 119.12, 117.94,
115.19, 114.88, 77.29, 44.82; ESI-MS: m/z, 393 [M+H]+; ESI-HRMS:
m/z calcd for C24H20N6 [M+H]+ 393.1814, found 393.1822.
ESI-HRMS: m/z calcd for C
25H21N5 [M+H]+ 392.1869, found
392.1884.
4.1.6.15. 1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-4-(4-methoxy-
phenyl)-1H-1,2,3-triazole (10o). Yield: 86%; white solid; m.p.:
188–190 °C; IR mmax (cmꢁ1): 3086, 2925, 1603, 1499, 1452, 1247,
1025, 829, 751, 689; 1H NMR (CDCl3): d 8.03 (s, 1H), 7.77 (dd,
J = 13.9, 7.8 Hz, 4H), 7.66 (s, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.51–
7.28 (m, 6H), 7.01 (d, J = 8.6 Hz, 2H), 5.68 (s, 2H), 3.87 (s, 3H);
13C NMR (CDCl3):
d 159.89, 151.46, 147.89, 139.51, 130.39,
129.44, 129.10, 128.75, 128.36, 128.13, 126.78, 125.60, 124.51,
119.23, 118.95, 114.32, 114.17, 55.29, 44.86; ESI-MS: m/z, 408
[M+H]+; ESI-HRMS: m/z calcd for C25H21ON5 [M+H]+ 408.1818,
found 408.1834.
4.1.6.10. 4-(1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,3-tria-
zol-4-yl)-N,N-dimethylaniline (10j). Yield: 74%; white solid; m.p.:
168–170 °C; IR mmax (cmꢁ1): 3419, 3100, 2926, 1618, 1506, 1449,
1224, 1067, 813, 751, 693; 1H NMR (CDCl3): d 7.99 (s, 1H), 7.73
(d, J = 7.9 Hz, 2H), 7.65 (dd, J = 7.7, 4.1 Hz, 4H), 7.51 (s, 1H), 7.50–
7.41 (m, 5H), 7.31 (t, J = 7.4 Hz, 1H), 6.73 (d, J = 8.8 Hz, 2H), 5.65
(s, 2H), 2.97 (s, 6H); 13C NMR (CDCl3): d 151.60, 150.38, 148.47,
4.1.6.16. 1-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)-4-(4-methoxy-2-
methylphenyl)-1H-1,2,3-triazole (10p). Yield: 89%; white solid; m.
p.: 138–140 °C; IR mmax (cmꢁ1): 3083, 2925 1602, 1455, 1253,
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C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
1174, 1022, 831, 756, 689; 1H NMR (CDCl3): d 8.06 (s, 1H), 7.76 (d,
J = 7.7 Hz, 2H), 7.68 (dd, J = 11.4, 4.7 Hz, 3H), 7.54–7.40 (m, 6H),
7.33 (t, J = 7.4 Hz, 1H), 6.86–6.74 (m, 2H), 5.71 (s, 2H), 3.82 (s,
3H), 2.33 (s, 3H); 13C NMR (CDCl3): d 159.32, 151.61, 147.12,
139.53, 137.04, 132.20, 130.09, 129.48, 128.92, 128.61, 128.40,
127.93, 126.92, 122.50, 120.84, 119.07, 116.16, 114.82, 111.34,
55.19, 44.73, 21.49; ESI-MS: m/z, 422 [M+H]+; ESI-HRMS: m/z calcd
for C26H23N5O [M+H]+ 422.1960, found 422.1975.
129.44, 129.07, 128.33, 128.13, 126.85, 119.09, 114.16, 53.34,
52.68, 45.16, 21.29; ESI-MS: m/z, 432 [M+H]+; ESI-HRMS: m/z calcd
for C23H21N5O4 [M+H]+ 432.1666, found: 432.1670.
4.1.7.5. Dimethyl 1-((3-phenyl-1-(p-tolyl)-1H-pyrazol-4-yl)methyl)-
1H-1,2,3-triazole-4,5-dicarboxylate (12e). Yield: 84%; white solid;
m.p.: 96–98 °C; IR mmax (cmꢁ1): 3036, 2957, 1752, 1517, 1356,
1230, 1230, 1140, 1059, 815, 765, 691; 1H NMR (CDCl3): d 7.88
(s, 1H), 7.70–7.64 (m, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.52–7.38 (m,
3H), 7.25 (t, J = 6.4 Hz, 2H), 5.88 (s, 2H), 3.95 (s, 3H), 3.74 (s, 3H),
2.37 (s, 3H); 13C NMR (CDCl3): d 160.38, 158.87, 151.50, 139.94,
137.31, 136.92, 132.12, 130.02, 128.82, 128.55, 128.45, 128.35,
119.14, 114.03, 53.42, 52.76, 45.16, 20.99; ESI-MS: m/z, 432 [M
+H]+; ESI-HRMS: m/z calcd for C23H21N5O4 [M+H]+ 432.1666, found
432.1671.
4.1.7. General procedure for the preparation of 1H-1,2,3-triazole-4,5-
dicarboxylates (12a-l)
Dimethyl but-2-ynedioate 11a (1.0 mmol) was added to a stir-
red solution of pyrazole azide 6a (1.0 mmol) in tert-butanol and
H2O (1:1) at room temperature, followed by CuSO4ꢀ5H2O
(0.1 mmol) and sodium ascorbate (0.05 mmol). The reaction mix-
ture was stirred for 6–12 h at room temperature. After completion
of the reaction (TLC), the reaction mixture was extracted with ethyl
acetate (2 ꢂ 5 mL). The organic extract was washed with H2O and
dried over anhydrous Na2SO4. The residue was purified by column
chromatography using silica gel (ethyl acetate/hexane) furnished
dimethyl 1-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,3-tria-
zole-4,5-dicarboxylate 12a. The 1H-1,2,3-triazole dicarboxylate
compounds 12b-l were synthesized from pyrazole azides 6a-f
under similar conditions.
4.1.7.6. Dimethyl 1-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)methyl)-1H-1,2,3-triazole-4,5-dicarboxylate (12f). Yield: 88%;
white solid; m.p.: 108–110 °C; IR mmax (cmꢁ1): 3133, 3057, 2955,
1731, 1461, 1224, 1180, 1060, 819, 752, 685; 1H NMR (CDCl3): d
7.92 (s, 1H), 7.69 (d, J = 7.7 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H), 7.45
(t, J = 7.9 Hz, 2H), 7.34–7.27 (m, 1H), 7.02 (d, J = 8.7 Hz, 2H), 5.87
(s, 2H), 3.96 (s, 3H), 3.87 (s, 3H), 3.79 (s, 3H); 13C NMR (CDCl3): d
160.37, 159.94, 158.94, 151.62, 139.94, 139.56, 130.02, 129.58,
129.49, 128.44, 126.87, 124.51, 119.09, 114.24, 114.01, 55.37,
53.44, 52.74, 45.23; ESI-MS: m/z, 448 [M+H]+; ESI-HRMS: m/z calcd
for C23H21N5O5 [M+H]+ 448.1615, found 448.1619.
4.1.7.1. Dimethyl 1-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-1H-
1,2,3-triazole-4,5-dicarboxylate (12a). Yield: 82%; white solid; m.
p.: 86–88 °C; IR mmax (cmꢁ1): 3123, 2953, 1741, 1448, 1349, 1224,
1134, 1055, 830, 757, 690; 1H NMR (CDCl3): d 7.93 (s, 1H), 7.73–
7.64 (m, 4H), 7.53–7.38 (m, 5H), 7.34–7.25 (m, 1H), 5.88 (s, 2H),
3.94 (s, 3H), 3.74 (s, 3H); 13C NMR (CDCl3): d 160.23, 158.69,
151.60, 139.81, 139.35, 131.87, 129.87, 129.37, 128.69, 128.48,
128.39, 128.19, 126.86, 119.00, 114.20, 53.29, 52.64, 44.97;
ESI-MS: m/z, 418 [M+H]+; ESI-HRMS: m/z calcd for C22H19N5O4
[M+H]+ 418.1510, found 418.1514.
4.1.7.7. Diethyl 1-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)-1H-1,2,3-
triazole-4,5-dicarboxylate (12g). Yield: 85%; colorless liquid; IR mmax
(cmꢁ1): 3065, 2923, 1730, 1453, 1213, 1060, 856, 759, 695; 1H
NMR (CDCl3): d 7.89 (s, 1H), 7.70 (d, J = 7.8 Hz, 4H), 7.47 (dt,
J = 11.6, 7.6 Hz, 5H), 7.31 (t, J = 7.4 Hz, 1H), 5.89 (s, 2H), 4.42 (q,
J = 7.1 Hz, 2H), 4.24 (q, J = 7.1 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H), 1.22
(t, J = 7.2 Hz, 3H); 13C NMR (CDCl3): d 159.95, 158.52, 151.61,
140.08, 139.47, 131.97, 130.09, 129.41, 128.72, 128.50, 128.34,
128.22, 126.87, 119.06, 114.41, 62.95, 61.82, 45.01, 14.10, 13.65;
ESI-MS: m/z, 446 [M+H]+; ESI-HRMS: m/z calcd for C24H23N5O4Na
[M+Na]+ 468.1750, found: 468.1682.
4.1.7.2. Dimethyl 1-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazole-4,5-dicarboxylate
(12b). Yield:
86%;
white solid; m.p.: 86–88 °C; IR mmax (cmꢁ1): 3134, 2953, 1727,
1461, 1344, 1225, 1060, 962, 819, 754, 687; 1H NMR (CDCl3): d
7.95 (s, 1H), 7.71–7.63 (m, 4H), 7.49–7.41 (m, 4H), 7.31 (t,
J = 7.4 Hz, 1H), 5.86 (s, 2H), 3.96 (s, 3H), 3.82 (s, 3H); 13C NMR
(CDCl3): d 160.35, 158.95, 150.68, 140.10, 139.43, 134.68, 130.55,
129.73, 129.64, 129.55, 129.02, 128.79, 127.16, 119.21, 114.30,
53.47, 52.78, 44.96; ESI-MS: m/z, 452 [M+H]+; ESI-HRMS: m/z calcd
for C22H18N5O4Cl [M+H]+ 452.1120, found 452.1131.
4.1.7.8. Diethyl 1-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazole-4,5-dicarboxylate (12h). Yield: 84%; col-
orless liquid; IR mmax (cmꢁ1): 2985, 1729, 1504, 1272, 1199, 1065,
836, 757, 683 cmꢁ1 1H NMR (CDCl3): d 7.95 (s, 1H), 7.69 (dd,
;
J = 8.7, 1.9 Hz, 4H), 7.51–7.41 (m, 4H), 7.31 (dd, J = 13.2, 5.8 Hz,
1H), 5.86 (s, 2H), 4.43 (q, J = 7.1 Hz, 2H), 4.30 (q, J = 7.1 Hz, 2H),
1.40 (t, J = 7.1 Hz, 3H), 1.27 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3): d
160.05, 158.66, 150.61, 140.30, 139.44, 134.63, 130.58, 129.88,
129.61, 129.55, 129.01, 128.78, 127.13, 119.16, 114.43, 63.09,
61.98, 44.91, 14.20, 13.78; ESI-MS: m/z, 480 [M+H]+; ESI-HRMS:
m/z calcd for C24H22N5O4Cl [M+Na]+ 480.1433, found 480.1451.
4.1.7.3. Dimethyl 1-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazole-4,5-dicarboxylate (12c). Yield: 92%; white
solid; m.p.: 96–98 °C; IR mmax (cmꢁ1): 3010, 2955, 1741, 1592,
1478, 1232, 1135, 1059, 779, 689; 1H NMR (CDCl3): d 7.95 (s,
1H), 7.71–7.64 (m, 4H), 7.46 (ddd, J = 8.4, 4.7, 2.1 Hz, 4H), 7.32 (t,
J = 7.4 Hz, 1H), 5.86 (s, 2H), 3.97 (s, 3H), 3.82 (s, 3H); 13C NMR
(CDCl3): d 160.29, 158.74, 152.11, 140.36, 140.03, 135.32, 131.68,
130.47, 129.81, 128.81, 128.74, 128.39, 128.28, 126.87, 119.37,
116.81, 114.93, 53.38, 52.71, 44.95; ESI-MS: m/z, 452 [M+H]+;
ESI-HRMS: m/z calcd for C22H18N5O4Cl [M+H]+ 452.1120, found
452.1126.
4.1.7.9. Diethyl 1-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazole-4,5-dicarboxylate (12i). Yield: 92%; color-
less liquid; IR mmax (cmꢁ1): 2983, 1729, 1593, 1488, 1210, 1060,
855, 776; 1H NMR (CDCl3): d 7.90 (s, 1H), 7.77 (t, J = 1.9 Hz, 1H),
7.71–7.67 (m, 2H), 7.55 (dd, J = 8.1, 1.2 Hz, 1H), 7.48 (t, J = 7.4 Hz,
2H), 7.43 (t, J = 7.3 Hz, 1H), 7.35 (t, J = 8.1 Hz, 1H), 7.25 (d,
J = 7.9 Hz, 1H), 5.87 (s, 2H), 4.42 (q, J = 7.1 Hz, 2H), 4.25 (q,
J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H); 13C
NMR (CDCl3): d 160.03, 158.55, 152.09, 140.43, 140.26, 135.35,
131.76, 130.53, 130.03, 128.85, 128.77, 128.40, 128.29, 126.87,
119.36, 116.83, 115.12, 63.07, 61.94, 44.97, 14.18, 13.75; ESI-MS:
m/z, 480 [M+H]+; ESI-HRMS: m/z calcd for C24H22N5O4Cl [M+Na]+
480.1433, found 480.1452.
4.1.7.4. Dimethyl 1-((1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl)-
1H-1,2,3-triazole-4,5-dicarboxylate (12d). Yield: 86%; white solid;
m.p.: 84–86 °C; IR mmax (cmꢁ1): 3016, 2923, 1741, 1446, 1227,
1135, 1057, 753, 683; 1H NMR (CDCl3): d 7.90 (s, 1H), 7.70–7.66
(m, 2H), 7.58–7.55 (m, 2H), 7.46–7.41 (m, 2H), 7.32–7.27 (m,
3H), 5.87 (s, 2H), 3.95 (s, 3H), 3.76 (s, 3H), 2.41 (s, 3H); 13C NMR
(CDCl3): d 160.32, 158.83, 151.75, 139.88, 139.52, 138.41, 129.99,
Please cite this article in press as: Dayakar C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.08.042

C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
13
4.1.7.10. Diethyl 1-((1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl)-
1H-1,2,3-triazole-4,5-dicarboxylate (12j). Yield: 88%; colorless liq-
uid; IR mmax (cmꢁ1): 3071, 2983, 1740, 1598, 1462, 1223, 1061,
757, 688; 1H NMR (CDCl3): d 7.88 (s, 1H), 7.68 (dt, J = 8.9, 1.8 Hz,
2H), 7.58 (d, J = 8.1 Hz, 2H), 7.46–7.41 (m, 2H), 7.29 (ddd, J = 7.6,
3.9, 1.6 Hz, 3H), 5.86 (s, 2H), 4.41 (q, J = 7.1 Hz, 2H), 4.23 (q,
J = 7.1 Hz, 2H), 2.40 (s, 3H), 1.38 (t, J = 7.1 Hz, 3H), 1.21 (t,
standard and control. The media, Petri dishes were autoclaved at
121 °C for 15 min. After sterilization the plates were poured with
appropriate medium left over for 30 min for solidification, later
the plates were inoculated with 60 mL of test inoculum using sterile
cotton swabs. An 8 mm width size wells were made with sterile
cork borer and in each well exactly 100 mL of sample were loaded.
Control and standard also placed in separate wells. The plates were
initially incubated for 20–30 min at 4 °C to allow the compounds to
diffuse into the agar, and then subsequently incubated for 24 h at
37 °C for bacteria and 48 h at 28 °C for fungi. Zone diameters were
expressed in mm using calibrated scale. Experiment was triplicate
to minimize the deviations.
J = 7.1 Hz, 3H); 13C NMR (CDCl3):
d 159.91, 158.50, 151.59,
139.96, 139.46, 138.28, 130.10, 129.34, 129.04, 128.22, 128.01,
127.67, 126.71, 118.95, 114.22, 62.88, 61.74, 45.04, 21.20, 14.04,
13.58; ESI-MS: m/z, 460 [M+H]+; ESI-HRMS: m/z calcd for
C
25H26N5O4 [M+H]+ 460.1985, found 460.1983.
4.2.2.1. Minimum inhibitory concentration. The compounds having
better anti-microbial activity 5c and 8f were selected for the MIC
(minimum inhibitory concentration) and MBC (minimum bacteri-
cidal concentration) studies against the positive strains (Salmonella
typhi, Salmonella paratyphi and Micrococcus luteus). The concentra-
tions of test samples were serially diluted from 500 to 1.9 mg/mL
and one tube without drug serves as control. All the tubes were
inoculated with 1 mL of respective cultures having an OD of 0.2
(ꢄMcFarland standard) and the tubes were incubated at 37 °C for
12–16 h. The turbidity of each tube is measured with respect to
control tube. MIC values are defined as the lowest concentration
of compound at which growth is completely inhibited. After incu-
bation the culture from each tube was plated in nutrient agar to
evaluate the MBC concentration. The concentration at which the
cells are completely dead was defined as minimum bactericidal
concentration.
4.1.7.11. Diethyl 1-((3-phenyl-1-(p-tolyl)-1H-pyrazol-4-yl)methyl)-
1H-1,2,3-triazole-4,5-dicarboxylate (12k). Yield: 84%; colorless liq-
uid; IR mmax (cmꢁ1): 3063, 2986, 1740, 1516, 1461, 1223, 1150,
1061, 844, 772; 1H NMR (CDCl3):
d 7.85 (s, 1H), 7.68 (d,
J = 6.9 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.51–7.39 (m, 3H), 7.24
(d, J = 8.3 Hz, 2H), 5.87 (s, 2H), 4.42 (q, J = 7.1 Hz, 2H), 4.23 (q,
J = 7.1 Hz, 2H), 2.38 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H), 1.21 (t,
J = 7.1 Hz, 3H); 13C NMR (CDCl3):
d 160.05, 158.63, 151.43,
140.12, 137.34, 136.88, 132.14, 130.23, 130.01, 128.80, 128.52,
128.36, 128.31, 119.12, 114.16, 63.04, 61.92, 45.13, 20.99, 14.20,
13.75; ESI-MS: m/z, 460 [M+H]+; ESI-HRMS: m/z calcd for
C
25H25N5O4 [M+H]+ 460.1979, found 460.1962.
4.1.7.12. Diethyl 1-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)
methyl)-1H-1,2,3-triazole-4,5-dicarboxylate (12l). Yield: 80%; color-
less liquid; IR mmax (cmꢁ1): 2926, 2848, 1727, 1552, 1460, 1197,
1062, 1012, 843, 757, 681; 1H NMR (CDCl3): d 7.87 (s, 1H), 7.67
(d, J = 7.9 Hz, 2H), 7.61 (t, J = 5.7 Hz, 2H), 7.42 (t, J = 7.9 Hz, 2H),
7.27 (t, J = 5.2 Hz, 1H), 6.99 (d, J = 8.7 Hz, 2H), 5.84 (s, 2H), 4.40
(q, J = 7.1 Hz, 2H), 4.24 (q, J = 7.1 Hz, 2H), 3.84 (s, 3H), 1.38 (t,
J = 7.1 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3): d 160.06,
159.96, 158.71, 151.58, 140.15, 139.61, 130.19, 129.57, 129.49,
128.37, 126.84, 124.56, 119.08, 114.25, 114.16, 63.04, 61.91,
55.38, 45.20, 14.20, 13.75; ESI-MS: m/z, 476 [M+H]+; ESI-HRMS:
m/z calcd for C25H25N5O5 [M+H]+ 476.1928, found 476.1945.
4.2.3. Anti-inflammatory activity
4.2.3.1. In-vitro assay by protein denaturation method. Protein
denaturation method was followed for performing anti-inflamma-
tory activity of synthesized pyrazole and pyrazolyltriazole com-
pounds 5a-h, 6a-h, 8a-h, 10a-p and 12a-l according to
Mizushima and Kobayashi 1968.18 Bovine serum albumin and
egg albumin proteins were used in the study and were solubilised
in 50 mM sodium phosphate buffer (pH 6.4) at a concentration of
1%. The reaction mixture consists of 0.1 ml of test sample (1 mg/ml),
0.2 ml of albumin protein and make up to a final volume of 5 ml
with buffer. Reaction mixture were incubated at 37 °C for 20 min
and then heated to 95 °C for 20 min. After cooling the samples to
room temperature, the turbidity was measured at 660 nm using
UV–visible Spectrophotometer (Model SL 210, Elico India Ltd.).
The experiment was performed in triplicate and average values
were reported. The percentage inhibition of protein denaturation
was calculated as follows:
4.2. Biology
4.2.1. Chemicals and kits
Dexamethasone (ꢃ95%), 2,3-bis(2-methoxy-4-nitro-5-sul-
fophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT sodium
salt), phorbol 12-myristate 13-acetate (PMA) and lipopolysaccha-
rides from Escherichia coli 055:B5 (LPS) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Human U937cells were pro-
cured from American Type Culture Collection (ATCC, Manassas,
USA). Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park
Memorial Institute medium (RPMI), Foetal bovine serum (FBS),
Trypsin-EDTA, l-glutathione and Penicillin-Streptomycin (Pen-
Strep) were obtained from Gibco, Life Technologies, Scotland, UK.
Percentage inhibition ¼ ½ðAbs control ꢁ Abs sampleÞ=Abs controlꢅ ꢂ 100
4.2.3.2. In vitro anti-inflammatory activity TNF-
a and IL-6. Anti-
inflammatory activity of the 5a-h and 8a-h were assessed using
reported protocol19 with slight modifications by employing LPS
induced inflammation model in human U937 cells. Briefly, human
U937 cells were incubated in RPMI1640 medium which was sup-
plemented with 10% foetal bovine serum, 2mM l-glutathione and
100U/ml Pen Strep and were incubated at 37 °C and 5% CO2. The
cells were treated with 30 ng/ml of Phorbol 12-myristate 13-acet-
ate (PMA) for 24 h and following differentiation, the U937 cells
were incubated with test compounds for 1 h. Subsequently, the
TNF-a and IL-6 ELISA kits (BD Opt EIA) were obtained from BD
Biosciences, San Diego, CA, USA. All other chemicals used were of
analytical grade unless otherwise specified.
4.2.2. Anti-microbial assay
Well plate method is followed for both antibacterial and anti-
fungal activities for measuring the zone of inhibitions.17 For
antibacterial activity test strains used are Micrococcus luteus, Sta-
phylococcus aureus (Gram positive) and Salmonella typhi, Salmonella
paratyphi (Gram negative), in nutrient agar. For antifungal studies
test strains used are Aspergillus niger and Aspergillus fumigatus and
the medium used is potato dextrose agar. The compounds 5a-h,
6a-h, 8a-h, 10a-p and 12a-l were used for activity studies and
the concentration of each compound is 1.0 mg/mL along with
cells were challenged with lipopolysaccharide (LPS, 1
were further incubated for 24 h. The supernatants were then
extracted and used for the estimation of the amount of TNF-
lg/ml) and
a
and IL-6 secreted using respective ELISA kits (BD Bioscience, San
Diego, CA, USA) as per the instructions provided by the
manufacturer.
Please cite this article in press as: Dayakar C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.08.042

14
C. Dayakar et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.2.3.3. Cell viability XTT assay. XTT (2,3-Bis(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) assay
was performed to assess the cell viability by using mitochondria-
dependent reduction of colorless tetrazolium XTT salt to orange
colored solution.30 The cells were seeded at 1 ꢂ 104 cells/well in
a 96 well plate and were incubated overnight. The cells were then
treated with different concentrations of test compounds 5a-h, 8a-h
and incubated for 24 h. After incubation, the cells were treated
with 50 ml of 1 mg/ml XTT in PBS solution and were further incu-
bated in RPMI 1640 medium without phenol red or serum for a
period of 4 h. The plates were then briefly mixed and the optical
density of the resultant orange colored solution was measured at
450 nm using a Bio-Tek multi mode microplate reader (Bio-Tek
Instruments, Inc., Winooski, VT, USA).
Group IV: (LPS + compound 8b (100 mg/kg), compound 8b
+ LPS): Mice were orally administered with a suspension of
100 mg/kg of test compound 8b, in 2% gum acacia (10 ml/kg)
intragastrically for 3 consecutive days. On day 3, 1 h following oral
administration, mice were injected LPS (10 mg/kg) intraperitoneally.
Group V: (LPS + compound 8h (100 mg/kg), compound 8h
+ LPS): Mice were orally administered with a suspension of
100 mg/kg of test compound 8h, in 2% gum acacia (10 ml/kg)
intragastrically for 3 consecutive days. On day 3, 1 h following
oral administration, mice were injected LPS (10 mg/kg)
intraperitoneally.
Six hours following LPS injection, blood samples were collected
from the retro-orbital plexus of the mice and were centrifuged at
4000 RPM for 15 min to separate the serum. The serum obtained
was used for the estimation of inflammatory cytokines, TNF-
a
4.2.4. Molecular docking studies
Docking of the compounds 5f, 8a-b, 8e-f and 8h to the both pro-
teins TNF-a (PDB ID- 2AZ5) and IL-6 (PDB ID- 1ALU) (https://www.
and IL-6 levels by enzyme linked immunosorbent assay (ELISA)
using respective kits (BD Opt EIA, BD Biosciences, San Diego, CA,
USA) as per manufacturer’s instruction. The % inhibition of inflam-
matory cytokines by the test compounds was calculated with
respect to the LPS control group.
rcsb.org/pdb/home/home.do) were performed by using auto dock
tool (ADT) 4.2 (http://autodock.scripps.edu/resources/adt).20 The
ADT graphics interface was employed for manual preparation of
the protein by adding polar hydrogens and merging non-polar
hydrogens. In the case of compounds, all compounds were
sketched in Tripo’s Sybyl6.7 and Gasteiger-Huckel charges were
added to minimize the molecules and non-polar hydrogens were
merged to give stability. Rotatable bonds were set for synthesized
compounds. GPF (grid parameter file) and DPF (docking parameter
file) files were prepared and the grid points for autogrid calcula-
tions were set as 60⁄60⁄60A° with the active site residues at the
centre of the grid box. Lamarckian genetic algorithm method is
used to calculate protein-fixed, ligand-flexible calculations.
Acknowledgments
The authors thank Dr. S. Chandrasekhar, Director, CSIR-IICT for
constant encouragement. CD thanks CSIR, New Delhi for fellow-
ship. BCR thank CSIR, New-Delhi for financial assistance through
XII year program ORIGIN (CSC0108).
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.08.042.
4.2.5. Animal experiments
Healthy male Balb/c mice, weighing 20–25 g were acclimatized
to the laboratory conditions for about seven days before initiation
of the study in the BIOSAFE, an animal quarantine facility of the
institute (Registration No: 97/GO/RBi/S/1999/CPCSEA). All animals
were given free access to standard pellet diet and fresh drinking
water ad libitum. They were housed under standard laboratory
conditions with 12 h/12 h of light/dark cycle, 25 2 °C tempera-
ture and 56 2% relative humidity throughout the study period.
All protocols involving experimental animals were performed as
per Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA) guidelines under the approval
and scrutiny of Institutional Animal Ethics Committee (IAEC) of
CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad,
India (Approval No: IICT/34/2016).
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Group I: (vehicle control, Control): Mice were orally administered
with a suspension of 2% gum acacia (10 ml/kg) intragastrically for 3
consecutive days. On day 3, 1 h following oral administration, mice
were injected normal saline (10 mg/kg) intraperitoneally.
Group II: (LPS control): Mice were orally administered with a
suspension of 2% gum acacia (10 ml/kg) intragastrically for 3 con-
secutive days. On day 3, 1 h following oral administration, mice
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was selected on the basis of previously reported literature.21,22
Group III: (LPS + dexamethasone (2 mg/kg), LPS + Dex): Mice
were orally administered with a suspension of 2 mg/kg of dexam-
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Please cite this article in press as: Dayakar C., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.08.042