Synthesis of new thiazolyl-pyrazolyl-1,2,3-triazole derivatives as potential antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2019.06.074

A series of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole derivatives (7a-y) have been synthesized by click reaction of 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-aryl-1,3-thiazole (5a-e) w

Full text of DOI:10.1016/j.ejmech.2019.06.074

Accepted Manuscript
Synthesis of new thiazolyl-pyrazolyl-1,2,3-triazole derivatives as potential
antimicrobial agents
Jitendra Nalawade, Abhijit Shinde, Abhijit Chavan, Sachin Patil, Manjusha
Suryavanshi, Manisha Modak, Prafulla Choudhari, Vivek D. Bobade, Pravin C.
Mhaske
PII:
S0223-5234(19)30601-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.074
EJMECH 11477
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2019
Revised Date: 26 June 2019
Accepted Date: 26 June 2019
Please cite this article as: J. Nalawade, A. Shinde, A. Chavan, S. Patil, M. Suryavanshi, M. Modak, P.
Choudhari, V.D. Bobade, P.C. Mhaske, Synthesis of new thiazolyl-pyrazolyl-1,2,3-triazole derivatives
as potential antimicrobial agents, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.06.074.
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ACCEPTED MANUSCRIPT
Synthesis of new thiazolyl-pyrazolyl-1,2,3-triazole derivatives as potential
antimicrobial agents
Jitendra Nalawade,a,b Abhijit Shinde,b Abhijit Chavan,b Sachin Patil,a Manjusha Suryavanshi,a Manisha
Modak,c Prafulla Choudhari,d Vivek D Bobade,a* Pravin C. Mhaskeb*
N
N
N
1,4-Disubstituted
1,2,3-triazole
N
R1
Click
Reaction
S
N
N
N
1,3,4-Trisubstituted
pyrazol
R
5a-e
CuSO4
Na Ascorbate
S
N
N
N3
R1
R
DMF-H2O
2,4,5-Trisubstituted
thiazole
6a-e
7a-y
Antifungal activity against A. niger
Compound 7b 7d 7g 7i
CH3 CH3 CH3
CH3 Cl Br
7j
7k
7l
7m
7n
F
7p
Cl
H
7v
Br
CH3
Ravuconazole
R
R1
H
H
F
F
F
CH3 Cl
H
CH3
F
Cl
MIC(µg/mL) 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25 31.25

ACCEPTED MANUSCRIPT
Synthesis of new thiazolyl-pyrazolyl-1,2,3-triazole derivatives as potential
antimicrobial agents
Jitendra Nalawade,a,b Abhijit Shinde,b Abhijit Chavan,b Sachin Patil,a Manjusha Suryavanshi,a
Manisha Modak,c Prafulla Choudhari,d Vivek D Bobade,a* Pravin C. Mhaskeb*
aPost-Graduate Department of Chemistry H. P. T. Arts and R. Y. K. Science College, Nashik
India 422005 (Affiliated to Savitribai Phule Pune University).
E-mail: v_bobade31@rediffmail.com
bPost-Graduate Department of Chemistry, S. P. Mandali’s Sir Parashurambhau College, Tilak
Road, Pune, India 411 030, (Affiliated to Savitribai Phule Pune University).
cDepartment of Zoology, S. P. Mandali’s Sir Parashurambhau College, Tilak Road, Pune, India
411 030, (Affiliated to Savitribai Phule Pune University).
dDepartment of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Kolhapur.
413016.
Fax: 91-020-24332479; Tel: 91-020-24331978; E-mail: mhaskepc18@gmail.com
Abstract
A series of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-
yl]-1H-1,2,3-triazole derivatives (7a-y) have been synthesized by click reaction of 5-(4-ethynyl-
1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-aryl-1,3-thiazole (5a-e) with substituted benzyl azide.
The starting compounds 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-aryl-1,3-thiazole
(5a-e) were synthesized from corresponding 3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1-phenyl-1H-
pyrazole-4-carbaldehyde (3a-e) by using Ohira-Bestmann reagent. All newly synthesized
thiazolyl-pyrazolyl-1,2,3-triazole derivatives were screened for antibacterial activity against two
Gram negative strains, Escherichia coli (NCIM 2574), Proteus mirabilis (NCIM 2388), a Gram
positive strain Staphylococcus albus (NCIM 2178) and in vitro antifungal activity against
Candida albicans (NCIM 3100), Aspergillus niger (ATCC 504) and Rhodotorula glutinis
(NCIM 3168). Ten thiazolyl-pyrazolyl-1,2,3-triazole derivatives, 7b, 7g, 7i, 7j, 7k, 7l, 7m, 7n,
7p and 7v exhibited promissing antifungal activity against A. niger with MIC 31.5 µg/mL.
Compounds 7g, 7i, 7k, 7l and 7m were further evaluated for ergosterol inhibition assay against
A. niger cells sample at 31.5 µg/mL concentration. The analysis of sterol inhibition assay
revealed that ergosterol biosynthesis is decreased in the fungal samples treated with azole
1

ACCEPTED MANUSCRIPT
derivatives. Promising antifungal activity suggested that, these compounds could be further
promoted for optimization and development which could have the potential to treat against
fungal infection.
Keywords: Thiazole, Pyrazole, 1,2,3-Triazole, Ohira-Bestmann reagent, Antibacterial activity,
Antifungal activity
1. Introduction
Over the last few decades, antimicrobial resistance has becomes one of the biggest
challenges to global health, food security, and development. Antimicrobial resistance threatens
the effective prevention and treatment of an increasing range of infections caused by bacteria,
fungi, parasites and viruses. In future, medical procedures such as organ transplantation, cancer
chemotherapy, diabetes management and major surgery become high risk without effective
antimicrobial drugs [1].
The hybrid architecture included two or more bioactive pharmacophore scaffolds is one
of the leading tools used in the new drug discovery [2]. Thiazole ring clubbed with pyrazole and
triazole rings are privileged scaffolds for the creation of lead molecules and have received much
attention in recent years [3]. The large number of natural and synthetic compounds containing
thiazole [4], are reported for promising antibacterial, antifungal, antitumor, anti-malarial and
antiviral activities [5,6]. Thiazole and its derivatives provide a wide spectrum of biological
activities such as antimicrobial [7-12], antimycobacterial [13-18], anti-inflammatory [19-21],
antiviral [22], CNS active agents [23], and anticancer activities [24,25]. Pyrazole nucleus
containing compounds are known for biological activities such as antimicrobial, antitubercular,
anticancer, anti-inflammatory and antipyretic activity [26-30]. 1,2,3-Triazole derivatives are
extremely potent inhibitors against certain enzymes, agonists, antagonist, ligand in
receptor−ligand binding studies for drug development [31]. They also exhibit significant
pharmacological activities such as antitubercular [32-36], anti-microbial [37-39], anti-neoplastic
[40], anti-proliferative [41], anti-viral activity [42], anti-cancer [43], fungicidal [44] activity and
many more. The clubbed thiazole-pyrazole, thiazole-triazole and pyrazole-triazole nucleus
containing heterocycles have received much attention due to their promising antibacterial
activity (Figure 1). Thiazole clubbed with pyrazole reported for antimicrobial [45] anti-
2

ACCEPTED MANUSCRIPT
inflammatory [46-47] and antitubercular [3,48,49] activities. Thiazole clubbed with triazole
reported for antimicrobial and antitubercular [3] activities. Substituted 2-amino thiazole clubbed
with 1,2,3-triazole reported as inhibitors of leukemia stem cells, [50] glucokinase activators,
[51] activities. Pyrazole clubbed with triazole were reported for antifungal [52],
antimycobacterial [53] and anticancer [54] activities.
The structural diversity and biological importance of thiazole, pyrazole and 1,2,3-
triazole have made them attractive target for new antimicrobial agents.
Figure 1: Representative biologically active thiazolyl-pyrazole (A-C), thiazolyl-triazole (D-G)
and our new proposed analogues (7a-y).
Keeping in mind, the biological significance of thiazole, pyrazole and 1,2,3-triazole
derivatives and in continuation of our search for new anti-infection agents, [11-15] we report
herein the synthesis of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-
1H-pyrazol-4-yl]-1H-1,2,3-triazole as potential antimicrobial agents.
2. Result and discussion
2.1. Chemistry
1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-
1H-1,2,3-triazole 7a-y were synthesized as illustrated in Scheme 1. Starting compounds 1-(4-
methyl-2-substituted phenylthiazol-5-yl)ethanone (1a-e) were synthesized as per our reported
procedure [13]. Acetyl thiazole 1a-e on reaction with phenyl hydrazine, (2) gave phenyl
hydrazone derivative which on reaction with DMF/POCl3 gave 3-(4-methyl-2-phenyl-1,3-
thiazol-5-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3a-e. Aldehyde 3a-e on reaction with
diethyl-1-diazo-2-oxopropylphosphonate (Ohira-Bestmann reagent) 4 and K2CO3 in methanol
gave 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-aryl-1,3-thiazole 5a-e. Alkyne 5a-e
on click reaction with substituted 1-(azidomethyl)benzene 6a-e in presence of sodium
3

ACCEPTED MANUSCRIPT
ascorbate, CuSO4 in DMF:Water (3:1) furnished 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-
2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole 7a-y. The structure of the newly
synthesized compounds was established by spectral analysis. All the synthesized compounds
were evaluated for antimicrobial activity.
O
O
OEt
P
CHO
OEt
N2
N
O
+
1. EtOH, Reflux
N
NHNH2
4
S
N
N
S
K2CO3, MeOH
0 oC to rt
2. DMF-POCl3,
DMF 0 oC to rt
R
R
3a-e
1a-e
2
N
N
N
N3
R1
N
N
Na Ascorbate, CuSO4
DMF:H2O (3:1) rt
+
N
S
N
N
S
N
R1
6a-e
R
R
5a-e
7a-y
Comp R R1
Comp R
7f
CH3
H CH3 7g
R1
H
Comp R R1
7k
F H
Comp
7p
R
R1
Cl H
Cl CH3 7v
Comp R R1
7u
Br
Br CH3
Br
7a
7b
7c
7d
7e
H H
H
CH3 CH3 7l
F CH3 7q
H F
H Cl
H Br
7h
7i
CH3
CH3 Cl
CH3 Br
F
7m
7n
7o
F F
F Cl
F Br
7r
7s
7t
Cl F
Cl Cl
Cl Br
7w
7x
7y
F
Br Cl
Br Br
7j
Scheme 1: Synthetic route of compounds 7a-y
The 1H NMR spectrum of 4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-
phenyl-1H-pyrazol-4-yl}-1-[(4-methylphenyl)methyl]-1H-1,2,3-triazole (7g) showed a signal at
δ 5.46 corresponds to triazole-CH2-aryl group confirmed the conversion of alkyne to 1,2,3-
triazole. The three singlets resonated in aliphatic region at δ 2.28, 2.32 and 2.41 corresponds to
the methyl protons of aryl and thiazole ring. The 1,2,3-triazole, pyrazole and aromatic protons
appeared at δ 7.10 to 8.55. The structure of compound 7g was further established by the 13C
NMR spectrum, signals appeared in the aliphatic and aromatic region and no signals found in
alkyne region confirmed the conversion of alkyne to 1,2,3-triazole. The three signals of methyl
and a methylene carbon appeared at δ 16.43 (thiazole-CH3), 21.11, 21.41 (Ar-CH3) and 53.95
(triazole-CH2-aryl), respectively. The thiazole, pyrazole and aromatic carbons appeared at δ
114.98 to 167.37 confirmed the formation of compound 7g. Further, a peak m/z = 503.2012
4

ACCEPTED MANUSCRIPT
(M+H)+ in the HRMS spectrum confirmed the formation of compound 7g. The structure of
newly synthesized compounds was established accordingly.
2.2. Antimicrobial activity
The in vitro antibacterial activity of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-
aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole 7a-y derivatives were evaluated
against Gram-negative bacteria E. coli and P. mirabilis and Gram-positive bacteria S. albus
using well diffusion method [55-57]. Standard drug Streptomycin was used as reference and
DMSO was used as negative control. The in vitro antifungal activity [55-57] was performed
against C. albicans, A. niger and R. glutinis using well diffusion method. The antifungal drugs
Fluconazole and Ravuconazole were used as reference. All the test solutions were prepared at
1000 µg/mL concentrations and the wells were filled with 80 µL (80 µg) of the samples. The
result of antibacterial and antifungal activity in zone of inhibition (mm) is presented in Table 1.
The analysis of antimicrobial activity of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-
2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole
derivatives
revealed
that,
compounds 7d, 7e, 7i, 7m, 7p, 7q and 7r showed moderate activity against E. coli and were
inactive against P. mirabilis. Compounds 7d, 7e, 7g, 7i, 7k, 7l, 7q, 7u, 7v and 7w reported
moderate activity against S. albus. Most of the compounds exhibited moderate activity against
antibacterial strains; therefore, they were not further screened for their minimum inhibitory
concentration (MIC).
The mechanism of action of azole containing antifungal compounds is the inhibition of
fungal cytochrome P450 enzyme, responsible for synthesis of ergosterol from lanosterol. The
azole compounds also have direct effect on the fatty acids of a cell membrane causing a leakage
of amino acids and proteins and interference with uptake of necessary nutrients [58].
The thiazolyl-pyrazolyl-1,2,3-triazole derivatives reported good activity against C.
albicans, A. niger and R. glutinis. The convincing antifungal activity of synthesized compounds
in our preliminary screening (Table 1) leads us to determine the minimum inhibitory
concentration. To further investigate this class of lead molecules, we has screened newly
synthesized thiazolyl-pyrazolyl-1,2,3-triazole,7a-y derivatives for inhibition of C. albicans, A.
niger and R. glutinis in a dose dependent way with the concentrations range from 500 to 3.90
µg/mL. The in vitro antifungal MIC screening results of synthesized compounds 7a-y is
presented in Table 2.
5

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Table 1: Antimicrobial activitya in zone of inhibition (mm) of compounds 7a-y
Comp.
R
R1
E.
coli
nd
P.
S.
albus
nd
C.
A.
R.
glutinis
nd
mirabilis
albicans niger
H
H
H
CH3
F
-
nd
11.8
15.8
11.5
16.4
15.8
13.0
12.5
10.0
14.0
16.2
12.2
12.8
10.4
14.4
15.2
13.8
13.8
11.3
17.6
14.6
14.6
12.0
14.8
15.0
-
nd
14.0
16.4
13.6
14.0
-
7a
7b
7c
7d
7e
7f
10.0
11.0
13.2
12.0
-
-
-
19.5
14.6
19.0
17.5
18.4
22.3
16.2
19.0
18.8
16.0
16.2
13.3
14.5
16.5
15.2
11.4
20.0
14.0
18.8
16.2
13.3
11.7
14.8
-
H
-
10.0
12.2
13.0
11.2
14.4
11.2
12.0
12.2
12.2
13.7
11.8
11.8
11.4
11.0
12.0
11.3
11.5
11.8
14.6
12.6
12.0
-
H
Cl
Br
H
-
H
-
CH3
-
CH3 CH3
10.2
10.6
13.6
11.0
11.8
9.6
-
-
7g
7h
7i
CH3
CH3
CH3
F
F
Cl
Br
H
-
16.3
16.0
14.4
15.8
13.0
11.0
13.5
16.8
12.3
11.4
12.5
12.3
12.0
13.4
12.4
17.0
13.5
-
-
-
7j
-
7k
7l
F
CH3
F
-
7m
7n
7o
7p
7q
7r
7s
F
12.2
-
-
F
Cl
Br
H
-
F
-
-
12.8
12.4
12.2
9.8
-
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
CH3
F
-
-
Cl
Br
H
-
11.0
11.5
11.6
11.8
11.0
-
-
7t
-
7u
7v
7w
7x
CH3
F
-
-
Cl
Br
-
-
-
7y
Streptomycin
25.0
-
18.52
21.6
-
-
-
Fluconazole
-
20.25
18.35
25.30
Ravuconazole
-
-
-
28.64
20.18
20.15
aThe concentration of test compounds and reference = 80 µg/well; NA = Not Applicable; (-) = Inactive.
6

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Table 2: Antifungal activity in MIC (µg/mL) of compounds 7a-y
Compound
R
H
R1
C. albicans A. niger
R. glutinis
-
H
-
-
-
7a
H
CH3
F
-
7b
31.25
125
H
125
-
125
62.5
62.5
125
125
125
62.5
62.5
62.5
62.5
62.5
125
62.5
125
125
125
62.5
62.5
62.5
125
62.5
125
-
7c
H
Cl
Br
H
7d
31.25
62.5
62.5
31.25
62.5
31.25
31.25
31.25
31.25
31.25
31.25
125
H
125
125
62.5
-
7e
CH3
CH3
CH3
CH3
CH3
F
7f
CH3
F
7g
7h
Cl
Br
H
-
7i
125
125
-
7j
7k
F
CH3
F
7l
F
125
-
7m
F
Cl
Br
H
7n
F
125
500
250
250
-
7o
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
7p
31.25
125
CH3
F
7q
7r
62.5
125
Cl
Br
H
7s
250
125
-
7t
62.5
62.5
31.25
62.5
62.5
-
7u
CH3
F
7v
125
125
-
7w
7x
Cl
Br
7y
7.81
7.81
7.81
31.25
7.81
15.625
Fluconazole
Ravuconazole
NA = Not Applicable; (-) = Inactive.
7

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The antifungal activity analysis provided some lead molecules that recorded excellent to good
antifungal activity. It is noteworthy that compounds 7b, 7g, 7i, 7j, 7k, 7l, 7m, 7n, 7p, and 7v
recorded comparable activity against A. niger with respect to standard drug Ravuconazole.
The structure activity relationship analysis revealed that, amongst the 1-substituted benzyl-4-[1-
phenyl-3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole
(7a-e),
compound 7b (R = H, R1 = CH3) showed excellent activity against A. niger, while compound
7d (R = H, R1 = Cl) showed excellent activity against A. niger and good activity against R.
glutinis, compound 7e (R = H, R1 = Br) showed good activity against A. niger and R. glutinis
strains. From compounds 1-substituted benzyl-4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-
5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole (7f-j), compound 7g (R = CH3, R1 = CH3)
showed good activity against C. albicans and excellent activity against A. niger, 7h (R = CH3,
R1 = F) showed good activity against A. niger, compound 7i (R = CH3, R1 = Cl) and 7j (R =
CH3, R1 = Br) showed excellent activity against A. niger and good activity against R. glutinis
strains. Among 1-substituted benzyl-4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-
phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole (7k-o), compounds 7k (R = F, R1 = H), 7l (R = F,
R1 = CH3) and 7m (R = F, R1 = F) reported excellent activity against A. niger and good activity
against R. glutinis. Compounds 7n (R = F, R1 = Cl) showed excellent activity against A. niger
and 7o (R = F, R1 = Br) showed good activity against R. glutinis. From the compounds 1-
substituted benzyl-4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-
yl}-1H-1,2,3-triazole (7p-t), compounds 7p (R = Cl, R1 = H) showed excellent activity against
A. niger, 7r (R = Cl, R1 = F) showed good activity against A. niger, compound 7s showed good
activity against R. glutinis and compound 7t showed good activity against A. niger and R.
glutinis strains. Among the compounds 1-substituted benzyl-4-{3-[2-(4-bromophenyl)-4-
methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole (7u-y), compounds 7u (R
= Br, R1 = H) and 7w (R = Br, R1 = F) showed good activity against A. niger and R. glutinis
strains. Compound 7v (R = Br, R1 = CH3) showed excellent activity against A. niger and
compound 7x (R = Br, R1 = Cl) showed good activity against A. niger.
According to analysis of antifungal activity, it is worth mentioning, most of the thiazolyl-
pyrazolyl-1,2,3-triazole derivatives were found comparable or only two fold less active against
A. niger than the standard drug Ravuconazole. It was also observed that R = H, R1 = Cl and
vice-versa, similarly R = CH3, R1 = Br and vice-versa reported similar activity against A. niger.
8

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Also R = F and R1 =H / CH3 / F or Cl in compounds 7k-n, showed excellent activity against A.
niger.
2.3. Ergosterol inhibition activity
The common mode of action of azole antifungal drugs is to disrupt the sterol
biosynthetic pathway which leads to reduction in ergosterol biosynthesis. Ergosterol is the
major component of fungal plasma membrane which maintains the cell integrity and
functionality. The compound which disrupts this biosynthesis has the potential of antifungal
activity [59,60]. The quantitative estimation ergosterol biosynthesis would be confirmatory way
to show the antifungal activity. In spectrophotometric analysis of ergosterol and 24(28)-
dehydroergosterol [24(28)DHE] gives a characteristic peak curve from 240 – 300 nm scan.
From the azole derivatives 7a-w, compounds 7g, 7i, 7k, 7l and 7m evaluated for
ergosterol inhibition assay against A. niger (NCIM 504) cells sample at 31.5 µg/mL
concentration (MIC). The spectrophotometric absorbance profile between 240 and 300 nm of
compounds 7g, 7i, 7k, 7l and 7m are shown in Figure 2. The absorption spectra revealed that,
the characteristic peaks of ergosterol in control are not observed in compounds 7g, 7i, 7k, 7l
and 7m. This clearly indicates that the ergosterol biosynthesis is decreased in the fungal
samples treated with azole derivatives. This was also confirmed by quantitative estimation of
ergosterol (Table 3). These results indicate that the ergosterol was synthesized in control fungal
samples but not detectable in rest fungal samples which are treated with azole derivatives. This
predicts that the ergosterol biosynthesis might be inhibited by azole derivatives.
9

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Figure 2: Spectrophotometric analysis of ergosterol composition of A. niger at 31.5µg/mL
concentration of 7g, 7i, 7k, 7l and 7m.
Table 3: Quantitative estimation of ergosterol.
Compound % ergosterone synthesis/g wet weight
0.0000290836
Not detected
Not detected
Not detected
Not detected
Not detected
Control
7g
7i
7k
7l
7m
2.4. Molecular docking
Molecular docking analysis was performed to confirm the mechanism of action of synthesised
derivatives. Crystal structure of sterol 14-alpha demethylase (CYP51) from Candida albicans
complexed with azole-based antifungal drug candidate was utilised for docking analysis. All the
synthesised derivatives are found to be interacting with CYP51 with binding energy ranges
from -20.20 kcal/mol to -24.97 kcal/mol. Compound 7b showed aromatic binding interactions
with TYR505, HIS373, TYR69 and hydrophobic interactions with GLU70 and GLN67 (Figure
S1). Compound 7d exhibited aromatic binding interactions with TYR505, HIS373 and
hydrophobic interactions with GLU70 and GLN67 (Figure S2), compound 7g showed hydrogen
bond interaction with TYR505, aromatic interactions with TYR69, HIS373, TYR505 and
hydrophobic interactions with GLN67, GLU70, SER412, GLU413 (Figure 3). Compound 7i
10

ACCEPTED MANUSCRIPT
interacted with protein via formation of hydrogen bond interaction with GLN67 aromatic
interactions with TYR69, TYR505 and hydrophobic interactions with GLN67, GLU70,
GLU413, ASP502, PRO503 (Figure S3). Compound 7j showed hydrogen bond interaction with
GLN67 aromatic interactions with TYR69, TYR505 and hydrophobic interactions with GLN67,
ASP502, PRO503 (Figure S4). Compound 7k interacted via formation of hydrogen bond with
SER74 and aromatic interaction with TYR69, hydrophobic interaction with GLU420, ASP502,
PRO503 (Figure S5). Azole derivative 7l showed hydrogen bond interaction with TYR505,
aromatic interaction with HIS373, TYR505 and hydrophobic interaction with GLN67, GLU70
(Figure S6). Compound 7m interacted via formation of hydrogen bond interaction with GLN67,
aromatic interaction with TYR69, TYR505 and hydrophobic interaction with GLN67, GLU70,
ASP502 (Figure S7). Compound 7n showed aromatic interaction with HIS373, TYR505 and
hydrophobic interaction with GLN67, ASP502, PRO503 (Figure S8), derivative 7p showed
aromatic interaction with TYR69 and hydrophobic interactions with GLU420, ASP502,
PRO503 (Figure S9). Compound 7v interacted via formation of aromatic interactions with
TYR69, HIS373 and hydrophobic interactions with MET372, THR411, GLU420, VAL500,
ASP502, PRO503 (Figure S10).
Figure 3: 4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-
[(4-methylphenyl)methyl]-1H-1,2,3-triazole (7g)
11

ACCEPTED MANUSCRIPT
3. Conclusion
A series of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-
yl]-1H-1,2,3-triazole (7a-y) have been synthesized. The antibacterial and antifungal screening
studies of compounds 7a-y were undertaken to evaluate the effects of substituent on the
activities. Most of the synthesized compounds exhibited good to excellent antifungal activity
against A. niger and R. glutinis. The plausible mode of action studies revealed that the
antifungal action of the azole compounds was through inhibition of ergosterol biosynthesis
pathways. It is concluded that, 1-substituted benzyl-4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-
thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole, (7k-o) except compound 7o, all 2-
(4-fluorophenyl)-4-methylthiazole substituted compounds reported comparable activity against
A. niger with respect to standard drug Ravuconazole.
4. Experimental
4.1. General procedure of 3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1-phenyl-1H-pyrazole-
4-carbaldehyde (3a)
The solution of 1-(4-methyl-2-phenylthiazol-5-yl)ethanone, 1a (10 mmol) and phenyl
hydrazine, 2 (11 mmol) and catalytic amount of acetic acid (1 drop) in absolute ethanol (30 mL)
was refluxed for two hours. After completion of reaction (TLC), the reaction mixture was
cooled and solvent was removed under vacuum. The phenylhydrazone product was
recrystallized
from
aqueous
ethanol.
1-(4-methyl-2-phenylthiazol-5-yl)ethanone
phenylhydrazone (5 mmol) in DMF (10 mL) was added drop wise to a solution of DMF-POCl3
(30 mmol - 15 mmol) at 0 °C for 30 minutes, and reaction mixture was stirred at room
temperature for 10 hours. After completion of the reaction (TLC) the reaction mass was poured
in ice cold water and stirred for 4-5 hours. The solid product was filtered, washed with water
and recrystallized from ethanol which gave pure 3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1-
phenyl-1H-pyrazole-4-carbaldehyde, 3a (Yield 76 %). Compounds 3b-3e were synthesized by
similar procedure. In the mass spectrum of all the compounds, the isotopic peak at (M+2) was
observed because of S, Br, and/or Cl.
4.1.1. 3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3a:
Yield: 76 %; mp: 178-180 °C; 1H NMR (500 MHz, CDCl3)
δ 2.67 (s, 3H, Thiazole-CH3), 7.48
– 7.39 (m, 4H, Ar-H), 7.53 (t, J = 8.0 Hz, 2H, Ar-H), 7.81 – 7.77 (m, 2H, Ar-H), 7.98 (dd, J =
12

ACCEPTED MANUSCRIPT
7.4, 2.1 Hz, 2H, Ar-H), 8.56 (s, 1H, Pyrazole C5-H), 10.03 (s, 1H, Aldehyde-H); 13C NMR (125
MHz, CDCl3)
δ 17.06, 119.70, 120.46, 123.44, 126.59, 128.29, 129.03, 129.81, 130.33, 130.56,
133.33, 138.80, 146.12, 153.56, 167.52, 184.46, LCMS: m/z = 346.1 (M+H)+.
4.1.2. 3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazole-4-carbaldehyde
1
3b: Yield: 72 %; mp: 230-231 °C (dec.); H NMR (500 MHz, CDCl3)
δ 2.40 (s, 3H, Ar-CH3),
2.65 (s, 3H, Thiazole-CH3), 7.26 (d, J = 7.9 Hz, 2H, Ar-H), 7.41 (dd, J = 10.7, 4.2 Hz, 1H, Ar-
H), 7.53 (t, J = 8.0 Hz, 2H, Ar-H), 7.80-7.76 (m, 2H, Ar-H), 7.87 (d, J = 8.1 Hz, 2H, Ar-H),
8.55 (s, 1H, Pyrazole C5-H), 10.03 (s, 1H, Aldehyde-H); 13C NMR (125 MHz, CDCl3)
δ 17.05,
21.49, 119.68, 119.91, 123.41, 126.50, 128.25, 129.72, 129.79, 130.47, 130.66, 138.80, 140.67,
146.24, 153.37, 167.74, 184.54; LCMS: m/z = 360.95 (M+H)+.
4.1.3. 3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazole-4-carbaldehyde
1
3c: Yield: 66 %; mp:240 241 °C (dec.); H NMR (500 MHz, CDCl3)
δ 2.65 (s, 3H, Thiazole-
CH3), 7.15 (t, J = 8.6 Hz, 2H, Ar-H), 7.42 (t, J = 7.4 Hz, 1H, Ar-H), 7.53 (t, J = 7.9 Hz, 2H, Ar-
H), 7.78 (d, J = 8.3 Hz, 2H, Ar-H), 7.97 (dd, J = 8.8, 5.3 Hz, 2H, Ar-H), 8.56 (s, 1H, Pyrazole
C5-H), 10.03 (s, 1H, Aldehyde-H); 13C NMR (125 MHz, CDCl3)
δ 17.04, 116.04, 116.22,
119.69, 123.41, 127.96, 128.31, 128.48, 128.55, 129.81, 130.73, 132.21, 138.77, 145.90,
153.61,163.04, 165.03, 166.24, 184.31; LCMS: m/z = 364.05 (M+H)+.
4.1.4. 3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazole-4-carbaldehyde
3d: Yield: 70 %; mp: 192-193 °C; 1H NMR (500 MHz, CDCl3)
δ 2.65 (s, 3H, Thiazole-CH3),
7.45-7.40 (m, 3H, Ar-H), 7.53 (t, J = 8.0 Hz, 2H, Ar-H), 7.80-7.77 (m, 2H, Ar-H), 7.92 (d, J =
13
8.6 Hz, 2H, Ar-H), 8.55 (s, 1H, Pyrazole C5-H), 10.02 (s, 1H, Aldehyde-H); C NMR (125
MHz, CDCl3)
δ 17.07, 119.68, 120.88, 123.41, 127.75, 128.32, 129.26, 129.82, 130.81, 131.84,
136.25, 138.76, 145.78, 153.77, 166.03, 184.23, LCMS: m/z = 380.05 (M+H)+, 382.00
(M+H+2)+.
4.1.5. 3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazole-4-carbaldehyde
1
3e: Yield: 75 %; mp: 184-185 °C; H NMR (500 MHz, CDCl3)
δ 2.66 (s, 3H, Thiazole-CH3),
7.42 (t, J = 7.4 Hz, 1H, Ar-H), 7.54 (dd, J = 10.7, 5.2 Hz, 2H, Ar-H), 7.60-7.57 (m, 2H, Ar-H),
7.78 (dd, J = 8.5, 0.9 Hz, 2H, Ar-H), 7.86-7.83 (m, 2H, Ar-H), 8.56 (s, 1H, Pyrazole C5-H),
10.02 (s, 1H, Aldehyde-H); 13C NMR (125 MHz, CDCl3)
δ 17.05, 119.67, 120.91, 123.38,
124.56, 127.94, 128.31, 129.80, 130.81, 132.19, 132.24, 138.73, 145.75, 153.78, 166.06,
184.21; LCMS: m/z = 424.03 (M+H)+, 426.01(M+H+2)+.
13

ACCEPTED MANUSCRIPT
4.2. General procedure of 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-phenyl-1,3-
thiazole (5a)
The solution of diethyl (1-diazo-2-oxopropyl)phosphonate 4 (13 mmol) and K2CO3 (20
mmol) in dry methanol (20 mL), 3-(4-methyl-2-phenylthiazol-5-yl)-1-phenyl-1H-pyrazole-4-
carbaldehyde (3a) (10 mmol) in methanol (20 mL) was added and reaction mixture was stirred
overnight. After completion of the reaction (TLC), solvent was distilled under vacuum and the
residue was dissolved in water (80 mL). The reaction mass was extracted by ethyl acetate (3 x
25 mL), ethyl acetate layer was washed with brine, dried over sodium sulphate and evaporated
on rotary evaporator. The product was purified by column chromatography using ethyl
acetate:hexane (2:8) as eluent gave 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-phenyl-
1,3-thiazole (5a), Yield 42 %). Compounds 5b-5e was synthesized by similar procedure.
4.2.1. 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-phenyl-1,3-thiazole 5a: Yield: 42 %;
1
mp: 88-90 °C; H NMR (500 MHz, CDCl3)
δ 2.79 (s, 3H, Thiazole-CH3), 3.26 (s, 1H, Alkyne-
H), 7.05 (d, J = 8.1 Hz, 2H, Ar-H), 7.30 (t, J = 7.4 Hz, 1H, Ar-H), 7.47 (t, J = 8.0 Hz, 2H, Ar-
H), 7.62-7.65 (m, 3H, Ar-H), 7.85 (d, J = 8.1 Hz, 2H, Ar-H), 8.17 (s, 1H, Pyrazole-H); 13C
NMR (125 MHz, CDCl3)
δ 17.71, 74.80, 82.72, 104.00, 119.10, 121.38, 126.30, 127.25,
128.95, 129.36, 129.45, 131.02, 131.13, 139.20, 147.47, 152.43, 166.30; LCMS: m/z = 342.06
(M+H)+.
4.2.2. 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-(4-methylphenyl)-1,3-thiazole 5b:
1
Yield: 40%; mp:93-94 °C; H NMR (500 MHz, CDCl3)
δ 2.40 (s, 3H, Ar-CH3), 2.78 (s, 3H,
Thiazole-CH3), 3.32 (s, 1H, Alkyne-H), 7.13 (t, J = 7.4 Hz, 2H, Ar-H), 7.25 (d, J = 8.1 Hz, 2H,
Ar-H), 7.31 (t, J = 7.4 Hz, 1H, Ar-H), 7.49 (t, J = 8.0 Hz, 2H, Ar-H), 7.73 (d, J = 7.6 Hz, 2H,
Ar-H), 7.87 (d, J = 8.1 Hz, 2H, Ar-H), 8.17 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ
17.71, 21.47, 74.82, 82.78, 104.02, 119.14, 121.42, 126.43, 127.27, 129.61, 129.63, 131.02,
131.13, 139.23, 140.20, 147.54, 152.70, 166.35; LCMS: m/z = 356.1 (M+H)+.
4.2.3. 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-2-(4-fluorophenyl)-4-methyl-1,3-thiazole 5c:
Yield: 48%; mp: 92-94 °C; 1H NMR (500 MHz, CDCl3)
δ 2.77 (s, 3H, Thiazole-CH3), 3.27 (s,
1H, Alkyne-H), 7.13 (t, J = 8.8 Hz, 2H, Ar-H), 7.29-7.34 (m, 1H, Ar-H), 7.46 (dd, J = 8.3, 7.6
Hz, 2H, Ar-H), 7.71 (d, J = 7.6 Hz, 2H, Ar-H), 8.12 (s, 1H, Pyrazole-H), 8.17 (dd, J = 8.9, 5.5
13
Hz, 2H, Ar-H); C NMR (125 MHz, CDCl3)
δ 17.71, 75.75, 81.32, 101.73, 115.34, 115.51,
14

ACCEPTED MANUSCRIPT
119.13, 119.22, 127.12, 128.43, 128.46, 128.81, 128.87, 129.57, 132.13, 139.37, 147.31,
152.22, 162.03, 164.00, 166.22; LCMS: m/z = 360.04 (M+H)+.
4.2.4. 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-2-(4-chlorophenyl)-4-methyl-1,3-thiazole 5d:
Yield: 38%; mp: 96-98 °C; 1H NMR (500 MHz, CDCl3)
δ 2.78 (s, 3H, Thiazole-CH3), 3.33 (s,
1H, Alkyne-H), 7.12-7.15 (m, 2H, Ar-H), 7.36 (t, J = 7.4 Hz, 1H, Ar-H), 7.42 (d, J = 8.7 Hz,
2H, Ar-H), 7.73 (d, J = 7.6 Hz, 2H, Ar-H), 7.92 (d, J = 8.6 Hz, 2H, Ar-H), 8.17 (s, 1H,
Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 17.69, 74.72, 82.93, 104.06, 119.16, 122.35,
127.37, 127.68, 128.11, 129.15, 129.66, 131.20, 135.84, 139.19, 147.24, 153.03, 164.63;
LCMS: m/z = 376.01 (M+H)+, 377.99 (M+H+2)+.
4.2.5. 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-2-(4-bromophenyl)-4-methyl-1,3-thiazole 5e:
Yield: 40%; mp: 104-106 °C; 1H NMR (500 MHz, CDCl3)
δ 2.76 (s, 3H, Thiazole-CH3), 3.28
(s, 1H, Alkyne-H), 7.15 (t, J = 8.8 Hz, 2H, Ar-H), 7.31-7.33 (m, 1H, Ar-H), 7.54 (d, J = 8.6 Hz,
13
2H, Ar-H), 7.71 (d, J = 7.6 Hz, 2H, Ar-H), 8.07 (d, J = 7.6 Hz, 2H), 8.13 (s, 1H); C NMR
(125 MHz, CDCl3)
δ 17.72, 75.59, 81.60, 101.92, 119.27, 122.72, 124.15, 127.23, 127.90,
128.52, 129.60, 131.19, 132.24, 139.32, 147.22, 151.97, 164.64; LCMS: m/z = 419.95 (M+H)+,
421.91 (M+H+2)+.
4.3. General method of 1-benzyl-4-[1-phenyl-3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1H-
pyrazol-4-yl]-1H-1,2,3-triazole (7a)
To the solution of 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-phenylthiazole,
5a (1 mmol) in DMF:Water (6 mL, 3:1), benzylazide 6a (1 mmol), copper sulphate (0.25
mmol) and sodium ascorbate (0.22 mmol) was added and reaction mixture was stirred for 24
hours. After completion of reaction (TLC), the reaction mixture was quenched in water and
extracted with ethyl acetate (3 x 15 mL). Organic layer was dried over sodium sulphate and
evaporated on rotary evaporator. The product was purified by column chromatography (ethyl
acetate:hexane) to furnish pure 1-benzyl-4-[1-phenyl-3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-
1H-pyrazol-4-yl]-1H-1,2,3-triazole 7a. Compounds 7b to 7y were synthesized by similar
experimental procedure.
4.3.1.
1-phenyl-4-[1-phenyl-3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H-
1
1,2,3-triazole 7a: Yield: 82 %; mp: 122-124 °C; H NMR (500 MHz, CDCl3):
δ 2.34 (s, 3H,
Thiazole CH3), 5.51 (s, 2H, Ar-CH2-N), 7.18-7.23 (m, 3H, Ar-H), 7.29 (dd, J = 8.4, 6.0 Hz, 3H,
Ar-H), 7.34 (s, 1H, Triazole -H), 7.45 (dd, J = 5.0, 1.8 Hz, 3H, Ar-H), 7.48-7.51 (m, 2H, Ar-H),
13
7.79 (d, J = 7.7 Hz, 2H, Ar-H), 7.89-7.91 (m, 2H, Ar-H), 8.56 (s, 1H, Pyrazole-H); C NMR
15

ACCEPTED MANUSCRIPT
(125 MHz, CDCl3)
δ
15.39, 53.10, 113.87, 118.07, 119.19, 121.01, 125.29, 125.43, 126.08,
126.72, 127.68, 127.94, 128.04, 128.56, 129.12, 132.41, 133.57, 138.48, 138.86, 140.69,
151.64, 166.12; HRMS: m/z = 475.1703 (M+H)+.
4.3.2.
4-[3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1-phenyl-1H-pyrazol-4-yl]-1-[(4-
1
methylphenyl)methyl]-1H-1,2,3-triazole 7b: Yield: 72 %; mp: 160-162 °C; H NMR (500
MHz, CDCl3) δ 2.35 (s, 3H, Thiazole CH3), 2.55 (s, 3H, Ar-CH3), 5.50 (s, 2H, Ar-CH2-N), 7.21
(d, J = 7.8 Hz, 2H, Ar-H), 7.26 (d, J = 7.8 Hz, 2H, Ar-H), 7.31-7.33 (m, 3H, Ar-H), 7.35 (s,
1H, Triazole -H), 7.42-7.45 (m, 3H, Ar-H), 7.79 (d, J = 7.7 Hz, 2H, Ar-H), 7.89-7.91 (m, 2H,
13
Ar-H), 8.56 (s, 1H, Pyrazole-H); C NMR (125 MHz, CDCl3)
δ 15.40, 21.12, 53.15, 113.90,
118.10, 119.21, 121.05, 126.10, 127.47, 127.51, 127.71, 128.03, 128.60, 128.88, 129.14,
132.65, 133.60, 133.15, 138.50, 138.87, 140.70, 151.66, 166.14; HRMS: m/z = 489.2563
(M+H)+.
4.3.3.
pyrazol-4-yl]-1H-1,2,3-triazole 7c: Yield: 78 %; mp: 168-170 °C; 1H NMR (500 MHz, CDCl3)
2.27 (s, 3H, Thiazole CH3), 5.40 (s, 2H, Ar-CH2-N), 6.92 (t, J = 8.6 Hz, 2H, Ar-H), 7.15-7.11
1-[(4-fluorophenyl)methyl]-4-[3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1-phenyl-1H-
δ
(m, 3H, Ar-H, Triazole-H), 7.27 (t, J = 7.4 Hz, 1H, Ar-H), 7.38 (dd, J = 5.1, 1.8 Hz, 3H, Ar-H),
7.42 (t, J = 8.0 Hz, 2H, Ar-H), 7.71 (d, J = 7.8 Hz, 2H, Ar-H), 7.86-7.81 (m, 2H, Ar-H), 8.49
(s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 15.39, 52.35, 113.79, 115.06, 118.08,
119.01, 120.96, 125.32, 125.34, 125.37, 126.12, 128.00, 128.57, 129.20, 129.46, 132.35,
138.47, 139.01, 140.66, 151.67, 161.75, 166.18; HRMS: m/z = 493.1614 (M+H)+.
4.3.4.
pyrazol-4-yl]-1H-1,2,3-triazole 7d: Yield: 78 %; mp: 168-170 °C; 1H NMR (500 MHz, CDCl3)
2.34 (s, 3H, Thiazole CH3), 5.48 (s, 2H, Ar-CH2-N), 7.14 (d, J = 8.4 Hz, 2H Ar-H), 7.22 (s,
1-[(4-chlorophenyl)methyl]-4-[3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1-phenyl-1H-
δ
1H, Triazole H), 7.28 (m, d, J = 8.4 Hz, 2H, Ar-H), 7.27 (t, J = 7.4 Hz, 1H, Ar-H), 7.47-7.43
(m, 3H Ar-H), 7.52-7.48 (m, 2H Ar-H), 7.80 (d, J = 7.7 Hz, 2H Ar-H), 7.91 (dd, J = 7.5, 1.9
13
Hz, 2H Ar-H), 8.57 (s, 1H, Pyrazole-H); C NMR (125 MHz, CDCl3)
δ 15.40, 52.33, 113.76,
118.09, 119.07, 120.96, 125.32, 125.38, 126.13, 128.01, 128.02, 128.26, 128.58, 129.21,
132.11, 132.35, 133.73, 138.46, 139.07, 140.66, 151.67, 166.21; HRMS: m/z = 509.1319
(M+H)+.
4.3.5.
1-[(4-bromophenyl)methyl]-4-[3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1-phenyl-1H-
pyrazol-4-yl]-1H-1,2,3-triazole 7e: Yield: 74 %; mp: 174-176 °C; 1H NMR (500 MHz, CDCl3)
δ
2.28 (s, 3H, Thiazole CH3), 5.38 (s, 2H, Ar-CH2-N), 7.01 (d, J = 8.3 Hz, 2H, 2H Ar-H), 7.14
16

ACCEPTED MANUSCRIPT
(s, 1H, Triazole H), 7.27 (t, J = 7.4 Hz, 1H, Ar-H), 7.31 – 7.49 (m, 7H, Ar-H), 7.72 (d, J = 7.8
13
Hz, 2H, Ar-H), 7.84-7.85 (m, 2H, Ar-H), 8.49 (s, 1H, Pyrazole-H); C NMR (125 MHz,
CDCl3) δ 15.38, 52.31, 113.75, 118.10, 119.07, 120.95, 122.03, 125.32, 125.40, 126.14, 128.00,
128.60, 129.22, 130.66, 131.10, 132.34, 133.02, 138.45, 139.05, 140.64, 151.65, 166.23;
HRMS: m/z = 553.080454 (M+H)+, 555.0782 (M+H+2) +.
4.3.6. 1-benzyl-4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-
1
yl}-1H-1,2,3-triazole 7f: Yield: 74 %; mp: 132-134 °C; H NMR (500 MHz, CDCl3)
δ 2.32 (s,
3H, Thiazole CH3), 2.41 (s, 3H, Ar-CH3), 5.51 (s, 2H, Ar-CH2-N), 7.19-7.22 (m, 3H, Ar-H,
Triazole H), 7.23-7.26 (m, 3H, Ar-H), 7.28-7.36 (m, 3H, Ar-H), 7.47-7.51 (m, 2H, Ar-H), 7.77-
13
7.80 (m, 4H, Ar-H), 8.56 (s, 1H, Pyrazole-H); C NMR (125 MHz, CDCl3)
δ 16.43, 21.48,
54.14, 114.92, 119.11, 120.22, 121.49, 126.28, 126.40, 127.09, 127.75, 128.72, 129.09, 129.60,
129.66, 130.82, 134.63, 139.54, 139.95, 140.47, 141.82, 152.50, 167.40; HRMS: m/z =
489.2567 (M+H)+.
4.3.7. 4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-[(4-
1
methylphenyl)methyl]-1H-1,2,3-triazole 7g: Yield: 84 %; mp: 158-160 °C; H NMR (500
MHz, CDCl3) δ 2.28 (s, 3H, Thiazole CH3), 2.32 (s, 3H, Ar-CH3), 2.41 (s, 3H, Ar-CH3), 5.46 (s,
2H, Ar-CH2-N), 7.10 (s, 4H, Ar-H), 7.19 (s, 1H, Triazole H), 7.26 (m, 3H, Ar-H), 7.34 (t, J =
7.4 Hz, 1H, Ar-H), 7.49 (t, J = 7.9 Hz, 2H, Ar-H), 7.80 (dd, J = 10.2, 8.5 Hz, 4H, Ar-H), 8.55
(s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 16.43, 21.11, 21.48, 53.95, 114.98, 119.10,
120.14, 121.51, 126.26, 126.38, 127.07, 127.78, 129.59, 129.66, 129.73, 130.85, 131.56,
138.61, 139.55, 139.84, 140.44, 141.82, 152.50, 167.37; HRMS: m/z = 503.2012 (M+H)+.
4.3.8.
1-[(4-fluorophenyl)methyl]-4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-
1
phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7h: Yield: 72 %; mp: 224-226 °C; H NMR (500
MHz, CDCl3) δ 2.33 (s, 3H, Thiazole-CH3), 2.41 (s, 3H, Ar-CH3), 5.47 (s, 2H, Ar-CH2-N), 6.99
(t, J = 8.6 Hz, 2H, Ar-H), 7.18 – 7.22 (m, 3H, Ar-H, , Triazole H), 7.26 (d, J = 7.9 Hz, 2H, Ar-
H), 7.34 (t, J = 7.4 Hz, 1H, Ar-H), 7.49 (t, J = 8.0 Hz, 2H,Ar-H), 7.78-7.81 (m 4H, Ar-H), 8.56
(s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 16.43, 21.48, 53.38, 114.84, 116.10,
119.11, 120.06, 121.44, 126.28, 126.33, 127.13, 129.61, 129.65, 129.71, 130.53, 130.76,
139.52, 140.08, 140.57, 141.79, 152.52, 162.80, 167.45; HRMS: m/z = 507.1761 (M+H)+.
4.3.9.
1-[(4-chlorophenyl)methyl]-4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-
1
phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7i: Yield: 76 %; mp: 184-168 °C; H NMR (500
MHz, CDCl3) δ 2.34 (s, 3H, Thiazole-CH3), 2.42 (s, 3H, Ar-CH3), 5.47 (s, 2H, Ar-CH2-N), 7.14
(d, J = 8.4 Hz, 2H, Ar-H), 7.21 (s, 1H, Triazole H), 7.26-7.28 (m, 4H, Ar-H), 7.35 (t, J = 7.4
17

ACCEPTED MANUSCRIPT
Hz, 1H, Ar-H), 7.50 (t, J = 8.0 Hz, 2H, Ar-H), 7.80 (dd, J = 7.8, 6.5 Hz, 4H, Ar-H), 8.57 (s,
13
1H, Pyrazole-H); C NMR (125 MHz, CDCl3)
δ 16.41, 21.47, 53.35, 114.79, 119.11, 120.08,
121.41, 126.29, 126.32, 127.13, 129.02, 129.28, 129.59, 129.72, 130.73, 133.15, 134.73,
139.51, 140.14, 140.56, 141.77, 152.51, 167.48; HRMS: m/z = 523.1471 (M+H)+.
4.3.10.
1-[(4-bromophenyl)methyl]-4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-
1
phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7j: Yield: 75 %; mp: 148-150 °C; H NMR (500
MHz, CDCl3)
δ 2.33 (s, 3H), 5.52 (s, 2H), 7.24-7.18 (m, 3H), 7.26 (s, 1H), 7.37-7.29 (m, 4H),
7.42 (d, J = 8.5 Hz, 2H), 7.50 (t, J = 7.9 Hz, 2H), 7.78 (d, J = 7.8 Hz, 2H), 7.84 (d, J = 8.5 Hz,
13
2H), 8.54 (s, 1H); C NMR (125 MHz, CDCl3)
δ 16.38, 21.45, 53.34, 114.74, 119.15, 120.10,
121.42, 122.06, 126.30, 126.30, 127.15, 129.59, 129.72, 130.70, 131.13,133.18, 133.05, 139.55,
140.16, 140.58, 141.74, 152.52, 167.50;HRMS: m/z = 567.0955 (M+H)+,569.0937 (M+H+2)+.
4.3.11. 1-benzyl-4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-
1
yl}-1H-1,2,3-triazole 7k: Yield: 68 %; mp: 154-156 °C; H NMR (500 MHz, CDCl3)
δ 2.32 (s,
3H, Thiazole CH3), 5.52 (s, 2H, Ar-CH2-N), 7.14 (t, J = 8.6 Hz, 2H, Ar-H), 7.23-7.19 (m, 3H,
Ar-H, Triazole H), 7.30-7.34 (m, 4H, Ar-H), 7.49 (t, J = 8.0 Hz, 2H, Ar-H), 7.78 (d, J = 7.8 Hz,
2H, Ar-H), 7.88 (dd, J = 8.8, 5.3 Hz, 2H, Ar-H), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz,
CDCl3) δ 16.42, 54.15, 114.86, 116.06, 119.12, 120.24, 122.12, 126.39, 127.16, 127.76, 128.38,
128.73, 129.09, 129.62, 129.84, 134.64, 139.51, 139.88, 141.64, 152.68, 163.94, 165.89;
HRMS: m/z = 493.1613 (M+H)+.
4.3.12.
4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenylpyrazol-4-yl}-1-[(4-
1
methylphenyl)methyl]-1,2,3-triazole 7l: Yield: 70 %; mp: 150-152 °C; H NMR (500 MHz,
CDCl3)
δ 2.29 (s, 3H, Thiazole CH3), 2.33 (s, 3H, Ar-CH3), 5.47 (s, 2H, Ar-CH2-N), 7.11 (s,
4H, Ar-H), 7.14 (t, J = 8.6 Hz, 2H, Ar-H), 7.19 (s, 1H, Triazole H), 7.47-7.51 (m, Ar-H), 7.78
(d, J = 7.4 Hz, 3H, Ar-H), 7.90 (m, 2H, Ar-H), 8.54 (s, 1H, Pyrazole-H); 13C NMR (125 MHz,
CDCl3)
δ 16.43, 21.14, 53.96, 114.92, 116.09, 119.11, 120.16, 126.38, 127.14, 127.79, 128.36,
129.61, 129.72, 129.87, 131.57, 135.38, 138.64, 139.51, 139.77, 141.64, 152.67, 163.93,
165.86; HRMS: m/z = 507.1761 (M+H)+.
4.3.13.
4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenylpyrazol-4-yl}-1-[(4-
1
fluorophenyl)methyl]-1,2,3-triazole 7m: Yield: 66 %; mp: 158-160 °C; H NMR (500 MHz,
CDCl3)
δ 2.33 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 7.00(t, J = 8.6 Hz, 2H, Ar-H),
7.15 (t, J = 8.6 Hz, 2H, Ar-H), 7.18-7.23 (m, 3H, Ar-H, Triazole H), 7.35 (t, J = 7.4 Hz, 1H,
Ar-H), 7.50 (t, J = 7.9 Hz, 2H, Ar-H), 7.78 (d, J = 7.7 Hz, 2H, Ar-H), 7.89 (dd, J = 8.7, 5.3 Hz,
2H, Ar-H), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 15.37, 52.36, 113.73,
18

ACCEPTED MANUSCRIPT
115.06, 115.07, 118.08, 119.01, 125.35, 126.15, 127.29, 128.58, 128.64, 128.72, 129.47,
131.16, 138.44, 138.97, 140.55, 151.65, 161.76, 162.93, 164.91; HRMS: m/z = 511.1508
(M+H)+.
4.3.14.
1-[(4-chlorophenyl)methyl]-4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-
1
phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7n: Yield: 74 %; mp: 180-182 °C; H NMR (500
MHz, CDCl3)
δ 2.34 (s, 3H, Thiazole-CH3), 5.49 (s, 2H, Ar-CH2-N), 7.12-7.18 (m, 4H, Ar-H),
7.23 (s, 1H, Triazole H), 7.32-7.27 (m, 2H, Ar-H), 7.35 (t, J = 7.4 Hz, 1H, Ar-H), 7.50 (t, J =
7.9 Hz, 2H, Ar-H), 7.79 (d, J = 7.8 Hz, 2H, Ar-H), 7.89 (dd, J = 8.6, 5.3 Hz, 2H, Ar-H), 8.55
(s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 16.42, 53.35, 114.71, 116.13, 119.10,
120.18, 126.42, 127.19, 128.31, 129.07, 129.26, 129.61, 129.75, 132.20, 133.18, 134.72,
139.45, 140.02, 141.61, 152.67, 163.93, 165.90; HRMS: m/z = 527.1216 (M+H)+, 529.1196
(M+H+2)+.
4.3.15.
1-[(4-bromophenyl)methyl]-4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-
1
phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7o: Yield: 70 %; mp: 148-150 °C; H NMR (500
MHz, CDCl3)
δ 2.34 (s, 3H, Thiazole-CH3), 5.46 (s, 2H, Ar-CH2-N), 7.08 (d, J = 8.4 Hz, 2H,
Ar-H), 7.15 (t, J = 8.6 Hz, 2H, Ar-H), 7.22 (s, 1H, Triazole H), 7.35 (t, J = 7.4 Hz, 1H, Ar-H),
7.44 (d, J = 8.4 Hz, 2H, Ar-H), 7.50 (t, J = 7.9 Hz, 2H, Ar-H), 7.78 (d, J = 7.8 Hz, 2H, Ar-H),
13
7.89 (dd, J = 8.7, 5.3 Hz, 2H, Ar-H), 8.55 (s, 1H, Pyrazole-H); C NMR (125 MHz, CDCl3)
δ
15.39, 52.39, 113.69, 115.14, 118.09, 119.08, 121.84, 125.38, 126.17, 126.75, 127.30, 128.29,
128.59, 128.72, 131.22, 132.63, 138.43, 139.05, 140.56, 151.66, 162.93, 164.95; HRMS: m/z
=571.0710 (M+H)+, 573.0690 (M+H+2)+.
4.3.16. 1-benzyl-4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-
1
yl}-1H-1,2,3-triazole 7p: Yield: 70 %; mp: 156-158 °C; H NMR (500 MHz, CDCl3)
δ 2.34 (s,
3H, Thiazole-CH3), 5.47 (s, 2H, Ar-CH2-N), 7.09 (d, J = 8.3 Hz, 2H, Ar-H), 7.21 (s, 1H,
Triazole H), 7.35 (t, J = 7.4 Hz, 1H, Ar-H), 7.47-7.42 (m, 4H, Ar-H), 7.50 (t, J = 7.9 Hz, 2H,
Ar-H), 7.79 (d, J = 7.9 Hz, 2H, Ar-H), 7.85 (d, J = 8.5 Hz, 2H, Ar-H), 8.55 (s, 1H, Pyrazole-
H); 13C NMR (125 MHz, CDCl3)
δ 16.36, 53.14, 114.67, 119.17, 120.05, 121.68, 125.35,
125.48, 125.98, 126.29, 127.89, 128.09, 129.68, 129.78, 132.38, 132.98, 133.15, 139.47,
140.08, 141.59, 152.16, 166.41; HRMS: m/z = 509.1320 (M+H)+.
4.3.17. 4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-[(4-
1
methylphenyl)methyl]-1H-1,2,3-triazole 7q: Yield: 66 %; mp: 154-156 °C; H NMR (500
MHz, CDCl3) δ 2.29 (s, 3H, Thiazole-CH3), 2.33 (s, 3H, Ar-CH3), 5.47 (s, 2H, Ar-CH2-N), 7.11
(s, 4H, Ar-H), 7.18 (s, 1H, Triazole H), 7.34 (t, J = 7.4 Hz, 1H, Ar-H), 7.43 (d, J = 8.5 Hz, 2H,
19

ACCEPTED MANUSCRIPT
Ar-H), 7.49 (t, J = 7.8 Hz, 2H, Ar-H), 7.78 (d, J = 7.9 Hz, 2H, Ar-H), 7.85 (d, J = 8.5 Hz, 2H,
13
Ar-H), 8.53 (s, 1H, Pyrazole-H); C NMR (125 MHz, CDCl3)
δ 16.34, 21.16, 54.02, 114.67,
119.21, 120.01, 121.70, 126.02, 126.34, 127.84, 128.12, 129.66, 129.70, 129.83, 133.01,
133.18, 134.98, 138.74, 139.45, 140.12, 141.48, 152.06, 166.41; HRMS: m/z = 523.1471
(M+H)+.
4.3.18. 4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-[(4-
1
fluorophenyl)methyl]-1H-1,2,3-triazole 7r: Yield: 72 %; mp: 164 °C; H NMR (500 MHz,
CDCl3)
δ 2.33 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 7.01 (t, J = 8.6 Hz, 2H, Ar-H),
7.19 (s, 1H, Triazole H), 7.21 (dd, J = 8.5, 5.2 Hz, 2H, Ar-H), 7.35 (t, J = 7.4 Hz, 1H, Ar-H),
7.43 (d, J = 8.5 Hz, 2H, Ar-H), 7.50 (t, J = 7.9 Hz, 2H, Ar-H), 7.78 (d, J = 7.8 Hz, 2H, Ar-H),
7.84 (d, J = 8.5 Hz, 2H, Ar-H), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ
16.32, 53.18, 114.72, 115.12, 119.20, 120.10, 121.70, 125.38, 126.01, 126.31, 128.12, 129.42,
129.71, 129.72, 132.92, 133.12, 139.49, 140.10, 141.65, 152.20, 161.81, 166.43; HRMS: m/z =
527.1216 (M+H)+.
4.3.19. 4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-[(4-
chlorophenyl)methyl]-1H-1,2,3-triazole 7s: Yield: 70 %; mp: 166-168 °C; 1H NMR (500 MHz,
CDCl3)
δ 2.34 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 7.15 (d, J = 8.4 Hz, 2H, Ar-H),
7.21 (s, 1H, Triazole H), 7.29 (d, J = 8.4 Hz, 2H, Ar-H), 7.35 (t, J = 7.4 Hz, 1H, Ar-H), 7.43 (d,
J = 8.5 Hz, 2H, Ar-H), 7.50 (t, J = 7.9 Hz, 2H, Ar-H), 7.78 (d, J = 7.8 Hz, 2H, Ar-H), 7.84 (d,
13
J = 8.5 Hz, 2H, Ar-H), 8.55 (s, 1H,Pyrazole-H); C NMR (125 MHz, CDCl3)
δ 16.30, 53.35,
114.59, 119.14, 120.14, 121.77, 126.04, 126.28, 128.16, 128.24, 128.34, 129.70, 129.85,
132.22, 132.96, 133.16, 133.79, 139.49), 140.16, 141.63, 152.12, 166.44; HRMS: m/z =
542.0929 (M+H)+.
4.3.20.
1-[(4-bromophenyl)methyl]-4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-
1
phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7t: Yield: 76 %; mp: 162-164 °C; H NMR (500
MHz, CDCl3)
δ 2.34 (s, 3H, Thiazole CH3), 5.46 (s, 2H, Ar-CH2-N), 7.08 (d, J = 8.3 Hz, 2H,
Ar-H), 7.22 (s, 1H, Triazole H), 7.28 (d, J = 7.4 Hz, 2H, Ar-H), 7.34 (t, J = 7.4 Hz, 1H, Ar-H),
7.43 (d, J = 8.4 Hz, 2H, Ar-H), 7.49 (t, J = 7.9 Hz, 2H, Ar-H), 7.80 (t, J = 7.9 Hz, 4H, Ar-H),
8.56 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 16.44, 53.41, 114.80, 119.13, 120.12,
121.42, 122.85, 126.31, 126.34, 127.14, 129.30, 129.61, 129.75, 130.75, 132.25, 132.87,
133.69, 139.52, 140.17, 141.80, 152.53, 167.49; HRMS: m/z = 587.0420(M+H)+, 589.0408
(M+H+2)+.
20

ACCEPTED MANUSCRIPT
4.3.21. 1-benzyl-4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-
yl}-1H-1,2,3-triazole 7u: Yield: 68 %; mp: 216-218 °C; 1H NMR (500 MHz, CDCl3)
δ 2.33 (s,
3H, Thiazole-CH3), 5.51 (s, 2H, Ar-CH2-N), 7.19-7.23 (m, 3H, Ar-H, Triazole H), 7.29-7.36
(m, 4H, Ar-H), 7.49 (t, J = 7.9 Hz, 2H, Ar-H), 7.57 (d, J = 8.5 Hz, 2H, Ar-H), 7.75-7.79 (m,
4H, Ar-H), 8.54 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 16.28, 53.54, 114.69,
119.16, 120.11, 122.55, 124.51, 126.48, 127.14, 127.20, 127.80, 127.89, 129.15, 129.68,
132.26, 132.37, 134.70, 139.47, 139.88, 141.55, 152.65, 165.81; HRMS: m/z = 553.0804
(M+H)+, 555.0782 (M+H+2)+.
4.3.22. 4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-[(4-
1
methylphenyl)methyl]-1H-1,2,3-triazole 7v: Yield: 78 %; mp: 182-184 °C; H NMR (500
MHz, CDCl3)
δ 2.31 (s, 3H, Thiazole-CH3), 2.38 (s, 3H, Ar-CH3), 5.50 (s, 2H, Ar-CH2-N),
7.17 (s, 1H, Triazole H), 7.22-7.26 (m, 4H, Ar-H), 7.34 (t, J = 7.4 Hz, 1H, Ar-H), 7.45 (d, J =
8.5 Hz, 2H, Ar-H), 7.51 (t, J = 7.8 Hz, 2H, Ar-H), 7.74 (d, J = 7.8 Hz, 2H, Ar-H), 7.82 (d, J =
13
8.4 Hz, 2H, Ar-H), 8.54 (s, 1H, Pyrazole-H); C NMR (125 MHz, CDCl3)
δ 16.46 (Thiazole-
CH3), 21.15, 53.46, 114.79, 119.18, 120.11, 122.55, 124.51, 126.43, 126.50, 127.28, 127.84,
129.68, 129.78, 130.90, 132.28, 132.36, 138.68, 139.50, 140.03, 141.57, 152.85, 166.11;
HRMS: m/z = 567.0955 (M+H)+, 569.0938 (M+H+2)+.
4.3.23. 4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-[(4-
1
fluorophenyl)methyl]-1H-1,2,3-triazole 7w: Yield: 66 %; mp: 194-196 °C; H NMR (500
MHz, CDCl3)
δ 2.33 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 6.98-7.04 (m, 2H, Ar-H),
7.18-7.22 (m, 3H, Ar-H, Triazole H), 7.35 (t, J = 7.4 Hz, 1H, Ar-H), 7.50 (t, J = 8.0 Hz, 2H,
Ar-H), 7.61-7.56 (m, 2H, Ar-H), 7.78 (dd, J = 7.9, 5.6 Hz, 4H, Ar-H), 8.55 (s, 1H, Pyrazole-H);
13C NMR (125 MHz, CDCl3)
δ 16.43, 53.42, 114.75, 116.13, 119.13, 120.06, 122.51, 124.47,
126.45, 127.22, 127.79, 129.63, 129.68, 130.49, 132.21, 132.32, 139.46, 139.98, 141.52,
152.88, 162.82, 165.81; HRMS: m/z = 571.0710 (M+H)+, 573.0695 (M+H+2)+.
4.3.24. 4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-[(4-
chlorophenyl)methyl]-1H-1,2,3-triazole 7x: Yield: 72 %; mp: 170-172 °C; 1H NMR (500 MHz,
CDCl3)
δ 2.34 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 7.15 (d, J = 8.4 Hz, 2H, Ar-H),
7.21 (s, 1H, Triazole H), 7.29 (d, J = 8.4 Hz, 2H, Ar-H), 7.35 (t, J = 7.4 Hz, 1H, Ar-H), 7.48-
7.52 (m, 2H, Ar-H), 7.59 (d, J = 8.5 Hz, 2H, Ar-H), 7.76-7.80 (m, 4H, Ar-H), 8.55 (s, 1H,
Pyrazole-H); 13C NMR (125 MHz, CDCl3)
δ 16.42, 53.45, 114.77, 119.13, 120.10, 122.53,
124.48, 126.42, 127.20, 127.80, 127.97, 128.27,129.66, 132.14, 132.22, 132.35, 133.77, 139.44,
140.00, 141.50, 152.80, 165.97; HRMS: m/z = 587.0421 (M+H)+, 589.0410 (M+H+2)+.
21

ACCEPTED MANUSCRIPT
4.3.25. 4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1-[(4-
bromophenyl)methyl]-1H-1,2,3-triazole 7y: Yield: 65 %; mp: 188-190 °C; 1H NMR (500 MHz,
CDCl3)
δ 2.34 (s, 3H, Thiazole-CH3), 5.46 (s, 2H, Ar-CH2-N), 7.08 (d, J = 8.4 Hz, 2H, Ar-H),
7.21 (s, 1H, Triazole H), 7.35 (t, J = 7.4 Hz, 1H, Ar-H), 7.42-7.46 (m, 2H, Ar-H), 7.48-52 (m,
13
2H, Ar-H), 7.57-7.62 (m, 2H, Ar-H), 7.75-7.80 (m, 4H, Ar-H), 8.55 (s, 1H, Pyrazole-H); C
NMR (125 MHz, CDCl3)
δ 16.38, 53.58, 114.80, 119.09, 120.13, 122.09, 122.54, 124.55,
126.57, 127.29, 127.85, 129.59, 130.70, 131.16, 132.18, 132.25, 133.13,139.40, 139.90, 141.48,
152.69, 165.93; HRMS: m/z = 630.9910 (M+H)+, 632.9888 (M+H+2)+, 634.9870 (M+H+4)+.
5. Biological activity
5.1. Antibacterial activity
The in vitro antibacterial screening of the synthesized pyrazolyl-thiazolyl-triazole derivatives
was done by the well diffusion method [55-57] against the standard strains Gram-negative
bacteria E. coli and Gram-positive bacteria S. albus. All the strains were procured from
National Collection of Industrial Microorganisms (NCIM) NCL, Pune, India. All cultures were
maintained at 4 ºC over nutrient agar slants throughout the experiment, for antibacterial activity,
the cultures incubated for overnight at 37 ºC in nutrient broth. Five hundred microliters of 24-48
hr old fresh bacterial culture were spread over the nutrient agar plates. The sterile cotton swab
was used for inoculation of the cultures in order to get a uniform microbial growth. With the
help of well borer, 5 mm diameter wells were punched on the agar plates. The synthesized
compounds were dissolved in DMSO. The wells were filled with 80 µL of respective
synthesized compounds in DMSO. As a vehicle control, DMSO was added to one agar plate.
The plates were incubated for a period of 24-48 hrs. at 37 ºC. After the incubation period, the
antimicrobial activity was evaluated by measuring the zone of inhibition in mm using a
measuring scale and the average was calculated. The experiments were carried out in 4
replicates.
5.2. Antifungal activity
The in vitro antifungal activity of the synthesized pyrazolyl-thiazolyl-triazole derivatives was
done by well diffusion method against Candida albicans (NCIM 3100), Aspergillus niger
(NCIM 504) and Rhodotorula glutinous (NCIM 3168). All the strains were obtained from
NCIM, NCL, Pune, India. The pure cultures were maintained by routine sub culturing after
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every one-month interval on Potato Dextrose Agar slants (Hi-Media lab. Pvt. Ltd, Mumbai,
India). Mueller Hinton agar plates were prepared by pouring 20 mL in each sterile petri - plates
for fungal assay and allowed to solidify. During the assay, standard fungal cultures were grown
on Potato-Dextrose broth. Five hundred microliters of 48-72 hrs. old fresh fungal spore
suspension was spread on the agar plates using a sterile cotton swab to get uniform growth.
With the help of well borer, 5 mm diameter wells were punched on the agar plates. The
synthesized compounds were dissolved in DMSO. The wells were filled with 80 µL of the
samples. As a vehicle control, DMSO was added to one agar plate. A standard plate with
Fluconazole and Rouconazole was used as appositive control. The plates were incubated for a
period of 48-72 hrs at 30 °C. After the incubation period, the plates were observed for the clear
zone of inhibition. The zones of inhibition were measured in mm using a measuring scale and
the mean was calculated. The experiments were carried out in five replicates.
The micro-dilution susceptibility test in Sabouraud Liquid Medium (Oxoid) was used for the
determination of minimum inhibition concentration (MIC). Stock solution of the test
compounds, Fluconazole and Ravuconazole were prepared in DMSO at concentration of 1000
µg/mL. Two fold serial dilutions of the test compounds solutions were prepared using broth.
The final concentration of the solutions was 500, 250, 125, 62.5, 31.25, 15.62, 7.81 and 3.90
µg/mL. The tubes were inoculated with the test organisms, grown in the Potato-Dextrose broth.
The tubes were kept for incubation for 48-72 hrs. at 30 °C. The lowest concentration showing
no growth was considered as minimum inhibition concentration (MIC). Control experiment
with DMSO and uninoculated media were run parallel to the test compounds under similar
conditions. All experiments were carried in triplicates.
5.3. Sterol profile of A. niger NCIM 504
Total intracellular sterols were extracted as reported by Breivik and Owades [60] with slight
modifications. Briefly, A. niger colony from an overnight grown culture inoculate 100 mL of
Potato dextrose broth containing 31.5 µg/mL (MIC) concentration of compounds 7g, 7i, 7k, 7l
and 7m for the treatment of azole compounds. In control the fungus was grown in absence of
compounds. The cultures were incubated for 72 h with shaking at 37 °C. The cells were
harvested by centrifugation at 5000 rpm, for 5 min and washed once with sterile distilled water.
The net wet weight of the cell pellet was determined. 3 mL of 25% alcoholic KOH was added
to each pellet and vortex mixed for 1 min. Cell suspensions were incubated in 85 °C water bath
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for 1 h. The tubes were allowed to cool to room temperature. Sterols were then extracted by
addition of a mixture of 1 mL of sterile distilled water and 3 mL of n-heptane followed by
vigorous vortex mixing for 3 min. The heptane layer was transferred to a clean borosilicate
glass screw-cap tube. A 20 mL aliquot of sterol extract was diluted five fold in 100% ethanol
and scanned spectrophotometrically between 240 and 300 nm. All the experiments were carried
out in duplicates.
Ergosterol content was calculated as a percentage of the wet weight of the cell by the following
equations: % ergosterol 1 % 24(28)DHE 5 [(A281.5/290) 3 F]/pellet weight, % 24(28)DHE 5
[(A230/518) 3 F]/pellet weight, and %ergosterol 5 [% ergosterol 1%24(28)DHE]
2%24(28)DHE, where F is the factor for dilution in ethanol and 290 and 518 are the E values
(in percentages per centimeter) determined for crystalline ergosterol and 24(28)DHE,
respectively.
5.4. Molecular docking
Molecular docking was performed to identify possible mode of action of the synthesized
derivatives. Crystal structure of sterol 14-alpha demethylase (CYP51) from Candida albicans
utilized for docking analysis (PDB ID 5TZ1), which was downloaded from free protein
database www.rcsb.org. Biopredicta module of Vlife MDS 4.4 was utilized for docking analysis
[61]. Prior to the docking analysis protein structure was refined via addition of hydrogen atoms
and removal of water molecule to retain its native geometry. Grip based docking analysis was
performed in which the protein was kept in rigid conformation while ligands in flexible
conformation.
Acknowledgments
Authors are thankful to Shikshana Prasaraka Mandali’s Bhide Foundation, Pune for lending
support with their biological activities. Central Instrumentation facility, Savitribai Phule Pune
University, Pune is also acknowledged for spectral analysis. A D Shinde is grateful to CSIR-for
award of JRF, Award No 08/319(0004/)17-EMR-1.
Conflicts of interest: There are no conflicts to declare
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