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

European Journal of Medicinal Chemistry 179 (2019) 649e659

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis of new thiazolyl-pyrazolyl-1,2,3-triazole derivatives as potential antimicrobial agents Jitendra Nalawade a , Abhijit Shinde b, Abhijit Chavan b, Sachin Patil a, Manjusha Suryavanshi a, Manisha Modak c, Prafulla Choudhari d, Vivek D. Bobade a, **, Pravin C. Mhaske b, * a

Savitribai Phule Pune University, Post-Graduate Department of Chemistry H. P. T. Arts and R. Y. K. Science College, Nashik, 422005, India Savitribai Phule Pune University, Post-Graduate Department of Chemistry, S. P. Mandali's Sir Parashurambhau College, Tilak Road, Pune, 411 030, India Savitribai Phule Pune University, Department of Zoology, S. P. Mandali's Sir Parashurambhau College, Tilak Road, Pune, 411 030, India d Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Kolhapur, 413016, India b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2019 Received in revised form 26 June 2019 Accepted 26 June 2019 Available online 27 June 2019

A series of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H1,2,3-triazole derivatives (7a-y) have been synthesized by click reaction of 5-(4-ethynyl-1-phenyl-1Hpyrazol-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 promising antifungal activity against A. niger with MIC 31.5 mg/mL. Compounds 7g, 7i, 7k, 7l and 7m were further evaluated for ergosterol inhibition assay against A. niger cells sample at 31.5 mg/mL concentration. The analysis of sterol inhibition assay revealed that ergosterol biosynthesis is decreased in the fungal samples treated with azole 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. © 2019 Elsevier Masson SAS. All rights reserved.

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].

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (V.D. Bobade), mhaskepc18@ gmail.com (P.C. Mhaske). https://doi.org/10.1016/j.ejmech.2019.06.074 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

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, antimalarial and antiviral activities [5,6]. Thiazole and its derivatives provide a wide spectrum of biological activities such as antimicrobial [7e12], antimycobacterial [13e18], anti-inflammatory [19e21], 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 [26e30]. 1,2,3Triazole derivatives are extremely potent inhibitors against

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certain enzymes, agonists, antagonist, ligand in receptor
ligand binding studies for drug development [31]. They also exhibit significant pharmacological activities such as antitubercular [32e36], anti-microbial [37e39], 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 (Fig. 1). Thiazole clubbed with pyrazole reported for antimicrobial [45] anti-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. 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 [11e15], we report herein the synthesis of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2-aryl-1,3thiazol-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-thiazol5-yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole 7a-y were synthesized as illustrated in Scheme 1. Starting compounds 1-(4-methyl-2substituted 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-1,3thiazol-5-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3a-e. Aldehyde 3a-e on reaction with diethyl-1-diazo-2oxopropylphosphonate (Ohira-Bestmann reagent) 4 and K2CO3 in methanol gave 5-(4-ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl2-aryl-1,3-thiazole 5a-e. Alkyne 5a-e on click reaction with substituted 1-(azidomethyl)benzene 6a-e in presence of sodium 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. 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 d 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 d 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 d 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 d 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 d 114.98 to 167.37 confirmed the formation of compound 7g. Further, a peak m/z ¼ 503.2012 (M þ H)þ 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-[1phenyl-3-(4-methyl-2-aryl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H1,2,3-triazole 7a-y derivatives were evaluated against Gramnegative bacteria E. coli and P. mirabilis and Gram-positive bacteria S. albus using well diffusion method [55e57]. Standard drug Streptomycin was used as reference and DMSO was used as negative control. The in vitro antifungal activity [55e57] 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 mg/ mL concentrations and the wells were filled with 80 mL (80 mg) 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,

Fig. 1. Representative biologically active thiazolyl-pyrazole (AeC), thiazolyl-triazole (DeG) and our new proposed analogues (7a-y).

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Scheme 1. Synthetic route of compounds 7a-y.

Table 1 Antimicrobial activitya in zone of inhibition (mm) of compounds 7a-y. Comp.

R

R1

E. coli

P. mirabilis

S. albus

C. albicans

A. niger

R. glutinis

7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 7s 7t 7u 7v 7w 7x 7y Streptomycin Fluconazole Ravuconazole

H H H H H CH3 CH3 CH3 CH3 CH3 F F F F F Cl Cl Cl Cl Cl Br Br Br Br Br

H CH3 F Cl Br H CH3 F Cl Br H CH3 F Cl Br H CH3 F Cl Br H CH3 F Cl Br

nd 10.0 11.0 13.2 12.0 e 10.2 10.6 13.6 11.0 11.8 9.6 12.2 e e 12.8 12.4 12.2 9.8 11.0 11.5 11.6 11.8 11.0 e 25.0 e e

e e e e e e e e e e e e e e e e e e e e e e e e e 18.52 e e

nd e 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 e e 21.6 e e

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 e 20.25 28.64

nd 14.0 16.4 13.6 14.0 e e 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 e e 18.35 20.18

nd 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 e e 25.30 20.15

a

The concentration of test compounds and reference ¼ 80 mg/well; NA ¼ Not Applicable; (
) ¼ Inactive.

we has screened newly synthesized thiazolyl-pyrazolyl-1,2,3triazole,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 mg/mL. The in vitro antifungal MIC screening results of synthesized compounds 7a-y is presented in Table 2. 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-2phenyl-1,3-thiazol-5-yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole (7a-e), compound 7b (R ¼ H, R1 ¼ CH3) showed excellent activity against A. niger, while compound 7d (R ¼ H, R1 ¼ Cl) showed excellent activity against A. niger and good activity against R. glutinis, compound 7e (R ¼ H, R1 ¼ Br) showed good activity against A. niger and R. glutinis strains. From compounds 1-substituted benzyl-4-{3-[4methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole (7f-j), compound 7g (R ¼ CH3,

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Table 2 Antifungal activity in MIC (mg/mL) of compounds 7a-y. Compound

R

R1

C. albicans

A. niger

R. glutinis

7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 7s 7t 7u 7v 7w 7x 7y Fluconazole Ravuconazole

H H H H H CH3 CH3 CH3 CH3 CH3 F F F F F Cl Cl Cl Cl Cl Br Br Br Br Br

H CH3 F Cl Br H CH3 F Cl Br H CH3 F Cl Br H CH3 F Cl Br H CH3 F Cl Br

e e 125 e 125 125 62.5 e e 125 125 e 125 e 125 500 250 250 e 250 125 e 125 125 e 7.81 7.81

e 31.25 125 31.25 62.5 62.5 31.25 62.5 31.25 31.25 31.25 31.25 31.25 31.25 125 31.25 125 62.5 125 62.5 62.5 31.25 62.5 62.5 e 7.81 31.25

e e 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 e 7.81 15.625

NA ¼ Not Applicable; (
) ¼ Inactive.

R1 ¼ CH3) showed good activity against C. albicans and excellent activity against A. niger, 7h (R ¼ CH3, R1 ¼ F) showed good activity against A. niger, compound 7i (R ¼ CH3, R1 ¼ Cl) and 7j (R ¼ CH3, R1 ¼ Br) showed excellent activity against A. niger and good activity against R. glutinis strains. Among 1-substituted benzyl-4-{3-[2-(4fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4yl}-1H-1,2,3-triazole (7k-o), compounds 7k (R ¼ F, R1 ¼ H), 7l (R ¼ F, R1 ¼ CH3) and 7m (R ¼ F, R1 ¼ F) reported excellent activity against A. niger and good activity against R. glutinis. Compounds 7n (R ¼ F, R1 ¼ Cl) showed excellent activity against A. niger and 7o (R ¼ F, R1 ¼ Br) showed good activity against R. glutinis. From the compounds 1- substituted benzyl-4-{3-[2-(4-chlorophenyl)-4methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3triazole (7p-t), compounds 7p (R ¼ Cl, R1 ¼ H) showed excellent activity against A. niger, 7r (R ¼ Cl, R1 ¼ 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 benzyl4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl1H-pyrazol-4-yl}-1H-1,2,3-triazole (7u-y), compounds 7u (R ¼ Br, R1 ¼ H) and 7w (R ¼ Br, R1 ¼ F) showed good activity against A. niger and R. glutinis strains. Compound 7v (R ¼ Br, R1 ¼ CH3) showed excellent activity against A. niger and compound 7x (R ¼ Br, R1 ¼ 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 ¼ H, R1 ¼ Cl and vice-versa, similarly R ¼ CH3, R1 ¼ Br and vice-versa reported similar activity against A. niger. Also R ¼ F and R1 ¼ H/CH3/F or Cl in compounds 7k-n, showed excellent activity against A. niger.

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 to 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 mg/mL concentration (MIC). The spectrophotometric absorbance profile between 240 and 300 nm of compounds 7g, 7i, 7k, 7l and 7m are shown in Fig. 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. 2.4. Molecular docking Molecular docking analysis was performed to confirm the mechanism of action of synthesized derivatives. Crystal structure of sterol 14-alpha demethylase (CYP51) from Candida albicans complexed with azole-based antifungal drug candidate was utilized for docking analysis. All the synthesized 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 (Fig. S1). Compound 7d exhibited aromatic binding interactions with TYR505, HIS373 and hydrophobic interactions with GLU70 and GLN67 (Fig. S2), compound 7g showed hydrogen bond interaction with TYR505, aromatic interactions with TYR69, HIS373, TYR505 and hydrophobic interactions with GLN67, GLU70, SER412, GLU413 (Fig. 3). Compound 7i interacted with protein via formation of hydrogen bond interaction with GLN67 aromatic interactions with

2.3. Ergosterol inhibition activity The common mode of action of azole antifungal drugs is to

Fig. 2. Spectrophotometric analysis of ergosterol composition of A. niger at 31.5 mg/mL concentration of 7g, 7i, 7k, 7l and 7m.

J. Nalawade et al. / European Journal of Medicinal Chemistry 179 (2019) 649e659 Table 3 Quantitative estimation of ergosterol. Compound

% ergosterone synthesis/g wet weight

Control 7g 7i 7k 7l 7m

0.0000290836 Not detected Not detected Not detected Not detected Not detected

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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, 1substituted benzyl-4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol5-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

TYR69, TYR505 and hydrophobic interactions with GLN67, GLU70, GLU413, ASP502, PRO503 (Fig. S3). Compound 7j showed hydrogen bond interaction with GLN67 aromatic interactions with TYR69, TYR505 and hydrophobic interactions with GLN67, ASP502, PRO503 (Fig. S4). Compound 7k interacted via formation of hydrogen bond with SER74 and aromatic interaction with TYR69, hydrophobic interaction with GLU420, ASP502, PRO503 (Fig. S5). Azole derivative 7l showed hydrogen bond interaction with TYR505, aromatic interaction with HIS373, TYR505 and hydrophobic interaction with GLN67, GLU70 (Fig. S6). Compound 7m interacted via formation of hydrogen bond interaction with GLN67, aromatic interaction with TYR69, TYR505 and hydrophobic interaction with GLN67, GLU70, ASP502 (Fig. S7). Compound 7n showed aromatic interaction with HIS373, TYR505 and hydrophobic interaction with GLN67, ASP502, PRO503 (Fig. S8), derivative 7p showed aromatic interaction with TYR69 and hydrophobic interactions with GLU420, ASP502, PRO503 (Fig. S9). Compound 7v interacted via formation of aromatic interactions with TYR69, HIS373 and hydrophobic interactions with MET372, THR411, GLU420, VAL500, ASP502, PRO503 (Fig. S10).

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 2 h. 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-(4methyl-2-phenylthiazol-5-yl)ethanone phenylhydrazone (5 mmol) in DMF (10 mL) was added dropwise to a solution of DMF-POCl3 (30 mmole15 mmol) at 0  C for 30 min, and reaction mixture was stirred at room temperature for 10 h. After completion of the reaction (TLC) the reaction mass was poured in ice cold water and stirred for 4e5 h. The solid product was filtered, washed with water and recrystallized from ethanol which gave pure 3-(4-methyl-2-phenyl1,3-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 (Mþ2) was observed because of S, Br, and/or Cl.

3. Conclusion A series of 1-substituted benzyl-4-[1-phenyl-3-(4-methyl-2aryl-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

4.1.1. 3-(4-Methyl-2-phenyl-1,3-thiazol-5-yl)-1-phenyl-1Hpyrazole-4-carbaldehyde 3a Yield: 76%; mp: 178e180  C; 1H NMR (500 MHz, CDCl3) d 2.67 (s, 3H, Thiazole-CH3), 7.48e7.39 (m, 4H, AreH), 7.53 (t, J ¼ 8.0 Hz, 2H, AreH), 7.81e7.77 (m, 2H, AreH), 7.98 (dd, J ¼ 7.4, 2.1 Hz, 2H, AreH), 8.56 (s, 1H, Pyrazole C5eH), 10.03 (s, 1H, Aldehyde-H); 13C NMR (125 MHz, CDCl3) d 17.06, 119.70, 120.46, 123.44, 126.59, 128.29,

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).

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129.03, 129.81, 130.33, 130.56, 133.33, 138.80, 146.12, 153.56, 167.52, 184.46, LCMS: m/z ¼ 346.1 (M þ H)þ. 4.1.2. 3-[4-Methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1-phenyl1H-pyrazole-4-carbaldehyde 3b Yield: 72%; mp: 230e231  C (dec.); 1H NMR (500 MHz, CDCl3) d 2.40 (s, 3H, Ar-CH3), 2.65 (s, 3H, Thiazole-CH3), 7.26 (d, J ¼ 7.9 Hz, 2H, AreH), 7.41 (dd, J ¼ 10.7, 4.2 Hz, 1H, AreH), 7.53 (t, J ¼ 8.0 Hz, 2H, AreH), 7.80e7.76 (m, 2H, AreH), 7.87 (d, J ¼ 8.1 Hz, 2H, AreH), 8.55 (s, 1H, Pyrazole C5eH), 10.03 (s, 1H, Aldehyde-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 360.95 (M þ H)þ. 4.1.3. 3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl1H-pyrazole-4-carbaldehyde 3c Yield: 66%; mp:240,241  C (dec.); 1H NMR (500 MHz, CDCl3) d 2.65 (s, 3H, Thiazole-CH3), 7.15 (t, J ¼ 8.6 Hz, 2H, AreH), 7.42 (t, J ¼ 7.4 Hz, 1H, AreH), 7.53 (t, J ¼ 7.9 Hz, 2H, AreH), 7.78 (d, J ¼ 8.3 Hz, 2H, AreH), 7.97 (dd, J ¼ 8.8, 5.3 Hz, 2H, AreH), 8.56 (s, 1H, Pyrazole C5eH), 10.03 (s, 1H, Aldehyde-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 364.05 (M þ H)þ. 4.1.4. 3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl1H-pyrazole-4-carbaldehyde 3d Yield: 70%; mp: 192e193  C; 1H NMR (500 MHz, CDCl3) d 2.65 (s, 3H, Thiazole-CH3), 7.45e7.40 (m, 3H, AreH), 7.53 (t, J ¼ 8.0 Hz, 2H, AreH), 7.80e7.77 (m, 2H, AreH), 7.92 (d, J ¼ 8.6 Hz, 2H, AreH), 8.55 (s, 1H, Pyrazole C5eH), 10.02 (s, 1H, Aldehyde-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 380.05 (M þ H)þ, 382.00 (M þ Hþ2)þ. 4.1.5. 3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1-phenyl1H-pyrazole-4-carbaldehyde 3e Yield: 75%; mp: 184e185  C; 1H NMR (500 MHz, CDCl3) d 2.66 (s, 3H, Thiazole-CH3), 7.42 (t, J ¼ 7.4 Hz, 1H, AreH), 7.54 (dd, J ¼ 10.7, 5.2 Hz, 2H, AreH), 7.60e7.57 (m, 2H, AreH), 7.78 (dd, J ¼ 8.5, 0.9 Hz, 2H, AreH), 7.86e7.83 (m, 2H, AreH), 8.56 (s, 1H, Pyrazole C5eH), 10.02 (s, 1H, Aldehyde-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 424.03 (M þ H)þ, 426.01(M þ Hþ2)þ. 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-(4methyl-2-phenylthiazol-5-yl)-1-phenyl-1H-pyrazole-4carbaldehyde (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  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-phenyl1H-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-phenyl1,3-thiazole 5a Yield: 42%; mp: 88e90  C; 1H NMR (500 MHz, CDCl3) d 2.79 (s, 3H, Thiazole-CH3), 3.26 (s, 1H, Alkyne-H), 7.05 (d, J ¼ 8.1 Hz, 2H, AreH), 7.30 (t, J ¼ 7.4 Hz, 1H, AreH), 7.47 (t, J ¼ 8.0 Hz, 2H, AreH), 7.62e7.65 (m, 3H, AreH), 7.85 (d, J ¼ 8.1 Hz, 2H, AreH), 8.17 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 342.06 (M þ H)þ. 4.2.2. 5-(4-Ethynyl-1-phenyl-1H-pyrazol-3-yl)-4-methyl-2-(4methylphenyl)-1,3-thiazole 5b Yield: 40%; mp:93e94  C; 1H NMR (500 MHz, CDCl3) d 2.40 (s, 3H, Ar-CH3), 2.78 (s, 3H, Thiazole-CH3), 3.32 (s, 1H, Alkyne-H), 7.13 (t, J ¼ 7.4 Hz, 2H, AreH), 7.25 (d, J ¼ 8.1 Hz, 2H, AreH), 7.31 (t, J ¼ 7.4 Hz, 1H, AreH), 7.49 (t, J ¼ 8.0 Hz, 2H, AreH), 7.73 (d, J ¼ 7.6 Hz, 2H, AreH), 7.87 (d, J ¼ 8.1 Hz, 2H, AreH), 8.17 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 356.1 (M þ H)þ. 4.2.3. 5-(4-Ethynyl-1-phenyl-1H-pyrazol-3-yl)-2-(4-fluorophenyl)4-methyl-1,3-thiazole 5c Yield: 48%; mp: 92e94  C; 1H NMR (500 MHz, CDCl3) d 2.77 (s, 3H, Thiazole-CH3), 3.27 (s, 1H, Alkyne-H), 7.13 (t, J ¼ 8.8 Hz, 2H, AreH), 7.29e7.34 (m, 1H, AreH), 7.46 (dd, J ¼ 8.3, 7.6 Hz, 2H, AreH), 7.71 (d, J ¼ 7.6 Hz, 2H, AreH), 8.12 (s, 1H, Pyrazole-H), 8.17 (dd, J ¼ 8.9, 5.5 Hz, 2H, AreH); 13C NMR (125 MHz, CDCl3) d 17.71, 75.75, 81.32, 101.73, 115.34, 115.51, 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 ¼ 360.04 (M þ H)þ. 4.2.4. 5-(4-Ethynyl-1-phenyl-1H-pyrazol-3-yl)-2-(4chlorophenyl)-4-methyl-1,3-thiazole 5d Yield: 38%; mp: 96e98  C; 1H NMR (500 MHz, CDCl3) d 2.78 (s, 3H, Thiazole-CH3), 3.33 (s, 1H, Alkyne-H), 7.12e7.15 (m, 2H, AreH), 7.36 (t, J ¼ 7.4 Hz, 1H, AreH), 7.42 (d, J ¼ 8.7 Hz, 2H, AreH), 7.73 (d, J ¼ 7.6 Hz, 2H, AreH), 7.92 (d, J ¼ 8.6 Hz, 2H, AreH), 8.17 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 376.01 (M þ H)þ, 377.99 (M þ Hþ2)þ. 4.2.5. 5-(4-Ethynyl-1-phenyl-1H-pyrazol-3-yl)-2-(4bromophenyl)-4-methyl-1,3-thiazole 5e Yield: 40%; mp: 104e106  C; 1H NMR (500 MHz, CDCl3) d 2.76 (s, 3H, Thiazole-CH3), 3.28 (s, 1H, Alkyne-H), 7.15 (t, J ¼ 8.8 Hz, 2H, AreH), 7.31e7.33 (m, 1H, AreH), 7.54 (d, J ¼ 8.6 Hz, 2H, AreH), 7.71 (d, J ¼ 7.6 Hz, 2H, AreH), 8.07 (d, J ¼ 7.6 Hz, 2H), 8.13 (s, 1H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 419.95 (M þ H)þ, 421.91 (M þ Hþ2)þ. 4.3. General method of 1-benzyl-4-[1-phenyl-3-(4-methyl-2phenyl-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)-4methyl-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 h. After completion of reaction (TLC), the reaction mixture was quenched in water and extracted with ethyl acetate (3  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-

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benzyl-4-[1-phenyl-3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1Hpyrazol-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-5yl)-1H-pyrazol-4-yl]-1H-1,2,3-triazole 7a Yield: 82%; mp: 122e124  C; 1H NMR (500 MHz, CDCl3): d 2.34 (s, 3H, Thiazole CH3), 5.51 (s, 2H, Ar-CH2-N), 7.18e7.23 (m, 3H, AreH), 7.29 (dd, J ¼ 8.4, 6.0 Hz, 3H, AreH), 7.34 (s, 1H, Triazole eH), 7.45 (dd, J ¼ 5.0, 1.8 Hz, 3H, AreH), 7.48e7.51 (m, 2H, AreH), 7.79 (d, J ¼ 7.7 Hz, 2H, AreH), 7.89e7.91 (m, 2H, AreH), 8.56 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 475.1703 (M þ H)þ. 4.3.2. 4-[3-(4-methyl-2-phenyl-1,3-thiazol-5-yl)-1-phenyl-1Hpyrazol-4-yl]-1-[(4-methylphenyl)methyl]-1H-1,2,3-triazole 7b Yield: 72%; mp: 160e162  C; 1H NMR (500 MHz, CDCl3) d 2.35 (s, 3H, Thiazole CH3), 2.55 (s, 3H, Ar-CH3), 5.50 (s, 2H, Ar-CH2-N), 7.21 (d, J ¼ 7.8 Hz, 2H, AreH), 7.26 (d, J ¼ 7.8 Hz, 2H, AreH), 7.31e7.33 (m, 3H, AreH), 7.35 (s, 1H, Triazole eH), 7.42e7.45 (m, 3H, AreH), 7.79 (d, J ¼ 7.7 Hz, 2H, AreH), 7.89e7.91 (m, 2H, AreH), 8.56 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 489.2563 (M þ H)þ. 4.3.3. 1-[(4-fluorophenyl)methyl]-4-[3-(4-methyl-2-phenyl-1,3thiazol-5-yl)-1-phenyl-1H-pyrazol-4-yl]-1H-1,2,3-triazole 7c Yield: 78%; mp: 168e170  C; 1H NMR (500 MHz, CDCl3) d 2.27 (s, 3H, Thiazole CH3), 5.40 (s, 2H, Ar-CH2-N), 6.92 (t, J ¼ 8.6 Hz, 2H, AreH), 7.15e7.11 (m, 3H, AreH, Triazole-H), 7.27 (t, J ¼ 7.4 Hz, 1H, AreH), 7.38 (dd, J ¼ 5.1, 1.8 Hz, 3H, AreH), 7.42 (t, J ¼ 8.0 Hz, 2H, AreH), 7.71 (d, J ¼ 7.8 Hz, 2H, AreH), 7.86e7.81 (m, 2H, AreH), 8.49 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 493.1614 (M þ H)þ.

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4.3.6. 1-Benzyl-4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7f Yield: 74%; mp: 132e134  C; 1H NMR (500 MHz, CDCl3) d 2.32 (s, 3H, Thiazole CH3), 2.41 (s, 3H, Ar-CH3), 5.51 (s, 2H, Ar-CH2-N), 7.19e7.22 (m, 3H, AreH, Triazole H), 7.23e7.26 (m, 3H, AreH), 7.28e7.36 (m, 3H, AreH), 7.47e7.51 (m, 2H, AreH), 7.77e7.80 (m, 4H, AreH), 8.56 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 489.2567 (M þ H)þ. 4.3.7. 4-{3-[4-methyl-2-(4-methylphenyl)-1,3-thiazol-5-yl]-1phenyl-1H-pyrazol-4-yl}-1-[(4-methylphenyl)methyl]-1H-1,2,3triazole 7g Yield: 84%; mp: 158e160  C; 1H NMR (500 MHz, CDCl3) d 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, AreH), 7.19 (s, 1H, Triazole H), 7.26 (m, 3H, AreH), 7.34 (t, J ¼ 7.4 Hz, 1H, AreH), 7.49 (t, J ¼ 7.9 Hz, 2H, AreH), 7.80 (dd, J ¼ 10.2, 8.5 Hz, 4H, AreH), 8.55 (s, 1H, Pyrazole-H); 13 C NMR (125 MHz, CDCl3) d 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 ¼ 503.2012 (M þ H)þ. 4.3.8. 1-[(4-fluorophenyl)methyl]-4-{3-[4-methyl-2-(4methylphenyl)-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H1,2,3-triazole 7h Yield: 72%; mp: 224e226  C; 1H NMR (500 MHz, CDCl3) d 2.33 (s, 3H, Thiazole-CH3), 2.41 (s, 3H, Ar-CH3), 5.47 (s, 2H, Ar-CH2-N), 6.99 (t, J ¼ 8.6 Hz, 2H, AreH), 7.18e7.22 (m, 3H, AreH, Triazole H), 7.26 (d, J ¼ 7.9 Hz, 2H, AreH), 7.34 (t, J ¼ 7.4 Hz, 1H, AreH), 7.49 (t, J ¼ 8.0 Hz, 2H,AreH), 7.78e7.81 (m 4H, AreH), 8.56 (s, 1H, PyrazoleH); 13C NMR (125 MHz, CDCl3) d 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 ¼ 507.1761 (M þ H)þ.

4.3.4. 1-[(4-chlorophenyl)methyl]-4-[3-(4-methyl-2-phenyl-1,3thiazol-5-yl)-1-phenyl-1H-pyrazol-4-yl]-1H-1,2,3-triazole 7d Yield: 78%; mp: 168e170  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s, 3H, Thiazole CH3), 5.48 (s, 2H, Ar-CH2-N), 7.14 (d, J ¼ 8.4 Hz, 2H AreH), 7.22 (s, 1H, Triazole H), 7.28 (m, d, J ¼ 8.4 Hz, 2H, AreH), 7.27 (t, J ¼ 7.4 Hz, 1H, AreH), 7.47e7.43 (m, 3H AreH), 7.52e7.48 (m, 2H AreH), 7.80 (d, J ¼ 7.7 Hz, 2H AreH), 7.91 (dd, J ¼ 7.5, 1.9 Hz, 2H AreH), 8.57 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 509.1319 (M þ H)þ.

4.3.9. 1-[(4-chlorophenyl)methyl]-4-{3-[4-methyl-2-(4methylphenyl)-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H1,2,3-triazole 7i Yield: 76%; mp: 184e168  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s, 3H, Thiazole-CH3), 2.42 (s, 3H, Ar-CH3), 5.47 (s, 2H, Ar-CH2-N), 7.14 (d, J ¼ 8.4 Hz, 2H, AreH), 7.21 (s, 1H, Triazole H), 7.26e7.28 (m, 4H, AreH), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.50 (t, J ¼ 8.0 Hz, 2H, AreH), 7.80 (dd, J ¼ 7.8, 6.5 Hz, 4H, AreH), 8.57 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 523.1471 (M þ H)þ.

4.3.5. 1-[(4-bromophenyl)methyl]-4-[3-(4-methyl-2-phenyl-1,3thiazol-5-yl)-1-phenyl-1H-pyrazol-4-yl]-1H-1,2,3-triazole 7e Yield: 74%; mp: 174e176  C; 1H NMR (500 MHz, CDCl3) d 2.28 (s, 3H, Thiazole CH3), 5.38 (s, 2H, Ar-CH2-N), 7.01 (d, J ¼ 8.3 Hz, 2H, 2H AreH), 7.14 (s, 1H, Triazole H), 7.27 (t, J ¼ 7.4 Hz, 1H, AreH), 7.31e7.49 (m, 7H, AreH), 7.72 (d, J ¼ 7.8 Hz, 2H, AreH), 7.84e7.85 (m, 2H, AreH), 8.49 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 553.080454 (M þ H)þ, 555.0782 (M þ Hþ2) þ.

4.3.10. 1-[(4-bromophenyl)methyl]-4-{3-[4-methyl-2-(4methylphenyl)-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H1,2,3-triazole 7j Yield: 75%; mp: 148e150  C; 1H NMR (500 MHz, CDCl3) d 2.33 (s, 3H), 5.52 (s, 2H), 7.24e7.18 (m, 3H), 7.26 (s, 1H), 7.37e7.29 (m, 4H), 7.42 (d, J ¼ 8.5 Hz, 2H), 7.50 (t, J ¼ 7.9 Hz, 2H), 7.78 (d, J ¼ 7.8 Hz, 2H), 7.84 (d, J ¼ 8.5 Hz, 2H), 8.54 (s, 1H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 567.0955 (M þ H)þ,569.0937 (M þ Hþ2)þ.

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4.3.11. 1-Benzyl-4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7k Yield: 68%; mp: 154e156  C; 1H NMR (500 MHz, CDCl3) d 2.32 (s, 3H, Thiazole CH3), 5.52 (s, 2H, Ar-CH2-N), 7.14 (t, J ¼ 8.6 Hz, 2H, AreH), 7.23e7.19 (m, 3H, AreH, Triazole H), 7.30e7.34 (m, 4H, AreH), 7.49 (t, J ¼ 8.0 Hz, 2H, AreH), 7.78 (d, J ¼ 7.8 Hz, 2H, AreH), 7.88 (dd, J ¼ 8.8, 5.3 Hz, 2H, AreH), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 493.1613 (M þ H)þ. 4.3.12. 4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenylpyrazol-4-yl}-1-[(4-methylphenyl)methyl]-1,2,3-triazole 7l Yield: 70%; mp: 150e152  C; 1H NMR (500 MHz, CDCl3) d 2.29 (s, 3H, Thiazole CH3), 2.33 (s, 3H, Ar-CH3), 5.47 (s, 2H, Ar-CH2-N), 7.11 (s, 4H, AreH), 7.14 (t, J ¼ 8.6 Hz, 2H, AreH), 7.19 (s, 1H, Triazole H), 7.47e7.51 (m, AreH), 7.78 (d, J ¼ 7.4 Hz, 3H, AreH), 7.90 (m, 2H, AreH), 8.54 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 507.1761 (M þ H)þ. 4.3.13. 4-{3-[2-(4-fluorophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenylpyrazol-4-yl}-1-[(4-fluorophenyl)methyl]-1,2,3-triazole 7m Yield: 66%; mp: 158e160  C; 1H NMR (500 MHz, CDCl3) d 2.33 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 7.00(t, J ¼ 8.6 Hz, 2H, AreH), 7.15 (t, J ¼ 8.6 Hz, 2H, AreH), 7.18e7.23 (m, 3H, AreH, Triazole H), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.50 (t, J ¼ 7.9 Hz, 2H, AreH), 7.78 (d, J ¼ 7.7 Hz, 2H, AreH), 7.89 (dd, J ¼ 8.7, 5.3 Hz, 2H, AreH), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 15.37, 52.36, 113.73, 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 ¼ 511.1508 (M þ H)þ. 4.3.14. 1-[(4-chlorophenyl)methyl]-4-{3-[2-(4-fluorophenyl)-4methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3triazole 7n Yield: 74%; mp: 180e182  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s, 3H, Thiazole-CH3), 5.49 (s, 2H, Ar-CH2-N), 7.12e7.18 (m, 4H, AreH), 7.23 (s, 1H, Triazole H), 7.32e7.27 (m, 2H, AreH), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.50 (t, J ¼ 7.9 Hz, 2H, AreH), 7.79 (d, J ¼ 7.8 Hz, 2H, AreH), 7.89 (dd, J ¼ 8.6, 5.3 Hz, 2H, AreH), 8.55 (s, 1H, Pyrazole-H); 13 C NMR (125 MHz, CDCl3) d 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 ¼ 527.1216 (M þ H)þ, 529.1196 (M þ Hþ2)þ. 4.3.15. 1-[(4-bromophenyl)methyl]-4-{3-[2-(4-fluorophenyl)-4methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3triazole 7o Yield: 70%; mp: 148e150  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s, 3H, Thiazole-CH3), 5.46 (s, 2H, Ar-CH2-N), 7.08 (d, J ¼ 8.4 Hz, 2H, AreH), 7.15 (t, J ¼ 8.6 Hz, 2H, AreH), 7.22 (s, 1H, Triazole H), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.44 (d, J ¼ 8.4 Hz, 2H, AreH), 7.50 (t, J ¼ 7.9 Hz, 2H, AreH), 7.78 (d, J ¼ 7.8 Hz, 2H, AreH), 7.89 (dd, J ¼ 8.7, 5.3 Hz, 2H, AreH), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 571.0710 (M þ H)þ, 573.0690 (M þ Hþ2)þ. 4.3.16. 1-Benzyl-4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7p Yield: 70%; mp: 156e158  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s,

3H, Thiazole-CH3), 5.47 (s, 2H, Ar-CH2-N), 7.09 (d, J ¼ 8.3 Hz, 2H, AreH), 7.21 (s, 1H, Triazole H), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.47e7.42 (m, 4H, AreH), 7.50 (t, J ¼ 7.9 Hz, 2H, AreH), 7.79 (d, J ¼ 7.9 Hz, 2H, AreH), 7.85 (d, J ¼ 8.5 Hz, 2H, AreH), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 509.1320 (M þ H)þ. 4.3.17. 4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenyl-1H-pyrazol-4-yl}-1-[(4-methylphenyl)methyl]-1H-1,2,3triazole 7q Yield: 66%; mp: 154e156  C; 1H NMR (500 MHz, CDCl3) d 2.29 (s, 3H, Thiazole-CH3), 2.33 (s, 3H, Ar-CH3), 5.47 (s, 2H, Ar-CH2-N), 7.11 (s, 4H, AreH), 7.18 (s, 1H, Triazole H), 7.34 (t, J ¼ 7.4 Hz, 1H, AreH), 7.43 (d, J ¼ 8.5 Hz, 2H, AreH), 7.49 (t, J ¼ 7.8 Hz, 2H, AreH), 7.78 (d, J ¼ 7.9 Hz, 2H, AreH), 7.85 (d, J ¼ 8.5 Hz, 2H, AreH), 8.53 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 523.1471 (M þ H)þ. 4.3.18. 4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenyl-1H-pyrazol-4-yl}-1-[(4-fluorophenyl)methyl]-1H-1,2,3triazole 7r Yield: 72%; mp: 164  C; 1H NMR (500 MHz, CDCl3) d 2.33 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 7.01 (t, J ¼ 8.6 Hz, 2H, AreH), 7.19 (s, 1H, Triazole H), 7.21 (dd, J ¼ 8.5, 5.2 Hz, 2H, AreH), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.43 (d, J ¼ 8.5 Hz, 2H, AreH), 7.50 (t, J ¼ 7.9 Hz, 2H, AreH), 7.78 (d, J ¼ 7.8 Hz, 2H, AreH), 7.84 (d, J ¼ 8.5 Hz, 2H, AreH), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 527.1216 (M þ H)þ. 4.3.19. 4-{3-[2-(4-chlorophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenyl-1H-pyrazol-4-yl}-1-[(4-chlorophenyl)methyl]-1H-1,2,3triazole 7s Yield: 70%; mp: 166e168  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 7.15 (d, J ¼ 8.4 Hz, 2H, AreH), 7.21 (s, 1H, Triazole H), 7.29 (d, J ¼ 8.4 Hz, 2H, AreH), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.43 (d, J ¼ 8.5 Hz, 2H, AreH), 7.50 (t, J ¼ 7.9 Hz, 2H, AreH), 7.78 (d, J ¼ 7.8 Hz, 2H, AreH), 7.84 (d, J ¼ 8.5 Hz, 2H, AreH), 8.55 (s, 1H,Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 542.0929 (M þ H)þ. 4.3.20. 1-[(4-bromophenyl)methyl]-4-{3-[2-(4-chlorophenyl)-4methyl-1,3-thiazol-5-yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3triazole 7t Yield: 76%; mp: 162e164  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s, 3H, Thiazole CH3), 5.46 (s, 2H, Ar-CH2-N), 7.08 (d, J ¼ 8.3 Hz, 2H, AreH), 7.22 (s, 1H, Triazole H), 7.28 (d, J ¼ 7.4 Hz, 2H, AreH), 7.34 (t, J ¼ 7.4 Hz, 1H, AreH), 7.43 (d, J ¼ 8.4 Hz, 2H, AreH), 7.49 (t, J ¼ 7.9 Hz, 2H, AreH), 7.80 (t, J ¼ 7.9 Hz, 4H, AreH), 8.56 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 587.0420(M þ H)þ, 589.0408 (M þ Hþ2)þ.

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4.3.21. 1-Benzyl-4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5yl]-1-phenyl-1H-pyrazol-4-yl}-1H-1,2,3-triazole 7u Yield: 68%; mp: 216e218  C; 1H NMR (500 MHz, CDCl3) d 2.33 (s, 3H, Thiazole-CH3), 5.51 (s, 2H, Ar-CH2-N), 7.19e7.23 (m, 3H, AreH, Triazole H), 7.29e7.36 (m, 4H, AreH), 7.49 (t, J ¼ 7.9 Hz, 2H, AreH), 7.57 (d, J ¼ 8.5 Hz, 2H, AreH), 7.75e7.79 (m, 4H, AreH), 8.54 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 553.0804 (M þ H)þ, 555.0782 (M þ Hþ2)þ. 4.3.22. 4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenyl-1H-pyrazol-4-yl}-1-[(4-methylphenyl)methyl]-1H-1,2,3triazole 7v Yield: 78%; mp: 182e184  C; 1H NMR (500 MHz, CDCl3) d 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), 7.22e7.26 (m, 4H, AreH), 7.34 (t, J ¼ 7.4 Hz, 1H, AreH), 7.45 (d, J ¼ 8.5 Hz, 2H, AreH), 7.51 (t, J ¼ 7.8 Hz, 2H, AreH), 7.74 (d, J ¼ 7.8 Hz, 2H, AreH), 7.82 (d, J ¼ 8.4 Hz, 2H, AreH), 8.54 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 567.0955 (M þ H)þ, 569.0938 (M þ Hþ2)þ. 4.3.23. 4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenyl-1H-pyrazol-4-yl}-1-[(4-fluorophenyl)methyl]-1H-1,2,3triazole 7w Yield: 66%; mp: 194e196  C; 1H NMR (500 MHz, CDCl3) d 2.33 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 6.98e7.04 (m, 2H, AreH), 7.18e7.22 (m, 3H, AreH, Triazole H), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.50 (t, J ¼ 8.0 Hz, 2H, AreH), 7.61e7.56 (m, 2H, AreH), 7.78 (dd, J ¼ 7.9, 5.6 Hz, 4H, AreH), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 571.0710 (M þ H)þ, 573.0695 (M þ Hþ2)þ. 4.3.24. 4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenyl-1H-pyrazol-4-yl}-1-[(4-chlorophenyl)methyl]-1H-1,2,3triazole 7x Yield: 72%; mp: 170e172  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s, 3H, Thiazole-CH3), 5.48 (s, 2H, Ar-CH2-N), 7.15 (d, J ¼ 8.4 Hz, 2H, AreH), 7.21 (s, 1H, Triazole H), 7.29 (d, J ¼ 8.4 Hz, 2H, AreH), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.48e7.52 (m, 2H, AreH), 7.59 (d, J ¼ 8.5 Hz, 2H, AreH), 7.76e7.80 (m, 4H, AreH), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 587.0421 (M þ H)þ, 589.0410 (M þ Hþ2)þ. 4.3.25. 4-{3-[2-(4-bromophenyl)-4-methyl-1,3-thiazol-5-yl]-1phenyl-1H-pyrazol-4-yl}-1-[(4-bromophenyl)methyl]-1H-1,2,3triazole 7y Yield: 65%; mp: 188e190  C; 1H NMR (500 MHz, CDCl3) d 2.34 (s, 3H, Thiazole-CH3), 5.46 (s, 2H, Ar-CH2-N), 7.08 (d, J ¼ 8.4 Hz, 2H, AreH), 7.21 (s, 1H, Triazole H), 7.35 (t, J ¼ 7.4 Hz, 1H, AreH), 7.42e7.46 (m, 2H, AreH), 7.48e52 (m, 2H, AreH), 7.57e7.62 (m, 2H, AreH), 7.75e7.80 (m, 4H, AreH), 8.55 (s, 1H, Pyrazole-H); 13C NMR (125 MHz, CDCl3) d 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 ¼ 630.9910 (M þ H)þ, 632.9888 (M þ Hþ2)þ, 634.9870 (M þ Hþ4)þ.

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5. Biological activity 5.1. Antibacterial activity The in vitro antibacterial screening of the synthesized pyrazolylthiazolyl-triazole derivatives was done by the well diffusion method [55e57] 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  C over nutrient agar slants throughout the experiment, for antibacterial activity, the cultures incubated for overnight at 37  C in nutrient broth. Five hundred microliters of 24e48 h 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 mL 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 24e48 h at 37  C. 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 pyrazolylthiazolyl-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 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 PotatoDextrose broth. Five hundred microliters of 48e72 h 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 mL 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 48e72 h 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 mg/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 mg/mL. The tubes were inoculated with the test organisms, grown in the Potato-Dextrose broth. The tubes were kept for incubation for 48e72 h 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.

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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 mg/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 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 nheptane 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 14alpha 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. Conflicts of interest There are no conflicts to declare. 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)/2017EMR-1. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.06.074. References [1] https://www.who.int/en/news-room/fact-sheets/detail/antimicrobial-

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