Synthesis, antimicrobial and antioxidant activity of triazole, pyrazole containing thiazole Derivatives and Molecular docking studies on COVID-19

doi:10.21203/rs.3.rs-1522886/v1

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Synthesis, antimicrobial and antioxidant activity of triazole, pyrazole containing thiazole Derivatives and Molecular docking studies on COVID-19

https://doi.org/10.21203/rs.3.rs-1522886/v1

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published 17 Jun, 2023

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An innovative heterocyclic biologically active series of triazole, pyrazole contain 2,4-disubstituted thiazole analogs 12a-l derived from p-hydroxy benzaldehyde and phenylhydrazine in excellent yields and purity. All the synthesized compounds were unambiguously identified on the basis of their spectral data analyses (IR, ¹H-NMR, ¹³C-NMR spectra, and HRMS). The final derivatives were evaluated for their in vitro anti-microbial activity after thorough purification. Among all tested compounds the compound 12e, 12f and 12k possess highest growth inhibitory activity at MIC values of 4.8, 5.1 and 4.0 μg/ml respectively. The antioxidant properties of these compounds demonstrated and revealed that remarkable activity compared to the standard antioxidant by using DPPH free radical-scavenging assay. Moreover, molecular docking studies to evaluate the probable interactions with the catalytic domain of the gram-positive S. Aureus topoisomerase IV enzyme may provide new insights for developing these new hybrids as potential antimicrobial agents. The binding affinities of compounds 12a-l were ranging from -10.0 to -11.0K.cal/mol with topoisomerase IV enzyme and with COVID-19 main protease binding affinities are ranging from-8.2 to-9.3 Kcal/mol. These docking studies reveal that the compounds 12a-l could be the best inhibitors for novel SARS Cov-2 virus and have more future in discovery of potent drug candidate.

3-triazole

Pyrazole

Thiazole

Antimicrobial activity

Antioxidant

Molecular docking

The 1,2,3-triazoles are well-known scaffolds, which can conjugate easily with other heterocyclic moieties. Therefore, these triazole conjugated structural motifs became a commonly increasing pharmaceutical target with a wide array of bioactive molecules with antimicrobial [1,2] anticancer [3] and anti-hypocholesterolemic [4,5] activities. On the other hand, compounds containing pyrazole scaffolds, with π-excessive monocyclic aromatic heterocyclic groups prone to possess an extensive biological activity like antimicrobial [6,7], antimalarial [8,9], anti-inflammatory [10,11], antiviral [12,13], antileshmanial [14-16], antiproliferative[17,18], anticancer activities[19]. Pyrazole-based prominent drugs were available in the market, such as Pyrazofurin, Encorafenib, Celecoxib, Crizotinib, Lonazolac, etc. [20-23]

Thiazole is a specific moiety that plays a vital role in the life of biochemistry as a part of vitamin B1; it plays a crucial role in metabolism. Thiazoles were always included in the preparation of pharmaceutical compounds containing flavourings, perfumes, and agrochemicals. Thiazole scaffold containing compounds vize thiamine (B1) [24], sulfathiazole [25], abafungin (antifungal drug) [25], anti-inflammatory [26], diabetes [27], analgesic [28], cancer [29] possess a wide range of biological activities. Given these significant inhibitory activities, the new triazole, pyrazole chemical entities based 2,4-disubstituted thiazole hybrids are flexible moieties with constant attraction in research prospects and drawing the attention of medicinal chemists due to its ease and synthetic feasibility, numerous biological activities when combined with other heterocyclic moieties [30-34].

Based on the literature, it is evident that the synthesis of biologically active compounds containing triazole, pyrazole, and thiazole moieties has a tremendous attractive individual interest among researchers by their applications in medicinal chemistry, as cited above. However, heterocyclic building blocks consisting of triazole, pyrazole, and thiazole moieties in a group together have been reported in few numbers. Because of the superior biological activity and inspiration from the above-cited findings, we have been directed towards synthesizing single-molecule with three (triazole, pyrazole, and thiazole) nuclei with an anticipated biological activity.

The above findings encouraged the synthesized compound that can be to develop novel antimicrobial, antifungal and antioxidant agents with improved therapeutic efficacy. Herein, we have described the synthesis of a series of 1,2,3-triazole pyrazole embedded substituted thiazole derivative (12a-l).

Synthesis and characterization

The reported compound (Z)-4-(1-(2-phenylhydrazono)ethyl)phenol derivative 3 synthesized in good yield by the nucleophile addition reaction of 1-(4-hydroxyphenyl)ethanone 1 with phenylhydrazine 2 in acetic acid at reflux temperature. Further compound 3 underwent Vilsmeier-Haackcyclization in the presence of DMF and POCl₃ to produce pyrazole-4-carbaldehyde derivative 4 in an excellent yield [35]. Additional, alkylation reaction of the pyrazole-4-carbaldehyde 4 with propargyl bromide 5 in presence of potassium carbonate at room temperature to obtained good yield of 1-phenyl-3-(4-(prop-2-yn-1-yloxy)phenyl)-1H-pyrazole-4-carbaldehyde 6. The targeted 1,4-disubstituted 1,2,3-triazole derivatives 8a–c were synthesized by the cycloaddition of 1-phenyl-3-(4-(prop-2-yn-1-yloxy)phenyl)-1H-pyrazole-4-carbaldehyde 6 with various substituted aromatic azides 7a–c individually by using of click chemistry in the presence of CuSO₄.5H₂O, sodium ascorbate [36]. All the compounds 8a–c was obtained in moderate to be good yields and the structures of all compounds 8a–c were confirmed by their spectroscopic data. In order to construct the thiazole unit, the 1,4-disubstituted 1,2,3-triazole derivatives 8a–c were treated with thiosemicarbazamide 9 individually to give the compounds 10a-c in excellent yield. Finally, The desired compounds 12a-l were prepared in moderate to good yields, via Hantzsch reaction of compounds 10a-c treated with various substituted phenacyl bromides 11a-d in presence of sodium bicarbonate, under reflux conditions [37]. The chemical structures of newly synthesized compounds 12a-l were confirmed by IR, ¹H NMR, ¹³C NMR, and HRMS, spectral data. The synthetic route to the target compounds is given in Scheme 1.

Anti-microbial activity

Antibacterial properties of synthesized target compounds 12a-l were evaluated using two strains of Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) and two strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa). Minimum Inhibitory Concentration (MIC)[38] of the compounds against a bacterial activity was determined using the Broth dilution method and represented in the below Table 1.

For the tested compounds, it is noted that all compounds were found to be very effective, potent, and active against the growth of microbial activity when compared with standard antibacterial agents. Among all 12e, 12f and12k are profound to be better agents with significant growth inhibitory activity against all the tested strains and when compared to the standard references Novobiocin and Ampicillinas shown in Table 1. As can be seen, compounds 12a, 12f and 12k containing para-Bromo attached phenyl group directly bound to the thiazole ring showed the highest inhibitory activity against microbial growth. Also, compounds with p-chloro positions and additional -CH₃ and -OCH₃ substituents in the -phenyl ring (-Ph) showed high activity against microbial survival. These results reveal that compounds containing electron-donating groups promote high antibacterial activity. The only exception is the 12i, which has an aromatic group attached compound, which did not show any significant inhibitory activity towards microbial spp. It can explain that more aromatic groups might cause a steric hindrance with many nonbonding interactions. It is also noticed that the presence of steric bulk groups may reduce the biological inhibitory activity of the compounds.

Table 1 MIC of synthesized 12a-l compounds (μg/ml) against gram-positive and gram-negative bacterial strains

Compound	Gram positive bacteria		Gram negative bacteria
	S. aureus	B. subtilis	P. aeruginosa	E. coli
	MTCC 96	MTCC 441	MTCC 424	MTCC 443
12a	6.7±0.06	6.7±0.06	9.5±0.06	10.0±0.08
12b	7.2±0.05	6.1±0.08	9.8±0.03	10.0±0.08
12c	NA	NA	NA	NA
12d	NA	NA	NA	NA
12e	4.8±0.03	6.2±0.03	9.8±0.03	10.0±0.06
12f	5.1±0.08	6.2±0.03	8.7±0.08	10.0±0.06
12g	8.0±0.03	7.2±0.05	9.8±0.03	10.0±0.06
12h	7.2±0.05	8.0±0.03	9.5±0.06	10.0±0.08
12i	9.8±0.03	7.2±0.05	9.8±0.03	>50
12j	8.7±0.08	8.0±0.03	10.0±0.06	>25
12k	4.1±0.03	6.2±0.03	9.5±0.06	>25
12l	9.5±0.06	6.2±0.03	9.5±0.06	>50
Novobiocin	3.9±0.03	NT	NT	NT
Ampicillin	10.0±0.08	10.0±0.06	10.0±0.08	3.9±0.03

NT- Not tested

The antifungal activities of the synthesized compounds 12a-l were tested against filamentous fungi Aspergillusniger MTCC 404 and Saccharomyces cerevisiae MTCC 1344 yeast cultures. The steak plate method was used to identify the minimum concentration of compound required to inhibit the fungal growth, which was determined on the zone of precise area length (mm) on the YEP agar plate. The experiments were performed in triplicates, represented the data in the form of Mean ± SEM, and shown in the below Table 2.

The results clearly suggested that among all synthesized analogs, 12a, 12f and 12k showed prominent antifungal activity against Aspergillusniger MTCC 404 and compounds 12a and 12f, 12g, 12i and 12k showed potent fungal growth inhibition against Saccharomyces cerevisiae MTCC 1344 are reported in Table 2.

Table 2 Zone of fungal growth inhibition (mm) upon incubation with compound at 10 and 20 µM concentrations after 2 days at 37 ^oC

Compound	*Aspergillusniger*		*Saccharomyces cerevisiae*
Compound	MTCC 404		MTCC 1344
	10 µM	20 µM	10 µM	20 µM
12a	3.0±0.11	5.5±0.05	6.0±0.08	6.5±0.05
12b	2.0±0.57	2.0±0.48	1.5±0.03	2.0±0.57
12c	NT	NT	NT	NT
12d	NT	NT	NT	NT
12e	3.0±0.11	3.0±0.11	1.5±0.14	3.0±0.11
12f	7.3±0.06	7.4±0.05	6.2±0.05	6.8±0.05
12g	3.0±0.11	3.0±0.11	6.0±0.08	6.0±0.08
12h	2.1±0.03	3.0±0.11	2.0±0.00	3.0±0.11
12i	1.5±0.05	2.0±0.00	6.0±0.08	6.2±0.05
12j	2.1±0.03	3.0±0.11	2.0±0.00	2.0±0.00
12k	6.8±0.00	7.0±0.08	6.2±0.05	6.6±0.05
12l	1.5±0.05	2.0±0.06	1.5±0.03	2.0±0.00
Miconazole	8.0±0.11	10.0±0.11	8.0±0.11	9.0±0.00

NT- Not tested

Antioxidant activity

The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical-scavenging assay is usually employed to evaluate the antioxidant ability of newly derived compounds. The present study measured the free radical scavenging activity of compounds by the DPPH assay method. 0.5 mM solution of DPPH in methanol was prepared, and 100 μl of this solution was added to various concentrations of DMSO dissolved compounds ranging from 10 µM to 200 µM. The absorbance was measured at 517 nm upon incubation in the dark at room temperature after 30 minutes [39]. All the tests were performed in triplicate, and the percent antioxidant inhibitory activities were calculated by comparing the values of absorbance of the control and test samples. Dose-responsive curves were plotted against the concentration of compound for percent free radical scavenging activity. The antioxidant activity results are represented in Table 3.

From the results, it is noticed that all synthesized 12a-l analogues were found to be potent in donating hydrogen free radicals except 12i which showed a poor radical scavenging capacity. Compound 12f and 12k proved to be more or comparatively potent than the standard reference ascorbic acidand areshown in Table 3 and figure 1.

Table 3 Percent antioxidant activity of synthesized 12a-l compounds.

Compound	Compound Concentration
	10 µM	50 µM	100 µM	200 µM
12a	39.9	41.6	41.9	55.5
12b	24.09	24.4	25.1	27.3
12c	NT	NT	NT	NT
12d	NT	NT	NT	NT
12e	-5.9	-5.8	14.5	30.9
12f	42.1	47.1	56.1	67.8
12g	22.5	29	33.8	42.2
12h	24.09	24.4	25.1	27.3
12i	29.6	39.2	39.4	40.2
12j	24.4	24.8	26.9	28.1
12k	51.8	58.6	59.2	67
12l	-18.9	-19	-19.9	-16.8
AA	44.1	44.5	45.8	79.3

NT- Not tested; AA- Ascorbic acid (Standard)

3.4 Molecular Docking studies on S.aureus topoisomerase IV enzyme

The molecular docking studies were performed using Vina in PyRx docking tool to predict the protein-ligand interactions at the molecular level. The co-crystal structure of Gram-positive S. aureus topoisomerase IV enzyme of the catalytic domain in complex with novobiocin (PDB ID: 4URN) is used as a target. The crystal structure of 4URN bound with novobiocin as inhibitor defined hydrogen bonding with Lys36, Arg37, Asn49, Asp52, Asp76, Arg79, Arg138 and hydrophobic interaction with Asp35, Lys36 and Ile96. It also showed π-cationic interaction with Lys36 and salt bridge with Arg79, water bridges found with Ser50, Gly78 and Thr168. All the synthesized compounds showed hydrogen bond interactions with the target protein except compound 12l. Amino acid residues Thr34, Glu45, Asn49, Asp52, Glu53, Asn56, Asp76 and Gly80 of 4URN were involved in H-bond interactions similar to that of novobiocin. And amino acid residues Asp35, Lys36, Ile46, Asn49, Asn56, Gly80, Met81, Pro82, Ile96, Gly121, Ala122, Val170, Lys180 and Ala181 were involved in hydrophobic interactions with topoisomerase IV enzyme. The binding affinities of compounds 12a-l were ranging from -10.0 to -11.0 Kcal/mol, values are shown in table 4.

Table 4 Binding affinity of Compounds 12a-l and interacting amino acid residues of topoisomerase IV enzyme (PDB ID: 4URN)

Compound	Binding Affinity (Kcal/mol)	Interacting Amino Acid
Compound	Binding Affinity (Kcal/mol)	H-bond	Hydrophobic
12a	-10.4	Thr34, Asn56, Asp76,	Asp35, Lys36, Ile46, Asn49, Asn56, Gly80, Met81, Pro82, Ile96, Gly121, Ala122, Val170, Lys180, Ala181
12b	-10.6	Asn49, Asp52	Lys36, Glu45, Asn56, Asp76, Arg79, Met81, Pro82, Ile96, Lys180, Ala181
12c	-10.4	Asn49, Asp52, Gly80	Thr34, Lys36, Asn49, Glu53, Met81, Ile96, Arg138, Lys180, Ala181
12d	-10.7	Asn49, Asp52	Lys36, Arg79, Met81, Pro82, Ile96, Arg138, Lys180
12e	-10.6	Asp52, Gly80	Lys36, Asn49, Glu53, Met81, Ile96, Arg138, Lys180, Ala181
12f	-10.3	Asp52	Lys36, Val51, Asp52, Glu53, Arg79, Pro82, Ile96, Thr177, Lys180, Ala181, Leu201
12g	-10.1	Glu53, Gly80	Thr34, Asp35, Lys36, Glu45, Asp52, Glu53, Met81, Pro82, Ile96, Arg138, Lys180, Ala181
12h	-11.0	Asp52, Glu53, Gly80	Lys36, Asn49, Glu53, Met81, Pro82, Ile96, Arg138, Lys180, Ala181
12i	-10.6	Asn49, Asp52, Gly80	Thr34, Lys36, Asn49, Glu53, Gly80, Met81, Pro82, Ile96, Ala122, Arg138, Ile178, Lys180, Ala181
12j	-10.7	Glu45, Asn49, Asn56	Thr34, Lys36, Ile46, Asn49, Asp52, Glu53, Met81, Pro82, Ile96, Phe97, Ala122, Val170, Ile178, Lys180, Ala181
12k	-10.6	Asp52, Gly80	Thr34, Lys36, Asn49, Glu53, Met81, Pro82, Ile96, Arg138, Lys180, Ala181
12l	-10.0	--	Lys36, Ile46, Glu53, Tyr58, Arg79, Met81, Pro82, Ile96, Phe97, Ala122, Arg138, Val170, Lys180, Ala181

Compound 12h was shown highest docking score about -11.0 Kcal/mol. It was involved in H-bond interactions with Asp52, Glu53 and Gly80 of 4URN and hydrophobic interactions with Lys36, Asn49, Glu53, Met81, Pro82, Ile96, Arg138, Lys180 and Ala181 of the same shown as (Fig. 2, 3, 4). The standard compound ascorbic acid involved in only H-bond interactions with Asn49, Glu53, Asp76 and Glu80 of topoisomerase and hydrophobic interactions were absent. These docking results support those compounds 12a-l are the potent inhibitors of S. aureus topoisomerase IV enzyme.

Molecular Docking studies on COVID-19 main protease

The crystal structure of COVID-19 main protease in complex with an N3 inhibitor was used as SARS CoV-2 target. In the crystal structure of 6LU7 in complex with N3 inhibitor defined H-bond interactions with Phe140, Gly143, His164, Glu166, Gln189 and Thr190 and also hydrophobic interactions with His41, Met49, Met165, Leu167, Pro168, His172 and Ala191. All the synthesized ligand molecules were shown H-bond interactions similar to N3 inhibitor. Any of the amino acid residues Phe140, Leu141, Gly143, His163, His164, Met165, Glu166, Leu167, Arg188, Gln189 and Thr190 were involved in H-bond interactions with ligands. The hydrophobic interactions were involved by Thr24, Leu27, His41, Met49, Phe140, Ser144, Cys145, Met165, Glu166, Pro168, Gln189, Thr190 and Ala191 of 6LU7. The binding affinities of compounds 12a-l were ranging from -8.2 to -9.3 Kcal/mol, values are shown table 5.

Table 5 Docking scores of compounds 10a -10k and interacting amino acids of COVID-19 main protease (PDB ID: 6LU7)

Compound	Binding Affinity (Kcal/mol)	Interacting Amino Acid
Compound	Binding Affinity (Kcal/mol)	H-bond	Hydrophobic
12a	-9.1	Phe140, Leu141, His163, Met165	His41, Ser144, Cys145, Glu166
12b	-8.8	Leu141, Gly143, His163	Met49, Phe140, Ser144, Cys145, Glu166, Pro168, Thr190, Ala191
12c	-8.3	Leu141, Gln189	Thr26, Leu27, His41, Met49, Ser144, Cys145, Pro168
12d	-9.2	Leu141, Glu166, Gln189	His41, Ser144, Cys145, Met165, Glu166
12e	-9.3	Phe140, Leu141, His163, Met165	His41, Phe140, Gly143, Cys145, Met165, Glu166, Gln189
12f	-9.2	Phe140, Leu141, His163, Met165, Arg188	His41, Phe140, Gly143, Cys145, Met165, Glu166, Gln189
12g	-8.8	Glu166, Leu167	Met49, Asn142, Cys145, Met165, Glu166, Gln189, Thr190, Ala191,
12h	-8.7	Glu166, Leu167, Thr190	Leu27, His41, Met49, Cys145, Met165, Glu166, Pro168, Gln189, Thr190, Ala191
12i	-8.7	Asn142, His164	Thr26, His41, Met49, Phe140, Gly143, Cys145, Met165, Glu166, Pro168
12j	-8.3	Asn142, Thr190	His41, Cys145, Met165, Glu166, Leu167, Pro168, Gln189
12k	-9.1	Glu166. Leu167	Leu27, His41, Met49, Cys145, Met165, Glu166, Thr190, Ala191
12l	-8.2	Asn142, Thr190	Cys145, Met165, Glu166, Pro168

Compound 12e was shown highest docking score about -9.3 Kcal/mol. It was involved in H-bond interactions with amino acid residues Phe140, Leu141, His163 and Met165 of 6LU7 and hydrophobic interactions with amino acid residues His41, Phe140, Gly143, Cys145, Met165, Glu166 and Gln189 of main protease (Fig. 5, 6, 7). The docking study reveals that all the newly synthesized molecules could be the inhibitors of COVID-19 main protease similar to N3 inhibitor.

All reactions were performed in oven-dried glassware. The melting points of the compounds 12a-l were measured in open capillaries and are uncorrected. The NMR spectral analysis was done on a Bruker AV 400 MHz instrument using deuterated samples with TMS as an internal standard. The chemical shifts were measured in ppm against internal TMS. The IR spectral analysis was done on a Perkin–Elmer 337 spectrophotometer for solid samples in KBr pellets. On Merck silica gel 60 F254 plates, the thin layer chromatography (TLC) was carried out, and the spots were visualized with UV light at 254 nm of wavelength or by staining with aqueous basic potassium permanganate. Flash column chromatography was carried out on a Merck silica gel 60A0 (100–200 mesh). We used commercially available reagents as supplied, and some of them were distilled if required.

2.6. Chemistry Gelation procedure

Synthesis of 3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4, C₁₆H₁₂N₂O₂)

The starting compound 3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde was synthesized permitting to our earlier reported methods. Briefly, 4-Hydroxy acetophenones (1.0 mol) (1) treated with substituted phenyl hydrazine (1.2 mol) (2) to yield corresponding hydrazones(3), followed by the Vilsmeier-Haack cyclization in the presence of DMF/POCl₃ to give desired compound in 85% yield, which is agreement with previous reported data.

Synthesis of 1-phenyl-3-(4-(prop-2-yn-1-yloxy)-phenyl)-1H-pyrazole-4-carbaldehyde (5, C₁₉H₁₄N₂O₂)

The compound (6), (1.510g, 1.0 mol) was prepared by the propargylation of compound 3-(3-hydroxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4), (1.321g, 1.0 mol), using the propargyl bromide, (0.515g, 1.5 mol) in DMF (30 mL), under nitrogen condition for 3 h at room temperature. After completion (monitored by TLC), the reaction mixture was extracted using ice-cold water (3 x 50 mL) and DCM (2 x 50 mL). Then the combined organic layer was dried with anhydrous sodium sulphate, concentrated in vacuo, and purified by silica gel column chromatography using 20% ethyl acetate in hexane as an eluent to afford 1-phenyl-3-(4-(prop-2-yn-1-yloxy)phenyl)-1H-pyrazole-4-carbaldehyde (5), as light-yellow solid in 83% yield. Spectral data of isolated compound 6 is in agreement with the reported data. Yield 83%; m.p.:104-106 ^oC;¹H NMR(400MHz, CDCl₃) δ= 10.1 (1H), 7.81 (d, 2H, J = 7.78Hz), 7.91 (t , 1H, J = 7.05 Hz), 7.46-7.43 (m, 3H), 7.40-7.42 (m, 1H), 7.10-7.08 (m, 1H), 4.79 (d,J = 5.5 Hz, 2H) ppm; ¹³CNMR(100 MHz, CDCl₃): δ =197.1, 151.2, 145.2, 138.2, 136.6, 130.4, 128.2, 125.7, 124.3, 121.7, 124.0, 119.0, 111.5, 111.5, 45.5, 26.9, 25.9 ppm; IR: v = 3295, 1698 cm^-1; ESI-MS: m/z calcd for C₁₉H₁₄N₂O₂302; found 303 [M+1]⁺.

General procedure for the Synthesis of substituted 1-phenyl-3-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-pyrazole-4-carbaldehyde (7 a-c)

To the compound1-phenyl-3-(4-(prop-2-yn-1-yloxy)-phenyl)-1H-pyrazole-4-carbaldehyde (5) (0.1 eq) in DMF was added aryl azides (6 a-c) (0.1 eq), in the presence of 10 mol% CuSO₄.5H2O, sodium ascorbate and the reaction mixture was allowed to react at room temperature for 3-4 h. The reaction condition was monitored by TLC. After completion, added the ice-cold water and extracted with ethyl acetate, and the combined organic layers were removed under reduced pressure and purified by column chromatograpy (eluent; ethyl acetate: hexane 30:70) to get pure compounds 7 a-c with a yield of 80-85%.

1-phenyl-3-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-pyrazole-4-carbaldehyde (7a, C₂₅H₁₉N₅O₂)

White solid; yield 80%; m.p.:144-146 °C; ¹H NMR (400 MHz, CDCl₃): δ = 9.98 (s, 1H), 8.45 (s, 1H), 7.76 (d, J = 4.7 Hz, 2H), 7.72 (d, J = 7.6 Hz, 3H), 7.50 – 7.40 (m, 7H), 7.31 (d, J = 6.9 Hz, 1H), 7.12 (s, 2H), 5.30 (s, 2H) ppm;¹³C NMR (100 MHz, CDCl₃): δ = 184.0, 138.5, 138.0, 136.6, 135.5, 130.3, 129.3, 128.9, 128.6, 128.0, 126.9, 123.6, 119.8, 118.7, 63.2 ppm; IR: v = 3452, 2929, 1730, 1602, 1592, 1450, 1235, 1045 cm^-1; Anal.Calcd. ForC₂₅H₁₉N₅O₂: C, 71.25; H, 4.54; N, 16.62; Found: C, 71.24; H, 4.52; N, 16.64. ESI-MS: m/z calcd forC₂₅H₁₉N₅O₂421; found 422 [M+1]⁺.

3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (7b, C₂₅H₁₈ClN₅O₂)

White solid; yield 80%; m.p.: 142-144; ¹H NMR (400 MHz, CDCl₃): δ = 9.99 (s, 1H), 8.46 (s, 1H), 7.78 (s, 2H), 7.72 (d, J = 7.8 Hz, 2H), 7.63 (d, J = 7.3 Hz, 1H), 7.36-7.38(m, 7H), 7.11 (s, 2H), 5.30 (s, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ =185.0, 139.0, 136.0, 131.4, 131.3, 131.0, 130.4, 129.7, 129.0, 127.9, 124.7, 121.1, 119.7, 118.8, 115.0, 63.0 ppm; IR: v = 3451, 2927, 1732, 1602, 1591, 1453, 1236, 1044 cm^-1; Anal.Calcd. ForC₂₅H₁₈N₅O₂Cl: C, 65.86; H, 3.98; N, 7.78; Found: C, 65.87; H, 3.99; N, 7.76. ESI-MS: m/z calcd forC₂₅H₁₈N₅O₂Cl 455; found 456 [M+1]⁺.

3-(4-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (7c, C₂₇H₂₃N₅O₂)

Light white solid; yield 80%; m.p.:148-150 °C; ¹H NMR (400 MHz, CDCl₃): δ = 10.08 (s, 1H), 8.55 (s, 1H), 7.88 – 7.80 (m, 5H), 7.62 (s, 1H), 7.54 (t, J = 7.7 Hz, 3H), 7.42 (t, J = 6.4 Hz, 1H), 7.31 (d, J = 3.8 Hz, 1H), 7.22 (s, 2H), 5.37 (s, 2H), 2.38 (s, 3H), 2.34 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ= 185.0, 159.1, 139.0, 138.4, 137.7, 131.3, 130.6, 130.3, 129.6, 127.9, 124.5, 122.3, 121.8, 119.7, 117.9, 115.0, 62.1, 19.9, 19.4 ppm; IR: v = 3443, 2927, 1733, 1600, 1583, 1451, 1237, 1040 cm^-1; Anal.Calcd. ForC₂₇H₂₃N₅O₂: C, 76.94; H, 5.50; N, 9.97; Found: C, 76.93; H, 5.51; N, 9.96.ESI-MS: m/z calcd forC₂₇H₂₃N₅O₂449; found 450 [M+1]⁺.

General procedure for the Synthesis of 2-((1-phenyl-3-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-pyrazol-4-yl)methylene)hydrazinecarbothioamide (8 a-c)

To a solution of substituted compound, 7a-c (1.0 mmol) and ethanol (25 mL) was added thiosemicarbazamide (1.0 mmol), the reaction mixture was refluxed for 4 hours. Progress of the reaction was monitored by TLC. After completion, EtOH was removed in vacuum. The crude product was purified by silica gel column chromatography in the ratio of ethyl acetate: hexane (25:75) to afford the pure compound 8 a-c.

General procedure for the Synthesis of 4-phenyl-2-(2-((1-phenyl-3-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)-ph-enyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10 a-l)

To a solution of substituted compounds 8 a-c (1.0 eq) and acetonitrile (25 mL) was added phenacyl bromide 9 a-d (1.0 eq) in the presence of sodium bicarbonate (0.5 eq), refluxed for 1-2 hours. After completion (using TLC), acetonitrile was removed in vacuo. The crude product was purified by silica gel column chromatography in the ratio of ethyl acetate: hexane (30:70) to obtain the pure compound 10 a-l in good yields.

4-(4-bromophenyl)-2-(2-((1-phenyl-3-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10a, C₃₄H₂₅BrN₈OS)

White solid; yield 86%; m.p.: 184-186 ^oC; ¹H NMR (400 MHz, CDCl₃): δ= ppm 8.25 (s, 1H), 8.09 (s, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.75 (d, J = 7.7 Hz, 2H), 7.70 (s, 1H), 7.60 (dd, J = 8.1, 5.4 Hz, 4H), 7.52 (d, J = 5.9 Hz, 2H), 7.49 – 7.42 (m, 6H), 7.34 (d, J = 7.4 Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.83 (s, 1H), 5.37 (s, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ= 175.4, 169.0, 158.3, 149.4, 139.5, 135.6, 131.7, 131.6, 130.9, 130.0, 129.8, 129.5, 129.0, 128.8, 127.5, 127.2, 127.0, 126.4, 120.94, 120.6, 119.2, 118.5, 115.1, 114.7, 103.7, 62.3 ppm; IR: v = 3668, 3451, 2929, 1692, 1593, 1451, 1237, 1045 cm^-1; Anal.Calcd. for C₃₄H₂₅N₈OSBr: C, 60.63; H, 3.74; N, 16.64; Found: C, 60.61; H, 3.70; N, 16.65.ESI-MS: m/z calcd for C₃₄H₂₅N₈OSBr671.7; found 672.85 [M+1]⁺.

4-(4-chlorophenyl)-2-(2-((1-phenyl-3-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10b, C₃₄H₂₅ClN₈OS)

White solid; yield 82%; m.p.: 188-190 ^oC; ¹H NMR (400 MHz, CDCl₃): δ = 8.35 (d, J = 5.2 Hz, 1H), 8.09 (s, 1H), 7.94 (s, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.75 (d, J = 6.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 3H), 7.54 – 7.47 (m, 8H), 7.35 (d, J = 6.2 Hz, 2H), 7.14 (d, J = 7.7 Hz, 2H), 6.81 (s, 1H), 5.38 (s, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃):δ = 168.3, 155.5, 145.1, 141.7, 138.5, 137.8, 130.9, 130.1, 129.5, 129.0, 128.8, 127.2, 127.1, 125.6, 122.4, 120.8, 119.3, 118.5, 114.9, 105.9, 103.4, 95.0, 62.0 ppm; IR: v = 3668, 3453, 2929, 1692, 1602, 1593, 1451, 1237, 1045 cm^-1; Anal.Calcd. forC₃₄H₂₅ClN₈OS: C, 64.91; H, 4.01; N, 17.81; Found: C, 64.90; H, 4.00; N, 17.82.ESI-MS: m/z calcd for C₃₄H₂₅ClN₈OS 628.8; found 629.0952 [M+1]⁺.

4-(4-methoxyphenyl)-2-(2-((1-phenyl-3-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)meth-oxy)-phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10c, C₃₅H₂₈N₈O₂S)

White solid; yield: 84%; m.p.: 182-184 ^oC; ¹H NMR (400 MHz, CDCl₃):δ = 8.90 (s, 1H), 8.55 – 8.52 (m, 1H), 8.12 – 8.10 (m, 1H), 7.83-7.80 (m, 4H), 7.73-7.68 (m, 7H), 7.49-7.46 (m, 3H), 7.15 (dd, J = 5.6&3.6 Hz, 2H), 7.05 – 6.99 (m, 4), 5.38 (s, 2H), 3.91 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ = 168.8, 159.0, 154.2, 151.2, 144.7, 133.3, 130.3, 130.1, 129.8, 129.6, 129.5, 128.9, 128.6, 127.9, 127.3, 127.0, 121.0, 121.0, 120.6, 119.6, 119.4, 119.2, 115.0, 114.9, 114.7, 114.2, 62.0, 55.3 ppm; IR: v = 3673, 3456, 2926, 1698, 1602, 1508, 1449, 1248, 1046 cm^-1; Anal.Calcd. forC₃₅H₂₈N₈O₂S: C, 67.29; H, 4.52; N, 17.94; Found: C, 67.30; H, 4.50; N, 17.93.ESI-MS: m/z calcd for C₃₅H₂₈N₈O₂S 624; found 625.0 [M+1]⁺.

4-phenyl-2-(2-((1-phenyl-3-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10d, C₃₄H₂₆N₈OS)

White solid; yield 80%; m.p.: 176-178 ^oC; ¹H NMR (400 MHz, CDCl₃): δ = 8.23 (s, 1H), 8.08 (s, 1H), 7.79 – 7.61 (m, 7H), 7.52 – 7.48 (m, 8H) 7.42 – 7.36 (m, 4H), 7.33 (d, J = 7.4 Hz, 1H), 7.09 (s, 1H), 6.83 (s, 1H), 5.37 (s, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ = 169.0, 158.4, 151.6, 144.8, 139.4, 136.9, 135.3, 133.4, 131.7, 130.0, 129.8, 129.5, 128.9, 127.5, 127.0, 126.3, 125.6, 121.0, 120.6, 119.3, 114.9, 103.8, 62.1 ppm; IR: v = 367, 3444, 2926, 1698, 1602, 1508, 1449, 1248, 1037 cm^-1; Anal.Calcd. forC₃₄H₂₆N₈OS: C, 68.67; H, 4.41; N, 18.84; Found: C, 68.65; H, 4.40; N, 18.83.ESI-MS: m/z calcd for C₃₄H₂₆N₈OS 594.7; found 595.1578 [M+1]⁺.

4-(4-chlorophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy) phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10e, C₃₄H₂₄Cl₂N₈OS)

White solid; yield: 84%; m.p.: 196-198 ^oC; ¹H NMR (400 MHz, CDCl₃):δ= 8.26 (s, 1H), 8.08 (s, 1H), 7.81 – 7.77 (m, 3H), 7.73 (s, 1H), 7.66 (t, J = 7.5 Hz, 4H), 7.59 (d, J = 8.4 Hz, 2H), 7.50 – 7.43 (m, 5H), 7.30 – 7.26 (m, 3H), 7.08 (d, J = 8.4 Hz, 1H), 6.82 (s, 1H), 5.36 (s, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ= 176.3, 163.7, 158.5, 152.6, 151.9, 144.7, 140.0, 136.9, 134.5, 130.8, 130.3, 130.1, 130.0, 129.8, 129.5, 129.4, 129.1, 129.1, 128.9, 128.7, 127.3, 127.1, 121.0, 120.9, 120.6, 119.7, 119.3, 119.2, 115.1, 114.9, 109.9, 62.0 ppm; IR: v = 3673, 3437, 2932, 1692, 1581, 1495, 1235, 1045 cm^-1; Anal.Calcd. forC₃₄H₂₄N₈OSCl₂: C, 61.54; H, 3.65; N, 16.89; Found: C, 61.53; H, 3.63; N, 16.88.ESI-MS: m/z calcd for C₃₄H₂₄N₈OSCl₂662.6; found 663.0325 [M+1]⁺.

4-(4-bromophenyl)-2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10f, C₃₄H₂₄BrClN₈OS)

White solid; yield 83%; m.p.: 192-194 ^oC; ¹H NMR (400 MHz, CDCl₃):δ = 8.29 (d, J = 8.5 Hz, 1H), 8.08 (s, 1H), 7.80 (dd, J = 4.2, 1.7 Hz, 2H), 7.78 (d, J = 1.7 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 1.9 Hz, 1H), 7.64 (d, J = 4.1 Hz, 2H), 7.61 (dd, J = 5.8&3.8 Hz, 2H), 7.55 – 7.52 (m, 1H), 7.49 – 7.45 (m, 6H), 7.36 (dd, J = 4.8&3.1 Hz, 1H), 7.10 (d, J = 6.4 Hz, 1H), 6.82 (s, 1H), 5.58 (s, 1H), 5.36 (s, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ =173.5, 168.9, 158.3, 145.1, 144.0, 136.3, 136.0, 135.6, 131.8, 130.9, 130.1, 130.0, 129.5, 129.2, 129.0, 128.7, 127.5, 127.0, 126.5, 125.6, 124.2, 123.2, 123.0, 120.8, 119.3, 118.5, 116.4, 114.9, 103.6, 62.0 ppm; IR: v = 3664, 3452, 2932, 1690, 1602, 1599, 1451, 1246, 1033 cm^-1; Anal.Calcd. forC₃₄H₂₄N₈OSBrCl: C, 57.68; H, 3.42; N, 15.83; Found: C, 57.67; H, 3.40; N, 15.82.ESI-MS: m/z calcd for C₃₄H₂₄N₈OSBrCl 708.1; found 709.10 [M+1]⁺.

2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole (10g, C₃₅H₂₇ClN8O₂S)

White solid; yield 82%; m.p.: 186-188 ^oC; ¹H NMR (400 MHz, CDCl₃): δ = 8.89 (s, 1H), 8.54 – 8.52 (m, 1H), 8.11 – 8.10 (m, 1H), 7.83-7.79 (m, 4H), 7.73 (m, 6H), 7.49-7.46 (m, 3H), 7.15 (dd, J = 5.6&3.6 Hz, 2H), 7.04 – 6.96 (m, 4), 5.39 (s, 2H), 3.92 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ =171.4, 163.1, 160.2, 156.4, 148.7, 140.0, 130.9, 129.8, 129.6, 129.5, 129.0, 127.2, 126.9, 126.8, 125.7, 124.8, 120.8, 119.3, 118.5, 116.2, 115.0, 114.8, 114.2, 113.9, 111.0, 61.9, 55.3 ppm; IR: v = 3664, 3452, 2932, 1690, 1599, 1503, 1451, 1246, 1033 cm^-1; Anal.Calcd. for C₃₅H₂₇N₈O₂SCl: C, 63.77; H, 4.13; N, 17.00; Found: C, 63.76; H, 4.14; N, 17.01.ESI-MS: m/z calcd for C₃₅H₂₇N₈O₂SCl 658.05; found 659.0424 [M+1]⁺.

2-(2-((3-(4-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-phenylthiazole (10h, C₃₄H₂₅ClN₈OS)

Yield: 85%,white solid; m.p.: 180-182 ^oC; ¹H NMR (400 MHz, CDCl₃): δ = 8.29 (s, 1H), 8.08 (s, 1H), 7.82 – 7.76 (m, 6H), 7.68 – 7.65 (m, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.53 – 7.42 (m, 5H), 7.39 – 7.28 (m, 4H), 7.10 (d, J = 8.8 Hz, 2H), 6.86 (s, 1H), 5.38 (d, J = 6.5 Hz, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ= 169.0, 158.3, 151.7, 145.3, 139.6, 137.8, 135.6, 134.4, 130.9, 129.9, 129.5, 129.0, 128.7, 127.9, 127.0, 126.4, 125.9, 120.8, 119.2, 118.5, 116.6, 114.9, 103.3, 62.0 ppm; IR: v = 3673, 3437, 2932, 1581, 1495, 1446, 1235, 1045 cm^-1; Anal.Calcd. forC₃₄H₂₅N₈OSCl: C, 64.91; H, 4.01; N, 17.81; Found: C, 64.90; H, 4.00; N, 17.82.ESI-MS: m/z calcd for C₃₄H₂₅N₈OSCl 628.2; found 629.1371 [M+1]⁺

2-(2-((3-(4-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)thiazole (10i, C₃₇H₃₂N₈O₂S)

White solid; yield 82%; m.p.: 170-172 ^oC; ¹H NMR (400 MHz, CDCl₃): δ= 8.31 (s, 1H), 8.02 (d, J = 5.0 Hz, 1H), 7.80 (t, J = 7.4 Hz, 3H), 7.74 – 7.68 (m, 3H), 7.61 (s, 2H), 7.56 – 7.46 (m, 4H), 7.43 (s, 1H), 7.32 (dd, J = 14.2&7.7 Hz, 3H), 7.12 (d, J = 8.1 Hz, 2H), 6.84 (s, 1H), 5.37 (s, 2H), 4.89 (s, 1H), 2.33 (s, 3H), 2.31 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃):δ= 164.4, 161.9, 158.4, 151.8, 142.8, 139.5, 138.4, 137.7, 134.8, 133.5, 130.9, 130.6, 130.0, 129.5, 128.8, 127.2, 127.0, 121.9, 119.2, 117.9, 114.9, 61.6, 55.8, 19.9, 19.5 ppm; IR: v = 367, 3436, 2926, 1691, 1602, 1509, 1247, 1035 cm^-1; Anal.Calcd. forb C₃₇H₃₂N₈O₂S: C, 68.08; H, 4.94; N, 17.17; Found: C, 68.05; H, 4.92; N, 17.15.ESI-MS: m/z calcd for C₃₇H₃₂N₈O₂S 652.2; found 653.1621 [M+1]⁺.

4-(4-chlorophenyl)-2-(2-((3-(4-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10j, C₃₆H₂₉ClN₈OS)

White solid; yield 80%; m.p.: 174-176 ^oC; ¹H NMR (400 MHz, CDCl₃): δ= 8.32 (s, 1H), 8.05 (d, J = 5.1 Hz, 1H), 7.79 (t, J = 7.6 Hz, 3H), 7.74 – 7.68 (m, 3H), 7.62 (s, 2H), 7.55 – 7.47 (m, 4H), 7.42 (s, 1H), 7.33 (dd, J = 14.5&7.6 Hz, 3H), 7.12 (d, J = 8.1 Hz, 2H), 6.84 (s, 1H), 5.37 (s, 2H), 2.34 (s, 3H), 2.32 (s, 3H )ppm; ¹³C NMR (100 MHz, CDCl₃):δ= 161.9, 158.4, 151.8, 139.5, 138.4, 137.7, 134.8, 133.5, 130.9, 130.6, 130.0, 129.5, 128.8, 128.8, 127.7, 127.2, 127.0, 126.3, 125.5, 121.7, 119.2, 117.9, 114.9, 61.6, 19.9, 19.5 ppm. IR: v = 3435, 3074, 2928, 1721, 1602, 1578, 1510, 1448, 1236, 1048 cm^-1; Anal.Calcd. forC₃₆H₂₉N₈OSCl: C, 65.79; H, 4.45; N, 17.05; Found: C, 65.78; H, 4.16; N, 17.04.ESI-MS: m/z calcd for C₃₆H₂₉N₈OSCl 656.1; found 657.1523 [M+1]⁺.

4-(4-bromophenyl)-2-(2-((3-(4-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazole (10k, C₃₆H₂₉BrN₈OS)

White solid; yield 84%; m.p.: 164-166 ^oC; ¹H NMR (400 MHz, CDCl₃):δ= 8.34 (s, 1H), 8.04 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.69 – 7.61 (m, 5H), 7.53 (s, 1H), 7.49 (dd, J = 7.8&5.3 Hz, 5H), 7.43 (d, J = 5.9 Hz, 2H), 7.34 (s, 1H), 7.14 (d, J = 8.7 Hz, 2H), 6.84 (s, 1H), 5.30 – 5.29 (m, 2H), 2.35 (d, J = 4.2 Hz, 3H), 2.32 (d, J = 2.9 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ= 161.9, 158.4, 151.8, 146.8, 138.4, 137.7, 134.8, 133.5, 130.9, 130.6, 130.0, 128.8, 127.2, 127.0, 125.5, 121.7, 121.1, 119.2, 117.9, 114.9, 61.6, 19.9, 19.5 ppm; IR: v= 3535, 3435, 2928, 1578, 1448, 1236, 1048 cm^-1; Anal.Calcd. forC₃₆H₂₉N₈OSBr: C, 61.63; H, 4.17; N, 15.97; Found: C, 61.62; H, 4.18; N, 15.98.ESI-MS: m/z calcd for C₃₆H₂₉N₈OSBr 700.0; found 701.0441 [M+1]⁺.

2-(2-((3-(4-((1-(3,4-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-phenylthiazole (10l, C₃₆H₃₀N₈OS)

White solid; yield 82%; m.p.: 168-170 ^oC; ¹H NMR (400 MHz, CDCl₃): δ = 8.32 (s, 1H), 8.03 (s, 1H), 7.79 (d, J = 7.6 Hz, 2H), 7.75 (d, J = 8.1 Hz, 2H), 7.53 (s, 3H), 7.48 (d, J = 8.2 Hz, 3H), 7.43 (d, J = 8.5 Hz, 3H), 7.36 (s, 1H), 7.31 (d, J = 6.8 Hz, 2H), 7.15 – 7.11 (m, 3H), 6.81 (s, 1H), 5.37 (d, J = 5.0 Hz, 2H), 2.34 (s, 3H), 2.32 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ= 170.8, 162.0, 153.4, 148.1, 145.4, 138.4, 137.7, 134.9, 134.6, 133.7, 133.3, 130.6, 130.3, 130.1, 129.7, 129.6, 129.4, 127.6, 127.2, 124.4, 121.8, 119.6, 119.3, 117.9, 115.1, 114.9, 114.3, 114.2, 62.1, 19.9, 19.5 ppm; IR: v= 3667, 3494, 3121, 2926, 1691, 1602, 1509, 1452, 1247, 1035 cm^-1; Anal.Calcd. for C₃₆H₃₀N₈OS: C, 69.43; H, 4.86; N, 17.99; Found: C, 69.43; H, 4.84; N, 17.98. ESI-MS: m/z calcd for C₃₆H₃₀N₈OS 621.7; found 622.85 [M+1]⁺.

Protocol for the evaluation of Antimicrobial Activity

All types of microbial cultures received were grown in Muller-Hinton Agar (20 ml/plate) streak plates at 37 degrees C for 24 hrs in sterile conditions were treated as stock cultures. The loop full of grown cultures were inoculated into sterile autoclaved 10 mL Luria-Bertani Broth, Miller (LB) broth medium (containing Tryptone 10 g/L, Yeast extract 5 g/L, Sodium chloride 10 g/L, pH 7.5) and kept aside in incubator shaker for 12 hrs at 37 ◦C with continuous shaking at 220 rpm for growth. After exact 12 hrs, the growth was observed by forming turbidity in cultures and it was confirmed by comparing with control which is not inoculated. Then 1% grown culture was reinoculated into fresh new LB broth and incubated in the same conditions as above for another 3.5 to 4 hrsupto reaching log phase. The culture grows and enters into log phase which is very active to participate in any reactions was confirmed by an absorbance of OD 0.4 to 0.6 at 600 nM. Therefore, it can be recommended for testing antibacterial activity.

Minimum Inhibitory Concentration (MIC) determination using Broth method

Luria-Bertani Broth, Miller (LB broth) a potent microbiological growth medium was prepared and sterilized in autoclave under aseptic conditions, 121 °C /15 Lbs pressure for 30 minutes and used for antibiotic susceptibility testing. Microbial strains to be tested were grown at 37 ◦C for 12 h with continuous shaking for 220 rpm and kept constant at lag phase by storing at 4 degrees C. The compounds synthesized to be tested for antimicrobial activities were dissolved in DMSO to produce a concentration of 1 mg/ml are treated as a stock solution. Subsequently different concentration of compound ranging from minimum to maximum was tested against bacterial culture growth to determine its MIC value. Antibacterial activity of the compounds was determined by treating various concentrations of compounds with 1ul/ml log phase cultures upon incubation for 12 hrs at 37 degrees C with continuous shaking at 220 rpm. Ampicillin a standard drug (1%stock concentration) is used as positive control for the antibacterial activity and the blank was prepared by adding 1ul/ml culture to the broth and it is maintained as a negative control. Each tube contains 2ml of fresh LB broth and 2ul of log phase bacterial culture constantly with varying concentrations of compounds to check its MIC. All the tests were performed in triplicate to validate the observed results (Mean±SEM) by measuring the variability of data.

Where X_i-X_nis the number of data point values in triplicates

Protocol for Antifungal activity determination and methodology

The antifungal activities of the synthesized compounds were tested against filamentous fungi Aspergillus niger MTCC 404 and Saccharomyces cerevisiae MTCC 1344 yeast cultures. They were obtained from the Culture Collection Division at Department of Microbiology, Osmania University and are chosen based on the nature of active most frequent contaminant of food. The fungal cultures were grown on yeast extract agar (YEP) media containing chloramphenicol (50 μg/mL) for 2-4 days at 28 °C. After 2 days, the black spores and lawn of cultures were collected and stored in an aqueous solution of 40% (v/v) glycerol at -80 °C. Antifungal assay can be done by cylinder plate or disc method and cross streak plate method, broth dilution method. Here in the present study we used cross steak plate method to identify the minimum concentration of compound required to inhibit the fungal growth.

YEP agar media (commonly used to assess susceptibility of diverse mutants) was prepared by mixing the bactopeptone, sodium chloride and maintained final pH 7 at 25°C. Sterilization of media is carried at 121⁰C/15 lbs pressure for 30 minutes, and cooled till room temperature and poured into sterile petriplates. Aqueous culture of stock fungal strains was spreaded evenly on plates and different diluted concentration of compounds was streaked on plate. The measured zone of clear area length (mm) on YEP agar plate determined the efficacy of compound for fungal growth inhibition. Experiments were performed in triplicates to represent the data in form of Mean ± SEM.

Antioxidant activity (DPPH free radical scavenging activity)

Antioxidant activity of test compounds was checked by transferring concentrations of the compound ranging from 10 µM to 200 µM separately into sterile eppendorf tubes. Each concentration was performed in triplicates to avoid mean difference. Transfer 100 μl of DPPH freshly prepared reagent constantly to an eppendorf tube and Add distilled water to make up the mixture volume up to 1 mL. Shake and vortex the mixture gently and stand in the dark at Room temperature for 30 mins. Further blank for the test is prepared by adding various concentrations of diluent compounds and constant volume of reagent to make the reaction up to 1 mL. Absorbance of test compounds was measured by using UV spectrophotometer at 517 nm.

Percent antioxidant activity calculation:

Molecular Docking studies

Molecular docking studies were performed by using Vina in PyRx docking tool to predict the protein-ligand interactions at molecular level. The co-crystal structure of Gram positive S. aureus topoisomerase IV enzyme of catalytic domain in complex with novobiocin (PDB ID: 4URN) and the crystal structure of COVID-19 main protease (6LU7) were used as targets. Topoisomerase IV is a known efficient DNA decatenase, it plays a crucial role in separation of daughter chromosomes during DNA replication. Antibiotics like novobiocin and ampicillin acts upon bacteria by targeting this topoisomerase IV [40, 41]. Target proteins were downloaded from Protein Data Bank and prepared by Biovia discovery studio tool. The water molecules were removed and polar hydrogens were added to the protein. The ligand molecules and protein structures determined were imported into PyRx and converted into PDBQT format. The active sites of target molecules were identified from the existing ligand molecules in crystal structures. The docking simulations were performed after assigning grid box and grid centers in Vina wizard. The docking results were visualized by using Pymol and Biovia Discovery Studio Visulaizer..

In summary, we have synthesized a new series of pyrazole, triazole, based 2,4-di substituted thiazole derivatives 12a-l. The synthesized compounds 12a-l was characterized by ¹H NMR, ¹³C NMR, ESI-Mass, HRMS spectral data. The anti-microbial activities of these compounds were evaluated against various bacterial and fungal strains. The synthesized compounds 12a, 12f, and 12k showed good activity against the tested bacterial and fungal stains, similarly these compounds have shown potent DPPH scavenging activity and it was emerged as potential molecules for further development. Moreover, the best dock scored compounds from our current in silico study were further evaluated for minimal microbial growth-inhibitory studies. The binding affinity of compounds 12a-l are ranging from 10.0 to -11.0 Kcal/mol with topoisomerase IV enzyme and with COVID-19 main protease binding affinities are ranging from -8.2 to -9.3 Kcal/mol. These docking studies reveal that the compounds 12a-l could be the best inhibitors for novel SARS Cov-2 virus and of more interested potent drug candidates.

Acknowledgement

The authors thank the Head, Department of Chemistry, Osmania University, Hyderabad, for providing lab facilities. PJ, DB, thankful to UGC-SERO Hyderabad and DST-PURSE OU. SB is grateful to UGC-SRF for the fellowship. The authors thank CFRD for analytical support. B.S. thanks the University Grants Commission in India for the award of a Dr. D. S. Kothari postdoctoral fellowship.

Authors’ contributions

DB, MR and BS were responsible in each stage in the preparation of this manuscript like carried the literature study, designing of synthetic schemes, organic synthesis and purification of compounds and characterization experiments with the direct supervision of PJ. SB carried out the antimicrobial activity. AGA was responsible for the docking study. All authors read and approved the final manuscript.

Conflict Of Interest

The authors hereby declare no conflict of interest.

Availability of data and materials

All data are fully available without restriction.

Consent for publication

The authors declare that the copyright belongs to the journal.

Ethics approval and consent to participate

Not applicable.

Supplementary data

Provided with supplementary data for ¹H NMR, ¹³C NMR, Mass and IR spectra of all the new synthesized compounds 12a-l, the experimental protocols

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Scheme 1 is available in the Supplemental Files section.

No competing interests reported.

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Scheme1.jpg
Scheme 1. Synthesis of triazole, pyrazole contain 2,4-disubstituted thiazole analogs 12a-l

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Synthesis, antimicrobial and antioxidant activity of triazole, pyrazole containing thiazole Derivatives and Molecular docking studies on COVID-19

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