Design and synthesis of pyrazole–oxadiazole–thioacetamide hybrids with antimicrobial and antioxidant properties

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The compounds were prepared via a multistep synthetic approach, and their structures were confirmed using spectroscopic techniques, including IR, ¹H NMR, and ¹³C NMR. IR analysis revealed characteristic bands corresponding to N–H, C = O, C = N, and C–S functionalities, while ¹H NMR spectra displayed signals for aromatic, pyrazole, oxadiazole, and thioacetamide protons. The ¹³C NMR spectra further supported the assigned structures, showing resonances for carbonyl, heterocyclic, and aromatic carbons. The synthesized hybrids were screened for antimicrobial activity against selected Gram-positive and Gram-negative bacterial strains as well as fungal pathogens. Several derivatives exhibited promising inhibitory activity, highlighting the potential of these scaffolds in combating microbial infections. Antioxidant potential was assessed using standard radical scavenging assays, demonstrating significant activity in selected compounds. Pyrazole 1 3 4-Oxadiazole Thioacetamide Antimicrobial activity Antioxidant activity Figures Figure 1 Introduction Heterocyclic frameworks containing nitrogen, oxygen, and sulfur heteroatoms are central to contemporary drug discovery due to their exceptional structural diversity, favorable physicochemical properties, and strong receptor-binding potential [ 1 – 3 ]. Among these, pyrazole is a privileged heterocycle that has been widely studied for its antimicrobial, antioxidant, anti-inflammatory, and enzyme inhibitory activities [ 4 , 5 ]. Recent investigations have demonstrated that pyrazole-containing compounds exhibit significant antimicrobial and antioxidant effects, often supported by spectroscopic characterization such as FT-IR, ¹H NMR, and ¹³C NMR, confirming key functional groups and scaffolds responsible for bioactivity [ 6 , 7 ]. In addition, hybrid molecules built around pyrazole cores have shown enhanced performance against resistant microbial strains and oxidative stress models due to synergistic structural interactions [ 8 ]. Similarly, the 1,3,4-oxadiazole ring has attracted considerable attention in medicinal chemistry. This heterocycle is valued for its electron-deficient aromatic nature and ability to act as a bioisostere for amides and esters, often leading to improved metabolic stability and enhanced drug-like properties [ 9 , 10 ]. Recent reviews and experimental studies have shown that 1,3,4-oxadiazole derivatives exhibit broad-spectrum antimicrobial activity against pathogenic bacteria and fungi, as well as significant antioxidant potential, making them promising candidates for therapeutic development [ 11 , 12 ]. The combination of these heterocycles in single molecular frameworks has been a focus of modern research due to their cooperative electronic effects and increased likelihood of interacting effectively with multiple biological targets [ 13 , 14 ]. Incorporating sulfur-containing moieties such as thioacetamide into heterocyclic systems has also been shown to enhance bioactivity by influencing lipophilicity, membrane permeability, and redox behavior, which contribute to improved pharmacological profiles [ 15 ]. Sulfur-modified heterocyclic hybrids frequently display enhanced antimicrobial efficacy and radical scavenging ability, while providing versatile opportunities for structural optimization [ 16 ]. However, while many studies focus on synthesis and activity, the integration of experimental antimicrobial and antioxidant evaluations remains critical for identifying promising bioactive candidates with potential therapeutic relevance. [ 17 , 18 ]. Oxadiazole- and pyrazole-based heterocycles have attracted significant attention in medicinal chemistry due to their versatile structural features and wide range of reported biological activities, including antimicrobial [ 19 ], antioxidant [ 20 ], anticancer [ 21 ], anti-inflammatory [ 22 ], and enzyme inhibitory properties.[ 20 ] Combining such computational profiling with in vitro antimicrobial and antioxidant assays allows for a holistic assessment of compound potential, reducing experimental burden and guiding lead optimization. Therefore, this study reports the design, synthesis, spectroscopic characterization (FT-IR, ¹H NMR, ¹³C NMR), and biological evaluation of new pyrazole–1,3,4-oxadiazole–thioacetamide hybrids. The goal is to identify promising multifunctional agents exhibiting potent antimicrobial and antioxidant activities with favorable pharmacokinetic predictions, advancing them as candidates for further medicinal chemistry exploration. RESULT AND DISCUSSION A series of 1,3,4-oxadiazole derivatives, namely N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide 5a-5f, was prepared in 70–80% yields through a conventional method as depicted in Scheme 1 . Using this method, the product can be separated from the reaction mixture in high purify and a large variety of substituted aromatic aldehydes can be used directly. IR, 1H NMR, 13C NMR and mass spectrometry methods are applied for the confirmation of synthesized compounds. Antibacterial activity. The antimicrobial potential of the newly prepared compounds 5a–5f was investigated against gram-positive ( Bacillus subtilis ) and gram-negative ( Escherichia coli ) bacterial strains at concentrations of 1000, 500, and 250 µg/mL. Chloramphenicol was used as the reference antibacterial agent for comparison. Among all the synthesized derivatives, compounds 5c (4-Cl) and 5f (4-NO₂) demonstrated superior inhibitory activity against Bacillus subtilis across all tested concentrations. The remaining compounds exhibited noticeable antibacterial effects, although their activity was comparatively lower than the standard drug, as summarized in Table 1 . A similar activity trend was observed against Escherichia coli . Compounds 5c (4-Cl) and 5f (4-NO₂) displayed strong antibacterial action at all tested concentrations, showing activity levels comparable to chloramphenicol. Other derivatives also showed measurable antibacterial potential but with relatively reduced effectiveness, as illustrated in Table 1 . The enhanced activity of compounds bearing chloro and nitro substituents suggests that electron-withdrawing groups at the para position play a significant role in improving antibacterial performance. Antifungal Activity: The synthesized derivatives were further evaluated for their antifungal efficacy against Aspergillus niger at concentrations of 1000, 500, and 250 µg/mL, using fluconazole as the standard antifungal drug. Compounds 5c (4-Cl) and 5f (4-NO₂) exhibited excellent antifungal activity at all concentration levels and demonstrated stronger inhibition than the reference drug. The other synthesized compounds also showed appreciable antifungal activity but were less potent compared to fluconazole, as detailed in Table 1 . These findings indicate that the presence of strong electron-withdrawing substituents enhances antifungal effectiveness within this compound series. SAR study. The structure–activity relationship (SAR) analysis revealed a distinct correlation between the electronic properties of substituents on the aromatic ring and the observed antimicrobial activity of the synthesized derivatives. Compounds bearing electron-withdrawing substituents exhibited markedly improved antibacterial and antifungal activities compared to those containing electron-donating groups. Among the investigated derivatives, substitution at the para (4) position of the aromatic ring significantly influenced biological potency. In particular, compounds containing nitro (4-NO₂) and chloro (4-Cl) substituents demonstrated the highest inhibitory activity against both bacterial and fungal strains. The enhanced biological performance of these derivatives can be attributed to the strong electron-withdrawing nature of nitro and chloro groups, which may modulate the electron density of the molecular scaffold, thereby facilitating improved interaction with microbial enzymatic or receptor binding sites. Additionally, these substituents may increase lipophilicity, promoting better penetration through microbial cell membranes and contributing to enhanced biological efficacy. Conversely, compounds containing electron-donating substituents displayed comparatively reduced activity, likely due to their diminished ability to stabilize interactions with biological targets. Overall, the SAR findings suggest that para-position substitution with strong electron-withdrawing groups plays a crucial role in enhancing antimicrobial and antifungal activity within this class of compounds. These observations provide valuable guidance for the rational design and optimization of more potent derivatives in future studies. Table 1 Antimicrobial activity of 1,3,4-oxadiazole derivatives No. -R Zone of inhibition (mm) (Gram-positive bacteria) (Gram negative) (Fungi) 1000 µg/ml 500 µg/ml 250 µg/ml 1000 µg/ml 500 µg/ml 250 µg/ml 1000 µg/ml 500 µg/ml 250 µg/ml A B A B A B A B A B A B A B A B A B 5a -H 10 14 06 08 - - 12 14 - - - - 16 19 14 15 - - 5b 4-OH 17 16 16 15 15 14 16 17 15 16 14 14 17 18 16 17 15 16 5c 4-Cl 21 22 17 18 14 15 17 22 13 19 12 15 21 23 16 17 12 14 5d 3-OH 18 19 19 17 16 15 16 18 14 14 13 12 18 17 16 15 13 12 5e 3-Cl 15 16 14 15 12 13 14 15 13 14 12 13 16 17 13 14 10 11 5f 4-NO 2 20 21 19 20 15 16 16 20 14 20 13 16 20 23 18 19 13 15 Fluconazole (Positive control) 24 25 22 23 21 21 Chloramphenicol (Positive control) 25 28 25 27 24 25 25 28 24 26 24 24 *Gram Positive bacteria: A : B. subtilis; B : S. aureus *Gram Negative bacteria: A : E.Coli; B : P. aeruginosa *Fungi: A : A.niger; B : F. Solani Free radical scavenging by the compounds determined using DPPH analysis In the presence of antioxidant compounds or extracts, stable DPPH free radicals are converted to DPPH-H (instead of DPPH alone), resulting in a decreased absorbance at 515 nm. Antioxidant compound or extract scavenging activity is identified by the visual appearance of the test solutions, which become more discolored during the test. The total colour shift from deep violet to light yellow is assessed using UV-VIS spectrophotometry at 515 nm, in combination with the percent change from DPPH to DPHH absorption. A stock solution of DPPH was made using 1 mg dissolved in 1 mL of methanol, which produced a DPPH solution with an absorbance of 515 nm prior to diluting into the different dilutions of 5a–f (i.e., 25, 50, 75, 100 or 125 µg) into 3 mL of methanol. Prior to testing, the DPPH solution had been prepared. After combining 1 mL of the DPPH solution with 3 mL of the diluted samples, the mixtures had been incubated for 30 minutes at 30°C. The absorbance values of the incubated samples and controls, which did not contain the test compound, were measured at 515 nm. A standard of ascorbic acid was used to determine antioxidant activity, and all test samples and standard compounds were tested in triplicate. The IC50 values (i.e., µg/mL concentration that produces 50% growth inhibition for this experimental system) of each compound are shown in Table 2 and were determined using GraphPad Prism software version 7.0. The radical scavenging activity (RSA) was determined using the formula: % Inhibition = [AB - AA / AB] × 100, where AB represents the absorbance of the blank solution and AA denotes the absorbance of the test sample. The antioxidant potential of the synthesized compounds 5a–5f was evaluated using the DPPH free radical scavenging assay, as illustrated in Fig. 1. The results summarized in Table 2 indicate that all tested derivatives exhibited notable radical scavenging activity when compared with the standard antioxidant, ascorbic acid. The DPPH assay, expressed in terms of IC₅₀ values, is a well-established and reliable method for assessing the antioxidant efficiency of 1,3,4-oxadiazole derivatives. Among the tested compounds, compound 5f demonstrated the highest antioxidant activity, reflected by its lowest IC₅₀ value, indicating superior free radical scavenging efficiency. This enhanced activity may be attributed to favorable structural features that facilitate effective electron or proton donation and stabilization of the resulting radical species. In contrast, the remaining compounds exhibited comparatively higher IC₅₀ values, suggesting reduced scavenging capacity, possibly due to less favorable electronic or substituent effects. Table 2 Antioxidant scavenging activity of compounds 5a-f on DPPH* free radical at different concentrations Entry Compounds Concentration µg/ml IC50 25µg/ml 50µg/ml 75µg/ml 100µg/ml 125µg/ml 1 5a 52.84 ± 0.68 63.51 ± 0.17 69.61 ± 0.24 73.67 ± 0.18 74.63 ± 0.12 15.01 ± 3 2 5b 56.39 ± 0.12 68.45 ± 0.12 74.43 ± 0.12 77.17 ± 0.11 78.41 ± 0.24 13.78 ± 3 3 5c 58.18 ± 0.12 69.29 ± 0.12 76.10 ± 0.12 76.56 ± 0.11 78.37 ± 0.12 13.08 ± 3 4 5j 59.25 ± 0.12 67.48 ± 0.27 71.91 ± 0.12 74.79 ± 0.12 75.61 ± 0.12 9.54 ± 3 5 5e 49.7 ± 0.12 59.21 ± 0.17 65.82 ± 0.11 69.53 ± 0.12 70.25 ± 0.22 15.34 ± 3 6 5f 55.59 ± 0.24 64.75 ± 0.12 71.56 ± 0.12 75.38 ± 0.12 76.23 ± 0.13 13.62 ± 3 11 Ascorbic acid 64.15 ± 0.12 76.1 ± 0.12 85.18 ± 0.12 88.17 ± 0.12 89.36 ± 0.12 14.42 ± 3 * Results are expressed as mean of triplicates ± standard deviation. ** The IC50 values defined as the concentration corresponding to 50% growth inhibition, µg/mL. EXPERIMENTAL Commercially available reagents and solvents were used. TLC plates were used for analytical thin layer chromatography. It has a silica gel G coating for reaction monitoring and retardation factor determination. In an iodine chamber, TLC spots were visible. On an electrothermal melting point apparatus, the melting points of newly synthesized derivatives were determined and found to be uncorrected. A perkin-Elmer 2400 CHN analyzer confirmed the element analysis (percent C,H and N). The SHIMADZU LC-MS 2010 spectrometer was used to obtain mass spectra. 13 C NMR spectra were recorded on a Varian Mercury-400, 100 MHz in DMSO-d 6 as a solvent and TMS as an internal standard using a 5 mm tube on a Bruker Advance Ⅱ 400 MHz. Step 1: Synthesis of 2-chloro-N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)acetamide (3) Substituted 6-amino-3-ethoxy-4-phenyl-1,3a, 4, 7a-tetrahydropyrano[2,3-c]pyrazole-5-carbonitrile (1 mol) was added in DMF with containing few drops of triethylamine (TEA) with continue stirred at room temperature. Then chloro-acetyl chloride (1.5 mol) was added above solution with continue stirring and maintained the temp 0–5 0 C. Then solution was stirred at room temperature for 4 hours. The completion of reaction was monitored periodically by using toluene: acetone (7:3) as mobile phase. Then the solution was added into crushed ice and the obtained product was filtered, washed with water, dried and recrystallized from ethanol. Step 2: General Procedure for the Synthesis of N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide : 2-chloro-N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)acetamide (0.01 mol) obtained was further reacted with 5-(pyridin-4-yl)-1,3,4- oxadiazole-2-thiol (0.01 mol) for 4 h at room temperature. In the presence of K 2 CO 3 (0.02 mol) and acetone (20 ml) was used as reaction medium. After the completion of reaction judged on TLC using Toluene: Acetone (7:3) as mobile phase. The solution was poured in ice-cold water and stirred for 30 min. The solid obtained was filtered off, washed with water, dried and recrystallized from ethanol to give 5a–5f. N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydrophyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5a) Yield 68%, mp 175°C. IR spectrum, ν, cm–1: 2954, 1720, 1464, 1212, 1050. 1H NMR spectrum, δ, ppm: 1.26 t (3H, CH 3 ), 2.3 d (1H, CH), 3.3 q (1H, CH), 3.66 q (2H, CH 2 ), 4.00 s (2H, CH 2 ), 5.1 s (1H, CH), 7.40–7.98 m (10 H, Ar-H), 9.13 s (1H, NH), 9.21 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.3, 15.9, 39.3, 48.0, 54.7, 61.4, 72.6, 117.3, 126.1, 127.5, 128.8, 129.2, 139.2, 158.2, 164.3, 167.8. Mass spectrum, m/z: 503.0 [M + H]+. N-(4-(4-chlorophenyl)-5-cyano-3-ethoxy-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5b) Yield 70%, mp 165°C. IR spectrum, ν, cm–1: 2925, 1715, 1445, 1210, 1087. 1H NMR spectrum, δ, ppm: 1.30 t (3H, CH 3 ), 2.45 d (1H, CH), 3.26 q (1H, CH), 3.71 q (2H, CH 2 ), 4.12 s (2H, CH 2 ), 5.6 s (1H, CH), 7.32–7.78 m (9 H, Ar-H), 9.10 s (1H, NH), 9.26 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.0, 15.7, 39.0, 48.6, 54.8, 61.7, 72.8, 117.6, 126.3, 127.6, 128.9, 129.3, 139.5, 158.7, 164.1, 167.6. Mass spectrum, m/z: 537.0 [M + H]+. N-(5-cyano-3-ethoxy-4-(4-hydroxyphenyl)-1,3a, 4, 7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5c) Yield 72%, mp 143°C. IR spectrum, ν, cm–1: 3412, 2922, 1720, 1416, 1200, 1048. 1H NMR spectrum, δ, ppm: 1.12 t (3H, CH 3 ), 2.59 d (1H, CH), 3.56 q (1H, CH), 3.89 q (2H, CH 2 ), 4.14 s (2H, CH 2 ), 5.68 s (1H, CH), 7.25–7.87 m (9 H, Ar-H), 9.00 s (1H, OH), 9.13 s (1H, NH), 9.30 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.2, 15.9, 39.2, 48.7, 54.3, 61.0, 72.7, 117.5, 126.7, 127.3, 128.7, 129.0, 139.4, 158.5, 164.0, 167.2. Mass spectrum, m/z: 519.0 [M + H]+. N-(5-cyano-3-ethoxy-4-(3-hydroxyphenyl)-1,3a, 4, 7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5d) Yield 75%, mp 126°C. IR spectrum, ν, cm–1: 3420, 2923, 1724, 1418, 1206, 1047. 1H NMR spectrum, δ, ppm: 1.10 t (3H, CH 3 ), 2.54 d (1H, CH), 3.45 q (1H, CH), 3.48 q (2H, CH 2 ), 4.41 s (2H, CH 2 ), 5.46 s (1H, CH), 7.20–7.89 m (9 H, Ar-H), 9.06 s (1H, OH), 9.15 s (1H, NH), 9.45 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.3, 15.7, 39.6, 48.8, 54.5, 61.2, 72.7, 117.9, 126.6, 127.4, 128.6, 129.3, 139.4, 158.7, 164.6, 167.3. Mass spectrum, m/z: 519.0 [M + H]+. N-(4-(3-chlorophenyl)-5-cyano-3-ethoxy-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5e) Yield 70%, mp 160°C. IR spectrum, ν, cm–1: 2920, 1714, 1459, 1223, 1078. 1H NMR spectrum, δ, ppm: 1.31 t (3H, CH 3 ), 2.41 d (1H, CH), 3.27 q (1H, CH), 3.76 q (2H, CH 2 ), 4.00 s (2H, CH 2 ), 5.23 s (1H, CH), 7.30–7.89 m (9 H, Ar-H), 9.00 s (1H, NH), 9.20 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.2, 15.9, 39.2, 48.7, 54.3, 61.4, 72.7, 117.3, 126.7, 127.6, 128.9, 129.3, 139.5, 158.7, 164.1, 167.6. Mass spectrum, m/z: 537.0 [M + H]+. N-(5-cyano-3-ethoxy-4-(4-nitrophenyl)-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5f) Yield 73%, mp 156°C. IR spectrum, ν, cm–1: 2912, 1715, 1448, 1256, 1070. 1H NMR spectrum, δ, ppm: 1.32 t (3H, CH 3 ), 2.47 d (1H, CH), 3.23 q (1H, CH), 3.56 q (2H, CH 2 ), 4.20 s (2H, CH 2 ), 5.56 s (1H, CH), 7.15–7.79 m (9 H, Ar-H), 9.01 s (1H, NH), 9.26 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.3, 15.98, 39.4, 48.0, 54.5, 61.6, 72.7, 117.2, 126.3, 127.7, 128.5, 129.4, 139.8, 158.6, 164.0, 167.8. Mass spectrum, m/z: 548.0 [M + H]+. Conclusion In conclusion, a series of pyrazole–1,3,4-oxadiazole–thioacetamide derivatives was successfully synthesized through a straightforward multistep protocol and unambiguously characterized by FT-IR, ¹H NMR, and ¹³C NMR spectroscopic analyses. The synthesized compounds were evaluated for their antimicrobial and antioxidant activities, and several derivatives exhibited moderate to good inhibitory effects against Gram-positive and Gram-negative bacteria as well as fungal strains. Compounds bearing electron-withdrawing substituents on the aromatic ring showed comparatively enhanced antimicrobial activity, indicating a clear substituent-dependent structure–activity relationship. Antioxidant screening using the DPPH assay revealed that all derivatives possessed measurable radical scavenging ability, with compound 5f displaying the most pronounced activity within the series. The present study highlights pyrazole–oxadiazole–thioacetamide hybrids as useful chemical intermediates with promising biological profiles, warranting further structural modification and investigation. Declarations Funding Sources: The authors received no financial support for the research. Conflict of Interest: There were no commercial or financial links that may be deemed a potential conflict of interest during the research. Author Contributions: Dr. Shweta A. Patel and Dr. Ami Patel conceptualized the study. Dr. Shweta A. Patel carried out the synthesis and characterization of the compounds, performed the biological activity studies, interpreted the data, and prepared the manuscript. Dr. Ami Patel contributed to data analysis, and critically reviewed and revised the manuscript. All authors read and approved the final manuscript. Ethics declaration: not applicable. References Kapoor K, Kaur N, Sohal HS, Kaur M, Singh K, Kumar A. Drugs and their mode of action: A review on sulfur-containing heterocyclic compounds. Polycycl Aromat Compd. 2025;45:136–75. Biswas T, Mittal RK, Sharma V, Kanupriya, Mishra I. Nitrogen-fused heterocycles: Empowering anticancer drug discovery. Med Chem. 2024;20:369–84. Omar A. 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Triazole- and oxadiazole-based hydrazide-hydrazone derivatives with antioxidant potential. BMC Chem. 2025;19:1–15. Salem MM, El-Adl K, El-Morsy A, Abulkhair HS. Oxadiazole derivatives as promising anticancer agents: A two-decade overview. RSC Adv. 2025;15:32778–95. Shaheen K, Alam A, Elhenawy AA, Khan IA, Rahman FU, Ali A, et al. In vivo anti-inflammatory evaluation of oxadiazole derivatives bearing flurbiprofen. Sci Rep. 2025;15:29144. Shah M, Singh C, Yadav MR, Nagani A. Oxadiazoles as multitarget therapeutic agents in Alzheimer’s disease. J Saudi Chem Soc. 2025;29:1–61. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8836359","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618317762,"identity":"9840f1ba-c2ed-44dc-b9d6-198b01d09744","order_by":0,"name":"Dr. Shweta Patel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie2SsQrCMBRFnxTiEnDNIO0vRAIiOPgrdunU4OpQxKmTOBf8Cae6RoKZ2r1Dt0IHcSgUipPYKjjWjII5w3tcuAdeIAAGwy9C3steCCm6ONZT2i6Ds1p2EWsr7lYmtMvfFeewmxRVkLNBmNTXLJhhGMrLsU+hecKoUKVt4TSe+6o9DHte1qsQHhOBJEMkjZmPWoXgaa/iRPx0Fw/p7pxbyfyHhgIZj+EcSjeCxCp4qKHQbFWTdF8yCmpq8T3B6NtbnMhzq3WT2xRkUfvNxh4Npeo/7I3oBnr9BKRR/yhWpdk2GAyGP+MJ3pRRmTTK7HEAAAAASUVORK5CYII=","orcid":"","institution":"Department of Chemistry, Faculty of science, Gokul Global University, Sidhpur-384151","correspondingAuthor":true,"prefix":"Dr.","firstName":"Shweta","middleName":"","lastName":"Patel","suffix":""},{"id":618317763,"identity":"bb378e87-8e49-4dba-9259-6d6d76fbce6c","order_by":1,"name":"Dr. Ami Patel","email":"","orcid":"","institution":"R. R. Mehta college of science \u0026 C. L. Parikh college of commerce, Palanpur, Gujarat, India.","correspondingAuthor":false,"prefix":"Dr.","firstName":"Ami","middleName":"","lastName":"Patel","suffix":""}],"badges":[],"createdAt":"2026-02-10 04:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8836359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8836359/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106464724,"identity":"d31e95de-f429-4a83-841a-06f618505409","added_by":"auto","created_at":"2026-04-08 21:55:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":255607,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8836359/v1/325b76b5be1daaa04189b2f5.png"},{"id":106726234,"identity":"436d72fd-6b47-40ec-9569-a2320b5bc58d","added_by":"auto","created_at":"2026-04-12 18:35:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1352157,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8836359/v1/8430d474-d37e-4616-a0c3-5c0b6e8de389.pdf"},{"id":106724509,"identity":"790ec9f7-b4d1-43eb-b07f-12c4a7c6109d","added_by":"auto","created_at":"2026-04-12 18:28:21","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8836359/v1/c5e354f18b759ca41ff5b461.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Design and synthesis of pyrazole–oxadiazole–thioacetamide hybrids with antimicrobial and antioxidant properties","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeterocyclic frameworks containing nitrogen, oxygen, and sulfur heteroatoms are central to contemporary drug discovery due to their exceptional structural diversity, favorable physicochemical properties, and strong receptor-binding potential [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among these, pyrazole is a privileged heterocycle that has been widely studied for its antimicrobial, antioxidant, anti-inflammatory, and enzyme inhibitory activities [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recent investigations have demonstrated that pyrazole-containing compounds exhibit significant antimicrobial and antioxidant effects, often supported by spectroscopic characterization such as FT-IR, \u0026sup1;H NMR, and \u0026sup1;\u0026sup3;C NMR, confirming key functional groups and scaffolds responsible for bioactivity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, hybrid molecules built around pyrazole cores have shown enhanced performance against resistant microbial strains and oxidative stress models due to synergistic structural interactions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilarly, the 1,3,4-oxadiazole ring has attracted considerable attention in medicinal chemistry. This heterocycle is valued for its electron-deficient aromatic nature and ability to act as a bioisostere for amides and esters, often leading to improved metabolic stability and enhanced drug-like properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Recent reviews and experimental studies have shown that 1,3,4-oxadiazole derivatives exhibit broad-spectrum antimicrobial activity against pathogenic bacteria and fungi, as well as significant antioxidant potential, making them promising candidates for therapeutic development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The combination of these heterocycles in single molecular frameworks has been a focus of modern research due to their cooperative electronic effects and increased likelihood of interacting effectively with multiple biological targets [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncorporating sulfur-containing moieties such as thioacetamide into heterocyclic systems has also been shown to enhance bioactivity by influencing lipophilicity, membrane permeability, and redox behavior, which contribute to improved pharmacological profiles [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Sulfur-modified heterocyclic hybrids frequently display enhanced antimicrobial efficacy and radical scavenging ability, while providing versatile opportunities for structural optimization [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, while many studies focus on synthesis and activity, the integration of experimental antimicrobial and antioxidant evaluations remains critical for identifying promising bioactive candidates with potential therapeutic relevance. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Oxadiazole- and pyrazole-based heterocycles have attracted significant attention in medicinal chemistry due to their versatile structural features and wide range of reported biological activities, including antimicrobial [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], antioxidant [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], anticancer [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], anti-inflammatory [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and enzyme inhibitory properties.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eCombining such computational profiling with in vitro antimicrobial and antioxidant assays allows for a holistic assessment of compound potential, reducing experimental burden and guiding lead optimization. Therefore, this study reports the design, synthesis, spectroscopic characterization (FT-IR, \u0026sup1;H NMR, \u0026sup1;\u0026sup3;C NMR), and biological evaluation of new pyrazole\u0026ndash;1,3,4-oxadiazole\u0026ndash;thioacetamide hybrids. The goal is to identify promising multifunctional agents exhibiting potent antimicrobial and antioxidant activities with favorable pharmacokinetic predictions, advancing them as candidates for further medicinal chemistry exploration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eRESULT AND DISCUSSION\u003c/h2\u003e \u003cp\u003eA series of 1,3,4-oxadiazole derivatives, namely N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide 5a-5f, was prepared in 70\u0026ndash;80% yields through a conventional method as depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Using this method, the product can be separated from the reaction mixture in high purify and a large variety of substituted aromatic aldehydes can be used directly. IR, 1H NMR, 13C NMR and mass spectrometry methods are applied for the confirmation of synthesized compounds.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibacterial activity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe antimicrobial potential of the newly prepared compounds 5a\u0026ndash;5f was investigated against gram-positive (\u003cem\u003eBacillus subtilis\u003c/em\u003e) and gram-negative (\u003cem\u003eEscherichia coli\u003c/em\u003e) bacterial strains at concentrations of 1000, 500, and 250 \u0026micro;g/mL. Chloramphenicol was used as the reference antibacterial agent for comparison. Among all the synthesized derivatives, compounds 5c (4-Cl) and 5f (4-NO₂) demonstrated superior inhibitory activity against \u003cem\u003eBacillus subtilis\u003c/em\u003e across all tested concentrations. The remaining compounds exhibited noticeable antibacterial effects, although their activity was comparatively lower than the standard drug, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eA similar activity trend was observed against \u003cem\u003eEscherichia coli\u003c/em\u003e. Compounds 5c (4-Cl) and 5f (4-NO₂) displayed strong antibacterial action at all tested concentrations, showing activity levels comparable to chloramphenicol. Other derivatives also showed measurable antibacterial potential but with relatively reduced effectiveness, as illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The enhanced activity of compounds bearing chloro and nitro substituents suggests that electron-withdrawing groups at the para position play a significant role in improving antibacterial performance.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntifungal Activity:\u003c/h3\u003e\n\u003cp\u003eThe synthesized derivatives were further evaluated for their antifungal efficacy against \u003cem\u003eAspergillus niger\u003c/em\u003e at concentrations of 1000, 500, and 250 \u0026micro;g/mL, using fluconazole as the standard antifungal drug. Compounds 5c (4-Cl) and 5f (4-NO₂) exhibited excellent antifungal activity at all concentration levels and demonstrated stronger inhibition than the reference drug. The other synthesized compounds also showed appreciable antifungal activity but were less potent compared to fluconazole, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These findings indicate that the presence of strong electron-withdrawing substituents enhances antifungal effectiveness within this compound series.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSAR study.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe structure\u0026ndash;activity relationship (SAR) analysis revealed a distinct correlation between the electronic properties of substituents on the aromatic ring and the observed antimicrobial activity of the synthesized derivatives. Compounds bearing electron-withdrawing substituents exhibited markedly improved antibacterial and antifungal activities compared to those containing electron-donating groups. Among the investigated derivatives, substitution at the para (4) position of the aromatic ring significantly influenced biological potency. In particular, compounds containing nitro (4-NO₂) and chloro (4-Cl) substituents demonstrated the highest inhibitory activity against both bacterial and fungal strains.\u003c/p\u003e \u003cp\u003eThe enhanced biological performance of these derivatives can be attributed to the strong electron-withdrawing nature of nitro and chloro groups, which may modulate the electron density of the molecular scaffold, thereby facilitating improved interaction with microbial enzymatic or receptor binding sites. Additionally, these substituents may increase lipophilicity, promoting better penetration through microbial cell membranes and contributing to enhanced biological efficacy. Conversely, compounds containing electron-donating substituents displayed comparatively reduced activity, likely due to their diminished ability to stabilize interactions with biological targets.\u003c/p\u003e \u003cp\u003eOverall, the SAR findings suggest that para-position substitution with strong electron-withdrawing groups plays a crucial role in enhancing antimicrobial and antifungal activity within this class of compounds. These observations provide valuable guidance for the rational design and optimization of more potent derivatives in future studies.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial activity of 1,3,4-oxadiazole derivatives\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"22\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c19\" colnum=\"19\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c20\" colnum=\"20\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c21\" colnum=\"21\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c22\" colnum=\"22\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e-R\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"20\" nameend=\"c22\" namest=\"c3\"\u003e \u003cp\u003eZone of inhibition (mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003e(Gram-positive bacteria)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c16\" namest=\"c9\"\u003e \u003cp\u003e(Gram negative)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c22\" namest=\"c17\"\u003e \u003cp\u003e(Fungi)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e1000 \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e250\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c14\" namest=\"c11\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c16\" namest=\"c15\"\u003e \u003cp\u003e250\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c20\" namest=\"c19\"\u003e \u003cp\u003e500\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c22\" namest=\"c21\"\u003e \u003cp\u003e250\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c17\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c18\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c19\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c20\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c21\"\u003e \u003cp\u003e\u003cb\u003eA\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c22\"\u003e \u003cp\u003e\u003cb\u003eB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4-Cl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e\u003cb\u003e21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e\u003cb\u003e23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5f\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e4-NO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e\u003cb\u003e23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e\u003cb\u003e18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFluconazole\u003c/p\u003e \u003cp\u003e(Positive control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"14\" nameend=\"c16\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c22\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChloramphenicol\u003c/p\u003e \u003cp\u003e(Positive control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c13\" namest=\"c12\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c22\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"22\"\u003e*Gram Positive bacteria: \u003cb\u003eA\u003c/b\u003e: \u003cb\u003eB. subtilis;\u003c/b\u003e \u003cb\u003eB\u003c/b\u003e: \u003cb\u003eS. aureus\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"22\"\u003e*Gram Negative bacteria: \u003cb\u003eA\u003c/b\u003e: \u003cb\u003eE.Coli;\u003c/b\u003e \u003cb\u003eB\u003c/b\u003e: \u003cb\u003eP. aeruginosa\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"22\"\u003e*Fungi: \u003cb\u003eA\u003c/b\u003e: \u003cb\u003eA.niger;\u003c/b\u003e \u003cb\u003eB\u003c/b\u003e: \u003cb\u003eF. Solani\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eFree radical scavenging by the compounds determined using DPPH analysis\u003c/h3\u003e\n\u003cp\u003eIn the presence of antioxidant compounds or extracts, stable DPPH free radicals are converted to DPPH-H (instead of DPPH alone), resulting in a decreased absorbance at 515 nm. Antioxidant compound or extract scavenging activity is identified by the visual appearance of the test solutions, which become more discolored during the test. The total colour shift from deep violet to light yellow is assessed using UV-VIS spectrophotometry at 515 nm, in combination with the percent change from DPPH to DPHH absorption. A stock solution of DPPH was made using 1 mg dissolved in 1 mL of methanol, which produced a DPPH solution with an absorbance of 515 nm prior to diluting into the different dilutions of 5a\u0026ndash;f (i.e., 25, 50, 75, 100 or 125 \u0026micro;g) into 3 mL of methanol. Prior to testing, the DPPH solution had been prepared. After combining 1 mL of the DPPH solution with 3 mL of the diluted samples, the mixtures had been incubated for 30 minutes at 30\u0026deg;C. The absorbance values of the incubated samples and controls, which did not contain the test compound, were measured at 515 nm. A standard of ascorbic acid was used to determine antioxidant activity, and all test samples and standard compounds were tested in triplicate. The IC50 values (i.e., \u0026micro;g/mL concentration that produces 50% growth inhibition for this experimental system) of each compound are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and were determined using GraphPad Prism software version 7.0.\u003c/p\u003e \u003cp\u003eThe radical scavenging activity (RSA) was determined using the formula:\u003c/p\u003e \u003cp\u003e% Inhibition = [AB - AA / AB] \u0026times; 100, where AB represents the absorbance of the blank solution and AA denotes the absorbance of the test sample.\u003c/p\u003e \u003cp\u003eThe antioxidant potential of the synthesized compounds 5a\u0026ndash;5f was evaluated using the DPPH free radical scavenging assay, as illustrated in Fig.\u0026nbsp;1. The results summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicate that all tested derivatives exhibited notable radical scavenging activity when compared with the standard antioxidant, ascorbic acid. The DPPH assay, expressed in terms of IC₅₀ values, is a well-established and reliable method for assessing the antioxidant efficiency of 1,3,4-oxadiazole derivatives. Among the tested compounds, compound 5f demonstrated the highest antioxidant activity, reflected by its lowest IC₅₀ value, indicating superior free radical scavenging efficiency. This enhanced activity may be attributed to favorable structural features that facilitate effective electron or proton donation and stabilization of the resulting radical species. In contrast, the remaining compounds exhibited comparatively higher IC₅₀ values, suggesting reduced scavenging capacity, possibly due to less favorable electronic or substituent effects.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant scavenging activity of compounds 5a-f on DPPH* free radical at different concentrations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEntry\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIC50\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e125\u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e69.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e73.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e74.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.01\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e77.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e78.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e76.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e78.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e5j\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e71.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e74.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e75.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e69.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e70.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e71.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e75.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13.62\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAscorbic acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e88.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.42\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* Results are expressed as mean of triplicates\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003cp\u003e** The IC50 values defined as the concentration corresponding to 50% growth inhibition, \u0026micro;g/mL.\u003c/p\u003e"},{"header":"EXPERIMENTAL","content":"\u003cp\u003eCommercially available reagents and solvents were used. TLC plates were used for analytical thin layer chromatography. It has a silica gel G coating for reaction monitoring and retardation factor determination. In an iodine chamber, TLC spots were visible. On an electrothermal melting point apparatus, the melting points of newly synthesized derivatives were determined and found to be uncorrected. A perkin-Elmer 2400 CHN analyzer confirmed the element analysis (percent C,H and N). The SHIMADZU LC-MS 2010 spectrometer was used to obtain mass spectra. \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded on a Varian Mercury-400, 100 MHz in DMSO-d\u003csub\u003e6\u003c/sub\u003e as a solvent and TMS as an internal standard using a 5 mm tube on a Bruker Advance Ⅱ 400 MHz.\u003c/p\u003e\n\u003ch3\u003eStep 1: Synthesis of 2-chloro-N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)acetamide (3)\u003c/h3\u003e\n\u003cp\u003eSubstituted 6-amino-3-ethoxy-4-phenyl-1,3a, 4, 7a-tetrahydropyrano[2,3-c]pyrazole-5-carbonitrile (1 mol) was added in DMF with containing few drops of triethylamine (TEA) with continue stirred at room temperature. Then chloro-acetyl chloride (1.5 mol) was added above solution with continue stirring and maintained the temp 0\u0026ndash;5 \u003csup\u003e0\u003c/sup\u003eC. Then solution was stirred at room temperature for 4 hours. The completion of reaction was monitored periodically by using toluene: acetone (7:3) as mobile phase. Then the solution was added into crushed ice and the obtained product was filtered, washed with water, dried and recrystallized from ethanol.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStep 2: General Procedure for the Synthesis of N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide\u003c/b\u003e: 2-chloro-N-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)acetamide (0.01 mol) obtained was further reacted with 5-(pyridin-4-yl)-1,3,4- oxadiazole-2-thiol (0.01 mol) for 4 h at room temperature. In the presence of K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (0.02 mol) and acetone (20 ml) was used as reaction medium. After the completion of reaction judged on TLC using Toluene: Acetone (7:3) as mobile phase. The solution was poured in ice-cold water and stirred for 30 min. The solid obtained was filtered off, washed with water, dried and recrystallized from ethanol to give 5a\u0026ndash;5f.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eN-(5-cyano-3-ethoxy-4-phenyl-1,3a,4,7a-tetrahydrophyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5a)\u003c/h2\u003e \u003cp\u003eYield 68%, mp 175\u0026deg;C. IR spectrum, ν, cm\u0026ndash;1: 2954, 1720, 1464, 1212, 1050. 1H NMR spectrum, δ, ppm: 1.26 t (3H, CH\u003csub\u003e3\u003c/sub\u003e), 2.3 d (1H, CH), 3.3 q (1H, CH), 3.66 q (2H, CH\u003csub\u003e2\u003c/sub\u003e), 4.00 s (2H, CH\u003csub\u003e2\u003c/sub\u003e), 5.1 s (1H, CH), 7.40\u0026ndash;7.98 m (10 H, Ar-H), 9.13 s (1H, NH), 9.21 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.3, 15.9, 39.3, 48.0, 54.7, 61.4, 72.6, 117.3, 126.1, 127.5, 128.8, 129.2, 139.2, 158.2, 164.3, 167.8. Mass spectrum, m/z: 503.0 [M\u0026thinsp;+\u0026thinsp;H]+.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eN-(4-(4-chlorophenyl)-5-cyano-3-ethoxy-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5b)\u003c/h3\u003e\n\u003cp\u003eYield 70%, mp 165\u0026deg;C. IR spectrum, ν, cm\u0026ndash;1: 2925, 1715, 1445, 1210, 1087. 1H NMR spectrum, δ, ppm: 1.30 t (3H, CH\u003csub\u003e3\u003c/sub\u003e), 2.45 d (1H, CH), 3.26 q (1H, CH), 3.71 q (2H, CH\u003csub\u003e2\u003c/sub\u003e), 4.12 s (2H, CH\u003csub\u003e2\u003c/sub\u003e), 5.6 s (1H, CH), 7.32\u0026ndash;7.78 m (9 H, Ar-H), 9.10 s (1H, NH), 9.26 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.0, 15.7, 39.0, 48.6, 54.8, 61.7, 72.8, 117.6, 126.3, 127.6, 128.9, 129.3, 139.5, 158.7, 164.1, 167.6. Mass spectrum, m/z: 537.0 [M\u0026thinsp;+\u0026thinsp;H]+.\u003c/p\u003e\n\u003ch3\u003eN-(5-cyano-3-ethoxy-4-(4-hydroxyphenyl)-1,3a, 4, 7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5c)\u003c/h3\u003e\n\u003cp\u003eYield 72%, mp 143\u0026deg;C. IR spectrum, ν, cm\u0026ndash;1: 3412, 2922, 1720, 1416, 1200, 1048. 1H NMR spectrum, δ, ppm: 1.12 t (3H, CH\u003csub\u003e3\u003c/sub\u003e), 2.59 d (1H, CH), 3.56 q (1H, CH), 3.89 q (2H, CH\u003csub\u003e2\u003c/sub\u003e), 4.14 s (2H, CH\u003csub\u003e2\u003c/sub\u003e), 5.68 s (1H, CH), 7.25\u0026ndash;7.87 m (9 H, Ar-H), 9.00 s (1H, OH), 9.13 s (1H, NH), 9.30 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.2, 15.9, 39.2, 48.7, 54.3, 61.0, 72.7, 117.5, 126.7, 127.3, 128.7, 129.0, 139.4, 158.5, 164.0, 167.2. Mass spectrum, m/z: 519.0 [M\u0026thinsp;+\u0026thinsp;H]+.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eN-(5-cyano-3-ethoxy-4-(3-hydroxyphenyl)-1,3a, 4, 7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5d)\u003c/h2\u003e \u003cp\u003eYield 75%, mp 126\u0026deg;C. IR spectrum, ν, cm\u0026ndash;1: 3420, 2923, 1724, 1418, 1206, 1047. 1H NMR spectrum, δ, ppm: 1.10 t (3H, CH\u003csub\u003e3\u003c/sub\u003e), 2.54 d (1H, CH), 3.45 q (1H, CH), 3.48 q (2H, CH\u003csub\u003e2\u003c/sub\u003e), 4.41 s (2H, CH\u003csub\u003e2\u003c/sub\u003e), 5.46 s (1H, CH), 7.20\u0026ndash;7.89 m (9 H, Ar-H), 9.06 s (1H, OH), 9.15 s (1H, NH), 9.45 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.3, 15.7, 39.6, 48.8, 54.5, 61.2, 72.7, 117.9, 126.6, 127.4, 128.6, 129.3, 139.4, 158.7, 164.6, 167.3. Mass spectrum, m/z: 519.0 [M\u0026thinsp;+\u0026thinsp;H]+.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eN-(4-(3-chlorophenyl)-5-cyano-3-ethoxy-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5e)\u003c/h2\u003e \u003cp\u003eYield 70%, mp 160\u0026deg;C. IR spectrum, ν, cm\u0026ndash;1: 2920, 1714, 1459, 1223, 1078. 1H NMR spectrum, δ, ppm: 1.31 t (3H, CH\u003csub\u003e3\u003c/sub\u003e), 2.41 d (1H, CH), 3.27 q (1H, CH), 3.76 q (2H, CH\u003csub\u003e2\u003c/sub\u003e), 4.00 s (2H, CH\u003csub\u003e2\u003c/sub\u003e), 5.23 s (1H, CH), 7.30\u0026ndash;7.89 m (9 H, Ar-H), 9.00 s (1H, NH), 9.20 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.2, 15.9, 39.2, 48.7, 54.3, 61.4, 72.7, 117.3, 126.7, 127.6, 128.9, 129.3, 139.5, 158.7, 164.1, 167.6. Mass spectrum, m/z: 537.0 [M\u0026thinsp;+\u0026thinsp;H]+.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eN-(5-cyano-3-ethoxy-4-(4-nitrophenyl)-1,3a,4,7a-tetrahydropyrano[2,3-c]pyrazol-6-yl)-2-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)acetamide (5f)\u003c/h2\u003e \u003cp\u003eYield 73%, mp 156\u0026deg;C. IR spectrum, ν, cm\u0026ndash;1: 2912, 1715, 1448, 1256, 1070. 1H NMR spectrum, δ, ppm: 1.32 t (3H, CH\u003csub\u003e3\u003c/sub\u003e), 2.47 d (1H, CH), 3.23 q (1H, CH), 3.56 q (2H, CH\u003csub\u003e2\u003c/sub\u003e), 4.20 s (2H, CH\u003csub\u003e2\u003c/sub\u003e), 5.56 s (1H, CH), 7.15\u0026ndash;7.79 m (9 H, Ar-H), 9.01 s (1H, NH), 9.26 s (1H, NH). 13C NMR spectrum, δC, ppm: 15.3, 15.98, 39.4, 48.0, 54.5, 61.6, 72.7, 117.2, 126.3, 127.7, 128.5, 129.4, 139.8, 158.6, 164.0, 167.8. Mass spectrum, m/z: 548.0 [M\u0026thinsp;+\u0026thinsp;H]+.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, a series of pyrazole\u0026ndash;1,3,4-oxadiazole\u0026ndash;thioacetamide derivatives was successfully synthesized through a straightforward multistep protocol and unambiguously characterized by FT-IR, \u0026sup1;H NMR, and \u0026sup1;\u0026sup3;C NMR spectroscopic analyses. The synthesized compounds were evaluated for their antimicrobial and antioxidant activities, and several derivatives exhibited moderate to good inhibitory effects against Gram-positive and Gram-negative bacteria as well as fungal strains. Compounds bearing electron-withdrawing substituents on the aromatic ring showed comparatively enhanced antimicrobial activity, indicating a clear substituent-dependent structure\u0026ndash;activity relationship. Antioxidant screening using the DPPH assay revealed that all derivatives possessed measurable radical scavenging ability, with compound 5f displaying the most pronounced activity within the series. The present study highlights pyrazole\u0026ndash;oxadiazole\u0026ndash;thioacetamide hybrids as useful chemical intermediates with promising biological profiles, warranting further structural modification and investigation.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Sources:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no financial support for the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were no commercial or financial links that may be deemed a potential conflict of interest during the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDr. Shweta A. Patel and Dr. Ami Patel conceptualized the study. Dr. Shweta A. Patel carried out the synthesis and characterization of the compounds, performed the biological activity studies, interpreted the data, and prepared the manuscript. Dr. Ami Patel contributed to data analysis, and critically reviewed and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u003c/strong\u003e not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKapoor K, Kaur N, Sohal HS, Kaur M, Singh K, Kumar A. Drugs and their mode of action: A review on sulfur-containing heterocyclic compounds. Polycycl Aromat Compd. 2025;45:136\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiswas T, Mittal RK, Sharma V, Kanupriya, Mishra I. Nitrogen-fused heterocycles: Empowering anticancer drug discovery. Med Chem. 2024;20:369\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOmar A. 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Mini Rev Med Chem. 2019;19:477\u0026ndash;509.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFei Q, Liu C, Luo Y, Chen H, Ma F, Xu S, Wu W. Design, synthesis and antimicrobial evaluation of triazole-substituted oxadiazole derivatives. Mol Diversity. 2025;29:255\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdo MA, Badawy SA, Fadda AA, Elmorsy MR. Triazole- and oxadiazole-based hydrazide-hydrazone derivatives with antioxidant potential. BMC Chem. 2025;19:1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalem MM, El-Adl K, El-Morsy A, Abulkhair HS. Oxadiazole derivatives as promising anticancer agents: A two-decade overview. RSC Adv. 2025;15:32778\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaheen K, Alam A, Elhenawy AA, Khan IA, Rahman FU, Ali A, et al. In vivo anti-inflammatory evaluation of oxadiazole derivatives bearing flurbiprofen. Sci Rep. 2025;15:29144.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah M, Singh C, Yadav MR, Nagani A. Oxadiazoles as multitarget therapeutic agents in Alzheimer\u0026rsquo;s disease. J Saudi Chem Soc. 2025;29:1\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pyrazole, 1,3,4-Oxadiazole, Thioacetamide, Antimicrobial activity, Antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-8836359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8836359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel series of pyrazole\u0026ndash;1,3,4-oxadiazole\u0026ndash;thioacetamide hybrids was designed, synthesized, and evaluated for antimicrobial, antioxidant, and pharmacokinetic properties. The compounds were prepared via a multistep synthetic approach, and their structures were confirmed using spectroscopic techniques, including IR, \u0026sup1;H NMR, and \u0026sup1;\u0026sup3;C NMR. IR analysis revealed characteristic bands corresponding to N\u0026ndash;H, C\u0026thinsp;=\u0026thinsp;O, C\u0026thinsp;=\u0026thinsp;N, and C\u0026ndash;S functionalities, while \u0026sup1;H NMR spectra displayed signals for aromatic, pyrazole, oxadiazole, and thioacetamide protons. The \u0026sup1;\u0026sup3;C NMR spectra further supported the assigned structures, showing resonances for carbonyl, heterocyclic, and aromatic carbons. The synthesized hybrids were screened for antimicrobial activity against selected Gram-positive and Gram-negative bacterial strains as well as fungal pathogens. Several derivatives exhibited promising inhibitory activity, highlighting the potential of these scaffolds in combating microbial infections. Antioxidant potential was assessed using standard radical scavenging assays, demonstrating significant activity in selected compounds.\u003c/p\u003e","manuscriptTitle":"Design and synthesis of pyrazole–oxadiazole–thioacetamide hybrids with antimicrobial and antioxidant properties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 21:55:50","doi":"10.21203/rs.3.rs-8836359/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60e6d070-d60b-458e-a3b5-fbbb999f5145","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T00:54:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 21:55:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8836359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8836359","identity":"rs-8836359","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}