SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF NOVEL5, 6-DIHYDRO-3-(SUBSTITUTED PHENYL) [1, 2, 4] TRIAZOLE (3, 4-B) [1, 3, 4] THIADIAZINE-7-ONE DERIVATIVES FOR ANTIMICROBIAL AND FREE RADICAL SCAVENGING ACTIVITY

SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF NOVEL5, 6-DIHYDRO-3-(SUBSTITUTED PHENYL) [1, 2, 4] TRIAZOLE (3, 4-B) [1, 3, 4] THIADIAZINE-7-ONE DERIVATIVES FOR ANTIMICROBIAL AND FREE RADICAL SCAVENGING ACTIVITY

Kirti Sharma, Tinku *, Vikas Jogpal, Mohit Sanduja and Prabhakar Kumar Verma

School of Medical and Allied Sciences, G. D. Goenka University, Sohna Road, Gurgaon, Haryana, India.

ABSTRACT: The present research aims to synthesize novel 5, 6-dihydro-3-(substituted phenyl) [1, 2, 4] triazolo (3, 4-b) [1, 3, 4] thiadiazine-7-one derivatives and their physiochemical evaluation for antibacterial and antioxidant activities. Novel synthetic schemes were designed to synthesize compounds (1-6), which were then characterized using physicochemical and spectroscopic methods (IR, 1HNMR). In-vitro antimicrobial activity was performed against Gram-positive (Staphylococcus aureus, Bacillius subtilis), Gram-negative (Escherichia coli) bacterial strains and fungal strains (Aspergilus niger, Candida albicans) of all synthesized compounds. In addition, Minimum inhibition concentration (MIC) and Minimum fungicidal concentration (MFC) values were determined using tube dilution methods using standards such as norfloxacin and fluconazole. The free radical scavenging activity was also assessed using the DPPH assay, with vitamin C serving as a reference molecule. Compound 3 was discovered to have the best antibacterial activity (IC₅₀- 80.27) of all the compounds, while the rest compounds showed modest scavenging activity.

Keywords: Triazole, Fluconazole, Norfloxacin, Tube dilution, Ascorbic acid, Minimum bactericidal concentration (MIC), Minimum fungicidal concentration (MFC)

INTRODUCTION: The incidence of systemic microbial infections has surged due to increased immune-compromised hosts. Furthermore, the rising reports of germ resistance to effective antibiotics are becoming a chief supply of concern. As a result, the excessive value of therapy, toxicities, and drug resistance offer a new dilemma that prompted microbial scientists and chemists to develop novel antimicrobial with extra unique action ⁹.

Hence, the hunt for novel antimicrobial will continue to be a crucial and hard attempt for medicinal chemists. Triazoles and their derivatives are a fascinating family of chemical compounds offering various potential pharmacological activities.

Scaffolds containing 1,2,4 triazole containing ring systems were reported to have anti-bacterial, anti-fungal ^{16, 18, 19}, anti-tubercular  ^1-15, antihyper-glycemic ¹⁷, analgesic, anti-inflammatory ⁷ free radical scavenging) ³, anti-cancer ¹¹, anticonvulsant (Kamboj et al., 2015), antiviral ¹⁰, anti-amoebiosis ¹⁵, anti-depressant and other central nervous system (CNS) activities ⁶. Treatment of infectious ailments is a critical and challenging task because of the growing infectious and developing range of multi-drug resistant microbial infections.

Despite the availability of a large range of antibiotics and chemotherapeutics for medical use, the upward thrust of old and novel antibiotic resistance evokes the discovery of a new class of antimicrobial drugs ¹⁸. Usually, the 1, 2, 4-triazole moiety appears often in the structure of different natural products and the synthesis of molecules integrating this moiety has picked the interest of chemists and biologists alike, owing to their varied biological activity in pharmacological and agrochemical sectors ⁹.

MATERIALS AND METHOD: Melting points were obtained using sonar melting point equipment in open capillary tubes and are uncorrected. Thin layer chromatography on a silica gel plate (Merck silica gel G) was used to monitor the completion of the reaction. ¹H-NMR spectra were obtained via Brucker Avance II 400 NMR spectrometer using suitable deuterated solvents and are represented in parts per million (6 ppm) downfield from tetramethylsilane (TMS-internal standard). IR spectra were acquired on a Brucker 1206-0280 using OPUS 7.2.139.1294 software.

1. Synthesis of 5,6-dihydro-3-(substituted phenyl)[1,2,4]triazolo(3,4-b) [1,3,4] thiadiazine-7-one: Initially, substituted aromatic acid (0.001 mol) and absolute alcohol (50 mL) were simultaneously placed in a 100 mL round bottom flask (RBF). In that mixture, a few drops of concentrated sulfuric acid (H₂SO₄) and a little porcelain chip were added. To maintain the anhydrous environment, a condenser with a calcium chloride guard tube was connected at the top. After refluxing for 6-8 hours, the reaction mixture was concentrated under reduced pressure to yield the ester. After successful completion of first stage, hydrazine hydrate (0.2 mol) and absolute alcohol (50 mL) were added and the reaction mixture was refluxed for 8-10 hours. Subsequently, the concoction was concentrated and poured over crushed ice. The solid substituted benzohydrazides mass was separated, filtered, and dried ¹³. Further, the reaction mixture of an ethanolic solution of substituted benzohydrazides (0.01mol), potassium hydroxide (0.015 mol), and carbon disulphide (0.015 mol) was refluxed for 7-8 hours. Following that, the mixture was acidified with dil. HCI, and the resulting solid were collected, washed with distilled water, recrystallized, and dried in a vacuum. Afterward, the product was refluxed with hydrazine hydrate for 6-8 h; this reaction leads to the cyclization of traizoles with the elimination of water molecules. Finally, chloroacetyl chloride is refluxed with the final product to obtain the bicyclic targeted heterocyclic derivative. Thin layer chromatography using mobile phase chloroform: Methanol (8.5:1.5) was used to confirm the completion of the reaction. Eventually, 5, 6-dihydro-3 [substituted, 2,4] triazolo (3, 4b) [1,3,4] thiadiazine-7-one derivatives were synthesized Fig. 1 washed, and recrystallized using an appropriate solvent.

1.1 In-vitro Study:

1.1.1 Free Radical Scavenging Activity: Free radical scavenging activity of synthesized compounds was evaluated using a Shimadzu UV-visible spectrophotometer using DPPH (2, 2-diphenyl-2-picrylhydrazyl hydrate) as standard. Different dilutions (25, 50, 75, and 100 μg/ml of the compounds were prepared using methanol as a diluting agent. From the stock solutions, 0.25, 0.50, 0.75, 0.1 ml were taken in different test tubes. Freshly prepared DPPH solution (1.3 mg/ml) was added to each test tube and kept dark for 30 minutes. The absorbance was measured at 517 nm using UV-spectrophotometer ². The percentage inhibition was calculated using the following formula:

%I = A_control – A_sample / A_control × 100

A_control equals the absorbance of the control reaction and A_sample equals the absorbance of the test chemical (1-6). The IC₅₀ of the substances was determined by plotting inhibition against the concentration on a graph. A calibration curve was generated for varying amounts of ascorbic acid, which was utilized as a positive control (standard). All of the experiments were done in triplicate.

1.1.2 Antimicrobial Activity: The synthesized compounds were tested for antimicrobial activity against gram-positive bacteria (S. aureus, B. subtilis), gram-negative bacteria (E. coli), and fungal strains (C. albicans, A. niger) using the tube dilution technique.

Compounds 1–6 and standard samples were dissolved in dimethylsulphoxide (DMSO) to a 100 g/ml concentration. Dilutions of the test and control chemicals (norfloxacin and fluconazole) were produced in double strength nutritional broth-I.P. for bacteria and Sabouraud dextrose broth I.P. for fungi ⁵. The samples were incubated at 37 °C for 24 hours for gram's bacteria, 25 °C for 7 days for fungal stain (A. niger) and 37 °C for 48 hours for Candida albicans. The results were reported in terms of the lowest inhibitor concentration.

RESULTS AND DISCUSSION:

2.1 Synthesis of 5,6-dihydro-3-(substituted phenyl)[1,2,4]triazolo(3,4-b) [1,3,4]thiadiazine-7-one: A series of 5,6-dihydro-3-(substituted phenyl) [1, 2, 4] triazolo (3, 4-b)[1, 3, 4] thiadiazine-one compounds(1-6) were synthesized as per the above-mentioned scheme in Fig. 1 for the antimicrobial and free radical scavenging activity concerning the 1, 2, 4 triazole. The physicochemical characteristics of synthesized compounds (1-6) are presented in Table 1. IR and 1HNMR characterized the structures of synthesized compounds.

Fig. 2 revealed the spectral details of 5, 6-dihydro-3-(substituted phenyl) [1,2,4] triazolo (3, 4-b) thiadiazine-7-one intermediates and synthesized compounds (1-6) was Table 2.

TABLE 1: PHYSICOCHEMICAL PROPERTIES OF THE SYNTHESIZED COMPOUNDS

FIG. 1: SYNTHESIS OF 5, 6-DIHYDRO-3-(SUBSTITUTED PHENYL) [1, 2, 4] TRIAZOLO (3, 4-B) [1, 3, 4] THIADIAZINE-7-ONE

Intermediate 1 (cm^-1) 3027(C-H str., aromatic), 1707 (C=O str., carbonyl), 1275 (C-O str., ester), 1663 (C=C str., aromatic ring), 1568 (C-C str., aromatic in ring), 1438 (C-H bend, alkane), 3490 (O-H str.)

Intermediate 2 (cm^-1) 3017 (C-H str., aromatic), 1751 (C=O str., carbonyl), 1250 (C-O str., ester), 1670 (C=C str., aromatic ring), 1560 (C-C str., aromatic in ring), 1400 (C-H bend, alkane), 3487 (O-H str.), 1098(C-N str.), 3309 (N-H str., hydrazide).

Intermediate 3 (cm^-1) 3013 (C-H str., aromatic), 1256 (C-O str., C-OH), 1672 (C=C str., aromatic ring), 1509 (C-C str., aromatic in ring), 1404 (C-H bend, alkane), 3487 (O-H), 1084 (C-N str.), 3348 (N-H str., amino), 2367 (S-H str., thiole).

Compound 1 (cm^-1) 2987(C-H str., aromatic), 1707 (C=O str., carbonyl), 1251 (C-O str., ester), 1660 (C=C str., aromatic ring), 1567 (C-C str., aromatic in ring), 3467 (O-H str.), 1063 (C-N str.), 3331 (N-H str.).

Compound 2 (cm^-1) 3000 (C-H str., aromatic), 1717 (C=O str., carbonyl), 1251 (C-O str., ester), 1649 (C=C str., aromatic ring), 1541 (C-C str., aromatic in ring), 1070 (C-N str.), 3335 (N-H str.).

Compound 3 (cm^-1) 3034 (C-H str., aromatic), 1731 (C=O str., carbonyl), 1257 (C-O str., ester), 1681 (C=C str., aromatic ring), 1571 (C-C str., aromatic in ring), 1033 (C-O str., ether), 1061 (C-N str.) 3407 (N-H str.).

Compound 4 (cm^-1) 3046 (C-H str., aromatic), 1730 (C=O str., carbonyl), 1256 (C-O str., ester), 1689 (C=C str., aromatic ring), 1571 (C-C str., aromatic in ring), 1020 (C-O str., ether), 1104 (C-N str.), 3478 (N-H str.)

Compound 5 (cm^-1)3097 (C-H str., aromatic), 1748 (C=O str., carbonyl), 1680 (C=C str., aromatic ring), 1565 (C-C str., aromatic in ring), 1108 (C-N str.), 3368 (N-H str.), 745 (C-Cl str. halide).

Compound 6 (cm^-1) 3064 (C-H str., aromatic), 1730 (C=O str., carbonyl), 1680 (C=C str., aromatic ring), 1571 (C-C str., aromatic in ring), 1125 (C-N str.), 3368 (N-H str. Amine), 1076 (C-N str.).

FIG. 2: SPECTRAL DETAILS OF 5, 6-DIHYDRO-3-(SUBSTITUTED PHENYL) [1, 2, 4] TRIAZOLO (3, 4-B) THIADIAZINE-7-ONE INTERMEDIATES AND SYNTHESIZED COMPOUNDS: A) COMPOUND (1), B) COMPOUND (2),C) COMPOUND (3),D) COMPOUND (4),E) COMPOUND (5), F) COMPOUND (6)

FIG. 3: % AGE INHIBITION VALUE OF ACTIVE ANTIOXIDANT COMPOUNDS

FIG. 4: ¹H NMR SPECTRA: A) COMPOUND 1 B) COMPOUND 2 C) COMPOUND 3

2.2 Free Radical Scavenging Activity: Free radical scavenging using DPPH is regarded as a reliable in-vitro approach for assessing free radical scavenging activity. The free radical scavenging activity of synthesized compounds (1-6) against DPPH was evaluated and the result in Table 4 was computed as the inhibition percentage (percent I). The percent inhibition of several substances was shown to vary with concentration. The activity of o-hydroxy phenyl (compound 1) was shown in Fig. 3 at 75 g/ml and 100 g/ml, with values of 53.69 percent and 68.23 percent, respectively. Compound 3 had the highest activity at 100 g/ml of 3,4, 5-trimethoxy phenyl derivative (68.39 %). At 100 g/ml, 2-hydroxy-5-nitro phenyl (compound4) had the greatest efficacy (76.39 percent).

At 100g/ml, p-amino phenyl (compound 6) has considerable free radical scavenging properties (68.65 percent). The results reveal that most compounds had excellent action at greater concentrations of up to 100 g/ml.

When the concentration is reduced, the free radical scavenging activity decreases in comparison to ascorbic acid, however, the percent inhibition of the produced molecule with ascorbic acid increases with decreasing concentration.

TABLE 2: DETAILS OF 5, 6-DIHYDRO-3-(SUBSTITUTED PHENYL) [1, 2, 4] TRIAZOLO (3, 4-B) THIADIAZINE-7-ONE SYNTHESIZED COMPOUNDS (1-6)

S. no.	R1	Chemical Name
1.		o-hydroxy phenyl (2-hydroxy phenyl)
2.		p- methyl phenyl (4-methyl phenyl)
3.		3,4,5-trimethoxy phenyl
4.		2-hydroxy-5-nitro phenyl
5.		2-chloro phenyl
6.		p-amino phenyl (4-amino phenyl)

TABLE 3: ANTIMICROBIAL ACTIVITY (pMIC in μM/ml) OF SYNTHESIZED COMPOUNDS

Norfloxacin* Fluconazole**

TABLE 4: PERCENTAGE INHIBITION (% I) AND IC₅₀ VALUE OF THE SYNTHESIZED COMPOUNDS

2.3 Antimicrobial Activity: The antibacterial activity of triazole derivatives was measured using the MIC, and the findings are shown in Table 3. Almost all of the synthesized compounds (1-6) outperformed norfloxacin and fluconazole as conventional medications in terms of antibacterial and antifungal activities. The chemicals were efficient against a variety of bacteria types, including E. coli, B. subtilis, S. aureus, and Candida albicans. Compound 1 showed excellent activity against B. subtilis and S. aureus with a MIC value of 06.25, whilst other strains demonstrated action with a MIC value of 06.25 & 12.50. Compound 2 demonstrated the most effective antibacterial action against S. aureus with a MIC value (3.12), as well as improved results against C. albicans, A. niger, and B. subtilis with MIC values 06.25,06.25,06.25, respectively) (6.25). It did not demonstrate satisfactory action against E. coli. Compound 3 had no similar antibacterial action against the strains except S. aureus, where the MIC was determined to be 06.25. Compound 4 had antibacterial efficacy comparable to compound 3. Compound 5 was not extremely efficient against E. coli, with a MIC (25.00) and did not yield statistically significant results compared to the standard. Compound 6 has demonstrated promising effectiveness against S. aureus and Candida albicans. As a result, compound 2 was discovered to have the most potent antibacterial activity among the six produced compounds.

The results showed that triazole had high antibacterial action against a variety of bacteria types. As a result, the chemical derivative of substituted p-methyl phenyl (o-hydroxy phenyl) was shown to be more efficient against antibacterial activity than compound 1. Compounds 3(3, 4, 5-trimethoxy phenyl) and 4 (2-hydroxy-5-nitro phenyl) have the same action as compound 2. Compound 6 (p-amino phenyl) showed equivalent efficacy to the o-hydroxy phenyl derivative, whereas the o-chloro phenyl derivative did not outperform the other compounds. Different free radical species are often created and quenched for certain metabolic activities by an effective free radical scavenging pathway in the body. When the production of these species exceeds the free radical scavenging mechanism's capacity, oxidative damage to tissues and biomolecules occurs, leading to disease conditions, particularly degenerative diseases ⁴ and the pathogenesis of certain human diseases such as cancer, aging, diabetes, and atherosclerosis ¹². Triazole derivatives have already gotten a lot of press because of their wide range of pharmacological properties, including anti-carcinogenic ²⁰, antiviral ¹⁴, analgesic, and anti-malarial properties.

CONCLUSION: Most triazole derivatives had moderate to good antibacterial activity, although the free radical scavenging activity of the various compounds varied at different doses. Compound 3 was shown to have the most efficient antibacterial activity (MIC=03.12) against E. coli, B. subtilis, S. aureus, C. albicans, and A. niger, as well as free radical scavenging activity against DPPH, among the synthesized compounds. We may and conclude from the aforementioned findings that 1, 2, 4-triazole has high antibacterial and free radical scavenging activity and can be employed in the future in the medical area.

ACKNOWLEDGMENT: I'd like to show my gratitude to the Department of Pharmaceutical Sciences, MD University Rohtak, Haryana-India for providing me with all research facilities during the study project.

CONFLICT OF INTEREST: No conflicts of interest were declared by any of the writers.

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    How to cite this article:

    Sharma K, Tinku, Jogpal V, Sanduja M and Verma PK: Synthesis, characterization and biological evaluation of novel 5, 6-dihydro-3-(substituted phenyl) [1, 2, 4] triazole (3, 4-b) [1, 3, 4] thiadiazine-7-one derivatives for antimicrobial and free radical scavenging activity. Int J Pharm Sci & Res 2023; 14(1): 444-52. doi: 10.13040/IJPSR.0975-8232.14(1).444-52.

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