% Yield 74%, M.P. 215°C, ¹HNMR (DMSO-d6,400 MHz) 3.579 (s, 1H,-CH2),9.906 (s, 1H, = C.H.), 6.99–7.88 (m, Aryl H =8H), 10.852 (s, 1H, -N.H.) ¹³CNMR 193.23, 173.43, 158.14, 157.78, I.R. t_max 3207 (-N.H.), 1657 (-C=N), 1276 (-C=S), Elemental analysis calc for C₁₈H₁₅N₅OS C, 61.86; H, 4.35; N, 20.08; O,4.53; S, 9.18 Found: C, 61.89, H,4.32; N, 20.12; O; 4.57; M.S. Est m/z 49.10 (100.0%), 350.10 (22.2%), 351.11(1.8%)

% Yield 72%, M.P. 213°C, ¹HNMR (DMSO-d6,400 MHz) 3.579 (s, 1H,-CH2), 9.906 (s, 1H, = C.H.),6.99–7.88 (m, Aryl H =8H), 10.852 (s, 1H, -N.H.) ¹³CNMR 193.23, 173.43, 158.14, 157.78, I.R. t_max 3207 (-N.H.), 657 (-C=N), 1276 (-C=S), Est m/z: 349.10 (100.0%), 350.10 (22.2%), 351.10 (5.3%), 351.11 (1.8%); Elemental Analysis for C₁₈H₁₅N₅OS: C, 61.89; H, 4.34; N, 20.08; O, 4.56; S, 9.18

% Yield 75%, M.P. 219°C, ¹HNMR (DMSO-d6,400 MHz) 3.579 (s, 1H,-CH2), 9.906 (s, 1H, = C.H.), 6.99–7.88 (m, Aryl H =8H), 10.852 (s, 1H, -N.H.) ¹³CNMR 193.23, 173.43, 158.14, 157.78, I.R. t_max 3207 (-N.H.), 657 (-C=N), 1276 (-C=S),m/z: 367.07 (100.0%), 369.06 (36.5%), 368.07 (20.4%), 370.07 (6.3%), 369.07 (2.3%), 368.06 (1.8%), 370.06 (1.8%), 371.06 (1.6%); Elemental Analysis for C18H15ClN5S: C, 58.72; H, 3.83; Cl, 9.62; N, 19.07; S, 8.79

% Yield 75%, M.P. 220°C, ¹HNMR (DMSO-d6,400 MHz) 3.579 (s, 1H,-CH2), 9.906 (s, 1H, = C.H.), 6.99–7.88 (m, Aryl H =8H), 10.852 (s, 1H, -N.H.) ¹³CNMR 193.23, 173.43, 158.14, 157.78, I.R. t_max 3207 (-NH) 657 (-C=N), 1276 (-C=S), Elemental analysis for C₁₈H₁₄N₆O₂S; C 57.18; H, 3.77; N, 22.29; O, 8.46; S,8.47; M.S. Est m/z: 378.09(100.0%), 379.09(22.6%), 380.11(5.5%), 380.11(1.8%).

% Yield 77%, M.P. 215°C, ¹HNMR (DMSO-d6,400 MHz) 3.693 (s, 1H, -CH2), 9.881 (s, 1H, = C.H.), 7.1–7.81 (m, Aryl H =8H), 10.863 (s, 1H, -NH) ¹³ CNMR 193.77, 173.80 158.30, 138.03, I.R. t_max 3207 (-N.H.), 1657 (-C=N), 1276 (-C=S), Elemental analysis calc for C₁₈H₁₅N₅OS C, 61.85; H, 4.36; N, 20.09; O,4.53; S, 9.15; M.S. Est m/z 349.10 (100.0%), 350.10 (22.2%), 351.11(1.8%)

% Yield 75%, M. P 219°C, ¹HNMR (DMSO-d6,400 MHz) 3.693 (s, 1H, -CH2), 9.881 (s, 1H, = C.H.), 7.1–7.81 (m, Aryl H =8H), 10.863 (s, 1H, -N.H.),¹³CNMR 193.77, 173.80 158.30, 138.03, I.R. t_max3207 (-N.H.), 1657 (-C=N), 1276 (-C=S), M.S. Est m/z: 349.10 (100.0%), 350.10 (22.2%), 351.11(1.8%)

% Yield 71%, M.P. 220°C, ¹HNMR (DMSO-d6,400 MHz) 3.693 (s, 1H, -CH2), 9.881 (s, 1H, = C.H.), 7.1–7.81 (m, Aryl H =8H), 10.863 (s, 1H, -N.H.) ¹³CNMR 193.77, 173.80 158.30, 138.03, I.R. t_max 3207 (-N.H.), 1657 (-C=N), 1276 (-C=S), Est m/z: 409.12 (100.0%), 410.12 (24.3%), 411.12 (5.1%), 411.13 (2.9%), 412.12 (1.1%; Elemental Analysis for C₂₀H₁₉N₅O₃S: C, 58.63; H, 4.64; N, 17.13; O, 11.77; S, 7.87

The DPPH assay was executed via the technique stated by (Bibi et al., 2011). DPPH(Diphenyl-1-picrylhydrazyl), a free radical, is bagged to inquire about the antioxidant ability of the synthesized compounds. 10 μL from the synthesized compound (1 mg/mL DMSO) is added to 190 μL DPPH solution., making up the final conc. of 50 μg/mL. Absorbance was observed at 517 nm using a microplate reader after incubating at 37 C for 30 min approx. the given formula was computed % F r e e   r a d i c a l   S c a v e n g i n g   A c t i v i t y = 1 − A s / A c   *   100

DPPH assay was repeated in triplicate using DMSO as -ve and ascorbic acid as + ve control; findings are expressed as means standard deviation.

This evaluation was based on the protocol by (Bibi et al., 2011). This mechanism’s key point is reducing Mo (VI) to Mo (V). A solution consisting of 4 mM Ammonium molybdate, 28 mM SodiumPhosphate, and 0.6 M Sulphuric acid is utilized, which forms Phosphate Molybdenum Complex which is green in color and gives absorption = 630 nm. 20 mL of the sample is added to 180 mL of reagent, which is incubated at 95°C for one and a half hours and cooled to 25°C.

The process used for TRP was illustrated by (Bibi et al., 2011). The potassium ferrocyanide calorimetric method is used for 100 μL of each solution, 200 μL Phosphate buffer (0.2 M, pH 6.6), and 250 μL of potassium ferricyanide was mixed and incubated at 50°C for 20 min, to this solution, 10 percent (200 μL) trichloroacetic acid was added, centrifugation was done at 3,000 rpm for 10 min, the supernatant was collected, added with FeCl₃ (50 μL), and shifted to the microplate. Absorbance was recorded at 700 nm. Ascorbic acid (100 μg/mL DMSO) is the +ve control for the test, and dimethyl sulfoxide (DMSO) is the blank.

The micro broth dilution method was used to establish the antibacterial potential of test samples against Staphylococcus aureus (ATCC-6538), Bacillus subtilis (ATCC-6633), Escherichia coli (ATCC-25922), Klebsiella pneumoniae (ATCC-1705), and Pseudomonas aeruginosa (ATCC-15442) and for the resistant strains of MRSA Staphylococcus aureus, P. aeruginosa and Escherichia coli. The procedure used is described by (Sarker et al., 2007). Bacterial inoculum was formed under aseptic conditions keeping the density near 5 × 10⁴ CFU/mL in pre-autoclaved nutrient broth. Test samples of an aliquot of 5 µL were shifted to labeled wells of 96 well plates, and after that addition of 195 µL of nutrient broth; was accomplished. After that, a 2-fold serial dilution of every sample was arranged to get final concentrations of 12.5 μg/mL. Then, with the help of a micropipette, 195 µL of bacterial culture of respective strains was poured into each well; further advancement was incubating of plates for 30 min at the temperature of 37°C. Zero-time readings at 630 nm were observed with the help of a microplate reader. Incubation of plates was again carried out at 37°C for 1 day, absorbance was noted, and net change in turbidity was the difference between two values of absorbance.

The antifungal response to these chemical moieties was evaluated through a procedure designated by (Zahra et al., 2017). Amphotericin B (4 mg/mL DMSO) is used as + ve control and DMSO as-ve one (5 μL/disc). Sterile SDA-coated plates were used, on which 100 µL of suspension of spores for each strain (previously reaped-in-Tween-20 solution) was spread. Discs of sterile filter paper containing 5 µL of sample solution (4 mg/mL DMSO) were kept on plates and incubated for 2 days at 28°C. Zones of inhibition were recorded.

The cytotoxic evaluation was accomplished using the decorum designed by (Khan et al., 2011). To a plate of 96 wells, artificial seawater and nauplii were added. Test samples were taken in wells (40, 20, 10, 5 μg/mL) after making up the final volume with seawater, the plates were left uncovered under the lamp, and after 24 h, the number of surviving larvae was calculated. For samples having ≥50% mortality, the median lethal conc was calculated using the table curve software, Doxorubicin was used as + ve control, and DMSO was the -ve. The percentage of killed organisms was found using the formula. P e r c e n t a g e   M o r t a l i t y = N o   o f   d e a d   s h r i m p s ÷ T o t a l   N u m b e r   o f   S h r i m p s × 100

The protocol followed for this is mentioned (Fatima et al., 2015). To assess the test compound’s inhibitory potential, Streptomyces 85 E strains were used, which were refreshed in tryptone soya broth at 37 for 1 day. Spores from refreshed cultures were utilized to prepare the bacterial culture. 100 μg compound was loaded on 5 mm discs of filter paper. These were kept on sowed plates. DMSO-permeated discs were the -ve control. The incubation period was 7 days, and outcomes were stated as bald and clear zones of inhibition.

It is one of the very commonly utilized methods for the assessment of the antioxidant ability of compounds. DPPH turns from purple to yellow, which indicates the synthetic compound’s scavenging potential (Rahman et al., 2017). DMSO and A.A. were used as -ve and +ve control. The antioxidant potential of compounds was assessed at 50 μg/mL, 25 μg/mL, 12.5 μg/mL, and 6.25 μg/mL. All the compounds showed very slight antioxidant potential, with 5b (32%) being the most active in the series, followed by 5a (22.55%), then 5c and 5d (11.1%), and the least active was 5e (8.3%). The results are summarized in Figure 5. Production of free radicals is an outcome of regular oxygen consumption in the body; whenever something is missing with the natural antioxidant mechanism, the accumulation of these free radicals may lead to cytotoxic interaction with the body systems and may cause damage to genomic content of various cells, and proteins in the body (Barbuceanu et al., 2014). Free radical accumulation is also associated with different diseases, e.g., coronary heart disease, neuronic disorders, diabetes, necrosis, and various tumors (Ismaili.et al., 2008). The antioxidant potential of the compound depends upon its reducing power; the introduction of hydroxy and methoxy groups on the Benzene ring led to better reducing power or promising antioxidant activity (Rakesh et al., 2015). In this study, compounds 5b and 5a exhibited good antioxidant potential as having -OCH₃ and groups at position three of the benzene ring. However, a change in position from three to four for Hydroxy benzaldehyde showed a significant loss in reducing potential. The basicity of the nitrogen atom also plays a significant role, i.e., the more basic is the nitrogen atom, the more active the compound will be in terms of its reducing potential (Hayun et al., 2018).

The antioxidant potential of test compounds was estimated by TAC &TRP assays. The spectrophotometric method was used to analyze the formation of the green-colored phosphomolibdenum compound. The newly synthesized compounds have shown noteworthy results in the TAC assay; 5b showed the maximum activity among the synthetic compounds, whereas 5c showed the least. The findings are depicted in Figure 6.

For estimation of reducing potential TRP assay was carried out, compound 5b (706.42 ± 0.004 µg AAE/mg) showed the highest activity, followed by 5g (642.67 ± 0.001 µg AAE/mg), 5e (619.18 ± 0.010 µg AAE/mg), and 5a (592.26 ± 0.007 µg AAE/mg). It is apparent from the results that compounds having hydroxy and methoxy in their structure showed promising activities as compared to those having nitro and chloro moieties at various positions. The primary mechanism of TRP assay is based on the compound’s capacity to reduce free radicals, which are generated during the procedure. i.e., Fe³⁺ is reduced to Fe²⁺ (Lee et al., 2015). Various factors determine the antioxidant potential of the compound; a few to explain include the chemical attributes, nature of antioxidant moieties, conditions and mechanism of the reaction, which accounts for the reason for a compound’s activity in one assay and none-to-less response in the other (Francenia Santos-Sanchez et al., 2019). However, compounds with hydroxyl and methoxy groups form the bases for potential candidates as an antioxidant (Rakesh et al., 2015).

Schiff base tri-azo compounds were investigated for antibacterial potential. The antibacterial assays were performed for nine different bacterial strains, which include; Gram-positive (, S. aureus, B. subtilis), Gram-negative (Pseudomonas aeruginosa, K. pneumoniae, E. coli), and resistant bacterial strains (S. aureus, MRSA, Pseudomonas aeruginosa, E. coli. Compounds that exhibited significant activity, producing inhibitory growth zone ≥10 mm, their MIC values were determined (through broth microdilution method), ranging from 25 to 3.12 μg/mL. The Antibacterial activity of compounds and their minimum inhibitory concentration values are mentioned in Tables 2, 3. These results indicate that all the compounds showed in-vitro anti-microbial activity to some extent. None of the compounds in the given series was able to show inhibitory activity for B. subtilis, and only 5g and 5f were effective against S. aureus with MIC 25 μg/mL (5a-5g) showed activity against K. pneumoniae, and exhibited different values for MIC, ranging from 12.5 μg/mL for 5a, 5c, 5d, and 5e. 6.25 μg/mL for 5b and 5g, and the least, Table 4 3.12 μg/mL was for 5f. 5a, 5b (MIC =12.5 μg/mL), 5e (MIC = 3.12 μg/mL, same as Ciprofloxacin) were active against Pseudomonas aeruginosa, and 5c, 5d, 5e (MIC = 25 μg/mL) were active against E. coli. These compounds were effective against only one resistant bacterial strain, i.e., MRSA, and showed a MIC value of 12.5 μg/mL. Substituents with electron-withdrawing attributes, e.g., -NO_2, -Cl, -OH on the aromatic ring, assert a great impact on the antibacterial activity of synthetic compounds. It is also observed that an increase in the number of -OH groups tend to enhance the activity (Hosamani and Shingalapur, 2011). Due to the difference in cell-wall composition of Gram + ve and -ve bacteria, different compounds show different features towards the same micro-organism (Goszczyn’ska et al., 2015).

The Synthetic compound library was subjected to an antifungal assay using five different strains of Fungai, i.e., Fusarium solani (FCBP-0291), Aspergillus fumigatus (FCBP-66), Aspergillus flavus (FCBP-0064), Aspergillus niger (FCBP-0198) and Mucor species (FCBP-0300). Amphotericin B was the standard drug used. None of the compounds showed antifungal activity, except 5e, which was active against only one strain of Fusarium solani. The outcomes are concise in Table 5.

These synthetic products were evaluated to estimate the inhibitory potential for protein kinase of Streptomycin 85 E. Protein kinase is involved with the mycelium production of this species, so the growth patterns were observed as the bald zone. The clear zone specifies no cytotoxic bacterial growth; as far as the bald zone is concerned, it shows inhibition of protein kinase. The results for this assay for compounds (5a-5g) are given in Table 5. One of the propitious targets in cancer treatment is the inhibition of protein kinases. Protein phosphorylation by protein kinases at various residues regulates diverse biological processes, which may include apoptosis, proliferation and differentiation of cells. Deregulation at any step in early tumorigenesis may lead to cancer (Yao et al., 2011). Aerial hyphae formation in Streptomyces depends upon protein kinase activity; this scenario is exploited in this test; to inquire about these compounds’ kinase inhibition potential and to look at their anti-cancer potential. The advantage of using Streptomyces for kinase inhibition assay is its predisposition to a wide range of eukaryotic cells. Whole-cell assay of Streptomyces not only identifies the compound’s cytotoxic potential but also identifies signal transduction inhibitors for various activities, e.g., antitumor, anti-mycobacterial, and and anti-infective (Waters et al., 2002). Among the tested compounds, only 5a, 5b, and 5g showed inhibitory activity. 5b showed the max. Activity with a bald zone of 10.5 mm and a clear zone of 7.5 mm, following is 5a with a bald zone of 9 mm and a clear zone of 6 mm, and for 5g, values are 7.5 and 6 mm, respectively. It means that these compounds have the potential for protein kinase inhibition as well as the toxicity of the compound.