Synthesis, characterization, and biological activities of substituted pyridine-based azomethine scaffolds European Journal of Chemistry 15 (3) (2024) 226-231 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2024 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.15.3.226-231.2532 European Journal of Chemistry View Journal Online View Article Online Synthesis, characterization, and biological activities of substituted pyridine-based azomethine scaffolds Gautam Prabhakar Sadawarte 1, Jamatsing Darbarsing Rajput 1, Amol Diliprao Kale 2, Rajendra Pralhadrao Phase 2, and Vasant Bhagwan Jagrut 3,* 1 Department of Chemistry, Faculty of Science, Bhagirathi Purnapatre Arts, Sitabai Mangilal Agrawal Science and Kasturbai Khandu Chaudhari Commerce College, Chalisgaon, Dist-Jalgaon 424101, Maharashtra State, India 2 Department of Chemistry, Faculty of Science, Lal Bahadur Shastri Senior College, Partur, Dist-Jalna 431501, Maharashtra State, India 3 Department of Chemistry, Faculty of Science, Swami Vivekanand Senior College, Mantha, Dist-Jalna 431504, Maharashtra State, India * Corresponding author at: Department of Chemistry, Faculty of Science, Swami Vivekanand Senior College, Mantha, Dist-Jalna 431504, Maharashtra State, India. e-mail: jagrutvb@gmail.com (V.B. Jagrut). 10.5155/eurjchem.15.3.226-231.2532 Received: 25 February 2024 Received in revised form: 19 April 2024 Accepted: 22 June 2024 Published online: 30 September 2024 Printed: 30 September 2024 The present research work describes the synthesis of a new series of heterocyclic compounds, namely, pyridine-based azomethine scaffolds. A total of eight derivatives were prepared, purified, and characterized by analytical methods such as 1H NMR, 13C NMR, and IR spectroscopic techniques. All compounds were used to investigate their alpha-amylase inhibition activity. We have also reported antimicrobial activity using a micro broth dilution assay, with microbial strains Pseudomonas aeruginosa (NCIM 5031), Escherichia coli (NCIM 2065), Bacillus subtilis (NCIM 2699), Aspergillus niger (NCIM 620), Aspergillus fumigatus (NCIM 902), and Aspergillus flavus (NCIM 549). Finally, we report the antioxidant activity of the synthesized derivatives using a DPPH free radical assay. Pyridine Synthesis Azomethine Antioxidant Antimicrobial Alpha-Amylase Cite this: Eur. J. Chem. 2024, 15(3), 226-231 Journal website: www.eurjchem.com 1. Introduction Pyridine nuclei have been reported to be found in a large number of naturally occurring compounds. Among nitrogen- based heterocycles, the compounds and content of pyridine scaffolds are valued for their biological, medicinal, optical, chemical, and physical properties [1]. One of the heterocycles used most frequently in the field of drug design is pyridine- based ring systems [2]. This is mainly due to their significant impact on pharmacological activity, which has resulted in the discovery of several broad-spectrum therapeutic agents [3]. Pyridine is added to ethyl alcohol as a solvent to render it intoxicating. Numerous biological activities of pyridine have been discovered, including those that are antiviral, anti- tubercular, anticancer, antibacterial, and antidiabetic. In this review, we have covered some more recent biological activities, including antidotes, antioxidants, antileishmanial, antichagasic, and antithrombin [4]. The pyridine core and its derivatives have shown broad biological effects, such as antimycobacterial [5,6], anti-inflammatory [7,8], analgesic activities [9], antibacterial activity against pathogenic bacterial strains (S. aureus and E. coli) [10], anticancer (leukemia, breast cancer and idiopathic pulmonary fibrosis) [11], antihypertensive in the treatment of angina pectoris [12], antihistamine [13], anticholinergic, antiulcerative [14], anti-HIV [15], antiviral agents [16-20], antidiabetic [21], and antioxidant potency [22,23]. Nicotinic and isoniazid derivatives are important classes of compounds based on pyridine having pharmacological proper- ties such as in vitro anti-tubercular activity against Mycobac- terium tuberculosis [24,25], S. aureus [26-28], anticancer against human cancer cells with potent cytotoxicity [29], anti- fungal activities in vitro against dimorphic fungus, Histoplasma capsulatum and var. capsulatum [30], potential multi-target profiles for the treatment of Alzheimer’s disease [31]. Further- more, Sadawarte et al. reported that thiopyridine derivatives have promising antidiabetic activity; recently, many more investigations reported hybrid pyridine nuclei in type I and type II diabetic research, on the basis of this vast literature, we have selected substituted scaffolds based on pyridine nuclei for the present investigation [32]. We designed and synthesized a series of new 3-azomethine hybrid derivatives 3a-h and charac- terized them using IR, 1H NMR, 13C NMR, and MS techniques, ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.15.3.226-231.2532 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.15.3.226-231.2532 mailto:jagrutvb@gmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.15.3.226-231.2532&domain=pdf&date_stamp=2024-09-30 Sadawarte et al. / European Journal of Chemistry 15 (3) (2024) 226-231 227 2024 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.15.3.226-231.2532 Table 1. Optimization for the synthesis of pyridine-based scaffolds. Compound R Time required for synthesis, h 3a H 4 3b 6-CH3 6 3c 6-Br 3 3d 4-F 3 3e 4-Cl 2 3f 3,4-Cl 2 3g 3-Cl, 4-CH3 4 3h 4-CF3 4 NHN NH2 H O OH R Ethanol, acetic acid 2 to 6 h Room temperature NN H N H HO R 1 2 3a-h Scheme 1. Synthesis of compounds 3a-h. and to explore their alpha-amylase inhibition and antimicrobial activities. 2. Experimental 2.1. Instrumentation The melting points were determined using the Tanco PLT- 276 Delux Model melting temperature apparatus. The IR spectra were measured as KBr pellets by using a Shimadzu double beam infrared spectrometer. The 1H NMR and 13C NMR spectra were recorded on an Advanced Bruker Neo AM 500 MHz spectrometer at room temperature in CDCl3 solution using tetramethyl silane (TMS) as an internal standard reference. Chemical shifts were expressed in delta (ppm) downfield from TMS, and the coupling constants were in Hertz (Hz). LC-MS spectra were run on a Q-TOF Micromass Spectrometer (ESI-MS) at 70 eV. 2.2. Synthesis We purchased high-purity chemicals from LOBA and Merck. N2-isopropyl-4-methylpyridine-2,6-diamine was solved in 5 mL of ethanol and later in this solution was added a solution of the particular different substituted aldehyde (3a: 2-hydroxybenz- aldehyde; 3b: 2-hydroxy-3-methylbenzaldehyde; 3c: 2- hydroxy-3-bromobenzaldehyde; 3d: 2-hydroxy-5-Fluorobenz- aldehyde; 3e: 2-hydroxy-5-chlorobenzaldehyde; 3f: 2-hydroxy- 5,6-dichlorobenzaldehyde; 3g: 2-hydroxy-5-methyl-6-chloro benzaldehyde; 3h: 2-hydroxy-5-trifluoromethylbenzaldehyde) in 5 mL of ethanol. A catalytic amount of glacial acetic acid (3 to 4 drops) was added. The resulting reaction mixture received was stirred at room temperature for 2-6 hours. The solvent was removed under reduced pressure, and the residue was then washed with water. Finally, the resulting solid product was collected and recrystallized from the solution of the ethanol: water mixture solution (80:20, v:v) to obtain the desired compound 3a-h (Scheme 1, Table 1). 2-((6-(Isopropylamino)-4-methylpyridin-2-ylimino) methyl) phenol (3a): Color: Pale yellow. Yield: 78%. M.p.: 102-104 °C. FT-IR (KBr, ν, cm): 3277 (O-H), 3277 (N-H), 2547 (C-H), 1610 (C=N), 1475, 1463 (C=C). 1H NMR (500 MHz, CDCl3, δ, ppm): 1.261 (d, 6H, J = 6.8 Hz, 2×CH3), 2.27 (s, 3H, CH3), 2.86-2.91 (m, 1H, CH), 6.88-6.89 (d, 2H, J = 7.2 Hz, ArH), 7.10-7.12 (dd, 2H, J = 7.2 Hz, Py), 7.55-7.58 (t, 2H, J = 7.2 Hz, Ar-H), 7.70-7.71 (s, 1H, CH), 13.04-13.07 (s(b), 1H, H-N), 13.30-13.33 (s (b), 1H, H-O). 13C NMR (125 MHz, CDCl3, δ, ppm): 16.21, 24.21, 35.6, 76.82, 77.06, 77.27, 77.32, 115.01, 119.63, 123.20, 126.87, 131.06, 136.22, 152.09, 155.30. MS (ESI, m/z): 269.23 [M]+. 2-((6-(Isopropylamino)-4-methylpyridin-2-ylimino) methyl)- 6-methylphenol (3b): Color: Brown. Yield: 72 %. M.p.: 98-100 °C. FT-IR (KBr, ν, cm): 3292 (O-H), 2919 (N-H), 2567 (C-H), 1612 (C=N), 1469, 1390 (C=C). 1H NMR (500 MHz, CDCl3, δ, ppm): 1.24-1.26 (d, 6H, J = 6.8 Hz, 2×CH3), 2.27 (s, 3H, CH3), 2.30 (s, 3H, CH3), 3.84-3.86 (m, 1H, CH), 6.80-6.81 (d, 2H, J = 7.2 Hz, ArH), 7.10-7.12 (dd, 2H, J = 7.2 Hz, Py), 7.52-7.54 (t, 1H, J = 7.2 Hz, ArH), 7.71-7.72 (s, 1H, CH), 13.04-13.07 (s (b), 1H, H-N), 13.30- 13.33 (s(b), 1H, H-O). 13C NMR (125 MHz, CDCl3, δ, ppm): 16.18, 24.19, 29.32, 32.82, 110.03, 115.91, 125.28, 133.40, 152.85, 154.48. MS (ESI, m/z): 282.26 [M]+. 2-((6-(Isopropylamino)-4-methylpyridin-2-ylimino) methyl)- 6-bromophenol (3c): Color: Red. Yield: 80 %. M.p.: 106-108 °C. FT-IR (KBr, ν, cm): 3169 (O-H), 2953 (N-H), 2767 (C-H), 1612 (C=N), 1483, 1384 (C=C). 1H NMR (500 MHz, CDCl3, δ, ppm): 1.22-1.26 (d, 6H, J = 6.8 Hz, 2×CH3), 2.23 (s, 3H, CH3), 2.87-2.89 (m, 1H, CH), 7.22-7.24 (s, 1H, J = 7.2 Hz, Py), 7.50-7.51 (s, 1H, J = 7.2 Hz, Py), 7.70-7.72 (s, 1H, J = 7.2 Hz, ArH), 7.83-7.85 (d, 2H, J = 7.2 Hz, ArH), 7.92-7.96 (s, 1H, CH), 13.04-13.06 (s(b), 1H, NH), 13.40-13.43 (s(b), 1H, H-O). 13C NMR (125 MHz, CDCl3, δ, ppm): 16.13, 23.86, 32.26, 110.47, 116.89, 122.63, 126.60, 132.71, 135.28, 151.25, 153.90. MS (ESI, m/z): 349.00 [M]+. 2-((6-(Isopropylamino)-4-methylpyridin-2-ylimino) methyl)- 4-fluorophenol (3d): Color: Yellow. Yield: 80 %. M.p.: 100-102 °C. FT-IR (KBr, ν, cm): 3288 (O-H), 2947 (N-H), 2713 (C-H), 1610 (C=N), 1467, 1368 (C=C). 1H NMR (500 MHz, CDCl3, δ, ppm): 1.24-1.26 (d, 6H, J = 6.8 Hz, 2×CH3), 2.23 (s, 3H, CH3), 2.80-2.81 (m, 1H, CH), 7.13-7.15 (s, 2H, J = 7.2 Hz, Py), 7.43-7.44 (s, 1H, J = 7.2 Hz, ArH), 7.61-7.62 (s, 1H, J = 7.2 Hz, ArH), 7.69- 7.70 (s, 1H, J = 7.2 Hz, ArH), 7.70-7.72 (s, 1H, CH), 13.07-13.09 (s(b), 2H, H-N, H-O). 13C NMR (125 MHz, CDCl3, δ, ppm): 15.06, 24.95, 32.80, 101.45, 112.99, 115.39, 118.06, 122.82, 125.32, 131.21, 133.40, 154.46, 154.46. MS (ESI, m/z): 286.00 [M]+. 2-((6-(Isopropylamino)-4-methylpyridin-2-ylimino) methyl)- 4-chlorophenol (3e): Color: Yellow. Yield: 82%. M.p.: 104-106 °C. FT-IR (KBr, ν, cm): 3273 (O-H), 2945 (N-H), 2738 (C-H), 1616 (C=N), 1467, 1309 (C=C). 1H NMR (500 MHz, CDCl3, δ, ppm): 1.26-1.28 (d, 6H, J = 6.8 Hz, 2×CH3), 2.24 (s, 3H, CH3), 2.86-2.89 (m, 1H, CH), 7.12-7.15 (s, 1H, J = 7.2 Hz, Py), 7.30-7.32 (s, 1H, J = 7.2 Hz, Py), 7.66-7.69 (s, 2H, J = 7.2 Hz, ArH), 7.72 - 7.74 (s, 1H, J = 7.2 Hz, ArH), 7.74-7.77 (s, 1H, CH), 13.22-13.24 (s(b), 2H, H-N, H-O). 13C NMR (125 MHz, CDCl3, δ, ppm): 15.78, 23.26, 32.72, 110.79, 119.32, 120.02, 123.22, 125.29, 126.80, 131.55, 136.35, 153.57, 154.62. MS (ESI, m/z): 302.00 [M]+. 2-((6-(Isopropylamino)-4-methylpyridin-2-ylimino) methyl)- 3,4-dichlorophenol (3f): Color: Creamy. Yield: 80 %. M.p.: 108- 110 °C. FT-IR (KBr, ν, cm): 3267 (O-H), 2954 (N-H), 2627 (C-H), 1610 (C=N), 1463, 1377 (C=C). 1H NMR (500 MHz, CDCl3, δ, ppm): 1.25-1.27(d, 6H, J = 6.8 Hz, 2×CH3), 2.24 (s, 3H, CH3), 2.88- 228 Sadawarte et al. / European Journal of Chemistry 15 (3) (2024) 226-231 2024 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.15.3.226-231.2532 2.89 (m, 1H, CH), 7.16-7.18 (s, 1H, J = 7.2 Hz, Py), 7.73-7.75 (s, 1H, J = 7.2 Hz, Py), 7.83-7.87 (d, 2H, J = 7.2 Hz, ArH), 7.91-7.93 (s, 1H, CH), 12.75-12.78 (s(b), 1H, H-N), 13.40-13.41 (s(b), 1H, H-O). 13C NMR (125 MHz, CDCl3, δ, ppm): 15.76, 23.94, 32.69, 110.50, 121.06, 125.39, 131.64, 138.45, 154.59, 154.54. MS (ESI, m/z): 338.24 [M]. 2-((6-(Isopropylamino)-4-methylpyridin-2-ylimino) methyl)- 3-chloro-4-methyl phenol (3g): Color: Dark brown. Yield: 74 %. M.p.: 100-102 °C. FT-IR (KBr, ν, cm): 3304 (O-H), 2954 (N-H), 2721 (C-H), 1610 (C=N), 1405, 1304 (C=C). 1H NMR (500 MHz, CDCl3, δ, ppm): 2.23-2.24 (d, 6H, J = 6.8 Hz, 2×CH3), 2.24 (s, 3H, CH3), 2.51 (s, 3H, CH3), 2.61-2.63 (m, 1H, CH), 7.44-7.45 (s, 2H, J = 7.2 Hz, ArH + Py), 7.76-7.78 (s, 1H, J = 7.2 Hz, Py), 7.78-7.79 (s, 1H, J = 7.2 Hz, ArH), 7.81-7.82 (s, 1H, CH), 13.00-13.01 (s(b), 1H, H-N), 13.23-13.24 (s(b), 1H, H-O). 13C NMR (125 MHz, CDCl3, δ, ppm): 10.11, 20.01, 32.71, 111.02, 124.47, 125.07, 133.20, 139.04, 152.16, 154.43. MS (ESI, m/z): 316.00 [M]+. 2-((6-(Isopropylamino)-4-methylpyridin-2-ylimino) methyl)- 4-(trifluoromethyl)phenol (3h): Color: Creamy. Yield: 80%. M.p.: 102-104 °C. FT-IR (KBr, ν, cm): 3394 (O-H), 3061 (N-H), 2881 (C-H), 1625 (C=N), 1433, 1334 (C=C). 1H NMR (500 MHz, CDCl3, δ, ppm): 2.25-2.26 (d, 6H, J = 6.8 Hz, 2×CH3), 2.33-2.34 (s, 3H, CH3), 2.81-2.82 (m, 1H, CH), 7.27-7.29 (s, 1H, J = 7.2 Hz, Py), 7.70-7.71 (s, 1H, J = 7.2 Hz, Py), 7.86-7.87 (s, 1H, J = 7.2 Hz, ArH), 7.87- 7.89 (s, 1H, J = 7.2 Hz, ArH), 8.01-8.04 (s, 1H, J = 7.2 Hz, ArH ), 8.10-8.11 (s, 1H, CH), 13.01-13.02 (s(b), 1H, H-N), 13.24- 13.25 (s(b), 1H, H-O). 13C NMR (125 MHz, CDCl3, δ, ppm): 16.24, 23.95, 32.01, 112.03, 121.22, 124.32, 133.35, 139.66, 152.52, 154.55. MS (ESI, m/z): 336.01 [M]+. 2.3. Biological activities 2.3.1. Evaluation of α-amylase inhibitory activity α-Amylase inhibition assay was performed using a method previously reported in the literature [33]. 250 μL of total solutions of compounds having various concentrations were placed in different hard glass tubes and 250 μL of 0.02 M sodium phosphate buffer (pH = 6.9) containing α-amylase solution was added. All solutions were pre-incubated at 25 °C for 10 min, after which 250 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH = 6.9) were added at time intervals and then again incubated at 25 °C for 10 min. The reaction was terminated by adding 500 μL of dinitrosalicylic acid (DNS) reagent. The tubes were then incubated in boiling water for 5 min and cooled to room temperature. The reaction mixture was diluted with 5 mL of distilled water and the absorbance was measured at 540 nm using a spectro- photometer. A control was prepared using the same procedure as described above, replacing the compounds with water. The concentrations of the samples that resulted in 50% inhibition of enzyme activity (IC50) were graphically determined. 2.3.2. Antimicrobial activity In vitro antimicrobial activities of the derivatives were determined using different microorganisms by microbroth dilution assay [34]. Microbial strains, Pseudomonas aeruginosa (NCIM 5031), Escherichia coli (NCIM 2065), Bacillus subtilis (NCIM 2699), Aspergillus niger (NCIM 620), Aspergillus fumigatus (NCIM 902), Aspergillus flavus (NCIM 549) were obtained from the National Chemical Laboratory, Pune, India. The bacteria were maintained in nutrient broth (NB) and the fungal strains were maintained in Sabouraud dextrose broth at 37 °C. 2.3.2.1. Preparation of inoculums For bacteria, the bacterial strains used as inoculums were grown at 37 ° C to obtain optical density 0.6 at 600 nm. Colony forming units (CFU) were counted using the serial plate dilution method and bacterial counts were adjusted to 1×105 - 1×106 CFU/mL for susceptibility testing. For the fungus, the fungal inoculums were prepared from cultures grown 10 days in potato dextrose agar medium. The Petri dishes were flooded with 8 to 10 mL of distilled water and the conidia were scraped using a sterile spatula. The spore density of each fungus was adjusted with a spectrophotometer (A595 nm) to obtain a final concentration of approximately 1×105 spores/mL. Micro-Broth dilution assay - The determination of the mini- mum inhibitory concentration (MIC) was carried out using the micro-broth dilution method. According to the NCCLS guidelines. The test was carried out in 96-well culture plates (Hi-media). The compounds were dissolved in dimethyl sulfoxide to produce eight different concentrations, viz. 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 mg/mL in the wells by a twofold dilution method. The negative control was prepared using dimethyl sulfoxide, and the same concentrations of tetracycline for bacteria and amphotericin B for the fungus were used as positive control. The 96 well plates were incubated for 24 h and 48 h at 37 °C for bacteria and fungus, respectively. The lowest concentration of each compound that inhibited visual growth was considered the MIC of that respective compound. All such experiments were repeated three times. 2.3.3. Antioxidant activity, DPPH assay The antioxidant activity of the compound and the standard was taken on the basis of the radical scavenging effect of the stable DPPH free radical. 1,1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical that can accept an electron or hydrogen radical to become a stable dual magnetic molecule. Due to its odd electron, the alcoholic solution of DPPH shows a strong absorption band at λmax = 517 nm. DPPH radicals react with suitable reactive agents, and then the electrons become paired off, and the solution loses color equally to the number of electrons taken up. The reduction of DPPH radicals can be observed by decreasing the absorbance at λmax = 517 nm. 100 ppm DPPH solution was prepared in alcohol and 1.0 mL of this solution was added to dilutions of the compound solution in water at different concentrations (25, 50, 100, 250, 500 ppm). Thirty minutes later, the absorbance was measured at λmax = 517 nm. Butylated hydroxytoluene was used as standard. 3. Results and discussion 3.1. Synthesis The N2-isopropyl-4-methylpyridine-2,6-diamine used in this study was prepared by direct reduction of the nitro group of pyridine with iron in HCl solution following the standard procedure given in the reference [21,35]. Pyridine derivatives (3a-h) were synthesized in a single step method. First, substituted pyridines were reacted with benzaldehyde molecules in ethanol using glacial acetic acid as a catalyst. A total of eight derivatives were prepared using the same protocol. Depending on the substitution benzaldehyde, the reaction time is varied for each derivative. Compounds 3a-h were synthesized according to the method given in the experimental section and their structures were confirmed with the characterization data provided in the experimental section. The pure compounds obtained were then examined to determine their biological activities. Sadawarte et al. / European Journal of Chemistry 15 (3) (2024) 226-231 229 2024 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.15.3.226-231.2532 Table 2. α-Amylase inhibition activity of compounds 3a-h at various concentrations with acarbose standard. Compounds / Concentration % Inhibition at different concentration 25 μL 50 μL 100 μL 250 μL 3a 12.00 16.40 20.20 20.80 3b 36.92 40.20 44.05 50.30 3c 8.20 12.10 12.90 16.20 3d 38.20 42.85 50.44 58.18 3e 23.20 26.80 32.40 24.80 3f 50.00 50.80 58.60 62.10 3g 4.20 10.50 12.20 12.90 3h 10.00 14.20 26.50 30.50 Acarbose 62.90 68.70 74.00 82.00 Table 3. Antimicrobial activity of compounds 3a-h. Compounds Minimum inhibitory concentration each microbial species (mg/mL) P. aeruginosa E. coli S. typhi B. subtilis A. niger A. fumigatus A. flavus 3a 2.5 2.5 2.5 5.0 2 2.5 2 3b 2.5 2.5 2.5 0.156 1.25 2.5 2.5 3c 5.0 5.0 5.0 0.312 1.25 5.0 2.5 3d 5.0 5.0 2.5 0.156 1.00 5.0 2.5 3e 10.5 5.0 2.5 5.0 1.25 2 2.5 3f 10.5 5.0 5.0 5.0 5.0 10.5 2.5 3g 5.0 5.0 5.0 5.0 5.0 5.0 2.5 3h 10.0 10.0 5.0 2.5 2.5 5.0 2.5 Tetracycline 0.00125 0.01 0.01 0.00125 - - - Amphotericin B - - - - 0.00125 0.000156 0.000156 Table 4. Antioxidant activity of compounds 3a-f at various concentrations. Compounds / Concentrations % Inhibition at different concentration 25 ppm 50 ppm 100 ppm 250 ppm 500 ppm 3a 40.12 44.58 48.6 56.70 65.74 3b 36.92 40.20 44.05 50.30 55.56 3c 55.98 60.50 70.12 76.20 80.00 3d 38.20 42.85 50.44 58.18 64.80 3e 37.12 40.01 44.08 55.20 60.12 3f 50.00 58.00 60.00 76.00 80.00 3g 40.80 45.20 48.98 57.20 66.14 3h 32.50 38.20 40.20 45.50 48.20 Butylated hydroxytoluene 62.90 68.70 74.00 82.00 86.00 Figure 1. Possible active sites for free radical generation. 3.2. Biological activities 3.2.1. Evaluation of α-amylase inhibitory activity α-Amylase inhibition activities of pyridine-based deriva- tives were carried out using α-amylase inhibition assay that shows inhibition at various concentrations (Table 2). Compounds 3b, 3d, 3e, and 3f had shown comparable α- inhibition at 25, 50, 100, and 250 μL, when compared to the standard acarbose molecule, while compounds 3a, 3c, and 3g had lower inhibition at all concentrations. Theoretically α- amylase can be inhibited by NH-C=N-C=C-N-C=C-OH cavity, but the obtained result shows the effect of substituent on the inhibition of α-amylase. Compounds 3a, 3c, 3g and 3h that have bulky substituents on the aromatic nucleus can create a barrier in the protein cavity for inhibition. For this reason, compounds 3a, 3c, 3g, and 3h show low inhibition at all concentrations. Compounds 3b, 3d, 3e, and 3f show considerable inhibition at all concentrations due to perfect inhibition of the protein cavity. Among all derivatives, 3d and 3f show excellent inhibition at all concentrations which are comparable to the standard molecule, acarbose. 3.2.2 Antimicrobial activity Different classes of microorganisms were selected from different habitats for antimicrobial activity. The microbial strains Pseudomonas aeruginosa (NCIM 5031), Escherichia coli (NCIM 2065), Salmonella typhi (NCIM 2501), exhibited a minimum inhibitory concentration (MIC) of 0.01 and 0.00125 mg/mL, respectively, for the standard antibacterial agent tetracycline. Whereas MICs of 0.00125, 0.000156, and 0.000156 mg/mL were shown by amphotericin B against Aspergillus niger (NCIM 620), Aspergillus fumigatus (NCIM 902) and Aspergillus flavus (NCIM 549), respectively. The minimum inhibitory concentration 2.5 mg/mL was shown in derivatives 3a and 3b for Gram-ve bacteria. E. Coli. by compounds 3a, 3b, 3d, 3e and 3j for S. Typhi, compounds 3b and 3d exhibited 0.156 mg/mL MIC for Gram +ve bacteria Bacillus subtilis. Compound 3f showed a MIC of 0.625 for the pathogenic plant fungus A. niger in antifungal tests. The derivatives 3a and 3b showed 2.5 mg/mL MIC for A. fumigatus while seven derivatives exhibited 2.5 mg/mL MIC against A. flavus. From the results (Table 3), it can be concluded that compound 3f has broad-spectrum antifungal and antibacterial activities, and compound 3f has more antifungal activity. In contrast, the 230 Sadawarte et al. / European Journal of Chemistry 15 (3) (2024) 226-231 2024 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.15.3.226-231.2532 activity of this compound is much less (MIC levels are very high) than that of the standard tetracycline and amphotericin B. 3.2.3. Antioxidant activity, DPPH assay All substituted pyridine-based azomethine scaffolds had shown remarkable antioxidant activity at concentrations of 25, 50, 100, 250, and 500 ppm (Table 4). The entire compound with four active sites (Figure 1) from which radicals may be generated after its interaction with DPPH and which exhibits considerable radical scavenging activity over DPPH at lower concentrations. As the concentration of the DPPH molecule increases to 100, 250, and 500 ppm, the radical scavenging activity increases. Compounds 3a, 3b, 3d, 3e, 3g, and 3h have comparable activity at higher concentrations due to the temperate reactive substitution on the phenol ring. In contrast, compounds 3c and 3f have notable activity at higher concentrations due to reactive substitution on the phenol ring. 4. Conclusions In summary, we present a simple synthetic method for preparing azomethine derivatives and characterizing them through various spectroscopic methods. A series of novel pyridine-based compounds labeled 3a-h were created, which incorporate an azomethine functional group. These compounds were synthesized using N2-isopropyl-4-methylpyridine-2,6- diamine as building blocks. Comprehensive spectral analyses (IR, 1H NMR, and 13C NMR) confirmed the structures of these newly synthesized compounds. Subsequently, their anti- bacterial, antifungal, antioxidant and α-amylase inhibitory properties against Gram-positive bacteria, Gram-negative bacteria, and fungi were evaluated. The results revealed that many of the newly developed compounds exhibited significant activity in these areas compared to standard commercial drugs. Acknowledgements The authors express their gratitude to all colleagues and contributors for their continuous support throughout this study. Gautam Sadawarte also thanks the Principal, Head, Department of Chemistry of Deogiri College Aurangabad and Principal of Bhagirathi Purnpatre Arts, Sitabai Mangilal Agrawal Science and Kasturbai Khandu Chaudhari Commerce College, Chalisgaon, India. Disclosure statement Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Vasant Bhagwan Jagrut; Methodology: Gautam Prabhakar Sadawarte; Software: Jamatsing Darbarsing Rajput; Validation: Amol Diliprao Kale; Formal Analysis: Jamatsing Darbarsing Rajput; Investigation: Amol Diliprao Kale; Resources: Rajendra Pralhadrao Phase; Data Curation: Rajendra Pralhadrao Phase; Writing - Original Draft: Gautam Prabhakar Sadawarte; Writing - Review and Editing: Gautam Prabhakar Sadawarte; Supervision: Vasant Bhagwan Jagrut. ORCID and Email Gautam Prabhakar Sadawarte gautamsadawarte@gmail.com https://orcid.org/0000-0003-2002-0052 Jamatsing Darbarsing Rajput jamatsingh50@gmail.com https://orcid.org/0000-0002-4588-1345 Amol Diliprao Kale kaleamo009@gmail.com https://orcid.org/0009-0004-5535-1964 Rajendra Pralhadrao Phase rpphase@gmail.com https://orcid.org/0009-0000-4829-7649 Vasant Bhagwan Jagrut jagrutvb@gmail.com https://orcid.org/0000-0002-3328-1991 References [1]. Amin, A.; Qadir, T.; Sharma, P. 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The full terms of this license are available at https://www.eurjchem.com/index.php/eurjchem/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution, or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (https://www.eurjchem.com/index.php/eurjchem/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://nopr.niscpr.res.in/%20handle/123456789/18779 http://nopr.niscpr.res.in/%20handle/123456789/18779 https://www.eurjchem.com/index.php/eurjchem/terms http://creativecommons.org/licenses/by-nc/4.0 https://www.eurjchem.com/index.php/eurjchem/terms 1. Introduction 2. Experimental 2.1. Instrumentation 2.2. Synthesis 2.3. Biological activities 2.3.1. Evaluation of α-amylase inhibitory activity 2.3.2. Antimicrobial activity 2.3.2.1. Preparation of inoculums 2.3.3. Antioxidant activity, DPPH assay 3. Results and discussion 3.1. Synthesis 3.2. Biological activities 3.2.1. Evaluation of α-amylase inhibitory activity 3.2.2 Antimicrobial activity 3.2.3. Antioxidant activity, DPPH assay 4. Conclusions Acknowledgements Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: