Synthesis, Type II diabetes inhibitory activity, antimicrobial evaluation, and docking studies of N'-arylidene-2-((7-methylbenzo[4,5]thiazolo[2,3-c] [1,2,4]triazol-3-yl)thio)acetohydrazides European Journal of Chemistry 13 (4) (2022) 426-434 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2022 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.13.4.426-434.2315 European Journal of Chemistry View Journal Online View Article Online Synthesis, Type II diabetes inhibitory activity, antimicrobial evaluation, and docking studies of N'-arylidene-2-((7-methylbenzo[4,5]thiazolo[2,3-c] [1,2,4]triazol-3-yl)thio)acetohydrazides Satbir Mor 1,*, Suchita Sindhu 1, Mohini Khatri 1, Ravinder Punia 1 and Komal Jakhar 2 1 Department of Chemistry, Guru Jambheshwar University of Science and Technology, Hisar-125001, Haryana, India 2 Department of Chemistry, Maharishi Dayanand University, Rohtak-124001, Haryana, India * Corresponding author at: Department of Chemistry, Guru Jambheshwar University of Science and Technology, Hisar-125001, Haryana, India. e-mail: satbir_mor@yahoo.co.in (S. Mor). 10.5155/eurjchem.13.4.426-434.2315 Received: 25 July 2022 Received in revised form: 01 September 2022 Accepted: 09 September 2022 Published online: 31 December 2022 Printed: 31 December 2022 N'-Arylidene-2-((7-methylbenzo[4, 5]thiazolo[2,3-c][1, 2, 4]triazol-3-yl)thio)acetohydrazides (6a-j) were prepared by condensation of 2-((7-methylbenzo[4,5]thiazolo[2,3-c][1,2,4] triazol-3-yl)thio)acetohydrazide with appropriately substituted benzaldehydes in dry methanol and a catalytic amount of glacial acetic acid. The prepared compounds tested for in vitro Type II diabetes inhibition and antimicrobial (antibacterial and antifungal) activities employing α-amylase inhibition assay and the serial dilution method, respectively. Type II diabetes inhibitory assay results of all the tested derivatives revealed that precursor 3 (IC50 = 0.16 μM) and acetohydrazide 6i (IC50 = 0.38 μM) showed comparable activity with standard drug acarbose (IC50 = 0.15 μM). The derivatives 6i against B. subtilis and E. coli with MIC values of 0.0300 μmol/mL, compound 6c against S. aureus (MIC = 0.0312 μmol/mL) and compound 6e against P. aeruginosa (MIC = 0.0316 μmol/mL) exhibited remarkable antibacterial activity, however, compound 6b was found to be more active against the fungal strain C. albicans with MIC value of 0.0135 μmol/mL. All acetohydrazides (6a-j) showed greater potency against all strains tested than their precursors 1-4, which is also supported by the results of molecular docking analysis. Furthermore, no general trend for structure activity relationships was established for Type II diabetes inhibitory activity, nor antimicrobial activities of the tested hydrazones (6a-j). α-Amylase Hydrazones Benzothiazole Docking studies Acetohydrazide Antimicrobial activity Cite this: Eur. J. Chem. 2022, 13(4), 426-434 Journal website: www.eurjchem.com 1. Introduction Diabetes mellitus is a metabolic disorder resulting from insufficient insulin secretion characterized by chronic hyper- glycemia due to high-calorie diets rich in carbohydrates, fats, and proteins [1]. Reports on this disease by the International Diabetes Foundation (IDF) have estimated that cases of diabetes may increase to 629 million by 2045 [2]. There are 352 million people at risk of developing Type II diabetes [3]. The emergent factors responsible for the spreading of Type II diabetes include a progressive technological society, food habits, and an inactive lifestyle [4]. Type II diabetes is associated with obesity, hypertension, dyslipidemia, cardiovas- cular disease, etc. It may cause tissue or vascular damage leading to severe diabetic problems such as retinopathy, neuro- pathy, and nephropathy [5]. α-Glucosidase and α-amylase are the key enzymes that tend to reduce postprandial hyper- glycemia seen in Type II diabetes mellitus (DM2) [6]. α-Amylase inhibits the absorption of dietary starch and lowers blood glucose into the body system. Fundamentally, α-amylase inhibitors are classified into two groups: (i) proteinaceous inhibitors and (ii) non-proteinaceous inhibitors [7]. Non- proteinaceous inhibitors include chalcones, flavones, benzo- thiazoles, etc. as potential antidiabetic agents [8]. Similarly, the development of bacterial resistance of pathogenic micro- organisms is rapidly becoming one of the most urgent public health challenges in the world [9]. Antibiotic resistance can disturb people at any stage of life, as well as the healthcare, and also has the potential to affect veterinary, and agriculture industries [10]. Therefore, the demand for newer antimicrobial agents is increasing dramatically day by day, and molecular architectures have gained a great deal of interest in the synthesis of safer and new molecules with excellent activity to combat this challenge [11]. The appearance of a benzothiazole nucleus in nature is rare, but is found in complex molecules [12]. Derivatives containing benzothiazole cores are recognized to exhibit a wide spectrum of pharmacological activities, viz. antifungal, antibacterial, antiviral, anti-inflammatory, anticancer, anti-diabetic, analge- sic, antileishmanial, anticonvulsant, anti-tubercular, antihel- mintic, antioxidant, antipsychotic, and many more [13-15]. Some of the important drugs marketed that contain the benzothiazole nucleus are riluzole, sibenadet hydrochloride (Viozan), zopolrestat, and pramipexole [16]. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.13.4.426-434.2315 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.13.4.426-434.2315 mailto:satbir_mor@yahoo.co.in http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.13.4.426-434.2315&domain=pdf&date_stamp=2022-12-31 Mor et al. / European Journal of Chemistry 13 (4) (2022) 426-434 427 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.4.426-434.2315 Scheme 1. Protocol for the preparation of N'-arylidene-2-((7-methylbenzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl)thio)acetohydrazides (6a-j). Similarly, 1,2,4-triazoles are among the important classes of heterocyclics known for their usefulness in the production of insecticides, herbicides, and fungicides [17]. The compounds containing 1,2,4-triazole nuclei are associated with various biological activities such as analgesic and anti-inflammatory, antibacterial, antifungal, antiviral, anticonvulsant, antituber- cular, antiparasitic, antihypertensive, antileishmanial, anti- cancer, etc. [18]. Various drugs such as letrozole, fluconazole, estazolam, etizolam, rizatriptan and many others are available in clinical use that contain 1,2,4-triazole as the main nucleus [19-22]. Several commercial plant protection fungicides containing the triazole moiety include metconazole, prothioco- nazole, tebuconazole, propiconazole, cyproconazole, epoxico- nazole, triadimenol, and triadimefon [23]. Similarly, hydra- zones containing the azomethine group (-NHN=CH-) have gained interest in drug development [24], which are associated with multiple pharmacological properties such as antidep- ressant, analgesic, antiinflammatory, antiplatelet, antimalarial, antimicrobial, antimycobacterial, vasodilatory, and antiviral. etc. [25]. Furthermore, the hydrazone core is an effective bioactive skeleton that acts as a part of the structures of several marketed drugs, such as dantrolene, nifuroxazide, nitrofura- zone, carbazochrome, nitrofurantoin, and azumolene [26]. Moreover, hydrazones are appreciated building blocks in organic synthesis and are used as precursors for the synthesis of a variety of heterocyclics. Molecular docking has become a significant component of the drug discovery process. It is a computational technique used for the study of molecular recognition, due to its ability to predict the binding mode and binding affinity of a complex formed by two or more constituent molecules with known structures [27]. Nowadays, it is mostly utilized in amalgamation with other computational approaches within integrated work- flows [28]. In addition, it has the most important role in virtual screening, bioremediation, and drug discovery. Therefore, this is a useful tool for a researcher to reduce the time and cost of research activity and also gives a better understanding of the study of the ligand and receptor complex [29]. Taking into account the above facts, and in continuation of our program directed for the development of new, simple, safer, and efficient procedures for the synthesis of biologically active heterocyclic compounds utilizing readily accessible starting substrates and intermediates [30-35], here we report synthesis, characterization, Type II diabetes inhibitory activity and antimicrobial evaluation and docking studies of N'-arylidene-2- ((7-methylbenzo[4, 5]thiazolo[2, 3-c][1, 2, 4]triazol-3-yl)thio) acetohydrazides (6a-j) and their precursors 1-4. 2. Experimental 2.1. Chemistry All chemicals used in this study were purchased from commercial suppliers and used without further purification. Melting points (M.p., °C) of the synthesized compounds were determined in open head capillaries with an Electrothermal Melting Point apparatus, LABCO Co, India, and are uncorrected. FT-IR spectra were taken on an IR Affinity-1 FTIR (Shimadzu) spectrophotometer in the region 500-4000 cm-1 using KBr, and peaks are reported in cm-1. NMR (1H and 13C) spectra were recorded on a Bruker AVANCE III NMR spectrometer operating at 400 MHz using tetramethyl silane (TMS) as internal standard (chemical shift in δ, ppm), and the values of the coupling constant (J) are presented in Hertz (Hz). HRMS analysis was performed using LC-MS on SCIEX 5600+QTOF operating in a positive full scan mode (120,000 FWMH) in the range of 100- 1000 m/z using the electrospray ionization (ESI) method. 2.2. Synthesis 2.2.1. General procedure for the synthesis of 2-hydrazinyl-6- methylbenzo[d]thiazole (1) Hydrazine hydrate (10 g, 0.2 mol) was taken in a 250 mL round bottom flask equipped with a reflux condenser and concentrated HCl (20 mL) was added dropwise to this under stirring on a magnetic stirrer keeping the temperature of the reaction mixture below 10 °C. Subsequently, ethylene glycol (40 mL) was added followed by 6-methylbenzo[d]thiazol-2-amine (8.21 g, 0.05 mol) gradually to the reaction mixture above and the resulting mixture was refluxed for 2 h. The solid thus obtained was filtered, dried, and recrystallized from aqueous ethanol to give 2-hydrazinyl-6-methylbenzo[d]thiazole (1) as colorless crystals, Yield: 59.6%, M.p.: 212-214 °C (Scheme 1) (Lit. M.p.: 218 °C [30]). 428 Mor et al. / European Journal of Chemistry 13 (4) (2022) 426-434 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.4.426-434.2315 2.2.2. General procedure for the synthesis of 7-methylbenzo [4,5]thiazolo[2,3-c][1,2,4]triazole-3-thiol (2) A suspension of 2-hydrazinyl-6-methylbenzo[d]thiazole (1, 7.16 g, 0.04 mol) in ethanol (40 mL) was prepared in a 250 mL round bottom flask equipped with a reflux condenser and a solution of potassium hydroxide (2.24 g, 0.04 mol) in ethanol (10 mL) was added to this suspension. Subsequently, carbon disulfide (12 mL, 0.2 mol) was added dropwise to the above reaction mixture and the contents were heated to reflux gently on a water bath for 7 h. Thereafter, the solvents were removed under reduced pressure to give a solid upon cooling. The aqueous solution of the solid thus obtained was acidified with conc. HCl to achieve pH = 5 of the solution to obtain a solid that was separated by filtration, washed with cold water several times and recrystallized from ethanol to give 7-methylbenzo [4,5]thiazolo[2,3-c][1,2,4]triazole-3-thiol (2) as a yellow solid (Scheme 1), Yield: 60%, M.p.: 308-310 °C (Lit. M.p.: 312 °C [36]). 2.2.3. General procedure for the synthesis of ethyl 2-((7- methylbenzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl)thio) acetate (3) To a solution of 7-methylbenzo[4,5]thiazolo[2,3-c][1,2,4] triazole-3-thiol (2, 0.5 g, 0.002 mol) in assortment of acetone (14.0 mL) and DMF (21.0 mL) taken in a 100 mL round bottomed flask equipped with a reflux condenser was added K2CO3 (0.345 g, 0.0025 mol) and ethyl bromoacetate (0.24 mL, 0.002 mol). The reaction mixture was heated to reflux while stirring on a magnetic stirrer for 7 h. Subsequently, the contents were poured onto ice while stirring. The precipitates thus separated out were filtered off, washed with cold water, and recrystallized from aqueous ethanol to yield compound 3 as a white solid (Scheme 1). Ethyl 2-((7-methylbenzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3- yl)thio)acetate (3): Color: White solid. Yield: 72%. M.p.: 115- 117 °C. FTIR (KBr, ν, cm-1): 804, 1023, 1228, 1741 (C=O stretch), 2910, 2982 (aliphatic C-H stretch), 3092 (aromatic C-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 1.33 (t, 3H, J = 6.96 Hz, - CO2CH2CH3), 2.67 (s, 3H, C7-CH3), 4.15-4.21 (m, 4H, -CO2CH2CH3, -SCH2-), 7.73 (d, 1H, J = 8.56 Hz, 6-H), 8.13 (d, 1H, J= 8.52 Hz, 5- H), 8.43 (s, 1H, 8-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 20.48 (-CO2CH2CH3), 21.38 (C7-CH3), 48.09 (-SCH2-), 57.69 (- CO2CH2CH3), 119.17, 127.27, 127.68, 128.80, 129.07, 129.62, 139.56, 151.35, 168.59 (C=O). HRMS (ESI-TOF, m/z) calcd. for C13H13N3O2S2 [M+H]+: 308.0527, Found: 308.0525. 2.2.4. General procedure for the synthesis of 2-((7-methyl benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl)thio)aceto- hydrazide (4) A mixture of ethyl 2-((7-methylbenzo[4,5]thiazolo[2,3-c] [1,2,4]triazol-3-yl)thio)acetate (3, 0.325 g, 0.0009 mol), hydrazine hydrate (10 mL) and ethanol (5 mL) was taken in a 100 mL round bottomed flask equipped with a reflux condenser and heated to reflux for 2 h on a water bath. The completion of the reaction was ascertained by TLC. After completion of reaction, the contents were cooled upon subsequent usual workup and recrystallization from ethanol gave the desired product 2-((7-methylbenzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3- yl)thio)acetohydrazide (4) as a white solid (Scheme 1). 2-((7-Methylbenzo[4,5]thiazolo[2, 3-c][1, 2, 4]triazol-3-yl) thio)aceto hydrazide (4): Color: White solid. Yield: 52%. M.p.: 280-282 °C. FTIR (KBr, ν, cm-1): 822, 1080, 1174, 1508, 1662 (C=O stretch), 2934 (aliphatic C-H stretch), 3061 (aromatic C-H stretch), 3206, 3304 (NH2, NH stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.39 (s, 3H, -CH3), 2.50 (s, 2H, NH2), 4.45 (s, 2H, -SCH2-), 7.30 (d, 1H, J = 8.32 Hz, 6-H), 7.72 (s, 1H, 8-H), 7.76 (s, 1H, NH), 7.93 (d, 1H, J = 8.32 Hz, 5-H). 13C NMR (100 MHz, CDCl3, δ, ppm): 21.21 (-CH3), 44.36 (-SCH2-), 115.16, 125.19, 127.67, 127.69, 131.29, 135.78, 151.11, 154.12, 169.29. HRMS (ESI- TOF, m/z) calcd. for C11H11N5OS2[M+H]+: 294.0483, Found: 294.0436. 2.2.5. General procedure for the synthesis of 7-methylbenzo [4,5]thiazolo[2,3-c][1,2,4]triazole based hydrazones (6a-j) A mixture of compound 4 (0.0002 mol), dry methanol (15 mL), appropriately substituted benzaldehyde (5, 0.0002 mol), and a catalytic amount of glacial acetic acid (2-3 drops) was taken in a 100 mL round bottom flask equipped with a reflux condenser. The reaction mixture was heated to reflux on a heating mantle for 3 h. The progress of the reaction was monitored by TLC using hexane:ethylacetate (30:70, v:v) on aliquots withdrawn from the reaction mixture at different intervals of time. After completion, the reaction mixture was poured onto crushed ice and the precipitates thus obtained were filtered under suction in a Büchner funnel and washed with water and cold ethanol to obtain the corresponding 7- methylbenzo[4,5]thiazolo[2,3-c][1,2,4]triazole based hydra- zones (6a-j) in high yields (Scheme 1). N'-Benzylidene-2-((7-methylbenzo[4, 5]thiazolo[2, 3-c][1, 2, 4]triazol-3-yl)thio)acetohydrazide (6a): Color: Yellow solid. Yield: 91%. M.p.: 264-266 °C. FTIR (KBr, ν, cm-1):755, 1035, 1255, 1355, 1508, 1596 (C=N stretch), 1654 (C=O stretch), 2868 (aliphatic C-H stretch), 3061 (aromatic C-H stretch), 3338 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.39 (s, 3H, C7- CH3), 4.11 (s, 2H, -SCH2-), 7.30 (d, 1H, J = 7.24 Hz, 6-H), 7.40 (d, 2H, J = 8.00 Hz, 3ʹ-H, 5ʹ-H), 7.62 (d, 1H, J = 6.68 Hz, 5-H), 7.71 (s, 1H, H-8), 7.80 (t, 1H, J = 7.60 Hz, 4ʹ-H), 7.93 (d, 2H, J = 8.12 Hz, 2ʹ-H, 6ʹ-H), 8.22 (s, 1H, -N=CH-), 11.10 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm):21.22 (-CH3), 49.07 (-SCH2-), 115.85, 125.46, 126.73, 127.85, 128.01, 128.88, 129.36, 131.65, 134.88, 136.38, 148.99, 149.58, 152.50, 168.25 (C=O). HRMS (ESI-TOF, m/z) calcd. for C18H15N5OS2 [M+H]+: 382.0796, Found: 382.0741. N'-(3-Bromobenzylidene)-2-((7-methylbenzo[4, 5]thiazol[2, 3-c][1,2,4]triazol-3-yl)thio)acetohydrazide (6b): Color: Yellow solid. Yield: 85%. M.p.: 267-269 °C. FTIR (KBr, ν,c m-1):770, 1079, 1230, 1508, 1585, 1638 (C=N stretch), 1650 (C=O stretch), 2789, 3441 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm):2.42 (s, 3H, C7-CH3), 4.10 (s, 2H, -SCH2-), 7.34 (d, 1H, J = 7.88 Hz, 6-H), 7.38 (d, 1H, J = 8.48 Hz, 5ʹ-H), 7.51 (d, 1H, J = 8.48 Hz, 4ʹ-H), 7.66 (d, 1H, J = 7.76 Hz, 5-H), 7.72-7.77 (m, 1H, 6ʹ-H), 7.83 (s, 1H, 8-H), 7.92 (d, 1H, J = 8.20 Hz, 2ʹ-H), 8.17 (s, 1H, - N=CH-), 11.28 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm):21.34 (-CH3), 48.14 (-SCH2-), 114.06, 121.83, 122.68, 125.00, 125.68, 126.65, 128.12, 128.98, 129.08, 131.01, 133.59, 135.58, 142.14, 147.13, 155.37, 169.22 (C=O). HRMS (ESI-TOF, m/z) calcd. for C18H14BrN5OS2 [M+H]+: 459.9901 (79Br), 461.9881 (81Br), Found: 460.0026 (79Br), 461.9847 (81Br). N'-(4-Fluorobenzylidene)-2-((7-methylbenzo[4, 5]thiazolo[2, 3-c][1,2,4]triazol-3-yl)thio)acetohydrazide (6c): Color: Yellow solid. Yield: 90%. M.p.: 260-262 °C. FTIR (KBr, ν, cm-1): 713, 1114, 1233, 1508, 1609 (C=N stretch), 1648 (C=O stretch), 2875 (aliphatic C-H stretch), 3109 (aromatic C-H stretch), 3398 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.35 (s, 3H, C7- CH3), 4.13 (s, 2H, -SCH2-), 7.19 (d, 2H, J = 9.04 Hz, 3ʹ-H, 5ʹ-H), 7.26 (d, 1H, J = 8.28 Hz, 6-H), 7.63 (d, 1H, J = 8.60 Hz, 5-H), 7.75 (s, 1H, 8-H), 7.84 (d, 2H, J = 8.44 Hz, 2ʹ-H, 6ʹ-H), 8.17 (s, 1H, - N=CH-), 11.04 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm):21.22 (-CH3), 45.80 (-SCH2-), 115.75, 116.38 (d, J= 21.73 Hz), 124.92, 125.46, 127.87, 128.80 (d, J= 8.21 Hz), 131.50 (d, J= 2.69 Hz), 131.64, 136.36, 142.99, 149.54, 152.51, 160.68 (J= 246.96 Hz), 164.38 (C=O). HRMS (ESI-TOF, m/z) calcd. for C18H14FN5OS2 [M+H]+: 400.0702, Found: 400.0676. N'-(4-Methoxybenzylidene)-2-((7-methylbenzo[4, 5]thiazolo [2,3-c][1,2,4]triazol-3-yl)thio)acetohydrazide (6d): Color: Yellow solid. Yield: 87%. M.p.: 258-260 °C. FTIR (KBr, ν, cm-1): 811, 1031, 1244, 1508, 1618 (C=N stretch), 1650 (C=O stretch), Mor et al. / European Journal of Chemistry 13 (4) (2022) 426-434 429 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.4.426-434.2315 2837 (aliphatic C-H stretch), 3109 (aromatic C-H stretch), 3344 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.35 (s, 3H, C7- CH3), 4.04 (s, 2H, -SCH2-), 3.73 (s, 3H, -OCH3), 6.93 (d, 2H, J = 8.80 Hz, 3ʹ-H, 5ʹ-H), 7.26 (d, 1H, J = 8.32 Hz, 6-H), 7.51 (d, 2H, J = 8.72 Hz, 2ʹ-H, 6ʹ-H), 7.70 (d, 1H, J = 8.52 Hz, 5-H), 7.74 (s, 1H, 8-H), 8.12 (s, 1H, -N=CH-), 10.86 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm):2 1.23 (-CH3), 47.77 (-SCH2-), 55.74 (-OCH3), 114.86, 125.44, 127.48, 127.82, 128.05, 128.26, 128.74, 131.63, 136.30, 144.16, 148.91, 149.87, 160.80, 167.61 (C=O). HRMS (ESI-TOF, m/z) calcd. for C19H17N5O2S2 [M+H]+: 412.0902, Found: 412.0875. 2-((7-Methylbenzo[4, 5]thiazolo[2, 3-c][1, 2, 4]triazol-3-yl) thio)-N'-(2-methylbenzylidene)acetohydrazide (6e): Color: Yellow solid. Yield: 82%. M.p.: 266-268 °C. FTIR (KBr, ν, cm-1): 713, 1090, 1258, 1603 (C=N stretch), 1654 (C=O stretch), 2868 (aliphatic C-H stretch), 3091 (aromatic C-H stretch), 3447 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.35 (s, 3H, C7-CH3), 2.38 (s, 3H, C2ʹ-CH3), 4.04 (s, 2H, -SCH2-), 7.17-7.22 (m, 2H, 3ʹ-H, 4ʹ-H), 7.27 (d, 2H, J = 8.52 Hz, 6-H, 5ʹ-H), 7.64 (d, 1H, J = 7.56 Hz, 5-H), 7.75 (s, 1H, 8-H), 7.89 (d, 1H, J = 8.32 Hz, 6ʹ-H), 8.46 (s, 1H, -N=CH-), 10.96 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 19.58 (-CH3), 21.23 (-CH3), 49.33 (-SCH2-), 115.85, 125.51, 126.71, 127.82, 127.95, 128.74, 129.60, 131.38, 131.65, 132.77, 136.37, 136.55, 143.01, 149.61, 152.85, 164.21 (C=O). HRMS (ESI-TOF, m/z) calcd. for C19H17N5OS2 [M+H]+: 396.0953, Found: 396.0912. N'-(2-Fluorobenzylidene)-2-((7-methylbenzo[4, 5]thiazolo[2, 3-c][1,2,4]triazol-3-yl)thio)acetohydrazide (6f): Color: Yellow solid. Yield: 84%. M.p.: 262-264 °C. FTIR (KBr, ν, cm-1): 764, 1086, 1234, 1500, 1636 (C=N stretch), 1651 (C=O stretch), 2868 (aliphatic C-H stretch), 3091 (aromatic C-H stretch), 3318 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.43 (s, 3H, C7- CH3), 4.13 (s, 2H, -SCH2-), 7.27 (d,1H, J = 7.28 Hz, 5ʹ-H), 7.35 (d, 2H, J = 8.32 Hz, 6-H, 3ʹ-H), 7.47 (dd, 1H, J = 7.60 Hz and 13.20 Hz, 4ʹ-H), 7.76 (s, 1H, 8-H), 7.80 (d, 1H, J = 6.96 Hz, 5-H), 7.96 (d, 1H, J = 8.28 Hz, 6ʹ-H), 8.46 (s, 1H, -N=CH-), 11.33 (s, 1H NH). 13C NMR (100 MHz, CDCl3, δ, ppm):21.22 (-CH3), 47.31 (-SCH2-), 114.19, 120.98 (d, J = 2.43 Hz), 123.96, 124.93, 126.18, 127.89 (d, J = 5.64 Hz), 128.12, 130.59 (d, J = 2.21 Hz), 131.66 (d, J = 6.14 Hz), 135.54, 136.71 (d, J = 4.66 Hz), 142.64, 149.22, 152.58, 160.63 (d, J = 247.81 Hz), 166.51 (C=O). HRMS (ESI-TOF, m/z) calcd. for C18H14FN5OS2 [M+H]+: 400.0702, Found: 400.0684. N'-(4-Chlorobenzylidene)-2-((7-methylbenzo[4, 5] thiazolo [2,3- c][1,2,4]triazol-3-yl)thio)acetohydrazide (6g): Color: Yellow solid. Yield: 83%. M.p.: 254-258 °C. FTIR (KBr, ν, cm-1): 734, 805, 1089, 1257, 1508, 1603 (C=N stretch), 1654 (C=O stretch), 2873 (aliphatic C-H stretch), 3097 (aromatic C-H stretch), 3435 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.40 (s, 3H, C7- CH3), 4.12 (s, 2H, -SCH2-), 7.31 (d, 1H, J = 7.96 Hz, 6-H), 7.48 (d, 2H, J = 8.64 Hz, 3ʹ-H, 5ʹ-H), 7.64 (d, 1H, J = 8.40 Hz, 5-H), 7.73 (s, 1H, 8-H), 7.89 (d, 2H, J = 8.20 Hz, 2ʹ-H, 6ʹ-H), 8.21 (s, 1H, -N=CH- ), 11.19 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 21.23 (- CH3), 47.24 (-SCH2-), 115.74, 125.49, 127.91, 128.33, 128.78, 128.94, 129.43, 131.65, 133.83, 136.40, 142.68, 147.43, 153.12, 165.62 (C=O). HRMS (ESI-TOF, m/z) calcd. for C18H14ClN5OS2 [M+H]+: 416.0407 (35Cl), 418.0377 (37Cl), Found: 416.3555 (35Cl), 418.3586 (37Cl). N'-(3-Fluorobenzylidene)-2-((7-methylbenzo[4,5]thiazolo [2, 3-c][1,2,4]triazol-3-yl)thio)acetohydrazide (6h): Color: Yellow solid. Yield: 83%. M.p.: 254-258 °C. FTIR (KBr, ν, cm-1): 771, 814, 1261, 1514, 1594, 1637 (C=N stretch), 1648 (C=O stretch), 2794 (aliphatic C-H stretch), 3094 (aromatic C-H stretch), 3435 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.34 (s, 3H, C7- CH3), 4.01 (s, 2H, -SCH2-), 7.12-7.17 (m, 1H, 4ʹ-H), 7.33-7.41 (m, 4H, 6-H, 2ʹ-H, 5ʹ-H, 6ʹ-H), 7.67 (d, 1H, J = 7.92 Hz, 5-H), 7.74 (s, 1H, 8-H), 8.15 (s, 1H, -N=CH-), 11.18 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm):21.23 (-CH3), 47.98 (-SCH2-), 109.97, 114.77, 116.43 (d, J = 15.32 Hz), 123.89 (d, J = 2.79 Hz), 124.98, 125.52, 128.09, 129.08 (d, J = 2.23 Hz), 130.81 (d, J = 1.47Hz), 135.53 (d, J = 2.54 Hz), 136.46, 142.52, 149.25, 153.29,162.82 (d, J = 201.42 Hz), 164.19 (C=O). HRMS (ESI-TOF, m/z) calcd. for C18H14FN5OS2 [M+H]+: 400.0702, Found: 400.0675. N'-(2-Chlorobenzylidene)-2-((7-methylbenzo[4, 5]thiazolo[2, 3-c][1,2,4]triazol-3-yl)thio)acetohydrazide (6i): Color: Yellow solid. Yield: 88%. M.p.: 268-270 °C. FTIR (KBr, ν, cm-1): 734, 809, 1241, 1514, 1635 (C=N stretch), 1651 (C=O stretch), 2864 (aliphatic C-H stretch), 3084 (aromatic C-H stretch), 3346 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.46 (s, 3H, C7-CH3), 4.09 (s, 2H, -SCH2-), 7.37-7.41 (m, 1H, 6-H), 7.45-7.48 (m, 1H, 4ʹ- H), 7.53-7.58 (m, 2H, 3ʹ-H, 5ʹ-H), 7.63 (dd, 1H, J = 8.08 Hz and J = 1.16 Hz, 5-H), 7.69-7.73 (m, 1H, 8-H), 7.88 (dd,1H, J = 7.68 Hz and J = 1.76 Hz, 6ʹ-H), 8.46 (s, 1H, -N=CH-), 10.36 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 21.44 (-CH3), 53.99 (-SCH2-), 100.96, 127.34, 128.36, 128.51, 129.93, 130.15, 130.67, 131.20, 132.52, 132.62, 134.70, 135.62, 136.73, 151.52, 155.38, 167.23 (C=O). HRMS (ESI-TOF, m/z) calcd. for C18H14ClN5OS2 [M+H]+: 416.0407 (35Cl), 418.0377 (37Cl), Found: 416.0387 (35Cl), 418.0345 (37Cl). 2-((7-Methylbenzo[4, 5]thiazolo[2, 3-c][1, 2, 4]triazol-3-yl) thio)-N'-(4-nitrobenzylidene)acetohydrazide (6j): Color: Yellow solid. Yield: 83%. M.p.: 271-272 °C. FTIR (KBr, ν, cm-1):749, 827, 1014, 1044, 1105, 1207, 1252, 1341 (symmetrical N-O stretch), 1508, 1548 (asymmetrical N-O stretch), 1590 (C=N stretch), 1652 (C=O stretch), 2928 (aliphatic C-H stretch), 3091 (aromatic C-H stretch), 3447 (N-H stretch). 1H NMR (400 MHz, CDCl3, δ, ppm): 2.47 (s, 3H, C7-CH3), 4.09 (s, 2H, -SCH2-), 7.46- 7.48 (m, 2H, 5-H, 6-H), 7.91 (s, 1H, 8-H), 8.17 (d, 2H, J = 8.84 Hz, 2ʹ-H, 6ʹ-H), 8.38 (d, 2H, J = 8.88 Hz, 3ʹ-H, 5ʹ-H), 8.87 (s, 1H,- N=CH-), 10.18 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 21.47 (-CH3), 50.04 (-SCH2-), 115.09, 119.97, 124.66, 126.49, 130.05, 131.37, 135.51, 136.88, 139.35, 140.77, 145.40, 148.14, 152.71, 164.84 (C=O). HRMS (ESI-TOF, m/z) calcd. for C18H14N6O3S2 [M+H]+: 426.0569, Found: 426.0538. 2.3. Biological evaluation 2.3.1. In vitro α-amylase inhibition All synthesized compounds 1-4 and 6a-j were tested for their in vitro α-amylase inhibitory activity against α-amylase enzyme using the protocol reported by Xiao et al. and Yoshikawa et al. with slight modifications [37,38]. The results of in vitro α-amylase inhibitory activity of the compounds 1-4 and 6a-j reported in terms of % inhibition and IC50 values are shown in Table 1. 2.3.2. Antimicrobial evaluation All synthesized compounds 1-4 and N'-arylidene-2-((7- methylbenzo[4,5]thiazolo[2 ,3-c][1, 2, 4]triazol-3-yl)thio)aceto hydrazides (6a-j) were evaluated for their antimicrobial (antibacterial and antifungal) activities in vitro using a serial dilution method according to the literature procedure [39]. The in vitro inhibitory activity results of the synthesized compounds 1-4 and 6a-j were expressed in terms of minimum inhibition concentration (MIC, μmol/mL) and are shown in Table 2. 2.4. Molecular docking study Molecular docking analysis of compounds 1-4 and 6a-j was performed to find the plausible mechanism of action of their antifungal activity against C. albicans (MTCC 227). The protocol utilized for molecular docking is reported as follows: The protein-ligand crystal structure has been optimized to its lower energy conformation using a protein preparation wizard where all water molecules were removed during pre-process and missing side chains of residues have been added using prime. 430 Mor et al. / European Journal of Chemistry 13 (4) (2022) 426-434 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.4.426-434.2315 Table 1. In vitro α-amylase inhibitory activity of compounds 1-4 and 6a-j. Compounds Percentage inhibition±SD IC50 (μM) 12.5 (μg/mL) 25 (μg/mL) 50 (μg/mL) 100 (μg/mL) 1 70.10±0.00 77.95±0.30 79.61±0.75 85.35±0.15 3.96 2 75.09±0.30 80.67±0.75 87.77±0.15 93.81±0.45 3.71 3 75.69±0.75 80.97±0.15 82.03±0.45 85.96±0.30 0.16 4 88.22±0.45 88.97±0.15 67.84±0.30 96.07±0.00 11.31 6a 73.58±0.30 77.80±0.45 84.75±0.30 90.64±0.15 2.17 6b 68.29±0.60 79.01±0.30 80.97±0.15 91.54±0.60 4.99 6c 74.18±0.15 82.78±0.00 84.75±0.45 87.77±0.30 0.80 6d 60.44±0.75 65.27±0.30 68.90±0.15 85.96±0.45 18.98 6e 58.33±0.00 81.88±0.45 83.99±0.60 91.54±0.15 17.14 6f 61.20±0.60 63.76±0.15 66.33±0.30 81.73±0.75 17.97 6g 73.73±0.75 77.80±0.45 82.78±0.00 88.82±0.30 1.23 6h 75.69±0.45 81.58±0.30 87.01±0.15 92.45±0.45 1.25 6i 79.46±0.15 82.48±0.75 85.05±0.45 92.60±0.00 0.38 6j 53.04±0.00 67.54±0.45 77.50±0.15 89.13±0.30 23.19 Acarbose 80.52±0.00 87.46±0.30 90.64±0.45 93.96±0.15 0.15 Table 2. In vitro antimicrobial activity of compounds 1-4 and 6a-j. Compounds Minimum inhibitory concentration (MIC in μmol/mL) Gram-positive bacteria * Gram-negative bacteria * Fungi * B. subtilis S. aureus E. coli P. aeruginosa C. albicans A. niger 1 0.1396 0.1396 0.1396 0.1396 0.0349 0.0698 2 0.1129 0.1129 0.1129 0.1129 0.0282 0.0564 3 0.0813 0.0813 0.0813 0.0813 0.0203 0.0406 4 0.0852 0.0852 0.0852 0.0852 0.0213 0.0426 6a 0.0655 0.0655 0.0655 0.0655 0.0163 0.0327 6b 0.0543 0.0543 0.0543 0.0543 0.0135 0.0271 6c 0.0312 0.0312 0.0312 0.0625 0.0625 0.0625 6d 0.0607 0.0607 0.0607 0.0607 0.0607 0.0303 6e 0.0632 0.0632 0.0316 0.0316 0.0632 0.0316 6f 0.0625 0.0625 0.0625 0.0625 0.1251 0.0312 6g 0.0601 0.0601 0.0601 0.0601 0.0601 0.0300 6h 0.0625 0.0625 0.0625 0.0625 0.0625 0.0312 6i 0.0300 0.0601 0.0300 0.0601 0.0150 0.0300 6j 0.0586 0.0586 0.0586 0.0586 0.0146 0.0293 Ciprofloxacin 0.0047 0.0047 0.0047 0.0047 - - Fluconazole - - - - 0.0102 0.0102 * Bacillus subtilis (MTCC 441); Staphylococcus aureus (MTCC 7443), Escherichia coli (MTCC 1652); Pseudomonas aeruginosa (MTCC 424), Candida albicans (MTCC 227); Aspergillus niger (MTCC 8189). Keeping in mind the appropriate ionization states for the acidic as well as basic amino acids, hydrogen atoms were added to the protein structure corresponding to the physiological pH = 7.0. Finally, energy minimization with a root mean square deviation (RMSD) value of 0.30 Å was carried out using optimized potentials for liquid simulations (OPLS-2005) force field after assigning charge and protonation state. Ligand preparation was done using the Schrodinger LigPrep utility (Schrodinger, LLC, USA), which generates a low energy 3D structure. The active site of the protein was defined by a bounding box (grid) that was centered on the native ligand in the crystal complex. Extra- precision glide docking (Glide XP), which docks ligands flexibly, was used to rank the docking poses and to gauze the binding affinity of these ligands towards the protein. 3. Results and discussion 3.1. Chemistry The procedure for the preparation of N'-arylidene-2-((7- methylbenzo[4,5]thiazolo[2, 3-c][1, 2, 4]triazol-3-yl)thio)aceto hydrazides (6a-j) is described in Scheme 1. Firstly, we prepared 2-hydrazinyl-6-methylbenzo[d]thiazole (1) from p-toluidine in accord with the literature procedure [40,41]. Thiazole (1) upon reaction with carbon disulfide in the presence of potassium hydroxide/ethanol under reflux conditions followed by acidi- fication with concentrated hydrochloric acid gave 7-methyl benzo[4,5]thiazolo[2,3-c][1,2,4]triazole-3-thiol (2) with good yield [42]. The synthesis of key intermediate 2-((7-methyl benzo[4,5]thiazolo[2, 3-c][1, 2, 4]triazol-3-yl)thio)acetohydra- zide (4) was achieved in two steps: (i) the thiol 2 on alkylation with ethyl bromoacetate in presence of potassium carbonate using acetone/DMF as solvent under stirring at 80 °C yielded 7- methylbenzo[4,5]thiazolo[2,3-c][1,2,4]triazole based ester (3), and subsequently (ii) the ester (3) was treated with hydrazine hydrate in presence of ethanol under reflux to give acid hydrazide (4) [43]. Finally, acid hydrazide (4) was condensed with differently substituted benzaldehydes 5 using a catalytic amount of acetic acid (glacial) in methanol under reflux conditions to provide the target N'-Arylidene-2-((7-methyl- benzo[4, 5]thiazolo[2, 3-c][1, 2, 4]triazol-3-yl)thio)acetohydra- zides (6a-j) in high yields (82-91%). The structures of ester 3 and acid hydrazide 4 were well confirmed from FTIR, 1H NMR, 13C NMR, and HRMS spectral data. FTIR spectra of compounds 3 and 4, in each case, exhibited strong absorption bands at 1741 and 1662 cm-1 due to >C=O stretchings of the -CO2CH2CH3 and -CONHNH2 groups, respect- tively [42]. The 1H NMR spectrum of compound 3 showed a triplet integration for three protons resonated at δ 1.33 ppm (3J = 6.96 Hz) due to the methyl protons of the ester group (- CO2CH2CH3). A signal obtained as a three-proton singlet at δ 2.67 ppm was safely assigned to C7-CH3. A multiplet that appeared in the region at δ 4.15-4.21 ppm integrated for four protons was attributed to the two protons each of the -SCH2- and methylene protons of the ester group (-CO2CH2CH3). The remaining aromatic protons demonstrated signals in the expected regions. In its 13C NMR spectrum, the signals due to the methyl carbons of -CO2CH2CH3 and C7-CH3 appeared at δ 20.48 and 21.38 ppm, respectively, while the carbon atom of -SCH2- and the methylene carbon of the -CO2CH2CH3 group displayed signals at δ 48.09 and δ 57.69 ppm, respectively. In the downfield region of the spectrum, the characteristic signal due to the carbonyl carbon of the ester group in compound 3 was obtained at δ 168.59 ppm. Mor et al. / European Journal of Chemistry 13 (4) (2022) 426-434 431 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.4.426-434.2315 Figure 1. Structure Activity Relationship (SAR) of compounds 6a-j for α-amylase inhibitory activity. The remaining aromatic carbon atoms exhibited signals in the expected regions. Similarly, the 1H NMR spectrum of compound 4 showed a singlet of three protons in the most up- field region at δ 2.39 due to C7-CH3. The characteristic signals due to -NH2 and NH were appeared at δ 2.50 and 7.76 ppm integrating for two protons and one proton, respectively. The remaining aromatic protons appeared in the expected regions. In 13C NMR spectrum of compound 4, the signals resonated at δ 21.21 and 44.36 ppm could easily be assigned to the carbons of C7-CH3 and -SCH2-group, respectively. The most significant feature of 13C NMR spectrum of compound 4 is the resonance exhibited in the most downfield region at δ 169.29 ppm due to the carbonyl carbon of -CONHNH2 group. Furthermore, the results of the HRMS analysis of compounds 3 and 4 were found to agree well with their molecular formulae (vide experi- mental). The FTIR spectra of all synthesized acetohydrazides (6a-j), in each case, exhibited an absorption band in the region at 1590-1638 cm-1 due to the C=N stretching while the strong absorption bands obtained in the range of 1648-1654 and 3318-3447 cm-1 were safely assigned to >C=O and N-H stretch- ings of the -CONHN= group, respectively. In the 1H NMR spectra of compounds 6a-j, in their aliphatic regions, in each case, the singlet integrated for three protons centered in the region at δ 2.34-2.47 ppm could undoubtedly be ascribed to the protons of C7-CH3. The characteristic signal due to the protons of -SCH2- as a two-protons singlet, in each case, appeared in the range of δ 4.01-4.13 ppm while a singlet of one proton obtained in the region at δ 8.12-8.87 ppm could easily be assigned to the proton of the -N=CH- group. In the most down-field region of the spectra, in each case, a broad singlet (exchangeable with D2O) integrated for one proton appeared in the region at δ 10.18- 11.33 ppm, which was undoubtedly assigned to the NH of hydrazono (-CONHN=) [38]. The resonances due to the remaining protons were appeared in the expected regions. The 13C NMR spectra of compounds 6a-j, in their aliphatic region, in each case, exhibited a signal in the range of δ 21.22-21.47 ppm which could be securely assigned to the carbon of C7-CH3, while the carbon atom of the -SCH2- group, in each case, resonated in the range of δ 45.80-53.99 ppm. The most significant feature of 13C NMR spectra of compounds 6a-j, in each case, was the resonance due to carbon atom of carbonyl group that exhibited signal in the most downfield region at δ 164.19-169.22 ppm [42,44]. The signals from the remaining carbons were observed in the expected regions. Furthermore, the structures of hydrazones (6a-j) were also supported by their HRMS analysis results. 3.2. Pharmacological assay 3.2.1. Antidiabetic evaluation All synthesized compounds 1-4 and 6a-j were evaluated for preliminary in vitro α-amylase inhibitory activity (α-amylase isolated from Malt) at various concentrations, that is, 12.5, 25, 50 and 100 μg/mL following the procedure developed by Xiao et al. [37] and Yoshikawa et al. [38] with slight modifications. The reagent solution without test samples was used as a control, and acarbose was taken as a standard for comparison. The absorbance was recorded on ELISA microplate reader at wavelength (λ) = 650 nm. The α-amylase inhibitory activity reported as a percentage (%) inhibition was calculated using Equation (1) as shown below.   − = − ×   −    2 1 4 3 Enzyme inhibitory activity (% Inhibition) 1 100Abs Abs Abs Abs (1) where Abs1 = Absorbance of incubated solution containing test sample, starch, and α-amylase, Abs2 = Absorbance of incubated solution containing test sample and starch, Abs3 = Absorbance of incubated solution containing starch and α-amylase, Abs4 = Absorbance of incubated solution containing starch. Each experiment was performed in triplicate and mean % inhibition±SD at each concentration of all the compounds were determined. The IC50 values of all compounds 1-4 and 6a-j were calculated by linear regression and the results obtained thus are depicted in Table 1. The results summarized in Table 1 inferred that all compounds 1-4 and 6a-j tested exhibited poor to excellent α- amylase inhibitory activity with IC50 values in the range of 0.16- 23.19 μM. Among all the tested derivatives (4, 6d, 6e, 6f and 6j) were found to demonstrate poor inhibitory activity whereas compounds 1, 2, 6a, 6b, 6g and 6h were found moderately active and the derivative 6c (IC50 = 0.80 μM) was found to display good inhibitory activity. Compounds 3 (IC50 = 0.16 μM) and 6i (IC50 = 0.38 μM) showed comparable activity with standard acarbose with IC50 values of 0.15 μM. From the α- amylase inhibitory evaluation results of compounds 6a-j, the following Structure Activity Relationship (SAR) may be deduced: (i) The compound 6i (R= 2-Cl) demonstrated to increase the α-amylase inhibitory activity followed by compounds 6c (R= 4-F), 6g (R = 4-Cl) and 6h (R = 3-F). (ii) It was found that the derivatives 6a (R = H), 6b (R = 3-Br), 6d (R = 4-OCH3), 6e (R = 2-CH3), 6f (R = 2-F) and 6j (R = 4-NO2) showed poor inhibition against α-amylase. In general, it is inferred that there are different structural requirements for a compound to be active against α-amylase and no general trend for SAR was established for compounds 6a-j against antidiabetic activity. The findings mentioned above are summarized in Figure 1. 432 Mor et al. / European Journal of Chemistry 13 (4) (2022) 426-434 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.4.426-434.2315 Figure 2. Structure Activity Relationships (SAR) of compounds 6a-j for antimicrobial activity. (a) (b) Figure 3. (a) 3D and (b) 2D docked pose of compound 6j with 5TZ1. 3.2.2. Antimicrobial evaluation All synthesized compounds 1-4 and acetohydrazides 6a-j were evaluated for their in vitro antibacterial activity against two Gram-positive bacteria, viz. Bacillus subtilis (MTCC 441) and Staphylococcus aureus (MTCC 7443), two Gram-negative bacteria, viz. Escherichia coli (MTCC 1652) and P. aeruginosa (MTCC 424) and two fungi viz. Candida albicans (MTCC 227) and Aspergillus niger (MTCC 8189) using the serial dilution method [39]. Ciprofloxacin and fluconazole were used as standard drugs for preliminary bioassay of bacteria and fungi, respectively, and the results of Minimum Inhibitory Concentra- tions (MIC) were reported in terms of μmol/mL as depicted in Table 2. The antimicrobial evaluation data summarized in Table 2 revealed that compounds 6c and 6i against B. subtilis; compound 6c against S. aureus; compounds 6c, 6e and 6i against E. coli, and compound 6e against P. aeruginosa demonstrated noticeable antibacterial activity compared to the reference drug ciprofloxacin. The derivatives 6a, 6i, and 6j against C. albicans exhibited substantial antifungal activity compared to the reference drug fluconazole. Interestingly, compound 6b (MIC, 0.0135 μmol/mL) against C. albicans showed activity comparable to the standard drug fluconazole (MIC, 0.0102 μmol/mL). However, the remaining tested derivatives were found to demonstrate moderate to poor inhibitory activity against all bacterial and fungal strains under investigation. Moreover, all synthesized compounds 6a-j showed more potency against all tested strains compared to compounds 1-4. From the results of the in vitro antimicrobial assay of compounds 1-4 and 6a-j, the following structure activity relationship (SAR) can be inferred: (i) The presence of 2-Cl on the benzene ring increased the antibacterial efficiency for B. subtilis and E. coli followed by 4-F, which increased the antibacterial potency against B. subtilis, S. aureus, and E. coli, (ii) The presence of 2-CH3 on the benzene ring improved the antimicrobial activity against E. coli and P. aeruginosa, and (iii) The derivatives bearing the 3-Br, 2-Cl and 4-NO2 group on the benzene ring augmented inhibition for the fungus C. albicans. Therefore, these results showed that there are different structural requirements for a compound to be active against different strains and a general trend for Structure Activity Relationships (SAR) could not be established for the antimicrobial (antibacterial and antifungal) activities of the tested acetohydrazides 6a-j. The findings mentioned above are shown in Figure 2. 3.3. Molecular docking Molecular docking analysis of the synthesized compounds 1-4 and 6a-j was performed to identify the probable mechanism of action for their antifungal activity against C. albicans. The primary target of azoles is the sterol 14α- demethylase/heme protein which co-catalyses cytochrome P- 450-dependent 14α-demethylation of sterol [45]. Therefore, in an attempt to find out possible mechanism of binding action of the synthesized compounds, molecular docking analysis was performed with sterol 14α-demethylase of C. albicans. Hargrove et al. [46] reported the latest crystal structure of sterol 14α-demethylase of C. albicans for molecular docking analysis employing PDB code 5TZ1. Initially, the validation of the docking protocol was performed by re-docking the native ligand to the active site of C. albicans sterol 14α-demethylase (PDB ID: 5TZ1) with a root mean square deviation (RMSD) of < 1 Å. Among the entire series, compound 6b and 6j demonst- rated the highest in vitro antifungal activity against C. albicans; therefore, an analysis of all synthesized compounds was performed to determine the interactions that could be responsible for the inhibition of sterol 14α-demethylase. The derivative 6j with a docking score of -7.79 showed two aromatic interactions, one each with TYR118 and HIE377 as shown in Figure 3. Mor et al. / European Journal of Chemistry 13 (4) (2022) 426-434 433 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.4.426-434.2315 (a) (b) Figure 4. (a) 3D and (b) 2D docked poses of fluconazole with 5TZ1. On the other hand, fluconazole with a docking score of -10.04 exhibited four aromatic interactions with residues HEM601, TYR118, PHE233, and HIE377 as shown in Figure 4. Comparison of the docked pose of compound 6j and fluconazole showed that there are two common interacting residues, i.e., TYR118 and HIE377. These common interactions exhibited in the synthesized compounds are responsible for the inhibition of sterol 14α-demethylase and thus antifungal activity. The docking simulations described above reveal that compounds 6a-j are more active than their precursors 1-4, which is in agreement with the experimental in vitro antifungal evaluation results. 4. Conclusions N'-Arylidene-2-((7-methylbenzo[4, 5]thiazolo[2,3-c][1,2,4] triazol-3-yl)thio)acetohydrazides (6a-j) reported in this paper were easy to prepare from their precursors 1-4. They were preliminary examined for their in vitro Type-II antidiabetic and antimicrobial activities. Among all synthesized compounds 3 (IC50 = 0.16 μM) and 6i (IC50 = 0.38 μM) against α-amylase were found to show comparable activity with the standard acarbose with an IC50 value of 0.15 μM of both the derivatives. Analogues 6c and 6i against B. subtilis; compound 6c against S. aureus; compounds 6c, 6e, and 6i against E. coli, and compound 6e against P. aeruginosa showed noticeable antibacterial activity compared to the reference drug ciprofloxacin. The derivatives 6a, 6i, and 6j against C. albicans demonstrated substantial antifungal activity. Interestingly, compound 6b (MIC, 0.0135 μmol/mL) against C. albicans showed activity comparable to the standard drug fluconazole with a MIC value of 0.0102 μmol/mL. In addition, the antifungal activity of the synthesized compounds 6a-j was also supported by the docking simulation. From the α-amylase inhibitory and antifungal activities, it is inferred that derivatives 3, 6i and 6b can be considered as potential antidiabetic and antifungal agents, respectively, for further drug development. Acknowledgements The authors are grateful to the Council of Scientific and Industrial Research, New Delhi, India, for providing financial support (CSIR No. 09/752(0060)/ 2016-EMR-I). Disclosure statement Conflict of interests: 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: Satbir Mor, Suchita Sindhu; Methodology: Suchita Sindhu; Validation: Suchita Sindhu; Formal Analysis: Suchita Sindhu; Investigation: Satbir Mor, Suchita Sindhu; Resources: Satbir Mor, Suchita Sindhu; Data Curation: Suchita Sindhu; Writing - Original Draft: Suchita Sindhu; Writing - Review and Editing: Mohini Khatri, Ravinder Punia, Komal Jakhar; Visualization: Satbir Mor; Funding acquisition: Suchita Sindhu; Supervision: Satbir Mor; Project Administration: Satbir Mor. Funding The authors are grateful to the Council of Scientific & Industrial Research, New Delhi (CSIR no. 09/752(0060)/2016-EMR-I) for providing financial support. ORCID and Email Satbir Mor satbir_mor@yahoo.co.in https://orcid.org/0000-0003-2114-0611 Suchita Sindhu suchita.sindhu@gmail.com https://orcid.org/0000-0002-3447-0831 Mohini Khatri mohinikhatri23193@gmail.com https://orcid.org/0000-0002-8283-7167 Ravinder Punia rpunia112@gmail.com https://orcid.org/0000-0001-5085-3376 Komal Jakhar komal.jakhar@rediffmail.com https://orcid.org/0000-0003-3063-0655 References [1]. Kumar, P.; Duhan, M.; Kadyan, K.; Sindhu, J.; Kumar, S.; Sharma, H. Synthesis of novel inhibitors of α-amylase based on the thiazolidine- 4-one skeleton containing a pyrazole moiety and their configurational studies. Medchemcomm 2017, 8, 1468–1476. [2]. Cho, N. H.; Shaw, J. E.; Karuranga, S.; Huang, Y.; da Rocha Fernandes, J. D.; Ohlrogge, A. W.; Malanda, B. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res. Clin. Pract. 2018, 138, 271–281. [3]. International Diabetes Foundation: Brussels IDF diabetes atlas. https://www.diabetesatlas.org (accessed August 24, 2022). [4]. Wagman, A. S.; Boyce, R. S.; Brown, S. P.; Fang, E.; Goff, D.; Jansen, J. M.; Le, V. P.; Levine, B. H.; Ng, S. C.; Ni, Z.-J.; Nuss, J. M.; Pfister, K. B.; Ramurthy, S.; Renhowe, P. A.; Ring, D. B.; Shu, W.; Subramanian, S.; Zhou, X. A.; Shafer, C. M.; Harrison, S. D.; Johnson, K. W.; Bussiere, D. E. Synthesis, Binding Mode, and Antihyperglycemic Activity of Potent and Selective (5-Imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridyl amino)ethyl]amine Inhibitors of Glycogen Synthase Kinase 3. J. Med. Chem. 2017, 60, 8482–8514. [5]. Keri, R. S.; Patil, M. R.; Patil, S. A.; Budagumpi, S. A comprehensive review in current developments of benzothiazole-based molecules in medicinal chemistry. Eur. J. Med. Chem. 2015, 89, 207–251. mailto:satbir_mor@yahoo.co.in https://orcid.org/0000-0003-2114-0611 mailto:suchita.sindhu@gmail.com https://orcid.org/0000-0002-3447-0831 mailto:mohinikhatri23193@gmail.com https://orcid.org/0000-0002-8283-7167 mailto:rpunia112@gmail.com https://orcid.org/0000-0001-5085-3376 mailto:komal.jakhar@rediffmail.com https://orcid.org/0000-0003-3063-0655 434 Mor et al. / European Journal of Chemistry 13 (4) (2022) 426-434 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.4.426-434.2315 [6]. Patil, V. S.; Nandre, K. P.; Ghosh, S.; Rao, V. J.; Chopade, B. A.; Sridhar, B.; Bhosale, S. V.; Bhosale, S. V. Synthesis, crystal structure and antidiabetic activity of substituted (E)-3-(Benzo [d]thiazol-2- ylamino) phenylprop-2-en-1-one. Eur. J. Med. Chem. 2013, 59, 304– 309. [7]. Bashary, R.; Vyas, M.; Nayak, S. K.; Suttee, A.; Verma, S.; Narang, R.; Khatik, G. L. An insight of alpha-amylase inhibitors as a valuable tool in the management of type 2 diabetes mellitus. Curr. Diabetes Rev. 2020, 16, 117–136. [8]. Chiasson, J.-L.; Josse, R. G.; Gomis, R.; Hanefeld, M.; Karasik, A.; Laakso, M.; STOP-NIDDM Trail Research Group Acarbose for prevention of type 2 diabetes mellitus: the STOP-NIDDM randomised trial. Lancet 2002, 359, 2072–2077. [9]. Larsson, D. G. J.; Flach, C.-F. Antibiotic resistance in the environment. Nat. Rev. Microbiol. 2022, 20, 257–269. [10]. Dhingra, S.; Rahman, N. A. A.; Peile, E.; Rahman, M.; Sartelli, M.; Hassali, M. A.; Islam, T.; Islam, S.; Haque, M. Microbial resistance movements: An overview of global public health threats posed by antimicrobial resistance, and how best to counter. Front. Public Health 2020, 8, 535668. [11]. Kim, M. B.; O’Brien, T. E.; Moore, J. T.; Anderson, D. E.; Foss, M. H.; Weibel, D.-L. B.; Ames, J. B.; Shaw, J. T. The synthesis and antimicrobial activity of heterocyclic derivatives of totarol. ACS Med. Chem. Lett. 2012, 3, 818–822. [12]. Le Bozec, L.; Moody, C. J. Naturally occurring nitrogen–sulfur compounds. The benzothiazole alkaloids. Aust. J. Chem. 2009, 62, 639. [13]. Asif, M.; Imran, M. A mini-review on pharmacological importance of benzothiazole scaffold. Mini Rev. Org. Chem. 2021, 18, 1086–1097. [14]. Sharma, P. C.; Sinhmar, A.; Sharma, A.; Rajak, H.; Pathak, D. P. Medicinal significance of benzothiazole scaffold: an insight view. J. Enzyme Inhib. Med. Chem. 2013, 28, 240–266. [15]. Mustafa, M.; Winum, J.-Y. The importance of sulfur-containing motifs in drug design and discovery. Expert Opin. Drug Discov. 2022, 17, 501– 512. [16]. Scott, K. A.; Njardarson, J. T. Analysis of US FDA-approved drugs containing sulfur atoms. In Sulfur Chemistry; Springer International Publishing: Cham, 2019; pp. 1–34. [17]. Maddila, S.; Pagadala, R.; Jonnalagadda, S. 1,2,4-triazoles: A review of synthetic approaches and the biological activity. Lett. Org. Chem. 2013, 10, 693–714. [18]. Aggarwal, R.; Sumran, G. An insight on medicinal attributes of 1,2,4- triazoles. Eur. J. Med. Chem. 2020, 205, 112652. [19]. Peyton, L. R.; Gallagher, S.; Hashemzadeh, M. Triazole antifungals: a review. Drugs Today (Barc.) 2015, 51, 705–718. [20]. H. Zhou, C.; Wang, Y. Recent researches in triazole compounds as medicinal drugs. Curr. Med. Chem. 2012, 19, 239–280. [21]. Russell, P. E. A century of fungicide evolution. J. Agric. Sci. 2005, 143, 11–25. [22]. Láinez, M. J. A. Rizatriptan in the treatment of migraine. Neuropsychiatr. Dis. Treat. 2006, 2, 247–259. [23]. Stresser, D. M.; Turner, S. D.; McNamara, J.; Stocker, P.; Miller, V. P.; Crespi, C. L.; Patten, C. J. A high-throughput screen to identify inhibitors of aromatase (CYP19). Anal. Biochem. 2000, 284, 427–430. [24]. Sonsona, I. G.; Alegre-Requena, J. V.; Marqués-López, E.; Gimeno, M. C.; Herrera, R. P. Asymmetric organocatalyzed Aza-Henry reaction of hydrazones: Experimental and computational studies. Chemistry 2020, 26, 5469–5478. [25]. Wahbeh, J.; Milkowski, S. The use of hydrazones for biomedical applications. SLAS Technol. 2019, 24, 161–168. [26]. Thota, S.; Rodrigues, D. A.; Pinheiro, P. de S. M.; Lima, L. M.; Fraga, C. A. M.; Barreiro, E. J. N-Acylhydrazones as drugs. Bioorg. Med. Chem. Lett. 2018, 28, 2797–2806. [27]. Kolb, P.; Ferreira, R. S.; Irwin, J. J.; Shoichet, B. K. Docking and chemoinformatic screens for new ligands and targets. Curr. Opin. Biotechnol. 2009, 20, 429–436. [28]. Stanzione, F.; Giangreco, I.; Cole, J. C. Use of molecular docking computational tools in drug discovery. Prog. Med. Chem. 2021, 60, 273–343. [29]. Maia, E. H. B.; Assis, L. C.; de Oliveira, T. A.; da Silva, A. M.; Taranto, A. G. Structure-based virtual screening: From classical to artificial intelligence. Front. Chem. 2020, 8, 343. [30]. Mor, S.; Sindhu, S. Synthesis, Type II diabetes inhibitory activity, antimicrobial evaluation and docking studies of indeno[1,2-c]pyrazol- 4(1H)-ones. Med. Chem. Res. 2020, 29, 46–62. [31]. Mor, S.; Sindhu, S.; Nagoria, S.; Khatri, M.; Garg, P.; Sandhu, H.; Kumar, A. Synthesis, biological evaluation, and molecular docking studies of SomeN-thiazolyl hydrazones and indenopyrazolones. J. Heterocycl. Chem. 2019, 56, 1622–1633. [32]. Mor, S.; Nagoria, S.; Sindhu, S.; Khatri, M.; Sidhu, G.; Singh, V. Synthesis of Indane-Based 1,5-Benzothiazepines Derived from 3-Phenyl-2,3- dihydro-1H-inden-1-one and Antimicrobial Studies Thereof: Synthesis and Antimicrobial Studies of Indane-Based 1,5- Benzothiazepines. J. Heterocycl. Chem. 2017, 54, 3282–3293. [33]. Mor, S.; Sindhu, S.; Khatri, M.; Singh, N.; Vasudeva, N.; Panihar, N. Synthesis, Type II Diabetes Inhibitory Activity, and Antimicrobial Tests of Benzothiazole Derivatives Bridged with Indenedione by Methylenehydrazone. Russian Journal of General Chemistry 2019, 89, 1867–1873. [34]. Mor, S.; Sindhu, S.; Khatri, M.; Punia, R.; Sandhu, H.; Sindhu, J.; Jakhar, K. Antimicrobial evaluation and QSAR studies of 3,6-disubstituted- 11H-benzo[5,6][1,4]thiazino[3,4-a]isoindol-11-ones. European Journal of Medicinal Chemistry Reports 2022, 5, 100050. [35]. Mor, S.; Sindhu, S. Convenient and efficient synthesis of novel 11H- benzo[5,6][1,4]thiazino[3,4-a]isoindol-11-ones derived from 2- bromo-(2/3-substitutedphenyl)-1H-indene-1,3(2H)-diones. RSC Adv. 2019, 9, 12784–12792. [36]. Abdelazeem, A. H.; Gouda, A. M.; Omar, H. A.; Alrobaian, M. Synthesis and Biological Evaluation of Novel Benzo[4,5]thiazolo[2,3-c][1,2,4] triazole Derivatives as Potential Anticancer Agents. Acta Poloniae Pharmaceutica 2018, 75, 625–636. [37]. Xiao, Z.; Storms, R.; Tsang, A. A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Anal. Biochem. 2006, 351, 146–148. [38]. Yoshikawa, M.; Nishida, N.; Shimoda, H.; Takada, M.; Kawahara, Y.; Matsuda, H. Polyphenol Constituents from Salacia Species: Quantitative Analysis of Mangiferin with α-Glucosidase and Aldose Reductase Inhibitory Activities. Yakugaku Zasshi 2001, 121, 371–378. [39]. Mosaddik, M. A.; Haque, M. E. Cytotoxicity and antimicrobial activity of goniothalamin isolated from Bryonopsis laciniosa. Phytother. Res. 2003, 17, 1155–1157. [40]. Wood, J. L.; Roger, A. Organic Reactions, Volume 3; John Wiley & Sons: Nashville, TN, 1946. [41]. Ajmal, M.; Mideen, A. S.; Quraishi, M. A. 2-hydrazino-6-methyl- benzothiazole as an effective inhibitor for the corrosion of mild steel in acidic solutions. Corros. Sci. 1994, 36, 79–84. [42]. Aboelmagd, A.; Ali, I. A. I.; Salem, E. M. S.; Abdel-Razik, M. Synthesis and antifungal activity of some s-mercaptotriazolobenzothiazolyl amino acid derivatives. Eur. J. Med. Chem. 2013, 60, 503–511. [43]. Kumar, P.; Kadyan, K.; Duhan, M.; Sindhu, J.; Singh, V.; Saharan, B. S. Design, synthesis, conformational and molecular docking study of some novel acyl hydrazone based molecular hybrids as antimalarial and antimicrobial agents. Chem. Cent. J. 2017, 11, 115. [44]. Haraguchi, R.; Tanazawa, S.-G.; Tokunaga, N.; Fukuzawa, S.-I. Palladium-catalyzed formylation of arylzinc reagents withS-phenyl thioformate. Org. Lett. 2017, 19, 1646–1649. [45]. Hitchcock, C. A.; Dickinson, K.; Brown, S. B.; Evans, E. G. V.; Adams, D. J. Interaction of azole antifungal antibiotics with cytochrome P-450- dependent 14α-sterol demethylase purified from Candida albicans. Biochem. J. 1990, 266, 475–480. [46]. Hargrove, T. Y.; Garvey, E. P.; Hoekstra, W. J.; Yates, C. M.; Wawrzak, Z.; Rachakonda, G.; Villalta, F.; Lepesheva, G. I. Crystal structure of the new investigational drug candidate VT-1598 in complex with Aspergillus fumigatus sterol 14α-demethylase provides insights into its broad-spectrum antifungal activity. Antimicrob. Agents Chemother. 2017, 61, e00570-17. Copyright © 2022 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/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 (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Chemistry 2.2. Synthesis 2.2.1. General procedure for the synthesis of 2-hydrazinyl-6-methylbenzo[d]thiazole (1) 2.2.2. General procedure for the synthesis of 7-methylbenzo [4,5]thiazolo[2,3-c][1,2,4]triazole-3-thiol (2) 2.2.3. General procedure for the synthesis of ethyl 2-((7-methylbenzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl)thio) acetate (3) 2.2.4. General procedure for the synthesis of 2-((7-methyl benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3-yl)thio)aceto- hydrazide (4) 2.2.5. General procedure for the synthesis of 7-methylbenzo [4,5]thiazolo[2,3-c][1,2,4]triazole based hydrazones (6a-j) 2.3. Biological evaluation 2.3.1. In vitro α-amylase inhibition 2.3.2. Antimicrobial evaluation 2.4. Molecular docking study 3. Results and discussion 3.1. Chemistry 3.2. Pharmacological assay 3.2.1. Antidiabetic evaluation 3.2.2. Antimicrobial evaluation 3.3. Molecular docking 4. Conclusions Acknowledgements Disclosure statement CRediT authorship contribution statement Funding ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: