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Design, synthesis of new novel quinoxalin-2(1H)-one derivatives incorporating hydrazone, hydrazine, and pyrazole moieties as antimicrobial potential with in-silico ADME and molecular docking simulation
⁎Corresponding authors. ahmed_ragab@azhar.edu.eg (Ahmed Ragab), Ahmed_ragab7@ymail.com (Ahmed Ragab), yossry@azhar.edu.eg (Yousry A. Ammar) yossry@yahoo.com (Yousry A. Ammar)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A series of 6-(morpholinosulfonyl)quinoxalin-2(1H)-one based hydrazone, hydrazine, and pyrazole moieties were designed, synthesized, and evaluated for their in vitro antimicrobial activity. All the synthesized quinoxaline derivatives were characterized by IR, NMR (1H /13C), and EI MS. The results displayed good to moderate antimicrobial potential against six bacterial, and two fungal standard strains. Among the tested derivatives, six quinoxalin-2(1H)-one derivatives 4a, 7, 8a, 11b, 13, and 16 exhibited a significant antibacterial activity with MIC values (0.97–62.5 µg/mL), and MBC values (1.94–88.8 µg/mL) compared with Tetracycline (MICs = 15.62–62.5 µg/mL, and MBCs = 18.74–93.75 µg/mL), and Amphotericin B (MICs = 12.49–88.8 µg/mL, and MFC = 34.62–65.62 µg/mL). In addition, according to CLSI standards, the most active quinoxalin-2(1H)-one derivatives demonstrated bactericidal and fungicidal behavior. Moreover, the most active quinoxaline derivatives showed a considerable antibacterial activity with bactericidal potential against multi-drug resistance bacteria (MDRB) strains with MIC values ranged between (1.95–15.62 µg/mL), and MBC values (3.31–31.25 µg/mL) near to standard Norfloxacin (MIC = 0.78–3.13 µg/mL, and MBC = 1.4–5.32 µg/mL. Further, in vitro S. aureus DNA gyrase inhibition activity were evaluated for the promising derivatives and displayed potency with IC50 values (10.93 ± 1.81–26.18 ± 1.22 µM) compared with Ciprofloxacin (26.31 ± 1.64 µM). Interestingly, these derivatives revealed as good immunomodulatory agents by a percentage ranging between 82.8 ± 0.37 and 142.4 ± 0.98 %. Finally, some in silico ADME, toxicity prediction, and molecular docking simulation were performed and showed a promising safety profile with good binding mode.
Keywords
Quinoxaline derivatives, Antimicrobial activity
MIC, MBC and MFC
Multi-drug resistance bacteria (MDRB)
DNA gyrase
In silico ADME and molecular docking
1 Introduction
Multidrug-resistant bacteria have become a serious concern in many countries around the world in recent decades. The medical community has been seriously affected by the infections caused by these bacteria, and the necessity for treatment has led to research into new antimicrobials agents (Cheesman et al., 2017; Wise et al., 1998). Infections as rheumatic, diarrhea, food poisoning, and salmonellosis are caused by multidrug-resistant gram-negative and gram-positive pathogens such as S. typhimurium E. coli, S. aureus, and S. pyogenes (Ayliffe, 1997; Khan and Asiri, 2011). These infections are responsible for a high mortality rate in both our community and hospitals (Almeida et al., 2021; Mitevska et al., 2021; Wanger and Chávez, 2021). As a result of these parasitic, bacterial infections, millions of people around the subtropical regions are infected, and 20,000 deaths every year. Ciprofloxacin, Amoxicillin, and Norfloxacin are the most commonly used antibiotics for treating bacterial infections since they are effective against intestinal and extra-intestinal wall infections (Guo et al., 2021; Ito and Budke, 2021). However, microbial infections have been increasing dramatically and are currently estimated to affect approximately 1.2 billion people globally (Denning and Bromley, 2015; Zhao et al., 2018).
The incidence of invasive fungal infections (IFIs) and the emergence of resistant fungal pathogens have increased markedly, leading to high morbidity and mortality in immune-compromised patients, such as patients receiving organ transplants, patients undergoing anticancer chemotherapy, and patients with AIDS (Campoy and Adrio, 2017; Liu et al., 2011). Clinically, the three fungal genera, aspergillus, candida, and cryptococcus account for most fungal infections (Denning and Hope, 2010). The common antifungal agents currently used in the clinic are Amphotericin B, Nystatin, Echinocandins, Caspofungin, and Micafungin (Langebrake et al., 2014; Surarit and Shepherd, 1987). As a result, discovering and developing a new class of antimicrobial drugs is critical to fighting the increasing danger of drug-resistant microbes (El-Attar et al., 2018).
Quinoxalines form an attractive biologically active molecule as these are a part of various antibiotics (Kim et al., 2004). The quinoxaline antibiotics of bicyclic showed activity against gram-positive bacteria (Shōji and Katagiri, 1961) and certain animal tumors (Xu et al., 2016) and also are potent inhibitors of RNA synthesis (Khatoon and Abdulmalek, 2021). The mechanism of action occurs by binding to DNA, in which they function as bifunctional intercalating agents. Two antibiotic families of the antibiotic Echinomycin (Kim et al., 2004) and the Triostins are well known. Both series are similar in composition; they consist of two quinoxaline-2-carboxylic acid moieties (Ughetto et al., 1985) (Fig. 1).
Rational design of the target quinoxaline derivatives and previously reported quinoxaline or azomethane groups containing drugs.
Furthermore, marketed drugs, such as Levomycin, Actinoleutin, Quinacillin, contain a quinoxaline ring (Fig. 1) (Bough et al., 1971; Christie et al., 1966; Salwan and Sharma, 2020). Many scientists have reported quinoxalinone derivatives as non-classical analogs of the antifolic agents as Methotrexate and Trimetrexate (Sanna et al., 1998). Additionally, the quinoxaline scaffold is known to be characterized by medically important derivatives with many therapeutical applications as anti-inflammatory (El-Sabbagh et al., 2009), antidiabetic (Yang et al., 2012), anthelmintic (Sakata et al., 1988), antiprotozoal (Guillon et al., 2011), antiviral (Ali et al., 2007), antidepressant (Sarges et al., 1990), antituberculosis (Ancizu et al., 2010), anticancer (Khan et al., 2009), and antimicrobial (Ammar et al., 2020a).
Sulfonamides are well-known antibiotics for treating bacterial infection, malaria, leprosy, etc. (Mondal et al., 2017). In addition, sulfonamides are commonly used antibacterial agents worldwide, owing to their low toxicity, low cost, and excellent efficacy against common bacterial diseases (Özbek et al., 2007). Sulfa drugs exert their bactericidal effect by inhibiting the metabolic pathway of the enzyme dihydropteroate synthetase (DHPS). Folate, a vital agent for forming nucleic acids (DNA, RNA) in the cells, is synthesized by direct participation of DHPS in a catalytic cycle (Epstein et al., 1997; Mondal et al., 2017; Smilack, 1999). Prolonged consumption of sulfonamides shows some adverse reactions to the liver, kidney, skin, lung, heart, and blood (Mondal et al., 2017). These side effects have demanded worldwide effort to search for new generation drugs. The literature reveals that the presence of a morpholine ring on a heterocyclic system contributes to enhanced pharmacological activities in many cases (El-sharief et al., 2019; Muhammad et al., 2017).
Furthermore, the hydrazone function, R1R2C = NR3–NR4 (R = alkyl, aryl or H), is an important pharmacophore in a variety of drugs, especially antibiotic drugs as Thioacetazone, Furazolidone, Nitrofurazone, and Rifampicin (Fig. 1) (Matson and Stupp, 2011). Related hydrazide-hydrazones have been shown to exhibit significant antibacterial (El-Sharief et al., 2016; Hassan et al., 2021), antifungal (Rahman et al., 2005), anticonvulsant (Fayed et al., 2021b; Ragavendran et al., 2007), anticancer (Ammar et al., 2018; Fayed et al., 2020), carbonic anhydrase inhibitors (Wassel et al., 2021b), anti-inflammatory (Salgın-Gökşen et al., 2007), and antimalarial activity (Verma et al., 2014).
Because of the findings mentioned earlier, and as a continuation of our effort in medicinal chemistry (Fayed et al., 2021a; Rizk et al., 2020; Selim et al., 2019; Wassel et al., 2021a) and identifying new candidates that may be of value in designing new, potent, selective, and less toxic antibacterial agents (Ibrahim et al., 2021a). We herein reported design, synthesis, and antimicrobial evaluation of novel structure hybrids incorporating the 6-(morpholinosulfonyl)quinoxaline derivatives with hydrazine, hydrazone, and pyrazole. The hybrid of both moieties in a single entity may result in worthwhile molecules with promising antibacterial activity. The antifungal and antibacterial actions of all novel synthetized quinoxaline derivatives were investigated in vitro using the agar well diffusion method to determine the inhibition zones (IZs). Besides, the most active quinoxaline derivatives were further evaluated to determine the MIC, MBC/MFC against the standard and multidrug-resistant strains and determine the inhibitory assay of in vitro S. aerates DNA gyrase. Besides, the molecular docking simulation inside the active site of DNA gyrase was achieved to determine the binding energy and binding mode. Finally, the in-silico prediction of physicochemical, drug-likeness, some pharmacokinetics, medicinal chemistry, and toxicity predictions were calculated using web tools.
2 Material and methods
2.1 Chemistry
With no further purifications, reagents and chemicals were acquired from Aldrich Chemicals, and solvent from Fisher. Melting points (MPs) of all the newly designed compounds were recorded on a digital Gallen Kamp MFB-595 instrument using open capillaries. Within the range of 400–4000 cm−1, IR spectra were calculated using the KBr disc methodology on a Shimadzu 440 spectrophotometer. In NMR spectra (1H / 13C), chemical shifts were calculated in δ/ppm relative to TMS as an internal default (ppm) that obtained on a JOEL spectrometer 400 / 101 MHz using DMSO‑d6 as solvents. The data was provided in the following format: chemical shift, multiplicity (br = broad, m = multiplet, q = quartet, t = triplet, d = doublet, and s = singlet), the coupling constant (J) in Hertz (Hz), and integration. Elemental analysis were carried out at Micro Analytical Unit in Cairo University, Cairo. The mass spectra were calculated at 70 eV using the DI-50 unit of a Shimadzu GC/MSQP5050A Spectrometer at Al-Azhar University's Regional Center for Biotechnology. 6-(Morpholinosulfonyl)-1,4-dihydroquinoxaline-2,3-dione (2) was prepared according to previously reported methods (Ammar et al., 2020a, 2020b).
2.1.1 Synthesis of 3-hydrazinyl-6-(morpholinosulfonyl)quinoxalin-2(1H)-one (3a)
To a solution of dihydroquinoxaline-2,3-dione derivative 2 (1 mmol) in EtOH (5 mL), the hydrazine hydrate (80%) (5 mL) was added dropwise, and the solution was stirred at room temperature for 0.5 hr. Additionally, the reaction mixture was heated under reflux for 3 hs (TLC), then allowed to cool. The solid precipitate that formed was collected by filtration and crystallized from EtOH to yield the desired product.
Yield 81% as yellow crystals; M.p. = 230–232 ˚C; IR (KBr, cm−1): 3312, 3245 (NH2, 2NH), 3025 (CH-Ar.), 2972, 2847 (CH-aliph.), 1678 (C = O), 1621 (C = N) 1332, 1155 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.19, 9.50 (s, 2H, 2NH; D2O exchangeable), 7.57 (s, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.38 (d, J = 8.2 Hz, 1H), 4.79 (s, 2H, NH2; D2O exchangeable), 3.62 (t, 4H, (CH2)2O), 2.88 (t, 4H, (CH2)2N); 13C NMR (101 MHz, DMSO‑d6) δ/ppm 155.23 (C = O), 151.21 (C = C-N), 150.44 (C = N), 135.08, 124.67, 122.98, 117.17, 115.05, 65.76 ((CH2)2O), 46.35 ((CH2)2N); MS : (Mwt = 325): m/z, 45.36 (48%), 53.09 (71%), 57.01 (100%), 192.16 (66%), 24.75 (43%), 227.69 (42%), 324 (M−1, 48%), 325.42 (M+, 49%); Anal. Calcd. for C12H15N5O4S (325.34): C, 44.30; H, 4.65; N, 21.53; Found: C, 44.35; H, 4.44; N, 21.41.
2.1.2 Synthesis of hydrazone derivatives linked 6-(morpholinosulfonyl)quinoxalin-2(1H)-one (4–7)
To a solution of 3-(hydrazinyl)quinoxalin-2(1H)-one derivative 3a (1 mmol) in ethanol (25 mL) and various substituted aromatic aldehydes (1 mmol) catalyzed with acetic acid (2 mL). The solution mixture was heated under reflux conditions for a period of 3–6 hs (TLC). The solid precipitate that formed was collected by filtration and crystallized from EtOH/DMF to yield the desired products.
2.1.3 3-(2-(4-Chlorobenzylidene)hydrazinyl)-6-(morpholinosulfonyl)quinoxalin-2(1H)-one (4a)
Yield 75% as light orange powder; M.p. = 325–327 ˚C; IR (KBr, cm−1): 3323, 3228 (2NH), 3067 (CH Ar.), 2946, 2843 (CH aliph.), 1689 (C = O), 1609 (C = N), 1337, 1153 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.22, 12.02 (s, 2H, 2NH; D2O exchangeable), 8.70 (s, 1H, CH = N), 7.87 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.47 (s, 1H), 7.42 (dd, J = 8.4, 1.9 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 3.61 (t, 4H, (CH2)2O), 2.83 (t, 4H, (CH2)2N); 13C NMR (101 MHz, DMSO‑d6): δ /ppm 161.15 (C = O), 155.70 (C = N), 155.33 (CH = N), 133.03, 130.51, 130.28, 129.58, 128.53, 126.60, 122.88, 116.16, 114.95, 65.72 ((CH2)2O), 46.29 ((CH2)2N); Anal. Calcd. for C19H18ClN5O4S (447.89): C, 50.95; H, 4.05; N, 15.64; Found: C, 50.82; H, 3.88; N, 15.79.
2.1.4 3-(2-(4-Fluorobenzylidene)hydrazinyl)-6-(morpholinosulfonyl)quinoxalin-2(1H)-one (4b)
Yield 61% as deep orange crystals; M.p. = 335–337 ˚C; IR (KBr, cm−1) = 3325, 3210 (2NH), 3068 (CH Ar.), 2955, 2858 (CH aliph.), 1688 (C = O), 1603 (CH = N), 1335, 1152 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.22, 12.02 (s, 2H, 2NH; D2O exchangeable), 8.70 (s, 1H, CH = N), 7.91 (d, J = 8.8 Hz, 2H), 7.47 (s, 1H), 7.41 (dd, J = 8.4, 2.0 Hz, 1H), 7.34 (d, J = 8.9 Hz, 2H), 7.28 (d, J = 8.4 Hz, 1H), 3.61 (t, 4H, (CH2)2O), 2.83 (t, 4H, (CH2)2N); 13C NMR (101 MHz, DMSO‑d6): δ/ppm 160.98 (C = O), 155.71 (C = N), 155.33 (CH = N), 131.21, 131.12, 130.87, 130.84, 128.53, 126.60, 122.87, 116.66, 116.44, 116.16, 114.95, 65.72 ((CH2)2O), 46.29 ((CH2)2N); Anal. Calcd. for C19H18FN5O4S (431.44): C, 52.89; H, 4.21; N, 16.23; Found: C, 52.98; H, 4.05; N, 16.07
2.1.5 3-(2-(4-Methoxybenzylidene)hydrzinyl)-6-(morpholinosulfonyl)quinoxalin-2(1H)-one (4c)
Yield 70% as yellow powder; M.p. = 296–298 ˚C; IR (KBr, cm−1): 3325, 3296 (2NH), 3057 (CH-Aro.), 2920, 2854 (CH-aliph.), 1677 (C = O), 1608 (CH = N), 1346, 1160 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 11.33, 10.68 (s, 2H, 2NH; D2O exchangeable), 8.61 (s, 1H, CH = N), 8.51 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.72 (s, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 3.81 (s, 3H, OCH3), 3.61 (t, 4H, (CH2)2O), 2.86 (t, 4H, ((CH2)2N); Anal. Calcd. For C20H21N5O5S (443.48): C, 54.17; H, 4.77; N, 15.79; Found: C, 53.92; H, 4.69; N, 15.55.
2.1.6 3-(2-(2-Hydroxybenzylidene)hydrazineyl)-6-(morpholinosulfonyl)quinoxalin-2(1H)-one (5)
Yield 61% as light yellow powder; M.p. = 318–320 ˚C; IR (KBr, cm−1): 3423 (br-OH), 3261 (2NH), 3061 (CH-Aro.), 2973, 2847 (CH-aliph.), 1687 (C = O), 1619 (CH = N), 1339, 1164 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 11.91, 11.72, 11.62 (s, 3H, OH; 2NH, D2O exchangeable), 8.76 (s, 1H, CH = N), 8.74 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.30 (t, 2H), 6.95 (d, 1H), 6.93 (d, 1H), 6.90 (d, 1H), 3.63 (t, 4H, (CH2)2O), 2.87 (t, 4H, (CH2)2N); MS : (Mwt = 429): m/z, 44.05 (40%), 123.92 (87 %), 137.73 (100%), 154.19 (57%), 333.42 (56%), 429.74 (M+, 13%); Anal. Calcd. For C19H19N5O5S (429.45): C, 53.14; H, 4.46; N, 16.31; Found: C, 53.29; H, 4.19; N, 16.15
2.1.7 3-(2-(4-Hydroxy-3-methoxybenzylidene)hydrazinyl)-6-(morpholinosulfonyl)quinoxalin-2(1H)-one (6)
Yield 63% as pale orange powder; M.p. = 346–348 ˚C; IR (KBr, cm−1): 3450 (OH), 3230 (2NH), 3052 (CH-Aro.), 2930, 2845 (CH-aliph.), 1680 (C = O), 1605 (CH = N), 1344, 1163 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 11.87, 11.27, 9.53 (s, 3H, OH, 2NH; D2O exchangeable), 8.47 (s, 1H, CH = N), 8.44 (s, 1H), 7.48 (dd, J = 8.4, 2.0 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.29 (s, 1H), 7.08 (dd, J = 8.4, 1.8 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 3.84 (s, 3H, OCH3), 3.61 (t, 4H, (CH2)2O), 2.86 (t, 4H, (CH2)2N); 13C NMR (101 MHz, DMSO‑d6): δ/ppm 158.00 (C = O), 157.87 (C = N), 155.70 (CH = N), 155.33, 151.97, 148.69, 145.11, 131.22, 129.85, 127.83, 125.24, 124.21, 117.43, 116.21, 111.80, 65.73 ((CH2)2O), 61.80 (OCH3), 46.35 ((CH2)2N); Anal. Calcd. For C20H21N5O6S (459.48): C, 52.28; H, 4.61; N, 15.24; Found: C, 52.23; H, 4.55; N, 15.19.
2.1.8 3-(2-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-6-(morpholinosulfonyl) quinoxalin-2(1H)-one (7)
Yield 77% as deep yellow powder; M.p. = 200–205 ˚C; IR (KBr, cm−1): 3356, 3287 (2NH), 3052 (CH-Aro.), 2993, 2913, 2852 (CH-aliph.), 1683 (C = O), 1616 (CH = N), 1360, 1161 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 11.52, 9.30 (s, 2H, 2NH; D2O exchangeable), 9.14 (s, 2H, CH = N, CH-pyrazole), 9.01 (d, J = 8.8 Hz, 1H), 8.83 (d, J = 8.8 Hz, 1H), 8.67 (s, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.73 (d, J = 6.8 Hz, 2H), 7.49 (t, 3H), 7.36 (t, 3H), 3.63 (t, 4H, (CH2)2O), 2.87 (t, 4H, (CH2)2N); 13C NMR (101 MHz, DMSO‑d6): δ/ppm 159.66 (C = N), 154.03 (C = O), 153.35 (CH = N), 151.36 (C = N-pyrazole), 148.67, 139.37, 132.24, 131.56, 130.15, 129.33, 129.27, 129.05, 127.74, 127.71, 126.22, 123.78, 120.12, 119.39 , 116.89, 114.77, 66.81 ((CH2)2O), 42.28 ((CH2)2N); Anal. Calcd. For C28H25N7O4S (555.61): C, 60.53; H, 4.54; N, 17.65; Found: C, 60.50; H, 4.51; N, 17.62.
2.1.9 General method for synthesis of hydrazone derivatives (8–11)
A mixture of 3-(hydrazinyl)quinoxaline derivative 3a (1 mmol) and substituted ketone derivatives (1 mmol) in EtOH (25 mL), acetic acid as catalyst (2 mL) was added. The solution mixture was heated under reflux for a period of 6–8 hs (TLC), then allowed to cool. The solvent was removed by rotary evaporator and the precipitate was quenched with crushed ice. The resulting precipitate was filtered off, dried and recrystallized from ethanol to yield (8–11).
2.1.10 3-(2-(1-(4-Bromophenyl)ethylidene)hydrazinyl)-6-(morpholinosulfonyl)quinoxalin-2(1H)-one (8a)
Yield 75% as red powder; M.p. = 183–185 ˚C; IR (KBr, cm−1): 3338, 3224 (2NH), 3071 (CH-Aro.), 2954, 2863 (CH-aliph.), 1691 (C = O), 1609 (C = N), 1337, 1153 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 11.72, 10.56 (s, 2H, 2NH; D2O exchangeable), 8.02 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.34–7.66 (m, 5H), 3.62 (t, 4H, (CH2)2O), 2.84 (t, 4H, (CH2)2N), 2.42 (s, 3H, CH3); 13C NMR (101 MHz, DMSO‑d6): δ/ppm 162.13 (C = O), 155.19 (C = N), 142.69, 137.51, 131.64, 129.74, 128.97, 126.90, 126.10, 124.03, 123.11, 115.82, 114.69, 65.74 ((CH2)2O, 46.32 ((CH2)2N), 15.14 (CH3); Anal. Calcd. for C20H20BrN5O4S (506.38): C, 47.44; H, 3.98; N, 13.83; Found: C, 47.31; H, 3.75; N, 13.99.
2.1.11 3-(2-(1-(4-Aminophenyl)ethylidene)hydrazinyl)-6-(morpholinosulfonyl)quinoxalin-2(1H)-one (8b)
Yield 69% as brown crystals; M.p. = ˃ 360 ˚C; IR (KBr, cm−1): 3421, 3352, 3278 (NH2, 2NH), 3089 (CH-Aro.), 2974, 2920, 2858 (CH-aliph.), 1685 (C = O), 1604 (C = N), 1388, 1157 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 11.79, 10.29 (s, 2H, 2NH; D2O exchangeable), 7.92 (s, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H), 6.58 (d, J = 8.8 Hz, 1H), 5.58 (s, 2H; D2O exchangeable), 3.61 (t, 4H, (CH2)2O), 2.84 (t, 4H, (CH2)2N), 2.36 (s, 3H, CH3); 13C NMR (101 MHz, DMSO‑d6): δ/ppm 164.84 (C = O), 152.22 (C = N), 151.49, 150.82, 136.86, 135.37, 129.25, 128.31, 127.37, 123.79, 123.03, 113.45, 112.90, 111.54, 111.46, 65.75 ((CH2)2O), 46.33 ((CH2)2N), 14.65 (CH3); Anal. Calcd. for C20H22N6O4S (442.49): C, 54.29; H, 5.01; N, 18.99; Found: C, 54.11; H, 4.85; N, 19.07
2.1.12 6-(Morpholinosulfonyl)-3-(2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)hydrazinyl)quinoxalin-2(1H)-one (9)
Yield 71% as deep red crystals; M.p. = 323–325 ˚C; IR (KBr, cm−1): 3356, 3280 (2NH), 3132 (CH-Aro.), 2950, 2857, 2775 (CH-aliph.), 1698 (C = O), 1619 (C = N), 1373, 1154 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 11.91, 11.71 (s, 2H, 2NH; D2O exchangeable), 8.74 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.54 (dd, J = 8.4, 2.0 Hz, 1H), 7.45 (t, J = 7.6 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.39 (s, 1H), 7.29 (t, 1H), 6.94 (d, J = 8.7 Hz, 1H), 3.62 (t, 4H, (CH2)2O), 3.31 (s, 3H, CH3), 2.86 (t, 4H, (CH2)2N); Anal. Calcd. for C23H21N5O6S (495.51): C, 55.75; H, 4.27; N, 14.13; Found: C, 55.71; H, 4.25; N, 14.10.
2.1.13 3-(2-(5-Methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-ylidene)hydrazinyl)-6-(morpholino-sulfonyl)quinoxalin-2(1H)-one (10)
Yield 65% as pale brown crystals; M.p. = 310–312 ˚C; IR (KBr, cm−1): 3315, 3219 (2NH), 3073 (CH Ar.), 2954, 2851 (CH aliph.), 1690 (C = O), 1608 (C = N), 1336, 1152 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.08, 12.27 (s, 2H, 2NH; D2O exchangeable), 8.69 (s, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.26 – 7.59 (m, 5H), 4.35 (s, 2H, CH2), 3.64 (t, 4H, (CH2)2O), 2.85 (t, 4H, (CH2)2N), 2.51 (s, 3H, CH3); Anal. Calcd. for C22H23N7O4S (481.53): C, 54.88; H, 4.81; N, 20.36; Found: C, 54.63; H, 4.97; N, 20.21.
2.1.14 6-(Morpholinosulfonyl)-3-(2-(5-(morpholinosulfonyl)-2-oxoindolin-3-ylidene)hydrazinyl)-quinoxalin-2(1H)-one (11a)
Yield 84% as reddish orange powder; M.p. = 273–275 ˚C; IR (KBr, cm−1): 3345, 3183 (3NH), 3056 (CH-Aro.), 2963, 2853 (CH-aliph.), 1697 (2C = O), 1614 (C = N), 1335, 1157 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.03, 11.80, 11.23 (s, 3H, 3NH; D2O exchangeable), 8.85 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 3.63 (t, 8H, 2(CH2)2O), 2.93 (t, 4H, (CH2)-N), 2.87 (t, 4H, (CH2)-N); 13C NMR (101 MHz, DMSO‑d6) δ/ppm 172.53 (C = O), 163.54 (C = O), 155.70 (C = N), 155.32 (C = C), 151.48, 135.60, 130.28, 128.57, 128.33, 126.60, 122.87, 121.23, 116.16, 114.95, 112.07, 110.73, 65.73 (CH2)2O), 46.42 (CH2)2N), MS : (Mwt = 603): m/z, 128.47 (40%), 257.98 (77%), 301.50 (100%), 559.11 (58%), 603.45 (M+, 13%); Anal. Calcd. for C24H25N7O8S2 (603.63): C, 47.76; H, 4.17; N, 16.24; Found: C, 47.93; H, 4.32; N, 16.10.
2.1.15 6-(Morpholinosulfonyl)-3-(2-(2-oxo-5-(piperidin-1-ylsulfonyl)indolin-3-ylidene)hydrazinyl)-quinoxalin-2(1H)-one (11b)
Yield 82% as orange crystals; M.p. = 288–290 ˚C; IR (KBr, cm−1):3365, 3193 (3NH), 3053 (CH-Aro.), 2970, 2850 (CH-aliph.), 1699 (br-2C = O), 1620 (C = N), 1346, 1152 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.24, 12.03, 11.19 (s, 3H, 3NH; D2O exchangeable), 8.82 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 3.62 (t, 4H, (CH2)2-O), 2.92 (t, 4H, (CH2)2-N), 2.86 (t, 4H, 2CH2-pip), 1.53 (m, 4H, 2CH2-pip), 1.35 (t, 2H, CH2-pip); 13C NMR (101 MHz, DMSO‑d6) δ/ppm 163.14 (C = O), 155.71 (C = O), 155.33 (C = N), 142.16, 130.30, 128.57, 127.14, 126.62, 124.86, 123.21, 122.87, 116.51, 116.17, 114.96, 110.64, 65.73 ((CH2)2O), 47.10 ((CH2)2N), 46.29 ((CH2)2N), 25.14 (2CH2-pip), 23.31 (CH2-pip); Anal. Calcd. for C25H27N7O7S2 (601.65): C, 49.91; H, 4.52; N, 16.30; Found: C, 49.85; H, 4.49; N, 16.27.
2.1.16 Synthesis of 2-(7-(morpholinosulfonyl)-3-oxo-3,4-dihydroquinoxalin-2-yl)-N-phenyl-hydrazine-1-carbothioamide (12)
To a solution of 3-(hydrazinyl)quinoxaline derivative 3a (1 mmol), phenyl isothiocyanate (1 mmol) in absolute ethanol (25 mL) with three drops of triethyl amine (TEA) was heated under reflux for 6 hs (TLC). The solid precipitate that formed was collected by filtration and crystallized from EtOH to yield the desired product.
Yield 51% as yellow powder; M.p. = 218–220 ˚C; IR (KBr, cm−1): 3201, 3121 (4NH), 3055 (CH– Aro.), 2985, 2972, 2902 (CH-aliph.), 1688 (C = O), 1600 (C = N), 1347, 1159 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.25, 12.05, 9.52, 9.35 (s, 4H, 4NH; D2O exchangeable), 8.32 (d, J = 9.1 Hz, 1H), 7.75 (s, 1H), 7.55 (t, 1H), 7.50 (d, 1H), 7.43 (d, 2H), 7.26 (t, 2H), 3.64 (t, 4H, (CH2)2O), 2.86 (t, 4H, (CH2)2N); MS: (Mwt = 460): m/z, 63.36 (43%), 74.02 (100%), 413.74 (58%), 454.93 (20 %), 460.23 (M+, 20%); Anal. Calcd. for C19H20N6O4S2 (460.53): C, 49.55; H, 4.38; N, 18.25; Found: C, 49.31; H, 4.54; N, 18.10.
2.1.17 Synthesis of 4-(2-(7-(morpholinosulfonyl)-3-oxo-3,4-dihydroquinoxalin-2-yl)hydrazinyl)-4-oxobutanoic acid (13)
An equimolar mixture of 3-(hydrazinyl)quinoxaline derivative 3a (1 mmol), and succinic anhydride (1 mmol) in absolute ethanol (25 mL), firstly the reaction mixture was heated under reflux for a period 5 hs (TLC). The resulting mixture was precipitated on hot, collected by filtration, dried and washed with hot ethanol or recrystallized from ethanol/DMF to give the desired product.
Yield 72% as yellow crystals; M.p. = 303–305 ˚C; IR (KBr, cm−1): 3350, 3207 (3NH), 3050 (CH Ar.), 2967, 2860 (CH aliph.), 1684 (C = O), 1601 (C = N), 1347, 1154 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.27, 12.09, 9.74, 8.96 (s, 4H, OH, 3NH; D2O exchangeable), 7.49 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 3.67 (t, 4H, (CH2)2O), 2.86 (t, 4H, (CH2)2N), 2.44 (t, 2H, CH2), 2.34 (t, 2H, CH2); 13C NMR (101 MHz, DMSO‑d6) δ/ppm 174.06 (C-OH), 170.38, 155.69 (2C = O), 155.32 (C = N), 130.29, 128.57, 126.61, 122.88, 116.16, 114.95, 65.73 ((CH2)2O), 46.29 ((CH2)2N), 29.24 (CH2), 28.42 (CH2); MS: (Mwt = 425): m/z, 79.33 (69%), 168.77 (73%), 218.28 (90%), 259.62 (100%), 330.04 (78%), 425.66 (M+, 58%); Anal. Calcd. for C16H19N5O7S (425.42): C, 45.17; H, 4.50; N, 16.46; Found: C, 45.32; H, 4.74; N, 16.23.
2.1.18 Synthesis of ethyl 5-amino-3-(methylthio)-1-(6-(morpholinosulfonyl)-2-oxo-1,2-dihydroqui-noxalin-3-yl)-1H-pyrazole-4-carboxylate (14)
To equimolar amount of 3-(hydrazinyl)quinoxaline derivative 3a (1 mmol) and ethyl 2-cyano-3,3-bis(methylthio)acrylate (1 mmol) in absolute ethanol (25 mL) was heated under reflux for 5 hs (TLC). The solid precipitate that formed was collected by filtration and crystallized from EtOH/DMF to yield the desired product (14).
Yield 68% as light yellow crystals; M.p. = 283–285 ˚C; IR (KBr, cm−1): 3410, 3350, 3260 (NH2, NH), 3034 (CH-Aro.), 2904, 2825 (CH-aliph.), 1690 (C = O), 1615 (C = N), 1357, 1162 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 13.16 (s, 1H, NH; D2O exchangeable), 8.12 (s, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 6.92 (s, 2H, NH2, D2O exchangeable), 4.23 (q, 2H, CH2), 3.62 (t, 4H, (CH2)2O), 2.91 (t, 4H, (CH2)2N), 2.36 (s, 3H, S-CH3), 1.27 (t, 3H, (CH3-CH2)); MS: (Mwt = 496): m/z, 82.42 (44%), 137.38 (100 %), 197.56 (44%), 357.35 (53%), 421.79 (57%), 459.09 (69%), 472.08 (51%), 494.96 (M+1, 15%); Anal. Calcd. for C19H22N6O6S2 (494.54): C, 46.15; H, 4.48; N, 16.99; Found: C, 46.43; H, 4.31; N, 16.84.
2.1.19 Synthesis of 5-amino-3-(cyanomethyl)-1-(7-(morpholinosulfonyl)-3-oxo-3,4-dihydroquinoxalin-2-yl)-1H-pyrazole-4-carbonitrile (16)
An equimolar amount of 3-(hydrazinyl)quinoxaline derivative 3a (1 mmol) and 2-amino-1,1,3-propenetricarbonitrile (1 mmol) in absolute ethanol (25 mL). The reaction mixture was allowed to cool after being heated under reflux for 8 hs (TLC). The solid precipitate that formed was collected by filtration and crystallized from EtOH to yield the desired product.
Yield 60% as light red crystals; M.p. = 200–202 ˚C; IR (KBr, cm−1): 3307, 3203 (NH2, NH), 3060 (CH-Aro.), 2971, 2902, 2860 (CH-aliph.), 2286 (CH2-C≡N), 2204 (C≡N), 1684 (C = O), 1608 (C = N), 1343, 1156 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 9.27 (s, 1H, NH; D2O exchangeable), 7.60 (s, 1H), 7.50 (s, 2H, NH2; D2O exchangeable), 7.43 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 3.63 (t, 4H, (CH2)2O), 3.17 (s, 2H, CH2-C≡N), 2.86 (t, 4H, (CH2)2N); MS: (Mwt = 440): m/z, 50.33 (45%), 65.43 (100%), 103.95 (53%), 234.40 (55%), 267.49 (51%), 440.00 (M+, 7%); Anal. Calcd. for C18H16N8O4S (440.44): C, 49.09; H, 3.66; N, 25.44; Found: C, 48.92; H, 3.83; N, 25.29.
2.1.20 General method for synthesis of hydrazone derivatives (17a, b)
An equimolar amount of 3-(hydrazinyl)quinoxaline derivative 3a (1 mmol) and requisite active methylene (ethyl acetoacetate or acetyl acetone) (1 mmol) in absolute ethanol (25 mL). The suspension was heated under reflux for a period 5–7 hs (TLC), then allowed to cool. The precipitate that formed was collected by filtration and crystallized from EtOH/ DMF to afford the desired product.
2.1.21 Ethyl-3-(2-(7-(morpholinosulfonyl)-3-oxo-3,4-dihydroquinoxalin-2-yl)hydrazineylidene) Butanoate (17a)
Yield 58% as yellowish crystals; M.p. = 205–208 ˚C; IR (KBr, cm−1): 3354, 3244 (2NH), 3065 (CH Ar.), 2970, 2901, 2862 (CH-aliph.), 1684 (br 2C = O), 1609 (C = N), 1347, 1160 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.18 (br s, 2H, 2NH; D2O exchangeable), 7.49 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 4.13 (q, 2H, (CH3-CH2)), 3.64 (t, 4H, (CH2)2O), 3.58 (s, 2H, CH2), 3.17 (s, 2H, CH2-C≡N), 2.89 (t, 4H, (CH2)2N), 2.18 (s, 3H, CH3), 1.21 (t, 3H, (CH3-CH2)); 13C NMR (101 MHz, DMSO‑d6) δ/ppm 155.71 (C = O), 155.33 (C = N), 153.58 (C = O), 130.74, 130.30, 128.51, 126.62, 122.88, 116.16, 114.94, 65.72 ((CH2)2O), 60.94 (CH3-CH2), 50.05 (CH2), 46.29 ((CH2)2N), 30.55 (CH3), 14.54 (CH3-CH2); MS: (Mwt = 437): m/z, 70.28 (84%), 119.37 (42%), 158.09 (44 %), 298.54 (42%), 352.95 (100%), 369.41 (77%), 437.07 (M+, 16%); Anal. Calcd. for C18H23N5O6S (437.47): C, 49.42; H, 5.30; N, 16.01; Found: C, 49.59; H, 5.15; N, 15.86.
2.1.22 6-(Morpholinosulfonyl)-3-(2-(4-oxopentan-2-ylidene)hydrazinyl)quinoxalin-2(1H)-one (17b)
Yield 62% as red crystals; M.p. = 310–312 ˚C; IR (KBr, cm−1): 3362, 3220 (2NH), 3054 (CH-Aro.), 2947, 2866 (CH aliph.), 1687 (br 2C = O), 1622 (C = N), 1335, 1164 (SO2); 1H NMR (400 MHz, DMSO‑d6) δ/ppm 12.22, 12.04 (s, 2H, 2NH; D2O exchangeable), 7.47 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 3.99 (s, 2H, CH2), 3.61 (t, 4H, (CH2)2O), 2.83 (t, 4H, (CH2)2N), 2.10 (s, 3H, COCH3), 1.89 (s, 3H, CH3); MS: (Mwt = 407): m/z, 53.49 (100%), 76.18 (50 %), 94.18 (65%), 215.21 (40%), 353.52 (32%), 407.45 (M+, 4%); Anal. Calcd. for C17H21N5O5S (407.45): C, 50.11; H, 5.20; N, 17.19; Found: C, 50.02; H, 5.03; N, 17.36
2.2 Biological activity (all details in supplementary material file)
The antimicrobial activity of the newly designed derivatives were evaluated against three gram-negative strains, namely (E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. typhi ATCC 6539) , three gram-positive strains (B. subtilis ATCC 6633, S. aureus ATCC 29213, and E. faecalis ATCC 29212), and two fungal strains as (C. albicans ATCC 10231, and F. oxysporum RCMB 008002). The inhibition zone represented as the diameter of the inhibition zones by mm were evaluated by the agar well diffusion method according to previously reported methods (A Ammar et al., 2016; Ammar et al., 2017).
For the most promising derivatives 4a, 7, 8a, 11b, 13, and 16 depending on the zone of inhibition, the minimal inhibitory concentration were performed and verified using the broth micro-dilution procedure outlined in the (CLSI) Laboratory Standards Institute guidelines (Wikler et al., 2008). Both Tetracycline and Amphotericin B were used as positive controls.
Additionally, the most active derivatives 4a, 7, 8a, 11b, 13, and 16 were screened and determine both MIC and MBC against multidrug resistance strains as gram-negative, namely (P. aeruginosa ATCC BAA-2111, and E. coli ATCC BAA-196), and gram-positive (S. aureus ATCC 43300, and S. aureus ATCC 33591) according to previously reported methods (Ammar et al., 2021; Ragab et al., 2021). Tetracycline, as well as Norfloxacin, were evaluated as positive controls.
The immunomodulatory activity using nitro-blue tetrazolium (NBT) reduction (R.L. Baehner, 1968) and in-vitro DNA gyrase inhibitory assay for the most active derivatives 4a, 7, 8a, 11b, 13, and 16 were evaluated according to our previous work (Alt et al., 2011).
2.3 Molecular docking study
The molecular docking study was performed inside the active site of S. aerates DNA gyrase (PDB: 2XCT) using the Molecular Operating Environmental (MOE) 10.2008 according to the previously reported methods (Ibrahim et al., 2021b; Ragab et al., 2021).
3 Results and discussion
3.1 Chemistry
The target quinoxaline derivatives were synthesized using the synthetic approaches depicted in Schemes 1-3. The starting material 3-hydrazino-7-(morpholinosulfonyl)-3,4-dihydroquinoxalin-2(1H)-one (3a) in a good yield was synthesized via interaction of 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride (1) with one mole of morpholine to yield the 6-(morphilonosulfonyl)-2,3-dioxoquinoxaline derivative 2 according to the reported method (Ammar et al., 2020b). The 6-(morpholinosulfonyl)quinoxaline derivative 2 underwent hydrazinolysis with an equivalent amount of hydrazine to get a sole product conceived as 3-hydrazine-6-morpholinosulfonylquinoxaline (3a) or 2-hydrazine-6-morpholinosulfonyl-quinoxaline (3b). Based on the electron-withdrawing property of the sulfonyl group and according to the theoretical calculation of energy (see supplementary material results for DFT calculation) and according to reported method (Elsisi et al., 2022), the isomer 3a structure is more favorable, and the reactions will be completed on the 3-carbon center via a nucleophilic substitution mechanism. The IR analysis of 3-(hydrazinyl)quinoxaline derivative 3a demonstrated absorption bands at υ 3312, 3245, 1678, and 1332, 1155 cm−1 characteristic for NH2, NH, C = O, and SO2 groups. Additionally, the 1H NMR spectrum showed three exchangeable signals at δ 12.19, 9.50, and 4.79 ppm corresponding to the protons of two NH and NH2, as well as two triplet signals at δ 3.62 and 2.88 ppm characteristic for the morpholinyl group protons ((CH2)2O) and (CH2)2N), respectively. Besides, the aromatic protons that appeared between δ 7.38–7.57 ppm. Moreover, the 13C NMR data of compound 3a revealed signals at δ 65.76, 46.35 ppm characteristic for the morpholine carbons ((CH2)2O) and (CH2)2N), respectively, as well as three signals at δ 155.23, 151.21, and 150.44 characteristics for carbonyl, C = C-N, and C = N groups, respectively. Also, the signals of aromatic carbons appeared in the range of δ 115.05–135.08 ppm. The mass spectrum demonstrated molecular ion peak at m/z = 325 and base peak at m/z = 57, which agrees with its calculated molecular formula C12H15N5O4S.
Synthesis of new Schiff base derivative 4a-c, 5, 6, and 7 incorporating quinoxaline pharmacophore moiety.
Reaction of 3-(hydrazinyl)quinoxaline derivatives 3a with some acetyl, ketone, and phenyl isothiocyanate to afford the corresponding hydrazones 7–11 and thiosemicarbazide derivative 12.
Synthesis of new quinoxaline derivatives containing pyrazole 14–16 or hydrazone 13, and 17a, b moiety.
Thus, refluxing of 3-(hydrazinyl)quinoxaline derivative 3a with formyl derivatives afforded the corresponding hydrazone derivatives 4–7. The elemental analysis and spectral data were used to elucidate the structure of the synthesized compounds. The IR spectra of hydrazono-quinoxaline derivative 4b demonstrated absorption bands at υ 3323, 3210, 1688 cm−1 characteristic for NH and carbonyl groups. The 1H NMR spectrum of hydrazono-quinoxaline derivative 4b showed two triplet signals at δ 2.83, 3.61 ppm for the morpholine protons ((CH2)2N) and ((CH2)2O) respectively, one singlet signal at δ 8.70 ppm for methine-H, and two exchangeable singlet signals at δ 12.02 and 12.22 ppm referred to the protons of two NH groups. 13C NMR spectra showed two signals at δ 46.29, 65.72 for morpholine carbons, aromatic carbons in the range of δ 114.95–131.21 ppm and three signals at δ 155.33, 155.71 and 160.98 ppm for CH = N, C = N and C = O carbons, respectively. Besides, the elemental analysis and spectroscopic data were used to elucidate the structure of hydrazone derivative 4b. Additionally, the IR spectra of hydrazine derivative 6 demonstrated absorption bands at υ 3450, 3230, 1680, and 1605 cm−1 characteristic for OH, NH, C = O, and C = N groups, respectively. Furthermore, the 1H NMR spectrum showed a significant signal at δ 3.84 ppm characteristic for methoxy group, and three exchangeable singlet signals at δ 11.87, 11.27, and 9.53 ppm characteristic for two NH and OH protons. Moreover, the 13C NMR spectra showed signals at δ 61.80 ppm characteristic for methoxy group, three signals at δ 158.00, 157.87 and 151.70 ppm related for C = O and two C = N groups, respectively, besides the signals of aromatic carbons ranged between δ 111.80–155.70 ppm (Scheme 1).
Moreover, condensation of 3-(hydrazinyl)quinoxaline derivative 3a with some acetyl derivatives afforded the corresponding 2-(1-(substituted-aryl)ethylidene)hydrazono-quinoxaline derivatives (8, 9). IR spectra of hydrazine derivative 8a demonstrated stretching vibration bands at υ 3224, 1691, and 1609 cm−1 characteristic for NH, C = O, and C = N groups, respectively. Also, its 1H NMR spectrum revealed a new signal owning to the methyl protons at δ 2.42 ppm and two exchangeable signals related to two NH protons at δ 10.56 and 11.72 ppm. Besides, signals are characteristic of morpholine and aromatic protons. Its 13C NMR spectra revealed the signal for the methyl group at δ 15.14 ppm, two signals at δ 155.19, and 162.13 ppm for carbonyl (C = O), and C = N, besides signals between δ 114.69–142.69 ppm related to the aromatic carbons.
Similarly, the hydrazide derivative was subjected to react with some selected keto heterocyclic compounds such as pyrazolone and isatin derivatives where hydrazone-quinoxaline derivatives 10, 11 were obtained. For the spectroscopic analysis of previous hydrazone templates, compound 11b was used as an example. Its IR spectrum showed characteristic stretching vibrational frequencies for N–H, C = O, and C = N at υ 3228, 3193, 1699, and 1612 cm−1, respectively. Moreover, the 1H NMR of compound 11b showed three singlet signals corresponding to three N–H at δ 11.19, 12.03, and 12.24 ppm that are exchangeable with D2O. Besides, three triplet signals and two multiplets were observed at δ 3.62, 2.92, 2.86, 1.53, and 1.35 ppm, characteristic of the morpholinyl and piperidinyl protons. Further, the 13C NMR revealed signals at δ 23.31, 25.14, 46.29, 47.10, and 65.73 ppm due to the piperidinyl and morpholinyl carbons, in addition to the aromatic carbons that ranged between δ 110.64–142.16 ppm and two carbonyl groups at δ 155.71, 163.14 ppm (Scheme 2).
The thiosemicarbazide derivative 12 was obtained upon treatment of the 3-(hydrazinyl)quinoxaline derivative 3a with phenyl isothiocyanate. The 1H NMR spectrum of thiosemicarbazide derivative 12 led to the appearance of four singlet signals at δ 9.35, 9.52, 12.05, 12.25 ppm for four NH protons, besides the signals related to aromatic protons appeared at δ 7.26–8.32 ppm. The mass spectrum of compound 12 exhibited a molecular ion peak at m/z = 460 (20%) with a base peak at m/z = 74, which confirmed the molecular formula.
Furthermore, the corresponding quinoxaline derivative containing butanoic acid 13 was obtained upon the reaction of succinic anhydride with 3-(hydrazinyl)quinoxaline derivative 3a in ethanol as solvent. The elemental analysis and spectroscopic data confirmed the prepared compound. The IR spectra of 4-oxo-butoric acid derivative 13 demonstrated absorption bands at υ 3350, 3207, and 1684 cm−1, assignable to the NH and carbonyl groups. Additionally, the 1H NMR spectra are characterized by the existence of four triplets at δ 2.34, 2.44, 2.86, and 3.67 ppm due to the four CH2 groups corresponding to butanoic acid and morpholinyl moieties. Also, the CH2 carbons of butanoic acid derivatives were observed at δ 28.42, 29.24 ppm, as well as the morpholinyl signals displayed at δ 46.29 and 65.73 ppm. Further, the two C = N and two C = O were detected at δ 155.32, 155.69, 170.38, and 174.06 ppm. The mass spectrum of 4-oxo-butric acid derivative 13 exhibited a molecular ion peak at m/z = 425 (58%) and a base peak at m/z = 259 assignable to the molecular formula C16H19N5O7S.
On the other hand, the starting material 3-(hydrazinyl)quinoxaline derivative 3a reacted with either ethyl 2-cyano-3,3-bis(methylthio)acrylate or 2-aminoprop-1-ene-1,1,3-tricarbonitrile to afford the 1-(1,2-dihydroquinoxalin-3-yl)-1H-pyrazole derivative 14, and 16. The spectral data of pyrazole derivatives 14, 16 are confirmed with the suggested structures. The 1-H-pyrazole-4-carbonitrile derivative 16 afforded stretching vibration bands of NH2, NH, and carbonyl groups at υ 3307 and 3203 and 1684 cm−1, in addition to 1H NMR data exhibited a new singlet signal at δ 3.17 ppm corresponding for CH2 of acetonitrile derivative. Also, new significant signals due to amino group at δ 7.50 ppm exchangeable with deuterated. The mass spectrum exhibited a molecular ion peak at m/z = 440 (7.0 %), characteristic of the molecular formula C18H16N8O4S.
Finally, the interaction of the 3-(hydrazinyl)quinoxaline derivative 3a with dicarbonyl compounds as (ethyl acetoacetate or acetylacetone) failed to obtain the pyrazole nucleus due to the cyclization was incomplete and the corresponding hydrazone derivatives 17a, b were obtained, as showed Scheme 3. For compound 17 a, 1H NMR spectra revealed the presence of ethoxy ester protons as triplet and quartet at δ 1.21 and 4.13 ppm, respectively. In addition, the morpholinyl protons displayed at δ 3.64, 2.89 ppm, as well as the methylene group of butanoate moiety at δ 3.58 ppm and a methyl group at δ 2.18 ppm. Further, the 13C NMR spectra exhibited signals at δ 14.54, 60.94, 65,72, 46.29, 50.05, and 30.55 ppm related to ethoxy, morpholinyl, methylene, and a methyl group, respectively. Besides, signals at δ 155.71, 155.33, and 153.58 ppm corresponding to two carbonyl groups and C = N, as well as the aromatic carbons that ranged between δ 114.94–130.74 ppm.
3.2 Biological activity evaluation
3.2.1 Antimicrobial activity
The newly synthesized nineteen quinoxaline derivatives containing hydrazone 4–11 and 17, hydrazinyl 12–13, and pyrazole 14–16 moieties were tested in vitro antimicrobial activity to evaluate and explore the relationship between antimicrobial activity and the structure. Six bacterial strains were used in this study and classified as three gram-negative strains (E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. typhi ATCC 6539), three gram-positive strains (B. subtilis ATCC 6633, S. aureus ATCC 29213, and E. faecalis ATCC 29212). Additionally, two fungal strains (C. albicans ATCC 10231, and F. oxysporum RCMB 008002) were evaluated to determine the antifungal activity. Both Tetracycline and Amphotericin B as abroad spectrum antibiotics were used as a positive control against bacterial and fungal pathogens. The antimicrobial activity was determined by measuring the inhibition zone diameters (mm) by agar well diffusion method according to the clinical and laboratory standard institute guidelines CLSI and previous methods (Ammar et al., 2016; Ammar et al., 2017). As represented in Table 1, the synthesized quinoxaline derivatives displayed good to moderate activity. *Na: No activity, *S1 = Tetracycline, S2 = Amphotericin B
Code
Inhibition zone represented by (mm)
Gram-positive
Gram-negative
B. subtilis
S. aureus
E. faecalis
E. Coli
P. aeruginosa
S. typhi
C. albicans
F. oxysporum
3a
20 ± 0.11
23 ± 0.29
21 ± 0. 54
22 ± 0.43
Na
15 ± 0. 36
17 ± 0.21
Na
4a
28 ± 0.16
23 ± 0. 55
25 ± 0. 3
21 ± 0.14
20 ± 0. 78
22 ± 0.12
21 ± 0. 2
17 ± 0. 45
4b
13 ± 0.25
20 ± 0.98
18 ± 0.65
16 ± 0.74
11 ± 0.54
15 ± 0.65
13 ± 0.54
15 ± 0.65
4c
14 ± 0.65
11 ± 025
15 ± 032
16 ± 0.24
14 ± 0.35
12 ± 0.68
14 ± 0.28
16 ± 0.35
5
22 ± 0.41
17 ± 0.78
25 ± 0. 14
18 ± 0.3
Na
14 ± 0. 52
19 ± 0.65
Na
6
13 ± 0.41
Na
14 ± 0. 47
19 ± 0. 33
12 ± 0. 63
Na
13.0 ± 0.2
Na
7
27 ± 0.5
26 ± 0. 14
25 ± 0. 33
23 ± 0. 14
25 ± 0. 85
23 ± 0. 11
24 ± 0. 3
20 ± 0. 82
8a
28 ± 0.5
24 ± 0. 12
29 ± 0. 55
25 ± 0.81
24 ± 0. 2
20 ± 0. 16
22 ± 0.56
18 ± 0.15
8b
15 ± 0.45
Na
12 ± 0. 74
14 ± 0.21
Na
15 ± 0. 2
12 ± 0. 65
Na
9
22 ± 0.18
22 ± 0. 34
21 ± 0. 72
23 ± 0. 44
Na
23 ± 0. 33
21 ± 0. 5
19 ± 0. 28
10
14 ± 0.24
17 ± 0.27
13 ± 0.47
20 ± 0.34
15 ± 0.46
19 ± 0.41
14 ± 0.53
17 ± 0.25
11a
23 ± 0.22
24 ± 0. 33
25 ± 0. 35
27 ± 0. 3
23 ± 0. 74
17 ± 0. 12
20 ± 0. 5
15 ± 0. 14
11b
32 ± 0.22
33 ± 0. 53
29 ± 0. 17
30 ± 0.29
27 ± 0. 73
29 ± 0.2
27 ± 0. 5
22 ± 0. 11
12
19 ± 0.65
20 ± 0.54
13 ± 0.25
17 ± 0.35
14 ± 0.45
19 ± 0.28
12 ± 0.24
16 ± 0.65
13
27 ± 0.50
25 ± 0. 77
23 ± 0.65
26 ± 0. 11
21 ± 0. 2
23 ± 0.65
25 ± 0.33
21 ± 0. 16
14
25 ± 0.21
21 ± 0. 17
19 ± 0. 14
22 ± 0. 18
17 ± 0.2
20 ± 0. 33
22 ± 0. 19
19 ± 0. 55
16
25 ± 0.87
21 ± 0.3
24 ± 0. 35
23 ± 0.2
21 ± 0.55
23 ± 0. 4
19 ± 0. 25
17 ± 0. 5
17a
18 ± 0.12
16 ± 0. 54
Na
15 ± 0. 96
Na
12 ± 0. 61
18 ± 0.2
14 ± 0. 38
17b
22 ± 0.4
20 ± 0.31
20 ± 0. 11
19 ± 0. 2
20 ± 0. 15
18 ± 0. 16
17 ± 0.35
21 ± 0.3
S1
25 ± 0.22
25 ± 0.11
22 ± 0.25
23 ± 0. 2
20 ± 0. 5
21 ± 0.55
Na
Na
S2
Na
Na
Na
Na
Na
Na
22 ± 0.2
18 ± 0.32
Firstly, the synthesized derivatives have higher antibacterial potential against gram-positive bacteria rather than gram-negative bacteria with the zone of inhibition (IZ) ranged between (12 ± 0.74 to 33 ± 0.53), (12 ± 0.61 to 30 ± 0.29) mm, respectively compared with Tetracycline (20 ± 0.50 to 25 ± 0.22) mm. Six quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 displayed better and broad antimicrobial activity against the tested strains. Among them, four quinoxaline derivatives 7, 8a, 11b, and 13 showed inhibition zones ranged between (23 ± 0.65 to 32 ± 0.22) mm for gram-positive bacteria and (20 ± 0.16 to 30 ± 0.29) mm for gram-negative bacteria compared with Tetracycline (22 ± 0.25 to 25 ± 0.22) mm, and (20 ± 0.50 to 23 ± 0.20) mm for gram-positive and negative bacteria, respectively. Quinoxaline derivatives 4a, 7, 8a, 11b, and 13 exhibited the most active derivatives against B. subtilis with inhibition zones (IZ) (27 ± 0.50 to 32 ± 0.22), equipotent or nearly with 2-(pyrazolyl)quinoxalin-3-one derivatives 14, 16 with IZs (25 ± 0.21, and 25 ± 0.87) in comparison to Tetracycline (25 ± 0.22). Further, 3-(hydrazono)quinoxaline-3-one derivatives 7 and 11b showed promising activity against S. aureus with IZs ranged between (33 ± 0.53 to 26 ± 0.14) mm compared with Tetracycline (25 ± 0.11) mm, while quinoxaline derivatives 6 and 8b displayed nonactivity. Besides, eight quinoxaline derivatives 4a, 5, 7, 8a, 11a, 11b, 13, and 16 revealed higher inhibition zones than Tetracycline against E. faecalis.
Furthermore, hybridization between isatin sulfonamide and quinoxaline derivatives 11a,b demonstrated the most active derivatives against E. coli with inhibition zones (27 ± 0.30, and (30 ± 0.29) mm followed by 8a and 13 (25 ± 0.81, and 26 ± 0.11) mm and compared with Tetracycline (23 ± 0.20) mm. Also, 3-(methylene-pyrazole)hydrazinyl-quinoxaline derivative 7, 3-(chromene-3-yl)ethylidene)hydrazinyl-quinoxaline derivative 9, and 2(pyrazolyl)quinoxaline derivative 16 displayed inhibition zones equipotent to Tetracycline. Moreover, quinoxaline derivatives 7, 8a, 11a, 11b, 13, and 16 exhibited the remarkable antibacterial activity toward P. aeruginosa with inhibition zones ranged between (21 ± 0.55 to 27 ± 0.73) mm in comparison to Tetracycline (20 ± 0.50) mm, while, quinoxaline derivatives 5, 8b, 9, and 17a exhibited no activity. On the other hand, quinoxaline derivatives 7, 11b, 13, and 16 revealed comparable activity against S. typhi with a zone of inhibition ranging between (23 ± 0.11 to 29 ± 0.20) mm compared with Tetracycline with only one derivative 6 that displayed no activity.
As for antifungal activity, all the synthesized derivatives displayed activity against C. albicans (ATCC 10231), while 3a, 5, 6, and 8b exhibited no activity against F. oxysporum (RCMB 008002). Besides, the other derivatives displayed a considerable antifungal activity. Furthermore, the quinoxaline derivatives 7, 8a, 9, 11a, 11b, and 13 revealed the highest antifungal activity against C. albicans (ATCC 10231) and F. oxysporum (RCMB 008002) pathogens with inhibition zones from (18 ± 0.15) to (27 ± 0.50) mm compared with Amphotericin B (18 ± 0.32 to 22 ± 0.20) mm.
3.2.2 Minimal inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC)
The most active quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 depending on the antimicrobial screening were selected to evaluate the minimal inhibitory concentrations (MIC) (µg/mL) and minimal bactericidal/fungicidal concentration (MBC/MFC) (µg/mL) as represented in Table 2. Both the MIC and MBC/MFC were determined by the conventional paper disk diffusion method and confirmed using broth microdilution procedure as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines and previously reported methods (Ammar et al., 2020a, 2020b; Dias et al., 2018; Wikler, et al., 2008). *Tetr. = Tetracycline, Amph.B = Amphotericin B.
Cpd.
No.Test Name
Gram-positive
Gram-negative
Fungi
B. subtilis
S. aureus
E. faecalis
E. Coli
P. aeruginosa
Salmonella typhi
C. albicans
F. oxysporum
4a
MIC
4.5
7.81
3.9
7.81
15.62
5.57
7.81
15.62
MBC
9.2
15.62
7.41
14.05
31.25
10.58
12.49
26.55
7
MIC
3.9
7.81
15.62
7.81
27.77
18.51
9.25
31.25
MBC
6.63
15.62
31.25
15.62
55.54
36.5
17.57
56.25
8a
MIC
5.57
1.95
3.9
7.81
31.25
3.9
15.62
31.25
MBC
10.58
3.7
6.63
12.49
59.37
6.63
28.11
46.87
11b
MIC
1.95
5.57
3.9
0.97
5.57
7.81
7.81
15.62
MBC
3.9
5.57
6.63
1.94
10.58
12.49
15.62
27.77
13
MIC
9.25
31.25
7.81
18.51
55.5
31.25
31.25
55.54
MBC
18.5
53.12
15.62
36.5
88.8
56.25
41.65
87.5
16
MIC
7.81
15.62
9.25
62.5
31.25
15.62
31.25
55.54
MBC
14.05
31.25
18.5
87.5
53.12
28.11
53.12
88.8
Tetr.
MIC
31.25
62.5
62.5
15.62
62.5
31.25
–
–
MBC
40.62
87.5
93.75
18.74
87.5
43.75
–
–
Amph. B.
MIC
–
–
–
–
–
–
15.62
31.25
MFC
–
–
–
–
–
–
34.62
65.62
The most active derivatives 4a, 7, 8a, 11b, 13, and 16 exhibited significant antibacterial activity with MIC values ranged between (1.95–31.25 µg/mL), (0.97–62.5 µg/mL) against gram-positive and gram-negative bacteria, respectively, and compared with Tetracycline as positive control (15.62–62.5 µg/mL). Surprisingly, 3-(2-(2-oxo-5-(piperidin-1-ylsulfonyl)indolin-3-ylidene) hydrazinyl)quinoxalin-2(1H)-one derivative 11b revealed highest antibacterial potential on B. subtilis (MIC of 1.95 µg/mL), and E. faecalis (MIC of 3.9 µg/mL) in comparison to Tetracycline (MIC of 31.25 & 62.5 µg/mL). Additionally, the quinoxaline derivatives 11b displayed the second promising derivatives against S. aureus with MIC value (5.57 µg/mL), after 3-(2-(1-(4-bromophenyl)ethylidene)hydrazineyl)quinoxalin-2(1H)-one derivative 8a (MIC = 1.95 µg/mL) compared with Tetracycline (MIC = 62.50 µg/mL). Interestingly, quinoxaline derivatives 4a, 7, 8a, 13, and 16 exhibited considerable antibacterial activity on B. subtilis (MIC = 4.5, 3.9, 5.57, 9.25, 7.81 µg/mL) than Tetracycline (MIC = 31.25 µg/mL). Moreover, the quinoxaline derivatives 4a, 7, 8a, 16 showed antibacterial potential S. aureus (MIC of 7.81, 7.81, 1.95 & 15.62 µg/mL) compared to Tetracycline (MIC = 62.5 µg/mL). Moreover, the hydrazono-quinoxaline derivatives 4a, 8a, and 11b revealed equipotent antibacterial activity on E. faecalis with inhibitory activity (MIC = 3.9 µg/mL) compared to Tetracycline (MIC = 62.5 µg/mL) (Fig. 2).
Minimum inhibitory concentrations (MIC) (µg/mL) of most active 3-hydrazono-quinoxaline derivatives against pathogenic microbes.
Furthermore, 3-(2-(2-oxo-5-(piperidin-1-ylsulfonyl)indolin-3-ylidene)hydrazinyl)-quinoxalin-2(1H)-one derivative 11b showed the better antibacterial activity against E. coli, and P. aeruginosa with MIC values (0.97 and 5.57 µg/mL), respectively, in comparison to Tetracycline (MIC of 15.62 & 62.5 µg/mL). On the other hand, 3-(2-(1-(4-bromophenyl)ethylidene)hydrazineyl)-quinoxalin-2(1H)-one derivative 8a demonstrated the best member antibacterial potential on S. typhi with inhibitory concentration (MIC = 3.9 µg/mL) in comparison to Tetracycline (MIC = 31.25 µg/mL). Additionally, 3- hydrazinylquinoxalin-2(1H)-one derivatives 4a, 7, and 8a that are containing 4-chlorobenzylidene, (1,3-diphenyl-1H-pyrazol-4-yl)methylene, and 1-(4-bromophenyl)ethylidene, respectively, as variable bioactive cores showed the same antibacterial activity against E. coli with MIC values equal 7.81 µg/mL. Besides, these quinoxaline derivatives 4a, 7, and 11b showed the best inhibitory ability against P. aeruginosa with MIC values (15.62, 27.77 & 5.57 µg/mL) in comparison to Tetracycline (MIC = 62.5 µg/mL). It’s interesting, the presence of 4-bromophenyl derivative in 3-(2-(1-(4-bromophenyl)ethylidene)hydrazineyl)quinoxalin-2(1H)-one derivative 8a exhibited the highest antibacterial activity against S. typhi with MIC value (3.9 µg/mL) followed by quinoxaline derivative 4a, and 11b (MIC = 5.57, and 7.81 µg/mL), respectively in comparison to Tetracycline (MIC of 31.25 µg/mL) (Fig. 2).
Whilst quinoxaline derivatives 4a, and 11b revealed strong antifungal potential with MIC values (7.81 µg/mL) against C. albicans and (15.62 µg/mL) against F. oxysporum in comparison to Amphotericin B (MIC of 15.62, 31.25, µg/mL). Further, 3-(2-(1-(4-bromophenyl)ethylidene)- hydrazineyl)quinoxalin-2(1H)-one derivative 8a demonstrated equipotent to Amphotericin B against to fungal strains C. albicans and F. oxysporum with MIC values (15.62, 31.25 µg/mL), respectively. Additionally, quinoxaline containing 4-oxo-butanoic acid derivative 13, and 5-aminopyrazole derivative 16 exhibited lower antifungal activity with MIC values (31.25 and 55.54 µg/mL), respectively. For the F. oxysporum pathogen, the most active two quinoxaline derivatives 4a and 11b that exhibited MIC values (15.62 µg/mL), while the other hydrazono-quinoxaline derivatives 7 and 8a showed equipotent activity in comparison to Amphotericin B with MIC value (31.25 µg/mL) (Table 2 and Fig. 2).
For further exploration, the minimal bactericidal concentration (MBC) of the most promising quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 were determined using the conventional paper disc diffusion method as described previously (Salem et al., 2020a, 2020b). As listed in Table 3, these derivatives revealed bactericidal/fungicidal activity with MBC values (3.7–53.12 µg/mL), (1.94–88.8 µg/mL), and MFC values (12.49–88.8 µg/mL) against gram-positive (B. subtilis, S. aureus, and E. faecalis), gram-negative (E. coli, P. aeruginosa, and S. typhi), and fungal strains (C. albicans and F. oxysporum) compared with Tetracycline (18.74–87.5 µg/mL), and Amphotericin B (34.62–65.62 µg/mL).
The 3-(2-(2-oxo-5-(piperidin-1-ylsulfonyl)indolin-3-ylidene)hydrazinyl)-quinoxalin-2(1H)-one derivative 11b displayed bactericidal concentration (MBC = 3.9, 5.57, and 6.63 µg/mL) against gram-positive strains. Besides, the hydrazono-quinoxaline derivative 11b showed MBC values (1.94, 10.58, and 12.49 µg/mL) when tested against gram-negative strains compared with Tetracycline (MBC = 18.74–93.75 µg/mL). Interestingly, the 3-(2-(1-(4-bromophenyl)ethylidene)-hydrazineyl)quinoxaline-2(1H)-one derivative 8a displayed better bactericidal activity on S. aureus (MBC = 3.7 µg/mL), E. faecalis (MBC = 6.63 µg/mL) and S. typhi (MBC = 6.63 µg/mL) compared to Tetracycline (MBC of 87.5, 93.75 & 43.75 µg/mL) (Fig. 3).
Minimum bactericidal/fungicidal concentrations MBC/MFC (µg/mL) of highest activity of synthesized compounds against pathogenic microbes.
The hydrazono-quinoxaline derivatives 4a, 7, 8a, and 11b revealed fungicidal activity with MFC values (12.49–56.25 µg/mL) lower than Amphotericin B (34.62–65.62 µg/mL). Furthermore, the hydrazine-quinoxaline 13, and 3-pyrazolyl-quinoxaline 16 showed higher MFC values (41.65, 54.12 µg/mL), (87.5, and 88.8 µg/mL) against C. albicans and F. oxysporum, respectively. Among the tested derivatives, 3-(2-(4-chlorobenzylidene)hydrazineyl)quinoxalin-2(1H)-one derivative 4a exhibited better fungicidal activity on C. albicans and F. oxysporum (MFC = 12.49, 26.55 µg/mL) compared to Amphotericin B (MFC = 34.62 & 65.62 µg/mL) (Fig. 3). According to the CLSI standards, it can be determined that the tested quinoxaline derivatives exhibited bactericidal/fungicidal or bacteriostatic/fungistatic depending on the values of (MBC or MFC /MIC) ratio, where if the (MBC or MFC)/MIC ratio ranged between 1 and 2 is considered as indicative cidal potential. On the other hand, for (MBC or MFC)/MIC ratio ≥ 8 is considered indicative of static behavior (Daschner, 1977; Guo et al., 2016; Kusakabe et al., 2019; Sun et al., 2019).
Finally, the MIC and MBC/ MFC values indicated that all the hydrazono-quinoxaline 4a, 7, 8a, 11b, hydrazine-quinoxaline 13, and 3-pyrazolyl-quinoxalin-2-one derivative 16 exhibited bactericidal and fungicidal behavior with MBC/MIC and MFC/MIC ratio ranged between 1 and 2.
3.2.3 Drug resistance study
The most active quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 were further evaluated toward multidrug-resistant bacterial strains classified as gram-negative (P. aeruginosa ATCC BAA-2111, and E. coli ATCC BAA-196), and gram-positive (S. aureus ATCC 43300, and S. aureus ATCC 33591) according to previously reported methods (Ammar et al., 2021; Ragab et al., 2021). In addition, Tetracycline and Norfloxacin as broad-spectrum antibiotics were used as a positive control.
As listed in Table 3, the most active quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 revealed potent activity against all the multi-drug resistance bacteria (MDRB) strains with MIC values ranged between (1.95–15.62 µg/mL), and MBC values (3.31–31.25 µg/mL). The 3-(2-(2-oxo-5-(piperidin-1-ylsulfonyl)indolin-3-ylidene)hydrazinyl)-quinoxalin-2(1H)-one derivative 11b exhibited the most active derivatives against three strains (S. aureus ATCC 43300, P. aeruginosa ATCC BAA-2111, and E. coli ATCC BAA-196) with MIC values (1.95, 1.95, and 3.9 µg/mL) ,and MBC values (3.9, 3.9, and 7.8 µg/mL) in comparison to Tetracycline that observed no activity and Norfloxacin MICs (1.25, 0.78, and 3.13 µg/mL), and MBCs (2.5, 1.40, and 5.32 µg/mL). Additionally, the 3-(2-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)hydrazineyl)quinoxalin-2(1H)-one derivative 7 showed the best antibacterial activity against S. aureus (ATCC 33591) with MIC values (1.95 µg/mL), and MBC values (3.9 µg/mL) comparison to Norfloxacin (MIC = 1.57 µg/mL, and MBC = 2.66 µg/mL).
Code
Mean diameter of inhibition zone (mm) and minimal inhibitory concentrations (MIC/MBC) (µg/mL)
S. aureus ATCC 43,300
S. aureus ATCC 33,591
E. coli ATCC BAA-196
P. aeruginosa ATCC BAA-2111
IZ
MIC
MBC
IZ
MIC
MBC
IZ
MIC
MBC
IZ
MIC
MBC
4a
26 ± 0.71
6.25
11.87
15 ± 0.4
8.88
15.98
20 ± 0.33
9.25
18.5
21 ± 0.3
7.81
15.62
7
24 ± 0. 5
4.44
8.88
25 ± 0.2
1.95
3.31
23 ± 0. 14
6.25
11.87
24 ± 0.18
5.2
8.61
8a
25 ± 0.45
3.9
7.41
23 ± 0.4
5.55
11.1
22 ± 0.2
7.81
15.62
24 ± 0.16
4.44
6.66
11b
27 ± 0.33
1.95
3.9
24 ± 0.2
3.9
7.8
25 ± 0.16
1.95
3.9
26 ± 0. 44
3.9
7.8
13
17 ± 0.45
15.62
31.25
22 ± 0.45
3.9
7.41
19 ± 0.81
9.25
18.5
23 ± 0. 5
15.62
31.25
16
20 ± 0. 3
7.81
15.62
21 ± 0. 15
9.25
18.5
22 ± 0.15
3.9
7.41
19 ± 0. 66
6.25
11.87
Tetr.
–
–
–
–
–
–
–
–
Nor.
25 ± 0.5
1.25
2.5
26 ± 0.5
0.78
1.4
27 ± 0.98
1.57
2.66
24 ± 0.47
3.13
5.32
Finally, the structure–activity relationship (SAR) indicated that 6-(morphilionsulfonyl)quinoxaline linked to hydrazine, hydrazone, and pyrazolyl moieties had a profound effect on the antibacterial action, especially multi-drug resistance bacteria strains. Furthermore, hydrazono-quinoxaline derivatives 11b revealed the best activity toward multi-drug resistance bacteria compared to Norfloxacin, which may be due to the presence of indolinyl and piperidinyl moieties in position three. Similarly, the other quinoxaline derivatives showed a considerable antibacterial potential against multi-drug resistance strains and displayed MIC and MBC values near-standard Norfloxacin. Moreover, the MBC/MIC ratios of the most active derivatives and Norfloxacin against MDRB exhibited bactericidal behavior.
3.2.4 DNA gyrase inhibition activity
DNA gyrase is an essential bacterial enzyme that belongs to topoisomerase enzymes involved in controlling topological transitions of DNA. It can inhibit bacterial growth by two different mechanisms as inhibiting the ATPase activity of gyrase blocks the introduction of negative supercoils in DNA as amino coumarin or by direct DNA gyrase inhibition as Ciprofloxacin (gyrase poisoning) that may have an impact on cell physiology and division (Collin et al., 2011).
To explore the mode of action for the most active quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16, the S. aureus DNA gyrase inhibition activity expressed by IC50 (µM) were performed and represented in Table 4 and Fig. 4. The Ciprofloxacin was used as positive control. The order of DNA gyrase inhibitory potential can be represented as 11b < 7 < 8a < 4a < 16 < 13. The 3-(2-(2-oxo-5-(piperidin-1-ylsulfonyl)indolin-3-ylidene)hydrazinyl)-quinoxalin-2(1H)-one derivative 11b and 3-(2-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)hydrazineyl)quinoxalin-2(1H)-one derivative 7 showed better activity with inhibitory (IC50 = 10.93 ± 1.81 & 15.83 ± 1.55 µM), respectively compared to Ciprofloxacin (IC50 = 26.31 ± 1.64 µM).
Compound
S. aureus DNA gyrase
Supercoiling IC50 (µM)
4a
20.05 ± 1.45
7
15.83 ± 1.55
8a
16.56 ± 1.12
11b
10.93 ± 1.81
13
26.18 ± 1.22
16
23.47 ± 1.23
Cip.
26.31 ± 1.64
Determination of the S. aureus DNA gyrase inhibitory activity of the most active quinoxaline derivatives.
Meanwhile, the 3-(2-(4-chlorobenzylidene)hydrazineyl)quinoxalin-2(1H)-one derivative 4a displayed (IC50 = 20.05 ± 1.45 µM), while the 3-(2-(1-(4-bromophenyl)ethylidene) hydrazineyl)quinoxalin-2(1H)-one derivative 8a showed DNA gyrase inhibitory potential (IC50 = 16.56 ± 1.12 µM). This difference in IC50 values (nearly 3.49 µM) and activity (MIC, MBC) between the two quinoxaline derivatives 4a, and 8a may be related to the presence of excess methyl group and replace the choro by bromo atom in hydrazono-quinoxaline derivative 8a. Additionally, the 3-(1H-pyrazole)-2-oxoquinoxaline derivative 16 showed DNA gyrase inhibitory activity (IC50 = 23.47 ± 1.23 µM), while 3-(hydrazino)quinoxaline derivative 13 revealed the lowest activity (IC50 = 26.18 ± 1.22 µM), but still more active than Ciprofloxacin (IC50 = 26.31 ± 1.64 µM).
3.2.5 Immunomodulatory activity for most potent compounds
Our work was extended to study the in vitro immunomodulatory activity of the most active quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 using nitro-blue tetrazolium (NBT) reduction according to the reported method (Baehner,0.1968, and Salem et al., 2020a). The immunomedioratory activity is expressed as the intracellular killing percentage (%) values represented in Table 5. The NBT assay was evaluated for most active compounds and the results represented an increase in neutrophil killing capabilities. Additionally, an increased intracellular killing percentage related to an enhancement in the killing ability toward neutrophils.
Compound
Intracellular killing activity %
4a
113.2 ± 0. 5
7
116.7 ± 0. 14
8a
136.5 ± 0.3
11b
142.4 ± 0.98
13
82.8 ± 0.37
16
98.7 ± 0. 19
The quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 revealed as good immunomedioratory agents by percentage ranged between 82.8 ± 0.37 to 142.4 ± 0.98 %. Interestingly, hydrazono-quinoxaline with isatin sulfonamide 11b showed the highest immunomedioratory derivative with intracellular killing percentages (142.4 ± 0.98) %. The order of intracellular killing percentages can be represented as 11b < 8a < 7 < 4a < 16 < 13. The quinoxaline derivatives 8a, 7, 4a, 16, and 13 displayed a good immunostimulatory potential with ratio (136.5 ± 0.3, 116.7 ± 0. 14, 113.2 ± 0. 5, 98.7 ± 0. 19, and 82.8 ± 0. 37) %, respectively.
3.2.6 In silico ADME study
Some physicochemical properties of the most active quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 were calculated using the Molinspiration cheminformatics web tool as represented previously (
https://www.molinspiration.com/) (Salem et al., 2020a). Among the physicochemical parameters, the molecular weight (M. wt.), n-octanol–water partition coefficient (MlogP), number of hydrogen bond acceptors (nHBA), number of hydrogen bond donor (nHBD), number of rotatable bonds (nRB), and topological polar surface area (TPSA) were calculated to determine if the most active derivatives obey Lipinski’s and Veber rule in Drug-likeness or not. According to Lipinski’s rule, the drug can follow this role when observed one or no violation. Lipinski’s rule involved (molecular weight < 500 Dalton, MLogP ≤ 4.15, nHBA ≤ 10, and nHBD ≤ 5). From Table 6, the results observed that the quinoxaline derivatives 4a, 8a, 13, and 16 obeyed Lipinski’s rule of five without violation, similarly to Norfloxacin and Ciprofloxacin as the positive control. In contrast, the hydrazono-quinoxaline derivatives 7, and 11b exhibited violations from Lipinski’s due to the molecular weight higher than 500 Dalton and the number of hydrogen bonds more than ten.
Cpd.
No.M.wt.
MLogP
nHBA
nHBD
nRB
TPSA
Bioavailability Score
Synthetic accessibility
4a
447.90
1.96
9
2
5
116.76
0.55
3.48
7
555.61
2.61
11
2
7
134.59
0.17
4.21
8a
506.37
2.54
9
2
5
116.76
0.55
3.60
11b
601.65
1.25
14
3
6
187
0.17
4.35
13
425.42
−1.28
12
4
7
170.79
0.11
3.58
16
440.44
−0.37
12
3
4
183.80
0.55
3.64
Nor.
319.33
−0.69
6
2
3
74.57
0.55
2.46
Cip.
331.34
−0.70
6
2
3
74.57
0.55
2.51
It’s interesting to know that both topological polar surface area (TPSA) and the number of rotatable bonds (nRB) are very useful physicochemical parameters for the prediction of drug transport properties and good descriptors of oral bioavailability of drugs (Z El-Attar et al., 2018). Additionally, for a drug that can obey the Veber rule when a number of rotatable bonds are less than ten and TPSA<140 Å2. Further, quinoxaline derivatives 4a, 7, and 8a follow the Veber rule, while 11b, 13, and 16 have one violation from the Veber rule by displayed TPSA > 140 Å2.
Furthermore, bioavailability score, synthetic accessibility, and some pharmacokinetic properties for the most active and positive control were calculated using the SwissADME web tool (http://swissadme.ch/index.php) according to the previously reported method (Fayed et al., 2020). The quinoxaline derivatives revealed bioavailability scores ranged between 0.11 and 0.55 compared with Norfloxacin and Ciprofloxacin 0.55. Besides, easy synthetic accessibility ranged between 3.48 and 4.35 compared to Norfloxacin (2.46) and Ciprofloxacin (2.51).
From Table 7, we found that all the quinoxaline derivatives are substrates of P-gp protein and, therefore, can efflux out of the cell except hydrazino-quinoxaline derivatives 13. Surprisingly, the most promising quinoxaline derivatives, Norfloxacin, and Ciprofloxacin showed no permeant to the blood–brain barrier. In addition, the quinoxaline derivatives 4a, and 8a besides Norfloxacin and Ciprofloxacin displayed Gastrointestinal high absorption, while the quinoxaline derivatives 7, 11b, 13, and 16 exhibited Gastrointestinal low absorption. Gastrointestinal absorption = GI Abs.; blood–brain barrier permeant = BBB Permeant; P-glycoprotein substrates = P-gp Substrate; Carcino. = Carcinogenicity; Immuno. = Immunotoxicity; Mutagen = Mutagenicity; Cyto. = Cytotoxicity
Cpd.
No.Pharmacokinetics
Oral toxicity prediction
GI
Abs.
BBB
Pert.
P-gp
Sub.
LD50
mg/kg
Toxicity Class
Carcino.
Immuno.
Mutagen.
Cyto.
4a
High
No
Yes
3000
V
Inactive
0.57Inactive
0.93Inactive
0.74Inactive
0.77
7
Low
No
Yes
1400
IV
Inactive
0.52Inactive
0.90Inactive
0.70Inactive
0.72
8a
High
No
Yes
1600
IV
Inactive
0.57Inactive
0.88Inactive
0.72Inactive
0.74
11b
Low
No
Yes
3000
V
Inactive00.52
Inactive
0.76Inactive
0.70Inactive
0.69
13
Low
No
No
1000
IV
Inactive
0.52Inactive
0.99Inactive
0.71Inactive
0.60
16
Low
No
Yes
1800
IV
Inactive
0.51Inactive
0.99Inactive
0.68Inactive
0.65
Nor.
High
No
Yes
1000
IV
Inactive
0.57Inactive
0.98Inactive
0.92Inactive
0.90
Cip.
High
No
Yes
2000
IV
Inactive 0.57
Inactive 0.91
Active 0.75
Inactive 0.92
The importance of toxicity prediction in drug design is related to reducing the number of animal experiments. The most active derivatives 4a, 7, 8a, 11b, 13, and 16 and Norfloxacin, as well as Ciprofloxacin, were exported as a smile to ProTox-II web tool ( https://tox-new.charite.de/protox_II/) (Banerjee et al., 2018), to evaluate carcinogenicity, immunotoxicity, mutagenicity, cytotoxicity, and lethal dosage 50 (LD50) expressed by mg/kg. The tested derivatives 4a, 7, 8a, 11b, 13, and 16 exhibited non-carcinogenic, non-immunotoxin, and non-cytotoxic with confidence values ranging between (0.51–0.57, 0.76–0.99, and 0.60–0.77), respectively. Additionally, these derivatives exhibited inactive against mutagenicity with confidence values ranged between (0.68–0.74) compared with Norfloxacin that displayed inactive with confidence value (0.92), while Ciprofloxacin was expected to have mutagenic properties.
Our work extended to study the lethal dosage 50 (LD50) meaning the dose at which 50% of test subjects die upon exposure to a drug. The LD50 expressed by mg/kg and according to the globally harmonized system of classification of labelling of chemicals (GHS) (Miyagawa, 2010) classified to six classes as [Class I: fatal if swallowed (LD50 ≤ 5), Class II: fatal if swallowed (5 < LD50 ≤ 50), Class III: toxic if swallowed (50 < LD50 ≤ 300), Class IV: harmful if swallowed (300 < LD50 ≤ 2000), Class V: may be harmful if swallowed (2000 < LD50 ≤ 5000), Class VI: non-toxic (LD50 > 5000)]. The most active quinoxaline derivatives demonstrated predicted LD50 values ranged between (1000–3000 mg/kg), compared with Norfloxacin (LD50 = 1000 mg/kg), and Ciprofloxacin (LD50 = 2000 mg/kg). In addition, the tested quinoxaline derivatives and positive controls belong to class IV, except hydrazono-quinoxaline derivatives 4a and 11b appertain to class V (Table 8). Cip. = Ciprofloxacin; (-) = meaning arene-cation interaction
Cpd. No.
S
(Kcal/mol)Residues
Interacting group
Type of
H-bondStrength
%Length
˚A
4a
−20.11
Arg1048
Arg1048
Ser1028
Asp510
Lys460Oxygen of morpholinyl
Oxygen of morpholinyl
Oxygen of sulfonyl group
NH of hydrazine derivative
4-chlorophenyl derivativeAcceptor
Acceptor
acceptor
Donor
-23
34
65
38
-3.16
3.04
2.71
3.36
-
7
–22.12
Arg1033
Arg1033
Arg1092NH of hydrazine derivative
Phenyl of quinoxaline
Phenyl at N1 of pyrazole derivativeDonor
-
-13
-
-2.90
-
-
8a
−21.20
Arg1048
Arg1048
Ser1028Oxygen of morpholinyl
Oxygen of morpholinyl
Oxygen of sulfonylAcceptor
Acceptor
Acceptor52
16
932.75
3.13
2.67
11b
–23.81
Lys1043
Lys460
Glu435
Arg1033Oxygen of morpholinyl
Carbonyl of quinoxaline
NH of isatin
Phenyl of quinoxalineAcceptor
Acceptor
Donor
-11
41
60
-3.66
2.47
2.70
-
13
−17.38
Arg1048
Oxygen of morpholinyl
Acceptor
60
2.55
16
−18.96
Arg1048
Arg1048
Arg1033Cyano of acetonitrile derivative
Cyano of acetonitrile derivative
Cyano at position four at pyrazole ringAcceptor
Acceptor
acceptor27
28
442.89
3.04
2.78
Cip
−13.65
His1081
Tyr580Oxygen of carboxylate
NH of piperazineAcceptor
Donor37
482.30
2.55
Finally, it can be concluded that the most active quinoxaline derivatives displayed good drug-likeness, some pharmacokinetics, and oral bioavailability properties, besides non-toxicity prediction with safety LD50 values.
3.2.7 Molecular docking study
Molecular docking simulations of most active quinoxaline derivatives 4a, 7, 8a, 11b, 13, and 16 were performed inside the active site of S. aureus DNA gyrase (PDB: 2XCT) according to the reported method (Ragab et al., 2021). The docking study was achieved using Molecular Operating Environmental (MOE) 10.2008 (Eissa et al., 2021). The docking results showed good binding between the tested derivatives and active site in the pocket with lower binding energy ranged between S = − 17.38 to –23.81 Kcal/mol, compared with co-crystallized ligand Ciprofloxacin S =- 13.65 Kcal/mol. Additionally, the quinoxaline derivatives displayed two types of intersections as Hydrogen bond or arene-cation interaction.
The most active hydrazone derivatives 11b depending on the IC50 values of DNA gyrase (IC50 = 10.93 ± 1.81 µM), that containing two bioactive cores (isatin sulfonamide and quinoxaline sulfonamide) exhibited the lowest binding energy S = –23.81 Kcal/mol with three hydrogen bonds and one arene-cation interaction. The hydrogen bonds formed between the resides Lys1043 with the oxygen of morpholinyl group, Lys460 with the carbonyl of quinoxaline, and Glu435 with NH of isatin derivative with bond length 3.66, 2.47, and 2.70 ˚A, respectively (Figs. 5a and b).
2D interaction between the quinoxaline derivative 11b and the active site of DNA gyrase (2XCT).
3D interaction between the quinoxaline derivative 11b and the active site of DNA gyrase (2XCT).
Furthermore, 3-(2-((1H-pyrazol-4-yl)methylene)hydrazineyl)quinoxalin-2(1H)-one derivative 7 demonstrated binding energy S = –22.12 Kcal/mol through only one hydrogen bond backbone donor between Arg1033 and NH of hydrazinyl quinoxaline derivative with bond length 2.90 ˚A, and strength 13%. Besides, two arene-cation interactions between Arg1033, Arg1092 with phenyl of quinoxaline, and phenyl at N1 of pyrazole derivatives, respectively.
Moreover, the 3-(2-(1-(4-bromophenyl)ethylidene)hydrazineyl)quinoxalin-2(1H)-one derivative 8a observed binding energy S = -21.20 Kcal/mol with three hydrogen bonds sidechain acceptor with bond length ranged between 2.67 and 3.13 ˚A through two residues Arg1048 and Ser1028. The residues Arg1048 formed two hydrogen bonds with the oxygen of morpholinyl with bond length and strength 2.75 ˚A (52%), and 3.13 ˚A (16%) (Figs. 6a and b). Additionally, the hydrazino-quinoxaline derivative 13 observed the less active member in our study with binding energy S = -17.38 Kcal/mol through forming one hydrogen bond between the residue Arg1048 and oxygen of morpholinyl with bond length 2.55 ˚A and strength 60%. On the other hand, the 2-pyrazolyl-2-oxoquinoxaline derivative 16 showed binding energy S = -18.96 Kcal/mol with three hydrogen bonds sidechain acceptor. Similarly, the 3-(2-(4-chlorobenzylidene)hydrazineyl)quinoxalin-2(1H)-one derivative 4a exhibited binding energy S = -20.11 Kcal/mol with two hydrogen bonds sidechain acceptor between Arg1048 and the oxygen of morpholinyl and one hydrogen bond acceptor between Ser1028 and oxygen of sulfonyl with bond length 3.16, 3.04, 2.71 ˚A, respectively. Besides, one hydrogen bond sidechain acceptor between Asp510 with NH of hydrazino-quinoxaline derivative with bond length 2.36 ˚A and strength 38%, as well as arene-cation interaction between Lys460 and phenyl of 4-chlorophenyl derivative. (All docking figures were represented in the supplementary material file).
2D interaction between the quinoxaline derivative 8a and the active site of DNA gyrase (2XCT).
3D interaction between the quinoxaline derivative 8a and the active site of DNA gyrase (2XCT).
4 Conclusion
The present study reported the synthesis of nineteen quinoxalin-2(1H)-one derivatives containing hydrazone, hydrazine, and pyrazole moieties were developed and synthesized. The newly synthesized nineteen quinoxaline derivatives containing hydrazone 4–11 and 17, hydrazinyl 12–13, and pyrazole 14–16 moieties were tested in vitro antimicrobial activity to evaluate the antimicrobial activity. The synthesized derivatives have higher antibacterial potential against gram-positive bacteria rather than gram-negative bacteria with a zone of inhibition (IZ) ranged between (12 ± 0.74 to 33 ± 0.53), (12 ± 0.61 to 30 ± 0.29) mm, respectively compared with Tetracycline (20 ± 0.50 to 25 ± 0.22) mm. Six quinoxaline derivatives 4a, 7, 8a, 11b, 13 and 16 displayed better and broad antimicrobial activity against the tested strains. The most active derivatives 4a, 7, 8a, 11b, 13 and 16 exhibited significant MIC values ranged between (1.95–31.25 µg/mL), (0.97–62.5 µg/mL) against gram-positive and gram-negative bacteria, respectively, and compared with Tetracycline as positive control (15.62–62.5 µg/mL). Additionally, these derivatives revealed bactericidal activity with MBC values (3.7–53.12 µg/mL) against gram-positive strains (B. subtilis, S. aureus, and E. faecalis), and MBC values (1.94–88.8 µg/mL) against gram-negative strains (E. coli, P. aeruginosa, and S. typhi) compared with Tetracycline (40.62–93.75 µg/mL), and (18.74–87.5 µg/mL), respectively. Besides, fungicidal activity with MFC values (12.49–88.8 µg/mL) against fungal strains (C. albicans and F. oxysporum) in comparison to Amphotericin B (34.62–65.62 µg/mL). The MBC/MIC and MFC/MIC ratio ranged between 1 and 2 and exhibited bactericidal and fungicidal potency. Also, the most active quinoxaline derivatives 4a, 7, 8a, 11b, 13 and 16 revealed potent activity against all the multi-drug resistance bacteria (MDRB) strains with MIC values ranged between (1.95–15.62 µg/mL), and MBC values (3.31–31.25 µg/mL). The hydrazono-quinoxaline derivatives 11b revealed the best activity toward multi-drug resistance bacteria with MIC values (1.95, 1.95, 3.9 µg/mL) ,and MBC values (3.9, 3.9, 7.8 µg/mL) in comparison to tetracycline that observed no activity and Norfloxacin MICs (1.25, 0.78, 3.13 µg/mL), ad MBCs (2.5, 1.40, and 5.32 µg/mL). This good activity may be a result of the presence of indolinyl and piperidinyl moieties in position three. The most active quinoxaline derivatives 4a, 7, 8a, 11b, 13 and 16 were evaluated against S. aureus DNA gyrase inhibition assay with IC50 values (10.93 ± 1.81–26.18 ± 1.22 µM) compared with Ciprofloxacin (26.31 ± 1.64 µM) and the order of DNA gyrase inhibitory potential can be represented as 11b < 7 < 8a < 4a < 16 < 13. Further, these quinoxaline derivatives could increase intracellular killing percentage and therefore display immunomedioratory activity. Furthermore, the most promising derivatives were performed in silico ADME and toxicity prediction. Most of them showed agreement to Lipinski’s and Veber’s rules with good drug-likeness, some pharmacokinetic, and oral bioavailability properties. Besides, these derivatives showed non-carcinogenic, non-immunotoxin, non-mutagenic, and non-cytotoxic prediction with safety LD values. Additionally, the molecular docking study displayed lower binding energy with good binding mode and different interaction types with bond length lower than 3.40 ˚A. Finally, this study identifies 6-(morpholinosulfonyl)quinoxalin-2(1H)-one derivatives that can contribute to developing new antibacterial agents with DNA gyrase inhibitory and immunomodulatory potential.
Acknowledgements
Taif University Researchers Supporting Project number (TURSP-2020/220), Taif University, Taif, Saudi Arabia.
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Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.103497.
Appendix A
Supplementary material
The following are the Supplementary data to this article:Supplementary Data 1
Supplementary Data 1
