Synthesis of novel N-substituted β-amino acid derivatives bearing 2-hydroxyphenyl moieties as promising antimicrobial candidates targeting multidrug-resistant Gram-positive pathogens

Povilas Kavaliauskas; Birutė Grybaitė; Birute Sapijanskaite-Banevič; Rūta Petraitienė; Ramunė Grigalevičiūtė; Andrew Garcia; Ethan Naing; Vytautas Mickevičius; Sergey Belyakov; Vidmantas Petraitis

doi:10.1371/journal.pone.0311715

Abstract

The increasing prevalence of antimicrobial resistance among ESKAPE group pathogens presents a significant challenge in the healthcare sector, contributing to higher morbidity and mortality rates globally. Thus, it is essential to develop novel antimicrobial agents effective against drug-resistant pathogens. In this study, we report the synthesis and in vitro antimicrobial activity characterization of novel N-substituted β-amino acid derivatives bearing 2-hydroxyphenyl core against multidrug-resistant bacterial pathogens. The synthesized compounds (2–26) exhibited promising antimicrobial activity specifically against Gram-positive bacteria, with minimum inhibitory concentrations (MIC) ranging from 4 to 128 µg/mL. Compounds 9 (R = 4-nitrophenyl), 17 (R = 5-nitro-2-thienyl), 18 (R = 5-nitro-2-furyl), thiosemicarbazide 16, and 26 exhibited the most promising activity against Staphylococcus aureus MRSA USA300 lineage strain TCH-1516, with MIC values between 4 and 16 µg/mL. Compound 26 demonstrated strong antimicrobial activity against both S. aureus TCH-1516 and E. faecalis AR-0781, with the activity comparable to control antibiotics. Furthermore, compound 26 exhibited antifungal activity drug-resistant against Candida albicans AR-0761 (MIC 16 µg/mL). These findings indicate that N-substituted β-amino acid derivatives with a 2-hydroxyphenyl core warrant further investigation as a potential scaffold for the further development of antimicrobial agents based on compound 26 targeting Gram-positive pathogens and drug-resistant C. albicans AR-0761.

Citation: Kavaliauskas P, Grybaitė B, Sapijanskaite-Banevič B, Petraitienė R, Grigalevičiūtė R, Garcia A, et al. (2025) Synthesis of novel N-substituted β-amino acid derivatives bearing 2-hydroxyphenyl moieties as promising antimicrobial candidates targeting multidrug-resistant Gram-positive pathogens. PLoS One 20(6): e0311715. https://doi.org/10.1371/journal.pone.0311715

Editor: Wagdy M. Eldehna, Kafrelsheikh University Faculty of Pharmacy, EGYPT

Received: September 23, 2024; Accepted: March 6, 2025; Published: June 12, 2025

Copyright: © 2025 Kavaliauskas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The growing prevalence of antimicrobial resistance represents a critical challenge in healthcare setting, with the increasing numbers of infections caused by ESKAPE group pathogens [1–3]. This group comprises of drug-resistant Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species [4,5]. These pathogens are especially challenging for their antimicrobial resistance to multiple first line antibiotics, thereby complicating treatment regimens and leading to higher rates of morbidity and mortality [5]. The clinical impact of ESKAPE pathogens is profound, with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) being prominent contributors to severe healthcare-associated infections [4,6,7]. Among clinically important Enterococcus, E. faecalis remains important clinical pathogen often acquiring multiple resistance mechanisms therefore making a treatment of infections caused by E. faecalis often challenging [6,7]. The high mortality rates associated with these infections, coupled with the prolonged hospital stays and increased healthcare costs, underscore the urgent need for the development of novel antimicrobial agents for further pre-clinical development and optimization [8,9]. Infections caused by MRSA are known to cause a variety of infections, including skin and soft tissue infections, pneumonia, endocarditis, osteomyelitis, and sepsis, with mortality rates ranging from 20% to 50% depending on the severity and site of infection [10–12]. VRE predominantly causes bloodstream infections, urinary tract infections, intra-abdominal infections, and wound infections, with mortality rates for VRE bacteremia reaching up to 60% [13–15].

The molecular mechanisms by which these pathogens evade antibiotics are complex and multifaceted [16]. MRSA confers resistance to β-lactam antibiotics primarily through the acquisition of the mecA gene, which encodes a penicillin-binding protein (PBP2a) with low affinity for β-lactams, thereby preventing the inhibition of cell wall synthesis [16,17]. Additionally, MRSA employs various other resistance mechanisms, such as efflux pumps and the production of various antibiotics-modifying enzymes, often conferring resistance to aminoglycosides [18,19]. VRE, on the other hand, utilizes modification in cell wall precursors biosynthesis to evade vancomycin-mediated killing [20]. The acquisition of vanA or vanB gene clusters results in the modification of the terminal D-alanine-D-alanine to D-alanine-D-lactate in the peptidoglycan precursor, significantly reducing vancomycin binding affinity in VRE cell wall biogenesis pathway [20–22]. This modification impedes the antibiotic’s ability to inhibit cell wall synthesis, thereby conferring resistance to vancomycin [20]. The clinical impact of these resistance mechanisms is profound, as they severely limit the therapeutic options available for treating MRSA and VRE infections, necessitating the development of new antimicrobial strategies [21,22].

Various amino acid derivatives have been extensively investigated in medicinal chemistry as novel pharmacophores for targeting a variety of diseases, including cancers, bacterial and fungal infections, and parasitic diseases (Fig 1) [22–26].

Download:

Fig 1. Pharmaceuticals containing β-amino acid structures.

https://doi.org/10.1371/journal.pone.0311715.g001

These derivatives are attractive candidates for drug design due to their inherent biological activity, structural diversity, and ability to mimic natural substrates in biological systems [23,25,26]. For instance, modifications of amino acid side chains or addition of various aromatic or heterocyclic substituents can enhance binding affinity to specific biological targets, improve selectivity, and increase metabolic stability [22,23,26]. Especially 1,3,4-oxadiazole-2(3H)-thione fragment are an important part of heterocyclic compounds with broad spectrum of biological activities. Substituted 1,3,4-oxadiazoles have revealed antibacterial, anti-mycobacterial, antifungal, anti-inflammatory, analgesic, anticonvulsant and anticancer properties [24–26]. Among substituted amino acid derivatives, N-substitution in β-amino acids offers promising scaffold with remarkable versatility in tailoring molecular properties. This N-substituted scaffold allows to incorporate a wide variety of substitutions that are crucial for further optimization of physicochemical attributes, influencing lipophilicity, electronic distribution, and overall bioactivity [22–27].

Our previous studies have demonstrated that amino acid derivatives bearing a 4-hydroxyphenyl group exhibit promising antimicrobial activity against multidrug-resistant bacterial pathogens [28]. Additionally, the synthesized compounds exhibited significant antifungal activity against highly multidrug-resistant fungal pathogens, including the emerging azole-resistant Candida auris [28]. Further in silico analyses indicated favorable predicted pharmacological and drug-like properties, establishing 3-((4-hydroxyphenyl)amino)propanoic acid as a promising scaffold for the development of antimicrobial candidates [28]. Building on these findings, the present study explores the synthesis pathways and in vitro antimicrobial properties of new N-substituted β-amino acid derivatives bearing a 2-hydroxyphenyl moiety, along with various aromatic and heterocyclic substituents. In this study, we successfully demonstrate that N-substituted β-amino acid derivatives with a 2-hydroxyphenyl core, as well as heterocyclic substituents, could serve as promising scaffolds for the development of novel antimicrobial candidates targeting Gram-positive pathogens.

Materials and methods

Chemical synthesis

General analytical procedures.

The reaction course and purity of the synthesized compounds were monitored by TLC using aluminum plates precoated with Silica gel with F254 nm (Merck KGaA, Darmstadt, Germany). Reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Melting points were determined with a B-540 melting point analyzer (Büchi Corporation, New Castle, DE, USA) and were uncorrected. IR spectra (υ, cm⁻¹) were recorded on a Perkin–Elmer Spectrum BX FT–IR spectrometer using KBr pellets. NMR spectra were recorded on a Brucker Avance III (400, 101 MHz) spectrometer. Chemical shifts were reported in (δ) ppm relative to tetramethylsilane (TMS) with the residual solvent as internal reference ([D₆]DMSO, δ = 2.50 ppm for ¹H and δ = 39.5 ppm for ¹³C). ¹⁹F NMR spectra (376 MHz, absolute referencing via the Ξ ratio) were obtained on a Bruker Avance III 400 instrument with a ′directly′ detecting broadband observe probe (BBO). Data were reported as follows: chemical shift, multiplicity, coupling constant (Hz), integration, and assignment. Elemental analyses (C, H, N) were conducted using the Elemental Analyzer CE-440; their results were found to be in good agreement (±0.3%) with the calculated values.

General procedures for preparation of compounds 2–26.

3,3’-((2-Hydroxyphenyl)azanediyl)dipropionic acid (2): A mixture of o-aminophenol (1) (10.9 g, 100 mmol), acrylic acid (18 g, 250 mmol) and water (100 mL) was heated under reflux for 14 h and cooled. Crystaline product 2 was filtered off, washed with propan-2-ol, and dried. White powder, yield 18.97 g (75%), m.p. 181−183 °C (from propan-2-ol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.29 (t, J = 7.1 Hz, 4H, 2x CH₂CO), 3.18 (t, J = 7.1 Hz, 4H, 2x NCH₂), 6.66–7.06 (m, 4H, H_Ar); 8.77 (br.s, 1H, OH); 12.06 (br.s, 2H, 2x OH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 32.4 (CH₂CO), 48.6 (NCH₂), 115.6, 119.4, 123.1, 124.9, 136.5, 152.7 (C_Ar), 173.8 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3045, 2971 (3x OH), 1698 (2x C=O). Anal. Calcd. for C₁₂H₁₅NO₅, %: C 56.91; H 5.97; N 5.53. Found: C 56.69; H 5.70; N 5.33.

3-(2-Oxo-3,4-dihydrobenzo[b][1,4]oxazepin-5(2H)-yl)-N-(4-sulfamoylphenyl)propenamide (3): The dipropionic acid 2 (0.5g 2, mmol) were dissolved in dimethylformamide (2 mL) by heating, then sulfanilamide (0.83g, 4.8 mmol,) were dissolved in dimethylformamide (2 mL) in another beaker and both prepared solutions were poured into a flask and triethylamine (12 mmol) were added dropwise, the reaction mixture was stirred 20 minutes at room temperature. Separately, HBTU (2.28 g, 6 mmol) were dissolved in 9 mL of dimethylformamide and were added dropwise to the reaction mixture. The reaction was stirred at room temperature for 24 h. After that, the reaction mixture were diluted with water (40 mL), the formed crystals were filtered off and were washed with 5% potassium carbonate solution, water and dried. White powder, yield 0.22 g (28%), m.p. 190−192 °C (from dioxane/water mixture). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.58 (t, J = 7.0 Hz, 4H, 2x CH₂CO), 3.39 (t, J = 7.0 Hz, 4H, 2x NCH₂), 6.99–7.16 (m, 2H, SO₂NH₂), 7.19–7.32 (m, 4H, H_Ar), 7.72 (q, J = 8.7 Hz, 4H, H_Ar), 12.21 (s, 1H, NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 31.7 (CH₂CONH), 35.0 (CH₂CONHO), 47.5 (NCH₂ CH₂CONH), 54.3 (NCH₂CH₂CONHO), 118.6, 119.5, 121.1, 123.9, 126.3, 126.6, 138.2, 138.4, 141.9, 146.9 (C_Ar), 169.9, 170.0 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3344 (NH₂), 3241 (NH), 1742, 1689 (2x C=O). Anal. Calcd. for C₁₈H₁₉N₃O₅S, %: C 55.52; H 4.92; N 10.79. Found: C 55.31; H 4.72; N 10.55. HRMS m/z calculated for C₁₈H₁₉N₃O₅S [M+H]+ : 390.1045; found: 390.1119.

Dimethyl 3,3’-((2-hydroxyphenyl)azanediyl)dipropionate (4): A mixture of dipropionic acid 2 (17.48 g, 69 mmol), conc. sulfuric acid (8.6 g, 4.7 mL, 87 mmol) and methanol (250 mL) was heated under reflux for 7 h. Then the solvent was evaporated under reduced pressure, and the residue was neutralized with 5% sodium carbonate solution to pH 7. The obtained solid was filtered off, washed with plenty of water and recrystallized from propan-2-ol. Light brown powder, yield 15.7 g (81%), m.p. 87–89 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.37 (t, J = 7.0 Hz, 4H, 2x CH₂CO), 3.22 (t, J = 7.0 Hz, 4H, 2x NCH₂), 3.54 (s, 6H, 2x CH₃), 6.68–7.05 (m, 4H, H_Ar); 8.66 (s, 1H, OH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 32.9 (CH₂CO), 48.4 (NCH₂), 51.2 (CH₃), 115.5, 119.2, 123.3, 124.8, 135.9, 152.6 (C_Ar), 172.4 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3308 (OH), 1727 (2x C=O). Anal. Calcd. for C₁₄H₁₉NO₅, %: C 59.78; H 6.81; N 4.98. Found: C 59.52; H 6.60; N 4.72.

3,3’-((2-Hydroxyphenyl)azanediyl)di(propanehydrazide) (5): A mixture of methyl ester 4 (7.28 g, 26 mmol), hydrazine hydrate (7.89 g, 157 mmol), and propan-2-ol (25 mL) was heated under reflux for 5 h and cooled. Crystalline product 5 was filtered off, washed with propan-2-ol, diethyl ether, and dried. White powder, yield 5.82 g (80%), m.p. 149 − 151 °C (from propan-2-ol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.13 (t, J = 7.1 Hz, 4H, 2x CH₂CO), 3.12 (t, J = 7.2 Hz, 4H, 2x NCH₂), 4.20 (s, 4H, 2x NH₂), 6.70–7.08 (m, 4H, H_Ar); 9.01 (s, 3H, OH, 2x NHNH₂); ¹³C NMR (101 MHz, DMSO-d₆) δ: 31.6 (CH₂CO), 49.0 (NCH₂), 115.7, 119.1, 122.6, 124.6, 136.7, 152.8 (C_Ar), 170.6 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3295 (OH), 3178 (NH₂), 3057 (NH₂), 1677, 1624 (C=O). Anal. Calcd. for C₁₂H₁₉N₅O₃, %: C 51.23; H 6.81; N 24.90. Found: C 51.01; H 6.62; N 24.69.

2-((2-(1H-benzo[d]imidazol-2-yl)ethyl)amino)phenol (6): Mixture of compound 2 (0.7 g, 0.0028 mol) and o-phenylenediamine (0.61 g, 0.0056 mol) in dilute hydrochloric acid (1:1, 8 mL) was heated at reflux for 24 h, then cooled down. The reaction mixture was neutralized with a 5% sodium carbonate solution to pH 8. The formed crystals was recrystallized from a mixture of 1,4-dioxane and water (1:1, 15 ml). Brown powder, yield 0.2 g (28%), m.p. 196 − 198 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 3.10 (t, J = 7.0 Hz, 2H, 2x CH₂CN), 3.52 (q, J = 7.0 Hz, 2H, 2x NCH₂), 4.86 (t, J = 7.0 Hz, 1H, NHCH₂), 6.23–6.74 (m, 4H, H_Ar); 7.01–7.22 (m, 2H, H_Ar), 7.29–7.68 (m, 2H, H_Ar), 9.23 (s, 1H, OH), 12.28 (s, 1H, NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 28.5 (CH₂CO), 41.3 (NCH₂), 109.8, 110.8, 113.5, 116.0, 118.1, 119.8, 120.9, 121.5, 134.2, 137.1, 143.3, 144.2, 153.4 (C_Ar). IR (KBr): ν_max (cm⁻¹) = 3310 (OH), 3055 (2x NH). Anal. Calcd. for C₁₅H₁₅N₃O, %: C 71.13; H 5.97; N 16.59. Found: C 70.95; H 5.70; N 16.32.

General procedure for the preparation of hydrazones 7–22: To a solution of hydrazide 5 (0.42g, 1.5 mmol) in 2-propanol (15 mL), the corresponding aromatic aldehyde was added (1.65 mmol) and the mixture was heated at reflux for 2 h, then cooled down, and the formed precipitate was filtered off, washed with methanol, diethyl ether and recrystallizing from 1,4-dioxane.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(benzylidene)propanehydrazide) (7): White powder, yield 0.57 g (83%), m.p. 223−225 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.34 and 2.77 (2t, J = 7.1 Hz, 2H, 2x CH₂CO), 3.17–3.41 (m, 4H, 2x NCH₂), 6.68–7.21 (m, 4H, H_Ar); 7.29–7.79 (m, 10H, H_Ar), 7.83–8.19 (m, 2H, 2x CH), 8.91, 8.96, 9.02 (3s, 1H, OH), 11.30, 11.31, 11.41 (3s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.2, 30.4, 31.6, 32.4, 32.6 (CH₂CO), 48.3, 48.5, 48.7, 48.9 (NCH₂), 115.6, 119.1, 119.2, 122.4, 122.8, 124.3, 124.6, 126.6, 126.7, 126.9, 128.7, 128.8, 129.7, 129.9, 134.2, 134.3, 136.2, 136.5, 136.9, 152.8, 145.9, 152.4, 152.5, 152.8, 167.5, 167.6 (C_Ar) 170.6, 173.4, 173.5 (C=O). IR (KBr): ν_max (cm⁻¹) = 3109 (OH), 3007 (2x NH), 1672 (2x C=O). Anal. Calcd. for C₂₆H₂₇N₅O₃, %: C 68.25; H 5.95; N 15.31. Found: C 68.00; H 5.73; N 15.11. HRMS m/z calculated for C₂₆H₂₇N₅O₃ [M+H]+ : 458.2114; found: 458.2184.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(2,4-difluorobenzylidene)propanehydrazide) (8): White powder, yield 0.57 g (83%), m.p. 224−226 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.23–2.39 and 2.64–2.87 (2m, 4H, 2x CH₂CO), 3.15–3.34 (m, 4H, 2x NCH₂), 6.68–7.37 (m, 8H, H_Ar); 7.58–7.95 (m, 2H, H_Ar), 7.99–8.11 and 8.20–8.30 (2m, 2H, 2x N=CH), 8.88, 8.90, 9.00 (3s, 1H, OH), 11.39, 11.41, 11.53 (3s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.1, 30.4, 32.4, 32.6 (CH₂CO), 48.2, 48.4, 48.6, 48.7 (NCH₂), 104.1, 104.4, 104.6, 112.5, 115.6, 118.5, 118.6, 119.01, 119.2, 122.3, 122.7, 124.4, 124.6, 127.5, 127.8, 127.9, 134.8, 136.5, 136.8, 137.9, 152.4, 152.5, 159.3, 159.5, 161.6, 161.9, 164.1, 167.6, 167.7 (C_Ar), 173.5, 173.6 (2x C=O). ¹⁹F NMR (376 MHz, DMSO-d₆) δ: −107.2, −107.5, −117.0, −117.3. IR (KBr): ν_max (cm⁻¹) = 3297 (OH), 3196 (2x NH), 1673 (2x C=O). Anal. Calcd. for C₂₆H₂₃F₄N₅O₃, %: C 58.98; H 4.38; N 13.23. Found: C 58.73; H 4.13; N 13.03. HRMS m/z calculated for C₂₆H₂₃F₄N₅O₃ [M+H]+ : 530.1737; found: 530.1810.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(4-nitrobenzylidene)propanehydrazide) (9): Light yellow powder, yield 0.55 g (67%), m.p. 225 − 227 °C (from propan-2-ol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.38 (t, J = 6.8 Hz, 2H, CH₂CO), 2.80 (t, J = 6.7 Hz, 2H, CH₂CO), 3.25 − 3.39 (m, 4H, 2x NCH₂), 6.73–8.30 (m, 14H, H_Ar, 2x N=CH); 8.90, 8.93, 9.06 (3s, 1H, OH), 11.58, 11.60, 11.71 (3s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.3, 30.5, 32.6 (CH₂CO), 48.1, 48.4, 48.7, 48.8 (NCH₂), 115.6, 115.8, 119.1, 119.3, 119.4, 122.3, 122.8, 123.1, 123.9, 124.3, 124.6, 127.4, 127.5, 127.8, 136.2, 136.5, 136.8, 140.4, 140.5, 140.5, 140.7, 143.8, 143.6, 147.4, 147.5, 147.6, 147.7, 152.3, 152.5, 152.8, 168.0 (C_Ar), 173.8, 173.9 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3265 (OH), 3092 (2x NH), 1675 (2x C=O). Anal. Calcd. for C₂₆H₂₅N₇O₇, %: C 57.04; H 4.60; N 17.91. Found: C 56.83; H 4.42; N 17.75.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(4-chlorobenzylidene)propanehydrazide) (10): White powder, yield 0.55 g (69%), m.p. 220−222 °C (from dioxane). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.34 (t, J = 6.8 Hz, 2H, CH₂CO), 2.76 (t, J = 7.1 Hz, 2H, CH₂CO), 3.25 − 3.36 (m, 4H, 2x NCH₂), 6.70–8.12 (m, 14H, H_Ar, 2x N=CH); 8.89, 8.94, 9.02 (3s, 1H, OH), 11.35, 11.36, 11.47 (3s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.2, 30.4, 32.4, 32.6 (CH₂CO), 48.2, 48.4, 48.7, 48.9 (NCH₂), 115.6, 115.7, 119.1, 119.2, 119.3, 122.4, 122.7, 123.1, 124.3, 124.6, 124.9, 128.2, 128.3, 128.6, 128.8, 133.1, 133.3, 134.1, 134.3, 136.2, 136.5, 136.9, 144.7, 144.8, 152.3, 152.5, 152.8, 167.6, 167.7 (C_Ar), 173.5 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3179 (OH), 3107 (2x NH), 1673 (2x C=O). Anal. Calcd. for C₂₆H₂₅ Cl₂N₅O₃, %: C 59.32; H 4.79; N 13.30. Found: C 59.11; H 4.54; N 13.17. HRMS m/z calculated for C₂₆H₂₅ Cl₂N₅O₃ [M+Na]+ : 548.1334; found: 548.1228.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(4-(dimethylamino)benzylidene)propanehydrazide) (11): White powder, yield 0.75 g (91%), m.p. 206−208 °C. ¹H NMR (400 MHz, DMSO-d₆) δ: 2.30 and 2.73 (2q, J = 6.5 Hz, 2H, 2x CH₂CO), 2.91, 2.93, 2.95 (3s, 12H, 4x CH₃); 3.20–3.33 (m, 4H, 2x NCH₂), 6.56–6.98 (m, 6H, H_Ar); 7.06–7.17 (m, 1H, H_Ar), 7.29–7.51 (m, 4H, H_Ar), 7.76–8.01 (m, 2H, 2x N=CH), 9.00, 9.02, 9.06 (3s, 1H, OH), 11.03, 11.12, 11.13 (3s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.3, 30.5, 32.3, 32.4, 32.5 (4x CH₃, CH₂CO), 48.2, 48.6, 49.0 (NCH₂), 111.7, 111.8, 115.6, 119.2, 121.6, 122.2, 122.6, 123.01, 124.3, 124.5, 127.9, 128.3, 128.4, 136.3, 136.7, 137.2, 143.8, 146.9, 147.0, 151.2, 151.4, 152.5, 166.9, 167.0 (C_Ar), 172.9 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3437 (OH), 3078 (2x NH), 1661 (2x C=O). Anal. Calcd. for C₃₀H₃₇N₇O₃, %: C 66.28; H 6.86; N 18.03. Found: C 66.04; H 6.65; N 17.86. HRMS m/z calculated for C₃₀H₃₇N₇O₃ [M+H]+ : 544.2957; found: 544.3025.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(4-hydroxybenzylidene)propanehydrazide) (12): White powder, yield 0.45 g (62%), m.p. 164−166 °C (from methanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.31 (t, J = 6.9 Hz, 2H, CH₂CO), 2.74 (t, J = 6.7 Hz, 2H, CH₂CO), 3.19−3.36 (m, 4H, 2x NCH₂), 6.69–8.07 (m, 14H, H_Ar, 2x N=CH); 8.98 (t, 1H, OH), 9.84, 9.87 (2s, 2H, 2x OH), 11.10, 11.21 (2s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.2, 30.4, 32.3, 32.5 (CH₂CO), 48.3, 48.6, 48.7, 49.0 (NCH₂), 115.6, 115.7, 119.1, 119.2, 119.3, 122.4, 122.7, 123.1, 125.2, 125.3, 128.4, 128.4, 128.8, 136.3, 136.6, 137.1, 143.2, 146.4, 146.5, 152.4, 152.6, 152.8, 159.1, 159.3, 167.2, 167.3 (C_Ar), 173.1, 173.2 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3577 (OH), 3088 (2x NH), 1654 (2x C=O). Anal. Calcd. for C₂₆H₂₇N₅O₅, %: C 63.79; H 5.56; N 14.31. Found: C 63.55; H 5.36; N 14.15. HRMS m/z calculated for C₂₆H₂₇N₅O₅ [M+Na]+ : 512.2012; found: 512.1903.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(3,4,5-trimethoxybenzylidene)propanehydrazide) (13): White powder, yield 0.8 g (83%), m.p. 214−216 °C (from methanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.33 and 2.77 (q, J = 6.9 Hz, 4H, CH₂CO), 3.12−3.36 (m, 4H, 2x NCH₂), 3.60–3.85 (m, 18H, 6x OCH₃), 6.66–7.17 (m, 8H, H_Ar); 7.83, 7.87, 8.02, 8.04 (4s, 2H, 2x N=CH); 8.81, 8.92, 9.02 (3s, 1H, OH), 11.31, 11.34, 11.36, 11.39 (4s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.2, 30.4, 32.4, 32.6 (CH₂CO), 48.7, 48.9, 55.8, 55.9, 60.1 (NCH₂, 6x OCH₃), 103.7, 103.8, 104.2, 115.3, 115.5, 119.0, 119.2, 122.9, 124.7, 129.7, 129.8, 136.3, 136.5, 136.7, 138.8, 138.9, 139.0, 142.5, 142.6, 145.9, 146.0, 152.7, 153.1, 167.4, 167.5 (C_Ar), 173.4 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3327 (OH), 3111 (2x NH), 1670 (2x C=O). Anal. Calcd. for C₃₂H₃₉N₅O₉, %: C 60.27; H 6.16; N 10.98. Found: C 60.03; H 5.94; N 10.71.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(naphthalen-1-ylmethylene)propanehydrazide) (14): White powder, yield 0.73 g (87%), m.p. 213−215 °C (from methanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.42 and 2.86 (t, J = 7.2 Hz, 4H, CH₂CO), 3.34−3.52 (m, 4H, 2x NCH₂), 6.71–6.99 (m, 3H, H_Ar); 7.11–7.25 (m, 1H, H_Ar); 7.44–8.05 (m, 12H, H_Ar); 8.47–8.85 (m, 4H, H_Ar, 2x N=CH); 8.94, 9.02, 9.10 (3s, 1H, OH), 11.35, 11.37, 11.53, 11.54 (4s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.3, 30.6, 32.4, 32.6 (CH₂CO), 48.5, 48.6, 48.8, 48.9 (NCH₂), 115.5, 115.6, 115.8, 119.2, 122.7, 122.9, 123.6, 124.3, 124.5, 124.7, 125.5, 126.1, 126.2, 127.0, 127.1, 127.2, 127.3, 127.9, 128.8, 129.3, 129.4, 129.5, 129.9, 130.1, 130.3, 133.4, 133.5, 136.5, 136.8, 142.6, 142.7, 145.9, 146.1, 152.6, 152.7, 167.5, 167.6 (C_Ar), 173.3, 173.4 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3169 (OH), 3092 (2x NH), 1675 (2x C=O). Anal. Calcd. for C₃₄H₃₁N₅O₃, %: C 73.23; H 5.60; N 12.56. Found: C 73.02; H 5.43; N 12.33.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(furan-2-ylmethylene)propanehydrazide) (15): White powder, yield 0.46 g (70%), m.p. 194−196 °C (from dioxane). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.23−2.40 (m, 2H, CH₂CO), 2.60−2.75 (m, 2H, CH₂CO), 3.17−3.38 (m, 4H, 2x NCH₂), 6.50–7.18 (m, 8H, H_Ar); 7.69–8.07 (m, 4H, H_Ar, N=CH); 8.95 (s, 1H, OH), 11.25, 11.27, 11.34 (3s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.2, 30.3 (CH₂CO), 47.9, 48.1, 48.6, 48.9 (NCH₂), 112.0, 112.1, 112.9, 113.2, 115.6, 115.7, 119.2, 119.3, 122.0, 122.5, 123.2, 124.2, 124.5, 124.9, 133.1, 135.9, 136.0, 136.2, 136.6, 136.9, 144.7, 144.8, 144.9, 149.2, 149.4, 152.2, 152.5, 152.8, 167.5, 167.6 (C_Ar), 173.3 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3227 (OH), 3098 (2x NH), 1671 (2x C=O). Anal. Calcd. for C₂₂H₂₃N₅O₅, %: C 60.40; H 5.30; N 16.01. Found: C 60.27; H 5.17; N 15.87. HRMS m/z calculated for C₂₂H₂₃N₅O₅ [M+H]+ : 438.1699; found: 438.1770.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(thiophen-2-ylmethylene)propanehydrazide) (16): White powder, yield 0.49 g (70%), m.p. 212−214 °C (from dioxane). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.31 and 2.68 (2t, J = 6.9 Hz, 4H, CH₂CO), 3.15−3.34 (m, 4H, 2x NCH₂), 6.67–7.63 (m, 10H, H_Ar,H_Het), 8.12, 8.14, 8.33 (3s, 2H 2x N=CH), 8.95 (t, J = 10.9 Hz, 1H, OH), 11.27, 11.29, 11.35 (3s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.1, 30.4, 32.4, 32.5 (CH₂CO), 48.1, 48.5, 48.6, 48.9 (NCH₂), 115.6, 115.7, 119.1, 119.2, 119.3, 122.3, 122.7, 123.1, 124.3, 124.5, 124.8, 127.8, 128.1, 128.2, 128.7, 129.9, 130.1, 130.6, 130.7, 136.2, 136.5, 136.8, 138.0, 138.1, 138.9, 139.0, 139.1, 141.2, 141.3, 152.3, 152.5, 152.7, 167.4, (C_Ar), 173.1 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3203 (OH), 3011 (2x NH), 1672 (2x C=O). Anal. Calcd. for C₂₂H₂₃N₅O₃S₂, %: C 56.27; H 4.94; N 14.91. Found: C 52.05; H 4.74; N 14.76. HRMS m/z calculated for C₂₂H₂₃N₅O₃S₂ [M+H]+ : 470.1242; found: 470.1314.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-((5-nitrothiophen-2-yl)methylene)propanehydrazide) (17): Yellow powder, yield 0.6 g (72%), m.p. 221−223 °C (from methanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.35 and 2.71 (2t, J = 7.2 Hz, 4H, CH₂CO), 3.16−3.47 (m, 4H, 2x NCH₂), 6.72–6.87 (m, 2H, H_Ar); 6.88–6.98 (m, 1H, H_Ar); 7.07–7.15 (m, 1H, H_Ar); 7.33–7.50 (m, 2H, H_Ar); 7.94–8.39 (m, 4H, H_Ar, N=CH); 8.81, 8.86, 9.02 (3s, 1H, OH), 11.68, 11.70, 11.74, 11.76 (4s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.0, 30.3, 32.5, 32.7 (CH₂CO), 48.1, 48.6 (NCH₂), 115.6, 115.7, 119.1, 119.3, 122.4, 122.9, 124.7, 128.7, 128.8, 129.3, 130.4, 136.0, 136.2, 136.5, 139.5, 146.8, 146.9, 150.3, 150.6, 152.3, 152.5, 168.1 (C_Ar), 173.6, 173.7 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3198 (OH), 3100 (2x NH), 1672 (2x C=O). Anal. Calcd. for C₂₂H₂₁N₇O₇S₂, %: C 47.22; H 3.78; N 17.52. Found: C 47.05; H 3.56; N 17.34.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(5-nitrofuran-2-ylmethylene)propanehydrazide) (18): Yellow powder, yield 0.47 g (59%), m.p. 188 − 190 °C (from dioxane). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.24−2.42 (m, 2H, CH₂CO), 2.74 (t, J = 6.7 Hz, 2H, CH₂CO), 3.20−3.41 (m, 4H, 2x NCH₂), 6.42–7.21 (m, 6H, H_Ar), 7.58–8.12 (m, 4H, H_Ar 2x N=CH), 8.84, 8.89, 9.01 (3s, 1H, OH), 11.68, 11.72, 11.76, 11.79 (4s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.1, 30.3, 32.5 (CH₂CO), 47.9, 48.4, 48.8 (NCH₂), 114.2, 114.4, 114.6, 114.7, 114.9, 115.5, 115.6, 115.8, 122.2, 122.7, 123.2, 124.3, 124.6, 124.9, 130.9, 131.0, 133.8, 133.9, 136.1, 136.4, 136.8, 151.6, 151.7, 151.8, 151.9, 152.2, 152.5, 168.1, 168.2 (C_Ar), 173.8, 173.9 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3212 (OH), 3153 (2x NH), 1676 (2x C=O). Anal. Calcd. for C₂₂H₂₁N₇O₉, %: C 50.10; H 4.01; N 18.59. Found: C 49.94; H 3.87; N 18.35. HRMS m/z calculated for C₂₂H₂₁N₇O₉ [M+H]+ : 528.1400; found: 528.1476.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(thiophen-3-ylmethylene)propanehydrazide) (19): White powder, yield 0.62 g (89%), m.p. 209−211 °C (from 2-propanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.31 and 2.72 (2t, J = 7.2 Hz, 4H, CH₂CO), 3.18−3.32 (m, 4H, NCH₂), 6.72–8.21 (m, 12H, H_Ar, H_Het, 2x CH), 8.90, 8.95, 8.98 (3s, 1H, OH), 11.19, 11.28 (2s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 30.2, 30.4, 32.4, 32.5 (CH₂CO), 48.3, 48.5, 48.7, 48.9 (NCH₂), 115.5, 115.6, 115.7, 119.1, 119.2, 119.3, 122.4, 122.7, 123.1, 124.4, 124.5, 124.6, 124.7, 124.8, 127.3, 127.4, 127.5, 127.9, 136.3, 136.5, 136.9, 137.4, 137.5, 138.6, 138.7, 141.7, 141.8, 152.4, 152.6, 152.7, 167.4, 167.5 (C_Ar), 173.3 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3287 (OH), 3088 (2x NH), 1670 (2x C=O). Anal. Calcd. for C₂₂H₂₃N₅O₃S₂, %: C 56.27; H 4.94; N 14.91. Found: C 56.05; H 4.71; N 14.84. HRMS m/z calculated for C₂₂H₂₃N₅O₃S₂ [M+H]+ : 470.1242; found: 470.1313.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(propan-2-ylidene)propanehydrazide) (20): White powder, yield 0.43 g (80%), m.p. 161−163 °C (from 2-propanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 1.81, 1.82, 1.86, 1.90 (4s, 12H, 4x CH₃); 2.32 (q, J = 6.9 Hz, 2H, CH₂CO), 2.60 (t, J = 6.9 Hz, 2H, COCH₂), 3.20 (q, J = 6.9 Hz, 4H, NCH₂), 6.70–6.81 (m, 2H, H_Ar), 6.85–6.95 (m, 1H, H_Ar), 7.03–7.11 (m, 1H, H_Ar), 8.93, 8.96, 9.01 (3s, 1H, OH), 10.02, 10.04 (2s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 17.0, 17.5, 24.9, 25.1 (4x CH₃), 30.5, 30.6, 31.9, 32.0 (CH₂CO), 47.9, 48.2, 48.8, 48.9 (NCH₂), 115.6, 118.9, 119.1, 121.8, 122.3, 122.8, 124.1, 124.4, 124.6, 136.4, 136.6, 137.1, 150.4, 152.3, 152.6, 154.8, 154.9, 167.5 (C_Ar), 173.5 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3271 (OH), 3198 (2x NH), 1660 (2x C=O). Anal. Calcd. for C₁₈H₂₇N₅O₃, %: C 59.81; H 7.53; N 19.38. Found: C 59.63; H 7.36; N 19.17. HRMS m/z calculated for C₁₈H₂₇N₅O₃ [M+H]+ : 362.2113; found: 362.2186.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(butan-2-ylidene)propanehydrazide) (21): Light brown powder, yield 0.49 g (84%), m.p. 81−83 °C (from 2-propanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 0.82–1.11 (m, 6H, CH₂CH₃); 1.79, 1.81, 1.84, 1.88 (4s, 6H, 2x CH₃); 2.09–2.29 (m, 4H, CH₂CH₃); 2.32 (t, J = 7.1 Hz, 2H, CH₂CO), 2.63 (t, J = 7.1 Hz, 2H, COCH₂), 3.21 (q, J = 6.9 Hz, 4H, NCH₂), 6.67–6.82 (m, 2H, H_Ar), 6.85–6.95 (m, 1H, H_Ar), 7.03–7.11 (m, 1H, H_Ar), 8.95 (1s, 1H, OH), 10.01, 10.09 (2s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 9.7, 9.8, 10.5, 10.8, 15.7, 15.9 (4x CH₃), 22.1, 22.9, 23.3 (CH₂CH₃), 30.5, 31.4, 31.7, 31.9, 32.1 (CH₂CO), 48.1, 48.5, 48.8, 48.9 (NCH₂), 115.4, 115.5, 119.1, 122.1, 122.4, 122.7, 124.2, 124.5, 124.6, 136.6, 137.0, 152.4, 152.5, 153.7, 153.8, 158.3, 167.6 (C_Ar), 173.72 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3240 (OH), 3178 (2x NH), 1671 (2x C=O). Anal. Calcd. for C₂₀H₃₁N₅O₃, %: C 61.67; H 8.02; N 17.98. Found: C 61.45; H 7.88; N 17.87. HRMS m/z calculated for C₂₀H₃₁N₅O₃ [M+H]+ : 390.2426; found: 390.2500.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-(1-phenylethylidene)propanehydrazide) (22): White powder, yield 0.61 g (84%), m.p. 187−189 °C (from 2-propanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.09–2.27 (m, 6H, CH₃); 2.40–2.55 (overlaps with DMSO, 2H, CH₂CO), 2.80 (t, J = 7.1 Hz, 2H, COCH₂), 3.22–3.42 (m, 4H, NCH₂), 6.68–6.99 (m, 3H, H_Ar), 7.07–7.18 (m, 1H, H_Ar), 7.27–7.47 (m, 6H, H_Ar), 7.52–7.85 (m, 4H, H_Ar); 8.93, 8.99 (2s, 1H, OH), 10.44, 10.45, 10.48, 10.50 (4s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 13.5, 14.0 (2x CH₃), 30.6, 30.8, 32.2, 32.4 (CH₂CO), 48.5, 48.7, 48.9 (NCH₂), 115.5, 115.6, 119.1, 119.2, 122.5, 122.8, 124.4, 125.9, 126.3, 128.2, 128.3, 128.8, 128.9, 129.1, 136.4, 136.9, 138.1, 138.3, 147.2, 147.3, 150.9, 152.6, 168.1, 168.2 (C_Ar), 174.3 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3242 (OH), 3158 (2x NH), 1677 (2x C=O). Anal. Calcd. for C₂₈H₃₁N₅O₃, %: C 69.26; H 6.44; N 14.42. Found: C 69.03; H 6.21; N 14.24.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N-(2,5-dimethyl-1H-pyrrol-1-yl)propanamide) (23): To a solution of dihydrazide 5 (0.5 g, 1.8 mmol) in 2-propanol (25 mL), hexane-2,5-dione (0.82 g, 7.2 mmol) and a catalytic amount of acetic acid (0.1 mL) were added, and the mixture was heated under reflux for 6 h, then cooled down, and was diluted with water (25 mL); the formed precipitate was filtered off, washed with water, and recrystallized from a mixture of 2-propanol and water. White powder, yield 0.53 g (67%), m.p. 200−202 °C (from 2-propanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 1.95 (s, 12H, 4x CH₃); 2.41 (t, J = 7.2 Hz, 4H, 2x COCH₂), 3.34 (t, J = 7.2 Hz, 4H, 2x NCH₂), 5.61 (s, 4H, 4x CH_pyr); 6.74–6.86 (m, 2H, H_Ar), 6.90–6.98 (m, 1H, H_Ar), 7.09–7.16 (m, 1H, H_Ar), 8.88 (1s, 1H, OH), 10.60 (1s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 10.9 (2x CH₃), 31.4 (CH₂CO), 47.8 (NCH₂), 102.8, 115.7, 119.3, 123.3, 124.8, 126.7, 136.1, 152.6 (C_Ar), 170.6 (C=O). IR (KBr): ν_max (cm⁻¹) = 3237 (OH), 3018 (2x NH), 1672 (2x C=O). Anal. Calcd. for C₂₄H₃₁N₅O₃, %: C 65.88; H 7.14; N 16.01. Found: C 65.63; H 6.95; N 15.87. HRMS m/z calculated for C₂₄H₃₁N₅O₃ [M+H]+ : 438.2426; found: 438.2498.

5-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)-3-oxopropyl)-4,5-dihydrobenzo[b][1,4]oxazepin-2(3H)-one (24): To a solution of dihydrazide 5 (0.5 g, 1.8 mmol) in 2-propanol (28 mL), pentane-2,4-dione (0.9 g, 9.0 mmol) and a catalytic amount of hydrochloric acid (0.05 mL) were added, and the mixture was heated under reflux for 5 h, then cooled down. The solvent was removed under reduced pressure, the residue was poured with water (30 mL), and the formed precipitate was filtered off, washed with water and diethyl ether, and was recrystallized from a mixture of 2-propanol and water. White powder, yield 0.35 g (62%), m.p. 133−135 °C (from 2-propanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.12 and 2.44 (2s, 6H, 2x CH₃); 2.56 and 3.22 (2t, J = 6.9 Hz, 4H, 2x COCH₂), 3.38 and 3.44 (2t, J = 6.9 Hz, 4H, 2x NCH₂), 6.14 (s, 1H, 4x CH_Het); 7.00–7.33 (m, 4H, H_Ar); ¹³C NMR (101 MHz, DMSO-d₆) δ: 13.4, 14.0 (2x CH₃), 31.6, 33.5 (CH₂CO), 46.7, 54.5 (NCH₂), 111.1, 119.5, 121.0, 123.9, 126.2, 138.2, 143.1, 146.9, 151.3, 169.9 (C_Ar), 171.6 (C=O). IR (KBr): ν_max (cm⁻¹) = 1748, 1729 (2x C=O). Anal. Calcd. for C₁₇H₁₉N₃O₃, %: C 65.16; H 6.11; N 13.41. Found: C 64.98; H 5.96; N 13.27. HRMS m/z calculated for C₁₇H₁₉N₃O₃ [M+Na]+ : 336.1426; found: 336.1321.

3,3’-((2-Hydroxyphenyl)azanediyl)bis(N’-((Z)-2-oxoindolin-3-ylidene)propanehydrazide) (25): To a solution of hydrazide 5 (0.3, 1.06 mmol) in methanol (10 mL), isatin (0.38, 2.55 mmol) and glacial acetic acid (1 drops) were added. The reaction mixture was heated under reflux for 4 h. Precipitate was filtered off, washed with methanol, and recrystallized from 2-propanol/H₂O mixture. Yellow powder, yield 0.48 g (83%), m.p. 156−158 °C (2-propanol/H2O mixture). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.57–2.99 (m, 4H, 2x COCH₂), 3.39 (t, J = 7.1 Hz, 4H, 2x NCH₂), 6.99–7.40 (m, 10H, H_Ar), 7.82–8.11 (m, 2H, H_Ar), 8.94 (s, 1H, OH), 10.75 and 11.09 (2s, 4H, 4x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 31.6 (CH₂CO), 48.2 (NCH₂), 110.5, 115.3, 115.7, 119.3, 121.6, 124.4, 126.1, 126.2, 132.4, 143.6, 152.2, 164.6 (C_Ar), 174.8, 184.9 (2x C=O). IR (KBr): ν_max (cm⁻¹) = 3220 (OH), 3202 (4x NH), 1682, 1618 (2x C=O). Anal. Calcd. for C₂₈H₂₅N₇O₅, %: C 62.33; H 4.67; N 18.17. Found: C 62.17; H 4.42; N 17.89. HRMS m/z calculated for C₂₈H₂₅N₇O₅ [M+Na]+ : 540.1917; found: 540.1995.

5,5’-(((2-Hydroxyphenyl)azanediyl)bis(ethane-2,1-diyl))bis(1,3,4-oxadiazole-2(3H)-thione) (26): A mixture of dihydrazyde 5 (0.7 g, 2.5 mmol), potassium hydroxide (2.19 g, 39 mmol), carbon disulfide (3.62 g, 47.5 mmol), and 60 mL methanol was refluxed for 24 h, and then the volatile fractions were separated under reduced pressure. The obtained residue was dissolved in water (20 mL), and the solution was acidified with acetic acid to pH 6. The formed solid was filtered off, washed with water, and recrystallized from a mixture of 2-propanol and water. White powder, yield 0.65 g (71%), m.p. 176−178 °C (from 2-propanol). ¹H NMR (400 MHz, DMSO-d₆) δ: 2.79 (t, J = 7.1 Hz, 4H, 2x COCH₂), 3.40 (t, J = 7.1 Hz, 4H, 2x NCH₂), 6.73 (t, J = 7.5 Hz, 1H, H_Ar), 6.79 (d, J = 7.8 Hz, 1H, H_Ar), 6.90 (t, J = 7.5 Hz, 1H, H_Ar), 6.97 (d, J = 7.8 Hz, 1H, H_Ar), 8.97 (s, 1H, OH), 14.24 (br s, 2H, 2x NH); ¹³C NMR (101 MHz, DMSO-d₆) δ: 23.9 (CH₂CO), 48.5 (NCH₂), 115.9, 119.2, 123.4, 124.7, 135.2, 152.3, 162.8 (C_Ar), 177.6 (2x C=S). IR (KBr): ν_max (cm⁻¹) = 3193 (OH), 2966 (2x NH). Anal. Calcd. for C₁₄H₁₅N5O₃S₂, %: C 46.02; H 4.14; N 19.17. Found: C 45.87; H 3.89; N 18.96. HRMS m/z calculated for C₁₄H₁₅N₅O₃S [M+Na]+: 388.0616; found: 388.0508.

X-ray crystallography.

Diffraction data of 24 were collected at 160 K on a Rigaku, XtaLAB Synergy, Dualflex, HyPix diffractometer using monochromatic Cu-Kα radiation (λ = 1.54184 Å). The crystal structure was solved using the direct method and refined with the ShelXLrefinement package using Least Squares minimization. All nonhydrogen atoms were refined in anisotropic approximation. The hydrogen atoms involved in the formation of H-bonds were refined isotopically; all other H-atoms were refined by riding model with Uiso(H) = 1.2Ueq(C). Crystal data: monoclinic, a = 8.42179(6), b = 22.5776(2), c = 8.60767(7) Å, β = 109.3386(8)°; V = 1544.35(2) Å³, Z = 4, μ = 0.770 mm^–1, Dcalc = 1.348 g·cm^–3; space group is P2₁/n. The final R₁ was 0.0344 (I > 2σ(I)) and wR₂ was 0.0909 (all data). For further details, see crystallographic data for this compound deposited at the Cambridge Crystallographic Data Centre. Deposition Number (https://www.ccdc.cam.ac.uk/services/structures) CCDC 2420814 contains the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.

Microbial strains and culture conditions.

The multidrug-resistant Stahphylococcus aureus strain TCH 1516 [USA 300-HOU-MR] and pan-susceptible S. aureus ATCC 25923 was obtained from the American Type Culture Collection (ATCC). Acinetobacter baumannii PKC-1027 and Enterobacter cloacaePKC-0122 are laboratory strains of clinical origin obtained from the Institute of Infectious Diseases and Pathogenic Microbiology collection (Prienai, Lithuania). Enterococcus faecalis AR-0781, Klebsiella pneumoniae AR-0153 and Pseudomonas aeruginosa AR-0054 were obtained from ARisolate bank at CDC Atlanta. Candida albicans AR-0761 and Candida auris AR-0381 were obtained from ARisolate bank at CDC Atlanta. Aspergillus fumigatus CEA10, Cuninghamella bertholiatiae NIH-182, and Rhizopus delamar 1221 were kindly provided by Dr. Vidmantas Petraitis (Weill Cornell Medicine of Cornell University). Candida krusei ATCC 32196 were obtained from ATCC. Prior to the experiments all microbial strains were stored in commercial cryopreservation systems at a temperature of −80 °C. The strains were cultivated on Columbia sheep blood agar for bacterial strains (Becton Dickinson, Franklin Lakes, NJ, USA). Fungal strains were cultured on Sabouraud Dextrose agar (Becton Dickinson, Franklin Lakes, NJ, USA).

Minimal inhibitory concentration determination.

The antimicrobial activity of synthesized compounds or control antibiotics was assessed using the broth microdilution method, following the guidelines outlined by the Clinical Laboratory Standards Institute (CLSI), with modifications [29–31]. The test compounds were dissolved in dimethylsulfoxide (DMSO) to obtain a final concentration of 25–30 mg/mL. Vancomycin hydrochloride and gentamycin sulphate were dissolved in sterile deionized water, while meropenem and cefazolin were dissolved in DMSO (MedChemExpress, Deer Park, United Stated). Dilution series were prepared in deep 96-well microplates (Nunc, Thermo Scientiffic, Waltham, United States) to achieve a two-fold concentration range of 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 µg/mL, utilizing cation-adjusted Mueller–Hinton broth (CAMHB) (Thermo Scientiffic, Waltham, United States) as the growth medium. For Candida or filamentous mold screening, dilutions of test compounds were performed in RPMIMOPS broth (Criterion, Hardy Diagnostics, West McCoy Lane Santa Maria). Amphotericin B, fluconazole (Sigma, St. Louis, United States), posaconazole, and voriconazole (MedChemExpress, Deer Park, United Stated) were dissolved in DMSO and further serially diluted using RPMI/MOPS broth. The microplates containing the dilution series were then inoculated with fresh cultures of each tested organism to reach a final concentration of 5 × 10⁴ CFU (colony-forming units) of the test organism in media containing 1% DMSO and 1 × compound or control antimicrobial concentration, with a volume of 200 µL per well. Wells that were inoculated with media containing 1% DMSO served as positive controls. Subsequently, the microplates were incubated at 35 ± 1 °C for 18 ± 2 h. Following the incubation period, the plates were examined using a manual microplate viewer (Sensititre Manual Viewbox, United States). The minimal inhibitory concentration (MIC) was defined as the lowest concentration (µg/mL) of the tested drug that completely inhibited the growth of the test organism. All experiments were conducted in duplicate with three technical replicates for each condition.

Minimal bactericidal concentration determination.

Following the determination of the minimum inhibitory concentration (MIC) for the test compounds and control antimicrobial agents, 10 µL aliquots were removed from each well of the 96-well microplates and transferred onto Columbia sheep blood agar plates (Becton Dickinson, Franklin Lakes, NJ, USA). The inoculated plates were allowed to dry for 10 minutes under laminar airflow to prevent pooling of the samples. Subsequently, the plates were inverted and incubated at 37 °C for 18hours to allow for microbial growth.

The minimal bactericidal concentration (MBC) was defined as the lowest concentration of the test compound or control antibiotic that resulted in no visible colonies on the agar plate, indicating complete bacterial killing. Each experiment was performed in triplicate.

Time-kill assay.

Prior to the time-kill experiments, E. faecalis AR-0781 and S. aureus TCH-1516 were subcultured on Mueller-Hinton agar to obtain well-separated colonies. One to two colonies were picked and suspended in 5 mL of cation-adjusted Mueller-Hinton broth (CAMHB) and cultured overnight at 37 °C. The overnight cultures were diluted 1:100 in 5 mL of fresh, pre-warmed CAMHB and incubated at 37 °C with shaking at 200 rpm until the OD₆₀₀ reached 0.5. The cultures were then diluted 1:50 in CAMHB containing 0.1% DMSO, which served as the compound-free control, or CAMHB supplemented with 0.1% DMSO and either compound 9 or compound 26 at sub-MIC, MIC and 1X MIC concentrations. The cultures were incubated at 37°C with shaking at 200 rpm, and at 2, 4, 6, and 24-hour time points, a 100 µL aliquot was taken, serially diluted, and plated on sheep blood agar. The plates were incubated overnight at 37°C, and the resulting colonies were counted.

Results

Synthesis of N-substituted β-amino acid derivatives

In the first stage of this work, by using a well-known methodology described in the publication [32], the initial compound 5 were prepared. According to the methodology, the reaction of 2-aminophenol (1) with acrylic acid in water at reflux afforded intermediates 3,3′-((2-hydroxyphenyl)azanediyl)di(propanoic)acid (2) (Scheme 1).

Download:

Scheme 1. Synthesis of compounds 2–6.

https://doi.org/10.1371/journal.pone.0311715.g004

The attempt to synthetize the diamide 3 led to unexpected product. However, the diacid 2 cyclizes under these reaction conditions to the oxadiazepine moiety bearing derivative 3. Compound 3 was prepared by direct coupling of acids 2 with the sulphanilamide using HBTU as the coupling reagent and triethylamine as the base. The reaction were performed in dimethylformamide at room temperature. The product 3 was isolated by the dilution of the reaction mixture with water and were characterised using NMR, HRMS, IR spectroscopy and elemental analysis. Comparison of the ¹H NMR spectra of product 3 with the compound 2 has revealed, that proton singlet of hydroxy group at 8.77 ppm in the spectra of diacid 2 have been dissapered with the formation of the oxazepine fragment. The structure of the obtained compound 3 is also confirmed by the results of the HRMS m/z calculated for C₁₈H₁₉N₃O₅S [M+H]⁺: 390.1045; found: 390.1119.

In continuation of our interest in the chemistry of N-substituted β-amino acids, dimethyl ester 4 was synthesized through esterification of 3,3′-((2-hydroxyphenyl)azanediyl)di(propanoic)acid (2) with an excess of methanol in the presence of a catalytic amount of sulfuric acid. Dihydrazide 5 was obtained through hydrazinolysis of dimethyl ester 4 in propan-2-ol under reflux.

Condensation of dihydrazide 5 with aromatic aldehydes and ketones gave the corresponding hydrazones 7–22. The structures of hydrazones 7–22 have been established mainly on the basis of ¹H and ¹³C NMR spectra (Scheme 2). The restricted rotation around the CONH led to the formation in an isomeric mixture of hydrazones where Z isomer predominates. The obtained hydrazones 7−22 show double sets of resonances for the N=CH and CONH fragment protons with the intensity ratio of 0.3:0.7 (1H NMR). No formation of geometrical isomers wasobserved.

Download:

Scheme 2. Synthesis of compounds 7–22.

https://doi.org/10.1371/journal.pone.0311715.g005

In the next stage of this work, condensation reactions of dihydrazide 5 with various dicarbonyl compounds and carbon disulfite were performed. The reaction of dihydrazide 5 with hexane-2,5-dione, isatin and carbon disulfide resulted in the formation of compounds 23, 25 and 26 of the expected structure, each containing two identical heterocyclic fragments, while in the reaction with acetylacetone, cyclization also took place with the participation of the hydroxy group in the o-position, forming the corresponding 5-(3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropyl)-4,5-dihydrobenzo[b][1,4]oxazepin-2(3H)-one (24) (Scheme 3).

Download:

Scheme 3. Synthesis of compounds 23–26.

https://doi.org/10.1371/journal.pone.0311715.g006

Comparison of the ¹H NMR spectra of product 24 with the compound 5 has revealed, that proton singlet of hydroxy group at 9.01 ppm in the spectra of dihydrazide 5 have been dissapered with the formation of the oxazepine fragment. The structure of the obtained compound 24 is also confirmed by the results of the HRMS – m/z calculated for C₁₇H₁₉N₃O₃ [M+Na]⁺: 336.1426; found: 336.1321 (S1–S50 Figs in S1 File).

X-ray crystallographic study

To confirm the structure of synthesized compound, we performed an X-ray diffraction analysis on compound 24 (Fig 2 and S2–S8 Tables in S1 File). The molecular structure is characterized by fully staggered conformation of -CH₂−CH₂- fragment; the torsion angle of N5−C11−C12−C13 is equal −161.9(1)°. The conformation of the seven-membered cycle is close to the boat; the deviations of the C3, C5a and C9a atoms from the least-square plane of O1, C2, C4, N5 are 0.663(2), 1.008(1) and 0.901(2) Å, respectively. Carbon atoms C4 and C11 connected to nitrogen N5 have increased electronegativity; due to this, weak intermolecular hydrogen bonds of CH···O type are formed in the crystal structure. The parameters of these bonds are follows: C4···O1 = 3.375(2) Å, H···O1 = 2.51(2) Å, C4−H···O1 = 145(1)°; C11···O10 = 3.699(2) Å, H···O10 = 2.74(2) Å, C11−H···O10 = 166(1)°. By means of these bonds, molecular chains are formed in the crystal structure along the crystallographic direction [1 0 1].

Download:

Fig 2. The ORTEP diagram of analysed compound 24 showing the numbering scheme used in this study.

https://doi.org/10.1371/journal.pone.0311715.g002

2-Hydroxyphenyl propanoic acid derivatives shows Gram-positive bacteria-directed activity

After successfully synthesizing and characterizing a series of 2-hydroxyphenyl propanoic acid derivatives (compounds 2–26), we evaluated their in vitro antimicrobial activity by determining minimal inhibitory concentration (MIC) as well as minimal bactericidal concentration (MBC). The compounds were screened using a laboratory strain collection of ESKAPE pathogens, such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. Due to rising antimicrobial resistance among Enterococcus faecalis, we selected a vancomycin resistant isolate and included it in our screening. These pathogens were selected due to their clinical significance and the presence of genetically defined and emerging antimicrobial resistance mechanisms, while the control antibiotics (vancomycin, gentamycin, meropenem, and cefazolin) were selected to represent the antibacterial agents used in the clinical setting to treat infections caused by Gram-negative and Gram-positive pathogens.

Starting compound 2 demonstrated no antimicrobial activity against tested bacterial and fungal strains (Table 1 and S1 Table in S1 File). Oxadiazepine derivative 3 demonstrated moderate antimicrobial activity against Enterococcus faecalis AR-0781 and Staphylococcus aureus TCH-1516, with a minimum inhibitory concentration (MIC) of 64 µg/mL. Dimethyl ester 4 showed no antimicrobial activity against any of the tested bacterial and fungal strains (Table 1 and S1 Table in S1 File). The dihydrazide 5 exhibited activity against S. aureus TCH-1516 (MIC 64 µg/mL) but not against E. faecalis AR-0781 or any tested Gram-negative pathogens. Benzimidazole 6 demonstrated moderate activity against E. faecalis AR-0781 (MIC 64 µg/mL) and weak activity against S. aureus TCH-1516 (MIC 128 µg/mL).

Download:

Table 1. The in vitro antimicrobial activity of N-substituted β-amino acid derivatives 2–26 against panel of multidrug-resistant bacterial strains.

https://doi.org/10.1371/journal.pone.0311715.t001

To characterize the in vitro structure-dependent effects of aromatic substitutions on antimicrobial activity, hydrazide 5 was used as a starting compound. The incorporation of an aryl substituent resulted in compound 7, which showed no antimicrobial activity against all tested bacterial and fungal isolates (MIC > 128 µg/mL) (Table 1 and S1 Table in S1 File). The incorporation of a 2,4-difluorophenyl substituent (compound 8) demonstrated activity against methicillin-resistant S. aureus TCH-1516 (MIC 32 µg/mL) (MRSA) but not vancomycin-resistant E. faecalis AR-0781 (MIC > 128 µg/mL) (VRE). Compound 8 also showed near-MIC range bactericidal activity against S. aureus TCH-1516 (MBC 64 µg/mL) (Table 1 and S2 Table in S1 File). Furthermore, compound 8 showed weak activity against the ESBL-producing E. cloacae PKC-0122 strain (MIC 128 µg/mL), but not other Gram-negative pathogens. In addition, the incorporation of a 4-nitrophenyl substituent (compound 9) greatly enhanced the in vitro antimicrobial activity against E. faecalis AR-0781 (MIC 16 µg/mL) and S. aureus TCH-1516 (MIC 8 µg/mL) with MBC 16 µg/mL respectively, although it resulted in a loss of activity against E. cloacae PKC-0122 (MIC > 128 µg/mL) (S2 Table in S1 File). Furthermore, the addition of a 4-chlorophenyl (compound 10) or 4-(dimethylamino)phenyl (compound 11) substituent resulted in a loss of antimicrobial activity against all tested isolates (MIC > 128 µg/mL). The incorporation of a 4-hydroxyphenyl substituent (compound 12) showed weak activity against E. faecalis AR-0781 (MIC 128 µg/mL) and favorable activity against S. aureus TCH-1516 (MIC 16 µg/mL) with MBC of 32 µg/mL (S2 Table in S1 File). The 3,4,5-trimethoxyphenyl derivative (compound 13) showed no antimicrobial activity, while the addition of a 1-naphthyl substituent (compound 14) resulted in favorable activity against S. aureus TCH-1516 (MIC 8 µg/mL) (Table 1).

To better understand the function of aromatic substitutions on antimicrobial activity, we further generated a series of compounds with heterocyclic substituents (compounds 15–19). Interestingly, compounds bearing heterocyclic substitutions demonstrated activity only against the S. aureus TCH-1516 strain. Compound 15, bearing a 2-furyl substituent, showed no antimicrobial activity (MIC > 128 µg/mL). The incorporation of a 2-thienyl substituent resulted in compound 16 with weak antimicrobial activity against the S. aureus TCH-1516 strain (MIC 64 µg/mL).

The incorporation of a 4-nitro group on the 2-thienyl (compound 17) enhanced antimicrobial bactericidal activity against S. aureus TCH-1516 (MIC and MBC 8 µg/mL respectively), while the replacement of the 4-nitro-2-thienyl with a 5-nitro-2-furyl (compound 18) reduced activity against S. aureus TCH-1516 (MIC 16 µg/mL) (Table 1 and S2 Table in S1 File). Surprisingly, the addition of a 3-thienyl substituent (compound 19) diminished antimicrobial activity against S. aureus TCH-1516 (MIC > 128 µg/mL), demonstrating that the 2-thienyl position is crucial for S. aureus TCH-1516-directed activity. Finally, the methyl group adjacent substitutions resulted in compounds 20–22, with no antimicrobial activity against the tested strains (Table 1).

To characterize the effect of heterocyclic substituents on the in vitro antimicrobial activity, we generated a series of compounds bearing known heterocyclic pharmacophores such as dimethylpyrole, isatin or thiosemicarbazide. Compound 23 bearing two identical dimethylpyrole substitutions demonstrated no antimicrobial activity against tested bacterial strains (MIC > 128 µg/mL). Oxadiazepine derivative 24 bearing dimethyl pyrazole substitution demonstrated weak activity against E. faecalis AR-0781(MIC 128 µg/mL) and favorable activity against S. aureus TCH-1516 (MIC 32 µg/mL). Compound 25 bearing isatin substituent showed activity against both E. faecalis AR-0781(MIC 64 µg/mL) and S. aureus TCH-1516 (MIC 32 µg/mL). Thiosemicarbazide derivative 26 showed potent antimicrobial activity against E. faecalis AR-0781(MIC 8 µg/mL) and S. aureus TCH-1516 (MIC 4 µg/mL) with MBC 16 and 8 µg/mL respectively (Table 1 and S2 Table in S1 File). Interestingly, compound 26 also demonstrated antifungal activity against Candida albicans AR-0671 strain (MIC 16 µg/mL) but not other Candida species or filamentous molds (S1 Table in S1 File).

These results demonstrates that N-substituted β-amino acid derivative 26 could be further explored as starting compound to generate a sub-library of compounds potentially targeting multidrug-resistant Gram-positive pathogens with emerging antimicrobial resistance mechanisms.

Time-kill kinetics and antimicrobial activity of most promising compound 9 and compound 26 against drug-resistant E. faecalis AR-0781 and S. aureus TCH-1516

After identifying the most promising compounds (9 and 26), we aimed to characterize their time-kill kinetics using sub-MIC, MIC, and 1 × MIC concentrations against vancomycin-resistant E. faecalis AR-0781 and methicillin-resistant (MRSA) S. aureus TCH-1516 strains (Fig 3).

Download:

Fig 3. The time-kill kinetics of compounds 9 and 26 against multidrug-resistant E. faecalis AR-0781 (panel A) and S. aureus TCH-1516 (panel B) isolates.

The bacterial strains were exposed to sub-MIC, MIC, and 1 × MIC concentrations of each compound and incubated for 24 hours. At designated time points, the microbial cultures were aliquoted, serially diluted, and plated on sheep blood agar plates. The colonies were counted, and bacterial burden (expressed as log₁₀ CFU/mL) was calculated. The data are presented as the mean ± SD of three experimental replicates.

https://doi.org/10.1371/journal.pone.0311715.g003

Time-kill assays demonstrated that compound 9 exhibits dose-dependent bacteriostatic to bactericidal activity against E. faecalis AR-0781 (Fig 2). At 8 µg/mL and 16 µg/mL, compound 9 initially slowed bacterial growth but did not induce substantial killing. However, at the highest concentration tested (32 µg/mL), a significant reduction in bacterial burden was observed over time, suggesting a bactericidal effect at or above the MIC. In contrast, compound 26 displayed a more potent antimicrobial activity against E. faecalis AR-0781, with bactericidal activity observed at all tested concentrations (4, 8, and 16 µg/mL). The highest concentration (16 µg/mL) resulted in the most rapid and sustained bacterial killing. These results indicate that compound 26 exhibits more potent antimicrobial activity compared to compound 9 against E. faecalis (Fig 3).

In contrast, compound 9 displayed a weaker antimicrobial effect against S. aureus TCH-1516, compared to its activity against E. faecalis AR-0781. At the lowest concentration (4 µg/mL), minimal inhibition was observed, while at 8 and 16 µg/mL, bacterial burden was reduced but not eliminated, indicating that the compound is primarily bacteriostatic at these concentrations. Conversely, compound 26 demonstrated greater activity against S. aureus TCH-1516, with a clear dose-dependent reduction in bacterial burden. The highest tested concentration (8 µg/mL) achieved significant bacterial killing, suggesting that compound 26 shows more potent, Gram-positive bacteria directed activity than compound 9 (Fig 3).

Discussion

In this study we describe the synthesis of novel N-substituted β-amino acid derivatives bearing 2-hydroxyphenyl moieties as promising antimicrobial candidates targeting drug-resistant Gram-positive priority pathogens with genetically defined resistance mechanisms. This study identifies a promising N-substituted β-amino acid-based scaffold with broad Gram-positive pathogens-directed activity for further hit to lead optimization.

The ESKAPE group pathogens, comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, are extremely challenging for their role in hospital-acquired infections and multidrug resistance [2,4,13]. Among these, vancomycin-resistant Enterococcus (VRE), including E. faecalis as well as methicillin-resistant Staphylococcus aureus (MRSA) pose significant clinical challenges due to their extensive resistance to multiple antibiotics, complicating therapeutic strategies and leading to fatal outcomes [10,13,30]. Epidemiologically, VRE and MRSA are highly prevalent in nosocomial environments, contributing to severe infections, increased morbidity and mortality rates, and substantial healthcare expenditures [30]. The persistent difficulties in managing infections caused by these pathogens highlight the critical need for the development of novel antimicrobial agents.

N-substituted β-amino acid derivatives bearing phenolic groups represent a promising class of compounds due to their potential as pharmacophores [25,26,32]. These derivatives exhibit strong hydrogen-bonding capabilities and the ability to potentially form stable interactions with microbial cellular targets, thereby leading to the antimicrobial activity [25,26,32]. The incorporation of phenolic groups confers additional bactericidal properties, while aromatic or heterocyclic substitutions can further augment the binding affinity and specificity of these molecules [25,28,30]. In this study, we successfully synthesized a series of N-substituted β-amino acid derivatives bearing a 2-hydroxyphenyl group. These compounds were designed to represent a range of aromatic and heterocyclic substituents and exhibited a structure-dependent antimicrobial activity, predominantly against multidrug-resistant Gram-positive pathogens. Among the synthesized compounds, compounds 9 (R = 4-nitrophenyl), 17 (R = 5-nitro-2-thienyl), 18 (R = 5-nitro-2-furyl), thiosemicarbazide 16, and 26 demonstrated the most potent activity against S. aureus MRSA USA300 strain TCH-1516, with minimum inhibitory concentration values ranging from 4 to 16 µg/mL. The enhanced antimicrobial activity observed for these compounds can be attributed to the physicochemical properties of the substituents as well as geenral N-substituted β-amino acid or 2-hydroxyphenyl pharmacophores. The nitro groups present in compounds 9, 17, and 18 are strong electron-withdrawing moieties, which increase the electrophilicity of the aromatic ring and may enhance the binding affinity to various bacterial target sites. Furthermore, the heterocyclic rings in compounds 17 (thienyl) and 18 (furyl) provide additional molecular properties for interaction through π-π stacking and hydrogen bonding with bacterial enzymes or membrane components. Compound 26, bearing a thiosemicarbazide group, demonstrated significant antimicrobial activity against vancomycin-resistant E. faecalis AR-0781, with an activity profile comparable to that of control antibiotics.

The thiosemicarbazide moiety, containing sulfur and nitrogen atoms, is potentially involved in the formation of strong intermolecular interactions, including hydrogen bonding and coordination with metal ions, which may contribute to its potent bioactivity. In our previous studies, we successfully synthesized N-substituted β-amino acid derivatives bearing a 3-hydroxyphenyl core, which exhibited notable antimicrobial activity against Gram-positive drug-resistant pathogens as well as drug-resistant Candida species [28]. These findings suggest that the N-substituted β-amino acid scaffold represents a promising platform for the discovery of novel antimicrobial agents. Importantly, the position of the hydroxyl group on the phenolic ring appears to play a critical role in modulating the antimicrobial spectrum [33,34]. Specifically, phenolic groups with hydroxyl substitutions at different positions on the aromatic ring may influence both antibacterial and antifungal activities. Substitutions in the meta-position, as seen in our previous derivatives, seem to enhance activity against Gram-positive bacteria, while the incorporation of hydroxyl groups at other positions could potentially broaden the spectrum to include fungal pathogens. The data presented in this study underscore the significant potential of N-substituted β-amino acid derivatives as novel antimicrobial agents, particularly due to their selective activity against multidrug-resistant Gram-positive pathogens. The structure-activity relationship observed, especially with regard to the position of the phenolic hydroxyl group and the nature of the substituents, highlights the critical role of molecular design in optimizing antimicrobial efficacy. The strong activity exhibited by compounds containing nitro-aromatic and heterocyclic moieties reinforces the importance of electron-withdrawing groups and π-stacking interactions in effectively targeting resistant Gram-positive bacterial strains. The broad-spectrum activity of compound 26 against both vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus suggests that this scaffold could serve as a foundation for further optimization to address other high-priority pathogens, including those identified by the World Health Organization as critical threats. However, despite the promising results, this study has several limitations. First, additional screening is needed against a broader range of bacterial strains with diverse drug-resistant phenotypes to fully assess the efficacy of the most promising compounds. Second, although strong activity was observed against S. aureus and E. faecalis, there remains a gap in our understanding of how these compounds perform against other clinically significant Gram-positive pathogens, such as Streptococcus species or other Staphylococcus strains with other resistance and virulence mechanisms.

Finally, further studies are required to elucidate the specific bacterial targets of these N-substituted β-amino acid derivatives, which will be crucial for further advancing their development as therapeutic candidates for the early pre-clinical screening.

Conclusions

In this study, we report the synthetic pathways for the generation of novel libraries of 3,3’-((2-hydroxyphenyl)azanediyl)dipro-pionic acid derivatives, incorporating functional groups such as ester, hydrazine, hydrazones, benzimidazole, dimethylpyrrole, dimethylpyrazole, and oxadezipine moieties. These compounds demonstrated promising structure-depended antimicrobial activity against multidrug-resistant Gram-positive pathogens, notably methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). Structure-activity relationship (SAR) analysis revealed that electron-withdrawing nitro-aromatic and heterocyclic substituents significantly enhanced antimicrobial efficacy. Compound 26 exhibited broad-spectrum antibacterial and antifungal activity, demonstrating the potential of this scaffold for further hit-to-lead optimization. While these initial results are encouraging, further studies are needed to understand the activity of compound 26 and various compound 26-based derivatives against a broader range of drug-resistant Gram-positive isolates. These findings highlights that N-substituted β-amino acid derivatives could be explored as a promising platform for the development of novel antimicrobial agents targeting predominantly multidrug-resistant Gram-positive pathogens.

Supporting information

S1 File. Figure S1–S49.

¹H and ¹³C NMR spectra of compounds 2 − 26 (in DMSO-d₆). Table S1. The in vitro antimicrobial activity of N-substituted β-amino acid derivatives 2–26 against panel of fungal pathogens.

https://doi.org/10.1371/journal.pone.0311715.s001

(DOCX)

Acknowledgments

We are thankful for the supportive staff of Weill Cornell Medicine of Cornell University and Kaunas University of Technology for their immense technical help and support during this study.

References

1. Septimus EJ. Antimicrobial resistance: an antimicrobial/diagnostic stewardship and infection prevention approach. Med Clin North Am. 2018;102(5):819–29. pmid:30126573
- View Article
- PubMed/NCBI
- Google Scholar
2. Huemer M, Mairpady Shambat S, Brugger SD, Zinkernagel AS. Antibiotic resistance and persistence-Implications for human health and treatment perspectives. EMBO Rep. 2020;21(12):e51034. pmid:33400359
- View Article
- PubMed/NCBI
- Google Scholar
3. Buckel WR, Veillette JJ, Vento TJ, Stenehjem E. Antimicrobial stewardship in community hospitals. Med Clin North Am. 2018;102(5):913–28. pmid:30126580
- View Article
- PubMed/NCBI
- Google Scholar
4. De Oliveira DMP, Forde BM, Kidd TJ, Harris PNA, Schembri MA, Beatson SA, et al. Antimicrobial Resistance in ESKAPE Pathogens. Clin Microbiol Rev. 2020;33(3):e00181-19. pmid:32404435
- View Article
- PubMed/NCBI
- Google Scholar
5. Mulani MS, Kamble EE, Kumkar SN, Tawre MS, Pardesi KR. Emerging strategies to combat ESKAPE pathogens in the era of antimicrobial resistance: a review. Front Microbiol. 2019;10:539. pmid:30988669
- View Article
- PubMed/NCBI
- Google Scholar
6. Martin EM, Colaianne B, Bridge C, Bilderback A, Tanner C, Wagester S, et al. Discontinuing MRSA and VRE contact precautions: defining hospital characteristics and infection prevention practices predicting safe de-escalation. Infect Control Hosp Epidemiol. 2022;43(11):1595–602. pmid:34847970
- View Article
- PubMed/NCBI
- Google Scholar
7. Shenoy ES, Paras ML, Noubary F, Walensky RP, Hooper DC. Natural history of colonization with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE): a systematic review. BMC Infect Dis. 2014;14:177. pmid:24678646
- View Article
- PubMed/NCBI
- Google Scholar
8. Wei MZ, Zhu YY, Zu WB, Wang H, Bai LY, Zhou ZS, et al. Structure optimizing of flavonoids against both MRSA and VRE. Eur J Med Chem. 2024;271:116401. pmid:38640870
- View Article
- PubMed/NCBI
- Google Scholar
9. Ferguson JK, Munnoch SA, Kozierowski K, Chiu S, Oldmeadow C. Reduced VRE and MRSA colonisation and infection following sustained reduction in broad spectrum antibiotic use in a large tertiary hospital. Med J Aust. 2019;211(3):126–7. pmid:31155720
- View Article
- PubMed/NCBI
- Google Scholar
10. Lakhundi S, Zhang K. Methicillin-resistant Staphylococcus aureus: molecular characterization, evolution, and epidemiology. Clin Microbiol Rev. 2018;31(4):e00020-18. pmid:30209034
- View Article
- PubMed/NCBI
- Google Scholar
11. Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, et al. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol. 2019;17(4):203–18. pmid:30737488
- View Article
- PubMed/NCBI
- Google Scholar
12. Chalmers SJ, Wylam ME. Methicillin-resistant Staphylococcus aureus infection and treatment options. Methods Mol Biol. 2020;2069:229–51. pmid:31523777
- View Article
- PubMed/NCBI
- Google Scholar
13. Cimen C, Berends MS, Bathoorn E, Lokate M, Voss A, Friedrich AW, et al. Vancomycin-resistant enterococci (VRE) in hospital settings across European borders: a scoping review comparing the epidemiology in the Netherlands and Germany. Antimicrob Resist Infect Control. 2023;12(1):78. pmid:37568229
- View Article
- PubMed/NCBI
- Google Scholar
14. Reyes K, Bardossy AC, Zervos M. Vancomycin-resistant enterococci: epidemiology, infection prevention, and control. Infect Dis Clin North Am. 2016;30(4):953–65. pmid:27660091
- View Article
- PubMed/NCBI
- Google Scholar
15. Ryan L, O’Mahony E, Wrenn C, FitzGerald S, Fox U, Boyle B, et al. Epidemiology and molecular typing of VRE bloodstream isolates in an Irish tertiary care hospital. J Antimicrob Chemother. 2015;70(10):2718–24. pmid:26142479
- View Article
- PubMed/NCBI
- Google Scholar
16. Peacock SJ, Paterson GK. Mechanisms of methicillin resistance in Staphylococcus aureus. Annu Rev Biochem. 2015;84:577–601. pmid:26034890
- View Article
- PubMed/NCBI
- Google Scholar
17. Nataraj BH, Mallappa RH. Antibiotic resistance crisis: an update on antagonistic interactions between probiotics and methicillin-resistant Staphylococcus aureus (MRSA). Curr Microbiol. 2021;78(6):2194–211. pmid:33881575
- View Article
- PubMed/NCBI
- Google Scholar
18. Yao CJ, Li YL, Pu MJ, Luo LH, Xiong Q, Xie FJ, et al. Aminoglycosides with anti-MRSA activity: a concise review. Curr Top Med Chem. 2021;21(27):2483–99. pmid:34607544
- View Article
- PubMed/NCBI
- Google Scholar
19. Papkou A, Hedge J, Kapel N, Young B, MacLean RC. Efflux pump activity potentiates the evolution of antibiotic resistance across S. aureus isolates. Nat Commun. 2020;11(1):3970. pmid:32769975
- View Article
- PubMed/NCBI
- Google Scholar
20. Miller WR, Murray BE, Rice LB, Arias CA. Resistance in vancomycin-resistant enterococci. Infect Dis Clin North Am. 2020;34(4):751–71. pmid:33131572
- View Article
- PubMed/NCBI
- Google Scholar
21. Bender JK, Cattoir V, Hegstad K, Sadowy E, Coque TM, Westh H, et al. Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature. Drug Resist Updat. 2018;40:25–39. pmid:30447411
- View Article
- PubMed/NCBI
- Google Scholar
22. Perl TM. The threat of vancomycin resistance. Am J Med. 1999;106(5A):26S-37S; discussion 48S-52S. pmid:10348061
- View Article
- PubMed/NCBI
- Google Scholar
23. Hansen T, Ausbacher D, Zachariassen ZG, Anderssen T, Havelkova M, Strøm MB. Anticancer activity of small amphipathic β²,²-amino acid derivatives. Eur J Med Chem. 2012;58:22–9. pmid:23085771
- View Article
- PubMed/NCBI
- Google Scholar
24. Xu J, Kim H, Dong J, Chen H, Xu J, Ma R, et al. Structure-activity relationship studies on O-alkylamino-tethered salicylamide derivatives with various amino acid linkers as potent anticancer agents. Eur J Med Chem. 2022;234:114229. pmid:35334447
- View Article
- PubMed/NCBI
- Google Scholar
25. Hansen T, Ausbacher D, Flaten GE, Havelkova M, Strøm MB. Synthesis of cationic antimicrobial β(2,2)-amino acid derivatives with potential for oral administration. J Med Chem. 2011;54(3):858–68. pmid:21218818
- View Article
- PubMed/NCBI
- Google Scholar
26. López-López LI, Rivera-Ávalos E, Villarreal-Reyes C, Martínez-Gutiérrez F, de Loera D. Synthesis and antimicrobial evaluation of amino acid naphthoquinone derivatives as potential antibacterial agents. Chemotherapy. 2022;67(2):102–9. pmid:34839283
- View Article
- PubMed/NCBI
- Google Scholar
27. Mercuro NJ, Davis SL, Zervos MJ, Herc ES. Combatting resistant enterococcal infections: a pharmacotherapy review. Expert Opin Pharmacother. 2018;19(9):979–92. pmid:29877755
- View Article
- PubMed/NCBI
- Google Scholar
28. Kavaliauskas P, Grybaitė B, Sapijanskaitė-Banevič B, Vaickelionienė R, Petraitis V, Petraitienė R, et al. Synthesis of 3-((4-Hydroxyphenyl)amino)propanoic Acid Derivatives as Promising Scaffolds for the Development of Antimicrobial Candidates Targeting Multidrug-Resistant Bacterial and Fungal Pathogens. Antibiotics (Basel). 2024;13(2):193. pmid:38391579
- View Article
- PubMed/NCBI
- Google Scholar
29. Petraitis V, Petraitiene R, Kavaliauskas P, Naing E, Garcia A, Sutherland C, et al. Pharmacokinetics, tissue distribution, and efficacy of VIO-001 (meropenem/piperacillin/tazobactam) for treatment of methicillin-resistant Staphylococcus aureus bacteremia in immunocompetent rabbits with chronic indwelling vascular catheters. Antimicrob Agents Chemother. 2021;65(11):e0116821. pmid:34460301
- View Article
- PubMed/NCBI
- Google Scholar
30. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. CLSI document M07-A8. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
31. Kavaliauskas P, Grybaitė B, Sapijanskaite-Banevič B, Anusevičius K, Jonuškienė I, Stankevičienė R, et al. Identification of 3-((4-hydroxyphenyl)amino)propanoic acid derivatives as anticancer candidates with promising antioxidant properties. Molecules. 2024;29(13):3125. pmid:38999077
- View Article
- PubMed/NCBI
- Google Scholar
32. Žukauskas M, Grybaitė B, Jonutė P, Vaickelionienė R, Gibieža P, Vaickelionis G, et al. Evaluation of N-aryl-β-alanine derivatives as anticancer agents in triple-negative breast cancer and glioblastoma in vitro models. Bioorg Chem. 2021;115:105214. pmid:34426161
- View Article
- PubMed/NCBI
- Google Scholar
33. Bąchor U, Mączyński M. Selected β2-, β3- and β2,3-amino acid heterocyclic derivatives and their biological perspective. Molecules. 2021;26(2):438. pmid:33467741
- View Article
- PubMed/NCBI
- Google Scholar
34. Kumar A, Singh AK, Singh H, Vijayan V, Kumar D, Naik J, et al. Nitrogen containing heterocycles as anticancer agents: a medicinal chemistry perspective. Pharmaceuticals (Basel). 2023;16(2):299. pmid:37259442
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Septimus EJ. Antimicrobial resistance: an antimicrobial/diagnostic stewardship and infection prevention approach. Med Clin North Am. 2018;102(5):819–29. pmid:30126573
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Huemer M, Mairpady Shambat S, Brugger SD, Zinkernagel AS. Antibiotic resistance and persistence-Implications for human health and treatment perspectives. EMBO Rep. 2020;21(12):e51034. pmid:33400359
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Buckel WR, Veillette JJ, Vento TJ, Stenehjem E. Antimicrobial stewardship in community hospitals. Med Clin North Am. 2018;102(5):913–28. pmid:30126580
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. De Oliveira DMP, Forde BM, Kidd TJ, Harris PNA, Schembri MA, Beatson SA, et al. Antimicrobial Resistance in ESKAPE Pathogens. Clin Microbiol Rev. 2020;33(3):e00181-19. pmid:32404435
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Mulani MS, Kamble EE, Kumkar SN, Tawre MS, Pardesi KR. Emerging strategies to combat ESKAPE pathogens in the era of antimicrobial resistance: a review. Front Microbiol. 2019;10:539. pmid:30988669
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Martin EM, Colaianne B, Bridge C, Bilderback A, Tanner C, Wagester S, et al. Discontinuing MRSA and VRE contact precautions: defining hospital characteristics and infection prevention practices predicting safe de-escalation. Infect Control Hosp Epidemiol. 2022;43(11):1595–602. pmid:34847970
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Shenoy ES, Paras ML, Noubary F, Walensky RP, Hooper DC. Natural history of colonization with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE): a systematic review. BMC Infect Dis. 2014;14:177. pmid:24678646
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Wei MZ, Zhu YY, Zu WB, Wang H, Bai LY, Zhou ZS, et al. Structure optimizing of flavonoids against both MRSA and VRE. Eur J Med Chem. 2024;271:116401. pmid:38640870
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Ferguson JK, Munnoch SA, Kozierowski K, Chiu S, Oldmeadow C. Reduced VRE and MRSA colonisation and infection following sustained reduction in broad spectrum antibiotic use in a large tertiary hospital. Med J Aust. 2019;211(3):126–7. pmid:31155720
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Lakhundi S, Zhang K. Methicillin-resistant Staphylococcus aureus: molecular characterization, evolution, and epidemiology. Clin Microbiol Rev. 2018;31(4):e00020-18. pmid:30209034
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, et al. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol. 2019;17(4):203–18. pmid:30737488
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Chalmers SJ, Wylam ME. Methicillin-resistant Staphylococcus aureus infection and treatment options. Methods Mol Biol. 2020;2069:229–51. pmid:31523777
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Cimen C, Berends MS, Bathoorn E, Lokate M, Voss A, Friedrich AW, et al. Vancomycin-resistant enterococci (VRE) in hospital settings across European borders: a scoping review comparing the epidemiology in the Netherlands and Germany. Antimicrob Resist Infect Control. 2023;12(1):78. pmid:37568229
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Reyes K, Bardossy AC, Zervos M. Vancomycin-resistant enterococci: epidemiology, infection prevention, and control. Infect Dis Clin North Am. 2016;30(4):953–65. pmid:27660091
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Ryan L, O’Mahony E, Wrenn C, FitzGerald S, Fox U, Boyle B, et al. Epidemiology and molecular typing of VRE bloodstream isolates in an Irish tertiary care hospital. J Antimicrob Chemother. 2015;70(10):2718–24. pmid:26142479
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Peacock SJ, Paterson GK. Mechanisms of methicillin resistance in Staphylococcus aureus. Annu Rev Biochem. 2015;84:577–601. pmid:26034890
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Nataraj BH, Mallappa RH. Antibiotic resistance crisis: an update on antagonistic interactions between probiotics and methicillin-resistant Staphylococcus aureus (MRSA). Curr Microbiol. 2021;78(6):2194–211. pmid:33881575
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Yao CJ, Li YL, Pu MJ, Luo LH, Xiong Q, Xie FJ, et al. Aminoglycosides with anti-MRSA activity: a concise review. Curr Top Med Chem. 2021;21(27):2483–99. pmid:34607544
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Papkou A, Hedge J, Kapel N, Young B, MacLean RC. Efflux pump activity potentiates the evolution of antibiotic resistance across S. aureus isolates. Nat Commun. 2020;11(1):3970. pmid:32769975
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Miller WR, Murray BE, Rice LB, Arias CA. Resistance in vancomycin-resistant enterococci. Infect Dis Clin North Am. 2020;34(4):751–71. pmid:33131572
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Bender JK, Cattoir V, Hegstad K, Sadowy E, Coque TM, Westh H, et al. Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature. Drug Resist Updat. 2018;40:25–39. pmid:30447411
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Perl TM. The threat of vancomycin resistance. Am J Med. 1999;106(5A):26S-37S; discussion 48S-52S. pmid:10348061
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Hansen T, Ausbacher D, Zachariassen ZG, Anderssen T, Havelkova M, Strøm MB. Anticancer activity of small amphipathic β²,²-amino acid derivatives. Eur J Med Chem. 2012;58:22–9. pmid:23085771
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Xu J, Kim H, Dong J, Chen H, Xu J, Ma R, et al. Structure-activity relationship studies on O-alkylamino-tethered salicylamide derivatives with various amino acid linkers as potent anticancer agents. Eur J Med Chem. 2022;234:114229. pmid:35334447
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Hansen T, Ausbacher D, Flaten GE, Havelkova M, Strøm MB. Synthesis of cationic antimicrobial β(2,2)-amino acid derivatives with potential for oral administration. J Med Chem. 2011;54(3):858–68. pmid:21218818
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. López-López LI, Rivera-Ávalos E, Villarreal-Reyes C, Martínez-Gutiérrez F, de Loera D. Synthesis and antimicrobial evaluation of amino acid naphthoquinone derivatives as potential antibacterial agents. Chemotherapy. 2022;67(2):102–9. pmid:34839283
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Mercuro NJ, Davis SL, Zervos MJ, Herc ES. Combatting resistant enterococcal infections: a pharmacotherapy review. Expert Opin Pharmacother. 2018;19(9):979–92. pmid:29877755
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Kavaliauskas P, Grybaitė B, Sapijanskaitė-Banevič B, Vaickelionienė R, Petraitis V, Petraitienė R, et al. Synthesis of 3-((4-Hydroxyphenyl)amino)propanoic Acid Derivatives as Promising Scaffolds for the Development of Antimicrobial Candidates Targeting Multidrug-Resistant Bacterial and Fungal Pathogens. Antibiotics (Basel). 2024;13(2):193. pmid:38391579
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Petraitis V, Petraitiene R, Kavaliauskas P, Naing E, Garcia A, Sutherland C, et al. Pharmacokinetics, tissue distribution, and efficacy of VIO-001 (meropenem/piperacillin/tazobactam) for treatment of methicillin-resistant Staphylococcus aureus bacteremia in immunocompetent rabbits with chronic indwelling vascular catheters. Antimicrob Agents Chemother. 2021;65(11):e0116821. pmid:34460301
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. CLSI document M07-A8. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.

[ref31] 31. Kavaliauskas P, Grybaitė B, Sapijanskaite-Banevič B, Anusevičius K, Jonuškienė I, Stankevičienė R, et al. Identification of 3-((4-hydroxyphenyl)amino)propanoic acid derivatives as anticancer candidates with promising antioxidant properties. Molecules. 2024;29(13):3125. pmid:38999077
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref32] 32. Žukauskas M, Grybaitė B, Jonutė P, Vaickelionienė R, Gibieža P, Vaickelionis G, et al. Evaluation of N-aryl-β-alanine derivatives as anticancer agents in triple-negative breast cancer and glioblastoma in vitro models. Bioorg Chem. 2021;115:105214. pmid:34426161
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref33] 33. Bąchor U, Mączyński M. Selected β2-, β3- and β2,3-amino acid heterocyclic derivatives and their biological perspective. Molecules. 2021;26(2):438. pmid:33467741
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref34] 34. Kumar A, Singh AK, Singh H, Vijayan V, Kumar D, Naik J, et al. Nitrogen containing heterocycles as anticancer agents: a medicinal chemistry perspective. Pharmaceuticals (Basel). 2023;16(2):299. pmid:37259442
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Chemical synthesis

General analytical procedures.

General procedures for preparation of compounds 2–26.

X-ray crystallography.

Microbial strains and culture conditions.

Minimal inhibitory concentration determination.

Minimal bactericidal concentration determination.

Time-kill assay.

Results

Synthesis of N-substituted β-amino acid derivatives

X-ray crystallographic study

2-Hydroxyphenyl propanoic acid derivatives shows Gram-positive bacteria-directed activity

Time-kill kinetics and antimicrobial activity of most promising compound 9 and compound 26 against drug-resistant E. faecalis AR-0781 and S. aureus TCH-1516

Discussion

Conclusions

Supporting information

S1 File. Figure S1–S49.

Acknowledgments

References