New 2-(Quinolin-4-yloxy)acetamides: Synthesis, Antitubercular Evaluation, and Structure-Activity Relationships

Abstract

Tuberculosis remains a significant global health concern, particularly due to the increasing prevalence of drug-resistant strains. In this work, a series of 2-(quinoline-4-yloxy)acetamides was synthesized and evaluated for antimycobacterial activity, aiming to advance the understanding of structure-activity relationships (SAR) within this scaffold. Variations at the quinoline core and the aryl moiety of the acetamide side chain resulted in distinct biological profiles, with SAR analysis revealing that small to moderate hydrophobic substituents at the 4-position of the aryl ring enhanced potency, while bulky or strongly electron-withdrawing groups reduced activity, likely due to steric or electronic effects. Notably, the lead compound exhibited a minimum inhibitory concentration of 0.80 µM against Mycobacterium tuberculosis H37Rv, representing more than a 2.8-fold increase in potency compared to isoniazid under identical experimental conditions. Furthermore, this molecule demonstrated no detectable cytotoxicity in HepG2 (human caucasian hepatocytes carcinoma) and Vero (African green monkey kidney) cell lines up to 20 µM, resulting in selectivity indices exceeding 25, and exhibited favorable solubility under acidic conditions (156 µM at pH 1.2). These findings indicate that the 2-(quinoline-4-yloxy)acetamide scaffold holds promise for further development and support the rational design of new candidates for the treatment of tuberculosis, potentially contributing to strategies aimed at overcoming the limitations of current therapies.

Keywords:
Mycobacterium tuberculosis ; quinoline; anti-TB; medicinal chemistry

Introduction

Tuberculosis (TB) is a communicable disease caused by Mycobacterium tuberculosis (Mtb), which continues to pose a major global health challenge. Despite the availability of effective treatments, TB is the leading cause of death from a single infectious agent, surpassing human immunodeficiency virus (HIV), and continues to rank among the 15 leading causes of death globally.¹ According to the World Health Organization (WHO), in 2023, approximately 8.2 million individuals developed TB, and 1.25 million died from the disease, reflecting the persistent burden TB imposes on public health systems globally.¹

A significant barrier to TB control is the emergence and spread of drug-resistant strains, particularly multidrug-resistant TB (MDR-TB) and rifampicin-resistant TB (RR-TB). These forms are defined by resistance to at least isoniazid (INH) and rifampicin (RIF), the two most potent first-line antitubercular drugs.² Drug resistance in TB represents a critical public health threat due to the complexity of managing affected patients and the limited availability of effective therapeutic options.²,³ Treatment of MDR-TB relies on second-line regimens that are less effective, substantially more toxic, and significantly more expensive than standard first-line therapies.³ The typical duration of MDR-TB treatment ranges from 9 to 20 months, often resulting in poor patient adherence, higher rates of adverse drug reactions, and disappointing treatment outcomes.⁴ Consequently, the global treatment success rate for MDR-TB remains approximately 68%, compared to over 88% for drug-susceptible TB.¹ The magnitude of the MDR-TB crisis is considerable. In 2023 alone, an estimated 400,000 new cases of MDR/RR-TB were reported worldwide, with the highest burdens concentrated in Eastern Europe, Central Asia, Africa, and Southeast Asia.¹,⁵ These regions frequently face substantial challenges, including fragile healthcare infrastructure, limited access to quality-assured second-line drugs, and insufficient laboratory capacity for accurate diagnosis and treatment monitoring.⁶ Despite recent advances in diagnostics, treatment regimens, and public health initiatives, the current tools remain insufficient to fully contain the TB epidemic, particularly in the context of drug-resistant strains. Achieving the targets set by the United Nations and the WHO to end TB by 2030 will require transformative innovations in drug discovery, development of shorter and safer treatment regimens, and more efficient strategies for disease management.⁷ Addressing this challenge demands an integrated research effort aimed at identifying novel therapeutic agents, improving the pharmacological profile of existing treatments, and developing innovative drug delivery systems capable of enhancing treatment efficacy and patient adherence.

In recent years, the development of novel pharmacological regimens has significantly advanced the treatment of MDR-TB. Notably, the BPaL regimen-comprising bedaquiline, pretomanid, and linezolid-and its variant BPaLM, which includes moxifloxacin, have demonstrated high efficacy, achieving cure rates exceeding 85% with treatment durations as short as six months.⁸-¹⁰ However, these regimens are associated with some limitations. Linezolid, a key component, is associated with serious adverse effects such as peripheral neuropathy, myelosuppression, and optic neuritis, often requiring dose adjustments or early discontinuation.⁸,¹⁰ Bedaquiline has been linked to increased of the total time from ventricular depolarization to complete repolarization (QT interval), raising concerns about cardiac safety.⁹,¹⁰ Additionally, pretomanid has been associated with hepatotoxicity and gastrointestinal disturbances.⁹ These safety concerns, combined with restricted availability and high costs in several regions, highlight the urgent need for the discovery of new therapeutic agents with improved efficacy, reduced toxicity, and simplified administration.¹¹

Within this context, our research group has significantly advanced the development of 2-(quinolin-4-yloxy)acetamides (QOAs) as promising antitubercular agents (Figure 1).¹²-¹⁵ These molecules were initially identified through phenotypic screening and are part of a set of 177 lead compounds discovered in an open-source drug discovery initiative targeting tuberculosis (Figure 1).¹⁶ Subsequent studies demonstrated that QOAs exhibit potent in vitro activity against both drug-susceptible and multidrug-resistant Mtb strains, with minimum inhibitory concentrations (MICs) observed in the submicromolar range.¹² These compounds also showed synergistic effects when combined with rifampicin and displayed significant activities in macrophage infection models.¹³ In addition, whole-genome sequencing of spontaneous resistant mutants revealed mutations in the qcrB gene, indicating that QOAs target the cytochrome bc₁ complex, a critical component of the mycobacterial respiratory chain.¹⁷ Further structure-activity relationship (SAR) studies led to the design of simplified 4-alkoxyquinoline derivatives with enhanced pharmacokinetic properties, including improved chemical stability and in vivo exposure, while maintaining potent antimycobacterial activity.¹⁸ These findings underscore the potential of QOAs as a promising chemical class in tuberculosis drug discovery.

Figure 1
Chemical structures of selected 2-(quinolin-4-yloxy)acetamide derivatives previously reported in the literature and their respective minimum inhibitory concentrations (MICs) against M. tuberculosis H37Rv strain.

Given the promising results previously reported for 2-(quinolin-4-yloxy)acetamides, this work aims to further expand the understanding of the SAR within this chemical series. Therefore, a new set of substituted QOAs was designed and synthesized, with the goal of generate additional SAR information that could guide future optimization efforts. Initially, antibacterial potency was assessed by determining the MICs against the reference strain M. tuberculosis H37Rv. Additionally, the most active compounds were evaluated for cytotoxicity in HepG2 (human hepatocellular carcinoma) and Vero (African green monkey kidney) cell lines, as well as for aqueous solubility under physiologically relevant pH conditions.

Experimental

All reagents and solvents were purchased from commercial suppliers and used without further purification. Ethanol, chloroform, diethyl ether, hexane, ethyl acetate, and dichloromethane were obtained from Química Moderna (Barueri, Brazil). Silica gel 60 (particle size 0.063-0.200 mm), ethyl acetoacetate, bromoacetyl chloride, DMSO (dimethyl sulfoxide, molecular biology grade), and DMSO-d₆ were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Dimethylformamide (DMF), glacial acetic acid, acetonitrile (high-performance liquid chromatography (HPLC) grade), and methanol (HPLC grade) were supplied by Merck KGaA (Darmstadt, Germany). Potassium carbonate was purchased from Acros Organics (Geel, Belgium), and anhydrous magnesium sulfate from LabSynth (Diadema, Brazil). Sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na₂HPO₄), and potassium dihydrogen phosphate (KH₂PO₄) for phosphate-buffered saline (PBS) preparation were also obtained from Sigma-Aldrich (Saint Louis, MO, USA). Melting points were determined using a Microquímica MQAPF-302 apparatus (Palhoça, Brazil). ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on an Avance III HD spectrometer (Bruker Corporation, Fällanden, Switzerland). Chemical shifts (δ) were reported in parts per million (ppm) relative to the residual solvent signal from DMSO-d₆ or referenced to tetramethylsilane (TMS) as an internal standard. NMR spectra were processed with MestReNova software, version 14.0.0-2329 (Mestrelab Research S.L.). High-resolution mass spectrometry (HRMS) analyses were performed using an LTQ Orbitrap Discovery mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray ionization (ESI) source operating in positive-ion mode. Samples were directly infused in a MeOH/MeCN (1:1, v/v) solution containing 0.1% formic acid at a flow rate of 10 μL min^-1. Elemental composition was determined using the Qual Browser module from the Xcalibur software package (Thermo Fisher Scientific, Bremen, Germany). Finaly, compound purity was assessed by ultra-high-performance liquid chromatography (UHPLC) using a Dionex Ultimate 3000 system (Germering, Germany) equipped with a reverse-phase Nucleodur C18ec column (5 μm, 250 × 4.6 mm). The mobile phase consisted of 100% water with 0.1% acetic acid (isocratic from 0 to 7 min), followed by a linear gradient to 90% acetonitrile/methanol (1:1, v/v) from 7 to 15 min, which was maintained until 30 min. The system was then returned to 100% water with 0.1% acetic acid over 5 min (30-35 min) and held for an additional 10 min (35-45 min). The flow rate was 1.5 mL min^-1, and detection was performed at 254 nm. Data processing and calculations were carried out using Chromeleon 6.80 SR11 software (Build 3160). Graphical abstract layout was supported by ChatGPT (OpenAI). Finally, all evaluated compounds exhibited purity ≥ 95%.

General procedure for the synthesis of 2-(quinolin-4-yloxy)acetamides 6

The appropriate 2-bromoacetamide (0.33 mmol, 1.0 equiv) was added to a solution of 4-hydroxyquinoline (0.33 mmol, 1.0 equiv) and potassium carbonate (K₂CO₃, 1.03 mmol, 3.12 equiv) in dry DMF (6 mL). The reaction mixture was stirred at room temperature (25 °C) for 18 h. After completion, the reaction was quenched by dilution with distilled water (10 mL), resulting in the precipitation of the crude product. The solid was isolated by centrifugation (18,000 rpm, 4 °C, 10 min), washed with water (3 × 15 mL), and dried under reduced pressure to afford the corresponding products, typically in high purity. When further purification was required, the crude products were either washed with diethyl ether or purified by column chromatography on silica gel using hexane/ethyl acetate as the eluent.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(o-tolyl)acetamide (6a)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 47%; mp 203-205 °C; t_R 14.7 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.23 (s, 3H, CH₃), 2.61 (s, 3H, CH₃), 5.02 (s, 2H, O-CH₂), 7.01 (s, 1H, Ar-H), 7.10-7.29 (m, 3H, Ar-H ), 7.42 (dd, 1H, J 7.8, 1.4 Hz, Ar-H), 7.71 (dd, 1H, J 9.0, 2.4 Hz, Ar-H), 7.88 (d, 1H, J 8.9 Hz, Ar-H), 8.34 (d, 1H J 2.5 Hz, Ar-H), 9.69 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 17.7, 25.3, 67.3, 103.0, 119.9, 121.0, 125.7, 125.9, 126.0, 129.4, 130.0, 130.2, 130.3, 132.6, 135.4, 146.8, 159.2, 160.5, 165.5; HRMS (ESI) m/z, calcd. for C₁₉H₁₈ClN₂O₂ [M + H]⁺: 341.1051, found: 341.1057.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(p-tolyl)acetamide (6b)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); light yellow solid; yield 45%; mp 218-220 °C; t_R 15.0 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.27 (s, 3H, CH₃), 2.59 (s, 3H, CH₃), 5.00 (s, 2H, O-CH₂), 6.97 (s, 1H, Ar-H), 7.09-7.20 (m, 2H, Ar-H), 7.48-7.57 (m, 2H, Ar-H), 7.71 (dd, 1H, J 9.0, 2.5 Hz, Ar-H), 7.88 (d, , 1H, J 9.0 Hz, Ar-H), 8.24 (d, 1H, J 2.5 Hz, Ar-H), 10.17 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 20.4, 25.3, 67.3, 102.9, 120.0 (2C), 120.8, 129.1 (2C), 129.3, 130.0, 130.1, 132.8, 135.7, 146.8, 159.5, 160.5, 165.1; HRMS (ESI) m/z, calcd. for C₁₉H₁₈ClN₂O₂ [M + H]⁺: 341.1051, found: 341.1056.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(3,4-dimethylphenyl)acetamide (6c)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 47%; mp 212-214 °C; t_R 15.7 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.17 (s, 3H, CH₃), 2.19 (s, 3H, CH₃), 2.59 (s, 3H, CH₃), 4.99 (s, 2H, O-CH₂), 6.97 (s, 1H, Ar-H), 7.08 (d, 1H, J 8.2 Hz, Ar-H), 7.35 (dd, 1H, J 8.1, 2.3 Hz, Ar-H), 7.42 (d, 1H J 2.2 Hz, Ar-H), 7.71 (dd, 1H, J 9.0, 2.5 Hz, Ar-H), 7.88 (d,1H, J 8.9 Hz, Ar-H), 8.23 (d,1H, J 2.5 Hz, Ar-H), 10.10 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 18.7, 19.5, 25.3, 67.3, 102.9, 117.5, 120.0, 120.8, 121.1, 129.3, 129.5, 130.0, 130.1, 131.6, 135.9, 136.3, 146.8, 159.5, 160.5, 165.1; HRMS (ESI) m/z, calcd. for C₂₀H₂₀ClN₂O₂ [M + H]⁺: 355.1208, found: 355.1209.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(4-ethylphenyl)acetamide (6d)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); light yellow solid; yield 49%; mp 197-199 °C; t_R 15.9 min; ¹H NMR (400 MHz, DMSO-d₆) δ 1.16 (t, 3H, J 7.6 Hz, CH₃), 2.51-2.60 (m, 2H, CH₂), 2.60 (s, 3H, CH₃), 5.01 (s, 2H, O-CH₂), 6.97 (s, 1H, Ar-H), 7.14-7.21 (m, 2H, Ar-H), 7.52-7.59 (m, 2H, Ar-H), 7.72 (dd, 1H, J 9.0, 2.5 Hz, Ar-H), 7.89 (d, 1H, J 9.0 Hz, Ar-H), 8.24 (d, 1H, J 2.5 Hz, Ar-H), 10.18 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 15.6, 25.3, 27.6, 67.3, 102.9, 120.0, 120.1 (2C), 120.8, 127.9 (2C), 129.4, 130.0, 130.2, 135.9, 139.3, 146.8, 159.5, 160.5, 165.1; HRMS (ESI) m/z, calcd. for C₂₀H₂₀ClN₂O₂ [M + H]⁺: 355.1208, found: 355.1207.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(4-propylphenyl)acetamide (6e)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); light yellow solid; yield 62%; mp 189-191 °C; t_R 16.7 min; ¹H NMR (400 MHz, DMSO-d₆) δ 0.88 (t, J 7.3 Hz, 3H, CH₃), 1.56 (h, 2H, J 7.4 Hz, CH₂), 2.48-2.55 (m, 2H, CH₂), 2.60 (s, 3H, CH₃), 5.01 (s, 2H, O-CH₂), 6.98 (s, 1H, Ar-H), 7.15 (d, 2H, J 8.4 Hz, Ar-H), 7.51-7.57 (m, 2H, Ar-H), 7.72 (dd, 1H, J 8.9, 2.5 Hz, Ar-H), 7.89 (d, 1H, J 8.9 Hz, Ar-H), 8.24 (d, 1H, J 2.5 Hz, Ar-H), 10.18 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 13.5, 24.1, 25.3, 36.6, 67.3, 102.9, 120.0 (2C), 120.8, 128.5 (2C), 129.4, 130.1, 130.2, 135.9, 137.6, 146.8, 159.5, 160.5, 165.1; HRMS (ESI) m/z, calcd. for C₂₁H₂₂ClN₂O₂ [M + H]⁺: 369.1364, found: 369.1364.

N-(4-Butylphenyl)-2-((6-chloro-2-methylquinolin-4-yl)oxy)acetamide (6f)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); light yellow solid; yield 27%; mp 174-176 °C; t_R 17.6 min; ¹H NMR (400 MHz, DMSO-d₆) δ 0.88 (t, 3H, J 7.3 Hz, CH₃), 1.29 (h, 2H, J 7.2 Hz, CH₂), 1.52 (p, 2H, J 7.4 Hz, CH₂), 2.53 (t, 2H, J 6.6 Hz, CH₂), 2.60 (s, 3H, CH₂), 5.01 (s, 2H, O-CH₂), 6.97 (s, 1H, Ar-H), 7.15 (d, 2H, J 8.2 Hz, Ar-H), 7.50-7.58 (m, 2H, Ar-H), 7.72 (dd, 1H, J 9.0, 2.5 Hz, Ar-H), 7.89 (d, 1H, J 9.0 Hz, Ar-H), 8.24 (d, 1H, J 2.5 Hz, Ar-H), 10.18 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 13.7, 21.6, 25.3, 33.1, 34.2, 67.3, 102.9, 120.0, 120.0 (2C), 120.8, 128.4 (2C), 129.4, 130.0, 130.2, 135.9, 137.9, 146.8, 159.5, 160.5, 165.1; HRMS (ESI) m/z, calcd. for C₂₂H₂₄ClN₂O₂ [M + H]⁺: 383.1521, found: 383.1523.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(3-chlorophenyl)acetamide (6g)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 63%; mp 220-222 °C; t_R 15.6 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.60 (s, 3H, CH₃), 5.04 (s, 2H, O-CH₂), 6.99 (s, 1H, Ar-H), 7.13-7.20 (m, 1H, Ar-H), 7.33-7.42 (m, 1H, Ar-H), 7.50-7.56 (m, 1H, Ar-H), 7.68-7.76 (m, 1H, Ar-H), 7.82-7.86 (m, 1H, Ar-H), 7.89 (dd,1H, J 9.0, 2.2 Hz, Ar-H), 8.23 (d, 1H, J 2.4 Hz, Ar-H), 10.43 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.2, 103.0, 118.2, 119.3, 119.9, 120.8, 123.5, 129.4, 130.0, 130.2, 130.4, 133.1, 139.7, 146.7, 159.4, 160.5, 165.8; HRMS (ESI) m/z, calcd. for C₁₈H₁₅Cl₂N₂O₂ [M + H]⁺: 361.0505, found: 361.0510.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(4-chlorophenyl)acetamide (6h)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 64%; mp 231-232 °C; t_R 15.3 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.59 (s, 3H, CH₃), 5.03 (s, 2H, O-CH₂), 6.98 (s, 1H, Ar-H), 7.36-7.43 (m, 2H, Ar-H), 7.64-7.75 (m, 4H, Ar-H), 7.88 (dd, 1H, J 8.9, 2.1 Hz, Ar-H), 8.23 (s, 1H, Ar-H), 10.36 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.2, 102.9, 119.9, 120.7, 121.4 (2C), 127.4, 128.6 (2C), 129.3, 130.0, 130.1, 137.1, 146.8, 159.4, 160.5, 165.5; HRMS (ESI) m/z, calcd. for C₁₈H₁₅Cl₂N₂O₂ [M + H]⁺: 361.0505, found: 361.0509.

N-(4-Bromophenyl)-2-((6-chloro-2-methylquinolin-4-yl)oxy)acetamide (6i)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 27%; mp 283-285 °C; t_R 15.7 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.59 (s, 3H, CH₃), 5.03 (s, 2H, O-CH₂), 6.98 (s, 1H, Ar-H), 7.53 (d, 2H, J 8.5 Hz, Ar-H), 7.62 (d, 2H, J 8.4 Hz, Ar-H), 7.68-7.77 (m, 1H, Ar-H), 7.88 (d,1H, J 9.0 Hz, Ar-H), 8.20-8.28 (m, 1H, Ar-H), 10.37 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.2, 103.0, 115.5, 119.9, 120.8, 121.8 (2C), 129.4, 130.1, 130.2, 131.6 (2C), 137.6, 146.8, 159.4, 160.5, 165.6; HRMS (ESI) m/z, calcd. for C₁₈H₁₅BrClN₂O₂ [M + H]⁺: 405.0000, found: 405.0003.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(4-(trifluoro methyl)phenyl)acetamide (6j)

Column chromatography on silica gel (hexane/ethyl acetate, 8:2); light yellow solid; yield 59%; mp 253-255 °C; t_R 16.00 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.59 (s, 3H, CH₃), 5.07 (s, 2H, O-CH₂), 6.99 (s, 1H, Ar-H), 7.65-7.78 (m, 3H, Ar-H), 7.82-7.93 (m, 3H, Ar-H), 8.21 (d, 1H, J 2.5 Hz, Ar-H), 10.61 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.2, 103.0, 119.7, 119.9 (2C), 120.7, 123.8 (q, J 32.2 Hz), 124.3 (q, J 271.4 Hz), 126.1 (q, J 3.7 Hz), 129.4, 130.1, 130.2, 141.9, 146.8, 159.4, 160.5, 166.1; HRMS (ESI) m/z, calcd. for C₁₉H₁₅ClF₃N₂O₂ [M + H]⁺: 395.0769, found: 395.0773.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(naphthalen-1-yl)acetamide (6k)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 42%; mp 232-234 °C; t_R 15.6 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.64 (s, 3H, CH₃), 5.18 (s, 2H, O-CH₂), 7.10 (s, 1H, Ar-H), 7.49-7.60 (m, 3H, Ar-H), 7.65-7.77 (m, 2H, Ar-H), 7.84 (d, 1H, J 8.2 Hz, Ar-H), 7.90 (d, 1H, J 9.0 Hz, Ar-H), 7.93-8.00 (m, 1H, Ar-H), 8.06 (dd,1H, J 6.2, 3.5 Hz, Ar-H), 8.40 (d, 1H, J 2.5 Hz, Ar-H), 10.32 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.4, 67.5, 103.1, 120.0, 121.0, 122.8, 122.9, 125.5, 125.9, 126.1, 126.1 (2C), 128.1, 128.4, 129.4, 130.0, 130.2, 132.8, 133.7, 146.8, 159.4, 160.5, 166.4; HRMS (ESI) m/z, calcd. for C₂₂H₁₈ClN₂O₂ [M + H]⁺: 377.1051, found: 377.1035.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(naphthalen-2-yl)acetamide (6l)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); light yellow solid; yield 46%; mp 251-252 °C; t_R 15.9 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.60 (s, 3H, CH₃), 5.10 (s, 2H, O-CH₂), 7.03 (s, 1H, Ar-H), 7.45 (dt, 2H, J 23.8, 7.2 Hz, Ar-H), 7.63-7.78 (m, 2H, Ar-H), 7.80-7.95 (m, 4H, Ar-H), 8.23-8.38 (m, 2H, Ar-H), 10.48 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.3, 103.0, 116.2, 120.0, 120.3, 120.8, 124.8, 126.4, 127.3, 127.4, 128.4, 129.4, 130.0, 130.1, 130.2, 133.3, 135.8, 146.8, 159.5, 160.5, 165.7; HRMS (ESI) m/z, calcd. for C₂₂H₁₈ClN₂O₂ [M + H]⁺: 377.1051, found: 377.1037.

2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(2,3-dihydro-1H-inden-5-yl)acetamide (6m)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 47%; mp 214-216 °C; t_R 16.3 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.00 (p, 2H, J 7.5 Hz, CH₂), 2.59 (s, 3H, CH₃), 2.76-2.89 (m, 4H, CH₂ and CH₂ ), 5.00 (s, 2H, O-CH₂), 6.97 (s, 1H, Ar-H), 7.16 (d,1H, J 8.1 Hz, Ar-H), 7.34 (d, 1H, J 8.1 Hz, Ar-H), 7.54 (s, 1H, Ar-H), 7.71 (d, 1H, J 9.0 Hz, Ar-H), 7.88 (d,1H, J 8.9 Hz, Ar-H), 8.23 (s, 1H, Ar-H), 10.14 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.1, 25.3, 31.8, 32.4, 67.3, 103.0, 116.2, 118.1, 120.0, 120.8, 124.1, 129.4, 130.0, 130.2, 136.4, 139.1, 144.1, 146.7, 159.5, 160.5, 165.1; HRMS (ESI) m/z, calcd. for C₂₁H₂₀ClN₂O₂ [M + H]⁺: 367.1208, found: 367.1205.

N-(Benzo[d][1,3]dioxol-5-yl)-2-((6-chloro-2-methylquinolin-4-yl)oxy)acetamide (6n)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); light brown solid; yield 27%; mp 212-214 °C; t_R 14.2 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.58 (s, 3H, CH₃), 3.38 (s, 2H, CH₂), 4.97 (s, 2H, O-CH₂), 6.00 (s, 2H, O-CH₂-O), 6.88 (d, 1H, J 8.4 Hz, Ar-H), 6.95 (s, 1H, Ar-H), 7.03 (dd, 1H, J 8.4, 2.1 Hz, Ar-H), 7.32 (d,1H, J 2.1 Hz, Ar-H), 7.69 (dd, 1H, J 8.9, 2.5 Hz, Ar-H), 7.87 (d, 1H, J 9.0 Hz, Ar-H), 8.22 (d, 1H, J 2.4 Hz, Ar-H), 10.16 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.3, 101.1, 102.2, 102.9, 108.0, 113.0, 120.0, 120.8, 129.4, 130.0, 130.2, 132.5, 143.4, 146.8, 147.1, 159.5, 160.5, 165.1; HRMS (ESI) m/z, calcd. for C₁₉H₁₆ClN₂O₄ [M + H]⁺: 371.0793, found: 371.0795.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(o-tolyl)acetamide (6o)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); white solid; yield 24%; mp 213-215 °C; t_R 15.3 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.23 (s, 3H, CH₃), 2.61 (s, 3H, CH₃), 5.04 (s, 2H, O-CH₂), 7.02 (s, 1H, Ar-H), 7.13-7.27 (m, 4H, Ar-H), 7.43 (d, 1H, J 7.5 Hz, Ar-H), 7.82 (s, 1H, Ar-H), 8.49 (s, 1H, Ar-H), 9.70 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 17.6, 25.3, 67.3, 103.0, 117.7, 120.4, 124.1, 125.5, 125.8, 126.0, 130.1, 130.3, 132.4, 132.7, 135.4, 146.9, 159.1, 160.5, 165.4; HRMS (ESI) m/z, calcd. for C₁₉H₁₈BrN₂O₂ [M + H]⁺: 385.0546, found: 385.0530.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(p-tolyl)acetamide (6p)

The solid was purified by successive washings with diethyl ether (3 × 10 mL); off-white solid; yield 88%; mp 221-222 °C; t_R 15.3 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.27 (s, 3H, CH₃), 2.59 (s, 3H, CH₃), 5.01 (s, 2H, O-CH₂), 6.98 (s, 1H, Ar-H), 7.11-7.19 (m, 2H, Ar-H), 7.48-7.56 (m, 2H, Ar-H), 7.77-7.88 (m, 2H, Ar-H), 8.36-8.42 (m, 1H, Ar-H), 10.18 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 20.4, 25.3, 67.2, 102.9, 117.7, 119.9 (2C), 120.4, 124.0, 129.1 (2C), 130.1, 132.7, 132.8, 135.7, 146.9, 159.3, 160.6, 165.1; HRMS (ESI) m/z, calcd. for C₁₉H₁₈BrN₂O₂ [M + H]⁺: 385.0546, found: 385.0541.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(3,4-dimethyl phenyl)acetamide (6q)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 56%; mp 219-220 °C; t_R 16.2 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.17 (s, 3H, CH₃), 2.20 (s, 3H, CH₃), 2.59 (s, 3H, CH₃), 5.00 (s, 2H, O-CH₂), 6.97 (s, 1H, Ar-H), 7.08 (d, 1H, J 8.2 Hz, Ar-H), 7.35 (dd, 1H, J 8.2, 2.3 Hz, Ar-H), 7.43 (d, 1H, J 2.3 Hz, Ar-H), 7.82 (t, 2H J 1.5 Hz, Ar-H), 8.38 (dd, 1H, J 1.9, 0.9 Hz, Ar-H), 10.13 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 18.7, 19.5, 25.3, 67.2, 102.9, 117.4, 117.7, 120.5, 121.1, 124.0, 129.5, 130.1, 131.6, 132.7, 135.9, 136.3, 146.9, 159.4, 160.6, 165.0; HRMS (ESI) m/z, calcd. for C₂₀H₂₀BrN₂O₂ [M + H]⁺: 399.0703, found: 399.0698.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(4-ethylphenyl)acetamide (6r)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); white solid; yield 62%; mp 214-216 °C; t_R 16.3 min; ¹H NMR (400 MHz, DMSO-d₆) δ 1.15 (t, 3H, J 7.6 Hz, CH₃), 2.55 (q, 2H, J 7.6 Hz, CH₂), 2.61 (s, 3H, CH₃), 5.04 (s, 2H, O-CH₂), 7.04 (s, 1H, Ar-H), 7.16 (d, 2H, J 8.4, Ar-H), 7.53 (d, 2H, J 8.1 Hz, Ar-H), 7.79-7.92 (m, 2H, Ar-H), 8.40 (d, 1H, J 2.3 Hz, Ar-H), 10.22 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 15.6, 24.8, 27.6, 67.4, 103.2, 118.1, 120.0 (2C), 120.4, 124.1, 127.9 (2C), 129.1, 133.3, 135.9, 139.3, 145.8, 160.2, 160.5, 165.0; HRMS (ESI) m/z, calcd. for C₂₀H₂₀BrN₂O₂ [M + H]⁺: 399.0703, found: 399.0698.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(4-propylphenyl)acetamide (6s)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 76%; mp 208-210 °C; t_R 17.0 min; ¹H NMR (400 MHz, DMSO-d₆) δ 0.87 (t, 3H, J 7.3 Hz, CH₃), 1.56 (h, 2H, J 7.4 Hz, CH₂), 2.50 (dd, 2H, J 8.3, 6.7 Hz, CH₂), 2.58 (s, 3H, CH₃), 5.00 (s, 2H, O-CH₂), 6.96 (s, 1H, Ar-H), 7.14 (d, 2H, J 8.1 Hz, Ar-H), 7.54 (d, 2H, J 8.4 Hz, Ar-H), 7.81 (d, 2H, J 1.8 Hz, Ar-H), 8.38 (d, 1H, J 1.5 Hz, Ar-H), 10.19 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 13.5, 24.1, 25.4, 36.6, 67.3, 102.9, 117.7, 119.9 (2C), 120.5, 124.0, 128.5 (2C), 130.2, 132.7, 135.9, 137.6, 147.0, 159.4, 160.6, 165.1; HRMS (ESI) m/z, calcd. for C₂₁H₂₂BrN₂O₂ [M + H]⁺: 413.0859, found: 413.0861.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(4-butylphenyl)acetamide (6t)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 73%; mp 201-203 °C; t_R 17.8 min; ¹H NMR (400 MHz, DMSO-d₆) δ 0.88 (t, 3H, J 7.3 Hz, CH₃), 1.28 (h, 2H, J 7.3 Hz, CH₂), 1.46-1.58 (m, 2H, CH₂), 2.54 (d, 2H, J 7.6 Hz, CH₂), 2.59 (s, 3H, CH₃), 5.01 (s, 2H, O-CH₂), 6.97 (s, 1H, Ar-H), 7.11-7.19 (m, 2H, Ar-H), 7.50-7.58 (m, 2H, Ar-H), 7.82 (d, 2H, J 2.0 Hz, Ar-H), 8.37-8.39 (m, 1H, Ar-H), 10.18 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 13.7, 21.6, 25.4, 33.1, 34.2, 67.3, 102.9, 117.7, 120.0 (2C), 120.5, 124.0, 128.4 (2C), 130.2, 132.7, 135.9, 137.8, 147.0, 159.4, 160.6, 165.1; HRMS (ESI) m/z, calcd. for C₂₂H₂₄BrN₂O₂ [M + H]⁺: 427.1016, found: 427.1021.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(3-chlorophenyl)acetamide (6u)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 31%; mp 238-239 °C; t_R 15.9 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.59 (s, 3H, CH₃), 5.05 (s, 2H, O-CH₂), 6.99 (s, 1H, Ar-H), 7.12-7.21 (m, 1H, Ar-H), 7.38 (t, 1H, J 8.1 Hz, Ar-H), 7.50-7.54 (m, 1H, Ar-H) 7.78-7.89 (m, 3H, Ar-H), 8.38 (d, 1H, J 1.8 Hz, Ar-H), 10.44 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.4, 67.2, 103.0, 117.7, 118.2, 119.3, 120.5, 123.5, 124.0, 130.2, 130.5, 132.8, 133.0, 139.7, 147.0, 159.3, 160.7, 165.8; HRMS (ESI) m/z, calcd. for C₁₈H₁₅BrClN₂O₂ [M + H]⁺: 405.0000, found: 404.9986.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(4-chlorophenyl)acetamide (6v)

The solid was purified by successive washings with diethyl ether (3 × 10 mL); off-white solid; yield 86%; mp 221-222 °C; t_R 15.9 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.59 (s, 3H, CH₃), 5.04 (s, 2H, O-CH₂), 6.98 (s, 1H, Ar-H), 7.36-7.46 (m, 2H, Ar-H), 7.61-7.76 (m, 2H, Ar-H), 7.82 (d, 2H, J 1.9 Hz, Ar-H), 8.38 (t, 1H, J 1.3 Hz, Ar-H), 10.40 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.4, 67.2, 102.9, 117.7, 120.4, 121.4 (2C), 124.0, 127.4, 128.6 (2C), 130.2, 132.7, 137.2, 146.9, 159.3, 160.6, 165.5; HRMS (ESI) m/z, calcd. for C₁₈H₁₅BrClN₂O₂ [M + H]⁺: 403.9927, found: 403.9942.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(4-bromophenyl)acetamide (6w)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 29%; mp 276-278 °C; t_R 16.0 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.59 (s, 3H, CH₃), 5.03 (s, 2H, O-CH₂), 6.99 (s, 1H, Ar-H), 7.53 (d, 2H, J 8.5 Hz, Ar-H), 7.61 (d, 2H, J 8.5 Hz, Ar-H), 7.77-7.88 (m, 2H, Ar-H), 8.38 (s, 1H, Ar-H), 10.38 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.2, 67.2, 103.0, 115.4, 117.7, 120.4, 121.7 (2C), 123.9, 130.0, 131.5 (2C), 132.8, 137.6, 146.7, 159.4, 160.6, 165.5; HRMS (ESI) m/z, calcd. for C₁₈H₁₅Br₂N₂O₂ [M + H]⁺: 448.9495, found: 448.9479.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(4-(trifluoro methyl)phenyl)acetamide (6x)

Column chromatography on silica gel (hexane/ethyl acetate, 8:2); off-white solid; yield 76%; mp 214-217 °C; t_R 16.3 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.60 (s, 3H, CH₃), 5.09 (s, 2H, O-CH₂), 7.00 (s, 1H, Ar-H), 7.72 (d, 3H, J 8.5 Hz, Ar-H), 7.83 (d, 2H, J 2.0 Hz, Ar-H), 7.88 (d, 3H, J 8.5 Hz, Ar-H), 8.38 (s, 1H, Ar-H), 10.64 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.2, 102.9, 117.7, 119.6 (2C), 120.4, 122.9 (q, J 271.1 Hz), 123.8 (q, J 31.8 Hz), 123.9, 125.6, 126.0 (q, J 3.9 Hz), 130.2, 132.7, 141.8, 147.0, 159.3, 160.6, 166.0; HRMS (ESI) m/z, calcd. for C₁₉H₁₅BrF₃N₂O₂ [M + H]⁺: 439.0264, found: 439.0247.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(naphthalen-1-yl)acetamide (6y)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 53%; mp 201-203 °C; t_R 15.6 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.64 (s, 3H, CH₃), 5.18 (s, 2H, O-CH₂), 7.10 (s, 1H, Ar-H), 7.50-7.60 (m, 3H,Ar-H), 7.69 (d, 1H, J 7.4, Ar-H), 7.78-7.88 (m, 3H, Ar-H), 7.93-8.01 (m, 1H, Ar-H), 8.03-8.10 (m, 1H, Ar-H), 8.54 (s, 1H, Ar-H), 10.32 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.5, 103.0, 117.7, 120.5, 122.7, 124.1, 125.5, 125.9, 126.0(2C), 126.0, 128.1, 128.3, 130.1, 132.7, 133.7, 147.0, 159.3, 160.6, 166.3; HRMS (ESI) m/z, calcd. for C₂₂H₁₈BrN₂O₂ [M + H]⁺: 421.0546, found: 421.0538.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(naphthalen-2-yl)acetamide (6z)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); off-white solid; yield 30%; mp 249-250 °C; t_R 16.4 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.60 (s, 3H, CH₃), 5.10 (s, O-CH₂), 7.03 (s, 1H, Ar-H), 7.40-7.45 (m, 1H, Ar-H), 7.46-7.51 (m, 1H, Ar-H), 7.66 (dd, 1H, J 8.8, 2.1 Hz, Ar-H), 7.79-7.94 (m, 5H, Ar-H), 8.30-8.36 (m, 1H, Ar-H), 8.39-8.45 (m, 1H, Ar-H), 10.48 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.2, 103.0, 116.1, 117.7, 120.3, 120.5, 123.9, 124.8, 126.4, 127.3, 127.4, 128.3, 129.9, 130.1, 132.7, 133.2, 135.8, 146.9, 159.4, 160.6, 165.6; HRMS (ESI) m/z, calcd. for C₂₂H₁₈BrN₂O₂ [M + H]⁺: 421.0546, found: 421.0541.

2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(2,3-dihydro-1H-inden-5-yl)acetamide (6aa)

The solid was purified by successive washings with diethyl ether (3 × 10 mL); off-white solid; yield 85%; mp 222-224 °C; t_R 16.4 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.00 (p, 2H, J 7.4 Hz, CH₂), 2.59 (s, 3H, CH₃), 2.76-2.89 (m, 4H, CH₂ and CH₂), 5.00 (s, 2H, O- CH₂), 6.97 (s, 1H, Ar-H), 7.17 (d, 1H, J 8.1 Hz, Ar-H), 7.33 (dd, 1H, J 8.1, 2.0 Hz, Ar-H), 7.52-7.57 (m, 1H, Ar-H), 7.77- 7.87 (m, 2H, Ar-H), 8.38 (dd, 1H, J 2.1, 0.8 Hz, Ar-H), 10.14 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.1, 25.3, 31.7, 32.4, 67.3, 102.9, 116.1, 117.7, 118.0, 120.4, 123.9, 124.1, 130.1, 132.7, 136.3, 139.0, 144.1, 146.9, 159.3, 160.6, 165.0; HRMS (ESI) m/z, calcd. for C₂₁H₂₀BrN₂O₂ [M + H]⁺: 411.0703, found: 411.0695.

N-(Benzo[d][1,3]dioxol-5-yl)-2-((6-bromo-2-methylquinolin-4-yl)oxy)acetamide (6ab)

Column chromatography on silica gel (hexane/ethyl acetate, 1:1); light yellow solid; yield 32%; mp 232-234 °C; t_R 14.4 min; ¹H NMR (400 MHz, DMSO-d₆) δ 2.60 (s, 3H, CH₃), 4.99 (s, 2H, O-CH₂), 6.01 (s, 2H, O-CH₂-O), 6.89 (d, 1H, J 8.4 Hz, Ar-H), 6.94-7.09 (m, 2H, Ar-H), 7.32 (d, 1H, J 2.1 Hz, Ar-H), 7.74-7.90 (m, 2H, Ar-H), 8.39 (d, 1H, J 2.0 Hz, Ar-H), 10.18 (s, 1H, N-H); ¹³C NMR (100 MHz, DMSO-d₆) δ 25.3, 67.3, 101.0, 102.1, 103.0, 108.0, 112.9, 117.8, 120.5, 124.0, 130.0, 132.5, 132.8, 143.4, 146.7, 147.0, 159.5, 160.6, 165.0; HRMS (ESI) m/z, calcd. for C₁₉H₁₆BrN₂O₄ [M + H]⁺: 415.0288, found: 415.0294.

Minimum inhibitory concentration

The minimum inhibitory concentrations (MICs) of the synthesized compounds were determined using the resazurin microtiter assay (REMA) in 96-well microplates.¹⁹ Isoniazid was employed as a positive control. Stock solutions of each compound were prepared in DMSO at 2 mg mL^-1 and diluted in Middlebrook 7H9 broth supplemented with 10% ADC (albumin, dextrose, and catalase) to a final concentration of 20 μg mL^-1, with 2% DMSO (Sigma-Aldrich, St. Louis, MO, USA).

Serial 2-fold dilutions of each compound were prepared in 100 μL of Middlebrook 7H9 medium containing 10% ADC (BD Diagnostics, Franklin Lakes, NJ, USA) directly in 96-well plates, starting from the highest soluble concentration of each compound. Growth controls (medium with bacterial inoculum, no drug or evaluating molecule) and sterility controls (medium only, no inoculum) were included on each plate. The assay was conducted against M. tuberculosis H37Rv. The bacterial strain was cultured in Middlebrook 7H9 broth supplemented with 10% OADC (oleic acid, albumin, dextrose, and catalase) and 0.05% Tween 80. Cell clumps were disrupted by vortexing the cultures with sterile 4 mm glass beads for 5 min, followed by sedimentation for 20 min. The supernatant was collected, and the optical density was adjusted to 0.006 at 600 nm using a spectrophotometer. A volume of 100 μL of the standardized inoculum was added to each well, except in the sterility control wells.

Plates were covered, sealed, and incubated at 37 °C for 7 days. After incubation, 60 μL of 0.01% resazurin solution was added to each well, and the plates were incubated for an additional 48 h at 37 °C. Bacterial viability was indicated by a color change from blue (non-viable) to pink (viable). The MIC was defined as the lowest compound concentration that prevented color change, indicating inhibition of bacterial growth. Each compound was tested in triplicate in independent experiments, and the reported MIC corresponds to the highest value observed among the three replicates.

Cytotoxicity investigation

Cellular viability determination after incubation with the test compounds was performed using two different methods: the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method²⁰ and neutral red (NR)²¹ uptake assay. First, Vero and HepG2 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% inactivated fetal bovine serum, 1% antibiotic (gentamicin) and 0.01% antifungal (amphotericin B). Cells were seeded at 4 × 10³ (HepG2) or 2 × 10³ cells per well (Vero) in a 96-well microtiter plate, and incubated for 24 h. Evaluated compounds were diluted in three different concentrations (1, 5 and 20 µM) using 2% DMSO, and were incubated with the cell lines for 72 h at 37 °C under 5% CO₂. For the MTT assay, the cultures were incubated with MTT reagent (5 mg mL^-1) for 4 h. The absorbance was measured using excitation and emission wavelengths of 570 and 655 nm, respectively (SpectraMax M2e, Molecular Devices, San Jose, USA). The precipitated purple formazan crystals were directly proportional to the number of live cells with active mitochondria. For the neutral red assay, after 72 h of incubation with the compounds, the cells were washed with PBS before the addition of 200 mL of neutral red dye solution (25 mg mL^-1, Sigma, Saint Louis, USA) prepared in serum-free medium. The plate was incubated for an additional 3 h at 37 °C under 5% CO₂. After incubation, cells were washed with PBS, followed by incubation with 100 mL of a desorb solution (CH₃COOH/EtOH/H₂O, 1:50:49) for 30 min, with gentle shaking to extract neutral red dye from the viable cells. The absorbance was measured at 562 nm using a microtiter plate reader. The percentage of cell viability for the treated groups was reported by considering the control wells (2% DMSO) as 100% of cell viability: cell viability (%) = (absorbance of treated wells/absorbance of control wells) × 100. Statistical analysis was performed using one-way analysis of variance using GraphPad Prism 5.0 software.²²

Aqueous solubility determination

The aqueous solubility of selected QOAs was determined at three pH values (1.2, 7.4, and 9.1) using the shake-flask method. Briefly, 1 mg of each compound was added to 1 mL of buffer solution (0.1 M HCl for pH 1.2, PBS 1× for pH 7.4, or 0.1 M NH₄HCO₃ for pH 9.1), resulting in an initial suspension of 1 mg mL^-1. Samples were vortexed for 30 s and then incubated under orbital shaking for 4 h at 25 °C to reach equilibrium. After incubation, the samples were centrifuged at 13,000 rpm for 20 min at 25 °C, and the resulting supernatant was collected. The concentration of dissolved compound was quantified by HPLC using a single-point calibration prepared from a stock solution of the corresponding molecule in DMSO.

Results and Discussion

The synthesis of the QOAs was accomplished through a three-step sequence. Initially, the disubstituted 4-hydroxyquinolines (4a-4b) were prepared via the Conrad-Limpach reaction following established protocols.¹³ Subsequently, the 2-bromo-N-arylacetamides (5a-5n) were synthesized by the acylation of substituted anilines with bromoacetyl chloride also in accordance with previously reported methods.¹²-¹⁵ In the final step, the target 2-(quinolin-4-yloxy)acetamides (6a-6ab) were obtained via O-alkylation of the 4-hydroxyquinolines 3 with the corresponding 2-bromo-N-arylacetamides 6, in the presence of potassium carbonate, using DMF as solvent (Scheme 1). The reaction mixtures were stirred at room temperature (25 °C) for 18 h, affording the desired products in 24-88% yields.¹²-¹⁵ All synthesized compounds exhibited spectroscopic (NMR) and spectrometric (HRMS) data consistent with the proposed structures (Supplementary Information section). Furthermore, the compounds were analyzed for purity by HPLC and displayed purity levels of ≥ 95%. Taken together, these analyses confirm the identity and integrity of the synthesized structures and their suitability for subsequent biological evaluation.

Scheme 1
Reagents and conditions: i = DMF, K₂CO₃, 25 °C, 18 h.

All synthesized derivatives were subjected to antimycobacterial evaluation using REMA, a colorimetric method widely employed for determining MICs against Mycobacterium tuberculosis.¹⁹ Isoniazid and rifampicin, first-line drugs used in the treatment of tuberculosis, were included as positive controls. The MIC values spanned a broad range, from submicromolar to values exceeding 22 μM, highlighting the influence of structural modifications on antimycobacterial potency (Table 1).

Table 1
Yields of 2-(quinoline-4-yloxy)acetamides 6a-6ab, ClogP values, and in vitro activities against M. tuberculosis H37Rv strain

entry	R1	R2	Yielda / %	ClogPb	MICc / µM
6a	Cl	2-Me-Ph	47	4.15	7.3
6b	Cl	4-Me-Ph	45	4.80	1.8
6c	Cl	3,4-(Me)2-Ph	47	5.25	3.5
6d	Cl	4-Et-Ph	49	5.33	1.8
6e	Cl	4-Pr-Ph	62	5.86	1.7
6f	Cl	4-Bu-Ph	61	5.38	1.6
6g	Cl	3-Cl-Ph	63	5.27	13.8
6h	Cl	4-Cl-Ph	64	5.27	3.5
6i	Cl	4-Br-Ph	27	5.42	> 12.3
6j	Cl	4-CF3-Ph	59	5.63	> 12.7
6k	Cl	1-naphthyl	42	5.47	1.8
6l	Cl	2-naphthyl	46	5.47	> 13.3
6m	Cl		47	5.31	3.4
6n	Cl		27	4.36	13.5
6o	Br	2-Me-Ph	24	4.30	0.8
6p	Br	4-Me-Ph	88	4.95	1.6
6q	Br	3,4-(Me)2-Ph	56	5.40	3.1
6r	Br	4-Et-Ph	62	5.48	1.7
6s	Br	4-Pr-Ph	76	6.01	1.5
6t	Br	4-Bu-Ph	73	6.54	2.9
6u	Br	3-Cl-Ph	31	5.42	6.2
6v	Br	4-Cl-Ph	86	5.42	3.1
6w	Br	4-Br-Ph	29	5.57	> 22.2
6x	Br	4-CF3-Ph	76	5.78	11.4
6y	Br	1-naphthyl	53	5.62	1.5
6z	Br	2-naphthyl	30	5.62	13.0
6aa	Br		85	5.46	1.5
6ab	Br		32	4.51	6.0
INH	–	–	–	–	2.3
RIF	–	–	–	–	0.2

• ^a
  Isolated yields under non-optimized synthetic conditions.
• ^b
  Logarithm of partition coefficient calculated by ChemBioDraw Ultra, version 12.0.2.1076.²³
• ^c
  Minimal inhibitory concentration against M. tuberculosis H37Rv strain. INH: isoniazid; RIF: rifampin.

Among the series, compounds bearing a chloro at the 6-position of the quinoline core (R¹ = Cl) showed variable activity depending on the acetamide substituent (R²). Derivatives featuring small alkyl groups at the 4-position of the phenyl ring, such as 6b (4-Me-Ph, MIC = 1.8 μM), 6d (4-Et-Ph, MIC = 1.8 μM), 6e (4-Pr-Ph, MIC = 1.7 μM), and 6f (4-Bu-Ph, MIC = 1.6 μM), exhibited notable potency, all achieving MICs below 2 μM. By contrast, molecule 6a, bearing a methyl group at the 2-position of the phenyl ring (2-Me-Ph), displayed reduced activity (MIC = 7.3 μM), suggesting that steric hindrance or electronic effects at this position, when chloro is attached at the heterocycle, may negatively influence antimycobacterial potency. Interestingly, shifting the methyl substituent from the 4- to the 2-position led to an approximate four-fold decrease in activity, highlighting the sensitivity of biological potency to substitution patterns on the aryl ring. Moreover, the introduction of two methyl groups at 3- and 4-positions of the phenyl ring, as in structure 6c (3,4-(Me)₂-Ph, MIC = 3.5 μM), led to a moderate decrease in activity compared to the mono-substituted couterparts. Taken together, these results suggested that moderate hydrophobic substitutions at the 4-position favor antimycobacterial activity. On the other hand, halogenated analogues such as 6g (3-Cl-Ph, MIC = 13.8 μM) and 6h (4-Cl-Ph, MIC = 3.5 μM) displayed reduced activity, indicating that electron-withdrawing groups can negatively affect potency in certain positions. Consistently, 2-(quinolin-4-yloxy)acetamide 6i (4-Br-Ph) and the trifluoromethylated derivative 6j (4-CF₃-Ph) were not effective at the highest concentrations tested, exhibiting MIC values exceeding 12.3 μM, further suggesting that bulky or strongly electron-withdrawing substituents at the 4-position can markedly impair antimycobacterial activity. Additionally, bulky aromatic substituents were tolerated to varying extents. While compound 6k, bearing a 1-naphthyl group, maintained good activity (MIC = 1.7 μM), its regioisomer 6l (2-naphthyl) showed no significant inhibition at the highest concentration evaluated (MIC > 13.3 μM), suggesting a potential steric hindrance effect. Interestingly, molecule 6m, featuring a non-aromatic five-membered ring fused at the 3,4-positions of the benzene ring, exhibited moderate activity with a MIC of 3.4 μM. This finding suggests that reducing ring size and disrupting aromaticity, compared to the 2-naphthyl analogue, led to a more potent structure. Finally, increasing polarity through the incorporation of two oxygen atoms in 2-(quinolin-4-yloxy)acetamide 6n resulted in diminished activity (MIC = 13.5 μM), representing an approximate four-fold reduction in potency relative to 6m, indicating, once more, that improved polarity in this region may be detrimental to antimycobacterial efficacy.

Replacement of the quinoline chlorine (R¹ = Cl) by bromine (R¹ = Br) generally retained or slightly improved activity across the series. Notably, compound 6o (R¹ = Br, R² = 2-Me-Ph) emerged as the most potent derivative, with a MIC of 0.80 μM, surpassing isoniazid (MIC = 2.3 μM) under the assay conditions. When compared directly to the first-line drug isoniazid, molecule 6o demonstrated more than 2.8-fold greater potency. Interestingly, despite sharing the same R² substituent, structure 6o exhibited significantly greater activity than its chloro-containing analogue 6a (MIC = 7.3 μM), suggesting that bromine at the quinoline core may induce subtle conformational changes that improve binding to the biological target and account for its superior potency. Other 2-(quinolin-4-yloxy)acetamides such as 6p (4-Me-Ph, MIC = 1.6 μM), 6r (4-Et-Ph, MIC = 1.7 μM), and 6s (4-Pr-Ph, MIC = 1.5 μM) also exhibited significant activity, reinforcing the beneficial impact of small hydrophobic substituents in R². However, similar to what was observed among the chloro-containing analogues, the introduction of two methyl groups at 3- and 4-positions of the benzene ring, as in compound 6q (3,4-(Me)₂-Ph), resulted in a reduction of activity, with a MIC of 3.1 μM. In a related trend, while small to moderate single alkyl substituents at the 4-position of the aryl ring contributed to good antimycobacterial activity among the brominated derivatives, molecule 6t, bearing a bulkier butyl group (4-Bu-Ph), exhibited reduced potency, with a MIC of 2.9 μM. These observations collectively may indicate that, when bromine is present at the quinoline core, excessive steric bulk either through multiple small substituents or a single larger group at the para-position may hinder optimal interactions with the biological target, thereby diminishing antimycobacterial potency.

Further insights into the structure-activity relationship were obtained from the evaluation of structures bearing electron-withdrawing substituents at the 4-position of the benzene ring. 2-(Quinolin-4-yloxy)acetamides 6u (3-Cl-Ph, MIC = 6.2 μM), 6v (4-Cl-Ph, MIC = 3.1 μM), 6w (4-Br-Ph, MIC > 22.2 μM), and 6x (4-CF₃-Ph, MIC = 11.4 μM) all exhibited reduced antimycobacterial activity compared to their alkyl-substituted counterparts. This pattern suggests that the introduction of electron-withdrawing groups in this region of the molecule is poorly tolerated, likely due to unfavorable electronic effects or influence compound permeability. It is noteworthy that compound 6w, containing a 4-bromo substituent, was inactive at the highest concentration tested, with a MIC exceeding 22.2 μM, underscoring the detrimental impact of increased polarity or steric bulk in this part of structure. This behavior was similar to that observed among the chloro-containing analogues, where electron-withdrawing substituents also led to decreased activity, highlighting a consistent trend across the synthesized series. Additionally, bulky aromatic substituents were also tolerated to varying extents among the brominated derivatives. Molecule 6y, bearing a 1-naphthyl group, maintained good activity (MIC = 1.5 μM), comparable to its chloro analogue 6k (MIC = 1.7). However, its regioisomer 6z (2-naphthyl) exhibited significantly reduced potency, with a MIC of 13.0 μM, suggesting a potential steric hindrance effect similar to that observed in the chloro series. Further, structure 6aa, featuring a non-aromatic five-membered ring fused at the 3,4-positions of the benzene ring, demonstrated good activity with a MIC value of 1.5 μM. This result suggests, once more, that reducing the ring size and disrupting aromaticity, as compared to the 2-naphthyl derivative, led to a more potent compound. It is plausible that, in 6aa, the five-membered ring may adopt a conformation partially out of the plane of the aromatic system, which could alleviate steric interactions and improve the molecule’s fit within the biological target site, thereby contributing to its higher efficacy. Finally, 2-(quinolin-4-yloxy)acetamide 6ab, incorporating two oxygen atoms into the five-membered ring, showed diminished activity (MIC = 6.0 μM), reflecting an approximate four-fold reduction in potency relative to 6aa, and once again indicating that increased polarity in this region may be detrimental to antimycobacterial efficacy.

Regarding physicochemical properties, the synthesized 2-(quinolin-4-yloxy)acetamide derivatives displayed ClogP values ranging from 4.15 to 6.54, indicating moderate to high lipophilicity. An analysis of the MIC data in relation to calculated ClogP values suggests that compounds with moderate hydrophobicity, generally within the range of 4.0 to 5.5, tended to exhibit better antimycobacterial activity. For example, molecule 6o, the most potent derivative in the series (MIC = 0.80 μM), showed a ClogP of 4.30, while other active structures such as 6p (MIC = 1.6 μM) and 6r (MIC = 1.7 μM) also fell within this lipophilicity window. In contrast, increasing the ClogP beyond this range did not consistently correlate with improved potency; 2-(quinolin-4-yloxy)acetamide 6t, with a ClogP of 6.54, demonstrated reduced activity (MIC = 2.9 μM). Moreover, compounds bearing strongly electron-withdrawing groups showed poor activity despite possessing moderate ClogP values, suggesting that electronic and steric factors may also play a critical role alongside lipophilicity. These findings indicate that while moderate hydrophobicity is generally favorable for activity, excessive lipophilicity or the presence of polar, electron-withdrawing substituents may negatively impact antimycobacterial efficacy in this chemical series.

Eleven QOAs derivatives were selected for further evaluation based on a cutoff MIC value of ≤ 1.8 µM against the Mycobacterium tuberculosis H37Rv strain. HepG2 (human hepatocellular carcinoma) and Vero (African green monkey kidney) cell viability assays were employed as initial indicators of potential cytotoxicity and to assess the selectivity of these compounds relative to their antimycobacterial activity (Table 2). This strategy allowed for the preliminary identification of molecules with promising therapeutic windows, prioritizing those capable of inhibiting the bacillus at low micromolar concentrations while maintaining acceptable levels of cytotoxicity toward mammalian cells. Cell viability was assessed using the MTT and the NR assays. The MTT assay evaluates mitochondrial activity by measuring the reduction of the tetrazolium salt MTT to formazan crystals, thus serving as an indicator of cellular metabolic viability.²⁰ The NR assay, in turn, assesses the integrity of lysosomal membranes by quantifying the uptake and retention of the Neutral Red dye within viable cells.²¹ These combined assays provide insights into different cellular compartments and mechanisms of potential cytotoxicity. In addition, the use of both techniques enhances the reliability of the results and reduces the likelihood of false negatives during the early stages of drug development. Results from both assays were expressed as the concentration required to reduce cell viability by 50% (CC₅₀), offering a quantitative measure of cytotoxicity for comparison with antimycobacterial potency and for calculating selectivity indices (Table 2).

Table 2
Evaluation of the viability of HepG2 and Vero cells in the presence of selected 2-(quinoline-4-yloxy)acetamides using the MTT and NR assays

entry	MICa / μM	CC50 HepG2b / μM	CC50 Verob / μM	SIe HepG2	SIe Vero
6b	1.8	> 20c; > 20d	> 20c; > 20d	> 11.1c; > 11.1d	> 11.1c; > 11.1d
6d	1.8	> 20c; > 20d	> 20c; > 20d	> 11.1c; > 11.1d	> 11.1c; > 11.1d
6e	1.7	> 20c; > 20d	> 20c; > 20d	> 11.8c; > 11.8d	> 11.8c; > 11.8d
6f	1.6	> 20c; > 20d	> 20c; > 20d	> 12.5c; > 12.5d	> 12.5c; > 12.5d
6k	1.7	> 20c; 13.0d	> 20c; 19.3d	> 11.8c; 7.6d	> 11.8c; 11.3d
6o	0.8	> 20c; > 20d	> 20c; > 20d	> 25c; > 25d	> 25c; > 25d
6p	1.6	> 20c; > 20d	> 20c; > 20d	> 12.5c; > 12.5d	> 12.5c; > 12.5d
6r	1.7	> 20c; > 20d	> 20c; > 20d	> 11.8c; > 11.8d	> 11.8c; > 11.8d
6s	1.5	> 20c; > 20d	> 20c; > 20d	> 13.3c; > 13.3d	> 13.3c; > 13.3d
6y	1.5	> 20c; > 20d	> 20c; > 20d	> 13.3c; > 13.3d	> 13.3c; > 13.3d
6aa	1.5	> 20c; > 20d	> 20c; > 20d	> 13.3c; > 13.3d	> 13.3c; > 13.3d

• ^a
  M. tuberculosis H37Rv strain.
• ^b
  The cell viability and selectivity index (SI) of the compounds were evaluated in HepG2 and Vero cell lines. Cell viability was measured using MTT and NR assays, with results expressed as the concentration required to reduce viability by 50% (CC₅₀).
• ^c
  Determined by the MTT method.
• ^d
  Determined by the NR assay.
• ^e
  Selectivity index (SI = CC₅₀/MIC_H37Rv). MIC: minimum inhibitory concentrations.

Overall, most structures exhibited low cytotoxicity, with CC₅₀ values exceeding 20 μM in both cell lines under both assay conditions, suggesting a favorable preliminary safety profile. An exception was observed for 2-(quinoline-4-yloxy)acetamide 6k, which exhibited moderate cytotoxicity in the NR assay, with CC₅₀ values of 13.0 μM in HepG2 cells and 19.3 μM in Vero cells, resulting in reduced selectivity indices (SI) of 7.6 and 11.3, respectively. It is noteworthy that values below 10 generally indicate cytotoxicity concerns and a potentially narrow therapeutic window. By contrast, compound 6o, which displayed the lowest MIC value in the series (0.8 μM), showed no detectable cytotoxicity up to 20 μM in both assays. This resulted in selectivity indices greater than 25, highlighting molecule 6o as the most promising of the series, with an encouraging in vitro selectivity profile that warrants further investigation. Additionally, structures 6s, 6y, and 6aa also demonstrated favorable profiles, exhibiting MIC values of 1.5 μM coupled with high CC₅₀ values above 20 µM, translating to selectivity indices exceeding 13.3 in both cell lines. These results indicate that multiple derivatives within this series possess promising selectivity.

To complement the MIC, SAR, and selectivity analyses, the aqueous solubility of the most active QOAs (6o, 6s, 6y, and 6aa) was evaluated under physiologically relevant pH conditions (Table 3). Overall, the compounds exhibited marked pH-dependent solubility, with lower values generally observed under neutral and alkaline conditions. Molecule 6aa was the least soluble across all conditions, reaching only 3.9 µM at pH 1.2 and ≤ 0.7 µM at higher pH values. Structures 6s and 6y presented intermediate solubility, with 16.4 and 32.1 µM at pH 1.2, respectively, but both dropped to ≤ 1.6 µM at physiological pH. Notably, 2-(quinoline-4-yloxy)acetamide 6o- the most potent derivative of the series, with a MIC of 0.8 µM - exhibited the highest solubility under acidic conditions (156 µM at pH 1.2), although its solubility decreased significantly at pH 9.1 (4.0 µM) and was below the detection limit at pH 7.4. These results highlight that 6o combines the highest potency within the synthesized series with a favorable selectivity profile and adequate solubility under gastric conditions, suggesting that it represents a suitable starting point for further structural optimization.

Table 3
Aqueous solubilitye of selected 2-(quinolin-4-yloxy)acetamides at different pH values mimicking gastric (pH 1.2), plasma (pH 7.4), and intestinal (pH 9.1) conditions

entry	pH 1.2b / µM	pH 7.4c / µM	pH 9.1d / µM
6o	156.0	−e	4.0
6s	16.4	0.7	0.7
6y	32.1	1.5	1.6
6aa	3.9	0.3	0.7

• ^a
  Concentration was determined by UHPLC-DAD after 4 h of shaking incubation at 25 °C.
• ^b
  Using 0.1 M HCl solution.
• ^c
  Using PBS.
• ^d
  Using 0.1 M NH₄HCO₃ solution.
• ^e
  Not detected under the experimental conditions.

Conclusions

In this study, a series of 28 QOAs was synthesized, and biologically evaluated to expand the understanding of the SAR within this promising chemical scaffold for antimycobacterial therapy. Modifications to both the quinoline core and the aryl moiety of the acetamide side chain were explored, revealing clear trends in the SAR. Such well-defined structure-activity relationships strongly suggest that the observed antimycobacterial activity is mediated by specific interactions with a biological target, rather than arising from nonspecific effects. Among the chloro- and bromo-substituted derivatives, small to moderate hydrophobic groups at the 4-position of the aryl ring generally correlated with increased antimycobacterial activity, whereas the presence of bulky or strongly electron-withdrawing substituents tended to diminish potency, likely due to steric hindrance or unfavorable electronic effects. The evaluation of non-aromatic and partially saturated substituents provided additional insights, suggesting that certain conformational flexibility or deviation from planarity, as observed in compound 6aa, may positively influence activity. Notably, molecule 6o, featuring a bromo substituent at the quinoline core and a 2-methylphenyl group at the aryl moiety, emerged as the most potent derivative, exhibiting a MIC of 0.80 µM against M. tuberculosis H37Rv. Although less potent than the first-line antitubercular drug rifampicin (MIC = 0.20 μM), 2-(quinolin-4-yloxy)acetamide 6o was more than 2.8-fold more potent than isoniazid (MIC = 2.3 µM) under the same assay conditions. Importantly, 6o demonstrated no detectable cytotoxicity in HepG2 and Vero cells up to 20 μM in both the MTT and Neutral Red assays, resulting in selectivity indices greater than 25. In addition, 6o displayed favorable solubility under acidic conditions (156 μM at pH 1.2), which, when combined with its potent antimycobacterial activity (MIC = 0.8 μM), further supports its potential as a promising lead structure. Overall, the data generated in this work contribute valuable SAR insights and highlight critical substituent effects that modulate the biological activity and selectivity of the 2-(quinolin-4-yloxy)acetamide scaffold. These results support ongoing efforts to optimize this series and develop new antitubercular agents capable of addressing the persistent challenges posed by TB disease.

Supplementary Information

All supplementary data (mass spectrum, LC-MS/MS data) are available free of charge at http://jbcs.sbq.org.br as PDF file.

Data Availability Statement

All data supporting the findings of this study are included in the manuscript.

Acknowledgments

This work was supported by National Institute of Science and Technology on Tuberculosis (Decit/SCTIE/MS-MCTCNPq-FNDTCCAPES-FAPERGS) (grant No. 421703/2017-2), Banco Nacional de Desenvolvimento Econômico e Social (BNDES/FUNTEC) (grant No. 14.2.0914.1) and FAPERGS (grant No. 17/1265 8 INCT-TB). C.V.B (CNPq, grant No. 311949/2019-3), L.A.B. (CNPq, grant No. 303499/2021-4), and P.M. (CNPq, grant No. 310888/2022-0) are Research Career Awardees of CNPq. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES), Finance Code 001.

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Edited by

• Editor handled this article:
  Editor handled this article: Giovanni Wilson Amarante (Associate)

Publication Dates

• Publication in this collection
  20 Oct 2025
• Date of issue
  2025

History

• Received
  23 July 2025
• Accepted
  02 Sept 2025