Unveiling the Antimycobacterial Potential of Novel 4-Alkoxyquinolines: Insights into Selectivity, Mechanism of Action, and In Vivo Exposure

Abstract

This work presents a comprehensive investigation into the design, synthesis, and evaluation of a novel series of 4-alkoxyquinolines as potential antimycobacterial agents. The design approach, which combined molecular simplification and chain extension, resulted in compounds with potent and selective activity against both drug-susceptible and multidrug-resistant Mycobacterium tuberculosis strains. The lead molecule, targeting the cytochrome bc₁ complex, exhibited favorable kinetic solubility and remarkable chemical stability under acidic conditions. Despite in vitro ADME evaluations showing low permeability and high metabolism in rat microsomes, the lead compound exhibited bacteriostatic activity in a murine macrophage model of TB infection and demonstrated promising in vivo exposure following gavage in mice, with an AUC_0–t of 127.5 ± 5.7 μM h. To the best of our knowledge, for the first time, a simplified structure from 2-(quinolin-4-yloxy)acetamides has shown such potential. These findings suggest a new avenue for exploring this chemical class as a source of antituberculosis drug candidates.

1. Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has been recognized as a global public health crisis by the World Health Organization (WHO) since the 1990s. In 2022, the WHO reported 10.6 million TB cases and 1.3 million deaths worldwide, making TB the second leading cause of death from a single infectious agent, following SARS-CoV-2.¹ The disease primarily affects the lungs but can also impact other parts of the body, causing severe health complications. Despite significant advances in TB research and treatment, the disease remains a major health challenge, especially in low- and middle-income countries.¹ Unfortunately, the COVID-19 pandemic has disrupted access to diagnosis, treatment, and public health efforts, potentially worsening the TB burden and jeopardizing years of progress in TB control.¹

The primary pharmacological approach to treating TB involves the use of drugs such as isoniazid (INH), rifampicin (RIF), pyrazinamide, and ethambutol.¹ Despite the efficacy of this recommended treatment regimen, the emergence of mono-, multi-, and polydrug-resistant strains has complicated TB management, leading to increased morbidity and mortality. Alarmingly, the WHO reports that a significant proportion of patients with drug-resistant TB have not received proper diagnosis and treatment.² Inadequate healthcare infrastructure, limited access to diagnostics, and a lack of effective drugs have been significant barriers to optimal clinical management. In response to these growing resistance challenges, the 21st century has seen the approval of three new anti-TB drugs: bedaquiline (2012),³ delamanid (2014),⁴ and pretomanid (2019),⁵ aimed at combating strains that are unresponsive to traditional treatments. It is noteworthy that pretomanid was approved in combination with bedaquiline and linezolid for the treatment of a specific subset of adult patients with extensively drug-resistant TB.⁵ These new drugs represent a critical advancement in TB therapy, offering hope for improved outcomes in patients with drug-resistant TB. However, the implementation of these new agents has required careful monitoring to prevent the development of resistance. Treatment failures and genotypic changes associated with resistance have already been documented for bedaquiline and delamanid.^6,7 Therefore, continuous research and development, along with global collaboration, are essential to address the evolving threat of drug-resistant TB and ensure effective treatment for all patients.

Within this context, our research group has focused on the design and synthesis of compounds with the potential to inhibit the growth of both drug-susceptible and drug-resistant Mtb strains.⁸⁻¹⁵ Among the chemical classes explored, 2-(quinolin-4-yloxy)acetamides have demonstrated potent and selective activity,⁸⁻¹¹ exerting their inhibitory effect by targeting the cytochrome bc₁ complex.^10,11,16 This class of compounds was discovered through high-throughput screening, leading to the identification of molecule 1, which inhibited Mtb growth with a minimum inhibitory concentration (MIC) of 0.44 μM (Figure 1).¹⁷ As part of our ongoing research program, this chemical class has been optimized using classical techniques in medicinal chemistry. Modifications aiming the improvement of liposolubility of the acetamide group resulted in compound 2, which exhibited a significantly enhanced ability to inhibit Mtb, with an MIC of 0.05 μM (Figure 1).⁸ Further structural variations revealed that the potency of the 2-(quinolin-4-yloxy)acetamide scaffold was not dependent on the planarity of the amide substituent. Notably, compound 3, featuring a propyl group attached to the 4-position of the benzene ring, maintained an MIC of 0.05 μM while offering significant improvements in aqueous solubility and metabolic stability (Figure 1).⁹ Interestingly, while the presence of the amide function attached at the 4-position of the quinoline ring has been described as a vulnerable point for hydrolysis reactions, leading to potential stability issues,¹⁸ its absence has resulted in compounds with significantly reduced activity against Mtb.^13,18 In our study, the most potent simplified molecules, 4 and 5, presented MIC values of 0.3 μM and 1.8 μM, respectively (Figure 1).¹³ This represents a 6- to 36-fold decrease in potency compared to 2-(quinolin-4-yloxy)acetamides 2 and 3, suggesting that the amide group plays a critical role in the bioactivity of these compounds. To enhance the stability and efficacy of these agents, further structural modifications and optimization strategies has been investigated. One of our experimental approaches involved introducing electron-donating groups to strategically reduce the electrophilicity of the amide carbonyl, thereby mitigating hydrolysis-mediated reactions.¹⁰ Encouragingly, the results indicate enhanced metabolic stability of the designed compounds while maintaining their inhibitory potency against the bacillus.

Figure 1.

Scaffold evolution starting from 2-(quinolin-4-yloxy)acetamide 1, discovered through high-throughput screening, led to compounds 2 and 3, which showed optimized antimycobacterial activity. Molecules 4 and 5 were derived by molecular simplification of the amide function and exhibited a significant reduction in potency. Finally, the innovative design of 4-alkoxyquinolines, combining molecular simplification with chain extension explored in this study.

In this study, the proposed molecules were designed by excluding the amide group while simultaneously extending the side chain through the incorporation of alkyl groups (Figure 1). This strategic alteration aimed to access the hydrophobic regions of the potential molecular target. As previously demonstrated through molecular docking, 2-(quinolin-4-yloxy)acetamides interact with the β-subunit of the cytochrome bc₁ complex, specifically engaging residues T313, M342, and L176.¹⁶ Notably, the alkyl and aryl substituents positioned at the quinoline ring’s 4-position were oriented toward a substantial hydrophobic region within the protein, forming van der Waals interactions with the L176 residue. This site could facilitate additional hydrophobic interactions, enhancing binding affinity through closer contacts. It is noteworthy that molecular simplification performed keeping the side chain size did not result in molecules as potent as 2-(quinolin-4-yloxy)acetamides.^13,18 Based on Structure–Activity Relationship (SAR) data, the antimycobacterial activity of this chemical class has been enhanced in the presence of bulky and hydrophobic substituents adjacent to the 4-position of the heterocycle.^8−11,18,19 Thus, we hypothesized that elongating the side chain could facilitate optimal positioning of hydrophobic groups, potentially leading to improved antimycobacterial activity by enhancing intermolecular interactions. Therefore, a new series of 4-alkoxyquinolines featuring extended side chains was synthesized and evaluated against the drug-susceptible M. tuberculosis H37Rv strain. The basic structural requirements for compound potency (SAR) were determined using MIC values. The most promising structures were further tested against a panel of multidrug-resistant strains. Additionally, the viability of HepG2 and Vero cells was assessed to provide preliminary indications of molecular toxicity and selectivity. The possible mechanism of action of these compounds was investigated using a spontaneous mutant strain.¹⁶ Further insights were obtained through assessments of kinetic solubility, chemical stability, passive permeability, metabolic stability, intracellular activity and in vivo absorption profiling of the most promising compound.

2. Results and Discussion

The designed compounds were synthesized in two alkylation steps from the 4-hydroxyquinolines (6a–e). The O-alkylation of the 6a–e was carried out using 1,3-dibromopropane as the alkylating agent in the presence of cesium carbonate (Cs₂CO₃) and sodium iodide (NaI). Reactants and reagents were stirred in acetonitrile for 24 h at 25 °C to give the desired products 7a–e in 51–68% yields (Scheme 1). Although quinolinic nitrogen alkylation may be also obtained,⁸ only the O-alkylation product was observed in the reactions performed under the described experimental conditions. In the second step, the alkylation of appropriately substituted anilines was carried out in the presence of potassium carbonate (K₂CO₃) and NaI using toluene as solvent. The reaction mixture was heated at 110 °C for 48–72 h to provide structures 8a–w in 13–81% yields after purification (Scheme 1). All compounds presented spectroscopic and spectrometric data consistent with the proposed chemical structures (Supporting Information).

Scheme 1. Conditions: i = 1,3-Dibromopropane, Cs₂CO₃, NaI, CH₃CN, 25 °C, 24 h; ii = Anilines, K₂CO₃, NaI, toluene, 110 °C, 48-72 h.

Yields were quantified after purification and were not optimized.

Synthesized 4-alkoxyquinolines (8a–w) were evaluated in a whole-cell assay for their ability to inhibit the M. tuberculosis H37Rv strain using isoniazid, rifampicin, and telacebec (Q203) as positive control.²⁰ In this experiment, the MIC was defined as the lowest concentration of the compound capable of preventing colorimetric alteration on resazurin-based assay. Overall, the molecules 8a–w showed good to excellent antimycobacterial activities with MICs values ranging from 12.32 μM to 0.03 μM under the evaluated experimental conditions (Table 1).

Table 1. In Vitro Activities of the 4-Alkoxyquinolines against the M.tuberculosis H37Rv Strain.

				MIC (H37Rv)a
Entry	R6	R2	R4	(μM)
8a	OCH3	CH3	H	0.25
8b	OCH3	CH3	F	0.47
8c	OCH3	CH3	Cl	0.22
8d	OCH3	CH3	Br	0.10
8e	OCH3	CH3	I	0.09
8f	OCH3	CH3	CF3	3.20
8g	OCH3	CH3	OCF3	0.20
8h	OCH3	CH3	Me	0.24
8i	OCH3	CH3	Et	0.06
8j	OCH3	CH3	iPr	0.11
8k	Cl	CH3	Cl	0.22
8l	Cl	CH3	Br	1.55
8m	Cl	CH3	OCF3	0.19
8n	Cl	CH3	Et	0.03
8o	Cl	Et	Cl	0.11
8p	Cl	Et	Br	0.38
8q	Cl	Et	Et	0.22
8r	Br	CH3	Cl	0.10
8s	Br	CH3	Br	0.35
8t	Br	CH3	Et	0.05
8u	Br	Et	Cl	0.19
8v	Br	Et	Br	0.34
8w	Br	Et	Et	0.10
INH	-	-	-	2.3
RIF	-	-	-	0.2
Q203	-	-	-	0.009

^aMinimum inhibitory concentration (MIC) against the M. tuberculosis H37Rv strain. INH, isoniazid. RIF, rifampin. Q203, telacebec.

Notably, the unsubstituted product 8a, obtained from the reaction with aniline, was able to inhibit the bacillus with MIC value of 0.25 μM. This preliminary finding denoted that molecular simplification combined with chain extension could lead to structures with high activity against Mtb. The introduction of a fluorine atom at the 4-position of the benzene ring led to an approximate 2-fold reduction in activity, as evidenced by the 4-alkoxyquinoline 8b with a MIC of 0.47 μM. In contrast, the incorporation of chlorine (8c), bromine (8d), and iodine (8e) at this part of the compound, resulting in increased atomic volume and polarizability, correlated with improved antimycobacterial activity. Indeed, molecules 8c, 8d, and 8e exhibited MIC values of 0.22 μM, 0.10 μM, and 0.09 μM, respectively, reflecting an enhancement of roughly 2-fold to more than 5-fold when compared to activity of the fluorinated structure 8b. The presence of a trifluoromethyl group attached to the 4-position of the benzene ring significantly reduced the inhibitory activity of compound 8f. This electron-withdrawing group yielded a molecule with a MIC value of 3.20 μM. Interestingly, the introduction of an oxygen atom, forming the trifluoromethoxy group in structure 8g, restored potency, resulting in a MIC of 0.20 μM. This observation could be attributed to a more effective positioning of the trifluoromethyl group, rather than its electronic characteristics. With the inclusion of oxygen, the trifluoromethyl group deviates from the main plane of the benzene ring, possibly contributing to the heightened inhibitory capacity of the trifluoromethoxy-substituted compound 8g. The incorporation of a methyl group at the 4-position of the benzene ring was well-tolerated, exhibiting no substantial alteration in antimycobacterial activity. The methylated compound 8h inhibited the bacilli with a MIC of 0.24 μM, a value comparable to that observed for unsubstituted (8a), chlorinated (8c), and trifluoromethoxylated (8g) molecules. The increase of the alkyl chain with the presence of an ethyl group resulted in the creation of a structure endowed with potent activity against Mtb. The 4-alkoxyquinoline 8i demonstrated a MIC of 0.06 μM, which was 4-fold more potent than methyl-containing analogue 8h. Remarkably, for the first time, a simplified analogue derived from 2-(quinoline-4-yloxy)acetamides exhibited a MIC within the submicromolar range. In contrast, the introduction of branching in the alkyl substituent, using an iso-propyl group, diminished the potency of compound 8j, leading to a MIC value of 0.11 μM.

In the second round of structural modifications, chlorine, bromine, trifluoromethoxy and ethyl groups were fixed as substituents at the 4-position aniline ring. These substituents generated molecules displaying significant activity against the bacillus, forming a collection with diverse steric, electronic, and physicochemical properties. It is noteworthy that while iodine yielded a derivative with substantial inhibitory capacity, bromine was selected for subsequent modifications due to its comparable outcomes. Alteration of the methoxy group attached at 6-position of the quinoline ring by a chlorine resulted in the maintenance of inhibitory potency against M. tuberculosis H37Rv strain. Specifically, structure 8k effectively reduced the bacilli exhibiting a MIC of 0.22 μM, a value identical to that exhibited by its methoxylated counterpart 8c. In contrast, when chlorine replaced the methoxy group in the 4-alkoxyquinoline 8l, a considerable decline in activity was observed leading to a MIC of 1.55 μM. This value denoted a 15.5-fold decrease in potency when comparing compound 8l with molecule 8d. The incorporation of chlorine at the 6-position of the quinoline ring and trifluoromethoxy as a side chain attachment in structure 8m resulted in the restoration of potency as this 4-alkoxyquinoline showed MIC of 0.19 μM against Mtb. Additionally, when ethyl substituted the trifluoromethoxy group in the compound 8n, a significant enhancement in antimycobacterial activity was observed. Molecule 8n displayed a MIC value of 0.03 μM, representing an increase of over 6-fold in activity when compared to trifluoromethoxy-substituted 8m. Furthermore, the exchange of the methoxy group at the 6-position of the heterocycle in 8i with a chlorine atom in 8n led to a molecule that was 2-fold more potent. The elongation of the alkyl chain attached to the 2-position of the quinolinic ring, with the introduction of an ethyl group, improved the potency of structures 8o and 8p compared to their methyl-substituted analogues, 8k and 8l. Notably, these 4-alkoxyquinolines demonstrated MIC values of 0.11 μM and 0.38 μM for 8o and 8p, respectively, while compounds 8k and 8l displayed values of 0.22 μM and 12.32 μM, respectively. Conversely, the inclusion of an ethyl group at the 2-position of the heterocycle led to a reduction in the activity of molecule 8q by more than 7-fold in comparison to the methylated analogue 8n. Specifically, structure 8q exhibited a MIC value of 0.22 μM, whereas 8n inhibited the growth of the bacilli with a MIC of 0.03 μM.

Subsequently, in a third round of structural modifications, bromine was introduced to substitute chlorine at the 6-position of the quinoline ring. Once again, for the sake of comparability in the SAR study, chlorine, bromine, and an ethyl group were chosen as side chain substituents on the 4-alkoxyquinolines. In compound 8r, bromine was positioned at the 6-position of the heterocycle, while chlorine was retained in the side chain, resulting in a MIC of 0.10 μM. This value indicated that molecule 8r was approximately 2-fold more potent compared to the methoxylated (8c) and chlorinated (8k) derivatives, which displayed MIC values of 0.22 μM. Interestingly, the dibrominated structure 8s exhibited a MIC of 0.35 μM, representing nearly a 4.4-fold increase in potency compared to the chlorinated derivative 8l, with a MIC of 1.55 μM. Once again, the incorporation of an ethyl group attached to the aniline moiety resulted in a 4-alkoxyquinoline capable of inhibiting the M. tuberculosis H37Rv strain within the submicromolar range. Compound 8t exhibited a MIC of 0.05 μM, comparable to its methoxylated (8i) and chlorinated (8n) counterparts, which displayed MIC values of 0.06 μM and 0.03 μM, respectively. However, the presence of an ethyl group at the 2-position of the heterocyclic ring in molecule 8u reduced its potency by nearly 2-fold compared to the methyl-substituted structure 8r. The MIC of 4-alkoxyquinoline 8u was 0.19 μM, while its methyl-containing counterpart 8r exhibited a MIC of 0.10 μM. Conversely, the same exchange between methyl and ethyl resulted in a compound with nearly identical activity; both molecules 8v and 8s yielded MIC values of 0.34 μM and 0.35 μM, respectively. Finally, structure 8w exhibited a MIC of 0.10 μM, indicating that the insertion of an ethyl group at the 2-position of the heterocyclic ring diminished the antimycobacterial activity by approximately 2-fold when compared to the MIC value of the methylated derivative 8t. Altogether, the initial SAR findings appear to suggest a more pronounced correlation between the steric effects and the compound’s activity, rather than the electronic and physicochemical characteristics of the assessed molecules.

It noteworthy that isoniazid and rifampin, which constitute the principal drugs of first-line tuberculosis therapy, showed MIC values of 2.3 μM and 0.2 μM, respectively, against the M. tuberculosis H37Rv strain when evaluated under the same experimental conditions. Additionally, the clinical candidate telacebec demonstrated potent inhibition of the bacilli, with an MIC of 0.009 μM.

Employing a MIC threshold of 0.10 μM, seven 4-alkoxyquinolines (8d–e, 8i, 8n, 8r, 8t, and 8w) were selected for assessment against a panel of clinical isolate strains (Table 2). Importantly, the genomes of these multidrug-resistant tuberculosis (MDR-TB) strains have been sequenced, elucidating the genotypic alterations associated with resistance.²¹ Notably, M. tuberculosis strains PT2, PT12, and PT20 have been characterized as resistant to a spectrum of drugs, including isoniazid, rifampin, streptomycin, ethionamide, and rifabutin. Furthermore, PT12 and PT20 exhibit resistance to pyrazinamide and ethambutol, with PT12 manifesting additional resistance to amikacin and capreomycin. Interestingly, all evaluated compounds were able to inhibit the growth of MDR-TB strains, displaying MIC values below 0.06 μM. These findings not only underscore the potential effectiveness of the molecules against drug-resistant clinical isolates, but also hint at their possibly superior performance compared to the inhibition of wild-type M. tuberculosis H37Rv strain. Furthermore, it is plausible to infer that the synthesized structures exhibit no in vitro cross-resistance with key and clinically significant anti-TB drugs. Altogether, these data imply a promising potential for 4-alkoxyquinolines against both drug-susceptible and drug-resistant Mtb strains, likely acting through different molecular targets than those of the classical antimycobacterial drugs.

Table 2. In Vitro Evaluation of the Selected 4-Alkoxyquinolines against M. tuberculosis H37Rv, MDR Strains, and Assessment of HepG2 and Vero Cell Viability.

Entry	MIC H37Rv (μM)	MIC PT2 (μM)	MIC PT12 (μM)	MIC PT20 (μM)	CC50aHepG2 (μM)	CC50aVero (μM)	SIeHepG2	SIeVero
8d	0.10	<0.05	<0.05	<0.05	15.3b; 16.8c	>20b; 12.8c	153b; 168c	>200b; 128c
8e	0.09	<0.05	<0.05	<0.05	10.6b; 12.8c	8.2b; 7.5c	118b; 142c	91b; 83c
8i	0.06	<0.06	<0.06	<0.06	0.3b; 8.1c	<1b; 4.2c	5b; 135c	<17b; 70c
8n	0.03	<0.06	<0.06	<0.06	-d	-d	-	-
8r	0.10	<0.05	<0.05	<0.05	>20b; > 20c	19.8b; 11.2c	>200b; > 200c	198b; 112c
8t	0.05	<0.05	<0.05	<0.05	>20b; > 20c	>20b; 11.8c	>400b; > 400c	>400b; 236c
8w	0.10	<0.05	<0.05	<0.05	>20b; 8.3c	>20b; 9.1c	>200b; 83c	>200b; 91c
INH	2.3	291.7	291.7	291.7	-	-	-	-
RIF	0.2	>48.6	>48.6	>48.6	-	-	-	-

^aThe toxicity and selectivity of the compounds were investigated using HepG2 and Vero cell lines. The outcomes were quantified as the concentration causing a 50% reduction in cell viability (CC₅₀) using MTT and Neutral Red assays.

^bDetermined by the MTT method.

^cDetermined by the Neutral Red method.

^dLimited solubility within the applied experimental conditions.

^eSelectivity index (SI = CC₅₀/MIC H37Rv). INH, Isoniazid. RIF, Rifampin.

Additionally, the selectivity and preliminary toxicity assessment of selected compounds were performed by evaluating the viability of HepG2 and Vero cell lines after exposure to the molecules (Table 2). It is noteworthy that the concentration required to reduce cell viability by 50% (CC₅₀) was determined using the MTT (methyl-thiazolyl-tetrazolium)²² and neutral red²³ protocols. The MTT approach probes mitochondrial activity, whereas the neutral red assay provides insights into the lysosomal integrity of the cells. Unfortunately, owing to solubility challenges arising at diminished cosolvent concentrations, was unfeasible to subject molecule 8n to the mentioned experiments. Among the evaluated structures, 4-alkoxyquinoline 8i exhibited a notable capacity to inhibit the viability of HepG2 and Vero cells. Notably, apparent cytotoxicity toward mammalian cells was observed in the MTT assay, but this was not evident in the assessment utilizing neutral red. When employing MTT conditions for the assessment of cell viability of 8i, the selectivity indices (CC₅₀/MIC H37Rv) were 5 for HepG2 and less than 17 for Vero cells. Conversely, under neutral red protocol conditions, the selectivity index values were 135 for HepG2 and 70 for Vero cell lines. These results underscore the significance of employing multiple methodologies for evaluating viability in cell assays. The other compounds did not exert substantial alterations in the viability of the investigated cell lines. The presented selectivity indices surpassed 83, exceeding the minimum 10-fold ratio commonly employed in drug discovery campaigns.²⁴ It is important to emphasize that among the molecules demonstrating low apparent toxicity to mammalian cells, structure 8t displayed the highest selectivity indices, with values equal to or exceeding 236 for both evaluated cell lines.

Furthermore, the aqueous solubility of the selected 4-alkoxyquinolines (8d–e, 8i, 8n, 8r, 8t, and 8w) was assessed in solutions simulating stomach (pH 1.2), plasma (pH 7.4), and intestinal (pH 9.1) pH conditions (Table 3). As expected, the compounds exhibited substantially greater solubility at pH 1.2 compared to pH 7.4 and pH 9.1. The selected molecules demonstrated solubility ranging from 200 μM to over 2,230 μM at pH 1.2. Under plasma-like pH conditions, the structures displayed solubility ranging from 0.06 μM to 71.2 μM. Remarkably, these values position certain synthesized 4-alkoxyquinolines with solubility profiles surpassing the threshold of 25 μM, which has been described in early TB drug discovery programs.²⁵ Finally, under pH 9.1, the aqueous solubilities of the compounds were obtained with values ranging from 0.37 μM to 61.4 μM. It is important to highlight that molecule 8n exhibited the lowest aqueous solubility among the evaluated structures, aligning with expectations due to the impracticability of conducting cellular assays for toxicity and selectivity. This 4-alkoxyquinoline demonstrated solubility of merely 0.06 μM and 0.37 μM at pH 7.4 and pH 9.1, respectively. Additionally, despite multiple attempts, compound 8w could not be quantified through any of the methods tested using UHPLC-DAD.

Table 3. Solubility Profiles of Selected 4-Alkoxyquinolines.

	Solubilitya
Entry	pH 1.2b(μM)	pH 7.4c(μM)	pH 9.1d(μM)
8d	>2,500	71.2	21.03
8e	>2,230	2.9	2.49
8i	2,650	70.6	61.37
8n	200	0.06	0.37
8r	>2,470	15.0	57.18
8t	>2,500	35.3	30.22
8w	-	-	-

^aConcentration was determined by UHPLC-DAD after shaking incubation of the compound suspensions at 25 °C for 4 h.

^bUsing a 0.1 M HCl solution.

^cUsing PBS.

^dUsing a 0.1 M NH₄HCO₃ solution.

Based on the outcomes from the antimycobacterial assays conducted on both susceptible and resistant Mtb strains, as well as the evaluation of selectivity using two mammalian cell lines and the solubility profile, molecule 8t was selected for subsequent investigations. The objective was to assess the drug-like attributes of this structure as a representative of its chemical class, thus assessing its potential for integration into future preclinical programs.

Initially, the involvement of the qcrB gene product in the antitubercular activity demonstrated by 4-alkoxyquinoline 8t was examined using a spontaneously generated mutant M. tuberculosis strain resistant to 2-(quinolin-4-yloxy)acetamides (Table 4). This resistant strain is known to harbor a mutation in the qcrB gene, leading to a T313A amino acid substitution.¹⁶ The MIC value observed against this resistant strain for the compound 8t was 3.13 μM, which was approximately 62.5-fold higher than the value exhibited against the M. tuberculosis H37Rv strain (MIC = 0.05 μM). These findings suggest that the β-subunit of the cytochrome bc₁ oxidase complex, a component of the respiratory electron transport chain, plays a central role in the antimycobacterial activity of this molecule. Therefore, in line with our previous findings¹³ and those of other research groups,²⁶ it appears that molecular simplification and chain extension preserves the antitubercular action mechanism exhibited by 2-(quinolin-4-yloxy)acetamides. Moreover, structure 8t underwent evaluation against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, as well as the multidrug-resistant clinical isolates Acinetobacter baumannii and Klebsiella pneumoniae (Table 4). At a concentration of 20 μM, the 4-alkoxyquinoline was not able to inhibit bacterial growth, suggesting a potential selectivity of this compound toward Mtb inhibition.

Table 4. In Vitro Activities of the Selected 4-Alkoxyquinoline 8t against a 2-(Quinolin-4-yloxy)acetamide-Resistant Strain, Gram-Positive and Gram-Negative Bacterial Strains, and Assessment of Its Chemical Stability, Permeability, and Metabolic Stability.

Entry	Properties
8t	MIC qcrB-T313Aa = 3.13 μM
	MIC S. aureus = > 20 μM
	MIC E. coli = > 20 μM
	MIC A. baumannii = > 20 μM
	MIC K. pneumoniae = > 20 μM
	Chemical Stabilityb pH 1.2c = 100%
	Chemical Stabilityb pH 7.4d = 5.3%
	Chemical Stabilityb pH 9.1e = 6.0%
	PAMPA = 0.2 × 10−6 cm/s
	Clintf = 58 mL/min/kg
	t1/2g = 7.0 min

^a2-(Quinolin-4-yloxy)acetamide-resistant spontaneous mutant containing a unique alteration in the qcrB gene (ACC to GCC at nucleotide position 937 or a T313A amino substitution).

^bPercentage of remaining compound after incubation at 37 °C for 24 h.

^c0.1 M HCl solution.

^dUsing PBS.

^eUsing a 0.1 M NH₄HCO₃ solution.

^fIntrinsic clearance of rat liver microsomes.

^gHalf-life.

Furthermore, some in vitro ADME properties were examined for molecule 8t (Table 4). It is noteworthy that the stability of structure 8t remained unaltered after 24 h of incubation at pH 1.2. Conversely, when subjected to neutral (pH 7.4) or alkaline (pH 9.1) conditions, the compound’s concentration was significantly reduced. This data suggests that utilizing hydrochloride as a counterion could serve as an alternative to stabilize the 4-alkoxyquinoline in aqueous solutions. In terms of passive permeability, compound 8t exhibited low permeability, as evaluated by parallel artificial membrane permeability assay (PAMPA). Specifically, molecule 8t exhibited a permeability of 0.2 × 10^–6 cm/s, whereas alprenolol, employed as a positive control, exhibited a permeability of 4.1 × 10^–6 cm/s under the applied experimental conditions. Lastly, the metabolic stability of structure 8t was assessed in the presence of rat liver microsomes. The 4-alkoxyquinoline exhibited a high metabolic rate with clearance of 58 mL/min/kg which resulted in a half-life of 7 min. Notably, these values closely resembled those exhibited by verapamil, utilized as a positive control in the experiment. Verapamil displayed a clearance of 56 mL/min/kg along with a half-life of 7.6 min under the applied experimental conditions.

Based on the results describing antimycobacterial activity against susceptible and drug-resistant strains, selectivity using eukaryotic cells, preliminary mechanism of action data, and in vitro ADME profile, 4-alkoxyquinoline 8t was evaluated in a murine macrophage model of TB infection using the first-line drug isoniazid as positive control (Table 5). The objective was to determine whether the physicochemical characteristics of the molecule allow it to pass through different cellular compartments and reduce the bacterial load within macrophages. Compared to the early control group, macrophages in the untreated group showed an increase of approximately 1.9 log₁₀ colony-forming units (log₁₀ CFU) over 5 days, indicating intracellular growth of Mtb. Treatment with 1 μM and 5 μM of the compound 8t prevented bacterial growth and maintained stable bacterial loads inside the macrophages. The groups treated with 1 μM and 5 μM reduced the Mtb load by 1.27 log₁₀ CFU and 1.07 log₁₀ CFU, respectively, compared to the untreated group. However, when compared to the early control group, the number of colonies was not reduced, indicating a bacteriostatic effect of 4-alkoxyquinoline 8t on the intracellular growth of the M. tuberculosis H37Rv strain. It is important to note the lack of a dose–response effect regarding the intracellular activity of the structure, as there was no significant difference in bacteriostatic effects between the groups of macrophages treated with 1 μM and 5 μM of the compound 8t. As expected, isoniazid effectively reduced macrophage infection compared to both the early control and untreated control groups. At a concentration of 1 μM, the drug lowered the bacterial load to 2.25 log₁₀ CFU, demonstrating its bactericidal effect under the given test conditions.

Table 5. Intracellular Activity of 4-Alkoxyquinoline 8t in Murine Macrophages Model of TB Infection^a.

Entry	Log10 CFU/well (Mean ± SD)
Early Control	3.28 ± 0.11
Untreated	5.17 ± 0.40b
8t (1 μM)	3.90 ± 0.30d
8t (5 μM)	4.10 ± 0.02d
INH (1 μM)	2.25 ± 0.15c,d

^aSD, standard deviation.

^bP < 0.0001 compared to early control group.

^cP < 0.002 compared to early control group.

^dP < 0.0001 compared to untreated group which was dosed with 0.5% DMSO. Multiple comparisons were performed with Tukey’s test. INH, isoniazid.

Considering the potential challenges in terms of pharmacokinetic exposure occasioned by its low permeability and high metabolic rate observed in vitro, an experiment was conducted to determine whether compound 8t could be effectively absorbed and reach systemic circulation in effective amounts after administration via gavage to mice (Table 6). Employing a concentration of 300 mg/kg, the presence of molecule 8t in plasma was quantified, revealing concentrations exceeding 7.49 μM for a span of up to 8 h. Notably, the peak plasma concentration (C_max) obtained for the structure 8t was 32.1 ± 3.9 μM, achieved 1 h after administration (T_max). The observed half-life (t_1/2) was 4.3 ± 1.2 h, and the exposure to the 4-alkoxyquinoline 8t from time 0 to 8 h was 127.5 ± 5.7 μM*h. Furthermore, the elimination rate constant (K_e) indicated that the concentration of compound 8t decreased at a rate of 0.16 (16%) over the evaluated time interval. It is noteworthy that incomplete drug elimination may influence the values reported for t_1/2, area under the curve (AUC), and K_e. Lastly, after an 8 h period after oral administration, the measured molecule concentration was 9.80 μM. Notably, this concentration stands 196-fold higher than the MIC exhibited by the compound against the M. tuberculosis H37Rv strain. These preliminary absorption findings underscore that structure 8t is probably capable of absorption, allowing it to penetrate the systemic circulation of the animals and maintain substantial concentrations for at least 8 h, significantly surpassing the levels required to inhibit the growth of the bacillus.

Table 6. Absorption Profile and Preliminary Pharmacokinetic Parameters of 4-Alkoxyquinoline 8t after Oral Gavage Administration to Mice.

	Concentrationa	Pharmacokineticsb
Time (h)	μM ± SD	Parameters	Mean ± SD
0.25	7.49 ± 2.72	Cmax (μM)	32.1 ±3.9
0.5	11.10 ± 1.24	Tmax (h)	1
0.75	15.73 ± 1.61	t1/2 (h)	4.3 ± 1.2
1	32.15 ± 3.94	AUC0-t (μM*h)	127.5 ± 5.7
4	13.45 ± 2.01	Ke (h–1)	0.16
8	9.80 ± 2.13

^aPlasma concentration was determined using UHPLC-DAD after a single dose (300 mg/kg) of 8t, the results were presented as the mean of two animals over time, accompanied by the standard deviation.

^bNoncompartmental pharmacokinetics analysis. C_max, peak concentration. T_max, time to peak plasma concentration. t_1/2, elimination half-life. AUC_0-t, area under the concentration–time curve from 0 to time t. K_e, elimination rate constant.

3. Conclusion

In this study, we present the design and synthesis of a novel series of 4-alkoxyquinolines and demonstrate their in vitro antimycobacterial activities. The synthetic procedures were executed using readily available reagents and reactants within well established and straightforward protocols. Additionally, the compounds exhibited potent activity against both drug-sensitive and MDR-TB strains. Interestingly, the lead molecule manifests its antitubercular activity by targeting the cytochrome bc₁ complex, thereby expanding the potential utility of this chemical class in addressing nonreplicating forms of Mtb. Furthermore, the design strategy, involving molecular simplification and chain extension, yielded a compound characterized by favorable kinetic solubility and chemical stability using acidic conditions. This molecule also exhibited low permeability and a high metabolism rate in the presence of rat microsomes, a metabolism profile akin to that of verapamil. Additionally, the leading structure demonstrated bacteriostatic activity in a murine macrophages model of TB infection. Lastly, a preliminary absorption assessment of the lead compound revealed, for the first time, a simplified structure derived from 2-(quinolin-4-yloxy)acetamides that exhibited promising in vivo exposure following oral administration to mice. This suggests the potential of this chemical class to yield novel anti-TB drug candidates. Ongoing efforts are focused on further structural modifications to achieve a more comprehensive SAR, and these results will be presented in due course.

4. Experimental Section

4.1. Synthesis and Structure: Apparatus and Analysis

All solvents and reagents used in this study were obtained from commercial suppliers and were employed in the experiments without any additional purification steps. The progress of the reactions was monitored by thin-layer chromatography (TLC). Melting points were determined using a Microquímica MQAPF-302 apparatus. ¹H and ¹³C NMR spectra were acquired on an Avance III HD Bruker spectrometer located at the Pontifical Catholic University of Rio Grande do Sul. Chemical shifts (δ) were reported in parts per million (ppm) with reference to DMSO-d₆ or CDCl₃, which served as the solvents, and to tetramethylsilane (TMS), used as an internal standard. NMR spectra were processed using MestReNova v.14.0.0–23239 (2019 Mestrelab Research S.L.). In the ¹H NMR spectra, the following abbreviations were used to denote splitting patterns: s for Singlet, d for Doublet, t for Triplet, q for Quartet, p for Pentet (or quintet), m for Multiplet, and dd for Doublet of doublets. High-resolution mass spectra (HRMS) were acquired using either an LTQ Orbitrap Discovery (Thermo Fisher Scientific, Bremen, Germany) or a MicroTof-QII (Bruker Corporation, Bremen, Germany) mass spectrometer, both equipped with an electrospray ionization source (ESI) operating in positive ionization mode. The purity of the compounds was assessed using a Dionex Ultimate 3000 UHPLC chromatograph (Dionex Corporation, Germering, Germany). This UHPLC system was equipped with a binary pump, an automatic injector, and a diode array (DAD) detector. Data acquisition and processing were performed using Chromeleon 6.80 SR11 (Build 3160) software. The analytical conditions included an RP column (5 μm Nucleodur C-18, 250 × 4.6 mm), a flow rate of 1.5 mL/min, and UV detection at 254–260 nm. The elution gradient consisted of 100% water (0.1% acetic acid) from 0 to 7 min, followed by a linear gradient from 100% water (0.1% acetic acid) to 90% acetonitrile/methanol (1:1, v/v) from 7 to 15 min (15–30 min). Subsequently, the system returned to 100% water (0.1% acetic acid) within 5 min (30–35 min) and was maintained for an additional 10 min (35–45 min). All evaluated compounds exhibited a purity of >95%.

4.2. General Procedure for the Synthesis of 4-Bromoalkoxyquinolines7a–e

The compounds 7a–e were synthesized from 4-hydroxyquinolines 6a–e. The reaction was carried out in a round-bottom flask containing the respective 4-hydroxyquinoline (2 mmol), 1,3-dibromopropane (1.21 g, 6 mmol), cesium carbonate (1.95 g, 6 mmol), sodium iodide (0.075 g, 0.5 mmol) in acetonitrile (50 mL). The reaction mixture was stirred at room temperature (25 °C) for 24 h. Subsequently, extraction was performed using chloroform (3 × 50 mL) and saturated ammonium chloride solution. The organic layer was dried over magnesium sulfate and then concentrated under reduced pressure. The purification of 4-bromoalkoxyquinolines 7a–e was accomplished by chromatography using silica gel as the stationary phase and a mobile phase consisting of either 100% ethyl acetate or a mixture of ethyl acetate and hexane in a 3:7 ratio.

4.3. General Procedure for the Synthesis 4-Alkoxyquinolines8a–w

The compounds 8a–w were synthesized from alkylation reaction involving appropriately substituted anilines. The reactions were conducted in a round-bottom flasks containing the 4-bromoalkoxyquinoline of interest (1 mmol), the respective aniline (2 mmol), potassium carbonate (0.41 g, 3 mmol), sodium iodide (0.15 g, 1 mmol), and toluene (20 mL). The reaction mixture was heated at 110 °C for 48–72 h. Afterward, extraction was performed using chloroform (3 × 50 mL) and saturated ammonium chloride solution. The organic layer was dried over magnesium sulfate and then concentrated under reduced pressure. The purification of the products was conducted by a recrystallization process using ethyl acetate, or by employing chromatographic separation with silica gel as the stationary phase and a mobile phase comprising mixtures of hexane and ethyl acetate in ratios of 3:7 or 1:1, or alternatively using a mixture of chloroform and methanol in ratios of 99:1, 98:2, or 97:3.

4.4. N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)aniline (8a)

column chromatography on silica gel (chloroform: methanol, 98:2), pinkish solid, yield: 0.148 g (46%); mp.: 165–167 °C; UHPLC: 96% (t_R = 13.54 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.23 (p, J = 6.3 Hz, 3H), 2.84 (s, 3H), 3.32 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 4.62 (t, J = 6.2 Hz, 2H), 6.56 (t, J = 7.3 Hz, 1H), 6.66 (d, J = 7.9 Hz, 2H), 7.05–7.12 (m, 2H), 7.51 (d, J = 3.2 Hz, 2H), 7.71 (dd, J = 9.3, 2.8 Hz, 1H), 8.09 (d, J = 9.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 20.25, 27.52, 55.94, 69.00, 101.06, 103.71, 112.36 (2C), 116.09, 120.15, 121.47, 125.65, 128.87 (2C), 133.81, 148.26, 156.04, 158.25, 165.49; HRMS (ESI) m/z: 323.1748 [M + H]⁺; calcd. for C₂₀H₂₃N₂O₂: 323.1754.

4.5. 4-Fluoro-N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)aniline (8b)

column chromatography on silica gel (chloroform: methanol, 97:3), white solid, yield: 0.129 g (38%); mp.: 185–187 °C; UHPLC: 96% (t_R = 13.82 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.22 (p, J = 6.4 Hz, 2H), 2.83 (s, 3H), 3.28 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 4.61 (t, J = 6.3 Hz, 2H), 6.60–6.66 (m, 2H), 6.88–6.95 (m, 2H), 7.47–7.53 (m, 2H), 7.69 (dd, J = 9.2, 2.8 Hz, 1H), 8.08 (d, J = 9.2 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 20.40, 27.54, 55.91, 68.92, 101.01, 103.65, 112.84, 112.92, 115.09 (2C), 115.31 (2C), 120.12, 121.72, 125.47, 134.14, 145.25, 156.06, 158.16, 165.28; HRMS (ESI) m/z: 341.1651 [M + H]⁺; calcd. for C₂₀H₂₂FN₂O₂: 341.1660.

4.6. 4-Chloro-N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)aniline (8c)

column chromatography on silica gel (chloroform: methanol, 98:2), yellowish solid, yield: 0.289 g (81%); mp.: 193–196 °C; UHPLC: 97% (t_R = 14.44 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.21 (p, J = 6.4 Hz, 2H), 2.84 (s, 3H), 3.29 (t, J = 6.5 Hz, 2H), 3.95 (s, 3H), 4.61 (t, J = 6.2 Hz, 2H), 6.59–6.66 (m, 2H), 7.04–7.10 (m, 2H), 7.47–7.54 (m, 2H), 7.70 (dd, J = 9.3, 2.8 Hz, 1H), 8.08 (d, J = 9.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 20.32, 27.46, 55.93, 68.92, 101.07, 103.69, 113.32 (2C), 118.83, 120.14, 121.56, 125.59, 128.50 (2C), 133.93, 147.59, 156.04, 158.22, 165.41. HRMS (ESI) m/z: 357.1381 [M + H]⁺; calcd. for C₂₂H₂₂ClN₂O₂: 357.1364.

4.7. 4-Bromo-N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)aniline (8d)

recrystallized from ethyl acetate, white solid, yield: 0.177 g (44%); mp.: 200–204 °C; UHPLC: 96% (t_R = 14.59 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.20 (p, J = 6.3 Hz, 2H), 2.82 (s, 3H), 3.28 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 4.60 (t, J = 6.2 Hz, 2H), 6.55–6.59 (m, 2H), 7.14–7.19 (m, 2H), 7.47 (s, 1H), 7.53 (d, J = 2.8 Hz, 1H), 7.70 (dd, J = 9.2, 2.8 Hz, 1H), 8.08 (d, J = 9.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 20.30, 27.44, 39.21, 55.87, 68.83, 101.18, 103.58, 106.14, 113.86 (2C), 120.16, 121.59, 125.47, 131.23 (2C), 133.98, 147.88, 156.02, 158.21, 165.40; HRMS (ESI) m/z: 401.0860 [M + H]⁺; calcd. for C₂₀H₂₂BrN₂O₂: 401.0859.

4.8. 4-Iodo-N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)aniline (8e)

recrystallized from ethyl acetate, yellowish solid, yield: 0.211 (47%); mp.: 198–199 °C; UHPLC: 96% (t_R = 14.87 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.19 (p, J = 6.4 Hz, 2H), 2.80 (s, 3H), 3.28 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 4.57 (t, J = 6.2 Hz, 2H), 6.45–6.49 (m, 2H), 7.30–7.33 (m, 2H), 7.45 (s, 1H), 7.51 (d, J = 2.8 Hz, 1H), 7.69 (dd, J = 9.3, 2.8 Hz, 1H), 8.03 (d, J = 9.2 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 20.74, 27.47, 55.90, 68.66, 75.95, 101.02, 103.55, 114.63 (2C), 120.15, 122.21, 125.36, 134.68, 137.08 (2C), 148.38, 156.16, 158.11, 165.08; HRMS (ESI) m/z: 449.0734 [M + H]⁺; calcd. for C₂₂H₂₂IN₂O₂: 449.0721.

4.9. N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)-4-(trifluoromethyl)aniline (8f)

recrystallized from ethyl acetate, yellow solid, yield: 0.098 g (25%); mp.: 199–203 °C; UHPLC: 96% (t_R = 14.69 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.24 (p, J = 6.4 Hz, 2H), 2.83 (s, 3H), 3.38 (t, J = 6.6 Hz, 3H), 3.95 (s, 3H), 4.62 (t, J = 6.2 Hz, 2H), 6.72 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.51–7.55 (m, 2H), 7.72 (dd, J = 9.2, 2.8 Hz, 1H), 8.07 (d, J = 9.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 20.31, 27.37, 55.95, 68.86, 101.18, 103.72, 111.27, 114.98, 115.29, 120.20, 121.53, 123.97, 125.74, 126.16, 126.20, 126.65, 133.84, 151.63, 156.10, 158.31, 165.56; HRMS (ESI) m/z: 391.1643 [M + H]⁺; calcd. for C₂₁H₂₂F₃N₂O₂: 391.1628.

4.10. N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)-4-(trifluoromethoxy)aniline (8g)

recrystallized from ethyl acetate, white solid, yield: 0.065 g (16%); mp.: 181–188 °C; UHPLC: 97% (t_R = 14.89 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.14 (p, J = 6.5 Hz, 2H), 2.54 (s, 2H), 3.28 (q, J = 6.5 Hz, 2H), 3.86 (s, 3H), 4.33 (t, J = 6.2 Hz, 2H), 6.04 (t, J = 5.6 Hz, 1H), 6.60–6.67 (m, 2H), 6.89 (s, 1H), 7.05 (d, J = 8.5 Hz, 2H), 7.32 (dd, J = 9.1, 2.9 Hz, 1H), 7.38 (d, J = 2.9 Hz, 1H), 7.76 (d, J = 9.1 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 25.49, 28.38, 55.78, 66.46, 100.23, 102.44, 112.69 (2C), 120.34, 121.77, 122.55 (2C), 130.02, 138.75, 138.77, 144.52, 148.57, 156.70, 157.57, 160.32; HRMS (ESI) m/z: 407.1570 [M + H]⁺; calcd. for C₂₁H₂₂F₃N₂O₃: 407.1577.

4.11. N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)-4-methylaniline (8h)

column chromatography on silica gel (chloroform: methanol, 98:2), brownwish solid, yield: 0.101 g (30%); mp.: 171–175 °C; UHPLC: 96% (t_R = 13.47 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.24 (s, 3H), 2.87 (s, 3H), 3.40 (p, 2H), 3.56 (t, J = 11.0 Hz, 3H), 3.96 (s, 3H), 4.62 (t, J = 5.8 Hz, 2H), 7.13 (d, J = 8.8 Hz, 4H), 7.46–7.54 (m, 2H), 7.66–7.73 (m, 1H), 8.34 (d, J = 8.7 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 20.38, 20.84, 21.01, 26.56, 56.62, 68.95, 101.63, 104.25, 120.67, 121.92, 123.28, 126.00, 130.22 (2C), 130.39 (2C), 134.39, 137.30, 156.29, 158.73, 165.62; HRMS (ESI) m/z: 337.1897 [M + H]⁺; calcd. for C₂₁H₂₅N₂O₂: 337.1911.

4.12. 4-Ethyl-N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)aniline (8i)

recrystallized from ethyl acetate, yellowish solid, yield: 0.088 g (25%); mp.: 174–178 °C; UHPLC: 95% (t_R = 14.19 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.10 (t, J = 7.6 Hz, 3H), 2.21 (p, J = 6.3 Hz, 2H), 2.44 (q, J = 7.5 Hz, 2H), 2.80 (s, 3H), 3.29 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 4.60 (t, J = 6.2 Hz, 2H), 6.54–6.61 (m, 2H), 6.88–6.95 (m, 2H), 7.47 (s, 1H), 7.53 (t, J = 2.8 Hz, 1H), 7.70 (ddd, J = 9.3, 2.9, 1.2 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 15.99, 20.58, 27.20, 27.57, 39.68, 55.88, 68.82, 101.00, 103.56, 112.45 (2C), 120.11, 121.93, 125.46, 128.08 (2C), 131.28, 134.31, 146.20, 156.09, 158.13, 165.25; HRMS (ESI) m/z: 351.2053 [M + H]⁺; calcd. for C₂₂H₂₇N₂O₂: 351.2067.

4.13. 4-Isopropyl-N-(3-((6-methoxy-2-methylquinolin-4-yl)oxy)propyl)aniline (8j)

recrystallized from ethyl acetate, yellowish solid, yield: 0.193 g (53%); mp.: 166–170 °C; UHPLC: 97% (t_R = 15.19 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.13 (d, J = 6.9 Hz, 7H), 2.22 (p, J = 6.4 Hz, 2H), 2.71 (h, J = 6.9 Hz, 1H), 2.82 (s, 3H), 3.30 (t, J = 6.6 Hz, 2H), 3.95 (s, 3H), 4.61 (t, J = 6.2 Hz, 2H), 6.57–6.65 (m, 2H), 6.93–7.00 (m, 2H), 7.46–7.54 (m, 2H), 7.70 (dd, J = 9.2, 2.8 Hz, 1H), 8.03 (d, J = 9.2 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 20.46, 24.13 (2C), 27.51, 32.39, 40.09, 55.91, 68.89, 101.05, 103.61, 112.62 (2C), 120.12, 121.72, 125.54, 126.58 (2C), 134.06, 136.34, 145.98, 156.06, 158.17, 165.36; HRMS (ESI) m/z: 365.2228 [M + H]⁺; calcd. for C₂₃H₂₉N₂O₂: 365.2224.

4.14. 4-Chloro-N-(3-((6-chloro-2-methylquinolin-4-yl)oxy)propyl)aniline (8k)

column chromatography on silica gel (hexane: ethyl acetate, 70:30, then 50:50), brown solid, yield: 0.166 g (46%); mp.: 118–121 °C; UHPLC: 96% (t_R = 16.21 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.13 (p, J = 6.1 Hz, 2H), 2.59 (d, J = 1.5 Hz, 3H), 3.25 (q, J = 6.3 Hz, 2H), 4.34 (t, J = 6.1 Hz, 2H), 6.03 (s, 1H), 6.57–6.66 (m, 2H), 7.00 (d, J = 1.8 Hz, 1H), 7.08 (dd, J = 8.7, 1.5 Hz, 2H), 7.69 (dd, J = 9.0, 2.4 Hz, 1H), 7.86 (dd, J = 8.9, 1.7 Hz, 1H), 8.04 (d, J = 2.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 25.27, 27.74, 66.46, 102.68, 113.28 (2C), 118.71, 120.08, 120.27, 128.54 (2C), 129.27, 130.07 (2C), 146.57, 147.72, 159.92, 160.74; HRMS (ESI) m/z: 361.0881 [M + H]⁺; calcd. for C₁₉H₁₉Cl₂N₂O: 361.0869.

4.15. 4-Bromo-N-(3-((6-chloro-2-methylquinolin-4-yl)oxy)propyl)aniline (8l)

column chromatography on silica gel (chloroform: methanol, 99:1, then 97:3), yellow solid, yield: 0.166 g (41%); mp.: 135–139 °C; UHPLC: 97% (t_R = 17.36 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 2.24 (p, J = 6.3 Hz, 2H), 2.69 (s, 4H), 3.41 (t, J = 6.6 Hz, 2H), 4.32 (t, J = 5.9 Hz, 2H), 6.49–6.54 (m, 2H), 6.63 (s, 1H), 7.22–7.26 (m, 3H), 7.60 (ddd, J = 9.0, 2.4, 0.7 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 2.4 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ ppm 25.29, 28.86, 41.02, 66.82, 102.13, 109.43, 114.49 (2C), 120.63, 120.84, 129.13, 131.14, 131.33, 132.16 (2C), 146.29, 147.04, 160.32, 161.27; HRMS (ESI) m/z: 405.0379 [M + H]⁺; calcd. for C₁₉H₁₉BrClN₂O: 405.0364.

4.16. N-(3-((6-Chloro-2-methylquinolin-4-yl)oxy)propyl)-4-(trifluoromethoxy)aniline (8m)

column chromatography on silica gel (chloroform: methanol, 99:1), white solid, yield: 0.082g (20%); mp.: 122–126 °C; UHPLC: 98% (t_R = 16.85 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 2.25 (p, J = 6.3 Hz, 2H), 2.66 (s, 3H), 3.43 (t, J = 6.6 Hz, 2H), 4.28 (t, J = 5.9 Hz, 2H), 6.59–6.62 (m, 3H), 7.01–7.07 (m, 2H), 7.59 (dd, J = 9.0, 2.4 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 2.4 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ ppm 25.70, 28.66, 41.00, 66.36, 101.87, 112.97 (2C), 119.46, 120.46, 120.69, 120.72, 121.99, 122.55 (2C), 129.64, 130.73, 130.83, 140.56, 140.58, 146.75, 146.90, 160.45, 160.63; HRMS (ESI) m/z: 411.1092 [M + H]⁺; calcd. for C₂₀H₁₉ClF₃N₂O₂: 411.1082.

4.17. N-(3-((6-Chloro-2-methylquinolin-4-yl)oxy)propyl)-4-ethylaniline (8n)

column chromatography on silica gel (hexane: ethyl acetate, 50:50), dark brown solid, yield: 0.124 g (35%); mp.: 113–118 °C; UHPLC: 98% (t_R = 21.58 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 1.19 (t, J = 7.6 Hz, 3H), 2.24 (p, J = 6.3 Hz, 2H), 2.54 (q, J = 7.6 Hz, 2H), 2.65 (s, 3H), 3.43 (t, J = 6.6 Hz, 2H), 4.28 (t, J = 6.0 Hz, 2H), 6.57–6.64 (m, 3H), 6.99–7.06 (m, 2H), 7.58 (dd, J = 9.0, 2.4 Hz, 1H), 7.87 (dd, J = 8.9, 0.5 Hz, 1H), 8.07–8.12 (m, 1H); ¹³C NMR (101 MHz, CDCl₃) δ ppm 15.96, 25.83, 27.92, 28.91, 41.13, 66.44, 101.85, 112.99 (2C), 120.55, 120.77, 128.70 (2C), 129.78, 130.59, 130.68, 133.59, 145.91, 147.14, 160.47, 160.63; HRMS (ESI) m/z: 355.1580 [M + H]⁺; calcd. for C₂₁H₂₄ClN₂O: 355.1572.

4.18. 4-Chloro-N-(3-((6-chloro-2-ethylquinolin-4-yl)oxy)propyl)aniline (8o)

column chromatography on silica gel (hexane: ethyl acetate, 70:30, then 50:50), brown solid, yield: 0.049 g (13%); mp.: 124–129 °C; UHPLC: 98% (t_R = 16.80 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.30 (t, J = 7.6 Hz, 3H), 2.13 (p, J = 6.5 Hz, 2H), 2.86 (q, J = 7.6 Hz, 2H), 3.26 (q, J = 6.3 Hz, 2H), 4.35 (t, J = 6.1 Hz, 2H), 5.97 (t, J = 5.6 Hz, 1H), 6.57–6.65 (m, 2H), 6.98 (s, 1H), 7.06–7.12 (m, 2H), 7.69 (dd, J = 9.0, 2.5 Hz, 1H), 7.88 (d, J = 9.1 Hz, 1H), 8.06 (d, J = 2.4 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 13.38, 27.75, 31.78, 66.36, 101.62, 113.25 (2C), 118.75, 120.25, 120.28, 128.54 (2C), 129.28, 129.97, 130.30, 146.63, 147.66, 160.01, 165.47; HRMS (ESI) m/z: 375.1036 [M + H]⁺; calcd. for C₂₀H₂₁Cl₂N₂O: 375.1025.

4.19. 4-Bromo-N-(3-((6-chloro-2-ethylquinolin-4-yl)oxy)propyl)aniline (8p)

column chromatography on silica gel (hexane: ethyl acetate, 70:30), black solid, yield: 0.067 g (16%); mp.: 115–119 °C; UHPLC: 96% (t_R = 18.20 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 1.37 (t, J = 7.6 Hz, 4H), 2.25 (p, J = 6.3 Hz, 2H), 2.91 (q, J = 7.6 Hz, 2H), 3.42 (t, J = 6.6 Hz, 2H), 4.30 (t, J = 5.8 Hz, 2H), 6.51–6.55 (m, 2H), 6.63 (s, 1H), 7.24–7.28 (m, 3H), 7.72 (dd, J = 8.9, 2.3 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 8.26 (d, J = 2.3 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ pmm 13.89, 28.66, 32.78, 40.97, 66.38, 100.67, 109.20, 114.35 (2C), 118.73, 121.20, 123.95, 130.14, 132.07 (2C), 133.16, 146.93, 147.34, 160.62, 165.74; HRMS (ESI) m/z: 419.0533 [M + H]⁺; calcd. for C₂₀H₂₁BrClN₂O: 419.0520.

4.20. N-(3-((6-chloro-2-ethylquinolin-4-yl)oxy)propyl)-4-ethylaniline (8q)

column chromatography on silica gel (hexane: ethyl acetate, 70:30), brown solid, yield: 0.122 g (33%); mp.: 76–82 °C; UHPLC: 95% (t_R = 22.36 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 1.19 (t, J = 7.6 Hz, 3H), 1.36 (t, J = 7.6 Hz, 3H), 2.24 (p, J = 6.3 Hz, 2H), 2.54 (q, J = 7.6 Hz, 2H), 2.91 (q, J = 7.6 Hz, 2H), 3.43 (t, J = 6.6 Hz, 2H), 4.29 (t, J = 5.9 Hz, 2H), 6.59–6.64 (m, 3H), 7.01–7.05 (m, 2H), 7.58 (dd, J = 9.0, 2.4 Hz, 1H), 7.90 (dd, J = 9.0, 0.5 Hz, 1H), 8.11 (d, J = 2.3 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ ppm 13.95, 15.96, 27.92, 28.92, 32.78, 41.16, 66.41, 100.65, 112.99 (2C), 120.75 (2C), 128.69 (2C), 129.97, 130.51, 130.65, 133.58, 145.91, 147.18, 160.80, 165.60; HRMS (ESI) m/z: 369.1733 [M + H]⁺; calcd. for C₂₂H₂₆ClN₂O: 369.1728.

4.21. N-(3-((6-bromo-2-methylquinolin-4-yl)oxy)propyl)-4-chloroaniline (8r)

column chromatography on silica gel (hexane: ethyl acetate, 70:30, then 50:50), brown solid, yield: 0.089 g (22%); mp.: 120–127 °C; UHPLC: 96% (t_R = 16.60 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.13 (p, J = 6.4 Hz, 2H), 2.60 (s, 3H), 3.26 (t, J = 6.6 Hz, 2H), 4.34 (t, J = 6.1 Hz, 2H), 6.61 (d, J = 8.6 Hz, 2H), 7.00 (s, 1H), 7.09 (d, J = 8.7 Hz, 2H), 7.75–7.86 (m, 2H), 8.22 (d, J = 2.1 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 25.01, 27.71, 66.59, 102.68, 113.25 (2C), 117.79, 118.75, 120.55, 123.54, 128.53 (2C), 129.69, 132.84, 146.18, 147.64, 160.13, 160.72; HRMS (ESI) m/z: 405.0373 [M + H]⁺; calcd. for C₁₉H₁₉BrClN₂O: 405.0364.

4.22. 4-Bromo-N-(3-((6-bromo-2-methylquinolin-4-yl)oxy)propyl)aniline (8s)

column chromatography on silica gel (hexane: ethyl acetate, 70:30), black solid, yield: 0.094 g (21%); mp.: 134–138 °C; UHPLC: 97% (t_R = 17.97 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 2.25 (p, J = 6.4 Hz, 3H), 2.67 (s, 3H), 3.42 (t, J = 6.6 Hz, 2H), 4.29 (t, J = 5.8 Hz, 2H), 6.49–6.56 (m, 2H), 6.62 (s, 1H), 7.22–7.29 (m, 2H), 7.73 (dd, J = 9.0, 2.2 Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 8.25 (d, J = 2.2 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ ppm 25.65, 28.64, 40.91, 66.54, 101.95, 109.24, 114.36 (2C), 118.88, 118.95, 120.98, 124.02, 129.61, 132.08 (2C), 133.39, 146.91, 146.93, 160.57; HRMS (ESI) m/z: 448.9860 [M + H]⁺; calcd. for C₁₉H₁₈Br₂N₂O: 448.9859.

4.23. N-(3-((6-bromo-2-methylquinolin-4-yl)oxy)propyl)-4-ethylaniline (8t)

recrystallized from ethyl acetate, yellow solid, yield: 0.124 g (31%); mp.: 156–164 °C; UHPLC: 98% (t_R = 17.84 min); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.11 (t, J = 7.6 Hz, 3H), 2.22 (p, J = 6.4 Hz, 2H), 2.47 (q, J = 7.6 Hz, 2H), 2.81 (s, 3H), 3.34 (t, J = 6.7 Hz, 2H), 4.57 (t, J = 6.0 Hz, 2H), 6.72 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 7.50 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H), 8.15 (dd, J = 8.9, 2.2 Hz, 1H), 8.42 (t, J = 2.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 15.95, 21.53, 27.12, 27.32, 40.98, 68.92, 104.27 (2C), 114.04, 120.23, 120.40, 123.35, 124.73, 128.29 (2C), 133.32, 136.30, 138.87, 144.30, 159.86, 164.82; HRMS (ESI) m/z: 399.1076 [M + H]⁺; calcd. for C₂₁H₂₄BrN₂O: 399.1067.

4.24. N-(3-((6-bromo-2-ethylquinolin-4-yl)oxy)propyl)-4-chloroaniline (8u)

column chromatography on silica gel (hexane: ethyl acetate, 70:30, then 50:50), brownwish solid, yield: 0.126 g (30%); mp.: 146–148 °C; UHPLC: 96% (t_R = 17.07 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 1.32–1.41 (m, 4H), 2.25 (p, J = 6.4 Hz, 2H), 2.94 (q, J = 7.6 Hz, 3H), 3.41 (t, J = 6.6 Hz, 2H), 4.31 (t, J = 5.9 Hz, 2H), 6.57 (dd, J = 8.4, 1.5 Hz, 2H), 6.64 (s, 1H), 7.12 (dd, J = 8.8, 1.7 Hz, 2H), 7.72 (dd, J = 9.0, 2.3 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 8.25 (s, 1H); ¹³C NMR (101 MHz, CDCl₃) δ ppm 13.89, 28.62, 32.36, 40.94, 66.63, 100.76, 113.83 (2C), 119.00, 121.13, 122.11, 124.03, 129.16 (2C), 129.45, 133.46, 146.52, 161.07, 165.60; HRMS (ESI) m/z: 419.0533 [M + H]⁺; calcd. for C₂₁H₂₄BrN₂O: 419.0520.

4.25. 4-Bromo-N-(3-((6-bromo-2-ethylquinolin-4-yl)oxy)propyl)aniline (8v)

column chromatography on silica gel (hexane: ethyl acetate, 70:30), greenwish solid, yield: 0.083 g (18%); mp.: 145–147 °C; UHPLC: 98% (t_R = 17.98 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 1.37 (t, J = 7.5 Hz, 3H), 2.25 (p, J = 6.4 Hz, 2H), 2.92 (q, J = 7.6 Hz, 2H), 3.42 (t, J = 6.6 Hz, 2H), 4.30 (t, J = 5.9 Hz, 2H), 6.49–6.56 (m, 2H), 6.63 (s, 1H), 7.23–7.28 (m, 2H), 7.59 (dd, J = 9.0, 2.3 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 2.3 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ ppm 13.92, 28.67, 32.72, 40.94, 66.33, 100.66, 109.19, 114.34 (2C), 120.66, 120.68, 129.96, 130.61, 130.77, 132.07 (2C), 146.93, 147.07, 160.75, 165.56. HRMS (ESI) m/z: 463.0022 [M + H]⁺; calcd. for C₂₀H₂₁Br₂N₂O: 463.0015.

4.26. N-(3-((6-bromo-2-ethylquinolin-4-yl)oxy)propyl)-4-ethylaniline (8w)

column chromatography on silica gel (hexane: ethyl acetate, 70:30), brownwish solid, yield: 0.112 g (27%); mp.: 92–95 °C; UHPLC: 96% (t_R = 22.26 min); ¹H NMR (400 MHz, CDCl₃) δ ppm 1.19 (t, J = 7.6 Hz, 3H), 1.36 (t, J = 7.6 Hz, 3H), 2.24 (p, J = 6.3 Hz, 2H), 2.54 (q, J = 7.6 Hz, 2H), 2.90 (q, J = 7.7 Hz, 2H), 3.43 (t, J = 6.6 Hz, 2H), 4.29 (t, J = 5.9 Hz, 2H), 6.61 (d, J = 8.6 Hz, 3H), 7.00–7.05 (m, 2H), 7.71 (dd, J = 9.0, 2.3 Hz, 1H), 7.83 (d, J = 8.9 Hz, 1H), 8.28 (d, J = 2.3 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ ppm 13.90, 15.96, 27.92, 28.89, 32.80, 41.16, 66.46, 100.65, 112.98 (2C), 118.63, 121.25, 124.04, 128.69 (2C), 130.11, 133.07, 133.57, 145.90, 147.37, 160.69, 165.74; HRMS (ESI) m/z: 413.1230 [M + H]⁺; calcd. for C₂₂H₂₆BrN₂O: 413.1223.

4.27. Mycobacterium tuberculosis Inhibition Assay

The procedure for determining the minimum inhibitory concentration (MIC) in Mtb aims to determine whether the molecule can inhibit bacterial growth under in vitro conditions. The dilutions were made in round-bottom microplates (96 wells). The solution of isoniazid (INH), rifampicin (RIF), and telacebec (Q203) (positive control), and the synthesized compounds were diluted in 100% dimethyl sulfoxide (DMSO) at a concentration of 2 mg/mL. For the assay, aliquots were diluted in Middlebrook 7H9 medium containing 10% ADC (albumin, dextrose, catalase) and 5% DMSO in a series of assays of varying concentrations, depending on the solubility of each compound, finishing at the maximum possible concentration. To determine the aforesaid solubility limit, the solutions were evaluated for the possible presence of crystals. If crystals were identified, the aliquot would be diluted once more with half of the previous concentration. Growth controls without antibiotics and sterility controls without inoculation were included in the procedures. Mycobacterial strains were cultured in Middlebrook 7H9 containing 10% OADC (oleic acid, albumin, dextrose, catalase) and 0.05% tween 80. Cells were vortexed with sterile glass beads (4 mm) for 5 min to disrupt clamps and then left to rest for 20 min. The supernatants were measured in a spectrophotometer at an absorbance of 600 nm. Mtb suspensions were aliquoted and stored at −20 °C. Each suspension was properly diluted in Middlebrook 7H9 broth containing 10% ADC to reach an optical density of 0.006 at 600 nm. Ultimately, 100 μL of this solution was added to each well of the plate, except for the sterility control one. The plates were covered, sealed, and incubated at 37 °C. After 7 days of incubation, 60 μL of 0.01% resazurin solution was added to each well and the plate was incubated for an additional 48 h at 37 °C. The MIC was defined by the lowest concentration of the molecule that did not change the color of the medium from blue to pink, which would have meant the reduction of resazurin and bacterial growth. Assays were performed three times for each structure on different dates and two concordant results or the highest MIC value between assays was showed in micromolar concentration (μM). The laboratory reference strains were: Mtb H37Rv (ATCC 27294) and three strains considered multidrug-resistant (MDR) isolates of Mtb named PT2, PT12, and PT20, which were obtained from patients in the Lisbon Health Region, Lisbon, Portugal. Finally, INH and RIF were used as control drugs in all experiments.

4.28. Cellular Viability Evaluation

The evaluation of Vero and HepG2 cell viability was conducted using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and neutral red uptake (NRU) assays. Both cell lines were grown in DMEM medium (Dulbecco’s Modified Eagle Medium, Gibco, Grand Island, NY, USA) supplemented with 10% inactivated fetal bovine serum from Invitrogen, 1% antibiotic (penicillin and streptomycin) from Gibco, and 0.1% fungizone from Gibco. Cells were seeded in a 96-well culture plate (4 × 10³ HepG2 and 2 × 10³ Vero) and incubated overnight at 37 °C to allow for cell attachment. The media was then removed and replaced with fresh media containing each compound at a final concentration of 1 μM, 5 μM, or 20 μM using 1% DMSO. Cells were incubated during 72 h at 37 °C under a 5% CO₂ atmosphere. After the incubation, medium was removed and replaced with MTT reagent (5 mg/mL), followed by additional 4 h incubation. Then, 100 μL of DMSO was added to dissolve the formazan crystals and the absorbance was measured at 570 nm (EZ Read 400 microplate reader, Biochrom, Cambridge, UK). Precipitated formazan crystals were used as a direct measure of living cells with active mitochondrial metabolism. For the NRU assay, after 72 h of cell incubation with the treatments, the cells were washed with PBS and 200 μL of neutral red dye solution (25 μg/mL, Sigma) prepared in serum-free medium was added to each well. The plate was then incubated for 3 h at 37 °C under 5% CO₂. Afterward, cells were washed with PBS, and 100 μL of a desorb solution (ethanol/acetic acid/water, 50:1:49) was added to each well and shaken gently for 30 min to extract the neutral red dye from viable cells. The absorbance was measured at 562 nm (EZ Read 400 microplate reader (Biochrom, Holliston, MA, USA)). The percentage of cell viability was calculated using the vehicle control wells (1% DMSO) as the maximum cell viability. The results were presented as concentration required to reduce cell viability by 50% (CC₅₀) using three independent experiments performed in triplicate.

4.29. Solubility

The solubility assay employed 1 mL of buffer solution (PBS 1X, pH 7.4, or 0.1 M HCl, pH 1.0, or NH₄HCO₃ 0.1 M, pH 9.1). Each sample was weighed at 1 mg. Subsequently, the final solutions were vortexed, and the remaining suspensions were agitated for 4 h at 25 °C. Following this, the solutions were centrifuged at 13,000 rpm for 20 min at 25 °C, resulting in the formation of a pellet. The analysis was conducted via ultra high-performance liquid chromatography (UHPLC-DAD) utilizing single-point calibration with a known concentration of the compounds in DMSO.

4.30. Evaluation of Possible Broad-spectrum Activity

Four nonmycobacterial species were used: the standard strains Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922, along with the clinical isolates Klebsiella pneumoniae and Acinetobacter baumannii, both exhibiting extensive drug resistance. The isolates were stored at −20 °C in a medium with 20% glycerol. To prepare the cultures, 100 μL of each frozen stock was inoculated in Brain Heart Infusion (BHI) broth and incubated for 24 h at 37 °C. Cultures were then streaked on BHI agar plates and incubated for an additional 24 h to obtain isolated colonies. The inoculum was prepared by suspending isolated colonies in 0.85% saline to achieve a turbidity of 0.5 on the McFarland scale (1.5 × 10⁸ CFU/mL). Compound 8t was diluted in DMSO and Mueller-Hinton (MH) broth to create a stock solution with a concentration of 40 μM and a final DMSO concentration of 4%. In a 96-well U-bottom polystyrene plate, 100 μL of MH broth were added to three wells as negative controls, while 100 μL of MH broth plus 1 μL of adjusted inoculum served as positive controls. For the test wells, 100 μL of MH broth, 100 μL of the diluted compound, and 1 μL of inoculum were combined to reach a final concentration of 20 μM of the compound and 2% DMSO. The plates were incubated at 37 °C for 24 h, and bacterial growth inhibition was assessed visually.

4.31. Chemical Stability Evaluation

The assessment of chemical stability at different pH levels provides insights into the quantity or concentration of the test compound that remains unchanged and, consequently, available for absorption. In this study, 4-alkoxyquinoline 8t (10 μM) was incubated at 37 °C for 24 h in the presence of a buffer solution at controlled pH levels: 1.2 (simulating the stomach pH with 0.1 M HCl), 7.4 (simulating plasma pH with PBS), and 9.1 (simulating intestinal pH with 0.1 M NH₄HCO₃). Following this incubation period, the test compounds were quantified using HPLC-MS/MS, with alprenolol serving as the analytical control. This experiment was conducted by CEMSA (São Paulo, SP, Brazil).

4.32. Permeability Assay

The Parallel Artificial Membrane Permeability Assay (PAMPA) utilizes an artificial membrane as an efficient and rapid model for studying and evaluating the passive permeation potential of drug candidates. In this study, 4-alkoxyquinoline 8t was quantified (HPLC-MS/MS) after incubation in two solutions separated by an artificial and porous lipid membrane. The results of this test were expressed in terms of diffusion rate (permeation). Initially, compound 8t at a concentration of 10 μM (in 2% DMSO) was added to the donor aqueous phase (pH 7.4). After 5 h at 25 °C, an aliquot of the receptor solution (pH 7.4), through which the compound was theoretically transported via passive diffusion, was withdrawn, and the concentration of the tested molecule was measured. Based on the concentration results, the permeation velocity value was determined, with alprenolol serving as the analytical control. Permeability is typically categorized as low (<1.0 × 10^–6 cm/s) or high (>1.0 × 10^–6 cm/s).²⁷ This experiment was conducted by CEMSA (São Paulo, SP, Brazil).

4.33. Metabolic Stability Assay

In brief, 4-alkoxyquinoline 8t at a concentration of 2 μM were subjected to incubation with a rat microsomes preparation containing NADPH at 37 °C. The consumption of the compound from the incubation mixture was monitored at 0, 5, 15, and 30 min using the HPLC-MS/MS technique to determine the in vitro disappearance half-life. Verapamil was employed as the positive control. Intrinsic clearance is typically categorized as low (<16 mL/min/kg), moderate (16–47 mL/min/kg), and high (>47 mL/min/kg).²⁷ This experiment was conducted by CEMSA (São Paulo, SP, Brazil).

4.34. Intracellular Activity in a Murine Macrophages Model of TB Infection

The experiment utilized murine macrophage RAW 264.7 cells cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), without antimicrobials or fungizone. Cells were seeded at a density of 5 × 10³ cells per well in a 24-well flat-bottom plate and incubated at 37 °C with 5% CO₂. After the incubation period, the medium was removed, and the cells were washed with preheated sterile PBS to remove nonadherent cells. M. tuberculosis strain H37Rv (ATCC 27294) was grown in 10 mL of Middlebrook 7H9 medium (Becton-Dickinson-BD) supplemented with 10% BD Difco BBL Middlebrook OADC enrichment (oleic acid, albumin, dextrose, catalase), 0.05% (v/v) Tween 80 (Sigma-Aldrich), and 0.2% (v/v) glycerol (Sigma-Aldrich) to mid log phase (OD600 ≅ 0.8). The culture was then diluted in RPMI medium with 10% FBS (without antimicrobials or fungizone) to obtain 1.5 × 10⁴ CFUs/well and 2.5 × 10⁴ CFUs/well of Mtb for MOI 3:1 and 5:1, respectively, in two independent experiments. The cells infection was caried out after adding Mtb to the plates, they were incubated for 3 h at 37 °C with 5% CO₂. The infected cells were then washed twice with sterile PBS to remove any remaining Mtb in the medium. Early control (EC) cells were lysed at the beginning of treatment with 1 mL of 0.025% SDS diluted in 0.9% saline. Serial dilutions were performed in 0.9% saline, and the lysed cells were plated on Middlebrook 7H10 (Becton-Dickinson-BD) supplemented with 10% OADC and 0.5% glycerol. Infected cells were treated with 1 μM or 5 μM of 8t in triplicate. A 4 mM solution of each compound was prepared in DMSO and diluted in RPMI medium to a final concentration of 1 μM or 5 μM, with a final DMSO concentration of 0.5%. The late control (LC) group was treated with 0.5% DMSO in RPMI medium. After 5 days of treatment, cells were washed twice with sterile PBS and lysed with 1 mL of 0.025% SDS diluted in 0.9% saline. The cell lysate was diluted in 0.9% saline and plated on 7H10 medium supplemented with 10% OADC and 0.5% glycerol. After an incubation period of 3 to 4 weeks at 37 °C, CFUs were counted, establishing a limit of detection (LOD) between 20 and 300 CFU per plate. Calculated CFU values were converted to log₁₀ CFU, and the results of the two independent experiments were combined. Finally, the results were expressed as the mean log₁₀ CFU per well ± standard deviation (mean log₁₀ CFU/well ± SD). The groups were compared by one-way analysis of variance (ANOVA), followed by Tukey’s post-test, using GraphPad Prism 9.0 (GraphPad, San Diego, CA, USA).

4.35. In Vivo Absorption Profiling

Male CF1 mice (4–5 weeks old) were used for the absorption profiling evaluation (n = 12). All animals were sourced from the Center of Experimental Biological Models at Pontifícia Universidade Católica do Rio Grande do Sul (CeMBE/PUCRS). The mice were housed under controlled conditions, with humidity levels of 40–60% and room temperature maintained at 24 ± 2 °C, following a 12 h light/dark cycle. Food and water were provided ad libitum. The study protocol was approved by the Animal Ethics Committee at Pontifícia Universidade Católica do Rio Grande do Sul (CEUA/PUCRS, Porto Alegre, RS, Brazil; protocol number 10649). The experiment also adhered to the Brazilian guidelines for the production, maintenance, and use of animals in teaching or scientific research, as established by the National Council for the Control of Animal Experimentation (CONCEA, Brazil).

The determination of the absorption profile of compound 8t was conducted by using gavage administration. A single dose of 300 mg/kg (≈ 750 μmol/kg) per animal was administered. The suspension was prepared in a saline solution using ultrasound for 1 h. Following administration, the animals were euthanized, and blood samples were collected at the specified time intervals: 0.25, 0.5, 0.75, 1, 4, and 8 h. Two animals were employed for each experimental time point, yielding independent duplicate results. Blood collected from each mouse underwent centrifugation for 30 min at 4 °C and 13000 rpm to separate the plasma. Subsequently, 100 μL of plasma was extracted per sample and mixed with 100 μL of acetonitrile, followed by vortexing for 1 min. The resulting mixture was then centrifuged for 15 min at 4 °C and 13000 rpm. After this process, the supernatant was separated and combined with 300 μL of dichloromethane in a vial. Once again, the resulting mixture was vortexed for 10 s. After phase separation, the organic phase was transferred to another vial, and the dichloromethane was evaporated under reduced pressure. Finally, the sample was reconstituted with 150 μL of a 1% acetic acid solution for the subsequent quantification of the compound. The quantification method was carried out using UHPLC-DAD Shimadzu LC-2060 3D equipment, and detection was performed at a wavelength of 254 nm. The analytical calibration curve in plasma encompassed six concentrations of the analyte, ranging from 1.25 ug/mL to 50 ug/mL of the compound. The R² value of the analytical curve was determined to be 0.9969.

Glossary

Abbreviations

ADC

Albumin dextrose catalase

ADME

Absorption, distribution, metabolism, and excretion

AUC_0-t

Area under the concentration–time curve from 0 to time t

CFU

Colony-forming unit

DMSO

Dimethyl sulfoxide

FBS

Fetal bovine serum

HPLC-MS/MS

High-performance liquid chromatography with tandem mass spectrometry

INH

Isoniazid

K_e

Elimination rate constant

MIC

Minimum inhibitory concentration

NRU

Neutral red uptake

PAMPA

parallel artificial membrane permeability assay

PBS

Phosphate-buffered saline

Q203

Telacebec

RIF

Rifampicin

RPMI

Roswell Park Memorial Institute medium

SAR

Structure–activity relationship

t_1/2

Half-life

TLC

Thin-layer chromatography

UHPLC-DAD

Ultra high-performance liquid chromatography with diode array detection

Data Availability Statement

Data will be made available on request.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01302.

• ¹H NMR and ¹³C NMR spectra of synthesized compounds (8a–w), UHPLC chromatogram for in vivo evaluated compound 8t (PDF)

• Molecular formula strings and assay data (CSV)

Author Contributions

All authors have materially participated in the research, and the manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The Article Processing Charge for the publication of this research was funded by the Coordination for the Improvement of Higher Education Personnel - CAPES (ROR identifier: 00x0ma614).

The authors declare no competing financial interest.