Mycobacterium tuberculosis Inhibitors Based on Arylated Quinoline Carboxylic Acid Backbones with Anti-Mtb Gyrase Activity

New antitubercular agents with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The present study profiles new arylated quinoline carboxylic acids (QCAs) having activity against replicating and non-replicating Mycobacterium tuberculosis (Mtb), the causative agent of TB. Thus, the synthesis, characterization, and in vitro screening (MABA and LORA) of 48 QCAs modified with alkyl, aryl, alkoxy, halogens, and nitro groups in the quinoline ring led to the discovery of two QCA derivatives, 7i and 7m, adorned with C-2 2-(naphthalen-2-yl)/C-6 1-butyl and C-2 22-(phenanthren-3-yl)/C-6 isopropyl, respectively, as the best Mtb inhibitors. DNA gyrase inhibition was shown to be exhibited by both, with QCA 7m illustrating better activity up to a 1 μM test concentration. Finally, a docking model for both compounds with Mtb DNA gyrase was developed, and it showed a good correlation with in vitro results.


Introduction
Tuberculosis (TB) poses serious health risks to humans. Annually, this deadly infectious disease slowly affects millions of people worldwide causing illness and devastating fatalities. The global burden for TB incidences is accounted for by Southeast Asia at 41%, with Indonesia (14%) and the Philippines (12%) having the biggest increases in the number of diagnosed TB infections between 2019 and 2020 [1]. These are among the countries which had significant impacts on the worrisome global prevalence of the disease and were estimated to reach 10 million in 2012, with 1.3 million people dying from TB-related causes in 2022 [2]. The alarmingly high death rate is mostly due to poor diagnostic tools, the lack of universal healthcare, and the rising cases of strains resistant to most if the currently available anti-TB antibiotics. A strain of TB that is resistant to the two first-line medications (such as rifampicin and isoniazid) is known as a multidrug-resistant strain (MDR-TB). The extremely drug-resistant TB strain (XDR-TB), on the other hand, is a variety of DR-TB that is resistant to first-line medications, as well as fluoroquinolones and one of the second-line injectable anti-TB medications (such as amikacin, capreomycin, and kanamycin) [3].
The constant need to search for novel anti-TB antibiotics grows as more Mycobacterium tuberculosis (Mtb) strains become resistant. The creation of new synthetic or semi-synthetic Scheme 1. Synthesis of substituted isatins 2a-2c via Sandmeyer reaction.

Synthesis of Quinoline Carboxylic Acids 1
The procedure outlining the synthesis of compounds 1a-1p, 6a-6r, and 7a-7n is shown in Scheme 2. The reaction highlights a microwave-assisted Pfitzinger reaction using substituted isatins (2) and substituted ketones (5) as precursors to afford quinoline-4-carboxylic acid (QCA, 1). The Pfitzinger reaction is a two-component reaction which mechanism shows the alkaline hydrolysis-driven ring-opening of the pyrrolidinone ring of isatin as an initial step to produce 2-(2-aminophenyl)-2-oxoacetic acid in situ. The keto-acid aniline intermediate reacts further with the enolate of ketone 5 to give an imine which is eventually converted to an enamine. The final cyclization occurs via hydrolysis of the enamine to give the desired substituted QCA 1 [16,17]. All synthesized quinoline carboxylic acid derivatives 1 were characterized using NMR and HR-ESIMS for structure characterization and/or verification. Scheme 2. Synthesis of quinoline carboxylic acid derivatives (1) via Pfitzinger reaction.

Antitubercular Activity of QCAs 1
The quinoline carboxylic acid (QCA, 1) derivatives were evaluated against Mtb H37Rv, using the in vitro colorimetric susceptibility Microplate Alamar Blue Assay (MABA). In this assay, resazurin, non-fluorescent blue dye, is reduced to the pinkish fluorescent dye resorufin by active cells. The potency of the test compounds is taken depending on the reduction of resazurin through quantitative measurements of each microtiter well [18]. Compounds exhibiting TB activity were subsequently subjected to an additional inhibitory assay to determine their effectiveness against non-replicating persistent (NRP) TB, using the low oxygen recovery assay (LORA). LORA employs a recombinant Mtb expressing luciferase as the reporter gene since its luminescent intensity is indicative of a cell's overall metabolic activity in hypoxic conditions. The NRP of Mtb is a contributing factor to its virulence, as it generally develops antibiotic resistance to existing medications [19,20]. The viability of the cells is dependent on the luminosity of the luciferase after the normoxic "recovery" period of the test compounds [21]. The antitubercular activity of all synthetic QCAs is expressed as the minimum inhibitory concentration (MIC90), as µg/mL, which is defined as the lowest drug concentration needed for the visible growth inhibition of 90% of bacteria in the colony in relation to those in the drug-free controls [22]. Thus, an initial library of sixteen synthetic QCAs 1a-1p functionalized with alkyl, aryl, and halogens at C-2, C-3, C-6, and C-8 were prepared. In general, most QCA derivatives (Table 1) Scheme 1. Synthesis of substituted isatins 2a-2c via Sandmeyer reaction.

Synthesis of Quinoline Carboxylic Acids 1
The procedure outlining the synthesis of compounds 1a-1p, 6a-6r, and 7a-7n is shown in Scheme 2. The reaction highlights a microwave-assisted Pfitzinger reaction using substituted isatins (2) and substituted ketones (5) as precursors to afford quinoline-4-carboxylic acid (QCA, 1). The Pfitzinger reaction is a two-component reaction which mechanism shows the alkaline hydrolysis-driven ring-opening of the pyrrolidinone ring of isatin as an initial step to produce 2-(2-aminophenyl)-2-oxoacetic acid in situ. The keto-acid aniline intermediate reacts further with the enolate of ketone 5 to give an imine which is eventually converted to an enamine. The final cyclization occurs via hydrolysis of the enamine to give the desired substituted QCA 1 [16,17]. All synthesized quinoline carboxylic acid derivatives 1 were characterized using NMR and HR-ESIMS for structure characterization and/or verification. The procedure outlining the synthesis of compounds 1a-1p, 6a-6r, and 7a-7n is shown in Scheme 2. The reaction highlights a microwave-assisted Pfitzinger reaction using substituted isatins (2) and substituted ketones (5) as precursors to afford quinoline-4-carboxylic acid (QCA, 1). The Pfitzinger reaction is a two-component reaction which mechanism shows the alkaline hydrolysis-driven ring-opening of the pyrrolidinone ring of isatin as an initial step to produce 2-(2-aminophenyl)-2-oxoacetic acid in situ. The keto-acid aniline intermediate reacts further with the enolate of ketone 5 to give an imine which is eventually converted to an enamine. The final cyclization occurs via hydrolysis of the enamine to give the desired substituted QCA 1 [16,17]. All synthesized quinoline carboxylic acid derivatives 1 were characterized using NMR and HR-ESIMS for structure characterization and/or verification. Scheme 2. Synthesis of quinoline carboxylic acid derivatives (1) via Pfitzinger reaction.

Antitubercular Activity of QCAs 1
The quinoline carboxylic acid (QCA, 1) derivatives were evaluated against Mtb H37Rv, using the in vitro colorimetric susceptibility Microplate Alamar Blue Assay (MABA). In this assay, resazurin, non-fluorescent blue dye, is reduced to the pinkish fluorescent dye resorufin by active cells. The potency of the test compounds is taken depending on the reduction of resazurin through quantitative measurements of each microtiter well [18]. Compounds exhibiting TB activity were subsequently subjected to an additional inhibitory assay to determine their effectiveness against non-replicating persistent (NRP) TB, using the low oxygen recovery assay (LORA). LORA employs a recombinant Mtb expressing luciferase as the reporter gene since its luminescent intensity is indicative of a cell's overall metabolic activity in hypoxic conditions. The NRP of Mtb is a contributing factor to its virulence, as it generally develops antibiotic resistance to existing medications [19,20]. The viability of the cells is dependent on the luminosity of the luciferase after the normoxic "recovery" period of the test compounds [21]. The antitubercular activity of all synthetic QCAs is expressed as the minimum inhibitory concentration (MIC90), as µg/mL, which is defined as the lowest drug concentration needed for the visible growth inhibition of 90% of bacteria in the colony in relation to those in the drug-free controls [22]. Thus, an initial library of sixteen synthetic QCAs 1a-1p functionalized with alkyl, aryl, and halogens at C-2, C-3, C-6, and C-8 were prepared. In general, most QCA derivatives (Table 1) Scheme 2. Synthesis of quinoline carboxylic acid derivatives (1) via Pfitzinger reaction.

Antitubercular Activity of QCAs 1
The quinoline carboxylic acid (QCA, 1) derivatives were evaluated against Mtb H 37 Rv, using the in vitro colorimetric susceptibility Microplate Alamar Blue Assay (MABA). In this assay, resazurin, non-fluorescent blue dye, is reduced to the pinkish fluorescent dye resorufin by active cells. The potency of the test compounds is taken depending on the reduction of resazurin through quantitative measurements of each microtiter well [18]. Compounds exhibiting TB activity were subsequently subjected to an additional inhibitory assay to determine their effectiveness against non-replicating persistent (NRP) TB, using the low oxygen recovery assay (LORA). LORA employs a recombinant Mtb expressing luciferase as the reporter gene since its luminescent intensity is indicative of a cell's overall metabolic activity in hypoxic conditions. The NRP of Mtb is a contributing factor to its virulence, as it generally develops antibiotic resistance to existing medications [19,20]. The viability of the cells is dependent on the luminosity of the luciferase after the normoxic "recovery" period of the test compounds [21]. The antitubercular activity of all synthetic QCAs is expressed as the minimum inhibitory concentration (MIC 90 ), as µg/mL, which is defined as the lowest drug concentration needed for the visible growth inhibition of 90% of bacteria in the colony in relation to those in the drug-free controls [22]. Thus, an initial library of sixteen synthetic QCAs 1a-1p functionalized with alkyl, aryl, and halogens at C-2, C-3, C-6, and C-8 were prepared. In general, most QCA derivatives (Table 1) exhibited poor anti-TB activity (MIC 90 > 64 µg/mL). Compound 1h, however, showed weak inhibition, with an MIC 90 of 62.57 µg/mL, which led us to explore more active derivatives by further functionalizing a 2-([1,1'-biphenyl]-4-yl) QCA scaffold ushering to a subset of derivatives adorned with halogens and alkyl groups. Eighteen additional derivatives based on 2-([1,1'-biphenyl]-4-yl) QCA 6a-6i functionalized with halogens at C-6, C-7, or C-8 positions of the quinoline ring are shown in Table 2. Thus, the introduction of halogens at C-8 (6b, 6d, and 6f) led to the loss of anti-TB activity, whereas the variation of halogens at C-6 (6a, 6c, 6e) gratifyingly showed a 2-fold increase in M. tb. H 37 Rv inhibition, with 6-chloro derivative 6a as the most inhibitory compound. Among the halogen substitutions at C-6, iodo derivative 6g exhibited a decrease in anti-TB activity (MIC 90 > 64.0 µg/mL). Overall, the effective inhibition of the compounds against M. tb. H 37 Rv is influenced by the correct positioning of the halogen atoms in the benzene ring, i.e., at C-6. We also attempted to prepare additional derivatives modified with nitro at C-6 (6h) and methoxy at C-7 (6i); however, both showed poor potency toward M. tb. H 37 Rv. We also explored the structural activity by varying the C-2 aryl groups based on the most active compound 6a.  However, this led to a significant drop in inhibition when the biphenyl group was replaced with 2-(naphthalen-2-yl), 2-(anthracen-2-yl) and 2-(phenanthren-3-yl) handles even by changing the position and type of halogen. Interestingly, the 2-(naphthalen-2-yl)and 2-(phenanthren-3-yl)-modified 6-bromo derivatives 6q and 6r showed moderate anti-TB activity.
Subsequently, compounds 6a, 6c, 6e, 6q, and 6r, which showed anti-TB activity in MABA, were further tested against non-replicating M. tb., using the LORA assay. The LORA assay is a high-capacity screening method that is used to assess the efficacy against the non-replicating persistent Mtb phenotype in low-oxygen conditions. Interestingly, the tested compounds, compounds 6a, 6c, 6q, and 6r, showed retention of inhibitory activity against non-replicating M. tb. However, the LORA evaluation of 6-bromo QCA modified with 2-([1,1'-biphenyl]-4-yl) 6e resulted in a loss of activity.
A second generation of 14 compounds, i.e., 7a-7n, founded on the structure of on 2-([1,1'-biphenyl]-4-yl) QCA with alkyl modifications at C-6 of the quinoline ring is shown in Table 3. We identified a promising Mtb activity on compound 7a, where a methyl group at C-6 is incorporated. Based on this finding, we attempted to investigate the effect of lengthening the alkyl chain of the C-6 position and also varying the aryl groups at the C-2 position to the anti-TB activity. The 1-biphenyl series (7a-7d) exhibited good MABA  However, this led to a significant drop in inhibition when the biphenyl group was replaced with 2-(naphthalen-2-yl), 2-(anthracen-2-yl) and 2-(phenanthren-3-yl) handles even by changing the position and type of halogen. Interestingly, the 2-(naphthalen-2yl)-and 2-(phenanthren-3-yl)-modified 6-bromo derivatives 6q and 6r showed moderate anti-TB activity.
Subsequently, compounds 6a, 6c, 6e, 6q, and 6r, which showed anti-TB activity in MABA, were further tested against non-replicating M. tb., using the LORA assay. The LORA assay is a high-capacity screening method that is used to assess the efficacy against the non-replicating persistent Mtb phenotype in low-oxygen conditions. Interestingly, the tested compounds, compounds 6a, 6c, 6q, and 6r, showed retention of inhibitory activity against non-replicating M. tb. However, the LORA evaluation of 6-bromo QCA modified with 2-([1,1'-biphenyl]-4-yl) 6e resulted in a loss of activity.
A second generation of 14 compounds, i.e., 7a-7n, founded on the structure of on 2-([1,1'-biphenyl]-4-yl) QCA with alkyl modifications at C-6 of the quinoline ring is shown in Table 3. We identified a promising Mtb activity on compound 7a, where a methyl group at C-6 is incorporated. Based on this finding, we attempted to investigate the effect of lengthening the alkyl chain of the C-6 position and also varying the aryl groups at the C-2 position to the anti-TB activity. The 1-biphenyl series (7a-7d) exhibited good MABA activity, except for the C-6 ethyl-substituted QCA derivative, 7b. The 2-naphthalene series (7e-7i) indicated poor activity irrespective of the alkyl substituent present, except however, with the 1-butyllated QCA 7i derivative, which showed high inhibitory anti-TB activity against M. tb. H 37 Rv (MIC 90 > 16 µg/mL). The 2-phenanthrene modified series (7j, 7l-7n)) showed similar potency featuring C-6 methyl and 1-butyl substituents. The ethyl substituted C-6 (7l) displayed the weakest activity (MIC 90 = 51.3 µg/mL) among the 2-phenanthrenylated series. Finally, the isopropyl substituted C-6 also showed the highest activity, with MIC 90 of >16 µg/mL. Based on the results, the pattern shows unfavorable activity when the C-6 position is ethylated. The overall trend indicates that, with the substitution of C-6 positions with 1-butyl, the effectiveness of the compound against Mtb is enhanced. Retention of LORA activity was observed on compounds 7a and 7c. The LORA activity of compound 7d improved slightly from MIC 90 > 32 to MIC 90 = 19.84, whereas compounds 7j, 7k, and 7n resulted in having a loss of inhibitory potency. The activity of hit compounds 7i and 7m was conserved in LORA. activity, except for the C-6 ethyl-substituted QCA derivative, 7b. The 2-naphthalene series (7e-7i) indicated poor activity irrespective of the alkyl substituent present, except however, with the 1-butyllated QCA 7i derivative, which showed high inhibitory anti-TB activity against M. tb. H37Rv (MIC90 > 16 µg/mL). The 2-phenanthrene modified series (7j, 7l-7n)) showed similar potency featuring C-6 methyl and 1-butyl substituents. The ethyl substituted C-6 (7l) displayed the weakest activity (MIC90 = 51.3 µg/mL) among the 2-phenanthrenylated series. Finally, the isopropyl substituted C-6 also showed the highest activity, with MIC90 of >16 µg/mL. Based on the results, the pattern shows unfavorable activity when the C-6 position is ethylated. The overall trend indicates that, with the substitution of C-6 positions with 1-butyl, the effectiveness of the compound against Mtb is enhanced. Retention of LORA activity was observed on compounds 7a and 7c. The LORA activity of compound 7d improved slightly from MIC90 > 32 to MIC90 = 19.84, whereas compounds 7j, 7k, and 7n resulted in having a loss of inhibitory potency. The activity of hit compounds 7i and 7m was conserved in LORA.

Mtb DNA Gyrase Inhibitory Activity
DNA gyrase is a type II DNA topoisomerase found in bacteria, including mycobacteria. This enzyme is crucial for the replication and transcription functions of bacterial DNA, making it an interesting target for discovering antitubercular agents. Quinolones, which are an oxidized derivative of quinolines, are known to target type II topoisomerase (DNA gyrase and topoisomerase IV). Since Mtb does not possess topoisomerase IV, DNA gyrase is the key quinolone target [23]. This class of antitubercular compounds acts on DNA gyrase by inducing breaks on the DNA strand which subsequently result in cell lysis

Mtb DNA Gyrase Inhibitory Activity
DNA gyrase is a type II DNA topoisomerase found in bacteria, including mycobacteria. This enzyme is crucial for the replication and transcription functions of bacterial DNA, making it an interesting target for discovering antitubercular agents. Quinolones, which are an oxidized derivative of quinolines, are known to target type II topoisomerase (DNA gyrase and topoisomerase IV). Since Mtb does not possess topoisomerase IV, DNA gyrase is the key quinolone target [23]. This class of antitubercular compounds acts on DNA gyrase by inducing breaks on the DNA strand which subsequently result in cell lysis because of accumulated oxidative stress in the strand [24,25]. With this premise, the bioactive inhibitory quinoline carboxylic acid derivatives were investigated for their inhibitory potentials against DNA gyrase in vitro, using a DNA gyrase supercoiling assay. Based on the in vitro activity data in Table 3, compounds 7i and 7m were evaluated for their effectiveness in inhibiting DNA gyrase ( Figure S55).
The assay involves a gyrase to create supercoiled DNA from a circular DNA plasmid called pBR322, which is initially in a relaxed state. The supercoiled and relaxed versions of the plasmid can be distinguished by running an agarose gel using electrophoresis. Herein, three concentrations of compounds 7i and 7m were tested on their inhibitory activity against DNA gyrase (Figure 1). The target enzyme was only inhibited by QCA 7i at a 100 µM concentration, while QCA 7m inhibited Mtb DNA gyrase at 1.0 µM, as evidenced by an absence of supercoiled bands in their respective lanes. These suggest that the active compounds can inhibit the mycobacterial enzyme, which can be inferred to act as a potential antimycobacterial agent.
because of accumulated oxidative stress in the strand [24,25]. With this premise, the bioactive inhibitory quinoline carboxylic acid derivatives were investigated for their inhibitory potentials against DNA gyrase in vitro, using a DNA gyrase supercoiling assay. Based on the in vitro activity data in Table 3, compounds 7i and 7m were evaluated for their effectiveness in inhibiting DNA gyrase (( Figure S55).
The assay involves a gyrase to create supercoiled DNA from a circular DNA plasmid called pBR322, which is initially in a relaxed state. The supercoiled and relaxed versions of the plasmid can be distinguished by running an agarose gel using electrophoresis. Herein, three concentrations of compounds 7i and 7m were tested on their inhibitory activity against DNA gyrase (Figure 1). The target enzyme was only inhibited by QCA 7i at a 100 µM concentration, while QCA 7m inhibited Mtb DNA gyrase at 1.0 µM, as evidenced by an absence of supercoiled bands in their respective lanes. These suggest that the active compounds can inhibit the mycobacterial enzyme, which can be inferred to act as a potential antimycobacterial agent.

Molecular Docking of 7i and 7m onto Mtb DNA Gyrase
Both quinoline carboxylic acids 7i and 7m were subjected to in silico molecular docking analysis to assess the potential binding interactions of the in vitro active compounds with the fluoroquinolone-binding site of Mtb DNA gyrase (Table 4). After the docking between the two compounds, 7m exhibited a stronger binding to the active site of the enzyme, with a binding energy of −8.0 kcal/mol (Figure 2). The quinoline core of the compound and its isopropyl substituent were stabilized by pi-alkyl interaction with Pro123. The 2-phenanthrene side chain had all three of its aromatic rings bound to Ala126 via pialkyl intermolecular forces. Moreover, the carboxylic acid moiety interacted through conventional hydrogen bonding (bond distance of 3.68 Å) with Ser462. Quinoline 7i, on the other hand, was nestled to the enzyme's active site through pi-alkyl/alkyl interplay with Pro123, Ala126, and Met127. A carbon-hydrogen bonding can also be observed between the 4-carboxylic acid group and Ala125.

Molecular Docking of 7i and 7m onto Mtb DNA Gyrase
Both quinoline carboxylic acids 7i and 7m were subjected to in silico molecular docking analysis to assess the potential binding interactions of the in vitro active compounds with the fluoroquinolone-binding site of Mtb DNA gyrase (Table 4). After the docking between the two compounds, 7m exhibited a stronger binding to the active site of the enzyme, with a binding energy of −8.0 kcal/mol (Figure 2). The quinoline core of the compound and its isopropyl substituent were stabilized by pi-alkyl interaction with Pro123. The 2-phenanthrene side chain had all three of its aromatic rings bound to Ala126 via pialkyl intermolecular forces. Moreover, the carboxylic acid moiety interacted through conventional hydrogen bonding (bond distance of 3.68 Å) with Ser462. Quinoline 7i, on the other hand, was nestled to the enzyme's active site through pi-alkyl/alkyl interplay with Pro123, Ala126, and Met127. A carbon-hydrogen bonding can also be observed between the 4-carboxylic acid group and Ala125.   Corroborating the results of both the in vitro and in silico analysis performed on the two hit quinoline compounds, we found that QCA 7m had a better binding energy score than compound 7i that was brought about by various pi-alkyl interactions with the amino acids in the enzyme, leading to the inhibition of Mtb DNA Gyrase even at a low concentration.

Materials and Instrumentations
All reagents and other materials were obtained from Sigma Aldrich (St. Louis, MO, USA) and Alfa Aesar (Heysham, Lancashire, UK) and used without additional Corroborating the results of both the in vitro and in silico analysis performed on the two hit quinoline compounds, we found that QCA 7m had a better binding energy score than compound 7i that was brought about by various pi-alkyl interactions with the amino acids in the enzyme, leading to the inhibition of Mtb DNA Gyrase even at a low concentration.

Synthesis
The synthetic approach for quinoline carboxylic acid derivatives in this study is outlined above, in Scheme 2. The starting isatin compounds, 5-ethylindoline-2,3-dione, 5-isopropylindoline-2,3-dione, and 5-butylindoline-2,3-dione, were synthesized using the method described in Section 3.2.1, and the rest of the isatins were obtained from commercial sources and used without additional purification.

General Approach for the Synthesis of Substituted Isatins 2
A mixture of substituted aniline (0.05 mmol), hydroxylammonium chloride (0.15 mmol), Na 2 SO 4 (0.35 mmol), hydrochloric acid (10 mmol), and chloral hydrate (0.06 mmol) in 50 mL water was stirred at 95 • C, over a 180 min period. The completion of this reaction was determined via TLC (1:1 petroleum ether/ethyl acetate). Next, the mixture was cooled at room temperature, filtered, and dried. The obtained precipitates were dissolved in concentrated sulfuric acid (20 mL), with vigorous stirring, at 50 • C. Then, the mixture was heated to 80 • C for 30 min. The reaction mixture was allowed to cool until room temperature, after which it was poured into ice. The solids were filtered and purified by dissolving them in 5% NaOH (100 mL), followed by the addition of 4M HCl (50 mL). The formed precipitates were filtered and air-dried to obtain isatin 2, which was subsequently used for the method described in Section 3.2.2, without additional purification.

2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)quinoline-4-carboxylic acid
The antitubercular inhibitory activity of the compounds against Mtb H 37 Rv (American Type Culture Collection, Rockville, MD, USA) was determined using a fluorescence reading at a 530 nm excitation wavelength and 590 nm emission wavelength in the Microplate Alamar Blue Assay (MABA), with Rifampin (MIC = 0.05 µM), isoniazid (MIC = 0.25 µM), linezolid (MIC = 1.16 µM), and bedaquiline (MIC = 0.23 µM) as positive drug controls. A 12.8 mM stock solution of the test compounds was first acquired by solubilizing the compounds in DMSO, and then it was diluted to its final test concentrations, which ranged from 128.0 µM to 0.5 µM. Then, in a Middlebrook 7H12 medium (7H9 broth constituted by 0.1% w/v casitone, 5.6 µg/mL of palmitic acid, 5 mg/mL bovine serum albumin, and 4mg/mL catalase, filter-sterilized), two-fold dilutions of the compounds were prepared in a 100 µL 96-well microplate. The Mtb strains were added to the microplate wells with a volume of 100 µL inoculum that contains 2 × 10 5 cfu/mL, resulting in a total volume of 200 µL. This microplate was then incubated at 37 • C for seven days. Then, 12.5 µL of Tween 80 and 20 µL of Alamar Blue (Trek Diagnostic, Westlake, OH, USA) were added to each well on the seventh day and incubated for another 16-24 h. The fluorescence measurements were taken to obtain MIC 90 values based on the average of two individual measurements of the wells. The inhibitory concentration was determined by looking at the lowest concentration exhibiting a 90% fluorescence inhibition compared to the untreated bacterial control [26].

Low Oxygen Recovery Assay
LORA cell stock preparation: Mtb reporter strain harboring pFCA-luxAB from a 7H11 agar and was inoculated into 20 mL Middlebrook 7H9 medium with kanamycin. The culture was incubated at 37 • C (150) rpm for 5 days. From the culture, 2 mL was transferred into 7H9 medium plus kanamycin. The cultures were incubated for another 5-7 days. An aliquot of 1 mL of the cell suspension was transferred into tubes and stored at −80 • C. Then, an amount of 100 µL was taken from the stock, and serial dilutions were prepared (10 −4 to 10 −6 ), which were plated on 7H11 plates. After that, 770 mL of Dubos broth medium with kanamycin and methylene blue was inoculated with a volume of the seed stock in a culture flask. The culture flask was stirred at 120 rpm at 37 • C for 10-13 days. Culture cells were harvested by centrifugation. The pellet was resuspended in 1 mL of PBS, diluted with 30 mL of PBS, and centrifuged. The supernatant was discarded. The pellet was resuspended again in 1 mL of PBS. The seed stock was stored at −80 • C.
The LORA assay was performed under anaerobic conditions, with oxygen concentrations of less than 0.16%; this condition for microplate cultures was achieved by using Anoxomat model WS-8080, followed by subsequent cycling of air evacuation and filling of 10% H 2 and 5% CO 2 and then balanced with N 2 gas thrice. The anaerobic conditions were confirmed upon the use of an anaerobic indicator strip. The plates were then incubated for 10 days at 37 • C and were recovered by transferring to a 5% CO 2 -enriched air incubator for 28 h. On the 11th day, 100 µL of the cultures was pipetted into 96-well microtiter plates. On the other hand, the microplate cultures for the aerobic assay were incubated in ambient gas conditions for 7 days, with the procedures performed for luminescence reading. In both microtiter wells, 100 µL of freshly prepared 10-fold dilution in PBS of n-decanal aldehyde from Sigma in ethanol was added, and the luminescence of these wells were read with a reading time of 1 s with a Victor multilabel reader from Perkin-Elmer Life Sciences. Rifampin (MIC = 0.22 µM), isoniazid (MIC > 128 µM), linezolid (MIC = 1.75 µM), and bedaquiline (MIC = 0.41 µM) were used as positive drug controls [27].

DNA Gyrase Inhibition
A MIX solution of the assay was prepared: relaxed pBR322 (0.5 µL) and water 17.5 µL per assay. A total of 3 µL of 0.01 µM, 1.0 µM, and 100 µM test compounds was added into each assay and mixed gently. The enzyme was diluted in a dilution buffer (1:30), and then 3 µL was transferred into each assay. The sample mixtures were mixed and incubated for 60 min at 37 • C. This was followed by the addition of 30 µL of STEB (40 % (w/v) sucrose, 100 mM of Tris-HCl pH8, 10 mM of EDTA, and 0.5 mg/mL of Bromophenol Blue) and 30 µL of chloroform/isoamyl alcohol (v:v, 24:1). The mixtures were then mixed and centrifuged. After that, 20 µL of aqueous (upper blue) phase was loaded onto a 1% (w/v) agarose gel. Electrophoresis was then run at 70V for approximately 90 min. The obtained gel was stained with 50 µg/mL ethidium bromide in water and destained in water. Samples were viewed with a transilluminator or gel documentation system for analysis [28].

Molecular Docking Analysis
Quinoline-4-carboxylic acid derivatives were subjected to molecular docking simulations with the Mtb DNA gyrase binding site (PDB ID: 5BTN) to assess their binding characteristics. The enzymes were fetched from the protein data bank as co-crystallized structures. USCF Chimera (version 1.13.1) was used to facilitate the removal of bound residues and the minimization of structures. Dock-prepping of ligand and protein structures was performed using Antechamber, and molecular docking was performed using the BFGS algorithm of AutoDock Vina (version 1.1.2). Validation of the docking protocol was performed via a redocking experiment of the co-crystallized ligand, 5-methylmoxifloxacin. The conformational protein-ligand structure was visualized and analyzed using Biovia Discovery Studios (version 4.1) [29][30][31].

Conclusions
In summary, we can assert that quinoline carboxylic acids are endowed with antimycobacterial activity. Surprisingly, similar to what happens in the case of fluoroquinolones, the presence of substituents such as halogens and alkyl groups in the classic 6 position of fluoroquinolones involves an increase in antitubercular activity in vitro against fast and non-replicating Mtb. Out of the 48 compounds synthesized, 6-1-butyl-2-(naphthalen-2yl)quinoline-4-carboxylic acid (7i) and 6-isopropyl-2-(phenanthren-3-yl)quinoline-4-carboxylic acid (7m) showed antitubercular activity. Both derivatives exhibited inhibition against Mtb DNA gyrase in vitro and in silico.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.