Potent Antitubercular Activity Research Articles

Synthesis and Antitubercular Evaluation of Diverse Glycosylated Ureas from D-Glucose

N, N-disubstituted glycosylated ureas have been synthesized in good-to-excellent yields by 1,4-conjugate addition of amines to glycosylated olefinic esters followed by reaction of resulting glycosyl β-amino esters with diisocyanates. The developed sugar-based molecules were well characterized by extensive standard spectroscopic analysis including NMR (1H, 13C, and DEPT), IR, MS, and elemental analysis and were screened for their biological activity against Mycobacterium tuberculosis (M. Tb.), where some of them displayed potent antitubercular activity. The molecules have been designed keeping in view the well-known inhibitors of the enzymes involved in the biosynthesis of mycobacterial cell wall polymers and may be further explored as lead molecules for the successful development of a potential antitubercular drug candidate.Dedicated to Dr. Rama P. Tripathi, Former Chief Scientist at CSIR-Central Drug Research Institute, Lucknow, India, for his extraordinary contribution to “Drug development against Tuberculosis”

New thiazolyl-isoxazole derivatives as potential anti-infective agents: design, synthesis, in vitro and in silico antimicrobial efficacy

Antimicrobial resistance threatens the efficacious prevention and treatment of infectious diseases caused by microorganisms. To combat microbial infections, the need for new drug candidates is essential. In this context, the design, synthesis, antimicrobial screening, and in silico study of a new series of 5-aryl-3-(2-arylthiazol-4-yl)isoxazole (9a-t) have been reported. The structure of new compounds was confirmed by spectrometric methods. Compounds 9a-t were evaluated for in vitro antitubercular and antimicrobial activity. Against M. tuberculosis H37Rv, fourteen compounds showed good to excellent antitubercular activity with MIC 2.01–9.80 µM. Compounds 9a, 9b, and 9r showed four-fold more activity than the reference drug isoniazid. Nine compounds, 9a, 9b, 9d, 9e, 9i, 9q, 9r, 9s, and 9t, showed good antibacterial activity against E. coli with MIC 7.8–15.62 µg/mL. Against A. niger, four compounds showed good activity with MIC 31.25 µg/mL. Against C. albicans, all twenty compounds reported excellent to good activity with MIC 7.8–31.25 µg/mL. Compounds 9c–e, 9g–j, and 9q–t showed comparable activity concerning the reference drug fluconazole. The compounds 9a-t were screened for cytotoxicity against 3t3l1 cell lines and found to be less or non-cytotoxic. The in silico study exposed that these compounds displayed high affinity towards the M. tuberculosis targets PanK, DprE1, DHFR, PknA, KasA, and Pks13, and C. albicans targets NMT, CYP51, and CS. The compound 9r was evaluated for structural dynamics and molecular dynamics simulations. The potent antitubercular and antimicrobial activity of 5-aryl-3-(2-arylthiazol-4-yl)isoxazole (9a–t) derivatives has recommended that these compounds could assist in treating microbial infections. Communicated by Ramaswamy H. Sarma

Ultrasound-assisted ionic liquid-mediated green method for synthesis of 1,3-diphenylpyrazole-based spirooxindolopyrrolizidines, their anti-tubercular activity, molecular docking study and ADME predictions

The aim of the study is to develop the synthesis of a novel series of potent anti-tubercular (anti-TB) activity of 1,3-diphenylpyrazole-based spirooxindolopyrrolizidine derivatives via an efficient green approach achieved by...

Design, synthesis and in silico docking techniques of new 1,2,3-triazolylpyrrolidines bearing chalcone derivatives: Discovery of potent antitubercular agents

Compounds with a pyrrolidine scaffold play an important role in organic synthesis and especially in the synthesis of bioactive organic compounds, therefore, the development of new methods for modifying this scaffold is a very interesting framework of this study. We developed a rational approach for the synthesis of 1,2,3-trazolylchalcone substituted pyrrolidines derivatives, which were then examined using a variety of spectroscopic techniques such as 1H NMR, 13C NMR, FT-IR, mass spectroscopy and elemental analysis. Biological profiles showed that compounds 5e, 5h had better antibacterial inhibitory potency against S. aureus, E. coli with zone of inhibition 34 ± 0.1, 33 ± 0.3 mm, whereas 5a, 5e showed potent antifungal activity against C. parapsilosis, A. flavus with dimeter zone of inhibition 26 ± 0.2, and 30 ± 0.2 mm respectively. Among the tested compounds 5b, and 5h were the most potent antitubercular activity against Mycobacterium tuberculosis H37Rv and showing MIC values 5.23 µg/mL, 6.85 µg/mL respectively, which are similar activity that of the standard Streptomycin (MIC = 5.02 µg/mL). The binding mode for compound 5 inside the catalytic pocket of M. tuberculosis cytochrome P450 CYP121A1 and produced a network of hydrophobic and hydrophilic interactions (6GEO). From docking results, 5b demonstrated highly stable binding amino acids SerA:237, ArgA:386, ArgA:286, CysA:345, MetA:62, GlnA:385, AspA:282, PheA:280, LeuA:284, ValA:83, ProA:285, AlaA:337, HisA:343, AsnA:74, and ThrA:65, which are plays a crucial role in ensuring efficient binding of the ligand in a crystal structure of tubercular receptor. Furthermore, the physicochemical and ADME (absorption, distribution, metabolism, and excretion) filtration molecular properties, estimation of toxicity, and bioactivity scores of these scaffolds were evaluated.

Synthesis, Antifungal, and Anti-tubercular Activity of some new 7-amino-2- (Substituted aryl)-5-oxo-1,5-dihydro-[1,2,4]Triazolo[1,5- a]Pyridine-6-carbonitrile Derivatives

A series of some 7-amino-2-(substituted aryl)-5-oxo-1,5-dihydro-[1,2,4]triazolo[1,5- a]pyridine-6-carbonitrile derivatives was synthesized by the reaction of cyanoacetohydrazone derivatives with malononitrile in presence of catalytic base piperidine and ethanol as solvent. Comparing the antifungal activity of all the investigated compounds to that of standard Amphotericin-B on Potato- Dextrose-Agar medium, compounds 1a, 1e, and 1i revealed excellent antifungal action against Aspergillus niger and Candida albicans. Using the Microplate-Alamar Blue Assay, the anti-tubercular activity of compounds 1a?1j was assessed against Mycobacterium tuberculosis H37Rv. Compared to the conventional isoniazid and ethambutol minimum inhibitory concentration of 1.6 ?g/mL, 1i and 1j had potent anti-tubercular activity at 1.6 ?g/mL, and 1b possessed moderate activity at 3.12 ?g/mL. Electron-donating groups have better anti-fungal and anti-tubercular efficacy than other substituents.. KEYWORDS :Anti-tubercular activity, Ethylcyanoacetate, Malononitrile, Microplate- Alamar Blue Assay, Potato-Dextrose-Agar medium, Triazolo[1,5-a]pyridines.

New azaaurone derivatives as potential multitarget agents in HIV-TB coinfection.

Tuberculosis (TB) disease, caused by Mycobacterium tuberculosis (Mtb) is the leading cause of death among people with human immunodeficiency virus (HIV) infection. No dual-target drug is currently being used to simultaneously treat both infections. This work aimed to obtain new multitarget HIV-TB agents, with the goal of optimizing treatments and preventing this coinfection. These compounds incorporate the structural features of azaaurones as anti-Mtb and zidovudine (AZT) as the antiretroviral moiety. The azaaurone scaffold displayed submicromolar activities against Mtb, and AZT is a potent antiretroviral drug. Six derivatives were synthetically generated, and five were evaluated against both infective agents. Evaluations of anti-HIV activity were carried out in HIV-1-infected MT-4 cells and on endogenous HIV-1 reverse transcriptase (RT) activity. The H37Rv strain was used for anti-Mtb assessments. Most compounds displayed potent antitubercular and moderate anti-HIV activity. (E)-12 exhibited a promising multitarget profile with an MIC90 of 2.82 µM and an IC50 of 1.98 µM in HIV-1-infected T lymphocyte cells, with an 84% inhibition of RT activity. Therefore, (E)-12 could be the first promising compound from a family of multitarget agents used to treat HIV-TB coinfection. In addition, the compound could offer a prototype for the development of new strategies in scientific research to treat this global health issue.

DISCOVERY OF NEW NAPHTHYRIDINE HYBRIDS AGAINST ENOYL-ACP REDUCTASE (InhA) PROTEIN TARGET OF MYCOBACTERIUM TUBERCULOSIS: MOLECULAR DOCKING, MOLECULAR DYNAMICS SIMULATIONS STUDIES

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and remains a significant global public health concern, with many new cases reported annually. Despite advancements in TB treatment and control, the emergence of drug-resistant strains has presented a considerable challenge to eradication efforts. One crucial area that necessitates further research in antituberculosis is the development of effective treatments for drug-resistant strains, particularly multidrug resistance tuberculosis (MDR-TB), extensively drug-resistant tuberculosis (XDR-TB), and totally drug–resistant tuberculosis (TDR-TB). The limited and often unsatisfactory treatment options available for drug-resistant strains require the exploration of novel drugs and treatment regimens to combat this escalating issue. This study focuses on designing and developing a series of new 1,8-naphthyridine derivatives for their potential antitubercular activity. The compounds were designed and evaluated through in-silico screening using Molinspiration Cheminformatics, Osiris Property Explorer, AdmetSAR, and SwissADME. The initial filtering process identified the top 16 hybrids, which were then subjected to docking using the AutoDock tool. These compounds' binding energies were higher than the standard drug isoniazid, indicating a potentially more substantial interaction with the target. Based on the promising in silico and docking results, further investigations were conducted on the top 3 compounds, namely ST03, ST09, and ST14. To assess the stability and binding energies observed during the initial docking process, molecular dynamics simulations were performed over a 100 ns period. Both the docking and simulation studies consistently demonstrated that the 1,8-naphthyridine hybrid ST09 exhibited stable and efficient binding energies, suggesting its potential as an effective antituberculosis agent.

Reassessing the putative molecular Target(s) of potent antitubercular 2-(Alkylsulfonyl)acetamides

Simple alkyl-sulfonylacetamides have potent antitubercular activity and significantly decrease mycolic acid levels in mycobacteria. Although these compounds were originally designed to inhibit the ketoacyl synthase domain of fatty acid synthase, structure-activity relationships and biochemical evidence do not fully support fatty acid synthase as the target. In 2004, an enzyme family involved in the activation and transfer of fatty acids as acyl-adenylates was identified in mycobacteria, separate from the universal acetyl-CoA carrier mechanism. These fatty acyl-AMP ligases (FAAL), encoded by the FadD family play important roles in the biosynthesis of mycolic acids along with fatty acid metabolism and are hypothesised here to be the molecular target of the sulfonylacetamides. Due to structural similarities with the ligase's natural substrate, it is believed these compounds are exerting action via competitive inhibition of these highly potent molecular targets. The primary aim of this investigation was to synthesize an extended library of sulfonylacetamide derivatives, building upon existing structural activity relations to validate the molecular mechanism with the aid of molecular modelling, while also attempting to explore novel structural isosteres for further drug design and development. Sulfonylacetamide derivatives were modified based on the putative molecular target resulting in derivatives with improved activities towards Mycobacteriumtuberculosis (H37Rv). The most active novel derivatives reported were 19, 22b, 22c and 46 displaying MIC90 levels of 1.4, 16.0, 13.0 and 5.9 μg/mL, respectively.

Microbial glycosylation of antitubercular agent chlorflavonin

Flavonoids have shown health-benefiting properties, such as antioxidative and anti-inflammatory activities, and are commonly used as nutraceuticals and pharmaceuticals. Although flavonoids are predominantly identified from plants, several filamentous fungal species have also been reported to produce bioactive flavonoids, including chlorflavonin from Aspergillus candidus, a novel halogenated flavonoid with potent antifungal and antitubercular (anti-TB) activities. Unfortunately, the low water-solubility of this molecule may hinder its bioavailability. Glycosylation is an effective method to enhance the polarity of natural products and alter their physicochemical properties. This work focuses on the development of novel water-soluble chlorflavonin derivatives to combat the threat of drug-resistant tuberculosis. In this study, we first increased the production titer of chlorflavonin in A.candidus NRRL 5214 by optimizing the fermentation and purification processes. Next, chlorflavonin-5-O-β-d-glucuronopyranoside (1) and chlorflavonin-7-O-4″-O-methyl-β-d-glucopyranoside (2) were produced from chlorflavonin using Streptomyces chromofuscus ATCC 49982 and Beauveria bassiana ATCC 7159, respectively. Compared to chlorflavonin (4.38 ± 0.54mg/L in water), the water solubility of the two new glycosides was determined to be 117.86 ± 4.81mg/L (1) and 124.34 ± 9.13mg/L (2), respectively. This study provides a promising method to create water-soluble glycosides of chlorflavonin for the development of novel anti-TB drugs.

In vitro anti-mycobacterial property and phytochemical profiling of Senna auriculata and Trachyspermum copticum

Mycobacterium tuberculosis is a contagious disease that is the leading cause of death by a single causative organism. The present study aims to investigate and assess the anti-mycobacterial property of some traditionally important medicinal plants and analyze their phytochemical constitution. The extracts of some plants based on their medicinal importance were checked for their activity against M. smegmatis using anti-mycobacterial disc diffusion plate assay. Aqueous extracts of Senna auriculata, Trachyspermum copticum and Musa spp. were tested against M. smegmatis. Further Senna auriculata and Trachyspermum copticum extracts were taken to bioactivity guided sequential extraction. On screening, ethyl acetate extract of S. auriculata, T. copticum and methanol extract of S. auriculata showed considerable inhibition with an average zone of inhibition of about 2 cm dia against M. smegmatis. Further phytochemical analysis was done to narrow down the class of compounds that might be responsible for the inhibitory activity. The tests revealed the presence of some important group of natural compounds like alkaloids, anthocyanins, tannins, β- cyanins, coumarins and terpenoid. GC-MS analysis of the samples showed some biologically important compounds with potent anti-tubercular activity.

Design and synthesis of 4-nitroimidazole derivatives with potential antitubercular activity

Objectives. To develop the procedures for synthesis of hybrid molecules with potential anti-tubercular activity containing heterocyclic cores of 4-nitroimidazole and 1,3,4-thiadiazole within the framework of a double-drug strategy and predict bioactivity of target structures and drug-likeness physicochemical parameters.Methods. Target compounds were prepared by classical organic synthesis methods. The structure of the obtained compounds was characterized by melting points, 1H and 13C nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry. The calculation of the physicochemical parameters of the target compounds and prediction of their biological activity were carried out using publicly available software for cheminformatics and molecular modeling.Results. Acylation of propargylamine with (2-methyl-4-nitro-1H-imidazol-1-yl)acetic and (4-nitro-1H-imidazol-1-yl)acetic acids provided the corresponding amides, which were cyclized with seven different benzylamines in the presence of zinc triflate. In this way, seven new compounds were obtained at 20–30% yields. Ten arylamines were acylated with chloroacetyl chloride and the resulting chloroacetamides were converted into corresponding thio-oxahydrazides by the Willgerodt–Kindler reaction. Following acylation by (4-nitro-1H-imidazol-1-yl)acetic acid, these compounds were converted into the target hybrid imidazolyl-thiadiazoles at 29–54% yields.Conclusions. Two series of new heterocyclic compounds with a hybrid structure including a privileged 4-nitroimidazole moiety linked to the second heterocycle, imidazole, or thiadiazole, were obtained. The synthesis and characterization of compounds by physicochemical methods was aimed at searching for anti-tuberculosis activity. The bioactivity potential of target compounds was demonstrated by preliminary calculations performed using public prognostic programs.

Design, synthesis and biological evaluation of novel oxindole analogs as antitubercular agents.

Aim: To design, synthesize and evaluate oxindole derivatives for antitubercular activity. Methodology: We synthesized the derivatives, confirmed their structures by 1H/13C NMR and mass spectrometry, and evaluated them for antitubercular activity against Mycobacterium tuberculosis H37Rv strain using the microplate alamarBlue™ assay. Results: Among all the synthesized derivatives, OXN-1, -3 and -7 exhibited excellent antitubercular activity (minimum inhibitory concentration [MIC]: 0.78μg/ml). Compounds with a MIC ≤1.56 were tested for cytotoxicity against human embryonic kidney cells and were found to be relatively nontoxic. Molecular docking analysis of OXN-1, -3 and -7 was performed to determine their binding patterns at the active site of DNA topoisomerase II (PDB-5BS8). In drug combination studies, OXN-1, 3 and 7 showed synergism with isoniazid. Conclusion: The obtained results reveal that oxindole derivatives exhibit potent antitubercular activity.

Coumarin as a Privileged and Medicinally important Scaffold in the Treatment of Tuberculosis.

Coumarin and its derivatives, which are abundant in nature, have a significant role in medicinal chemistry due to their ability to bind with different targets or receptors. In addition, these possess a wide range of biological activity. Thus coumarin-based scaffold has inspired even further research into coumarin and its substituted derivatives, allowing for the creation of a huge variety of structurally different substituted products. In recent, these were reported to have potent antitubercular activity. Tuberculosis (TB) is a serious deadly infectious bacterial disease caused by gram-positive Mycobacterium tuberculosis. This review discusses various developments going on in the field of medicinal chemistry towards designing, synthesizing, and discovering coumarin-based antitubercular agents all across the globe.

Tandem LC-MS Identification of Antitubercular Compounds in Zones of Growth Inhibition Produced by South African Filamentous Actinobacteria.

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7-151.6 nM against M. tuberculosis H37RvTin vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique.

Design, synthesis, and biological evaluation of 1,2,4-triazole derivatives as potent antitubercular agents

Inhibition of mycobacterial membrane protein large 3 (MmpL3) thereby affecting the mycolic acid biosynthetic pathway has been proven to be an effective strategy for developing antitubercular drugs. Based on the X-ray crystal structure of MmpL3 inhibitor complexes, a series of novel 1,2,4-triazole derivatives were designed, synthesized and evaluated antitubercular activity against Mtb strain H37Rv. Comprehensive structure–activity relationship exploration resulted in the identification of compounds 21 and 28, which possess potent antitubercular activity against Mtb strain H37Rv [minimum inhibitory concentration (MIC) = 0.03–0.13 µg/mL] and the clinical isolates of multidrug resistance (MDR) and extensive drug resistance (XDR) tuberculosis (MIC = 0.06–1.0 µg/mL). Moreover, compounds 21 and 28 showed neglectable cytotoxicity (IC50 ≥ 32 µg/mL) to the mammalian Vero cells and favorable physicochemical and pharmacokinetic properties according to the in silico absorption, distribution, metabolism and excretion (ADME) prediction. Finally, the potential target of representative 1,2,4-triazole 28 was identified to be MmpL3 using a microscale thermophoresis (MST) assay.

Discovery of Anti-tubercular Analogues of Bedaquiline with Modified A-, B- and C-Ring Subunits.

To date, the clinical use of the anti-tubercular therapy bedaquiline has been somewhat limited due to safety concerns. Recent investigations determined that modification of the B- and C-ring units of bedaquiline delivered new diarylquinolines (for example TBAJ-587) with potent anti-tubercular activity yet an improved safety profile due to reduced affinity for the hERG channel. Building on our recent discovery that substitution of the quinoline motif (the A-ring subunit) for C5-aryl pyridine groups within bedaquiline analogues led to retention of anti-tubercular activity, we investigated the concurrent modification of A-, B- and C-ring units within bedaquiline variants. This led to the discovery that 4-trifluoromethoxyphenyl and 4-chlorophenyl pyridyl analogues of TBAJ-587 retained relatively potent anti-tubercular activity and for the 4-chlorophenyl derivative in particular, a significant reduction in hERG inhibition relative to bedaquiline was achieved, demonstrating that modifications of the A-, B- and C-ring units within the bedaquiline structure is a viable strategy for the design of effective, yet safer (and less lipophilic) anti-tubercular compounds.

Synthesis, characterization and in vitro anti mycobacterial activity of some novel benzothiazole derivatives

World Health Organization considers Mycobacterium tuberculosis is the most dangerous chronic transmittable disease. Development of resistance by Mycobacterium tuberculosis is a common problem world Wide. To combat these problems there could be a solution that we should search newer molecules with different mechanism of action to fight against such diseases. A series of some novel benzothiazole derivatives (I-X) have been synthesized and evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using Microplate Alamar Blue Assay Method (MABA). Structures of the synthesized compounds were supported by means of IR, NMR and MASS spectral studies. Among the synthesized compounds, compound [IX], 2-[5-(4-Bromophenyl)-2-furylcarboxamido]-6-methoxy-l,3-benzothiazole exhibited a significant activity when compared with the standard drug Isoniazid. This could be the remarkable starting point to develop new lead molecules with potential antitubercular activity.

Nitrothiazole-Thiazolidinone Hybrids: Synthesis and in Vitro Antimicrobial Evaluation.

Herein we report the synthesis of novel compounds inspired by the antimicrobial activities of nitroazole and thiazolidin-4-one based compounds reported in the literature. Target compounds were investigated in vitro for antitubercular, antibacterial, antifungal, and overt cell toxicity properties. All compounds exhibited potent antitubercular activity. Most compounds exhibited low micromolar activity against S. aureus and C. albicans with no overt cell toxicity against HEK-293 cells nor haemolysis against human red blood cells. Notably, compound 3b exhibited low to sub-micromolar activities against Mtb, MRSA, and C. albicans. 3b showed superior activity (0.25 μg/ml) against MRSA compared to vancomycin (1 μg/ml).

Discovery of newer pyrazole derivatives with potential anti-tubercular activity via 3D-QSAR based pharmacophore modelling, virtual screening, molecular docking and molecular dynamics simulation studies.

Tuberculosis is one of the leading causes of death of at least one million people annually. The deadliest infectious disease has caused more than 120 million deaths in humans since 1882. The cell wall structure of Mycobacterium tuberculosis is important for survival in the host environment. InhA is the foremost target for the development of novel anti-tubercular agents. Therefore, we report pharmacophore-based virtual screening (ZINC and ASINEX databases) and molecular docking study (PDB Code: 4TZK) to identify and design potent inhibitors targeting to InhA. A five-point pharmacophore model AADHR_1 (with R2 = 0.97 and Q2 = 0.77) was developed by using 47 compounds with its reported MIC values. Further, to identify and design potent hit molecules based on lead identification and modification, generated hypothesis employed for virtual screening using ZINC and ASINEX databases. Predicted pyrazole derivatives further gauged for drug likeliness and docked against enoyl acyl carrier protein reductase to categorize the essential amino acid interactions to the active site of the enzyme. Structure elucidation of these synthesized compounds was carried out using IR, MS, 1H-NMR and 13C-NMR spectroscopy. Amongst all the synthesized compounds, some of the compounds 5a, 5c, 5d and 5e were found to be potent with their MIC ranging from 2.23 to 4.61µM. Based on preliminary anti-tubercular activity synthesized potent molecules were further assessed for MDR-TB, XDR-TB and cytotoxic study.

Synthesis and Assessment of the In Vitro and Ex Vivo Activity of Salicylate Synthase (Mbti) Inhibitors as New Candidates for the Treatment of Mycobacterial Infections.

Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of M. tuberculosis (Mtb) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new anti-TB agents is the salicylate synthase MbtI, the first enzyme of the mycobacterial siderophore biochemical machinery, absent in human cells. In this work, a set of analogues of 5-(3-cyanophenyl)furan-2-carboxylic acid (I), the most potent MbtI inhibitor identified to date, was synthesized, characterized, and tested to further elucidate the structural requirements for achieving an efficient MbtI inhibition and potent antitubercular activity. The structure–activity relationships (SAR) discussed herein evidenced the importance of the side chain linked to the phenyl moiety to improve the in vitro antimycobacterial activity. In detail, 1f emerged as the most effective analogue against the pathogen, acting without cytotoxicity issues. To deepen the understanding of its mechanism of action, we established a fluorescence-based screening test to quantify the pathogen infectivity within host cells, using MPI-2 murine cells, a robust surrogate for alveolar macrophages. The set-up of the new assay demonstrates significant potential to accelerate the discovery of new anti-TB drugs.