Structure-directed identification of pyridine-2-methylamine derivatives as MmpL3 inhibitors for use as antitubercular agents.

Mycobacterial membrane protein Large 3 (MmpL3), an inner membrane protein, plays a crucial role in the transport of mycolic acids that are essential for the viability of M. tuberculosis and has been a promising therapeutic target for new anti-TB agents. Herein, we report the discovery of pyridine-2-methylamine antitubercular compounds using a structure-based drug design strategy. Compound 62 stands out as the most potent compound with high activity against M. tb strain H37Rv (MIC = 0.016 µg/mL) as well as the clinically isolated strains of MDR/XDR-TB (MIC = 0.0039-0.0625 µg/mL), low Vero cell toxicity (IC50 ≥ 16 µg/mL), and moderate liver microsomal stability (CLint = 28 µL/min/mg). Furthermore, the resistant mutant of S288T due to single nucleotide polymorphism in mmpL3 was resistant to pyridine-2-methylamine 62, demonstrating compound 62 is likely target to MmpL3.

Synthesis and Biological Evaluation of 3-Amino-4,4-Dimethyl Lithocholic Acid Derivatives as Novel, Selective, and Cellularly Active Allosteric SHP1 Activators.

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1), a non-receptor member of the protein tyrosine phosphatase (PTP) family, negatively regulates several signaling pathways that are responsible for pathological cell processes in cancers. In this study, we report a series of 3-amino-4,4-dimethyl lithocholic acid derivatives as SHP1 activators. The most potent compounds, 5az-ba, showed low micromolar activating effects (EC50: 1.54-2.10 µM) for SHP1, with 7.63-8.79-fold maximum activation and significant selectivity over the closest homologue Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) (>32-fold). 5az-ba showed potent anti-tumor effects with IC50 values of 1.65-5.51 µM against leukemia and lung cancer cells. A new allosteric mechanism of SHP1 activation, whereby small molecules bind to a central allosteric pocket and stabilize the active conformation of SHP1, was proposed. The activation mechanism was consistent with the structure-activity relationship (SAR) data. This study demonstrates that 3-amino-4,4-dimethyl lithocholic acid derivatives can be selective SHP1 activators with potent cellular efficacy.

Therapeutic potential of coumestan Pks13 inhibitors for tuberculosis.

Polyketide synthase 13 (Pks13) is an important enzyme found in Mycobacterium tuberculosis (M. tuberculosis) that condenses two fatty acyl chains to produce α-alkyl ß-ketoesters, which in turn serve as the precursors for the synthesis of mycolic acids that are essential building blocks for maintaining the cell wall integrity of M. tuberculosis Coumestan derivatives have recently been identified in our group as a new chemotype that exert their antitubercular effects via targeting of Pks13. These compounds were active on both drug-susceptible and drug-resistant strains of M. tuberculosis as well as showing low cytotoxicity to healthy cells and a promising selectivity profile. No cross-resistance was found between the coumestan derivatives and first-line TB drugs. Here we report that treatment of M. tuberculosis bacilli with 15 times the MIC of compound 1, an optimized lead coumestan compound, resulted in a colony forming unit (CFU) reduction from 6.0 log10 units to below the limit of detection (1.0 log10 units) per mL culture, demonstrating a bactericidal mechanism of action. Single dose (10 mg/kg) pharmacokinetic studies revealed favorable parameters with a relative bioavailability of 19.4%. In a mouse infection and chemotherapy model, treatment with 1 showed dose-dependent mono-therapeutic activity, whereas treatment with 1 in combination with rifampin showed clear synergistic effects. Together these data suggest that coumestan derivatives are promising agents for further TB drug development.

Structure-Based Optimization of Coumestan Derivatives as Polyketide Synthase 13-Thioesterase(Pks13-TE) Inhibitors with Improved hERG Profiles for Mycobacterium tuberculosis Treatment.

Pks13 was identified as a key enzyme involved in the final step of mycolic acid biosynthesis. We previously identified antitubercular coumestans that targeted Pks13-TE, and these compounds exhibited high potency both in vitro and in vivo. However, lead compound 8 presented potential safety concerns because it inhibits the hERG potassium channel in electrophysiology patch-clamp assays (IC50 = 0.52 µM). By comparing the Pks13-TE-compound 8 complex and the ligand-binding pocket of the hERG ion channel, fluoro-substituted and oxazine-containing coumestans were designed and synthesized. Fluoro-substituted compound 23 and oxazine-containing coumestan 32 showed excellent antitubercular activity against both drug-susceptible and drug-resistant Mtb strains (MIC = 0.0039-0.0078 µg/mL) and exhibited limited hERG inhibition (IC50 ≥ 25 µM). Moreover, 32 exhibited improved metabolic stability relative to parent compound 8 while showing favorable bioavailability in mouse models via serum inhibition titration assays.

Design, synthesis, and biological evaluation of indole-based hydroxamic acid derivatives as histone deacetylase inhibitors.

The equilibrium between histone acetylation and deacetylation plays an important role in cancer initiation and progression. The histone deacetylases (HDACs) are a class of key regulators of gene expression that enzymatically remove an acetyl moiety from acetylated lysine ε-amino groups on histone tails. Therefore, HDAC inhibitors have recently emerged as a promising strategy for cancer therapy and several pan-HDAC inhibitors have globally been approved for clinical use. In the present study, we designed and synthesized a series of substituted indole-based hydroxamic acid derivatives that exhibited potent anti-proliferative activities in various tumor cell lines. Among the compounds tested, compound 4o, was found to be among the most potent in the inhibition of HDAC1 (half maximal inhibitory concentration, IC50 = 1.16 nM) and HDAC6 (IC50 = 2.30 nM). It also exhibited excellent in vitro anti-tumor proliferation activity. Additionally, compound 4o effectively increased the acetylation of histone H3 in a dose-dependent manner and inhibited cell proliferation by inducing cell cycle arrest and apoptosis. Moreover, compound 4o remarkably blocked colony formation in HCT116 cancer cells. Based on its favorable in vitro profile, compound 4o was further evaluated in an HCT116 xenograft mouse model, in which it demonstrated better in vivo efficacy than the clinically used HDAC inhibitor, suberanilohydroxamic acid. Interestingly, compound 4k was found to have a preference for the inhibition of HDAC6, with IC50 values of 115.20 and 5.29 nM against HDAC1 and HDAC6, respectively.

Synthesis and antitumour evaluation of indole-2-carboxamides against paediatric brain cancer cells.

Paediatric glioblastomas are rapidly growing, devastating brain neoplasms with an invasive phenotype. Radiotherapy and chemotherapy, which are the current therapeutic adjuvant to surgical resection, are still associated with various toxicity profiles and only marginally improve the course of the disease and life expectancy. A considerable body of evidence supports the antitumour and apoptotic effects of certain cannabinoids, such as WIN55,212-2, against a wide spectrum of cancer cells, including gliomas. In fact, we previously highlighted the potent cytotoxic activity of the cannabinoid ligand 5 against glioblastoma KNS42 cells. Taken together, in this study, we designed, synthesised, and evaluated several indoles and indole bioisosteres for their antitumour activities. Compounds 8a, 8c, 8f, 12c, and 24d demonstrated significant inhibitory activities against the viability (IC50 = 2.34-9.06 µM) and proliferation (IC50 = 2.88-9.85 µM) of paediatric glioblastoma KNS42 cells. All five compounds further retained their antitumour activities against two atypical teratoid/rhabdoid tumour (AT/RT) cell lines. When tested against a medulloblastoma DAOY cell line, only 8c, 8f, 12c, and 24d maintained their viability inhibitory activities. The viability assay against non-neoplastic human fibroblast HFF1 cells suggested that compounds 8a, 8c, 8f, and 12c act selectively towards the panel of paediatric brain tumour cells. In contrast, compound 24d and WIN55,212-2 were highly toxic toward HFF1 cells. Due to their structural resemblance to known cannabimimetics, the most potent compounds were tested in cannabinoid 1 and 2 receptor (CB1R and CB2R) functional assays. Compounds 8a, 8c, and 12c failed to activate or antagonise both CB1R and CB2R, whereas compounds 8f and 24d antagonised CB1R and CB2R, respectively. We also performed a transcriptional analysis on KNS42 cells treated with our prototype compound 8a and highlighted a set of seven genes that were significantly downregulated. The expression levels of these genes were previously shown to be positively correlated with tumour growth and progression, indicating their implication in the antitumour activity of 8a. Overall, the drug-like and selective antitumour profiles of indole-2-carboxamides 8a, 8c, 8f, and 12c substantiate the versatility of the indole scaffold in cancer drug discovery.

Enantioselective synthesis of chiral 3-alkyl-3-nitro-4-chromanones via chiral thiourea-catalysed intramolecular Michael-type cyclization.

Herein we report an enantioselective method for the rapid construction of chiral 3-nitro-4-chromanones via a chiral thiourea-catalyzed intramolecular Michael-type cyclization reaction. With this method, a series of 3,3-disubstituted-3-nitro-4-chromanones bearing contiguous C2/C3 stereocenters were obtained with high diastereoselectivities and good to excellent enantioselectivities. In vitro biological evaluations indicated that the chiral amide derivative of the product showed more potent antitumor activities than both the racemic and the corresponding enantiomers, showcasing the high influence of enantioselective methodology development toward medicinal studies.

[Characteristics of Partial Denitrification in Biofilm System].

As an intermediate form of microbial denitrification, nitrite serves as a key substrate for anaerobic ammonium oxidation (ANAMMOX). This study investigated the partial dentification (PD) characteristics and the coupling feasibility of PD+ANAMMOX in the biofilm system, using a moving bed biofilm reactor which was operated for 120 days. After 40 days of operation with a C/N ratio of 3.0 and filling fraction of 20%, the nitrate-to-nitrite transformation ratio (NTR) reached (69.38±3.53)%, and enzymatic assays indicated that the activities of nitrate reductase (NAR) had increased from 0.03 to 0.45 µmol·(min·mg)-1 while the activities of nitrite reductase (NIR) had decreased from 0.18 to 0.02 µmol·(min·mg)-1. Illumina high-throughput sequencing analysis revealed that the proportion of genus of Thauera bacteria to total microorganism increased from 0.3% (d1) to 37.27% (d64). Finally, the effluent had a total nitrogen (TN) concentration of (6.41±1.50) mg·L-1, indicating a total nitrogen removal ratio of (88.16±2.71)% and confirming the feasibility of PD+ANAMMOX in the biofilm system.

Sigma-1 Receptor Agonist TS-157 Improves Motor Functional Recovery by Promoting Neurite Outgrowth and pERK in Rats with Focal Cerebral Ischemia.

Sigma-1 (σ-1) receptor agonists are considered as potential treatment for stroke. TS-157 is an alkoxyisoxazole-based σ-1 receptor agonist previously discovered in our group. The present study describes TS-157 profile in a battery of tests for cerebral ischemia. Initial evaluation demonstrated the compound's safety profile and blood-brain barrier permeability, as well as its ability to induce neurite outgrowth in vitro. The neurite outgrowth was shown to be mediated via σ-1 receptor agonism and involves upregulation of ERK phosphorylation (pERK). In particular, TS-157 also significantly accelerated the recovery of motor function in rats with transient middle cerebral artery occlusion (tMCAO). Overall, the results herein support the notion that σ-1 receptor agonists are potential therapeutics for stroke and further animal efficacy studies are warranted.

[Seasonal Effects of Influent Ammonia Oxidizing Bacteria of Municipal Wastewater Treatment Plants on Activated Sludge System].

The specific ammonia uptake rates (SAUR) and ammonia oxidizing bacteria (AOB) community of influent sewage and activated sludge in the 2nd wastewater treatment plant (WWTP) of Xi'an without the primary settling tank were analyzed over multiple years to explore the seasonal effects of the influent AOB on the activated sludge systems. During the experiment, the SAUR of the raw sewage and activated sludge were 0.48-3.02 mg·(g·h)-1 and 0.68-2.25 mg·(g·h)-1, respectively. Meanwhile, the correlation analysis indicated that the monthly SAUR of the raw sewage was highly correlated with that of the activated sludge of the following month (r=0.862,P<0.05), which indicated that influent nitrifiers had a significant effect on the nitrification performance of activated sludge. Considering that the estimated AOB seeding intensities based on the ammonia oxidizing activity were 0.21-0.92 g·(g·d)-1, the nitrifier immigration from the raw sewage should added to the design of WWTP and the activated sludge modeling. Moreover, the qPCR results revealed that the AOB abundance of activated sludge in winter decreased but remained at 1010 cells·g-1, indicating that the immigration of influent nitrifiers could partially compensate for the reduction of the AOB abundance in the activated sludge caused by decreasing temperatures. Finally, the Illumina MiSeq sequencing demonstrated that the shared dominant AOB between the raw sewage and activated sludge were Nitrosomonas sp. Nm58, Nitrosomonas sp. JL21, and bacterium CYCU-0253. These findings can provide theoretical support for the design and operation of a WWTP.

Bedside cardiopulmonary ultrasonography evaluates lung water content in very low-weight preterm neonates with patent ductus arteriosus.

BACKGROUND: Patent ductus arteriosus (PDA) is a common congenital heart abnormality in preterm neonates with a high incidence in neonates with very low birth weights. When PDA persists, interstitial lung water content increases, which could lead to abnormal circulation hemodynamics and pulmonary edema. It is important to perform early and reliable assessment of lung water content in very low-weight preterm neonates with persistent PDA. AIM: To evaluate the role of bedside cardiopulmonary ultrasonography in the lung water content assessment in very low-weight preterm neonates with persistent PDA. METHODS: From January 2018 to March 2020, 69 very low-weight preterm neonates with echocardiography-confirmed PDA were selected as the PDA group. At the same time, 89 very low-weight preterm neonates without PDA were randomly selected as the control group. All neonates underwent echocardiography and 6-segment lung ultrasonography on the fourth day after birth. The clinical characteristics and main ultrasonography results were compared between the two groups. Pearson's analysis was used to analyze the correlation between lung ultrasonography score (LUS) and other related clinical and ultrasonography results in all neonates. In the PDA group, PDA diameters were recorded, and the correlation with LUS and left atrium to aortic (LA/AO) dimension ratio were also analyzed. LA/AO ratio is one of the ultrasonic diagnostic criteria for hemodynamically significant PDA. When the ratio is ≥ 1.5, it suggests the possibility of hemodynamic changes in persistent PDA. A receiver operating characteristic curve was established using the sensitivity of LUS to predict the hemodynamic changes in neonates with PDA as the ordinate and 1-specificity as the abscissa. RESULTS: A total of 158 neonates were enrolled in this study, including 69 in the PDA group and 89 in the control group. There were no statistical differences in sex, gestational age, birth weight, ventilator dependence, hospitalization length and left ventricular ejection fraction between the two groups (P > 0.05). The LUS and LA/AO ratio in the PDA group were higher than those in the control group (P < 0.05), but there was no difference of LUS in neonates with or without use of the ventilator (t = 0.58, P = 0.16). In all cases, LUS was negatively correlated with gestational age (r = -0.28, P < 0.01) and birth weight (r = -0.36, P < 0.01), while positively correlated with the LA/AO ratio (r = 0.27, P < 0.01). In the PDA group, PDA diameter was positively correlated with the LA/AO ratio (r = 0.39, P < 0.01) and LUS (r = 0.31, P < 0.01). Receiver operating characteristic results showed that LUS had the moderate accuracy for predicting hemodynamic changes in PDA (area under the curve = 0.741; sensitivity = 93.75%; specificity = 50.94%). CONCLUSION: Bedside cardiopulmonary ultrasonography can evaluate lung content in neonates with PDA and predict the possibility of hemodynamic changes in persistent PDA.

Design and synthesis of mycobacterial pks13 inhibitors: Conformationally rigid tetracyclic molecules.

We previously reported a series of coumestans-a naturally occurring tetracyclic scaffold containing a δ-lactone-that effectively target the thioesterase domain of polyketide synthase 13 (Pks13) in Mycobacterium tuberculosis (Mtb), resulting in superior anti-tuberculosis (TB) activity. Compared to the corresponding 'open-form' ethyl benzofuran-3-carboxylates, the enhanced anti-TB effects seen with the conformationally restricted coumestan series could be attributed to the extra π-π stacking interactions between the benzene ring of coumestans and the phenyl ring of F1670 residue located in the Pks13-TE binding domain. To further probe this binding feature, novel tetracyclic analogues were synthesized and evaluated for their anti-TB activity against the Mtb strain H37Rv. Initial comparison of the 'open-form' analogueues against the tetracyclic counterparts again showed that the latter is superior in terms of anti-TB activity. In particular, the δ-lactam-containing 5H-benzofuro [3,2-c]quinolin-6-ones gave the most promising results. Compound 65 demonstrated potent activity against Mtb H37Rv with MIC value between 0.0313 and 0.0625 µg/mL, with high selectivity to Vero cells (64-128 fold). The thermal stability analysis supports the notion that the tetracyclic compounds bind to the Pks13-TE domain as measured by nano DSF, consistent with the observed SAR trends. Compound 65 also showed excellent selectivity against actinobacteria and therefore unlikely to develop potential drug resistance to nonpathogenic bacteria.

Synthesis and Pharmacological Evaluation of σ2 Receptor Ligands Based on a 3-Alkoxyisoxazole Scaffold: Potential Antitumor Effects against Osteosarcoma.

Since its initial discovery as the basis for nicotinic acetylcholine receptor ligands, the 3-alkoxyisoxazole scaffold has been shown to be a versatile platform for the development of potent σ1 and σ2 receptor ligands. Herein we report a further SAR exploration of the 3-alkoxyisoxazole scaffold with the aim of obtaining potent σ2 receptor ligands. Various substitutions on the benzene ring and at the basic amino regions resulted in a total of 21 compounds that were tested for their binding affinities for the σ2 receptor. In particular, compound 51 [(2S)-1-(4-ammoniobutyl)-2-(((5-((3,4-dichlorophenoxy)methyl)isoxazol-3-yl)oxy)methyl)pyrrolidin-1-ium chloride] was identified as one of the most potent σ2 ligands within the series, with a Ki value of 7.9 nM. It demonstrated potent antiproliferative effects on both osteosarcoma cell lines 143B and MOS-J (IC50 values of 0.89 and 0.71 µM, respectively), relative to siramesine (IC50 values of 1.81 and 2.01 µM). Moreover, compound 51 inhibited clonal formation of osteosarcoma 143B cells at 1 µM, corresponding to half the dose required of siramesine for similar effects. The general cytotoxicity profile of compound 51 was assessed in a number of normal cell lines, including HaCaT, HAF, and LO2 cells. Furthermore, FACS analysis showed that compound 51 likely inhibits osteosarcoma cell growth by disruption of the cell cycle and promotion of apoptosis.

From a Designer Drug to the Discovery of Selective Cannabinoid Type 2 Receptor Agonists with Favorable Pharmacokinetic Profiles for the Treatment of Systemic Sclerosis.

Synthetic cannabinoids, as exemplified by SDB-001 (1), bind to both CB1 and CB2 receptors and exert cannabimimetic effects similar to (-)-trans-Δ9-tetrahydrocannabinol, the main psychoactive component present in the cannabis plant. As CB1 receptor ligands were found to have severe adverse psychiatric effects, increased attention was turned to exploiting the potential therapeutic value of the CB2 receptor. In our efforts to discover novel and selective CB2 receptor agonists, 1 was selected as a starting point for hit molecule identification and a class of 1H-pyrazole-3-carboxamide derivatives were thus designed, synthesized, and biologically evaluated. Systematic structure-activity relationship investigations resulted in the identification of the most promising compound 66 as a selective CB2 receptor agonist with favorable pharmacokinetic profiles. Especially, 66 treatment significantly attenuated dermal inflammation and fibrosis in a bleomycin-induced mouse model of systemic sclerosis, supporting that CB2 receptor agonists might serve as potential therapeutics for treating systemic sclerosis.

[Adaptability of Nitrifying Biofilm Systems to Low Temperature: MBBR and IFAS].

The long-term effects of a decreasing temperature on the nitrification performance, biofilm characteristics, and nitrifier community in a moving-bed biofilm reactor (MBBR) and integrated fixed-film activated sludge (IFAS) system were investigated at various temperatures (20, 15, and 10℃) to explore the adaptability of nitrifying biofilm systems to low temperatures. During the experiment, the extracellular polymeric substances (EPS) in the biofilms increased with decreasing temperature, which resulted in an increased biofilm mass and thickness. As there was only a biofilm phase in the MBBR to remove ammonia, the part of the carriers in the MBBR at 10℃ became plugged, which partially led to a deterioration in the effluent water quality. This indicated that the IFAS system was more adaptable to low temperatures than was the MBBR. Meanwhile, the results for the nitrifier activities showed that, although the nitrification contribution rate of the suspended phase in the IFAS system always dominated during the experiment, that of the fixed phase with regards to the ammonia uptake rate (AUR) gradually increased from 30.72% at 20℃ to 39.85% at 10℃. This indicated that the biofilm played an enhanced role in nitrification in the IFAS system. Moreover, the qPCR results revealed that the nitrifier copies of the number of biofilms increased slightly with decreased temperature, and coincided with an increase in biomass, which partially compensated for the decreased nitrification activity. These findings provide a theoretical basis for the application of the biofilm systems to wastewater treatment.

Capsaicin derivatives with nitrothiophene substituents: Design, synthesis and antibacterial activity against multidrug-resistant S. aureus.

To address the emergency caused by multi-drug resistant Staphylococcus aureus, a series of novel capsaicin derivatives with nitrothiophene substituents have been designed and evaluated for the antibacterial activities against S. aureus Newman and multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108, and NRS-271). The structure-activity relationship was further revealed. Compound 13c, 13f, and 13g were highly active against staphylococcal growth, with minimal inhibition concentration (MIC) values of 0.39-1.56 µg/mL. The oxadiazole-derived compound 21, a bioisostere of ester 13f, is the most potent candidate for anti-growth of five multidrug-resistant S. aureus strains with MICs of 0.20-0.78 µg/mL, which is more active compared with vancomycin in vitro. Notably, these anti-staphylococcal compounds are much less cytotoxic to the normal kidney epithelial cell line (HK293T).

Design, Synthesis, and In Vitro Evaluation of Benzofuro[3,2-c]Quinoline Derivatives as Potential Antileukemia Agents.

Herein, we design and synthesize an array of benzofuro[3,2-c]quinolines starting from 3-(2-methoxyphenyl)quinolin-4(1H)ones via a sequential chlorination/demethylation, intramolecular cyclization pathway. This sequential transformation was efficient, conducted under metal-free and mild reaction conditions, and yielded corresponding benzofuro[3,2-c]quinolines in high yields. In vitro biological evaluation indicated that such type of compounds showed excellent antileukemia activity and selectivity, and therefore may offer a promising hit compound for developing antileukemia compounds.

1-Phenyl-N-(benzothiazol-2-yl)methanimine derivatives as Middle East respiratory syndrome coronavirus inhibitors.

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to human health, and currently there are no effective or specific therapies available to treat it. Herein a series of 1-phenyl-N-(benzothiazol-2-yl)methanimine derivatives with inhibitory activity against MERS-CoV are described. The compound 4f with a 50% inhibition concentration value of 0.09 µM is a promising inhibitor that warrants further evaluation, towards the development of potential anti-MERS-CoV drugs.

Synthesis and anticancer activity of novel 9,13-disubstituted berberine derivatives.

Novel berberine derivatives with disubstituents on positions C9 and C13 were synthesized and evaluated for antiproliferative activities against human prostate cancer cell lines (PC3 and DU145), breast cancer cell line (MDA-MB-231) and human colon cancer cell lines (HT29 and HCT116). All compounds showed significantly enhanced antiproliferative activities compared with berberine. Notably, compound 18e exhibited the strongest cytotoxicity against PC3 cells with an IC50 value of 0.19 µM, and the highest selectivity index (SIPC3 > 20). Further studies showed that 18e could arrest the cell cycle at G1 phase, and significantly inhibit tumor cell colony forming and migration even at low concentrations. Interestingly, 18e could significantly induce cytoplasmic vacuolation, suggesting a different mode of action from berberine.

Identification of Novel Coumestan Derivatives as Polyketide Synthase 13 Inhibitors against Mycobacterium tuberculosis. Part II.

Our group recently reported the identification of novel coumestan derivatives as Mycobacterium tuberculosis ( Mtb) Pks13-thioesterase (TE) domain inhibitors, with mutations observed (D1644G and N1640K) in the generated coumestan-resistant Mtb colonies. Herein, we report a further structure-activity relationships exploration exploiting the available Pks13-TE X-ray co-crystal structure that resulted in the discovery of extremely potent coumestan analogues 48 and 50. These molecules possess excellent anti-tuberculosis activity against both the drug-susceptible (MIC = 0.0039 µg/mL) and drug-resistant Mtb strains (MIC = 0.0078 µg/mL). Moreover, the excellent in vitro activity is translated to the in vivo mouse serum inhibitory titration assay, with administration of coumestan 48 at 100 mg/kg showing an 8-fold higher activity than that of isoniazid or TAM16 given at 10 or 100 mg/kg, respectively. Preliminary ADME-Tox data for the coumestans were promising and, coupled with the practicality of synthesis, warrant further in vivo efficacy assessments of the coumestan derivatives.