hERG, Plasmodium Life Cycle, and Cross Resistance Profiling of New Azabenzimidazole Analogues of Astemizole.

Toward addressing the cardiotoxicity liability associated with the antimalarial drug astemizole (AST, hERG IC50 = 0.0042 µM) and its derivatives, we designed and synthesized analogues based on compound 1 (Pf NF54 IC50 = 0.012 µM; hERG IC50 = 0.63 µM), our previously identified 3-trifluoromethyl-1,2,4-oxadiazole AST analogue. Compound 11 retained in vitro multistage antiplasmodium activity (ABS PfNF54 IC50 = 0.017 µM; gametocytes PfiGc/PfLGc IC50 = 1.24/1.39 µM, and liver-stage PbHepG2 IC50 = 2.30 µM), good microsomal metabolic stability (MLM CLint < 11 µL·min-1·mg-1, EH < 0.33), and solubility (150 µM). It shows a ∼6-fold and >6000-fold higher selectivity against human ether-á-go-go-related gene higher selectively potential over hERG relative to 1 and AST, respectively. Despite the excellent in vitro antiplasmodium activity profile, in vivo efficacy in the Plasmodium berghei mouse infection model was diminished, attributable to suboptimal oral bioavailability (F = 14.9%) at 10 mg·kg-1 resulting from poor permeability (log D7.4 = -0.82). No cross-resistance was observed against 44 common Pf mutant lines, suggesting activity via a novel mechanism of action.

Antimalarial Imidazopyridines Incorporating an Intramolecular Hydrogen Bonding Motif: Medicinal Chemistry and Mechanistic Studies.

We previously identified a novel class of antimalarial benzimidazoles incorporating an intramolecular hydrogen bonding motif. The frontrunner of the series, analogue A, showed nanomolar activity against the chloroquine-sensitive NF54 and multi-drug-resistant K1 strains of Plasmodium falciparum (PfNF54 IC50 = 0.079 µM; PfK1 IC50 = 0.335 µM). Here, we describe a cell-based medicinal chemistry structure-activity relationship study using compound A as a basis. This effort led to the identification of novel antimalarial imidazopyridines with activities of <1 µM, favorable cytotoxicity profiles, and good physicochemical properties. Analogue 14 ( PfNF54 IC50 = 0.08 µM; PfK1 IC50 = 0.10 µM) was identified as the frontrunner of the series. Preliminary mode of action studies employing molecular docking, live-cell confocal microscopy, and a cellular heme fractionation assay revealed that 14 does not directly inhibit the conversion of heme to hemozoin, although it could be involved in other processes in the parasite's digestive vacuole.

Novel 3-Trifluoromethyl-1,2,4-oxadiazole Analogues of Astemizole with Multi-stage Antiplasmodium Activity and In Vivo Efficacy in a Plasmodium berghei Mouse Malaria Infection Model.

Iterative medicinal chemistry optimization of an ester-containing astemizole (AST) analogue 1 with an associated metabolic instability liability led to the identification of a highly potent 3-trifluoromethyl-1,2,4-oxadiazole analogue 23 (PfNF54 IC50 = 0.012 µM; PfK1 IC50 = 0.040 µM) displaying high microsomal metabolic stability (HLM CLint < 11.6 µL·min-1·mg-1) and > 1000-fold higher selectivity over hERG compared to AST. In addition to asexual blood stage activity, the compound also shows activity against liver and gametocyte life cycle stages and demonstrates in vivo efficacy in Plasmodium berghei-infected mice at 4 × 50 mg·kg-1 oral dose. Preliminary interrogation of the mode of action using live-cell microscopy and cellular heme speciation revealed that 23 could be affecting multiple processes in the parasitic digestive vacuole, with the possibility of a novel target at play in the organelles associated with it.

Total Synthesis of the Antimycobacterial Natural Product Chlorflavonin and Analogs via a Late-Stage Ruthenium(II)-Catalyzed ortho-C(sp2)-H-Hydroxylation.

The continuous, worldwide spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) endanger the World Health Organization's (WHO) goal to end the global TB pandemic by the year 2035. During the past 50 years, very few new drugs have been approved by medical agencies to treat drug-resistant TB. Therefore, the development of novel antimycobacterial drug candidates to combat the threat of drug-resistant TB is urgent. In this work, we developed and optimized a total synthesis of the antimycobacterial natural flavonoid chlorflavonin by selective ruthenium(II)-catalyzed ortho-C(sp2)-H-hydroxylation of a substituted 3'-methoxyflavonoid skeleton. We extended our methodology to synthesize a small compound library of 14 structural analogs. The new analogs were tested for their antimycobacterial in vitro activity against Mycobacterium tuberculosis (Mtb) and their cytotoxicity against various human cell lines. The most promising new analog bromflavonin exhibited improved antimycobacterial in vitro activity against the virulent H37Rv strain of Mtb (Minimal Inhibitory Concentrations (MIC90) = 0.78 µm). In addition, we determined the chemical and metabolic stability as well as the pKa values of chlorflavonin and bromflavonin. Furthermore, we established a quantitative structure-activity relationship model using a thermodynamic integration approach. Our computations may be used for suggesting further structural changes to develop improved derivatives.

Spiropyrimidinetriones: a Class of DNA Gyrase Inhibitors with Activity against Mycobacterium tuberculosis and without Cross-Resistance to Fluoroquinolones.

Described here is a series of spiropyrimidinetrione (SPT) compounds with activity against Mycobacterium tuberculosis through inhibition of DNA gyrase. The SPT class operates via a novel mode of inhibition, which involves Mg2+-independent stabilization of the DNA cleavage complex with DNA gyrase and is thereby not cross-resistant with other DNA gyrase-inhibiting antibacterials, including fluoroquinolones. Compound 22 from the series was profiled broadly and showed in vitro cidality as well as intracellular activity against M. tuberculosis in macrophages. Evidence for the DNA gyrase mode of action was supported by inhibition of the target in a DNA supercoiling assay and elicitation of an SOS response seen in a recA reporter strain of M. tuberculosis. Pharmacokinetic properties of 22 supported evaluation of efficacy in an acute model of M. tuberculosis infection, where modest reduction in CFU numbers was seen. This work offers promise for deriving a novel drug class of tuberculosis agent without preexisting clinical resistance.

Structure-Activity Relationship Studies Reveal New Astemizole Analogues Active against Plasmodium falciparum In Vitro.

In the context of drug repositioning and expanding the existing structure-activity relationship around astemizole (AST), a new series of analogues were designed, synthesized, and evaluated for their antiplasmodium activity. Among 46 analogues tested, compounds 21, 30, and 33 displayed high activities against asexual blood stage parasites (PfNF54 IC50 = 0.025-0.043 µM), whereas amide compound 46 additionally showed activity against late-stage gametocytes (stage IV/V; PfLG IC50 = 0.6 ± 0.1 µM) and 860-fold higher selectivity over hERG (46, SI = 43) compared to AST. Several analogues displaying high solubility (Sol > 100 µM) and low cytoxicity in the Chinese hamster ovary (SI > 148) cell line have also been identified.

1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis Agents Targeting Cell Wall Biosynthesis in Mycobacterium tuberculosis.

Phenotypic whole cell high-throughput screening of a ∼150,000 diverse set of compounds against Mycobacterium tuberculosis (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of <0.5 µM, and a plausible pharmacophore model was developed to describe the chemical space of active compounds. Compounds are bactericidal in vitro against replicating Mtb and retained activity against multidrug-resistant clinical isolates. Initial biology triage assays indicated cell wall biosynthesis as a plausible mode-of-action for the series. However, no cross-resistance with known cell wall targets such as MmpL3, DprE1, InhA, and EthA was detected, suggesting a potentially novel mode-of-action or inhibition. The in vitro and in vivo drug metabolism and pharmacokinetics profiles of several active compounds from the series were established leading to the identification of a compound for in vivo efficacy proof-of-concept studies.

Benzoheterocyclic Oxime Carbamates Active against Mycobacterium tuberculosis: Synthesis, Structure-Activity Relationship, Metabolism, and Biology Triaging.

Screening of a library of small polar molecules against Mycobacterium tuberculosis (Mtb) led to the identification of a potent benzoheterocyclic oxime carbamate hit series. This series was subjected to medicinal chemistry progression underpinned by structure-activity relationship studies toward identifying a compound for proof-of-concept studies and defining a lead optimization strategy. Carbamate and free oxime frontrunner compounds with good stability in liver microsomes and no hERG channel inhibition liability were identified and evaluated in vivo for pharmacokinetic properties. Mtb-mediated permeation and metabolism studies revealed that the carbamates were acting as prodrugs. Toward mechanism of action elucidation, selected compounds were tested in biology triage assays to assess their activity against known promiscuous targets. Taken together, these data suggest a novel yet unknown mode of action for these antitubercular hits.

Antimalarial Benzimidazole Derivatives Incorporating Phenolic Mannich Base Side Chains Inhibit Microtubule and Hemozoin Formation: Structure-Activity Relationship and In Vivo Oral Efficacy Studies.

A novel series of antimalarial benzimidazole derivatives incorporating phenolic Mannich base side chains at the C2 position, which possess dual asexual blood and sexual stage activities, is presented. Structure-activity relationship studies revealed that the 1-benzylbenzimidazole analogues possessed submicromolar asexual blood and sexual stage activities in contrast to the 1H-benzimidazole analogues, which were only active against asexual blood stage (ABS) parasites. Further, the former demonstrated microtubule inhibitory activity in ABS parasites but more significantly in stage II/III gametocytes. In addition to being bona fide inhibitors of hemozoin formation, the 1H-benzimidazole analogues also showed inhibitory effects on microtubules. In vivo efficacy studies in Plasmodium berghei-infected mice revealed that the frontrunner compound 41 exhibited high efficacy (98% reduction in parasitemia) when dosed orally at 4 × 50 mg/kg. Generally, the compounds were noncytotoxic to mammalian cells.

Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite Plasmodium falciparum and Optimization Efforts.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

Synthesis and In Vitro Antiprotozoan Evaluation of 4-/8-Aminoquinoline-based Lactams and Tetrazoles.

A second generation of 4-aminoquinoline- and 8-aminoquinoline-based tetrazoles and lactams were synthesized via the Staudinger and Ugi multicomponent reactions. These compounds were subsequently evaluated in vitro for their potential antiplasmodium activity against a multidrug-resistant K1 strain and for their antitrypanosomal activity against a cultured T. b. rhodesiense STIB900 strain. Several of these compounds (4a-g) displayed good antiplasmodium activities (IC50 = 0.20-0.62 µM) that were comparable to the reference drugs, while their antitrypanosomal activity was moderate (<20 µM). Compound 4e was 2-fold more active than primaquine and was also the most active (IC50 = 7.01 µM) against T. b. rhodesiense and also exhibited excellent aqueous solubility (>200 µM) at pH 7.

Identification of 2,4-Disubstituted Imidazopyridines as Hemozoin Formation Inhibitors with Fast-Killing Kinetics and In Vivo Efficacy in the Plasmodium falciparum NSG Mouse Model.

A series of 2,4-disubstituted imidazopyridines, originating from a SoftFocus Kinase library, was identified from a high throughput phenotypic screen against the human malaria parasite Plasmodium falciparum. Hit compounds showed moderate asexual blood stage activity. During lead optimization, several issues were flagged such as cross-resistance against the multidrug-resistant K1 strain, in vitro cytotoxicity, and cardiotoxicity and were addressed through structure-activity and structure-property relationship studies. Pharmacokinetic properties were assessed in mice for compounds showing desirable in vitro activity, a selectivity window over cytotoxicity, and microsomal metabolic stability. Frontrunner compound 37 showed good exposure in mice combined with good in vitro activity against the malaria parasite, which translated into in vivo efficacy in the P. falciparum NOD-scid IL-2Rγnull (NSG) mouse model. Preliminary mechanistic studies suggest inhibition of hemozoin formation as a contributing mode of action.

Synthesis, Structure-Activity Relationship, and Mechanistic Studies of Aminoquinazolinones Displaying Antimycobacterial Activity.

Phenotypic whole-cell screening against Mycobacterium tuberculosis (Mtb) in glycerol-alanine-salts supplemented with Tween 80 and iron (GASTE-Fe) media led to the identification of a 2-aminoquinazolinone hit compound, sulfone 1 which was optimized for solubility by replacing the sulfone moiety with a sulfoxide 2. The synthesis and structure-activity relationship (SAR) studies identified several compounds with potent antimycobacterial activity, which were metabolically stable and noncytotoxic. Compound 2 displayed favorable in vitro properties and was therefore selected for in vivo pharmacokinetic (PK) studies where it was found to be extensively metabolized to the sulfone 1. Both derivatives exhibited promising PK parameters; however, when 2 was evaluated for in vivo efficacy in an acute TB infection mouse model, it was found to be inactive. In order to understand the in vitro and in vivo discrepancy, compound 2 was subsequently retested in vitro using different Mtb strains cultured in different media. This revealed that activity was only observed in media containing glycerol and led to the hypothesis that glycerol was not used as a primary carbon source by Mtb in the mouse lungs, as has previously been observed. Support for this hypothesis was provided by spontaneous-resistant mutant generation and whole genome sequencing studies, which revealed mutations mapping to glycerol metabolizing genes indicating that the 2-aminoquinazolinones kill Mtb in vitro via a glycerol-dependent mechanism of action.

Semisynthetic Antimycobacterial C-3 Silicate and C-3/C-21 Ester Derivatives of Fusidic Acid: Pharmacological Evaluation and Stability Studies in Liver Microsomes, Rat Plasma, and Mycobacterium tuberculosis culture.

Fusidic acid (FA), a natural product fusidane triterpene-based antibiotic with unique structural features, is active in vitro against Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). While possessing good pharmacokinetics in man, FA is rapidly metabolized in rodents, thus complicating proof-of-concept studies in this model. Toward the repositioning of FA as an anti-TB agent, we herein describe the synthesis, activity, and metabolism of FA and semisynthesized ester derivatives in rat liver microsomes, rat plasma, and mycobacterial cell culture. FA and derivative molecules with a free C-3 OH underwent species-specific metabolism to the corresponding 3-OH epimer, 3-epifusidic acid (3-epiFA). FA was also metabolized in rat plasma to form FA lactone. These additional routes of metabolism may contribute to the more rapid clearance of FA observed in rodents. C-3 alkyl and aryl esters functioned as classic prodrugs of FA, being hydrolyzed to FA in microsomes, plasma, and Mycobacterium tuberculosis culture. In contrast, C-3 silicate esters and C-21 esters were inert to hydrolysis and so did not act as prodrugs. The antimycobacterial activity of the C-3 silicate esters was comparable to that of FA, and these compounds were stable in microsomes and plasma, identifying them as potential candidates for evaluation in a rodent model of tuberculosis.

Azaaurones as Potent Antimycobacterial Agents Active against MDR- and XDR-TB.

Herein we report the screening of a small library of aurones and their isosteric counterparts, azaaurones and N-acetylazaaurones, against Mycobacterium tuberculosis. Aurones were found to be inactive at 20 µm, whereas azaaurones and N-acetylazaaurones emerged as the most potent compounds, with nine derivatives displaying MIC99 values ranging from 0.4 to 2.0 µm. In addition, several N-acetylazaaurones were found to be active against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical M. tuberculosis isolates. The antimycobacterial mechanism of action of these compounds remains to be determined; however, a preliminary mechanistic study confirmed that they do not inhibit the mycobacterial cytochrome bc1 complex. Additionally, microsomal metabolic stability and metabolite identification studies revealed that N-acetylazaaurones are deacetylated to their azaaurone counterparts. Overall, these results demonstrate that azaaurones and their N-acetyl counterparts represent a new entry in the toolbox of chemotypes capable of inhibiting M. tuberculosis growth.

Antimalarial Pyrido[1,2- a]benzimidazole Derivatives with Mannich Base Side Chains: Synthesis, Pharmacological Evaluation, and Reactive Metabolite Trapping Studies.

A novel series of pyrido[1,2- a]benzimidazoles bearing Mannich base side chains and their metabolites were synthesized and evaluated for in vitro antiplasmodium activity, microsomal metabolic stability, reactive metabolite (RM) formation, and in vivo antimalarial efficacy in a mouse model. Oral administration of one of the derivatives at 4 × 50 mg/kg reduced parasitemia by 95% in Plasmodium berghei-infected mice, with a mean survival period of 16 days post-treatment. The in vivo efficacy of these derivatives is likely a consequence of their active metabolites, two of which showed potent in vitro antiplasmodium activity against chloroquine-sensitive and multidrug-resistant Plasmodium falciparum ( P. falciparum) strains. Rapid metabolism was observed for all the analogues with <40% of parent compound remaining after 30 min of incubation in liver microsomes. RM trapping studies detected glutathione adducts only in derivatives bearing 4-aminophenol moiety, with fragmentation signatures showing that this conjugation occurred on the phenyl ring of the Mannich base side chain. As with amodiaquine (AQ), interchanging the positions of the 4-hydroxyl and Mannich base side group or substituting the 4-hydroxyl with fluorine appeared to block bioactivation of the AQ-like derivatives though at the expense of antiplasmodium activity, which was significantly lowered.

Structure-Activity Relationship Studies and Plasmodium Life Cycle Profiling Identifies Pan-Active N-Aryl-3-trifluoromethyl Pyrido[1,2- a]benzimidazoles Which Are Efficacious in an in Vivo Mouse Model of Malaria.

Structure-activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2- a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.