Syntheses and studies of deuterated Imdiazo[1,2-a]pyridine-3-carboxamides with potent anti-tuberculosis activity and improved metabolic properties.

The imidazo[1,2-a]pyridine-3-carboxyamides (IAPs) are a unique class of compounds endowed with impressive nanomolar in vitro potency against Mycobacterium tuberculosis (Mtb) as exemplified by clinical candidate Telacebec (Q203). These compounds target mycobacterial respiration through inhibition of the QcrB subunit of cytochrome bc1:aa3 super complex resulting in bacteriostatic efficacy in vivo. Our labs have had a long-standing interest in the design and development of IAPs. However, some of these compounds suffer from short in vivo half-lives, requiring multiple daily dosing or the addition of a cytochrome P450 inhibitor for murine efficacy evaluations. Deuteration has been shown to decrease metabolism as the C-D bond is stronger than the CH bond. Herein we describe our efforts on design and synthesis of potent deuterated IAPs and the effect that deuteration has upon metabolism through microsomal stability studies.

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b.

The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e-/2H+ from each propionate to an activated form of H2O2 Time-resolved EPR spectroscopy revealed that Tyr-145 formed a radical species within 30 s of the reaction of the enzyme-coproheme complex with H2O2 This radical disappeared over the next 270 s, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 s after initiating the reaction with H2O2, nor did it change the ∼270 s required for the radical signal to recede to ≤10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145• was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV)=O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of ∼2 on the decarboxylation rate constant, indicating that cleavage of the propionate Cß-H bond was partly rate-limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate Cß-H to Tyr-145•.

PvdF of pyoverdin biosynthesis is a structurally unique N10-formyltetrahydrofolate-dependent formyltransferase.

The hydroxyornithine transformylase from Pseudomonas aeruginosa is known by the gene name pvdF, and has been hypothesized to use N10-formyltetrahydrofolate (N10-fTHF) as a co-substrate formyl donor to convert N5-hydroxyornithine (OHOrn) to N5-formyl- N5-hydroxyornithine (fOHOrn). PvdF is in the biosynthetic pathway for pyoverdin biosynthesis, a siderophore generated under iron-limiting conditions that has been linked to virulence, quorum sensing and biofilm formation. The structure of PvdF was determined by X-ray crystallography to 2.3 Å, revealing a formyltransferase fold consistent with N10-formyltetrahydrofolate dependent enzymes, such as the glycinamide ribonucleotide transformylases, N-sugar transformylases and methionyl-tRNA transformylases. Whereas the core structure, including the catalytic triad, is conserved, PvdF has three insertions of 18 or more amino acids, which we hypothesize are key to binding the OHOrn substrate. Steady state kinetics revealed a non-hyperbolic rate curve, promoting the hypothesis that PvdF uses a random-sequential mechanism, and favors folate binding over OHOrn.

Imidazopyridine Compounds Inhibit Mycobacterial Growth by Depleting ATP Levels.

The imidazopyridines are a promising new class of antitubercular agents with potent activity in vitro and in vivo We isolated mutants of Mycobacterium tuberculosis resistant to a representative imidazopyridine; the mutants had large shifts (>20-fold) in MIC. Whole-genome sequencing revealed mutations in Rv1339, a hypothetical protein of unknown function. We isolated mutants resistant to three further compounds from the series; resistant mutants isolated from two of the compounds had single nucleotide polymorphisms in Rv1339 and resistant mutants isolated from the third compound had single nucleotide polymorphisms in QcrB, the proposed target for the series. All the strains were resistant to two compounds, regardless of the mutation, and a strain carrying the QcrB T313I mutation was resistant to all of the imidazopyridine derivatives tested, confirming cross-resistance. By monitoring pH homeostasis and ATP generation, we confirmed that compounds from the series were targeting QcrB; imidazopyridines disrupted pH homeostasis and depleted ATP, providing further evidence of an effect on the electron transport chain. A representative compound was bacteriostatic against replicating bacteria, consistent with a mode of action against QcrB. The series had a narrow inhibitory spectrum, with no activity against other bacterial species. No synergy or antagonism was seen with other antituberculosis drugs under development. In conclusion, our data support the hypothesis that the imidazopyridine series functions by reducing ATP generation via inhibition of QcrB.

Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ).

Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H2O2 as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C-C bond and results in net loss of hydride, via steps that are not clear. Solution and solid-state structural characterization of the protein in complex with a substrate analog revealed a highly unconventional H2O2-activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggested that, in contrast to direct C-H bond cleavage catalyzed by a high-valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr•. This residue then abstracts a net hydrogen atom (H•) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO2 products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an extended relay of heme-mediated e-/H+ transfers and a novel route for the conversion of carboxylic acids to alkenes.

Imidazo[1,2-a]Pyridine-3-Carboxamides Are Active Antimicrobial Agents against Mycobacterium avium Infection In Vivo.

A panel of six imidazo[1,2-a]pyridine-3-carboxamides (IAPs) were shown to have low-micromolar activity against Mycobacterium avium strains. Compound ND-10885 (compound 2) showed significant activity in the lung, spleen, and liver in a mouse M. avium infection model. A combined regimen consisting of ND-10885 (compound 2) and rifampin was additive in its anti-M. avium activity in the lung. Our data indicate that IAPs represent a new class of antibiotics that are active against M. avium and could potentially serve as an effective addition to a combined treatment regimen.

Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ.

A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ), the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though amounts significantly greater than the expected 2 equiv were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high-valent iron intermediates cannot be ruled out.

Scaffold-switching: an exploration of 5,6-fused bicyclic heteroaromatics systems to afford antituberculosis activity akin to the imidazo[1,2-a]pyridine-3-carboxylates.

A set of 5,6-fused bicyclic heteroaromatic scaffolds were investigated for their in vitro anti-tubercular activity versus replicating and non-replicating strains of Mycobacterium tuberculosis (Mtb) in an attempt to find an alternative scaffold to the imidazo[1,2-a]pyridine and imidazo[1,2-a]pyrimidines that were previously shown to have potent activity against replicating and drug resistant Mtb. The five new bicyclic heteroaromatic scaffolds explored in this study include a 2,6-dimethylimidazo[1,2-b]pyridazine-3-carboxamide (7), a 2,6-dimethyl-1H-indole-3-carboxamide (8), a 6-methyl-1H-indazole-3-carboxamide (9), a 7-methyl-[1,2,4]triazolo[4,3-a]pyridine-3-carboxamide (10), and a 5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxamide (11). Additionally, imidazo[1,2-a]pyridines isomers (2 and 12) and a homologous imidazo[1,2-a]pyrimidine isomer (6) were prepared and compared. Compounds 2 and 6 were found to be the most potent against H37Rv Mtb (MIC's of 0.1 µM and 1.3 µM) and were inactive (MIC >128 µM) against Staphylococcus aureus, Escherichia coli and Candida albicans. Against other non-tubercular mycobacteria strains, compounds 2 and 6 had activity against Mycobacterium avium (16 and 122 µM, respectively), Mycobacterium kansasii (4 and 19 µM, respectively), Mycobacterium bovis BCG (1 and 8 µM, respectively) while all the other scaffolds were inactive (>128 µM).

Thiolates chemically induce redox activation of BTZ043 and related potent nitroaromatic anti-tuberculosis agents.

The development of multidrug resistant (MDR) and extensively drug resistant (XDR) forms of tuberculosis (TB) has stimulated research efforts globally to expand the new drug pipeline. Nitroaromatic compounds, including 1,3-benzothiazin-4-ones (BTZs) and related agents, are a promising new class for the treatment of TB. Research has shown that the nitroso intermediates of BTZs that are generated in vivo cause suicide inhibition of decaprenylphosphoryl-ß-D-ribose 2' oxidase (DprE1), which is responsible for cell wall arabinogalactan biosynthesis. We have designed and synthesized novel anti-TB agents inspired from BTZs and other nitroaromatic compounds. Computational studies indicated that the unsubstituted aromatic carbons of BTZ043 and related nitroaromatic compounds are the most electron-deficient and might be prone to nucleophilic attack. Our chemical studies on BTZ043 and the additional nitroaromatic compounds synthesized by us and others confirmed the postulated reactivity. The results indicate that nucleophiles such as thiolates, cyanide, and hydride induce nonenzymatic reduction of the nitro groups present in these compounds to the corresponding nitroso intermediates by addition at the unsubstituted electron-deficient aromatic carbon present in these compounds. Furthermore, we demonstrate here that these compounds are good candidates for the classical von Richter reaction. These chemical studies offer an alternate hypothesis for the mechanism of action of nitroaromatic anti-TB agents, in that the cysteine thiol(ate) or a hydride source at the active site of DprE1 may trigger the reduction of the nitro groups in a manner similar to the von Richter reaction to the nitroso intermediates, to initiate the inhibition of DprE1.

Allylic thiocyanates as a new class of antitubercular agents.

TB is a global public health emergency in which new drugs are desperately needed. Herein we report on the synthesis of a diverse panel of 41 aryl allylic azides, thiocyanates, isothiouronium salts, and N,N'-diacetylisothioureas that were evaluated for their in vitro activity against replicating and non-replicating Mycobacterium tuberculosis (Mtb) H(37)Rv and toxicity to VERO cells. We found a selective group of new and promising compounds having good (micromolar) to excellent (sub-micromolar) potency against replicating Mtb H(37)Rv. Allylic thiocyanates bearing halophenyl (halo=2-Br, 4-Br, 4-Cl, 4-F), 4-methylphenyl and 2-naphthyl moieties were the most active as antitubercular agents. In particular, the 2-bromophenyl-substituted thiocyanate showed MIC=0.25 µM against replicating Mtb, MIC=8.0 µM against non-replicating Mtb and IC(50)=32 µM in the VERO cellular toxicity assay.

Syntheses and biological studies of novel spiropiperazinyl oxazolidinone antibacterial agents using a spirocyclic diene derived acylnitroso Diels-Alder reaction.

Several novel oxazolidinone antibiotics with a spiropiperazinyl substituent at the 4'-position of the phenyl ring were synthesized through nitroso Diels-Alder chemistry and the in vitro antibacterial activities were evaluated against various Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and mycobacteria (Mycobacterium vaccae, Mycobacterium tuberculosis). Analogs (8a and 12) were active against selected drug resistant microbes, like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and had no mammalian toxicity in a Hep-2 cellular assay (CC(50) >100 µM).

Generation and exploration of new classes of antitubercular agents: The optimization of oxazolines, oxazoles, thiazolines, thiazoles to imidazo[1,2-a]pyridines and isomeric 5,6-fused scaffolds.

Tuberculosis (TB) is a devastating disease resulting in a death every 20s. Thus, new drugs are urgently needed. Herein we report ten classes of compounds-oxazoline, oxazole, thiazoline, thiazole, pyrazole, pyridine, isoxazole, imidazo[1,2-a]pyridine, imidazo[1,2-a]pyrimidine and imidazo[1,2-c]pyrimidine-which have good (micromolar) to excellent (sub-micromolar) antitubercular potency. The 5,6-fused heteroaromatic compounds were the most potent with MIC's as low as <0.195 µM (9 and 11). Overall, the imidazo[1,2-a]pyridine class was determined to be most promising, with potency similar to isoniazid and PA-824 against replicating Mtb H(37)Rv, clinically relevant drug sensitive, multi- and extensively resistant Mtb strains as well as having good in vitro metabolic stability.

Syntheses and Structure-Activity Relationships of N-Phenethyl-Quinazolin-4-yl-Amines as Potent Inhibitors of Cytochrome bd Oxidase in Mycobacterium tuberculosis.

The development of cytochrome bd oxidase (cyt-bd) inhibitors are needed for comprehensive termination of energy production in Mycobacterium tuberculosis (Mtb) to treat tuberculosis infections. Herein, we report on the structure-activity-relationships (SAR) of 22 new N-phenethyl-quinazolin-4-yl-amines that target cyt-bd. Our focused set of compounds was synthesized and screened against three mycobacterial strains: Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Rv and the clinical isolate Mycobacterium tuberculosis N0145 with and without the cytochrome bcc:aa 3 inhibitor Q203 in an ATP depletion assay. Two compounds, 12a and 19a, were more active against all three strains than the naturally derived cyt-bd inhibitor aurachin D.

Structure guided generation of thieno[3,2-d]pyrimidin-4-amine Mycobacterium tuberculosis bd oxidase inhibitors.

Cytochrome bd oxidase (Cyt-bd) is an attractive drug target in Mycobacterium tuberculosis, especially in the context of developing a drug combination targeting energy metabolism. However, currently few synthetically assessable scaffolds target Cyt-bd. Herein, we report that thieno[3,2-d]pyrimidin-4-amines inhibit Cyt-bd, and report an initial structure-activity-relationship (SAR) of 13 compounds in three mycobacterial strains: Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis clinical isolate N0145 in an established ATP depletion assay with or without the cytochrome bcc : aa 3 (QcrB) inhibitor Q203. All compounds displayed activity against M. bovis BCG and the M. tuberculosis clinical isolate strain N0145 with ATP IC50 values from 6 to 54 µM in the presence of Q203 only, as expected from a Cyt-bd inhibitor. All derivatives were much less potent against M. tuberculosis H37Rv compared to N0145 (IC50's from 24 to >100 µM and 9-52 µM, respectively), an observation that may be attributed to the higher expression of the Cyt-bd-encoding genes in the laboratory-adapted M. tuberculosis H37Rv strain. N-(4-(tert-butyl)phenethyl)thieno[3,2-d]pyrimidin-4-amine (19) was the most active compound with ATP IC50 values from 6 to 18 µM against all strains in the presence of Q203, making it a good chemical probe for interrogation the function of the mycobacterial Cyt-bd under various physiological conditions.

Hydride-induced Meisenheimer complex formation reflects activity of nitro aromatic anti-tuberculosis compounds.

The formation efficiency of hydride-induced Meisenheimer complexes of nitroaromatic compounds is consistent with their anti-TB activities exemplied by MDL860 and benzothiazol N-oxide (BTO) analogs. Herein we report that nitro cyano phenoxybenzenes (MDL860 and analogs) reacted slowly and incompletely which reflected their moderate anti-TB activity, in contrast to the instantaneous reaction of BTO derivatives to quantitatively generate Meisenheimer complexes which corresponded to their enhanced anti-TB activity. These results were corroborated by mycobacterial and radiolabelling studies that confirmed inhibition of the DprE1 enzyme by BTO derivatives but not MDL860 analogs.

Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis.

The approval of bedaquiline has placed energy metabolism in the limelight as an attractive target space for tuberculosis antibiotic development. While bedaquiline inhibits the mycobacterial F1 F0 ATP synthase, small molecules targeting other components of the oxidative phosphorylation pathway have been identified. Of particular interest is Telacebec (Q203), a phase 2 drug candidate inhibitor of the cytochrome bcc:aa3 terminal oxidase. A functional redundancy between the cytochrome bcc:aa3 and the cytochrome bd oxidase protects M. tuberculosis from Q203-induced death, highlighting the attractiveness of the bd-type terminal oxidase for drug development. Here, we employed a facile whole-cell screen approach to identify the cytochrome bd inhibitor ND-011992. Although ND-011992 is ineffective on its own, it inhibits respiration and ATP homeostasis in combination with Q203. The drug combination was bactericidal against replicating and antibiotic-tolerant, non-replicating mycobacteria, and increased efficacy relative to that of a single drug in a mouse model. These findings suggest that a cytochrome bd oxidase inhibitor will add value to a drug combination targeting oxidative phosphorylation for tuberculosis treatment.

UTILIZTION OF THE SUZUKI COUPLING TO ENHANCE THE ANTITUBERCULOSIS ACTIVITY OF ARYL OXAZOLES.

Potent antituberculosis aryl oxazoles can be made in an efficient three step process--formation of ß-hydroxy amides with serine benzyl ester; cyclization to afford oxazolines; and then dehydration to give the corresponding oxazoles. Furthermore, incorporation of an appropriate aryl halide allows utilization of the Suzuki cross coupling reaction to access new chemical space and more elaborate analogs. The compounds prepared by this method were shown to possess improved activity against M. tuberculosis, extremely low toxicity toward VERO cells and, as a result, high therapeutic indexes.

Intracellular and in vivo evaluation of imidazo[2,1-b]thiazole-5-carboxamide anti-tuberculosis compounds.

The imidazo[2,1-b]thiazole-5-carboxamides (ITAs) are a promising class of anti-tuberculosis agents shown to have potent activity in vitro and to target QcrB, a key component of the mycobacterial cytochrome bcc-aa3 super complex critical for the electron transport chain. Herein we report the intracellular macrophage potency of nine diverse ITA analogs with MIC values ranging from 0.0625-2.5 µM and mono-drug resistant potency ranging from 0.0017 to 7 µM. The in vitro ADME properties (protein binding, CaCo-2, human microsomal stability and CYP450 inhibition) were determined for an outstanding compound of the series, ND-11543. ND-11543 was tolerable at >500 mg/kg in mice and at a dose of 200 mg/kg displayed good drug exposure in mice with an AUC(0-24h) >11,700 ng·hr/mL and a >24 hr half-life. Consistent with the phenotype observed with other QcrB inhibitors, compound ND-11543 showed efficacy in a chronic murine TB infection model when dosed at 200 mg/kg for 4 weeks. The efficacy was not dependent upon exposure, as pre-treatment with a known CYP450-inhibitor did not substantially improve efficacy. The ITAs are an interesting scaffold for the development of new anti-TB drugs especially in combination therapy based on their favorable properties and novel mechanism of action.

Carbon metabolism modulates the efficacy of drugs targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis.

The influence of carbon metabolism on oxidative phosphorylation is poorly understood in mycobacteria. M. tuberculosis expresses two respiratory terminal oxidases, the cytochrome bc1:aa3 and the cytochrome bd oxidase, which are jointly required for oxidative phosphorylation and mycobacterial viability. The essentiality of the cytochrome bc1:aa3 for optimum growth is illustrated by its vulnerability to chemical inhibition by the clinical drug candidate Q203 and several other chemical series. The cytochrome bd oxidase is not strictly essential for growth but is required to maintain bioenergetics when the function of the cytochrome bc1:aa3 is compromised. In this study, we observed that the potency of drugs targeting the cytochrome bc1:aa3 is influenced by carbon metabolism. The efficacy of Q203 and related derivatives was alleviated by glycerol supplementation. The negative effect of glycerol supplementation on Q203 potency correlated with an upregulation of the cytochrome bd oxidase-encoding cydABDC operon. Upon deletion of cydAB, the detrimental effect of glycerol on the potency of Q203 was abrogated. The same phenomenon was also observed in recent clinical isolates, but to a lesser extent compared to the laboratory-adapted strain H37Rv. This study reinforces the importance of optimizing in vitro culture conditions for drug evaluation in mycobacteria, a factor which appeared to be particularly essential for drugs targeting the cytochrome bc1:aa3 terminal oxidase.

Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc1:aa3. While the cytochrome bc1:aa3 is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc1:aa3 to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment.