High-Throughput Crystallography Reveals Boron-Containing Inhibitors of a Penicillin-Binding Protein with Di- and Tricovalent Binding Modes.

The effectiveness of ß-lactam antibiotics is increasingly compromised by ß-lactamases. Boron-containing inhibitors are potent serine-ß-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) ß-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by ß-lactamase-driven resistance.

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.

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.

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Pyrrolo[3,4-c]pyridine-1,3(2H)-diones: A Novel Antimycobacterial Class Targeting Mycobacterial Respiration.

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.

Carbon-14 radiolabeling and in vivo biodistribution of a potential anti-TB compound.

A potential anti-TB compound bearing a nitroimidazole moiety from iThemba Pharmaceuticals TB chemical library exhibits promising in vitro activity in the microplate almar blue assay (MABA) with a minimum inhibitory concentration (MIC) value of 3 µg/mL. It is equipotent to the front-line drug Isoniazid, but the compound is less toxic with an IC50 of >100 µg/mL. Therefore, this potential iThemba nitroimidazole, 4-([1,1'-[(14)C6]biphenyl]-4-ylmethyl)-9-nitro-3,4,5,6-tetrahydro-2H-imidazo[2,1-b][1,3,6]oxadiazocine, was radiolabeled with the C-14 isotope. The synthesis of the (14)C-labeled nitroimidazole was accomplished in seven steps from diethanolamine with a final specific radioactivity of 3.552 GBq/mmol, a radiochemical yield of 87%, and a radiochemical purity of ≥96%. The source of the C-14 radiolabel was bromobenzene which was introduced by the Suzuki-Miyaura reaction. Tissue distribution results showed that the radiotracer has a high accumulation in the lungs of TB-infected mice, statistically significantly higher than in healthy mice. However, the clearance (for both TB-infected and non-TB-infected mice) from all organs (except the small intestine) from 1 to 2 h as well as the low percentage of injected dose per gram values achieved indicates breakdown of the compound in vivo and subsequent clearance from the body. The latter suggests that the compound might not be useful as an anti-TB drug in humans.

Enantioselective synthesis of the carbocyclic nucleoside (-)-abacavir.

An enantiopure ß-lactam with a suitably disposed electron withdrawing group on nitrogen, participated in a π-allylpalladium mediated reaction with 2,6-dichloropurine tetrabutylammonium salt to afford an advanced cis-1,4-substituted cyclopentenoid with both high regio- and stereoselectivity. This advanced intermediate was successfully manipulated to the total synthesis of (-)-Abacavir.

NMR elucidation of a novel (S)-pentacyclo-undecane bis-(4-phenyloxazoline) ligand and related derivatives.

The NMR elucidation of a novel ligand (S)-pentacyclo-undecane bis-(4-phenyloxazoline) and related pentacyclo-undecane (PCU) derivatives is reported. Two-dimensional NMR proved to be a powerful technique in overcoming the difficulties associated with the elucidation of these compounds when only one-dimensional NMR data is utilized. A chiral substituent was introduced to both 'arms' of the PCU skeleton to produce derivatives 1-3. These derivatives display C(1) symmetry with all thecage atoms being nonequivalent. Owing to overlapping of peaks in the (1)H spectra, identification of these diastereomeric protons was very difficult. The (13)C spectra gave rise to clear splitting of the nonequivalent carbons. This is unusual compared to similar PCU derivatives with chiral substituents as splitting of all the diastereomeric cage carbons has not yet been reported. Nuclear Overhauser enhancement spectroscopy (NOESY) correlations of derivatives 1-3 confirm the different conformations of the molecule in which the side 'arms' occupy different orientations with respect to cage moiety.

(4-Hydr-oxy-3-nitro-benz-yl)methyl-ammonium chloride.

The title compound, C(8)H(11)N(2)O(3) (+)·Cl(-), was synthesized as an inter-mediate in the development of a new sugar sensor. The structure displays N-H⋯Cl and O-H⋯O hydrogen bonding, as well as weak O-H⋯Cl inter-actions and π-π stacking (3.298 Å). There are two formula units in the asymmetric unit.

N-(2-Hydroxy-ethyl)-N-(tricyclo-[3.3.1.1]dec-2-yl)benzamide.

The title adamantane derivative, C(19)H(25)NO(2), was synthesized as part of a study into potential anti-tuberculosis agents. The adamantane skeleton displays shorter than normal C-C bond lengths ranging between 1.5230 (15) and 1.5329 (16) Å. The structure displays O-H⋯O hydrogen bonding and an inter-digitated layered packing structure with distinct hydro-philic and hydro-phobic regions.

2-(Tricyclo[3.3.1.1]dec-2-ylamino)ethanol hemihydrate.

The title adamantane derivative, C(12)H(21)NO·0.5H(2)O, was synthesized as part of an investigation into the biological activities of cage amino-alcohol compounds as potential anti-tuberculosis agents. The structure displays inter-molecular O-H⋯N, N-H⋯O, O-H⋯O hydrogen bonding and a layered packing structure with distinct hydro-philic and hydro-phobic regions. The water molecule lies on a twofold rotation axis.

8-(Biphenyl-4-yl)-8-hydroxy-penta-cyclo-[5.4.0.0.0.0]undecan-11-one ethyl-ene ketal.

The title compound, C(25)H(24)O(3), synthesized as a potential chiral catalyst, exhibits a range of C-C bond lengths in the penta-cyclo-undecane cage between 1.5144 (18) and 1.5856 (16) Å. The two benzene rings are not planar with respect to each other, but rather are twisted at a torsion angle of 34.67 (17)°. The mol-ecule has an intra-molecular O-H⋯O inter-action and participates in two C-H⋯O inter-molecular inter-actions to form a one-dimensional chain.