Usage GuidelinesPlease refer to usage guidelines at http://researchonline.lshtm.ac.uk/policies.html or alternatively contact researchonline@lshtm.ac.uk.Available under license: http://creativecommons.org/licenses/by-nc-nd/2.5/ 7-Substituted 2-nitro-5,6-dihydroimidazo [2,1-b] [1,3] that was present in 6-nitroimidazooxazole 5. This was equivalent to a one carbon expansion of the oxazole ring between C-2 and C-3 ( Figure 2). The rationale for this design concept stemmed from initial evidence 21 that delamanid (5) was highly stable towards metabolism, as well as from a report 22 that 7-methyl derivatives of 6 retained excellent antitubercular potency, suggesting that such an approach merited investigation.Serendipitously, we soon discovered 23 that this novel "7-substituted oxazine" class not only showed considerable promise for TB (as later confirmed by others 24,25 ), it also displayed potent antileishmanial activity, comparable to the 6-nitroimidazooxazoles in early screening assays. Therefore, following the success with 4, this new series was similarly repositioned for VL as part of an extensive backup program, run in collaboration with DNDi. In this paper, we first highlight some critical VL hit to lead assessments on the original subset of compounds that had been prepared for TB. We then detail the findings of our lead optimisation study directed at developing backups to 4 having an improved physicochemical/pharmacological profile and better safety, which culminated in the selection of a new preclinical candidate for VL. Finally, in light of these encouraging results and the excellent activities of this novel 7-substituted 2-nitroimidazooxazine class against both TB and Chagas disease, we point to related analogues that might be worthy of further assessment for the latter applications. 5 CHEMISTRYIn order to rapidly access some initial examples, the racemic 7-H and 7-methyl alcohol intermediates, 13 and 20, were first sought (Scheme 1A). These could be obtained in very good overall yield (62-79%) via similar 5 step reaction sequences, starting with base catalysed alkylation of 2-bromo-4-nitroimidazole (8) For more efficient synthesis of 7-H biaryl analogues having a proximal 3-pyridine ring, an epoxide-opening strategy (Scheme 3A) was preferred over the Mitsunobu route described above. Epoxide 67 was obtained in 72% optimised yield from 2-chloro-4-nitroimidazole (40), via alkene 66; in this case, the slow epoxidation step was best achieved under non-buffered conditions at higher concentration (with initial cooling). Ring opening of 67 with 6-bromopyridin-3-ol (68) (K2CO3, MEK, 81-82 °C) gave mainly alcohol 69 (51% using 2 equiv for 35 h, or 57% from 4 equiv and 14 h), together with small amounts of the oxazine 70 (6-12%). Ring closure of purified 69 (NaH, DMF, 0-20 °C) then gave additional 70 in excellent 7 yield (91%). Comparable results were obtained for scale-up of 39 from 67 (62%), as well as for reaction of epoxide 42 with pyridinol 68 and ring closure, leading to oxazine 89 (Scheme 3D). As expected, br...

ObjectivesDrugs for Neglected Diseases initiative (DNDi) has identified three chemical lead series, the nitroimidazoles, benzoxaboroles and aminopyrazoles, as innovative treatments for visceral leishmaniasis. The leads discovered using phenotypic screening, were optimised following disease- and compound-specific criteria. Several leads of each series were progressed and preclinical drug candidates have been nominated. Here we evaluate the efficacy of the lead compounds of each of these three chemical classes in in vitro and in vivo models of cutaneous leishmaniasis.MethodsThe in vitro activity of fifty-five compounds was evaluated against the intracellular amastigotes of L. major, L. aethiopica, L. amazonensis, L. panamensis, L. mexicana and L. tropica. The drugs demonstrating potent activity (EC50 < 5 μM) against at least 4 of 6 species were subsequently evaluated in vivo in different L. major – BALB/c mouse models using a 5 or 10-day treatment with either the oral or topical formulations. Efficacy was expressed as lesion size (measured daily using callipers), parasite load (by quantitative PCR – DNA) and bioluminescence signal reduction relative to the untreated controls.ResultsThe selected drug compounds (3 nitroimidazoles, 1 benzoxaborole and 3 aminopyrazoles) showed consistent and potent activity across a range of Leishmania species that are known to cause CL with EC50 values ranging from 0.29 to 18.3 μM. In all cases, this potent in vitro antileishmanial activity translated into high levels of efficacy with a linear dose-response against murine CL. When administered at 50 mg/kg/day, DNDI-0690 (nitroimidazole), DNDI-1047 (aminopyrazole) and DNDI-6148 (benzoxaborole) all resulted in a significant lesion size reduction (no visible nodule) and an approximate 2-log-fold reduction of the parasite load as measured by qPCR compared to the untreated control.ConclusionsThe lead compounds DNDI-0690, DNDI-1047 and DNDI-6148 showed excellent activity across a range of Leishmania species in vitro and against L. major in mice. These compounds offer novel potential drugs for the treatment of CL.

ObjectivesThree new chemical series (bicyclic nitroimidazoles, aminopyrazoles and oxaboroles) were selected by Drugs for Neglected Diseases initiative as potential new drug leads for leishmaniasis. Pharmacodynamics studies included both in vitro and in vivo efficacy, cross-resistance profiling against the current antileishmanial reference drugs and evaluation of their cidal activity potential.MethodsEfficacy against the reference laboratory strains of Leishmania infantum (MHOM/MA(BE)/67/ITMAP263) and L. donovani (MHOM/ET/67/L82) was evaluated in vitro on intracellular amastigotes and in vivo in the early curative hamster model. Cidal activity was assessed over a period of 15 days in an in vitro ‘time-to-kill’ assay. Cross-resistance was assessed in vitro on a panel of L. infantum strains with different degrees of resistance to either antimony, miltefosine or paromomycin.ResultsAll lead compounds showed potent and selective in vitro activity against the Leishmania strains tested and no cross-resistance could be demonstrated against any of the current antileishmanial drugs. Cidal activity was obtained in vitro for all series within 15 days of exposure with some differences noted between L. donovani and L. infantum. When evaluated in vivo, all lead compounds showed high efficacy and no adverse effects were observed.ConclusionsThe new lead series were shown to have cidal pharmacodynamic activity. The absence of cross-resistance with any of the current antileishmanial drugs opens possibilities for combination treatment to reduce the likelihood of treatment failures and drug resistance.

SummaryAntibiotic resistance continues to reduce the number of available antibiotics, increasing the need for novel antibacterial drugs. Since the seminal work of Sir Alexander Fleming, antibiotic identification has been based exclusively on the inhibition of bacterial growth in vitro. Recently, inhibitors of bacterial virulence which interfere with bacterial pathogenesis mechanisms have been proposed as an alternative to antibiotics, and a few were discovered using assays targeting specific virulence mechanisms. Here we designed a simple surrogate host model for the measurement of virulence and systematic discovery of anti-virulence molecules, based on the interaction of Tetrahymena pyriformis and Klebsiella pneumoniae cells. We screened a library of small molecules and identified several inhibitors of virulence. In a mouse pneumonia model we confirmed that an anti-virulence molecule displayed antibacterial activity against Klebsiella pneumoniae and Pseudomonas aeruginosa, by reducing dramatically the bacterial load in the lungs. This molecule did not inhibit bacterial growth in vitro but prevented biosynthesis of the Klebsiella capsule and lipopolysaccharides, a key requirement for virulence. Our results demonstrate that anti-virulence molecules represent an alternative to antibiotics and those can be discovered using nonanimal host models.

Visceral leishmaniasis is a severe parasitic disease that is one of the most neglected tropical diseases. Treatment options are limited, and there is an urgent need for new therapeutic agents. Following an HTS campaign and hit optimization, a novel series of amino-pyrazole ureas has been identified with potent in vitro antileishmanial activity. Furthermore, compound 26 shows high levels of in vivo efficacy (>90%) against Leishmania infantum, thus demonstrating proof of concept for this series.

The nitroimidazole DNDI-0690 is a clinical drug candidate for visceral leishmaniasis (VL) that also shows potent in vitro and in vivo activity against cutaneous leishmaniasis (CL). To support further development of this compound into a patient-friendly oral or topical formulation for the treatment of CL, we investigated the free drug exposure at the dermal site of infection and subsequent elimination of the causative Leishmania pathogen. This study evaluates the pharmacokinetics (PK) and pharmacodynamics (PD) of DNDI-0690 in mouse models of CL. Skin microdialysis and Franz diffusion cell permeation studies revealed that DNDI-0690 permeated poorly (<1%) into the skin lesion upon topical drug application (0.063% [wt/vol], 30 μl). In contrast, a single oral dose of 50 mg/kg of body weight resulted in the rapid and nearly complete distribution of protein-unbound DNDI-0690 from the plasma into the infected dermis (ratio of the area under the curve [0 to 6 h] of the free DNDI-0690 concentration in skin tissue to blood [fAUC0-6 h, skin tissue/fAUC0-6 h, blood] is greater than 80%). Based on in vivo bioluminescence imaging, two doses of 50 mg/kg DNDI-0690 were sufficient to reduce the Leishmania mexicana parasite load by 100-fold, while 6 such doses were needed to achieve similar killing of L. major; this was confirmed by quantitative PCR. The combination of rapid accumulation and potent activity in the Leishmania-infected dermis indicates the potential of DNDI-0690 as a novel oral treatment for CL.

Discovery of the potent antileishmanial effects of antitubercular 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]oxazoles and 7-substituted 2-nitro-5,6-dihydroimidazo[2,1-b][1,3]oxazines stimulated the examination of further scaffolds (e.g., 2-nitro-5,6,7,8-tetrahydroimidazo[2,1-b][1,3]oxazepines), but the results for these seemed less attractive. Following the screening of a 900-compound pretomanid analogue library, several hits with more suitable potency, solubility, and microsomal stability were identified, and the superior efficacy of newly synthesized 6R enantiomers with phenylpyridine-based side chains was established through head-to-head assessments in a Leishmania donovani mouse model. Two such leads (R-84 and R-89) displayed promising activity in the more stringent Leishmania infantum hamster model but were unexpectedly found to be potent inhibitors of hERG. An extensive structure–activity relationship investigation pinpointed two compounds (R-6 and pyridine R-136) with better solubility and pharmacokinetic properties that also provided excellent oral efficacy in the same hamster model (>97% parasite clearance at 25 mg/kg, twice daily) and exhibited minimal hERG inhibition. Additional profiling earmarked R-6 as the favored backup development candidate.

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