A practical drug discovery project at the undergraduate level.

In this article, we describe a practical drug discovery project for third-year undergraduates. No previous knowledge of medicinal chemistry is assumed. Initial lecture workshops cover the basic principles; then students, in teams, seek to improve the profile of a weakly potent, insoluble phosphatidylinositide 3-kinase delta (PI3Kδ) inhibitor (1) through compound array design, molecular modelling, screening data analysis and the synthesis of target compounds in the laboratory. The project benefits from significant industrial support, including lectures, student mentoring and consumables. The aim is to make the learning experience as close as possible to real-life industrial situations. In total, 48 target compounds were prepared, the best of which (5b, 5j, 6b and 6ap) improved the potency and aqueous solubility of the lead compound (1) by 100-1000 fold and ≥tenfold, respectively.

Nonsteroidal glucocorticoid agonists: tetrahydronaphthalenes with alternative steroidal A-ring mimetics possessing dissociated (transrepression/transactivation) efficacy selectivity.

The synthesis and biological activity of tetrahydronaphthalene derivatives coupled to various heterocycles are described. These compounds are potent glucocorticoid receptor agonists with efficacy selectivity in an NFkappaB glucocorticoid receptor (GR) agonist assay (representing transrepression effects) over an MMTV GR agonist assay (representing transactivation effects). Quinolones, indoles, and C- and N-linked quinolines are some of the heterocycles that provide efficacy selectivity. For example, the isoquinoline 49D1E2 has NFkappaB agonism with pIC50 of 8.66 (89%) and reduced efficacy in MMTV agonism (6%), and the quinoline 55D1E1 has NFkappaB agonism with pIC50 of 9.30 (101%) and reduced efficacy in MMTV agonism with pEC50 of 8.02 (47%). A description of how a compound from each class is modeled in the active site of the receptor is given.

Non-steroidal glucocorticoid agonists: the discovery of aryl pyrazoles as A-ring mimetics.

Starting from an established series of non-steroidal glucocorticoid receptor (GR) agonists, a large array was designed where a metabolically labile benzoxazinone moiety was replaced. Initial hits bound to GR but lacked agonist activity. Following two further iterations, potent GR agonists were discovered with 20D1E1 having NFkappaB agonism pIC(50) 8.8 (103%). Other analogues such as 23D1E1 display a dissociated profile (NFkappaB pIC(50) 8.1 (103%), MMTV pEC(50) 7.02 (36%)). The tetrahydronaphthalene moiety can also be replaced with substituted aryls such as 24E1 and 25E1.

Carbon-carbon bond forming reactions with substrates absorbed non-covalently on a cellulose chromatography paper support.

Reactions and purifications, including carbon-carbon bond forming reactions, can be carried out on a cellulose support on which the substrates are non-covalently absorbed.

Dissociated nonsteroidal glucocorticoid receptor modulators; discovery of the agonist trigger in a tetrahydronaphthalene-benzoxazine series.

The tetrahydronaphthalene-benzoxazine glucocorticoid receptor (GR) partial agonist 4b was optimized to produce potent full agonists of GR. Aromatic ring substitution of the tetrahydronaphthalene leads to weak GR antagonists. Discovery of an "agonist trigger" substituent on the saturated ring of the tetrahydronaphthalene leads to increased potency and efficacious GR agonism. These compounds are efficacy selective in an NFkB GR agonist assay (representing transrepression effects) over an MMTV GR agonist assay (representing transactivation effects). 52 and 60 have NFkB pIC(50) = 8.92 (105%) and 8.69 (92%) and MMTV pEC(50) = 8.20 (47%) and 7.75 (39%), respectively. The impact of the trigger substituent on agonism is modeled within GR and discussed. 36, 52, and 60 have anti-inflammatory activity in a mouse model of inflammation after topical dosing with 52 and 60, having an effect similar to that of dexamethasone. The original lead was discovered by a manual agreement docking method, and automation of this method is also described.

Dimeric zanamivir conjugates with various linking groups are potent, long-lasting inhibitors of influenza neuraminidase including H5N1 avian influenza.

The synthesis, antiviral and pharmacokinetic properties of zanamivir (ZMV) dimers 8 and 13 are described. The compounds are highly potent neuraminidase (NA) inhibitors which, along with dimer 3, are being investigated as potential second generation inhaled therapies both for the treatment of influenza and for prophylactic use. They show outstanding activity in a 1 week mouse influenza prophylaxis assay, and compared with ZMV, high concentrations of 8 and 13 are found in rat lung tissue after 1 week. Retention of compounds in rat lung tissue correlated both with molecular weight (excluding 3 and 15) and with a capacity factor K' derived from immobilized artificial membrane (IAM) chromatography (including 3 and 15). Pharmacokinetic parameters for 3, 8 and 13 in rats show the compounds have short to moderate plasma half-lives, low clearances and low volumes of distribution. Dimer 3 shows NA inhibitory activity against N1 viruses including the recent highly pathogenic H5N1 A/Chicken/Vietnam/8/2004. In plaque reduction assays, 3, 8 and 13 show good to outstanding potency against a panel of nine flu A and B virus strains. Consistent with its shorter and more rigid linking group, dimer 8 has been successfully crystallized.

Potent and long-acting dimeric inhibitors of influenza virus neuraminidase are effective at a once-weekly dosing regimen.

Dimeric derivatives (compounds 7 to 9) of the influenza virus neuraminidase inhibitor zanamivir (compound 2), which have linking groups of 14 to 18 atoms in length, are approximately 100-fold more potent inhibitors of influenza virus replication in vitro and in vivo than zanamivir. The observed optimum linker length of 18 to 22 A, together with observations that the dimers cause aggregation of isolated neuraminidase tetramers and whole virus, indicate that the dimers benefit from multivalent binding via intertetramer and intervirion linkages. The outstanding long-lasting protective activities shown by compounds 8 and 9 in mouse influenza infectivity experiments and the extremely long residence times observed in the lungs of rats suggest that a single low dose of a dimer would provide effective treatment and prophylaxis for influenza virus infections.

Discovery of further pyrrolidine trans-lactams as inhibitors of human neutrophil elastase (HNE) with potential as development candidates and the crystal structure of HNE complexed with an inhibitor (GW475151).

Described herein is a modern approach to the rapid preparation and evaluation of compounds as potential back-up drug candidates. GW311616A, 1, a derivative of pyrrolidine trans-lactams, has previously been described as a potent, orally active inhibitor of human neutrophil elastase (HNE) for the treatment of respiratory disease. These properties made it a suitable candidate for development. Described here is the discovery of three further derivatives of pyrrolidine trans-lactams, which fulfill the criteria required for back-up candidates 28, 29, and 32. These include increased activity in inhibiting HNE in human whole blood (HWB) and comparable pharmacokinetic properties, in particular clearance, in two species. To provide a rapid assessment of clearance, cassette dosing in dog was used. Modern array techniques, including the synthesis of mixtures, were used to synthesize compounds rapidly. Having selected three potential compounds as back-up candidates, they were prepared as single enantiomers and profiled in in vitro and in vivo assays and evaluated pharmacokinetically in rat and dog. These compounds are highly potent and selective HNE inhibitors, with a prolonged pharmacodynamic action. Pharmacokinetically, these compounds are comparable with 1 while they are more potent in HWB. Compound 28, however, has a higher clearance. One of these compounds, 32, was cocrystallized with HNE, and features of this structure are described and compared with the cocrystal structure of 1 in porcine pancreatic elastase.

A Flexible, Practical, and Stereoselective Synthesis of Enantiomerically Pure trans-5-Oxohexahydropyrrolo[3,2-b]pyrroles (Pyrrolidine-trans-lactams), a New Class of Serine Protease Inhibitors, Using Acyliminium Methodology.

A flexible, practical, and stereoselective synthesis of enantiomerically pure trans-5-oxohexahydropyrrolo[3,2-b]pyrroles (pyrrolidine-trans-lactams) is described. The key reaction involves addition of Z-ketene acetal 24 to the acyliminium ion derived from 48. This reaction is mediated by BF3·OEt2 and introduces the 6S and 6aS stereocenters stereoselectively. The acyliminium precursor was prepared in four different ways: from racemic 2,4-diaminobutyric acid 8, from (R)-asparagine, from (R)-methionine, and via a crystallization-induced dynamic resolution of a salt of the racemic amine 56. (R)-Methionine is the preferred starting material for the preparation of enantiomerically pure material. The best conditions for addition of the ketene acetal to the acyliminium ion derived from 48 were determined by systematically screening a range of ketene acetals and Lewis acids. The best ketene acetal was Z-(1-ethoxy-3-methylbut-1-enyloxyl)triisopropylsilane 24. In this series, the bulk of the silyl group of the Z-ketene acetal can be correlated with increased 6S isopropyl product. Use of the E-ketene acetal does not lead to a significant change in stereoselectivity for the 6R isopropyl product. In contrast, variation of the Lewis acid has a considerable effect on the product stereochemistry. While BF3·OEt2 gives predominantly 6S,6aS product, AlCl3 and TiCl4 give predominantly mixtures of the 6R,6aS and 6S,6aS products and TMSOTf gives 6aR material with predominantly one unknown isopropyl isomer (trans-lactam numberings used). The synthesis can conveniently be carried out on a large scale to produce multigram quantities of the trans-lactam 28, which is a key precursor of pharmacologically active molecules such as 1, a selective and orally active human neutrophil elastase inhibitor. The overall chemical yield of 1 is 1.3%, corresponding to an average of >70% yield for each of the 14 steps, and the synthesis contains only one chromatographic purification.