Mark Ladlow

Anti-MRSA Agent Discovery Using Diversity-Oriented Synthesis†

Antibacterial drugs have played an essential role in the global increase in quality of life and life expectancy. However, these gains are at serious risk owing to bacterial drug resistance by so-called “superbugs”, such as methicillin-resistant Staphylococcus aureus (MRSA). The discovery of new antibiotics with novel modes of action is vital to tackle the threat of multidrug-resistant bacteria. Traditionally, antibiotics have been discovered from natural sources; however, there are many disadvantages to using extracts (e.g. limited availability, bioactive constituent identification, and complex analogue synthesis). These problems have led to a complementary approach of synthesizing structurally diverse, natural-product-like small molecules directly and efficiently, an approach known as diversity-oriented synthesis (DOS). Whereas compound collections of a common scaffold decorated with diverse building blocks have been synthesized efficiently, there are limited examples of the synthesis of small molecules with a high degree of skeletal diversity (usually by a build–couple–pair strategy). Previously, we have used a diazoacetate starting unit to mimic nature8s divergent synthetic strategy with acetyl CoA (by a pluripotent functional-group strategy) to synthesize compounds with natural-product scaffolds (e.g. cocaine and warfarin). Herein, we report the use of a solid-supported phosphonate unit to synthesize 242 drug-like compounds based on 18 natural-product-like scaffolds in two to five steps and their use in discovering a new structural class of antibiotic with anti-MRSA activity. The solid-supported phosphonate 1 (Scheme 1) was identified as an attractive DOS starting unit for three key reasons. First, the reactive phosphonate functionality permits the stereoselective formation of a,b-unsaturated acyl imidazolidinones (2) that could be used to generate enantioselectively a wide range of scaffolds that can be diversified further. Second, the imidazolidinone linker not only enables twopoint binding of chiral catalysts but also permits divergent cleavage of the exocyclic acyl group (hydrolysis, reduction, esterification, and amide formation). Thirdly, immobilization of 1 on a silyl polystyrene support simplified reaction optimization and work-up procedures in the multistep parallel synthesis (total of over 1000 individual steps), thereby allowing the efficient production of milligram quantities of 242 compounds without the requirement for automation equipment. In the first step of the diversity-oriented synthesis, 1 was treated with aldehyde building blocks (aryl, heteroaryl, and alkyl; see the Supporting Information) to deliver twelve a,bunsaturated acyl imidazolidinones (2). The second steps of the solid-supported synthesis exploited three catalytic, enantioselective, divergent reaction pathways (Scheme 1): 1) [2+3] cycloaddition (reaction b, ee 60–65%, de 7899%), 2) dihydroxylation (reaction c, ee 88–91%), and 3) [4+2] cycloaddition (reaction d, ee 89–98%, de 74– 74%). Similar selectivities were observed when repeating the reactions in solution with a triisopropylsilyl-protected linker (as opposed to the diisopropylpolystyrene group; see the Supporting Information). The reactions were also conducted with achiral catalysts to give racemic products, which were used for the later steps of the synthesis. This procedure enabled the diversity-oriented synthesis to be streamlined to half the size, yet permitted the enantioselective synthesis of hits during the structure–activity relationship stages of this [*] Dr. G. L. Thomas, R. J. Spandl, F. G. Glansdorp, Dr. M. Ladlow, Dr. D. R. Spring Department of Chemistry, University of Cambridge Lensfield Road, Cambridge, CB2 1EW (UK) Fax: (+44) 1223-336362 E-mail: drspring@ch.cam.ac.uk Homepage: http://www-spring.ch.cam.ac.uk/

Enzyme Accessibility and Solid Supports: Which Molecular Weight Enzymes Can Be Used on Solid Supports? An Investigation Using Confocal Raman Microscopy

The accessibility of various solid supports (TentaGel, PEGA 1900, and beaded controlled pore glasses (CPGs)) to a range of enzymes was investigated. The different beaded materials were loaded with the peptide 4-cyanobenzamide-Gly-Pro-Leu-Gly-Leu-Phe-Ala-Arg-OH and incubated with the enzymes MMP-12 (22 kDa), thermolysin (35 kDa), MMP-13 (42.5 kDa), clostridium collagenase (68 kDa), and NEP (90 kDa). The absence/presence of the cyano stretching frequency was measured by means of confocal Raman microscopy. It was found that none of the investigated enzymes could enter the polymer matrices of TentaGel. PEGA 1900 was compatible only with the two smallest enzymes, while beaded CPG was successful even with NEP (90 kDa), proving its superiority over other materials in terms of bio-compatibility.

Immunomodulatory effects of Pseudomonas aeruginosa quorum sensing small molecule probes on mammalian macrophages

Pseudomonas aeruginosa produces the quorum sensing signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). This natural product not only coordinates production of virulence factors by the bacterium, but also has immunomodulatory effects on the host organism. Immunomodulatory small molecules are valuable for immunology research and are potential therapeutics for autoimmune diseases such as rheumatoid arthritis, and immunosuppressive drugs following organ transplants. We describe the total synthesis of OdDHL using solid-supported reagents and scavengers, which has the potential to be used for automated analogue synthesis. OdDHL and four analogues were tested for their ability to activate or inhibit release of the pro-inflammatory mediators tumour necrosis factor alpha (TNFalpha) and nitric oxide (NO) from equine or murine macrophages (immune cells). Two of the analogues showed substantial immunomodulatory activity with these macrophages. One analogue showed differing species selectivity, being a potent antagonist in mouse cells, but a partial agonist in horse-derived macrophages. These compounds have the therapeutic potential to be used for protecting animals from bacterial septic shock.

Continuous flow based catch and release protocol for the synthesis of α-ketoesters

Summary Using a combination of commercially available mesofluidic flow equipment and tubes packed with immobilised reagents and scavengers, a new synthesis of α-ketoesters is reported.

Novel synthesis of fused isoxazolidines via a palladium catalysed allene insertion–intramolecular 1,3-dipolar cycloaddition cascade reaction

A one pot, three component palladium catalysed allenation of aryl iodides, in combination with a nitrone cycloaddition, leads to formation of fused isoxazolidines, creating two rings, two stereocentres and one tetrasubstituted carbon centre.

Effect of chain length on transfection properties of spermine-based gemini surfactants

The synthesis and associated structure-activity relationships for gene transfection of a series of spermine-derived cationic gemini surfactants incorporating diamino acid headgroups and either identical (symmetrical) or different (unsymmetrical) lipophilic tailgroups is described. Transfection activity is found to depend critically upon the structural elements present.

Fluorous tagged small molecule microarrays

The affinity fluorous interaction between fluorous tagged small molecules and a fluoroalkyl modified glass surface was shown to facilitate the detection of protein-ligand binding interactions in the fabrication and screening of small molecule microarrays.

The use of a continuous flow-reactor employing a mixed hydrogen–liquid flow stream for the efficient reduction of imines to amines

Imines have been reduced to amines in high yield, and with excellent chemoselectivity, by catalytic hydrogenation in a continuous flow-reactor, utilising an electrochemically-generated hydrogen source to produce a mixed hydrogen-liquid flow stream.

3D small-molecule microarrays

A PEG based 3D hydrogel slide was developed specifically for small-molecule microarraying purposes, which displayed improved loading capacity, signal sensitivity and spot morphology compared with a commercially available slide and comparative 2D slide.

Complete functionalisation of small and large diameter bromopolystyrene beads; applications for solid-supported reagents, scavengers and diversity-oriented synthesisElectronic supplementary information (ESI) available: experimental techniques, apparatus, characterisation and spectroscopic data. See http://www.rsc.org/suppdata/ob/b4/b406488g/

Bromopolystyrene beads with diameters of up to 600 microm have been derivatized completely, via bromine-magnesium exchange and interception with electrophiles, to yield high quality solid-supported reagents, scavengers and solid supports for use in diversity-oriented synthesis.