Development of benzo[1,4]oxazines as biofilm inhibitors and dispersal agents against Vibrio cholerae

Bacterial biofilms are surface associated bacterial communities of sessile cells encased in a matrix of polysaccharides, extracellular DNA and proteins. Such biofilms are of significant concern in nosocomial infections, where it is attributed to over $1 billion in increased hospital costs per annum in the US alone. Unlike cells in the planktonic state, bacterial biofilms do not exert their antimicrobial resistance through mutation or acquisition of resistance functions by horizontal gene transfer. Instead, resistance is largely driven by the formation of latent cells within the biofilm matrix that reduce cellular turnover and therefore remove the susceptibility of targets associated with traditional antimicrobials. V. cholerae is a diarrheal pathogen that naturally inhabits both fresh and saltwater environments. In spite of its prevalence, no clinical therapeutics have been approved for use in the US or elsewhere that directly target biofilm formation and persistence. A limited number of small molecule inhibitors of V. cholerae biofilms have been reported in the literature, both from natural product screening campaigns and medicinal chemistry development efforts. However in the majority of cases these compounds have been shown to impact quorum sensing (QS) rather than directly targeting processes involved with biofilm matrix production or regulation. We recently reported the development of two high throughout image-based screens capable of identifying biofilm inhibitors against the Gram-negative pathogens V. cholerae and Pseudomonas aeruginosa. Screening of our natural product library, compromising of over 6000 prefractions, identified the aureomycin chromophore 1 as a moderate inhibitor of V. cholerae biofilms (biofilm inhibitory concentration (BIC50) = 63 mM). Given the structural novelty of this scaffold compared with other biofilm inhibitors, and the unusual biofilm inhibitory phenotype observed in the primary screening images, we elected to develop the benzo[1,4]oxazine scaffold through medicinal chemistry optimization in order to identify key elements of the required pharmacophore, and generate analogues with improved potency and pharmacological properties. Key to this approach was the formation of the a-keto-amide 7 and its subsequent application in a debenzylation– cyclization strategy to form hemi-acetal 8. Gratifyingly, treatment of the a-ketoamide 7 (formed in 5 steps from the commercially available ester 2) with 2%Pd(OH)2 on charcoal and four equivalents of 1,4-cyclohexadiene in ethanol at 50 1C enabled formation of the cyclic hemi-acetal 8 in excellent yield on a multi-gram scale with reaction times of less than 5 minutes. Dehydration of the acetal afforded the target molecule in 7 steps on a multigram scale (Scheme 1). To identify key elements of the pharmacophore for this compound class, a library of 41 derivatives were prepared using a divergent strategy to diversify both the exocyclic alkene and the methyl ester moiety of the original scaffold (Fig. 1, see ESI† for a comprehensive list of synthesized compounds). Initial examination of the lead compound 1 highlighted the a,b unsaturated carbonyl (C9–C11) as aMichael acceptor with potential involvement in the mechanism of action. In line with the behavior of other Michael acceptors in the literature, modification of the exocyclic methylene unit (compounds 8–12) eliminated activity in all cases. Introduction of any substituent onto the double bond (compounds 13–17) also resulted in a pronounced decrease in biofilm inhibition, indicating a tight steric limitation for modifications at this position. To probe whether the increase in steric size of the Michael acceptor directly correlated with the ability of the compound to undergo Michael addition, both the original oxazine 1 and phenyl a Department of Chemistry and Biochemistry, University of California Santa Cruz, California, 95064, USA. E-mail: [email protected] b Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, California, 95064, USA † Electronic supplementary information (ESI) available: Experimental procedures and analytical data along with protocols for all biological experiments. See DOI: 10.1039/c4cc07003h Received 10th September 2014, Accepted 27th November 2014