Claisen-Schmidt condensation Novel Ferrocenyl Chalcone Compounds as Possible Antimicrobial Agents Elecia Henrya, Robert B Smithb, Michael Collinsc, Susan J Birda, Pauline Gowlanda, John P Cassellaa a School of Sciences, Staffordshire University, Stoke-on-Trent, Staffordshire, ST4 2DF, United Kingdom b School of Forensic and Investigative Sciences, University of Central Lancashire, Preston, Lancashire, PR1 2HE, United Kingdom c Royal Chesterfield Hospital NHS Foundation Trust, Calow, Chesterfield, Derbyshire, S44 5BL, United Kingdom Introduction Results The increasing occurrence of drug-resistant bacteria and the spread of these infections from healthcare settings to those in the wider community has quickly become a global concern (WHO, 2015). Infections caused by bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are partly caused as a consequence of misuse and abuse of conventional drug therapies and a decreased development of new drugs (Hadley, 2014). The development of novel drugs is of paramount importance and chalcones, which are plant-based flavonoids, have shown favourable antibacterial activity, especially against MDRs (Cushnie & Lamb, 2011). These flavonoids can be modified and the ferrocene group in ferrocenyl chalcones are have been proposed as inhibitors of cellular respiration within the nematode C. elegans (Attar et al, 2011). The current study evaluates the antimicrobial ability of a range of newly developed ferrocenyl chalcones containing alkyl residues of increasing chain lengths (see Figure 1) on three (3) types of drug-resistant S. aureus and other non-resistant Gram-positive bacteria. The potential effect on respiration was assessed by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) to measure the percentage of actively respiring cells following chalcone treatment (Moodley, 2004). Scanning electron microscopy (SEM) was used to show morphological changes to the cells. A B Figure 4 – Mean (±0.000, n=6) MIC values of ferrocenyl chalcone compounds against A) non-resistant bacteria and B) resistant bacteria (RCH = Royal Chesterfield Hospital). Fe N + O A B I - R Substituted Ferrocenyl Chalcone Fe N + O A B I - R Substituted Ferrocenyl Chalcone O Chalcone Figure 5 – The estimated mean (±SD, n=3) percentage of actively respiring cells present when treated with ferrocenyl chalcones at MIC. Base-catalyzed A B Claisen-Schmidt condensation Figure 1 – The basic structure of chalcones followed by nitrogen substitution and alkyl iodide addition in ring A, and a ferrocenyl group substitution on ring B with alkyl side chain R. Materials & Method A B Figure 6 – SEM images of A) Untreated S. aureus NCIMB 8244 and B) S. aureus NCIMB 8244 treated with decyl ferrocenyl chalcone at MIC. Conclusions Ferrocenyl chalcone compounds with longer alkyl chains (hexyl to decyl) were more effective against resistant and non-resistant Gram-positive bacteria (pre-hexyl results not shown). The MTT assay indicate that a potential mode of action is inhibition of respiration resulting in cell death. SEM images also indicate that most microbial cells were severely destroyed. Figure 2 – Rosys Anthos 2010 microplate reader used in measuring the absorbance values of chalcone-treated (1 mg/ml stock) and antibiotic-treated (1 mg/ml stock) bacteria for 2-fold broth microdilution, measured at 620 nm, and for MTT assay measurements recorded at 540 nm. Further work Further investigations will include time-kill assays to show the time taken for death to occur for treated cells. Additional evidence of antimicrobial mechanism(s) will be assessed by the validation of the molecular features of the compounds by Infrared spectroscopy (IR) and nuclear resonance microscopy (NMR). Figure 3 – JOEL JSM 6610V variable pressure scanning electron microscope with Oxford Mmax50 x-ray EDS system used in bacterial SEM, where chalcone-treated and untreated samples were fixed in 2% glutaraldehyde, dehydrated in a graded ethanol series and viewed under high vacuum. References Attar, S., O’Brien, Z., Alhaddad, H., Golden, M.L. & Calderón-Urrea, A., 2011. Ferrocenyl Chalcones versus Organic Chalcones: A Comparative Study of Their Nematocidal Activity. Bioorganic & Medicinal Chemistry, 19(6), pp.2055–73. Cushnie, T.P.T. & Lamb, A.J., 2011. Recent Advances in Understanding the Antibacterial Properties of Flavonoids. International Journal of Antimicrobial Agents, 38(2), pp.99–107. Hadley, S., 2014. Resistant Gram-Positive Infections: Where Have We Been, Where Are We Now, and Where Are We Going? Clinical Therapeutics, 36(10), pp.1298–1302. Moodley, S., Koorbanally, N. a, Moodley, T., Ramjugernath, D. & Pillay, M., 2014. The 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyl Tetrazolium Bromide (MTT) Assay Is a Rapid, Cheap, Screening Test for the in Vitro Anti-Tuberculous Activity of Chalcones. Journal of Microbiological Methods, 104, pp.72–8. WHO, 2015. Worldwide Country Situation Analysis: Response to Antimicrobial Resistance. World Health Organisation Press.