N-Thiolated b-lactams: A new family of anti-Bacillus agents Edward Turos, a, * Timothy E. Long, a Bart Heldreth, a J. Michelle Leslie, a G. Suresh Kumar Reddy, a Yang Wang, a Cristina Coates, a Monika Konaklieva, a,  Sonja Dickey, b Daniel V. Lim, b Eduardo Alonso c and Javier Gonzalez c a Department of Chemistry, 4202 East Fowler Avenue, CHE 207, University of South Florida, Tampa, FL 33620, USA b Department of Biology, 4202 East Fowler Avenue, University of South Florida, Tampa, FL 33620, USA c Departamento de Quimica Organica, Universidad de Oviedo, 37006 Oviedo, Spain Received 16 December 2005; revised 13 January 2006; accepted 13 January 2006 Available online 13 February 2006 Abstract—This report describes the evaluation of N-thiolated b-lactam antibiotics as potential anti-Bacillus agents. N-Thiolated b-lactams are a new family of antibacterials that previously have been found to selectively inhibit the growth of Staphylococcus bacteria over many other genera of microbes. From the data presented herein, these lactams similarly inhibit a variety of Bacillus species, including Bacillus anthracis. The preliminary structure–activity studies suggest that there is a need to balance the lipophilic character of the C 3 /C 4 groups in order to obtain optimal anti-Bacillus activity. Elongation or extensive branching of the organothio substitutent diminishes antibacterial effects, with the sec-butylthio derivative providing the strongest activity. Ó 2006 Elsevier Ltd. All rights reserved. Bacillus anthracis is a rod-shaped Gram-positive bacteri- um that is the causative agent of anthrax infections. 1,2 If inhaled, spores of B. anthracis rapidly migrate to lym- phonodes of the lungs, where they begin to germinate and release toxins that cripple the immune response, causing bacteremia, toxemia, and frequently, death. 3 Concerns about the possible use of B. anthracis as a bio- logical weapon have led to widespread efforts to prevent or treat anthrax infections with vaccine or antibacterial drug development, and to detect the microbe. 4,5 Our laboratory has recently identified a new family of anti-MRSA agents, N-thiolated b-lactams 1, which have a mode of action distinct from that of all other b-lactam antibiotics. 6 Rather than interfering directly with cell wall biosynthesis through irreversible acylation of penicillin binding transpeptidases, these compounds seem to affect cellular processes through transfer of the N-organothio group to a bacterial thiol. We also note that these lactams exert anti-proliferative properties against only a narrow range of bacterial genera, most significantly, Staphylococ- cus (including MRSA), Micrococcus, and Neisseria. This selectivity seems to be related to the levels and types of cel- lular thiols present in each microbe that is sensitive to the lactams, not to whether the microbes are Gram-positive or Gram-negative classes. Given that Staphylococcus and Bacillus are both prominent members of the Bacillales taxonomic order of bacteria, we therefore turned to inves- tigate whether these compounds could possess antibacte- rial properties against Bacillus spp. The lead compound in this study was N-methylthio- substituted lactam 1a, which in previous studies was found to have one of the most potent antigrowth activ- ities against Staphylococcus bacteria. 7 Prior studies on the overall structure–activity features of additional ana- logues of 1a determined that substituents at the C 3 and C 4 centers of the lactam ring exerted rather subtle effects on anti-MRSA activity, 8 while relative and absolute ste- reochemistry at these locations was largely inconsequen- tial. This led to the suggestion that the mode of action of the lactams requires passage of the lactam molecule through the bacterial membrane prior to interaction with a cytoplasmic thiol. Bioorganic & Medicinal Chemistry Letters 16 (2006) 2084–2090 N O S R 1 R 2 R 1 0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2006.01.070 Keywords: N-Thiolated b-lactams; Anthrax; Bacillus; SAR; Antibiotics. * Corresponding author. Tel.: +1 813 974 7312; fax: +1 813 974 1733; e-mail: eturos@shell.cas.usf.edu   Present address: Department of Chemistry, American University, Washington, DC 20016, USA.