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Combinatorial libraries of N-acylated 5-(S)-aminomethyloxazolidinone derivatives of S-oxide and S,S-dioxide tetrahydro-4(2H)-thiopyranyl and thiomorpholine phenyloxazolidinone series have been synthesized on a solid phase and evaluated for antimicrobial activity. Several novel potent leads have been identified, including orally active oxazolidinones with enhanced activity against respiratory tract infection pathogens Haemophilus influenzae and Moraxella catarrhalis.

Fluorine-19 nuclear magnetic resonance (NMR) studies of cells and perfused organs loaded with fluorinated ion chelators represent a new approach to determining cytosolic free calcium levels in situ. The molecular basis for this approach and the relative advantages and disadvantages of the NMR technique are discussed in this paper. Results obtained on perfused normoxic and ischemic rat hearts are presented, indicating that ischemia is associated with an elevation in the level of cytosolic free calcium before the onset of irreversible cell injury. The results are therefore consistent with this elevation playing a causative role in the mediation of myocardial cell injury resulting from ischemia.

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.

Negamycin 1 is a bactericidal antibiotic with activity against Gram-negative bacteria, and served as a template in an antibiotic discovery program. An orthogonally protected β-amino acid derivative 3a was synthesized and used in parallel synthesis of negamycin derivatives on solid support. This advanced intermediate was also used for N- and C-terminal modifications using solution-phase methodologies. The N-terminal modifications have resulted in the identification of active analogues, whereas the C-terminal modifications resulted in complete loss of antibacterial activity. The N -methyl negamycin analogue, 19a , inhibits protein synthesis (IC 50 =2.3 μM), has antibacterial activity ( Escherichia coli , MIC=16 μg/mL), and is efficacious in an E. coli murine septicemia model (ED 50 =16.3 mg/kg).

Changes in cytosolic free magnesium ion concentration (Mgi) during myocardial ischemia were measured by 19F NMR in perfused rat hearts loaded with fluorine-labeled derivatives of the magnesium chelator o-aminophenol-N,N,O-triacetate. The perfused rat hearts were loaded intracellularly with the appropriate magnesium indicator by perfusion with 200-400 ml of Krebs-Henseleit buffer containing 5 microM acetoxymethyl ester of the indicator. Basal Mgi concentrations measured by three different indicators averaged 0.85 +/- 0.10 mM (n = 9) and showed no correlation with the KD of the indicator used. 31P NMR measurements of the magnesium-dependent shift between alpha- and beta-phosphates of ATP demonstrate that there is no measurable lowering of Mgi during loading with fluorinated o-aminophenol-N,N,O-triacetate. Between 10 and 15 min of ischemia, Mgi rose nearly 3-fold to 2.1 +/- 0.4 mM. This increase in Mgi occurred over the same time course as the decrease in ATP. After 20 min of reperfusion with Krebs-Henseleit buffer, Mgi declined to 1.5 +/- 0.5 mM. This sustained elevation of Mgi above basal levels may inhibit calcium release from sarcoplasmic reticulum, thereby contributing to the well documented impairment of mechanical function that occurs after a reversible period of ischemia.

The previously developed chelator O-aminophenol-N,N,O-triacetic acid (APTRA) (L. A. Levy, E. Murphy, B. Raju, and R. E. London. Biochemistry 27: 4041-4048, 1988) has been modified to yield a fluorescent analogue which can be utilized as an intracellular probe for ionized Mg2+. The fluorescent analogue, FURAPTRA, with a magnesium dissociation constant of 1.5 mM, is structurally analogous to the calcium chelator fura-2 and exhibits a similar excitation shift on magnesium complexation. Hence, data on the intracellular Mg2+ concentration can be obtained using an analogous ratio method. The acetoxymethyl form of the chelator is readily loaded into cells and has been used to determine a cytosolic free Mg2+ concentration of 0.59 mM for isolated rat hepatocytes. As a consequence of the relatively high levels of cytosolic Mg2+, the problem of ion buffering is much less severe than for the analogous calcium indicators.