Quantitative structure-activity relationships have been found among macrolide antibacterial agents in their potencies against the bacterial pathogen Pasteurella multocida both in vitro and in mouse infections. To obtain these relationships we measured, among other things, the pK(a)'s and log P's of 15 known macrolides of diverse structures. Among these compounds, in vitro potency [log(1/MIC)] is a function of log P, log D, and CMR (R = 0.86). In vivo potency is a function of the higher pK(a), the HPLC chromatographic capacity factor log k', log(1/MIC) and pNF (R = 0.93). pNF is defined as the negative logarithm of the fraction of neutral drug molecules present in aqueous solution at pH 7.4. The same physical properties were determined for 14 macrolides not used in developing the original QSAR models. Using the in vivo model, we calculated the mouse protection potency ranges for these new compounds. Ten estimates agreed with those observed, three were lower by a half-order of magnitude, and one was calculated to be active in the range of 15-50 mg/kg, but in fact was not active at 50 mg/kg, the highest level tested. When these new compounds were combined with the original 15, and the QSAR's updated, the new equations for the in vitro and in vivo potencies were essentially the same as those originally found. Hence, the physical properties indicated above are major determinants of macrolide antibacterial potencies.

Antigenic properties of two mutants of Pasteurella haemolytica, strains 59B0071 and 59B0072, that do not produce detectable leukotoxin were investigated. Western blot (immunoblot) analysis with a number of polyclonal sera from animals recovering from pasteurellosis revealed that both mutants secreted a variety of antigens that were also present in cultures of several wild-type strains. These antigens ranged from about 100 to 15 kDa. Mutant strain 59B0071 was found to be totally deficient in leukotoxin, as judged not only by Western blotting but also by cytotoxicity assays with bovine lymphoma (BL-3) cells or bovine polymorphonuclear cells as targets. The mutant strain 59B0071 had normal levels of a secreted sialylglycoprotease, however. When strains were tested for virulence in goat and cattle challenge experiments, a reduction in mortality and lung lesions was observed with the mutant 59B0071 in comparison with results obtained with wild-type strains. These results are consistent with an important role for leukotoxin in P. haemolytica virulence and suggest that leukotoxin-negative mutants may be useful tools in the investigation of other virulence properties involved in P. haemolytica infections.

Reductive amination of repromicin with polyfunctional amines has led to new macrolide antibacterial agents, some of which are highly potent against the Gram-negative pathogen Pasteurella multocida both in vitro and in a mouse infection model. A key element in this discovery was the recognition that among certain known macrolides increasing lipophilicity results in diminished in vivo activity. One repromicin derivative, 20-[N-[3-(dimethylamino)-propyl]-N-L-alanylamino]-20-deoxorepro micin (35), was selected for advanced evaluation. At 5 mg/kg, a single subcutaneous dose was found to control induced pasteurellosis in swine and induced respiratory disease in cattle.

Two mutants of Pasteurella haemolytica A1 that do not produce leukotoxin were isolated. Following mutagenesis, colonies were screened with antiserum by a filter assay for absence of the secreted leukotoxin. The two mutants both appeared to produce normal amounts of other antigens, as judged by reactivity with polyclonal serum from an animal with pasteurellosis, and were not altered in beta-hemolytic activity as seen on blood agar plates. There was no evidence of either cell-associated or secreted leukotoxin protein when Western blots (immunoblots) were carried out with the polyclonal serum or with a monoclonal antibody directed against the leukotoxin. Southern blots revealed that both mutants show the wild-type restriction pattern at the leukotoxin locus, although the strain with the lktA2 mutation showed differences in other regions of the chromosome on analysis by pulsed-field gel electrophoresis. The strain with the lktA2 mutation grew more slowly than did the wild-type strain, while the strain with the lktA1 mutation was indistinguishable from the wild-type strain in its growth properties. The strain with the lktA1 mutation should be valuable in determining the role of the leukotoxin in virulence as well as in identifying other virulence factors of P. haemolytica.

A novel antibiotic, UK-69,753, has been isolated from a submerged fermentation of Amycolatopsis orientalis strain N731-15. UK-69,753 has been assigned the structure 1 using spectroscopic means, primarily by NMRanalysis. UK-69,753is a glycoside of factumycin (A40A), a previously reported member of a small groupof antibiotics related to aurodoxand efrotomycin. UK-69,753wasshownto have potent activity both in vitro and in vivo against the swine pathogen Treponemahyodysenteriae.In the preceding paper1}, we have described the taxonomy, fermentation and isolation of UK-69,753 (1), a novel memberof the efrotomycin2) family of antibiotics. In this paper we describe the structure elucidation of UK-69,753 along with its antibacterial activity. Results and DiscussionStructure Determination of UK-69,753 The structure of UK-69,753 (1) has been determined by spectroscopic means, relying heavily on the results ofNMRanalysis.^H, 13C, carbon multiplicity (distortionless enhancement by polarization transfer: DEPT)3) and 2D (homonuclear correlation: COSY4) and heteronuclear correlation: HETCOR5)) NMR spectra were recorded on UK-69,753in CD3OD, and the results of these experiments are summarizedin Table 1. The 13C NMRspectrum showed 57 distinct resonances with one line at 130.41 ppm accounting for two carbons, indicating that UK-69,753 contains 58 carbon atoms. Using the data available primarily from the two correlation experiments, eight distinct spin systems comprising 47 carbons were identified which correspond to the molecular fragments 2 through 9. The olefmic proton resonances of fragments 2, 7, and 8 were unambiguously assigned and measurement of the vicinal coupling constants of these resonances in the 1H NMRspectrum at 500 MHz (Fig. 1) allowed for assignment of the configuration of each disubstituted olefin in these fragments. The configuration of the two trisubstituted olefins (fragments 7 and 8) were both assigned as drawn on the basis of nuclear Overhauser effect (NOE) experiments1. The carbonyl and quaternary olefinic carbons in fragments 7 and 8 were assigned via multiple bond heteronuclear correlation (MBHC)to nearby carbons bearing protons.Consideration of the part structures 2~9 assembled from an analysis of NMRdata lead to the hypothesis that UK-69,753 is a novel memberof a small group of antibiotics characterized by compoundsThe nuclear Overhauser polarizations, observed at 500 MHz,were consistently negative.