A new antitumor antibiotic is produced in fermentation liquors of Streptomyces zelensis sp.n. The antibiotic is biologically active at extremely low concentrations. At 40 pg/ml, it inhibited 90% of the growth of L1210 cells in culture in tube dilution assays. The minimal inhibitory concentrations against Gram-positive bacteria is between 1-10 ng/ml, while these values for Gram-negative bacteria and fungi are mostly under 1 μg/ml. A microbiological assay with Bacillus subtilis can detect concentrations of 1-2 ng/ml.
A taxonomic study of Streptomyces X-14077 (NRRL 8144), which produces a water-soluble purple-red pigment complex, revealed it to be a new species which has been named Streptomyces echinoruber sp. nov. The pigment complex was produced in deep culture fermentation and isolated by solvent extraction and concentration. The major pigment component, rubrolone, has low toxicity and may have potential as a food coloring agent. It appears to be devoid of antibiotic activity.
Streptomyces echinoruber sp. nov. produces several red pigments. The major component, rubrolone, has been identified as 8(R), 9(R), 10(S), 10a(R)-tetrahydro-9, 10, 1Oa, 11-tetrahydroxy-3, 8-dimethyl-l-propyl-6aH(S)-pyrano[2'', 3'': 5', 4]furo[2', 3': 5, 6]azuleno[2, 3-c]pyridine-5, 13-di one (1) by single crystal X-ray analysis of a suitable derivative. A second pigment, B, is probably structurally closely related.
A key protected streptidine derivative (3) useful for the synthesis of antibiotics of streptomycin series was prepared by hydrolysis of an acylated dihydrostreptomycin (DHSM) derivative (2), and it was condensed with a protected dihydrostreptobiosaminyl chloride (5) to give two condensation products (6 and 7). By deblocking, 6 was led to DHSM and 7 to a biologically inactive isomer (8) of DHSM. From the PMR spectrum of 4-O-mesyl derivative (4) of 3, the benzyloxycarbonyl and acetyl groups were concluded to be attached to the end nitrogens of the guanidine groups.
CP-45, 899 [3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo(3, 2, 0)heptane-2-carboxylic acid, 4, 4-dioxide, [2S-(2α, 5α)]] has low intrinsic activity against most Gram-positive cocci, Enterobacteriaceae and Pseudomonas . It inhibits Neisseria at concentrations of 0.1-6.2 μg/ml. The combination of CP-45, 899 and ampicillin inhibited Staphylococcus aureus and Enterobacteria-ceae resistant to ampicillin by virtue of β-lactamase activity. Combination of CP-45, 899 and cephalothin was synergistic less often, and CP-45, 899 did not act synergistically with carbenicillin or ticarcillin against Pseudomonas resistant to these agents. CP-45, 899 acted synergistically with ampicillin against Bacteroides. Synergy of CP-45, 899 and ampicillin was demonstrated at varying concentrations suggesting that it may significantly enlarge the antibacterial activity of ampicillin against resistant bacteria.
The synthesis and antimicrobial profile of a series of 7-(β-ketoacylamino)cephalosporins (1) bearing an acetoxymethyl or a heterocyclicthiomethyl group at the 3-position are described. Of this series, 3-[[(1-methyl-1H-tetrazol-5-yl)thio]methyl]-7-(3-oxobutyrylamino)ceph-3-em-4- carboxylic acid (11) showed moderate antibacterial activities in in vitro and in vivo tests.
New cephalosporins modified in the acyl part of 7-(3'-oxobutyrylamino)cephalosporins (1), which have been described in the preceding paper, were synthesized by thiolation at the 2'-or the 4'-position, or by transforming the 3'-oxo group into a 3'-imino group. The most active compound in vitro was 3-[[(1-methyl-1 H-tetrazol-5-yl)thio]methyl]-7-(4-methylthio-3-oxobutyrylamino)ceph-3-em-4-carboxylic acid (7c), which showed superior in vitro activity against Gram-positive and Gram-negative bacteria compared to the parent cephalosporin (1b) with the same 3-substituent. The ED50 value for 7c, however, was essentially equal to that of 1b in mice infected with Escherichia coli O-111.
The synthesis and the in vitro and in vivo antimicrobial activities of a series of 7-[2-(2-aminothiazol-4-yl)acetamido]cephalosporins (1) having varied 3-substituents, such as methyl, hydroxymethyl, acetoxymethyl, pyridiniomethyl and heterocyclicthiomethyls, are described. The derivatives having five membered heterocyclicthiomethyls exhibited strong inhibitory activities against Gram-negative organisms including some strains of Escherichia coli and Proteus morganii which are insensitive to cefazolin and cephaloridine. Pronounced activities were noted with 7-[2-(2-aminothiazol-4-yl)-acetamido]-3-[[[1-(2-dimethylaminoethyl)-1H-tetrazol-5-yl]thio]methyl]ceph-3-em-4-carboxylic acid (1y; SCE-963).
A single dose of 20 mg/kg of SCE-963 [7β-[2-(aminothiazol-4-yl)acetamido]-3-[[[1-(2-dimethylaminoethyl)-1H-tetrazol-5-yl]thio]methyl]ceph-3-em-4-carboxylic acid] was administered subcutaneously to mice, intramuscularly to rats, rabbits and dogs. Plasma and tissue levels of SCE-963 reached a peak in 15-30 minutes after administration. In mice, rats and dogs, SCE-963 was distributed at high concentration in the descending order in the kidney, liver, plasma, lung and spleen, and in rabbits, in the kidney, plasma, lung, liver and spleen. The SCE-963 levels in the liver of mice, rats and dogs were higher than those of cefazolin, cephaloridine and cephalothin. The plasma and tissue levels of SCE-963 in mice and rats diminished rapidly, but those in rabbits and dogs declined gradually. SCE-963 was mainly excreted in the urine. The rate of excretion of SCE-963 in the bile was two to three times higher than that of cefazolin.
Membrane-bound penicillin-binding proteins of an Escherichia coil carrying an R factor which mediated the resistance to penicillins were examined by slab gel electrophoresis and fluorography using β-lactamase inhibitors such as methicillin, clavulanic acid and MC-696-SY2-A, and by affinity chromatography. By fluorography, it appeared that the penicillin-binding proteins of the strain carrying the R factor could not be distinguished from those of the parent strain. In both strains, methicillin had a preferential affinity for penicillin-binding proteins 2 and 3, clavulanic acid for 2 and 4, and MC-696-SY2-A for 1A at the concentration which was needed to inhibit about 75-80% of β-lactamase activity of the membrane fraction from a strain carrying an R factor. This with other facts indicates that MC-696-SY2-A has a unique character in the binding to penicillin-binding proteins. By affinity chromatography using cephalexin-CH-Sepharose 4B column, two major cephalexin-binding proteins were detected. Their molecular weights were found to be 110, 000 and 32, 000, respectively. These two proteins corresponded to penicillin-binding proteins 1 and 5/6. From these results it was suggested that the R factor had no influence on the penicillin-binding proteins in the E. coli strain examined.
The affinities of cefoxitin, a cephamycin antibiotic, to penicillin-binding proteins of Escherichia coil were reexamined using a recently developed method for separating penicillin-binding proteins. The inhibitions by this antibiotic of four measurable penicillin-sensitive enzymatic reactions, the reactions of D-alanine carboxypeptidases IA and IB, cross-bridge formation and concomitant release of D-alanine, were also measured. An approximate correlation was found between the affinities of cefoxitin to the penicillin-binding proteins responsible for these reactions and its rates of inhibition of the respective penicillin-sensitive reactions.
The stability of eight antimicrobial agents in WILKENS-CHALGREN broth was evaluated. The activities of only carbenicillin and benzyl penicillin were significantly decreased after storage for eight weeks at -20°C. Anaerobic reduction of the susceptibility testing broth prior to inoculation was found to be unnecessary.
Microorganisms were plated on agar plates containing various concentrations of an antimicrobial drug and inhibition of growth was determined at each drug concentration. The ID50 value and the gradient of the line were calculated by the least square method. When 200-800 bacterial cells were inoculated on an agar plate, growth inhibition corresponded linearly with the log concentration of a drug within the range of 5-95% inhibition. The ID50 value and the gradient obtained were reproducible and reliable using microorganisms at stationary phase of growth with all tested bacterial species and all tested antimicrobial agents. It was found that the ID50 values of drugs were more reproducible and may be more reliable than the MIC (minimum inhibitory concentration) values of the drugs.