Methylotrophic bacteria: biochemical diversity and genetics.

Bacteria that are able to use methane as a sole carbon and energy source also carry out a broad range of biotransformations, some of which have industrial and environmental significance. Genetic studies on methylotrophs, including the use of recombinant DNA techniques, show promise for the isolation and cloning of genes coding for specific functions.

Dopamine D(3) receptor antagonists. 1. Synthesis and structure-activity relationships of 5,6-dimethoxy-N-alkyl- and N-alkylaryl-substituted 2-aminoindans.

5,6-Dimethoxy-2-(N-dipropyl)-aminoindan (3, PNU-99194A) was found to be a selective dopamine D(3) receptor antagonist with potential antipsychotic properties in animal models. To investigate the effects of nitrogen substitution on structure-activity relationships, a series of 5,6-dimethoxy-N-alkyl- and N-alkylaryl-substituted 2-aminoindans were synthesized and evaluated in vitro for binding affinity and metabolic stability. The results indicate that substitution at the amine nitrogen of the 2-aminoindans is fairly limited to the di-N-propyl group in order to achieve selective D(3) antagonists. Thus, combinations of various alkyl groups were generally inactive at the D(3) receptor. Although substitution with an N-alkylaryl or N-alkylheteroaryl group yields compounds with potent D(3) binding affinity, the D(2) affinity is also enhanced, resulting in a less than 4-fold preference for the D(3) receptor site, and no improvements in metabolic stability were noted. A large-scale synthesis of the D(3) antagonist 3 has been developed that has proven to be reproducible with few purification steps. The improvements include the use of 3,4-dimethoxybenzaldehyde as a low-cost starting material to provide the desired 5,6-dimethoxy-1-indanone 5c in good overall yield (65%) and the formation of a soluble silyl oxime 17 that was reduced efficiently with BH(3).Me(2)S. The resulting amino alcohol was alkylated and then deoxygenated using a Lewis acid and Et(3)SiH to give the desired product 3 in good overall yield of ( approximately 65%) from the indanone 5c.

Anthelmintic activity of dioxapyrrolomycin.

Dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 produced by UC 11065 were evaluated as anthelmintics. Assays used to examine these compounds included effects on the free-living nematode Caenorhabditis elegans, ability to clear target nematodes (Haemonchus contortus and Trichostrongylus colubriformis) from jirds, and clearance of Haemonchus contortus from lambs. A crude extract of UC 11065 containing dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 was active against C. elegans and against H. contortus in the jird. Purified and/or synthetic samples of dioxapyrrolomycin, pyrrolomycin C, pyrrolomycin D, and piericidin C2 were tested in the jird model; only dioxapyrrolomycin exhibited appreciable activity against H. contortus (greater than or equal to 90.9% clearance at 0.33 mg/jird), while none of the compounds showed appreciable activity against T. colubriformis. Dioxapyrrolomycin cleared 99.9% of H. contortus from lambs at 12.5 mg/kg. An in vitro migration study using susceptible and closantel-resistant H. contortus showed there is cross-resistance between dioxapyrrolomycin and closantel. Dioxapyrrolomycin appears to be a narrow-spectrum anthelmintic which works through a closantel-like mode-of-action.

Development of a mechanism of action-based screen for anthelmintic microbial metabolites with avermectinlike activity and isolation of milbemycin-producing Streptomyces strains.

A high-volume screen for anthelmintic microbial metabolites with an avermectinlike mode of action was developed. The primary screen used the free-living nematode Caenorhabditis elegans in a whole-organism assay. The specificity for avermectinlike compounds resides in the secondary screen, which takes advantage of the chloride channel-opening properties of the avermectins. By using standard microelectrode techniques, membrane conductance changes following exposure to extracts of microbial cultures were measured in the walking leg stretcher muscle fibers of the lined shore crab Pachygrapsus crassipes. The avermectins and related milbemycins give a characteristic response of rapid loss of membrane resistance coupled with a slight hyperpolarization of the membrane. This is partially (near 50%) reversible with the chloride channel blocker picrotoxinin. Four morphologically similar cultures that produced avermectinlike activities were identified by this screen. Isolation of the active components from one of these cultures (strain UC 8984) followed by nuclear magnetic resonance spectroscopy resulted in the identification of milbemycins alpha 1 and alpha 3. These metabolites are members of a large family of milbemycins produced by Streptomyces hygroscopicus subsp. aureolacrimosus NRRL 5739. Systematic studies revealed that strain UC 8984 is also a S. hygroscopicus strain, but which is taxonomically distinct from NRRL 5739.