NotesYol. 28 leads to the speculation that a common biogenetic intermediate such as 6 may explain the observed formation of both tetracycline and 5-hydroxytetracycline by various Streptomyces strains. Such an intermediate as 6 might be visualized as undergoing either fermentative reduction or hydration (peroxidation-reduction) in the final step of biogenesis. We feel this route to be an attractive possibility, although the conversion of 5a,6-anhydro-5-hydroxytetracycIine to 5-hydroxytetracycline has been accomplished by an S. aureofaciens strain.m Experimental Isolation of Dehydrochlorotetracycline.-A Streptomyces aureofaciens mutant was grown on a medium similar to that used for the production of chlorotetracycline.2 This medium included 75 g. of cornstarch, 25 g. of corn steep liquor, and 10 ml. of soybean oil per liter, plus the usual organic salts and calcium carbonate. After a 2-day inoculum incubation, a 250-gal. tank was run for 5 davs at 26°with 12 cu. ft. of sterile air/hr./gal.; terminal pH, 7.3.The broth, 157 gal., was adjusted to pH 2 with sulfuric acid; 75 lb. of Supercel was added, filtered, and filtrate adjusted to pH 8.5 with sodium hydroxide. The precipitate which formed was filtered on a press and washed with water to yield 16 kg. of wet cake. This cake contained small amounts of chlortetracycline as well as dehydroaureomycin, as a metal complex. The wet broth precipitate (16 kg.) was slurried in isopropyl alcohol (17 1.). After the slurry was acidified to pH 1.9 with concentrated hydrochloric acid, sodium chloride (3 kg.), butanol (34 1.), and Supercel (650 g.) were added. The mixture was filtered and the phases were separated. To the aqueous phase was added the filtration residue, isopropyl alcohol (8.5 1.), butanol (17 1.), and sufficient concentrated hydrochloric acid to adjust the pH to 1.5. After filtration the phases were separated. The combined organic phases were concentrated under reduced pressure to a volume of 15 1. and filtered from precipitated solids. To the filtrate were added 0.01 ,Y hydrochloric acid (3 1.) and hexane (34 1.). After separation of the phases the organic phase was extracted twice with 0.01 N hydrochloric acid (1.5 1.). The combined acid extracts were freeze dried; residue, 416 g., was dissolved in methanol (2.5 1.) and filtered from insolubles. On addition of ethylacetate (1.11.) crystals formed on cooling and standing for 2 days.The crystals were collected and washed with ethanol; yield, 107 g. A small sample was recrystallized from methanol-ethanol. Its infrared spectrum was identical with that of an authentic sample.5
Analogues of bromazepam [7-bromo-1,3-dihydro-5(2-pyridyl)-2H-1,4-benzodiazepin-2-one, A], which is a clinically useful minor tranquilizer, have been prepared by replacing the 2-pyridyl group at position 5 with 4-pyrimidyl (5), 2-pyrazinyl (8), 2,5-dimethylpyrazin-3-yl (10), and 2-pyrimidyl (12) groups. Low to moderate CNS activities in both mice and cat were found for all the new compounds. For the screening procedures used, the 2-pyrimidyl-substituted derivatives were found to be the most active new analogues although none of the activities exceeded those observed for bromazepam.
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