Патент USA US3099673код для вставки
United States Patent 0 ” ice " 3,099,663 Patented July so, 1963 1 2 3,099,663 signi?cant number of day-old chicks are arti?cially in fected with the organism by the oral route. One group S-NlTRU-FUIJKFURAL AZINES James D. Johnston, New London, Conn, assignor to Chas. P?zer & (10., Inc., Brooklyn, N.Y., a corporation serves as a control and receives no treatment. This group is fed a nutritionally adequate diet containing sufficient protein, carbohydrate, fat, vitamins and minerals to pro mote growth in healthy chicks. The other group receives of Delaware [No Drawing. Filed Apr. 1, 1959, Bar. No. 863,376 6 Claims. (Cl. 260—-347.5) this same basic diet except that the compound under test is admixed with the feed in sufficient quantity to provide a level of 10.1% by weight of active ingredient. The sur This invention is concerned with antimicrobial agents and more particularly with a series of novel azine com 10 vival time of all chicks is recorded and from this data is calculated the ST5Q> that is the 50% survival time in days pounds which exhibits valuable activity against a variety at the 95% con?dence limit. The experiment is con of microorganisms. tinued for seven days, at which time the surviving birds I have made the discovery that compounds having the are sacri?ed and the heart, spleen and liver recovered for general formula 15 quantitilve determination of viable Salmonella organisms. ‘Compounds which are found to be particularly eifec tive in this test include, for example, 1~(5-nitro-2-fur O are remarkably effective antimicrobial agents showing activity against a variety of microorganisms, among them 20 organisms which cause disease in animals, including man, and fungi of industrial signi?cance. A may advantageously be selected from keto-substituted alkanoic and alkanedioic acids, less the keto oxygen, or frural) - 2 - (dimethyl - ,6 - keto - g1utarate)tazine, nitro - 2 - furfural) ~ 2 - (ethyl 3 - ketobutyrate) 1 - ethyl pyr'uvate - 2 - (5 - nitro - 2 - furfural) and 1 - (5 - nitro - 2 - furfural) - 2 - (dimethyl 2 - methyl - glutarate) azine. 1 - (5 azine, azine, 3 - keto Especially valuable are 1 - (5 - nitro - 2 - fur?ural) - 2 - (diethyl I3 - ketoglu tarate) azine, and l - (.5 - nitro - 2 - furfural) - 2 - (di from formyl-substituted alkanoic acids, less the carboxal 25 methyl 2,4-dimethyl-3-ketoglutarate) azine. These new compounds are conveniently prepared by the dehyde oxygen. Such substituents will preferably con condensation of S-nitro?urfural hydrazone with a carbonyl tain a total of up to 12 carbon atoms and will include, for compound, in a solvent such as ethanol or acetonitrile at example, re?uxing temperature, according to the following general 30 reaction ' and 35 Rz-(iH-COOH where R1 is hydrogen or .alkyl, R2 is alkyl, and x and y are zero or integers. where R1 and R2 are selected to conform to the structures In addition, the salts, such as the alkali 40 hereinabove described. Alternatively, they may be pre metal and amine salts, and esters, wherein the esterifying group is alkyl containing up to about 18 carbon atoms, may be employed in place of the corresponding acids. The novel compounds of this invention exhibit in vitro pared by condensing the hydrazone derived from the car bonyl compound with S-nitrofurfural, or its diacetate. The carbonyl compounds are in general readily available or may be synthesized by methods well known to those activity against a wide variety of microorganisms, includ 45 skilled in the art. ing gram positive and gram negative bacteria. Effective For anti-infective application, these new compounds ness is found, for example, against such organisms as may be blended with excipients or dispersed in diluents Micrococcus pyogenes var. aureus, including antibiotic-re sistant strains, Streptococcus pyogenes, Erysipelothrix rhusiopalhiae, Corynebacterium diphtheriae, Bacillus sub tilis, Clostridium perfringens, Escherichia coli, Vibrio including Water, isotonic saline, oils such as sesame oil, 50 and the like. Many modes of administration are pos sible, including oral, subcutaneous, intramuscular, intra vaneous and topical application, the choice being dictated by the type and severity of the infection. For adminis tration to poultry in the treatment of Salmonella infec ited against a variety of other microorganisms, for exam tions, the compounds will ordinarily be administered 55 ple, protozoa such as Endomeba histolytica and Tricho orally, and may be admixed with feed to provide a con monas vaginalz's, and fungi such as Alternarz'a solani, centration of at least about 0.001%, and preferably Cladosporium cladosporoides, Trichophyton rubrum, 0.01% or more by weight of active ingredient. Pythium debarynum, Aspergillus niger, and Penicillium The following examples are given solely for the pur comma, Pasteurella multocida, Mycobacterium 607, and Mycobacterium berolinense. Effectiveness also is exhib funiculosum. pose of illustration and are not to be construed as limi The compounds of this invention also exhibit activity 60 tations of this invention, many variations of which are against various species of Salmonella. Among these, a vpossible without departing from the spirit or scope thereof. number of compounds are particularly effective in the treatment of Salmonella infections in poultry. Each year EXAMPLE I a signi?cant number of mortalities occur among poultry A mixture of 0.1 mole S-nitrofurfural hydrazone and ?ocks, especially chickens, as a result of these infections, 65 0.1 mole of ‘dimethyl-2,4-dimethyl-3-keto-glutarate in 500 with a large economic loss resulting. The most impor ml. acetonitrile is heated at re?uxing temperatures for about four hours. The resulting solution is ?ltered and the ?ltrate treated with activated carbon and evaporated phoid, and S. pullorum, which produces white diarrhea in in vacuum. The clear gum is treated with 250 ml'. meth 70 chicks. anol and refrigerated. Filtration of the resulting crys Compounds are evaluated for activity against these or— talline slurry and recrystallization from methanol yields ganisms in the following manner. An experimentally tant diseases of this nature in poultry result from infec~ tions by Salmonella gallinaru‘m, which causes fowl ty 3,099,663 ll I-(S-nitro-Z-furfural)-2~(idimethy1-2,4-dirnethyl - 3 - keto monella according to the same procedures, with the fol glutarate) azine in the form of a. crystalline solid melting lowing results: Minimum Inhibitory Concentration‘ at about 155° C. EXAMPLE II Meg/ml. Following the procedure of Example I, diethyl acetone S. typhosa _________________________________ __ 100 S. pullorum ________________________________ __ 25 S. gallinarum _______________________________ __ 50 dicarboxylate is caused to react with 5-nitrofurfural hydrazone, yielding 14.6 g. of 1-(5-nitro-2-furfural)-2 ('diethyl-?-ketoglutanate) azine in the ‘form of orange plates melting at about 126° C. and-having the follow ing elemental analysis: carbon, 49.42%; hydrogen, 5.04%; nitrogen, 12.4%. Ultraviolet absorption maxi EXAMPLE VII 10 composition: ma are observed at 3100 A. (e=15,100) and 4060 A. Percent (s=29,250 in methaol). An infrared absorption maxi mum is observed at 5.755 microns. A typical poultry feed is prepared having the following Ground yellow corn ______________________ __ 51.28 15 Soybean oil meal (51%) ___________________ __ 38.15 Corn oil _________________________________ __ Calcium carbonate ________________________ __ 6.10 1.20 EXAMPLE ‘ III Following the procedure of Example I, the following series of compounds is prepared: Dicalcium phosphate ______________________ .._ 1.35 Salt ____________________________________ __ 0.61 20 Delmix (commercially available mineral mix con taining CaCOs and small amounts of iron, zinc, manganese and other salts—Limestone Products Corporation of America, New Jersey) . Vitamin A (5305 IU/lb.) __________________ __ 0.1 25 Vitamin D3 (681 ICU/lb.) _________________ __ 0.05 Klotogen F (commercially available form of vita min K-Abbott Laboratories) ____________ __ 0.0003 Pyridoxine hydrochloride __________________ __ 0.0006 30 D,L—methion-ine __________________________ __ 0.140 Niacin USP ______________________________ __ 0.0025 Choline chloride (25%) ___________________ __ Ribo?avin ___.. _____ Calcium pantothenate (45%) ______________ __ 0.2 0.06 0.002 Myvamix (commercially available form of vita 35 min E) _______________________________ __ 0.05 The products. of Examples I and II are added to dif ferent samples of this feed to provide compositions con taining 0.1% by weight of active ingredient. These com The product of Example I is subjected to standard in positions are successfully employed in the treatment of vitro plate tests against a variety of microorganisms. 40 chicks infected with S. gallinarum, no toxic eifects of the The medium employed is prepared by adding 37 grams azine being observed. At the conclusion of the experi of dehydrated Bacto brain-heart infusion B37 (purchased ment the birds are sacri?ced and the heart, spleen and from Difco Laboratories of Detroit) to a liter of distilled liver found to be free of viable Salmonella. water and sterilizing the resulting solution in an auto~~ When 5-nitrofurfura1 hydnazone is employed in the clave. The compound under test is added to the brain 45 same test, it is found to be toxic, causing Weight loss and heart broth in various concentrations; up to 200 mcg. early death. At levels below 0.1%, it is inactive. per ml., and the solutions are applied to agar plates seeded with one of the organisms. In this manner the EXAMPLE VIII minimum concentration of active ingredient necessary 1 (5 nitro Z-furfural)-2<(dimethyl-B-keto-glutarate) to inhibit organism growth for 24 hours at 37° C. is de 50 azine is tested in vivo against S. gallinarnm and S. pullo termined. Results are as follows: rum according to the procedure described in Example Minimum Inhibitory Concentration VIII at levels of 0.1, 0.05 and 0.01%. Signi?cant activity against both infections is noted at all levels with no toxic Mcg./ml. effects. Bacillus subtilis _____________________________ __ 25 55 What is claimed is: Clostridium perfringens ______________________ __ 25 l. 1~(5-nitro-2-furfural)-2-(ethyl 3-ketobutyrate) azine of Bacterium ammoniagenes _____________________ .._ 200 the formula Aerobacter aerogenes ________________________ __ 200 Proteus vulgaris ____________________________ _._ 100 Pseudomonas aeruginosa _____________________ __ 100 Erwinizz amylovora __________________________ __ 100 60 Dcsulfovibrio desulfuricans; __________________ -._ 200 Vibrio comma ______________________________ __ 100 2. 1-(5-nitro-2-furfural)-2-(dimethyl - ,6 - keto - gluta When S-nitrofurfur-al hydrazone is subjected to the 65 rate) azine of the formula same series of tests, it does not inhibit organism growth. EXAMPLE V The products of Examples II and III are subjected to 3. 1-(5-nitro-2-furfural) - 2 - (diethyl ,B-ketoglutarate) similar screening tests and are found to be active against 70 azine of the formula a wide variety of Gram-positive and Gram-negative or ganisms. EXAMPLE VI The product of Example I is screened‘again'st Sal 75 3,099,663 '~ 5 6 atoms, alkali-metal salts of said acids and esters of said 4. 1-(5-nitro-2-furfural) - 2 - idimethyl 2,4 dimethy1-3 acids wherein the esterifying group is alkyl containing up ketoglutarate) azine of the formula to 18 carbon atoms. References Cited in the ?le of this patent FOREIGN PATENTS CH3 3,225 5. 1-(S-nitro-2-furfural)-2-(dimethy1 3 - keto-2-methy1 180,357 glutarate) azine of the formula Japan ________________ __ May 14, 1955 Japan _______________ __ Sept. 12, 1949 OTHER REFERENCES Dann st ‘211.: Chemische Berichte, volume 82, pages 84 to 88 (1949). Dreizen et al.: Journal of Dental Research, volume 28, pages 288 to 298 (1949). 15 Chemical Abstracts, volume 45, page 10302 (1951). Dodd et -al.: J. Am. Pharm. Assoc., volume 39', pages 10 313-315 (1950). Chemical Abstracts, volume 47, page 6885 (1953). Dunlop et al.: “The Furans” (ACS Monograph No. wherein A is a substituted methylidene moiety derived from a carbonyl compound selected from the group con sisting of keto-alkanoic acid, keto-alkanedioic acid and formylalkanoic acid, each acid ‘containing up to 12 carbon 20 119), pages 164 to 165 and 362 to 36-3, Reinhold Publish ing Corp. (1953). Derwent French Patents Abstracts, volume 1, No. 11, Group 3, page 1 (Sept. 29, 1961).