Imines as antibacterial agents
United States Patent [1 1 Seng et al.  
 [ Apr, 29, 1975 IMINES AS ANTIBACTERIAL AGENTS [22] Filed: Aug. 16, 1973 [21] Appl. No.1 388.840  
 Related US. Application Data [62] Division of Ser. No. 323,953. Jan. 15, I973. which is a division of Scr. No. l3().007 March 3], 197] [30] Foreign Application Priority Data Apr. 2 1970 Germany 20l5676 52 us. cl. 424/250 [51 Int. Cl A6lk 27/00 [58] Field oi Search 424/250 [56] References Cited UNITED STATES PATENTS 3,;47L090 2/l968 Johnston 260/240 G FOREIGN PATENTS OR APPLICATIONS [215,815 l2/l970 United Kingdom 260/250 R Primary E.\&#39;aminer.lerome D. Goldberg [5 7] ABSTRACT lmines of 2-formylquinoxaline-3-carboxylic acid-1,4- dioxides and their salts are obtained through treatment of the lactone or a salt of 2- dihydroxymethylquinoxaline-N,N-dioxide-Ii-carboxylic acid .with a reactant bearing a free primary amino group. Theresultant compounds and their non-toxic salts are antibacterial agents and can be incorporated in pharmaceutical compositions and feedstuffs for this use. A typical embodiment is 2- (carbomethoxyhydrazonomethyl)-quin0xaline-3- carboxylic acid-1.4-dioxide.  
 32 Claims. No Drawings [MINES AS ANTIBACTERIAL AGENTS The new imines and salts have the general formula:  
 i COOY Amid-4R wherein Y is hydrogen, an alkali metal cation or the cation R --NH and each of R and R is identical to or different from the other and is selected from the group consisting of a. alkyl, substituted alkyl or cycloalkyl;  
 in which each of R and R when taken independently is identical to or different from the other, and is selected from the group consisting of hydrogen, alkyl or substituted alkyl, or when R and R are taken together with the nitrogen atom to which they are attached a 5- to 7- membered heterocyclic ring optionally containing as a ring member oxygen, sulphur, S0 or N-alkyl;  
 in which x is o, s or NH and, R and R are as above defined;  
 in which R is alkyl or substituted alkyl;  
 in which R is phenyl, pyridyl or norbornyl, and  
 X is as defined above;  
 in which R, R and X are as defined above; or  
 v N r AQI DQ l i A preferred group of the imines and salts of the invention are those of the above general formula (1), in which: Y has the meanings given above;  
 each of R and R is identical to or different from the other and is selected from the group consisting of a. alkyl or hydroxyalkyl of from l to 4 carbon atoms or a o-membered or 7-membered monocycloor bi-cycloalkyl group;  
 in which each ofR and R when taken independently is identical to or different from the other and is selected from the group consisting of hydrogen, alkyl or hydroxyalkyl of from 1 to 4 carbon atoms, or when R and R are taken together with the nitrogen atom to which they are attached, a 6membered heterocyclic ring optionally containing as a ring member oxygen or S0 in which X is O. S or NH, and R and R are as herein defined;  
 NH R  
 in which R is phenyl, pyridyl or norbornyl, and  
 X is as herein defined.  
  Alphatic groups embraced by R and R include straightchain or branched alkyl groups of from l to 6, preferably 1 to 4, carbon atoms. Cycloaliphatic radicals contain from 3 to 7, preferably 5 to 7, carbon atoms and include both monocyclic and bicyclic ring systems.  
  These aliphatic or cycloaliphatic groups can be optionally substituted, for example, by hydroxy, alkoxy, or acyloxy, the alkoxy and acyloxy groups containing 1 to 4, preferably 1 or 2, carbon atoms. The hydroxy group is the preferred substituent. Typical aliphatic and cycloaliphatic groups thus include methyl, ethyl, npropyl, isopropyl, n-butyl, isobutyl, tert.-butyl, npentyl, isopentyl, hexyl, 2-hydroxyethyl, cyclopropyl, cyclopentyl, cyclohexyl, bicyclo-(2,2,l) heptyl (norbornyl), and the like.  
  The substituents R and R are hydrogen or alkyl of from 1 to 4, preferably 1 or 2, carbon atoms. These alkyl groups can be optionally substituted with hydroxy, alkoxy or acyloxy, alkoxy and acyloxy groups containing 1 to 4, preferably 1 or 2, carbon atoms. Thus included are ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert.-butyl, as well as the corresponding groups substituted by hydroxy.  
  R and R when taken together with the nitrogen atom to which they are attached, can also form a heterocyclic ring, preferably containing 6 ring members, and preferably with an oxygen atom, a sulphur atom, an N- -alkyl group containing 1 to 4, preferably 1 or 2, carbon atoms, or the S0 group, as a ring member in the pposition relative to the nitrogen atom to which R and R are attached.  
  R is an alkyl of from 1 to 4, preferably 1 or 2, carbon atoms which may also be optionally substituted by hydroxy, alkoxy, acyloxy, alkoxy and acyloxy containing 1 to 4, preferably 1 or 2, carbon atoms. The hydroxy group is a particularly preferred substituent. R thus embraces such groups as methyl, ethyl, Z-hydroxyethyl and the like.  
  The alkali metal cation Y is, for example, that of sodium or potassium, preferably that of sodium.  
  R is phenyl, pyridyl or norborn-2-yl. When R is pyridyl, it can be bonded in the 2-, 3- or 4-position relative to the pyridyl nitrogen atom.  
  The above class of imines are obtained according to the process of the present invention by treatment of loXo-3-hydrdoxyl ,3-dihydro-furo-( 3,4-b )-quinoxaline- 4,9-dioxide, which has the formula:  
 or a salt thereof of the formula /Oll N ll with an amine of the general formula H NR, in which R is as previously defined, M is an alkali metal or alkaline earth metal cation and x is l or 2.  
  The salts of the formula can be obtained from the lactone of formula through treatment with alkali metal or alkaline earth metalhydrogen carbonates.  
  M is preferably the cation of sodium, potassium or most preferably calcium.  
  Both inorganic and organic polar solvents can be used as diluents for the reaction according to the invention, such as for example water, lower aliphatic alcohols of 1 to 4 carbon atoms, lower aliphatic nitriles such as acetonitrile, tetrahydrofurane, dioxane, dimethoxyethane, pyridine, dimethylformamide and the like.  
  The reaction according to the invention is carried out at a temperature of about 0 C to about 50 C, preferably 20 C to about 35 C.  
  In practice, the lactone or lactone salts are dissolved or suspended in the diluent, and this solution or suspension is then treated with an appropriate quantity of the amine. The formation of the imine or of the imine salt takes place in a weakly exothermic reaction and the final product is then isolated through conventional methods.  
  The imine salts 1) can also be prepared by the reaction of the free acids with amines.  
  The salts may be obtained in a subsequent step by conventional techniques or directly in the reaction of the lactone with the amine. If about 2 mols of the amine per mol of lactone are employed, the product will be in the form of that amine salt. If an alkali metal salt or alkaline earth metal salt of the lactone is employed, or if it is desired to obtain the free acid from the lactone, only about 1 mol of the amine per mol of the lactone or salt is required.  
  The course of the process according to the invention can thus be illustrated by the following equations:  
 \ OONa T 3 OH H -CH 0 2 1 3 The following examples will serve to further typify the nature of this invention without being a limitation on the scope thereof.  
 EXAMPLE 1 i o (9 H3 COOl-l H N/ as in the form of yellow crystals. which after recrystallisai i t g ZHZW INN/ c l l l 69 b00 1 N( a tion from isopropanol melt at l2325 C, with decomposition.  
 Analysis: C H N O (molecular weight 334) Calculated: C 57.5% H 6.6% N 16.8% Found: C 57.1% H 5.8% N 16.8%  
 EXAMPLE 2 27.4 g (0.1 mol) of the Na salt of 2-(dihydroxymethyl)-3-carboxylic acid-quinoxaline-di-N- oxide are dissolved in ml of water and 7.3 g (0.1 mol) of tert.-butylamine are added. The temperature is kept at 25 C by cooling. After 1 hour, the solution is evaporated in vacuo and 32 g of the compound of the are obtained in the form of yellow crystals which after recrystallisation from acetonitrile/water melt at 228 C, with decomposition.  
 Analysis: C, ,H N; N&#39;.\O (molecular weight 31 1) Calculated: C 54.0% H 4.57r N Na 7.4%  
  13.5% Found: C 53.7% H 4.9% N Na 6.9%  
 The 1-oxo-3-hydroxy-l ,3-dihydro-furo( 3,4-b  
 Example The compound is purified by dissolving it in sodium bicarbonate solution, filtering and acidifying the filtrate. The purified compound melts at 156 159 C, whilst foaming.  
 Analysis: C ,H N O (235) Calculated: C 51.3% H 2.6% N 12.0% 10 Found: C 52.0% H 2.8% N 12.6%  
  The alkali metal salts or alkaline earth metal salts of l-oxo-3-hydroxyl ,3-dihydro-furo-( 3 ,4-b )quinoxaline- 4,9-dioxide can be manufactured as follows:  
  The lactone (9) is suspended in water and approximately the stoichiometrically required amount of the alkali metal hydrogen carbonate or alkaline earth metal hydrogen carbonate is added at room temperature. The  
 20 salt of the lactone (10), thus produced, precipitates,  
 after evaporation of the solution if necessary, and can be isolated in the usual manner.  
  The following are obtained analogously to Examples 1 and 2:  
 Melting Point (C) N o Compound idecompos it: ion) 3 143-44 69 O0 11 N-CH Continued Example Melting Point (C) N Compound (decompositionL N// \CH==N-NHC/ N i OONa fig Q KZMA CH=N-N HOOC 2H5 As has already been mentioned, the new compounds of the invention surprisingly show an excellent chemotherapeutic activity. Their chemotherapeutic action was examined both. in animal experiments (oral and subcutaneous administration) with acute bacterial infections, and in vitro. In both cases the compounds show a very good antibacterial action, and the range of action encompasses both Gram-negative and Grampositive bacteria. The chemotherapeutic activity of the compounds according to the invention permits their use in human medicine and in veterinary medicine. Furthermore, the compounds can be employed as feedstuff additives, especially in raising young animals or fatstock. The good in vitro and in vivo activity of the compounds according to the invention can be seen from Tables I, 2 and 3 below.  
  The minimum inhibitory concentrations in vitro for some of the new compounds shown in Table 1 (MIC) were determined by the plate test in an agar medium of the following composition:  
 proteose peptone veal extract (solids) dextrose sodium chloride disodium phosphate sodium acetate adenine sulphate guanine hydrochloride uracil xanthin neutral agar per litre I,  
  5.10 X germswere inoculated per plate. Readings were taken after 24 and 48 hours, and the incubation temperature was about 37 C.  
  Table l MIC in ylml of medium Minimum inhibitory concentration (MIC) in y/ml of medium, measured by the series dilution test (complete medium), incubation temperature: 37 C, determination of the MIC after I8, 24 and 48 hours.  
 Compound of Bacterium Example 13 9700 8564 8580 8698 8699 8711 ATCC B. H. 4  
  Blaschke ,1 .t 13  
 species Iiquef. durans Streptococcus faecalis ATC C l I I I Escherichia coli C I65 H 2 55 B 5 I4 12 -25 41 A 26l G 2935 I2 3400 I017 Pseudomonas aeruginosa W Proteus mirahilis Proteus vulgaris I I KIebsieIla )mcasured in u PPLO medium For the compound of Example 3, the following minimal inhibitory concentrations (MIC) (&#39;y/ml of nutrient medium) were measured by the series dilution test (PPLO medium), incubation temperature 37 C, determination after I8, 24 and 48 hours.  
 Bacterium MIC M ycoplasma gallise licum M ycoplasma granu arum 25 M yvoplasma bovirhinis 200 In animal experiments on mice, the effective 100% dose (ED in mg/Kg was determined for certain compounds of the invention after intraperitoneal infection and subcutaneous (s.c.) or oral (p.o.) administration of the preparation.  
 Table 3 Compound Compound Compound Compound of Ex. 3 of Ex. 8 of Ex. 13 of Ex. I4 Bacterium s.c. p.c. s.c. p.c. s.c. p.c. s.c. p.c.  
  Escherichia 50 I00 50 I00 25 25 50 I00 coll C I65 Staph ylor&#39;oc- 25 50 (Us aureus In general, it has proved advantageous, in acute general infections, to administer amounts of about 5 mg to about 200 mg per kilogram, preferably about 25 to about 50 mg per kilogram of body weight per day, to achieve effective results. Nevertheless it can at times be necessary to deviate from the amounts mentioned, in particular depending on the body weight of the test animal or patient or on the nature of the method of administration, but also because of the type of animal and its individual behaviour towards the medicament, or because of the nature of the formulation of the latter, and the point in time or interval at which administration takes place. Thus it can, in some cases, suffice to use less than the abovementioned minimum amount, whilst in other cases the upper limit mentioned must be exceeded. In the case of the administration of larger amounts it can be advisable to divide these into several individual doses over the course of the day. The same range of dosages is envisaged for administration in human medicine. The other comments made above also apply in a general sense.  
  Accordingly, the present invention provides a pharmaceutical composition containing as an active ingredient at least one of the new compounds of the general formula (1) given above in admixture with a pharmaceutically acceptable solid or liquid diluent or carrier as hereinafter defined.  
  In the present specification the expression pharmaceutically acceptable diluent or carrier means a nontoxic substance that when mixed with the active ingredient or ingredients renders it suitable for administration. The expression preferably excludes water and low-molecular weight organic solvents commonly used in chemical synthesis, except in the presence of other pharmaceutically necessary ingredients such as salts in correct quantities to render the composition isotonic, buffers, surfactants, colouring and flavouring agents, and preservatives. Examples of suitable liquid diluents and carriers are vegetable oils, glycerol, propylene glycol, polyols, buffered aqueous solutions, isotonic saline aqueous solutions, syrups and lotion bases. Examples of suitable solid diluents and carriers are starches, cellulose and its derivatives, sugars, stearates and stearic acid, talc, and ointment bases. Examples of pharmaceutical compositions according to the invention are ointments, pastes, creams, sprays, lotions, aqueous and non-aqueous suspensions, emulsions, and solutions (including parenterally injectable solutions), elixirs and syrups, and granulates and powders either free-flowing or compressed into tablets.  
  Pharmaceutical compositions of the invention adapted for oral-administration are a preferred embodiment of the invention. The diluents and carriers used are preferably therefore those that adapt the active ingredient or ingredients for oral administration. Examples of such diluents and carriers are solid vehicles, excipients and lubricants such as glucose, lactose and sucrose, corn and potato starch, sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate, powdered gum tragacanth, gelatin, alginic acid, agar, talc, stearic acid and sodium, calcium and magnesium stearates, sodium lauryl sulphate, polyvinyl-pyrrolidone, sodium citrate, calcium carbonate, and dicalcium phosphate.  
  The pharmaceutical compositions of the invention may also contain other non-toxic adjuvants and modifiers such as dyes, surfactants, perfurmes, flavouring agents, such as sweeteners, preservatives and biocides.  
  Pharmaceutical compositions of the invention adapted for parenteral injection are another preferred embodiment of the invention. The diluents and carriers used are therefore preferably those that adapt the active ingredient for parenteral administration. Examples of diluents and carriers that adapt the active ingredient for parenteral administration are solvents and suspending diluents such as water, vegetable fatty oils, such as sesame oil, groundnut oil, corn oil, and cottonseed oil, aqueous propylene glycol, N,N-dimethylformamide, and dimethyl sulphoxide. In general, any non-aqueous diluent can be used that does not reduce the activity of the active ingredient and is non-toxic in the dose employed.  
  For the administration of the water-soluble compounds of the invention by parenteral injection sterile aqueous solutions can be employed, and are within the scope of the pharmaceutical compositions of the invention. Such aqueous solutions should preferably when necessary be buffered in the usual manner, and the liquid diluent should preferably before administration be rendered isotonic by adding the requisite amount of salt or glucose. Such sterile buffered isotonic solutions are especially suitable for intravenous, intramuscular and intraperitoneal injections. These pharmaceutical compositions of the invention can further contain local anaesthetics or substances that promote the diffusion of the active ingredient, for example hyaluronidase.  
  The pharmaceutical compositions of the invention preferably contain 0.5 to wt. of at least one new compound of the invention.  
  The present invention also provides medicaments in dosage unit form as hereinafter defined comprising as an active ingredient at least one compound of general formula (1) given above either along or in admixture with a pharmaceutically acceptable solid or liquid diluent or carrier. In thiscase the diluent or carrier is preferably as defined above but can also be water or another common solvent.  
  The expression medicament in dosage unit form&#34; as used in the present specification means a medicament in the form of discrete portions each containing a unit dose or a multiple or sub-multiple of a unit dose of the active ingredients(s); for example, 1, 2, 3 or 4 unit doses or a half, a third or a quarter of a unit dose. A unit dose is the amount of the active ingredient(s) to be administered on one occasion and will usually be a daily dose, or for example a half, a third, or a quarter of a daily dose depending on whether the medicament is to be administered once or, for example, 2, 3 times, or 4 times a day.  
  The discrete portions constituting the medicament in dosage unit form can include a protective envelope. The active ingredient can be undiluted and contained in such an envelope, or can be mixed with a pharmaceutically acceptable solid or liquid diluent or carrier as defined above. Such portions can for example be in monolithic coherent form, such as tablets, lozenges, pastilles, pills, suppositories, or dragees; in wrapped or concealed form, the active ingredients being within a protective envelope, such as wrapped powders, cachets, sachets, capsules, or ampoules; or in the form of a sterile solution suitable for parenteral injection, such as ampoules of buffered, isotonic, sterile, pyrogenfree aqueous solution; or in any other form known in the art.  
  As stated above, peroral administration is a preferred mode of administration. Preferred medicaments in dosage unit form according to the invention are therefore those adapted for oral administration, such as tablets, pills, dragees, capsules, and cachets, as well as wrapped powders containing the active ingredient in powdered form with a powdered diluent or carrier for suspension in water before being taken.  
  As also stated above a further preferred mode of administration is parenteral administration. Preferred medicaments in dosage unit form according to the invention are therefore those adapted for parenteral injection, such as ampoules containing a measured quantity of a sterile isotonic saline injectable aqueous solution of the new active ingredient, which may be buffered with a pharmaceutically acceptable buffer and are preferably free of pyrogens.  
  The preferred unit dose for administration of the medicaments of the invention is 250 l6,000 mg. of active ingredient, preferably 1,250 4,000 mg. This will usually be administered once daily.  
  The invention further provides a method of combatting bacterial infection in an animal which comprises administering to the animal (preferably parenterally or perorally) an effective amount of one of the new compounds, either alone, as a pharmaceutical composition according to the invention, or as a medicament in dosage unit form according to the invention.  
  Indications envisaged in human medicine are especially general infections, and infections of the efferent urinary tract, caused by Gram-positive and Gramnegative bacteria and by mycoplasma, and in veterinary medicine are general infections caused by Gramnegative and Gram-positive bacteria and my mycoplasma. infections of the respiratory passages in poultry, especially in chicks, and mastitis of cows, may be mentioned particularly The new compounds can, as has already been mentioned, also be employed as a feedstuff additive, predominantly in raising young animals, especially chicks and fatstock.  
  The preparations can be administered in the feedstuff, special feedstuff preparations and feedstuff concentrates, but also via the drinking water.  
  The invention therefore also provides animal feedstuffs and feedstuff concentrates containing at least one of the new compounds of general formula (1).  
  The administration of the new compounds together with the feedstuff or feedstuff preparations and/or with the drinking water makes it possible to prevent or treat infections by both Gram-negative and Gram-positive bacteria and mycoplasmagand can furthermore contribute to better utilization of the feedstuff. As examples of frequently occurring veterinary illnesses which cause considerable economic damage and which can be prevented or treated by adminstering the new compounds in the feedstuff or in the drinking water, there may be mentioned, in addition to general infections, infection of the air sac in chicks, and mastitis in cows.  
 What is claimed is:  
  1. An antibacterial composition which comprises an antibacterially effective amount of a compound of the formula OOY CH -H or a pharmaceutically acceptable non-toxic salt thereof wherein Y is hydrogen, an alkali metal cation or the cation R -NH and each of R and R is NHT 0-11 wherein R is alkyl of l to 4 carbon atoms or hydroxyalkyl of l to 4 carbon atoms, in combination with a pharmaceutically acceptable non-toxic inert diluent or carrier.  
  2. An antibacterial composition according to claim 1 wherein the alkyl group and the hydroxyalkyl group have 1 or 2 carbon atoms.  
  3. An antibacterial composition according to claim 1 wherein R is alkyl of l to 4 carbon atoms.  
  4. An antibacterial composition according to claim 1 wherein R is alkyl of l or 2 carbon atoms.  
  5. An antibacterial composition according to claim 1 wherein R is hydroxyalkyl of 1 to 4 carbon atoms.  
  6. An antibacterial composition according to claim 1 wherein R is hydroxyalkyl of 1 or 2 carbon atoms.  
  7. An antibacterial composition according to claim 1 wherein Y is hydrogen.  
  8. An antibacterial composition according to claim 1 wherein Y is sodium or potassium.  
  9. An antibacterial composition according to claim 1 in oral administration form.  
  10. An antibacterial composition according to claim 1 in subcutaneous administration form.  
 11. An antibacterial composition according to claim 1 wherein the compound is l cm-uamwcu K 12. An antibacterial composition according to claim 1 wherein the compound is 13. An antibacterial composition according to claim .1 wherein the compound is N OONa 14. An antibacterial composition according to claim 1 wherein the compound is 15. An antibacterial composition according to claim 1 wherein the compound is i WCOOIla Xl CIF=N-&#34;NH-COCH HT 16. An antibacterial composition according to claim 1 wherein the compound is 17. A method of treating bacterial infections in humans and animals which comprises administering to said human or animal an antibacterially effective amount of a compound of the formula or a pharmaceutically acceptable non-toxic salt thereof wherein Y is hydrogen, an alkali metal cation or the cation R -NH and each of R and R is R is alkyl of l to 4 carbon atoms or hydroxyalkyl of l to 4 carbon atoms, in combination with a pharmaceutically acceptable non-toxic inert diluent or carrier.  
  18. A method according to claim 17 wherein the alkyl group and the hydroxyalkyl group have 1 or 2 carbon atoms.  
  19. A method according to claim 17 wherein R is alkyl of l to 4 carbon atoms.  
  20. A method according to claim 17 wherein R is alkyl of l or 2 carbon atoms.  
  21. A method according to claim 17 wherein R is hydroxyalkyl of l to 4 carbon atoms.  
  22. A method according to claim 1 1 wherein R is hydroxyalkyl of l or 2 carbon atoms.  
  23. A method according to claim 17 wherein Y is hydrogen.  
  24. A method according to claim 17 wherein Y is sodium or potassium cation.  
  25. A method according to claim 17 wherein the administration is oral.  
  26. A method according to claim 17 wherein the administration is by subcutaneous injection.  
  27. The method according to claim 17 wherein the compound is 28. The method according to claim 17 wherein the compound is CCONa 29. The method according to claim 17 wherein the compound is 30. The method according to claim 17 wherein the compound is 31. The method according to claim 17 wherein the compound is 32. The method according to claim 17 wherein the UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3, 1, 9 Dated p i 9, 975  
 Inventor(s) FlOIill Seng et a1.  
  It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:  
 On the Title page, in item &#34;P 2O 15 676&#34; should read C Baigncd and Eacalcd this second Day Of September 1975 [SEAL] Arrest:  
 RUTH C. MASON C. MARSHALL DANN Arresting Officer (ummissimur ofParenrs and Trademarks FORM PO-1050 (10-69) uscoMM-Dc 60376-P69 U S. GOVERNMENT PRINTING OFFICE: 869- 930