IL45073A - 3-(5-nitro-imidazol-2-yl)pyrazoles - Google Patents

Abstract

Cpds. of formula (I): where R = 1-3C alkyl, 1-3C haloalkyl or 1-3C hydroxyalkyl; R1 = -NR3R4 or -N=CR5-X; R2 = H, -CN, -COOH or -CONHR6; R3, R4 = same or different H, 1-3C alkyl, 1-4C alkanoyl or -SO2N(CH3)2; R5 = H or 1-3C alkyl; X = -OR7, phenyl, -CH=CH-CH3, -CH=CH-C6H5, -CCl3, 2-furyl, 3-pyridyl, 3,4-methylenedioxyphenyl, -NH-NH-1-4C alkyl or -NR8R9; R6 = H or 1-4C alkanoyl; R7 = 1-8C alkyl, 2-5C alkenyl or 1-3C haloalkyl; R8, R9 = same or different 1-3C alkyl. Specif. claimed are (I) where R2 = CN and (a) R=CH3 and R1 = -NH2, -N=CH-OCH3 and -N=CH-OC4H9 and (B) R = C2H5 and R1 = -NH2. (I) are useful as antibacterials, antifungals and antiprotozoals active against Escherichia coli, Pasteurella multocida, Salmonella Typhimurium, Treponema hyodysentereae and coccidiosis etc. [FR2240009A1]

Description

FORMULA II ! ! wherein R is Ci-C3 alkyl or hydrox ( Ci -C3 ) alkyl with malono- nitrile in the presence of an acid acceptor to provide a compound of Formula I wherein R is Ci-C3 alkyl or hydroxy (Ci-C3) alkyl, R1 is -NH2 and R2 is -CN, and (A) when R is to be halo (C1-C3) alkyl reacting a compound of Formula I wherein R is hydroxy (C1-C3) alkyl, R1 is -NH2 and R2 is -CN with a halogenating agent; and (B) when R2 optionally is to be other than -CN hydrolyzing substituents a compound of Formula I wherein R is any of the xx,±3nxx defined above, R1 is -NH2 and R2 is -CN to form the corresponding compound wherein R2 is an amide group or an acid group and, if desired, acylating the resulting amide or decarboxy lating the resulting acid; and (c) when R1 optionally is to be other than -NH2 , subjecting a compound of Formula I wherein R and R2 are any of the values defined above to (l) acylation, (2) alkylation, (3) condensation- -( ) with an orthoester to form a corresponding imidate or (b) with an aldehyde to form the corresponding Schiff base, or (h) any combination of steps 1, 2 and 3 thereof, in any order.

A great deal of research has been conducted to develop agents for the control of bacterial and protozoal infections of poultry, swine and cattle. Thus, compounds and methods for controlling Es cher i chia coli , Pasteurella multocida , Salmonella typhimurium, and coccidiosis in chickens, as well as Treponema hyodys enter iae in swine have been the subject of extended research for many years. 2. Description of the Prior Art In the prior art, Belgian Patent No. 751,^53 (June k , 1970), teaches substituted 5-nitrofuran compounds, including 5-ami o- k -cyano-l-methyl-3-( -nitro-2-furyl)pyrazole, methods for preparation of the compounds, and medicaments containing the substituted nitrofurans as the active ingredient. The compounds are taught as possessing activity as antimicrobial, anthelmintic and antiprotozoal agents, and as also being active as c occ idi os tat s , trypanocides , and antimalarial agents.

Also in the prior art is Belgian Patent No. 782,851 (August 28, 197 ), which teaches 2- ( 5 - i t o -2- imi da z oly1 ) -pyrimidines, and methods for their preparation. The compounds are taught as being active as tr ichomonacides , bactericides, and antifungals, as well as being useful for the treatment of vaginal infections.

Tomcuf cik _et al. , U.S. 3; 52,03ί+ (June 2 h , I969), teach substituted 2 - ( 1 , 3 , thiadia z ol- 2- 1 ) - k - ( 5 ) - n i t r o-imidazoles and the preparation thereof. The compounds are taught as being useful as antibacterials , amoebicides, tricho-monacides, and coccidi ostats .

Papaioannou, U.S.. 3,682,9^-2 (August 8, 1972), teaches methods of preparing 2 - ( 2 -ami o- 1 , 3 th i adia z ol- 5- 1 ) - 1-s ub s ti tuted- 5 - ni tr oimida zole s . The products are taught as being useful for the control of bacterial, parasitic, and protozoal infections in poultry and animals. In particular, the compounds are said to be effective against Trichomonas )·, o- on ns ¬ ty antimicrobial properties and to be used in the same manner as described in the above two Howarth _et a_i. patents.

Sarett et al. , U.S. 3,719,759 (March 6, 1973), s directed to anti rotozoal compositions containing nitro- imidazoles. The nitroimidazole compounds taught include 1- s ub s t i tut ed -2 - ary 1 - 5 - i tr oi mi daz ole s , wherein aryl represents phenyl or naphthyl, substituted or uns ub s t i tut e d , and the 1 - s ub s t i tuent can be lower alkyl. The compounds are taught as being effective against protozoal infections such as hi s t omoni a s i ε , trichomoniasis, amoebiasis, and trypanosomiasis; as well as against helminths such as Heterakis and Ascarid species; bacteria such as Sa lmone 1 la sp., Streptococcus sp. , and Es che i chi a c oli . ; and pleuro pneumonia - like organisms (PPLO) In addition, Sarett et al. , U.S. 3,399,211 (August 2 , I968) teach methods for preparing the compounds utilized in the anti rotozoal compositions of U.S. 3,719,759, discussed supra.

Another prior art reference is Japanese Patent No. 7339935, also identified by Derwent No. 7^H5U, directed to 3 - ( -ni tr o-2 -furyl ) pyrazole derivatives, taught as being useful as fungicides, antiseptics, and food additives.

The compounds described in the prior art set forth above differ si nificantly structur ewise from those of the instant application.

In the substituent groups of Formula I, Ci-C8 alkyl represents a straight or branched chain saturated hydrocarbon radical such as methyl, ethyl, _n-propyl, isopropyl, n-butyl, _se_c. -butyl, isobutyl, _t-butyl, _n-amyl, isoamyl, s ec. -amy 1 , t-amyl, _n-hexyl, isohexyl, sec . -hexyl , _n-heptyl, isoheptyl, n-octyl, or isooctyl, and the like.

C2-C5 alkenyl represents a straight or branched chain unsaturated hydrocarbon such as vinyl, allyl, crotyl, methallyl, 1- pentenyl, 1 -methylbut eny1 , 2-pentenyl, and the like.

Halo( Ci -C3 ) alkyl represents a halogenated straight or branched chain saturated hydrocarbon such as halomethyl, 2-haloethyl, 3-halopropyl, 2 -ha lopropy1 , and the like, wherein halo is bromo, chloro, iodo, or fluoro.

Hydroxy ( Co. -C3 ) alky1 represents hydroxymethyl , β-hydroxy-ethyl, and 7-hydroxypropyl.

When practicing the process described above, typical suitable acid acceptors include organic amines such as methyl-amine, ethylamine, triethylamine and the like.

The compounds coming within the scope of Formula I, supra , have demonstrated in vitro activity against the following group of microorganisms, many of which are important animal pathogens : Escherichia coli Salmonella dub lin Arizona paracolon Ps eudomonas -6A- X- 821+ Pasteurella multocida (bovine isolate) (avian isolate) (swine isolate) Pasteurella heraolytica Streptococcus (Mastitis) Staphylococcus (Mastitis) Group E st eptococcus Spherophorus necrophorus B ordet e lla b r onchi s ept i ca Erysipelothrix rhusiopathiae Mycoplasma galli septic urn Mycoplasma synoviae Mycoplasma hyopneumoniae Mycoplasma hyosynoviae Mycoplasma hyorhinis T-strain Mycoplasma (bovine) Bacteroides frag i lis The novel compounds coming within the scope of the generic formula, supra, have also been found to possess in vivo activity. Thus, they have been found useful in controlling a number of infections in animals, such as Escherichia coli in poultry, Pasteurella multocida in chickens, turkeys, and mice, and Salmonella typhimurium in chickens.

The control of these organisms is accomplished by administering an effective amount of the compound to the poultry or mice either by injection, or orally such as in the feed. The compounds have also been found effective as growth promoters ng s e , These authors teach the use of an oxidizing system comprising an alkali metal periodate and osmium tetroxide in a suitable aqueous solvent medium, preferably water and 1 , 2-dimethoxy- ethane, at a temperature of from about 20 to 35°C., for a period of about 10 to 20 hours.

The l-methyl-5-nitroimidazole-2-carb oxaldehyde obtained by either of the two oxidation processes set forth above is the foundation stone, the basic starting material, for the preparation of the novel compounds disclosed in this invention .

The next step in the preparation of the novel compounds involves allowing the 1-methyl- 5-nitroimidazole-2- carb oxaldehyde , prepared as described supra, to react with a substituted hydrazine of the formula H2N-NHR, wherein R represents 0χ-03 alkyl or hydroxy( Cx- C3) alkyl . The reaction is carried out in a suitable solvent such as chloroform, at reflux temperature, to yield a 1-methy 1- 5- itr oimidazole -2- carb oxaldehyde alkyl or hydroxy alkyl hydrazone. Suitable substituted hydrazines for use in this reaction include methyl hydrazine, ethyl hydrazine, j -propyl hydrazine, isopropyl hydrazine, 2-hydr oxyethyl hydrazine, and the like. The reaction conditions are the same for all the hydrazines. Thus, for example, when methyl hydrazine is allowed to react with l-methyl-5-nitroimidazole-2-carboxaldehyde in chloroform solvent, there is obtained 1-methyl- 5-nit oimidazole-2- carb oxaldehyde methyl hydrazone.

The hydrazones formed in this manner are in turn allowed to react with N-bromosuccinimide at about room temperature in a suitable solvent, such as chloroform, to yield 1-methyl- 5- itroimidazole-2-carb onyl bromide alkyl or hydroxy-alkyl hydrazones. The reaction with N-bromosuccinimide is applicable to any of the substituted hydrazones to yield the bromo-substituted hydrazones. As a specific example, when 1-methyl- 5-nitr oimidazole-2-carb oxaldehyde methyl hydrazone is allowed to react with N-bromosuccinimide at room temperature in chloroform solvent, there is obtained 1-methyl- 5-nitro-imidazole-2-carbonyl bromide methyl hydrazone.

This bromo-substituted hydrazone is unstable and has vesicant and lachrimatory properties. It is therefore used immediately without isolation or extensive purification. The bromo-substituted hydrazone is suspended in a suitable solvent, for example absolute methanol, and malon onitr ile is added thereto. To the mixture thus formed is added t r iethylamine dissolved in absolute methanol, while the temperature of the reaction mixture is maintained at about 10-20°C by suitable cooling means. This reaction is slightly exothermic and some cooling is required to maintain the desired temperature. As the reaction proceeds, the initial yellow suspension dissolves and is replaced by another suspension during a period of about 1 to about 2 hours. The solid material in this second suspension is the desired product, and is filtered off, washed with methanol and then with water, and dried. For example, when 1-methyl- 5- nitr oimidazole-2- carb onyl bromide methyl hydrazone is used, this solid material is identified by elemental analyses and NMR spectrum as 5-amino-l-methyl-3-( 1-methyl- 5- nitr 0-2- imidazolyl) pyrazole- - carb onitr ile . The homologous 5-amino-l-( alkyl or hydr oxy- alkyl ) -3 - ( 1-methyl- 5-nitro-2-iraidazolyl)pyrazole-l+-carbonitriles can be prepared from the corresponding bromo-subst ituted alkyl or hydr oxy-alkyl hydrazones by following the same general procedure for reaction with malononi t i le .

The preparation of a 5-amino-l- ( haloalkyl ) -3- ( 1-methyl-5-nitr o-2-imidazolyl)pyrazole-^-carbonitrile is accomplished by reacting the corresponding hydroxy-alkyl compound with a halogenating agent such as phosphorus trichloride, phosphorus tribromide, phosphorus trifluoride, thionyl chloride and the like. Thus, 5-amino-l-( β-hydroxy-ethyl)-3-(l-methyl-5-nitro-2-imidazolyl) pyrazole- - carb onitrile, is allowed to react with thionyl chloride in an inert solvent, such as benzene, in the presence of a small amount of dimethyl-formamide to yield 5-a∞ino- 1- ( p- chlor oe thy l) -3- ( 1-methyl- 5-nitro-2-imida zolyl)pyrazole- -carb onitrile.

The 5-amino group of the carbonitriles synthesized as disclosed hereinabove readily lends itself to the preparation of derivatives. Thus, the 5-amino substituent readily reacts with an alkanoyl halide in the presence of a tertiary amine using an inert solvent such as dimethylformamide .

Suitable alkanoyl halides include acetyl chloride, propionyl chloride, butyryl chloride and the like. Suitable tertiary amines include pyridine, triethylamine and the like. The reaction is exothermic, and the products obtained depend on control of the reaction during the addition of the alkanoyl halide to the 5-amino compound in dimethylformamide as solvent. Thus, illustratively, acetyl chloride is added slowly to a -12- X-382^ mixture of 5-amino-l-ra ethyl-3- ( 1-methyl- 5-nitr o-2- imidazolyl ) -pyrazole-l4--carbonitrile and dimethylformamide , with little or no control of the exothermic reaction which occurs, followed by refluxing the reaction mixture for about two hours. The reaction product mixture is worked up by cooling and adding ice water thereto. The precipitate that forms is filtered off, recrystalli zed , and identified by elemental analyses as N- -cyano- 1-methy1-3- ( 1-meth l- 5- nitro- 2- imidazolyl) -pyrazol-5-yl_^cetamide .

Using the same reactants, but changing the procedure, yields different substituents on the 5-amino group. Thus, the acetyl chloride is added at such a rate that the exothermic reaction is avoided, followed by refluxing the reaction product mixture for about two hours. The reaction product mixture is worked up by adding water and filtering off the solid which precipitates. The solid is subjected to thin-layer chromatography. Two spots appear, one slow and one fast. The slow spot is identified as the monoacetyl compound described, supra. The fast spot is crystallized by fractional crystallization using a suitable solvent, such as commercial absolute ethanol. This product is identified by elemental analyses as - Γ_( dimethyl amino) methylene_j4mino7- 1-methy1-3- ( 1-methyl- 5- nitro- 2- imidazolyl) pyrazole-^-carbonitrile . This product comes about from the interreaction of the solvent dimethylformamide with the two principal reactants, the acetyl chloride and the compound In addition, derivatives of the 5-amino substituent can readily be obtained by reaction with organic acid anhydrides such as acetic anhydride, propionic anhydride, trifluoroacetic e l) or different.

Another method of preparing alkyl derivatives of the 5-amino group involves heating to refluxing a mixture of a 5-amino carbonitrile , concentrated sulfuric acid, and absolute alkanol for a period of time. At the end of the heating period, usually about 2 hours, the reaction product mixture is added to water, the mixture cooled and made basic with, for example, concentrated ammonium hydroxide. The dark solid which precipitates is filtered off. The solid is chr omatographed over silica gel using a suitable solvent such as ethyl acetate. There are obtained two products, identified as the 5-dialkylamino and the 5-monoalkylamino compounds, respectively. Thus, when 5-amino-l-methyl-3- ( 1-methyl- 5- nitro-2- imidazolyl ) pyr zole- k- carbonitrile is allowed to react with concentrated sulfuric acid and absolute ethanol as set forth above, the products obtained are identified as 5- ( ethylami o) -1 -methyl- 3- ( 1-methyl- 5-nitro-2- imidazolyl) - pyrazole, and 5- ( diethylamino) -l-methyl-3- ( 1-methyl- 5- nit ro- 2-imidazolyl)pyrazole , respectively. The process by which these compounds are prepared also serves to completely remove the carbonitrile group.

Formimidates , acetimidates , propionimidates, or butyr imidates are obtained from the 5-amino group by reaction with ortho esters such as triethyl orthoacetate , tri( n-propyl) orthoacetat e , triethyl orthopr opionate , triethyl orthoformate , trimethyl orthobutyrate , tri(n-butyl) orthoformate , or the like, in the presence of a small amount of acetic anhydride. Thus, for example, when the reactants are 5-amino-l-methyl-3- -15- X-382½. ( 1-methy1- 5- itr o- 2- imidazolyl) pyrazole carb onitrile , triethyl orthoacetate , and a small amount of acetic anhydride, the product obtained is ethyl Ν- ¾·- cyano- l-methyl-3- ( 1-methyl- 5-nitr o-2-imidazolyl) yrazol- 5-yl .cet imidate . Other ortho esters which can be reacted with any of the 5-aminopyrazole- -carbonitriles include triallyl orthoformate , tri( 2-chlor oethyl) orthoformate, tributyl orthoacetate, triisobutyl orthoformate, tri(2-ethyl-hexyl) orthoformate, triallyl orthoacetate, tributyl ortho-propionate, tripropyl orthobutyrate , and the like.

An unexpected and unique variation occurs in the reaction between tricrotyl orthoformate and 5-amino-l-methyl-3- ( 1-methyl- 5 -nitro- 2- imidazolyl) pyrazole - k - carb onitrile in the presence of acetic anhydride. There is obtained a product having a melting point of about l87-190°C., and identified by elemental analyses and NMR spectrum as 5-( -butenylideneamino) -1-methyl-3- ( 1-methyl- 5- nitro-2- imidazolyl) pyra z ole - k - carb onitrile Other alkylideneamino or arylideneamino derivatives are obtained by heating an alkyl or aryl aldehyde with 5-amino-1-methyl-3-(l-methyl-5-nitro-2-imidazolyl)pyrazole-¾-- carb onitrile in the presence of acetic anhydride for a period of time. At the end of the heating period, usually about 16 to about 2k hours, the reaction product mixture is cooled, and the solid which separates is filtered off. The solid is recrystallized from a suitable solvent, for example, dimethylformamide , to yield the product, which is identified by elemental analysis and NMR spectrum. Thus, a mixture of 5-a∞ino-l-methyl-3-( 1-methyl- 5 -nitro -2- imidazolyl) pyraz ole- k - carb onitrile , fur ural, and acetic anhydride is refluxed overnight. The reaction product id which separates is filtered off, eth Iformamide to yield a crystalline int of about 238-2l+2°C. This product analyses and NMR spectrum as 5-(2- l-3-( 1-meth l- 5 -nitro- 2- imidazolyl ) - present in these novel compounds, *' roup, can also be utilized to carbonitrile group can be readily mido grouping by allowing the concentrated sulfuric acid in tively, when 5-amino-l-methyl-3- lyl ) pyrazole- k - carb onitrile is ntrated sulfuric acid in absolute rature for about one hour, there l-3-( 1-methyl-5-nitro-2- imidazolyl) - e can also be completely removed le under acidic conditions, for f concentrated sulfuric acid and t 2 hours. Thus, when 5-araino- o- 2- imidazolyl) pyrazole-U- carb o- qual volumes of concentrated sulfuric for 2k hours, there is obtained l- 5-nitr o-2- imidazolyl ) pyrazole . -carbonitrile group to the carboxyl er reaction conditions present when l- 5-nitr o- 2- imidazolyl )pyrazole-4- ic anhydride and a about 8 hours at ion of the 5-amino group occurs, and 1-methy1- 5- itr o- 2- is identified by amples more fully ounds of this d to limit the le) of 2-methyl-5- zene, there was added of dimethyl sulfate e was allowed to there was added arbonate in 6 ml. of ur and filtered. The were separated and l. portions of with the original nesium sulfate. The te was concentrated point of about 135- 5- nitroimidazole .

X- 82 0-liter round-bottom flask equipped with er, reflux condenser, and dropping funnel, 705 g. (5·0 mole) of 1,2-dimethyl- 5-nitro- 75 liters of absolute ethanol. Solution was h stirring. To the solution thus prepared, ) of benzaldehyde was added rapidly. To this dly added 150 g. of sodium dissolved in 2.5 1 *■ addition being carried out at room temperature. ture was stirred and heated to about 70°C. about 90 minutes. At a temperature of ko°C*, d to dark brown. At the end of the 90 minutes, duct mixture was allowed to cool for 90 minutes reaction vessel in an ice water bath. A ed. The brown mixture was filtered. The uct was washed four times with a mixture of ethanol, in a 1:1:1 ratio using one liter of e crystalline product was air dried at 100°C. d as 1-methyl- -nitro-2-styrylimidazole . It int of about 191-192°C. Weight = 82 g. hree-neck, round-bottom, 5-liter flask equipped nd gas introduction tube, there was prepared a g. (2.0 mole) of 1-methyl- - itro- 2- styryl- ixture of 2.5 liters of methanol, 1.5 liters ne, and 200 ml. of water. The flask was om temperature by means of a water bath. A and oxygen (3 percent 03 at 1.1 liters per ed through the solution. The formation of the tored at intervals by gas-liquid chromatography aphy (TLC). The total ozonolysis end of which time the solution of sodium iodide in 2 liters acid was stirred in a round- e ozonolysis solution was ng the temperature below h o a C .

After stirring the mixture n of sodium metab isulfite (192 ded to remove the free iodine n to turn yellow. The mixture our. The mixture was then s discarded. The filtrate was one-third its volume and he addition of solid sodium s required about 300 g. of the e was extracted with k x TOO The combined ethyl acetate ous magnesium sulfate for about t was filtered off and the to yield a sticky solid. This liters of n-hexane and the inutes. The residual brown . On cooling, the filtrate were filtered off and dried was repeatedly used to reflux bove in the same manner as before e carried out. There was obtaine - a total of about 1^6 g. of product having a melting point of about 8l-83°C, and identified as 1-methyl- 5-nitroimidazole-2-carb oxaldehyde .

Example _1 5-Amino- 1-meth 1-3 -( l-meth 1- 5-nitro-2- im dazolyl) pyra ole- -carbonitrile A mixture of 23-5 g. (0.152 mole) of l-methyl-5-nitroimidazole-2-carboxaldehyde and Τ·0 g. (0.152 mole) of methyl hydrazine in 300 ml. of chloroform was boiled under reflux for about 2 hours. The reaction product mixture was evaporated to dryness to yield a bright yellow solid weighing about 25-5 g. The small sample crystallized from ethanol had a melting point of about 175°C. It was identified as 1-methyl-5-nitroimidazole-2-carboxaldehyde methyl hydrazone.

To a stirred solution of 23.5 g. (0.128 mole) of 1-methyl- 5-nitroimidazole-2-carboxaldehyde methyl hydrazone in 200 ml. of chloroform, there was added slowly, at room temperature, 22.9 g- (0.128 mole) of N-bromosuccinimide . The reaction was slightly exothermic and the internal temperature was kept below 30°C. by occasional external cooling. After stirring for about 2 hours, the solvent chloroform was removed _i_n vacuo and the residue extracted with 5 x 100 ml. of hot carbon tetrachloride. The insoluble residue was discarded and the combined carbon tetrachloride extracts were concentrated to give a bright yellow solid, identified as 1-methyl- 5-nitr o-imidazole-2-carbonyl bromide methyl hydrazone. Thin-layer chromatography showed the compound was almost pure. Yield 31.0 g.

This compound is unstable and has vesicant and lach imat ory properties. It was used immediately in the next step of preparation.

The bromo hydrazone thus prepared, 3 -0 g. ( 0. Il8 mole) was suspended in 250 ml. of absolute methanol, and 7.80 g. (O.II8 mole) of redistilled malononitrile was added. To the mixture was added dropwise a solution of 12 g. (0.1l8 *· mole) of tri ethylamine in 5 ml. of methanol while maintaining the reaction mixture at a temperature of about 10-20°C. The reaction was slightly exothermic.

The initial yellow suspension dissolved and was replaced by another suspension during a period of about 1 to 2 hours. At the end of that time, the reaction product mixture was filtered and the solid material collected. The solid was washed with methanol, and then with water, and dried. This solid product had a melting point greater than 300°C> and weighed about 23 g. It was identified as 5-amino-l-methyl-3-(l-∞ethyl-5-nitro-2-imidazolyl)pyrazole-¾--carbonitrile The product was analytically pure as isolated.

Example _2 5-Amino-l-methyl-3-(l-methyl-5-nitro-2-imidazolyl)pyrazole-¾--carb oxamide To a mixture of 3 ml. of concentrated sulfuric acid and 2 ml. of absolute ethanol was added 1.0 g. of 5-amino-l-methyl-3- ( 1-methyl- - itro-2- imidazolyl) pyrazole- -carb onitrile The mixture was heated on the steam bath for about 1 hour and then 10 ml. of water was added and the reaction mixture was allowed to stand overnight at ambient room temperature. A -22- X- 82U precipitate formed which was filtered off. The precipitate was recrystallized from commercial absolute ethanol to yield a solid having a melting point of about 255-256°c. The solid was identified by elemental analyses and NMR spectrum as 5-ami o-l-methyl-3-( 1-methyl- 5-nitr o-2-imidazolyl) py zole-Λ-carb oxamide .

Example 3 5-Amino- l-methyl-3-( 1-methyl- 5-nitr o-2-imid zolyl) yrazole A mixture of 1.0 g. of 5-amino-l-methyl-3-( 1-methyl-5-nitro-2-imidazolyl)pyrazole-14--carbonitrile , 10 ml. of water, and 10 ml. of concentrated sulfuric acid was refluxed for 2. hours. The reaction product mixture was cooled and 50 ml. of ether added. The pH of the mixture was adjusted to about pH 8 with concentrated ammonium hydroxide. The ether layer was separated and filtered to give a gold colored solid. The solid was chromatographed over a silica gel column, using acetone as solvent. A faster moving impurity (m.p. >36o°C.) was first eluted. Subsequently, a yellow solid, melting at about 221-226°C, was isolated and identified by IR and NMR spectra as 5-amino-l-raethyl-3-( 1- methyl- 5-nitr o-2-im d zol l) pyrazole . Example k Ethyl N- --cyano-l-meth l-3-( 1-methyl- 5-nitro-2-imidazolyl) -pyrazol- -ylI/formimidate A mixture consisting of 1 ml. of acetic anhydride, 12 ml. of triethyl orthoformate , and 1 g. of 5-amino- 1-methyl-3- ( 1-methyl- 5- nitr o-2-imidazolyl) pyrazole-h-ca bonitrile was refluxed for about 5 hours. The reaction product mixture was allowed to cool to. room temperature overnight. The precipitate which formed was filtered off and recrystallized from commercial absolute ethanol to yield product having a melting point of about lkj-lk9°C., with decomposition. The product was identified by elemental analyses and NMR spectrum as ethyl N-/ -cya o-l-methy 1-3- ( 1-methyl- 5-nitr o-2- imidazolyl ) pyrazol- 5-yl__7- ormimidate .

Example 5 Ν-Γ - Cyano-1-methyl- 3- ( 1-methyl- 5- nitr o-2- imida zolyl) pyrazol-5-yl liacetamide A mixture of 25 ml. of acetic anhydride and 1 g. of 5-amino-l-methyl-3-( 1 -methyl- 5 - nitro- 2 -imida zolyl) pyrazole-^-carbonitrile was refluxed overnight. The reaction product mixture was concentrated in vacuo and the residue was recrystallized from commercial absolute ethanol. Brown needles were obtained having a melting point of about 1T8-18O°C. The product was identified by elemental analyses and NMR spectrum as N-/ --cya o- 1- meth 1-3- ( 1-me th 1- 5-nitr o- - imidazolyl) pyrazol- -yl -diacetamide .

Example 6 Methyl N-/3--cyano-l-methyl-3-( l-methyl- 5 - nitro-2 - imida zolyl )-pyrazol- -ylZf>orniimi(la'ke A mixture of 1 g. of 5-amino-l-methyl-3-( l-methyl-5-nitro-2-imidazolyl)pyrazole-i(--carbonitrile, 12 ml. of trimethyl orthoformate , and 1 ml. of acetic anhydride was refluxed for about 2h hours. The reaction product mixture was filtered.

The solid material which was filtered off was recrystallized from commercial absolute ethanol to yield product having a melting point of about l8 -l85°C. The product was identified - - - by elemental analyses and NMR spectrum as methyl N- --cyano-l-methyl-jj- ( 1-methyl- 5- nitro-2- imidazolyl) pyrazol- formimidate .

Example 7 Butyl N- --cyano- 1-methy1-3- ( l-me thy1- 5 - n i tr o-2 - imida zolyl ) -pyrazol- 5-yl ¾Ormimidate A mixture of 1 g. of 5-amino-l-methyl-3- ( 1-methyl- 5 nitro-2-imidazolyl)pyrazole-i4--carbonitrile , 12 ml. of tri-n-butyl orthoformate, and 1 ml. of acetic anhydride was refluxed for 2h hours and then cooled. The reaction product mixture was filtered. The solid material which was isolated was recrystal-lized from commercial absolute ethanol to yield a product having a melting point of about 120-121°C. The product was identified by elemental analyses and NMR spectrum as butyl H-Th- cya o- l-methy1-3- (l-methyl-5-nitro-2-imidazolyl) pyrazol- Example 8 Ν- ¾- Cyano- l-me thyl-3-(l-methyl- -nitro-2- imida zolyl) pyrazol- -yl -cetamide A mixture of 1 g. of 5-amino-l-methyl-3-( l-methyl-5-nitro- 2- imida zolyl) pyraz ole- -carbonitr ile , 8 ml. of dimethyl-formamide, and 8 ml. of dry pyridine was prepared. To this mixture at room temperature was added very slowly 0.9 g« of acetyl chloride. After the addition was complete, the reaction mixture was refluxed for about 2 hours, cooled, and 25 ml. of ice water was added. The precipitate which formed was filtered off. It was r ecrystalli zed from commercial absolute ethanol to yield a product having a melting point of about 241- 2^2 °C . dec . The product was identified by elemental analyses as N- ¾-cyano-l-methyl- 3 - ( 1-methyl- 5-nitro- 2 -imidazolyl) pyrazol- 5-ylI/kcetamide .

Example nitro- 2-imidazolyl) pyrazole- - carb onitr ile The procedure of Example 8 was repeated using the same quantities of reactants but the addition of the acetyl chloride was carried out much more slowly to avoid the exothermic reaction previously experienced. The reaction mixture was refluxed for about 2 hours. At the end of the reaction period, water was added to the reaction product mixture. The precipitate which formed was filtered off.

Acidification of the mother liquor gave no additional precipitate .

The solid was subjected to thin-layer chromatography which showed a slow spot and a fast spot. The slow spot was identified as the monoacetyl compound described in Example 8 . The fast spot was crystallized by fractional crystallization from commercial absolute ethanol to yield material having a melting point of about 231- 233°C. This material was identified by elemental analyses as -/7~( dime thy lamino) me thyle e~¾.mi o~7-l-methyl-3 - ( l-methyl- 5-nit o- 2-imidazolyl) pyrazole- -carbo-nitr ile .

Example 10 5 -Ami no- l-me thyl- - ( 1-methyl- 5 -nitro- 2 - imidazolyl)pyrazole-U-carboxylic acid and ( acetyl carbamoyl ) - l-methyl- 3 - ( 1-methyl- 3- nitro- 2 -imidazolyl) pyrazol- 5-ylIiiace"tarai<3-e <« A mixture of 1 g. of 5-ami o- l-methyl-3- ( 1-methyl-5-nitro-2-imidazolyl)pyrazole- -carbonitrile , 0.^· g. of p-t oluenesulfonic acid monohydrate, and 30 ml. of acetic anhydride was refluxed for about 2 hours. The reaction product mixture was concentrated V vacuo and the partially crystalline residue was recrystall.i zed from commercial absolute ethanol to give two products, A and B.

Product A, which was insoluble in the commercial absolute ethanol, was re crystalli zed from dimethyIformamide to yield material having a melting point of about 250-251°C, dec. This product was identified by elemental analyses and NMR and IR spectra as 5-amino- l-methyl-3 -( 1- methyl- 5-nitr o-2-imidazolyl)pyrazole- -carboxylic acid.

Product B was recrystalli zed again from commercial absolute ethanol to yield material having a melting point of about 200-202°C. The material was identified by elemental analyses as N- -( acetylcarbamoy1 ) -l-methyl-3-( l-methyl-5-nitro-2-imidazolyl)pyrazol- 5-yl[]liacetamide · Example 11 5-( Ethylamino) -l-methyl-3-( l-methyl- -nitr o- 2- imidazolyl) -pyrazole and 5- ( Diethylamino) -l-meth l-3-(l-methyl-$-nitro-2-im da . -'.yl -pyrazole A mixture of 10 g. of 5-amino- l-methyl-3- ( 1-methyl-5-nitro-2-imidazolyl) pyrazole-^- carbonitrile , 30 ml. of concentrated sulfuric acid, and 20 ml. of absolute ethanol was refluxed for about 1 hour. To the reaction product mixture thus obtained was added 100 ml. of water and the dark mixture es )- - t .0 g. of 5-ami no- 1-methy 1-3- ( 1-me thyl- azole-14--carbonitrile , 12 ml. of nd 1 ml. of acetic anhydride was urs and then allowed to cool overnight. ture was filtered and the solid was ercial absolute ethanol to yield product f about l67-l68°C. The product was analyses and NMR spectrum as ethyl -me thyl- 5-nitro-2-imidazolyl) yrazol- same general procedure as set forth appropriate starting materials, the pounds were prepared: -cyano-l-methyl-3- ( 1-methyl- 5-nitro- lZ¾>ormimi(ia'te > having a melting point ntified by elemental analyses and hyl N-/ -cyano-l-methyl-3-( 1-raethyl- 5- ol- , having a melting . Identified by elemental analyses. l-methyl-5- ol- , having a melting . Identified by elemental analyses. yl N- ¼--cyano-l-methyl-3-( l-methyl-5- ol- e , having a melting Identified by elemental analyses.

Example ik- 5 -Ami no -1- ( -hydr oxyethyl) -3 - ( 1 -meth l- 5-nitr o-2-imidazolyl) -pyrazole-¾--carbonitrile A mixture of 15-5 g- (0.1 mole) of l-methyl-5-nitro-2-imidazolecarboxaldehyde and 7.6 g. (0.1 mole) of 2-hydroxy-ethyl hydrazine in 300 ml. of chloroform was refluxed for about 2 hours. The reaction product mixture was evaporated to dryness under vacuum to yield product weighing 21.3 g- a d having a melting point of about 122-129°C. A small sample recrystallized from water had a melting point of about 136-11-0°C.

To a stirred solution of 21.3 g- (0.1 mole) of 1-methyl-5-nitro-2-imidazolecarboxaldehyde 2-hydr oxyethyl h drazone (prepared above) in 200 ml. of chloroform, there was added, in small portions, while keeping the temperature below 30°C. , 17.8 g. (0.1 mole) of W-br omosuccinimide . The reaction mixture was stirred for about 3 hours at about 25°C. The reaction product mixture was concentrated iri vacuo and the residue was extracted eight times with 500 ml. portions of hot carbon tetrachloride. The combined carbon tetrachloride extracts were concentrated in vacuo to yield a bright yellow solid, identified as 1-methyl- 5-nitroimidazole-2-carbonyl bromide 2-hydr oxyethyl hydrazone. The product had a melting point of about 85-9 °C., and weighed 20.7 g.

The bromo hydrazone thus prepared, 20.7 g. (O.OTl mole), was suspended in 150 ml. of anhydrous methanol and k. g. (O.07I mole) of malononitrile was added. To the mixture was added dropwise a solution of 7·3 g- of triethylamine in 15 ml. of absolute methanol, while maintaining the reaction The reaction product mixture was cooled, slurried with about 25 ml. of water and the mixture filtered to yield a yellow solid. Thin-layer chromatography of a sample of the yellow solid, using ethyl acetate-benzene in 1:1 ratio, showed one main yellow spot plus two faster moving trace impurities.

The product, 1-me thyl- 5- itr oimidazole- 2- carb oxaldehyde ethyl hydrazone, was used without further purification.

To a stirred mixture of 6.8 g. (0.0322 mole) of 1-methyl-5-nitroimidazole-2-carboxaldehyde ethyl hydrazone and 100 ml. of chloroform was added portionwise 5·8 g- (0.0322 mole) of N-bromosuccinimide , while keeping the temperature of the reaction mixture at less than 30°C. After stirring at ambient room temperature for about 3 hours, the reaction product mixture was concentrated in vacuo . The residue which was obtained was extracted three times with 250 ml. portions of hot carbon tetrachloride. The combined carbon tetrachloride extracts were concentrated iri vacuo to yield a yellow solid which weighed 6.3 g- This compound, 1-me thyl- 5- nitroimidazole- 2- carbonyl bromide ethyl hydrazone, was used without purification in the next step of the preparation.

The bromo hydrazone thus prepared, 6.3 g- (0.0228 mole), was suspended in 75 ml* of absolute methanol and 1.5 g. (0.0228 mole) of malononitrile was added portionwise. The temperature was maintained at about 10-20°C. using an ice water bath. After addition was complete, the reaction product mixture was stirred for about 1 hour at ambient room temperature. The reaction product mixture was filtered. The solid material which was recovered was recrystalli zed from dimethyl- ic A mixture of 1.0 g. of 5-amino-l-methyl-3-( 1-methyl-5-nitro-2-imidazolyl )pyrazole-i)-- carbonitr i le , 12 ml. of tricrotyl orthoformate , and 1 ml. of acetic anhydride was refluxed for about 2 h hours, cooled and filtered. The solid obtained thereby was recrystallized two times from commercial absolute ethanol to yield product having a melting point of about l87-190°C. The product was identified by elemental analyses and NMR spectrum as 5- ( 2-butenylideneamino ) -1-methyl-3-( 1-methyl- 5-nitro-2-iraidazolyl) pyrazole- -carbonit ile .

Example 19 5- ( -Fur furylide eamino) -1-me thy1-3- ( 1-meth l- nitro-2-imidazolyl)pyrazole-¾--carbonitrile A mixture of 2.0 g. of 5-amino-l-methyl-3-( 1-methyl-5-nitro-2- imidazolyl )pyrazole-¼-- carb onitrile , 2 ml. of acetic anhydride, and 25 ml. of furfural was refluxed overnight. The reaction product mixture was cooled and the precipitate which formed was filtered off. The solid thus obtained was recrystallized from dimethylformamide and washed with cold commercial absolute ethanol. The crystalline product, having a melting point of about 238-2^2 °C . , was identified by elemental analyses and NMR spectrum as 5-( -furfurylideneamino) -1-methyl-3- ( 1-me thy1- 5-nitro- 2- imidazol l) pyrazole- h - carb onitri le .

Following the same general procedure as set forth in Example 19 and using appropriate starting materials, the following additional compounds were prepared: A. 5-(3-Pyridylideneamino)-l-methyl-3-(l-methyl-5-nitro-2-imidazolyl) yrazole-^-carbonitrile, having a melting point of about 257-262°C. Identified by elemental analyses and NMR spectrum. , a mixture of dimethylformamide and water to yield product having a melting point of about 250-251°C. dec . The product was identified by elemental analyses as N ' - --cyano-l-methyl-3- ( 1-meth l - 5-nitroimidazol-2-yl) pyrazol-2-yl 7- , N-dimethy1-sulfamide .

Example 21 Ν-Γ - Cyano-l-methyl-3- ( l-methyl-5-nitroimidazol-2-yl)pyrazol-$-yl]jformimidi c acid, 2-methyl hydrazide A mixture of 1.0 g. of methyl N-/ --cyano-l-methyl-3-( 1-methyl- 5-nitro-2- imida zolyl) pyrazol- 5-yl ¾"ormimidate , 1 ml. of methyl hydrazine, and 10 ml. of ethanol was stirred at ambient room temperature for about 1 hour. The reaction product mixture was filtered. The solid which was recovered was recrystalli zed from ethanol to yield product in the form of yellow needles having a melting point of about 232-23^°C. The product was identified by WMR spectrum and elemental analyses as N-/ 1+-cyano-l-methyl-3-( 1-methyl- 5-nitroimidazol-2-yl)pyrazol-5-ylZ7formimidi c acid, 2-methyl hydrazide.

Example 22 -Ami o-l- (_n- propyl )-3-(l-methyl-5-nitro-2-imidazolyl)pyrazole-¾--carbonitrile A mixture of 5 g. (0.0322 mole) of l-methyl-5-nitro-imidazole-2-carboxaldehyde , 3-8 g. (32.2 mmol.) of propyl hydrazine oxalate, 100 ml. of chloroform, and 3.3 g- (32.2 mmol.) of tri ethylamine was refluxed for about 3· 5 hours. The reaction mixture was cooled and concentrated in vacuo . The residue was extracted three times with 250 ml. portions of hot chloroform. The chloroform extracts were combined and concentrated _i_n vacuo to yield 5·3 g. of crude ydrazone, identified as l-methyl-5-nitro-2-imidazolecarboxaldehyde n-propyl hydrazone, which was used without further purification.

To a stirred suspension of 5·3 g. (0.0251 mole) of the hydrazone prepared above in 100 ml. of chloroform, there was added k .6 g. (0.0251 mole) of N-br omosuccinimide . The addition was carried out portionwise, keeping the temperature *· of the mixture at below 30°C. by use of an ice water bath.

The reaction mixture was then stirred at room temperature for about 3 hours. The reaction product mixture was concentrated in vacuo to remove the solvents and the residue was extracted three times with 250 ml. portions of hot carbon tetrachloride. The carbon tetrachloride extracts were combined and concentrated in vacuo to leave behind a crude bromo hydrazone identified as l-methyl-5-nitroimidazole-2-carbonyl bromide n-propyl hydrazone. This crude hydrazone was used as is without further purification.

The bromo hydrazone thus prepared was suspended in 75 ml. of anhydrous methanol, and 1.6 g. (0.025 mole) of malononitrile was added as a solid in portions. To the mixture was added dropwise 3.8 g« of tr iethylamine while maintaining the temperature of the mixture at about 10 to 20°C. by use of an ice bath. The reaction product mixture was stirred for about 1 hour at ambient room temperature. The reaction product mixture was then filtered and the solid which was thus isolated was r ecrystalli zed from dimethylformamide to yield product having a melting point of about 215-2l6°C. The product was identified by NMR spectrum and elemental analyses as 5-amino-l- (_n- ropyl) -3-( l-methyl-5-nitro-2-imidazolyl) -pyrazole-^-carbonitrile .

Example 23 1-Methyl- 5-[Tj) methylenedi oxy )benzy lidene^mino^^- ( 1-methyl-5-ni roimidazol-2-yl)pyrazole- -carbonitrile A mixture of 1.0 g. of 5-amino- l-methyl-3- ( l-methyl-5-nitro-2-imidazolyl)pyrazole- -carbonitrile , 0.7 g. of piperonal, and 1 ml. of acetic anhydride in 15 ml. of dimethyl-formamide was refluxed overnight. The reaction product mixture was cooled, and the precipitate which formed was filtered off. This solid was recrystalii zed from dime thyIformamide to yield product having a melting point of about 173-175°C. The product was identified by elemental analyses and NMR spectrum as 1-methyl- 5-Γ 3 - ( 1-methyl-5-nitroimidazol-2-yl)pyrazole-'+-carbonitrile.

The novel compounds of this invention have important antimicrobial properties, in particular antibacterial, antifungal, and antiprotozoal activity, useful in veterinary medicine. The compounds are particularly valuable in the treatment of infection of poultry caused by Escherichia coli , Pasteurella multocida, and Salmonella typhimurium . The compounds are also active as growth promoters in chicks.

Thus, for the treatment of E. coli-caused infection in chicks, the administration in the feed of a compound coming within the scope of the generic formula, supra , at a rate of from about 50 g. to about 200 g. per ton of feed is effective.

In chickens and turkeys, control of infections caused by P. multocida is effected by administration of one of the novel compounds in the feed at a rate of about 100 g. per ton.

Also in chickens j control of infections caused by S. typhimur ium can be accomplished by the oral administration of one of the novel compounds at the rate of from about 25 to abou† I 100 g. of compound per ton of feed. Thus, infections in chickens and turkeys caused by E. coli , P. multocid , and S_. typhimur ium are controlled by the oral administration of one of the novel compounds in the feed at rates of from about 25 g. to about 200 g. per ton of feed. 1 i Control of the infections caused by _S. ty—p—h—i—m—ur—_i_.um- i in chickens can also be accomplished by administration of the novel compounds subcutaneously at a dosage of from about 3 mg · to about 6o mg . per kilogram of body weight.

Infections in mice caused by P. multocida can be controlled by administration by intraperitoneal injection of as little as 2 . 5 mg . of compound per kilogram of mouse body weight. P. multocida infections in mice can also be controlled by the oral administration of about 100 g. of compound per ton of feed. 5-Amino-l-methyl- 3 - ( 1-methy 1- -nitr o- 2-imidazolyl) -pyrazole-¾--carbonitrile, exemplary of the novel compounds of the genus, has been tested and found to be effective as a growth promoter when administered orally to chicks at the rate of about 50 g. per ton of feed.

The above-described antimicrobial activity of the i compounds has been demonstrated in a variety of tests as i described hereinbelow.

Experiment 1 The efficacy of the novel compounds against Pasteurella multocida infections in mice when administered by injection was studied.

Female Swiss mice weighing from 15 to about 20 g. were used. The test compound, 5-0 m . , was weighed out and dissolved in 0.5 ml- of dimethyl sulfoxide, and this solution was added to 9-5 ml* of sodium carb oxymethylcellulose suspension. Groups of five mice each were injected intraperit oneally with 0.1 ml. each of the above-prepared test composition. This preparation gave a test rate of 2.5 mg . per kilo of body weight of, mouse. The compounds were tested at 2.5, 5.0, or 7.5 mg . per kilo of body weight of mouse, and the test preparations for rates of 5·0 and 7·5 mg./kg. were prepared in a similar manner. Immediately after receiving the test compound, the mice were challenged using loglD dilutions of 10~4, 10"5, 10~s, and 10~7 of a l6 to 20-hour tryptose broth culture of Pasteurella multocida at 0.1 ml. per mouse subcutaneously.

Similar groups of nontreated control mice were also challenged, the challenge being administered subcutaneously. The groups of mice were observed daily for mortality and the total mortality in treated groups compared with that which occurred in the nonmedicated control groups. The results are set forth in Chart 1, which follows. Each test compound is identified by the number of the example describing its preparation .

In the chart, column 1 lists the test compound; column 2, the dosage of test compound in mg./kg. of mouse body weight; column , the dilution of the challenge organism; number of mice surviving the nge dilution to the number of challenge dilution, called Survival Ratio 1/10 0/10 8/10 in cks vided ion pended sub- d ged 1 ml. chick in the leg d for 6 days; the group was compared e chicks which are set forth in s are identified in t 1. the compounds tested mg./kg. ; and column Chart 2 ( Cont irtued) 3.75 5 3-75 10 7-5 1/10 Chart 2 (Co tinued) Surviva 1 Ratio 7.5 V 5 Infected Controls 9/50 Experiment J5 The oral activity of the novel compounds coming within the scope of the generic formula supra , against Pasteurella multocida , bovine strain, in mice as the host animal was demonstrated in the following manner.

White Swiss female mice weighing 15-20 g. each were used in the test. Seven groups of ten mice each were treated at a level of 100 g. of test compound per ton of feed. The animals received the medication in the feed for 2 days prior to challenge and for 7 days post challenge. Similar groups of mice on a ration without the antimicrobial agent were used as controls. Both the treated mice and the control groups of mice were challenged with log1Q dilutions ranging from 10~e through 10"8 of a 16 to 20-hour culture of Pasteurella multocida , bovine strain, at 0.1 ml. per mouse, administered subcutaneously . The mice were observed for 7 days, then the LD5o for the various treated groups of mice and the LDs0 for the control group of mice were calculated. By subtracting the LD5o of each of the treated groups from that of the control group of mice, there was obtained a figure called the protective index. The higher the protective index in value, the more active the compound was against the infection.

The results of this test for three of the most active compounds are shown in the following Chart 3· In the chart, column 1 lists the test compounds, identified in the same manner as set forth in Experiment 1; and column 2 lists the protective index.

Chart 3 Cpd . Protective Index 1 > 5-6 6 .3 T 3 Experiment _ The activity of certain of the nitroimidazoles against Salmonella typhimurium in chicks when administered in the feed was determined.

One-day-old chicks, divided into groups of 12, were given compounds 1, 6, or 7, in the feed at levels of 100 g. per ton, 50 g. per ton, 5 g. per ton, and 10 g. per ton icates Later se d for ved as nd ent, Chart k , the chart, level sts the ge level inal - rmine the st, all t 5 ompound ate ; column B. This same test was run in turkeys. Eight-week-old turkeys were utilized, the test compounds were administered to the turkeys in the feed and 2k hours after the start of the feed medication,, the turkeys were challenged with 0.5 ml. _ 4 of a 10 dilution of a broth culture of P. multocida . The experiment continued for seven days after challenge. The number of turkeys which died in the treated groups was compared with the number which died in the control group. As in the test with chickens, the heart and liver were removed from the turkeys which died during the test, and cultured for P. multocida The turkeys which survived the test were sacrificed, the livers and hearts removed and cultured.

The results are recorded in Chart 6 which follows.

In the chart, column 1 lists the test compound; column 2 , the dosage of test compound in grams per ton of feed; column 3, the ratio of the number of turkeys from which P. multocida was cultured to the number of turkeys tested; and column k , the survival ratio.

Chart _6 Control of P. multocida in Turkeys Dosage Culture Isolation Survival Cpd. g . /ton P. multocida Ratio Infected Controls -- k/ 1/5 evaluation of the effectiveness of selected es for their oral activity against E. c oli chickens was accomplished as follows. iler type, five-week-old chickens were wing-banded lly weighed. Replicates of eight chickens, each ing the same average chicken weight, were formed.* tes were run for each treatment. Treatment was the feed 2k hours prior to challenge. The enge strain was No. 2-1-8. The challenge isted of 0.5 ml. of 10 1 dilution of a 15-18 broth culture administered into the right sac of each chicken. Eight to ten days following l surviving chickens were weighed and examined Any chicken that died during the experiment ined for lesions such as air sac lesions, lesions, and perihepatitis lesions, and a culture e -treatment average weight per bird was 790 g. ounds were the same as before, identified as results are shown in Chart 7 which follows. s the test compound; column 2, the dosage rate; ratio of the number of chickens with lesions of chickens tested; column k , the average icken; and column 5, the survival ratio. dazolyl)- ler chicks on of a of 8 y.

All the birds in the tests were pre-brooded for seven days in floor pens before being placed on treatment. The treatment period extended for 21 days in starter batterie! Feed and water were provided a_d lib itum at all times. A nontreated feed was fed during the pre-brood period.

The results of the study are presented in Chart 8. In the chart, column 1 gives the identification of the test compound; column 2, the level of administration of the test compound in the feed; column 3, the average weight gain in grams; column h , the percent improvement in weight gain; column 5 , the feed conversion, which is the ratio of the feed consumed (F) to the weight gained (G); and column 6, the percent improvement in feed conversion.

Chart 8_ Level of Avg .

Admin. Wt .

Compound g . /ton Gain Impr . F/G Impr . 1 50 56¼ g. 6.21 I.582 6.28 Control -- 531 -- 1.688 -- Experiment 8 A number of representative compounds, namely, 1, 5, 6, 7, 12, 13D> lh , and l8 , were tested for their in vitro activity against Treponema hyodysenteriae . The treponeraa were propagated on blood agar plates. Each plate which was prepared contained 20 ml. of trypticase soy agar (Baltimore Biological Laboratory ΓβΈ, ]) with 5.0 g./l. of yeast extract (Difco) and 5 percent sterile defibrinated horse blood. The plates were sterile plastic 15 x 100 mm. petri plates. Incubation was carried out at 37 °C. for h days under anaerobic conditions using the BBL Gas Pak system.

The concentrations of test compounds in this initial test were prepared in dilutions ranging from 2000 meg. /ml. to 7.8 meg. /ml. The test procedure was carried out in the following manner. Five ml. of a twenty-fold concentration in tryptose broth (Difco) of each ilter- sterili zed test compound dilution was mixed in a series of flasks containing 90 ml. of melted trypticase soy agar with yeast extract. Five ml. of horse blood was then added per flask. The contents of these flasks were then used to prepare a series of blood agar plates in doubling decreasing dilutions ranging from 100 meg. /ml. to The treponema for inoculation of these plates were obtained by adding 3.0 ml. of sterile tryptose broth to the surface of a plate on which the treponema had been propagated. A sterile cotton-tipped swab was used to loosen the treponema from the agar. This suspension was then diluted 100-fold and each of the plates containing the test compounds was inoculated with 0.01 ml. along with the control plates which did not contain test compounds. The inoculum was spread so that it covered the surface of each plate, a bent sterile plastic pipette being used for this purpose for each plate. The plates were incubated at about 36 °C. for about k days and then examined for growth of the treponema. The minimum inhibitory concentration (MIC) of each test compound was the concentration that prevented growth of treponema.

These representative compounds all inhibited growth of the treponema at each of the levels tested, and the MIC for these compounds was o.39 meg. /ml. doubling 0 8 est to be Treponema r. urchased as to the ly and were ean pig ed to were given razole-¾-- f 100 m um tical after the ated ntery a swine nd ery: A nonmedicated controls killed at the same time. The average weight gain of the pigs in the medicated group killed 28 days post- challenge was 35-9 l · > while the infected, nonmedicated controls killed 28 days post-challenge had an average weight gain of 21.8 lb. None of the pigs receiving the test compound died during the trial, whereas four of the infected control pigs died. Diarrhea was not detected in the pigs receiving the test compound, but was seen for a total of 23 days in the nonmedicated control pigs.

The first signs of mucus and blood typical of swine dysentery appeared in the nonmedicated control pigs approximately one week following the challenge, and were noted for a total o 17 days. Mucus and blood were not observed at any time during the trial in the groups of pigs receiving the test compound. Recurrence of swine dysentery was not observed in the group of pigs receiving the test compound and continued on the basal ration without medication from day IT until day 28 (replicate 2), whereas swine dysentery did continue in the nonmedicated pigs which were continued on the basal ration from day IT until day 28.

There was a marked difference in the lesions found at necropsy of the medicated pigs and the infected nonmedicated control pigs. The colons of three of the four control pigs which died were hemorrhagic. Five of the eight surviving infected controls were found, at necropsy, to have muco- hemorrhagic colons. Blood and/or mucus were not found in the colon of any pig receiving the test compound. Slight areas of hemorrhage in the colon and/or stomach were seen in most of the pigs receiving the test compound. This type of lesion has been observed on occasion in other groups of pigs not receiving this test compound and is not considered related to swine dysentery.

The test compound was shown to be highly effective in the control of induced swine dysentery based upon prevention of death, maintenance of weight gain, prevention of diarrhea and mucohemorrhagic stools, and prevention of colon lesions typical of swine dysentery.

The results of the in vivo test are shown in the table which follows. g t ©o Chart 9 Efficacy of Compound 1 Against Swine Dysentery Initial Avg. t. of Pigs ( Treat- Group Wt.1 Survivors (Days) ment ITo. (All pigs) 7 Ik 19 21 28 O Cpd. 1 100 g./ton2 1 35-3 ^3.3 51.0 57.3 22.0 Infected Control 1 35-7 kl.o hi.o J+0.5 k.8 Cpd. 1 100 g./ton2 2 35-3 ¼2.1 50.0 - 59 71.2 35-9 Infected Control 2 35-2 hi.6 - .6 - k6 56.0 21.8 1 Weight at time of challenge 2 Treatment was continuous from two days before challenge throug NOTE: Pigs in Group 1, both medicated and nonmedicated, were ne X CO IV) Experiment 10 The susceptibility of anaerobic bacteria, both gram positive and gram negative, to a representative number of the novel compounds of this application was determined using the agar dilution method. The bacteria used were isolated from patients hospitalized at the Indiana University Medical Center. The isolates were identified in the anaerobe laboratory of the Medical Center by the criteria established by Dowell et al. , Lab orat ory Methods in Anaerob i c Bacteri olog . Public Health Serv. Publ. Wo. l803 (1968). Tcenter for Disease Control, Atlanta, Ga._; and by Smith et al., T e Pathogenic Anaerobic Bacteria, pages 96-I36 (1968) /^Charles C. Thomas, Publisher, Springfield, 111. · All the organisms were maintained in chopped meat glucose medium (Scott Labs) at room temperature.

The method used for the agar dilution tests was based on the studies of Sutter jet al_. , Ant imicrob . Ag . Chemother . _3_, 188-193 (1973)· The strains of anaerobic bacteria were grown in l6 x 120 mm. tubes for ΐβ to l8 hours in Thioglycolate medium without indicator--135C (Becton, Dickinson and Co.), to which was added 5 meg. of hemin per milliliter prior to autoclaving, plus 1 mg . of sodium bicarbonate and 0.1 meg. of filter-sterilized menadione per milliliter after autoclaving. The growth tubes for the bacteria Veillonella alcalescens were additionally supplemented with 5 mg . of sodium lactate per milliliter. The strains were adjusted to the turbidity of the Wo. 1 McFarland standard in Thioglycolate medium without indicator--135C containing an additional 0.13 percent Bacto-agar (Difco). Each strain was then applied to the surface of the petri plate ( 100 ram. x 15 mm.) by means of a replicator.

For the agar dilution studies, two-fold dilutions of the test compounds were prepared in deionized water to which was then added laked sheep blood and filter-sterilized menadione so the concentrations after the agar dilution were 5 percent and 10 meg. /ml. respectively. An equal volume of double strength Brucella agar (Becton, Dickinson and Co.) was mixed with each dilution in a water bath held at 50°C. and then poured onto the petri plates.

All of the procedures were done inside a plastic glove ox, except for the incubation of the growth tubes and the pouring of the plates. After the plates hardened, they were put inside the glove box for a minimum of two hours before they were inoculated. Generally, the glove box procedures of Aranki ejb aJL. , Applied Microbiology IT, 568-576 (1 69), were followed, but the following modifications were used. Dry materials were introduced into the glove box by evacuating the lock to 28 inches of mercury and filling once with nitrogen gas, followed by evacuating in the same manner and filling with an 80 percent nitrogen plus 10 percent CO2 plus 10 percent hydrogen gas mixture. Liquid media and agar plates were introduced into the glove box by evacuating the lock to 10 inches of mercury and filling with gas four times. The nitrogen gas was used to fill the lock for the first two cycles of evacuation, with the gas mixture described above used for the final two fillings.

All the plates were incubated at 37 °C. in Gas Pak jars (Becton, Dickinson and Co.) containing palladiura-coated alumina catalysts (reactivated after each use). The results of the agar dilution tests were read after about 2k hours of incubation. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration of test compound at which there was no growth.

The results of these in vitr o tests are recorded in Charts 10 and 11 which follow. The test compounds are identified in the same manner as in the previous experiments. The antibiotics cephalothin and erythromycin were also run simultaneously with the novel compounds of this application, for comparison purposes, and the results are included in Charts 10 and 11 below. -89 " οε -ρ- ro H Compound Λ A A Α A A A ■ρ- ο ι- υ ο o Actinomyces Η ο Η Η Η Η H H ro ro ro ΓΟ ro ro Γ0 bovis 1368*4- \-Π Λ Λ Λ A A A Λ ο ο Η ο ο ο o O o Clostridium Η Ο Η Η Η H H H inocuum 1373 ο ■Ρ" Η Ο Η ro Η Η H H M septicum 1128 !3 o ΓΟ VJ1 ΓΟ ro ΓΟ ro ro ro i» VJ1 νπ vn fl> P O c+ Λ A Λ A A Λ A o Η· Η ο Η ο ο ο o o o Clostridium Γ3 O O Η Η Η H H H septicum bovine Ο Η Ο CD ω ΓΟ ΓΟ ro ro ro ro Γ0 C3 νπ νη η η VT) \Jl rt- o c+ A A A A A to o Cha t 10 ( Continued) Susceptibility of Anaerobic Bacterial 2k Hr . Minimum Inhibitory Concentration 11A 2 0.125 0.03 0.5 0.06 o.o6 X 18 <0.125 < 0.125 <0.125 <0.125 <0.125 <0.125 <0.

I r g Chart 10_ (Continued) Susceptibility of Anaerobic Bacteria 2k Hr . Mimimum Inhibitory Concentratio O 20 2 16 8 8 8 8 I 21 <0.125 <0.125 <0.125 <0.125 <0.125 <0.125 <0.1 22 <0.125 <0.125 <0.125 <0.125 <0.125 <0.125 <0.1 Erythromycin 1.0 >128 1.0 <0.5 1.0 Cephalo- thin 2 8 8 1.0 I Co ΓΟ 4=- -T L- CO v ro Compound Λ A A A A A A o O o o o o o o Propi onibacte r i urn H o H H H H H H ro VH ro ro ro ro ro ro acnes 79 ~~ vn vn n vn vn vn vn Λ A A Λ A A A o o o o o o o o Bacteroides ro ro ro ro ro ro ro vn V vn vn vn p. thetaio- O vn vn s taomicron 1*4-38 Η· !3 o (B (0 A A A A 4 A A o O o o o o o o Bacteroides O CT ro ro H H H H H H fragili s O H- E3 O vn vn ro ro Γ0 ro ro ro v-n v_n vn vn vn vn sp . vulgatus 1563 (0 bJ 3 P t- o c+ A A A Λ A Λ A Φ CO t © o Chart 11 ( Continued) Suscepti ility of Anaerobic Bacteria 2k Hr . Minimum Inhibitory Concentratio 11A <0. 0. 06 2 0. 06 <0.03 <0. 12 1. k 16 2 1.0 2 15A <0. -25 0. 5 ■ 2 0. 25 0.25 <0. 13D 2 2 0. 5 1. 0 0.5 1. l I 16 CO ro 8 t o Chart 11 ( Continued) Susceptibilit of Anaerobic Bacteria 2k Hr . Minimum Inhibitory Concentratio I 18 <0.125 <0.125 <0.125 <0.125 <0.125 <0. 20 1.0 k 2 k 1.0 2 21 <0.125 <0.125 <0.125 <0..¾25 <0.125 < C 22 <0.125 <0.125 <0.125 <0.125 <0.125 <0.

Erythromycin k Cephalo- thin 1.0 > 128 > 128 6k I Co ΓΟ ■p-

Claims (1)

1. FORMULA I r hydrox 0 10 is or 3 R R4 are the same or different and are or is hydrogen or X is J or is hydrogen or is or alky 20 ί and Re and R9 are the same or different and are A compound as in Claim said compound being A compound as in Claim said compound being but A compound as in Claim said compound being Λ compound as Claim said compound being pyrazole ormula FORMULA I ox alky formula FORMULA II th vide a compound alkyl when R is to be reacting a compound of Formula I wherein R is is and is with a halogenating and when optionally is to be other than hydrolyzing a compound of Formula I wherein R is any of the values defined is and R2 is to form the corresponding compound wherein R2 is an amide group or an acid group if acylating the resulting amide or decarbox lat ing the resulting and when optionally is to be other than subjecting a compound of Formula I wherein R and R2 are any of the values defined above to a with an orthoester to form a corresponding imidate or with an aldehyde to form the corresponding Schiff or any combination of steps 2 and 3 in any e which dehyde cting acting nitrile which presence id which id e with es c which comprises reacting 2 with tri j formate and acetic j A process as in Claim 7 for preparing 5 j which comprises reacting 5 j yrazole onitri with i dimethyl and dry and slow addition of acetyl A process as in Claim 7 for preparing ethyl j 5 ί date which comprises reacting 5 pyrazole carbonitri with triethyl j orthoacetate and acetic j A process as in Claim 7 for preparing pyrazol date which comprises reacting with allyl ί J and acetic j ί A process as in Claim 7 for preparing j j j formimidate which comprises reacting 1 le with j orthoformate and acetic A process as in Claim 7 for preparing isobutyl 5 which comprises reacting 5 j with triisobutyl orthoformatej and acetic A process as in Claim 7 for preparing i formimidate which comprises reacting Ί 3 pyrazole with and acetic A process as in Claim 7 for preparing j ί which comprises reacting 5 j with tricrotyl orthoformate and acetic A process as in Claim 7 for preparing fur furylidene which comprises reacting le carbo wi ί furfural and acetic A process as in Claim 7 for preparing 5 j which comprises reacting pyrazole le carboxaldehyde and acetic A process as in Claim 7 for preparing 5 lidene carbonitrile which comprises reacting yraz ole with benzaldehyde and acetic A process as in Claim 7 for preparing trile which comprises reacting with cinnamyl aldehyde and acetic A process as in Claim 7 for preparing 2 which comprises reacting pyrazole carbonitri with trichloroacetaldehyde and acetic A process as in Claim 7 for preparing benzylidene yrazole le which comprises reacting pyrazole le with piperonal and acetic anhydride in the presence of dimethyl formamide A process as in Claim 7 for preparing formimidie hydrazide which comprises reacting with methyl and acetic anhydride followed by reacting the resulting formimidate with methyl hydrazine and ethanol process as in Claim 7 for preparing which comprises reacting 5 carbonitrile with acetic A process as in Claim 7 for preparing acetamide which comprises reacting with dimethyl formamide and A process as in Claim for the preparation of propi which comprises reacting pyrazole with propionic anhydride and sulfuric A process as in Claim 7 for preparing which comprises reacting 5 with pionic anhydride and sulfuric A process as in Claim 7 for preparing which comprises reacting pyrazole le with sodium hydride and tetrahydr A process as in Claim 7 for preparing pyrazole whi ch comprises reacting with sulfuric acid and absolute A process as in Claim 7 for preparing pyrazole comprises reacting le with sulfuric acid and absolute A for controlling infections in poultry caused by bacteria selected from the group consisting of Salmonella Escherichia coli and Pasteurella multocida which comprises the administration to the poultry of a effective amount of a compound of Claim 1 The method of Claim wherein the effective compound is administered orally at the rate of from about 25 to about 200 per ton of The method of Claim wherein the effective compound is administered subcutaneous at the rate of from about 3 to about 60 The method of Claim wherein the effective compound is 1 5 i le The method of Claim wherein the effective compound is butyl 3 1 pyrazol mi The method of Claim wherein the bacteri effective compound is methyl A method for promoting the growth poultry which comprises administering thereto a amount a compound of Claim 1 The method of Claim wherein the compound is administered orally at the rate of about 5 0 per ton of The method of Claim 8 wherein the compound is 5 3 insufficientOCRQuality