Patent Publication Number: US-3876790-A

Title: Dihalonitromethylpyridines as antimicrobials

Description:
United States Patent [191 Friedman [4 1 Apr. 8, 1975 Dll-IALONITROMETHYLPYRIDINES AS ANTIMICROBIALS Henry Friedman, Indianapolis, Ind.  
 Eli Lilly and Company, Indianapolis, Ind.  
 Filed: Oct. 25, 1973 App]. No.: 409,556  
 Inventor:  
 Assignee:  
 US. Cl 424/263; ll7/l54 Int. Cl A0ln 9/22 Field of Search 424/263; 260/290 HL References Cited UNITED STATES PATENTS 9/1973 Gum, Jr. et a1. 424/349 X 9/1973 Gum, Jr. et al 424/349 X OTHER PUBLICATIONS Chem. Abst., Vol. 68 (I968), Zalukaev et al. 871 l2p. Feuer et al., J. Am. Chem. Soc., 91, 1856(1969).  
 Primary E.\&#39;aminerSam Rosen Assistant ExaminerAlIen J. Robinson Attorney. Agent, or Firm-Joseph A. Jones; Everet F. Smith [57] ABSTRACT 7 Claims, N0 Drawings DIHALONITROMETHYLPYRIDINES AS ANTIMICROBIALS BACKGROUND OF THE INVENTION This invention concerns a new method of controlling bacteria, fungi, and protozoa by contacting the microorganisms with a member of a class of dihalonitromethylpyridines.  
 The nitration of active methylene compounds con taining an activating group such as an ester, ketone or cyano group has been previously described. See Feuer et al.. J. Am. Chem. $00., 78. 4364 (1956). 117121.. 81. 5826 (1959), J. Org. Chenr. 29. 939 (1964). 111121.. 31. 3152 (1966). Ibid.. 34. 991 (1969); and Klager. Ibid., 20. 646 (1955).  
  In addition. p-anisylnitromethane and its ring nitro derivatives have been prepared by Zalukajevs ct al.. Latvijas P.S.R. Zinatnu Akad. Vesn&#39;s. 109. (1956). The same author also prepared a-naphthylnitromethane. J. Gen. Chem. U.S..S&#39;.R., 26, 657 (1956).  
  Primary or secondary nitro compounds form nitronate salts which react with bromine to form bromonitro compounds. In the case of oz-nitrocyclic ketones. cleavage can occur upon bromination. See Feuer et al., J.  
 Org. Chem.. 29. 939 (1964). Ibid.. 33. 3622 (1968), lhid.. 34. 991 (1969). The preparation of halo derivatives of various nitromethyl heterocycles was recently disclosed by Feuer et al.. J. Org. Chenr, 37, 3662 1972). Zalukajevs et al. prepared the halo derivatives of 2-nitromethylquinoline. Zhur. ()bslzchei Khim., 28, 483 (1958).  
  There is no suggestion in any of the above-described publications that any of the compounds possess antimicrobial activity.  
  Belgian Patent 702.570 discloses l-aryl-2- nitrohaloethanes useful in the control of bacteria. fungi and algae in water and aqueous compositions. Gum et al.. U.S. Pats. Nos. 3.703.515 and 3.754.042. disclose dihalonitromethyl-substituted quinoxalines and cycloalkanes. respectively. which are said to have antimicrobial activity.  
 SUMMARY This invention provides a new and superior method of killing bacteria. fungi. and protozoa which comprises contacting the microorganisms with an antimicrobiallyeffective amount of a dihalonitromethyl compound. The method is useful in such in vitro environments as paints, disinfecting compositions. cosmetic formulations. bodies of water and the like. The method makes use of compounds having the formula l (R) t... n  
 wherein X represents bromo or chloro;  
 each of the R groups independently represents C -C alkyl;  
 n represents -2;  
 provided that when the CX NO group is in the 3- position, n represents 0;  
 or the l-oxides or hydrohalide salts thereof.  
 DESCRIPTION OF THE PREFERRED EMBODIMENT The compounds used in this invention are characterized by a dihalonitromethyl group attached to a pyridine ring. When thedihalonitromethyl group is in the 3-position, it is the&#39;only ring substituent. The ring may be additionally substituted with alkyl groups when the dihalonitromethyl group is in the 2- or 4-position.  
  In the above formula. the term C -C alkyl refers to alkyl groups such as methyl. ethyl, propyl, and isopropyl. The term hydrohalide salts refers to such salts as hydrochlorides. hydrobromides, and hydroiodides.  
  While the formula above describes the compounds unambiguously. the following specific compounds are named to assure that those skilled in the art understand the scope of the invention.  
 2-dichloronitromethyl-3-picoline 2-dibromonitromethyl-3-ethylpyridine 4-dichloronitromethyl-2.6-diethylpyridine 2-dichloronitromethyl-4-isopropylpyridine 3-dichloronitromethylpyridine 4-dibromonitromethylpyridine 3-dibromonitromethylpyridinel -oxide 4-dibromonitromethyl-3.5-lutidine hydrochloride 4-dichloronitromethyl-Z-propylpyridine hydrobromide 2-dibromonitromethyl-4-picoline hydroiodide 2dibromonitromethyl-3-propylpyridine 2dibromonitromethyl-4-ethylpyridine 2dibromonitromethyl-4-picoline hydrobromid 2-dichloronitromethyl-3.S-dipropylpyridine The compounds with which this invention is preferably carried out are 6-dibromonitromethyl-2-picoline. 6-dichloronitromethyl-2-picoline. 4- dichloronitromethyl-3-picoline. 4-dichloronitromethylpyridine. and 4-dichloronitromethylpyridine-l-oxidc.  
  The pyridines used in this invention are easily prepared in a two-step synthesis from picolines which are commercially available or can be prepared using known procedures in the chemical literature.  
  Compounds bearing the diahlonitromethyl group at the 2- or 4-position are made differently than the 3- substituted compounds. The first step of the synthesis of 2- and 4-substituted compounds is the formation of a nitronate salt having the formula Q CH NOJM wherein O represents the pyridine ring, and M represents alkali metal, preferably potassium.  
  Nitronate salts are formed by the procedure of Feuer et al., J. Am. Chem. 500., 91, 1856 (l969).&#39;wherein a picoline is treated with an alkali metal amide and liquid ammonia, followed by n-propyl nitrate as the nitrating agent. Alternatively, such salts can be made by treating a picoline with a nitrating agent in the presence of butyllithium and tetrahydrofuran. Both processes are carried out at low temperatures from about to 30C.  
  Either process forms nitronate salts of picolines having only one methyl group. The metal amide process is preferential forr the 4-position. however. and the butyllithium process is preferential for the 2-position, when both positions are available. Either salt of a 2,4-lutidine can therefore be made by choosing the right process.  
  The 3-dihalonitromethylpyridines useful in this invention are prepared by a method described by Feuer et al., J. Org. Chem, 37, 3662 1972). A 3-pyridyl acetate is nitrated in liquid ammonia and acetic acid with potassium amide and an alkyl nitrate at about -50 to 30C. to produce 3-nitromethylpyridine.  
  The nitronate salts, and the 3-nitromethylpyridines as well, are converted to the dihalonitromethyl compounds by treatment with a halogenating reagent. Dichloro compounds can be prepared by treating the intermediate with an agent such as potassium hypochlorite at a temperature between lC. and 20C. The corresponding dibromo compounds are best prepared by treatment with bromine in potassium hydroxide solution under similar conditions. The use of a two-fold excess of the halogenating reagent forms the dihalonitro compound, usually in less than one hour.  
  The pyridine l-oxides used in this invention are easily prepared by using the corresponding picoline l-oxides as starting compounds. The oxide has no effect on the synthesis of the dihalonitromethyl compounds.  
  Hydrohalide salts of the compounds are easily prepared by the methods usually used for preparing salts. For example, the dihalonitromethyl compound can be dissolved in ether, and contacted with the anhydrous hydrohalide. Hydrohalides can also be made by contacting the compound with the corresponding acids in solvents such as aqueous alcohols or aqueousacetone.  
  The preparative examples below illustrate the methods by which the compounds are made, and are not intended to limit the scope of the invention in any way. The first two examples show the synthesis of typical nitronate salts.  
 EXAMPLE 1 Lithium 2-methyl-4-picolinenitronate A 10.7 g. portion of 2,4-lutidinewas dissolved in 250 ml. of dry tetrahydrofuran and the solution was cooled to about 50C. Then about 0.10 mole of nbutyllithium in about 50 ml. of tetrahydrofuran was added at-a rate such that the temperature of the mixture did not rise above 50C. The reaction mixture was then stirred for two hours at 55C. A 20.9 g. portion of n-propyl nitrate was then added at a rate such that the temperature remained below -50C. during the addition. After the mixture was warmed to room temperature, the solvent was removed under vacuum and dry ether was added. The solids which did not dissolve in the ether were collected, washed with more ether, and vacuum dried. The yield was 8.4 g. of lithium 2-methyl-4-picolinenitronate in a sufficiently pure form for use in the halogenation step without further purification.  
 EXAMPLE 2 Sodium 4-methyl-2-picolinenitronate Liquid ammonia. 250 ml., was added to a flask equipped with a stirrer, thermometer, and Dry Ice condenser. A 5.3 g. portion of sodium was added in small pieces and dissolved, followed by a catalytic amount of ferric nitrate hydrate. While the mixture was stirred and kept below 35C., 16.1 g. of 2,4-lutidine was added. The mixture was then stirred for minutes, and 31.2 g. of n-propyl nitrate was then added, holding the temperature below -35C. The ammonia was then allowed to evaporate and was replaced with ether. The solids which formed were collected by filtration and washed with ether. The yield was 28.4 g. of sodium 4-methyl-2-picolinenitronate.  
  The intermediate compounds are converted to the antimicrobial dihalonitromethylpyridines by processes typified by the following example.  
 EXAMPLE3 4-dichloronitromethyl-Z-picoline A 5.0 g. portion of sodium 4-methyl-2- picolinenitronate was dissolved in 50 ml. of water and the solution was filtered. The solution was then added dropwise to 83 ml. of 2.2 molar potassium hypochlorite at 510C. An oily phase formed which was extracted from the aqueous phase with ether. The ether extract was dried over magnesium sulfate, and evaporated under vacuum leaving a yellow oil. The oil was chromatographed over silica gel with 2:1 benzenezethyl acetate. The yield, after the product-containing fractions were evaporated under vacuum, was 2.40 g. of 4- dichloronitromethyl-Z-picoline (XIV), an oily liquid. Identity of the compound was confirmed by nuclear magnetic resonance analysis and elemental analysis.  
  The following exemplary compounds were made by the processes of Examples 1-3 with small modifications which can readily be supplied by one skilled in the art. 6-dibromonitromethyl-Z-picoline (1), mp. 485 1C. o-dichloronitromethyl-2-picoline (11), oil Z-dichloronitromethyl-5-ethylpyridine (I11), oil 4-dichloronitromethylpyridine (1V), oil 4-dichloronitromethyl-3-picoline (V), oil 4-dichloronitromethylpyridine-l-oxide (V1), oil Z-dibromonitromethylpyridine-l-oxide (V11). m.p.  
  l61168C. 4-dibromonitromethylpyridine-l-oxide (V111), m.p.  
  98-102C. 2-dichloronitromethylpyridine-1-oxide 1 l31 16C. 4-dichloronitromethylpyridine hydrochloride (X),  
  softened 145C; dec. at 240C. 4-dibromonitromethyl-2-picoline (X1), m.p. l26l28C. dec. 4-dichloronitromethyl-2,6-lutidine hydrochloride (X11), softened 145C; dec. at 210C.  
 (1X), m.p.  
 hydrochloride 4-dichloronitromethy1-2,6-1utidine (X111), m.p.  
 4-dibromonitromethyl-2-picoline (XV), m.p.  
 4-dibromonitromethyl-2,6-lutidine (XVI), m.p.  
 2-dichloronitromethyl-4-picoline (XVll), oil 2- dibromonitromethyl-4-picoline (XVlll), oil 2-dichloronitromethyl-4,6-lutidine (XIX), m.p.  
 2-dibromonitromethyl-4,6-lutidine (XX), m .p.  
 4-dibromonitromethylpyridine (XXl), m.p.  
  The invention described here is a method of killing bacteria, protozoa, and fungi in vitro which comprises contacting the microorganisms with a compound described above. The method is effective in many differ ent ways, and those skilled in the microbiological arts are aware of a great variety of ways in which the method can be advantageously applied.  
  The environment in which the microorganisms are killed in vitro may be any environment suitable for the growth of the microorganisms, away from a living host. For example. the method can be applied by adding the compounds described above to bodies of water such as cooling towers and ponds, lagoons, lakes and the like for the control of protozoa and slime-forming bacteria and fungi. Another desirable variant of the method is the addition of the compounds to such compositions animal feed, adhesives, inks, plasticizers. latices, polymers, resins, fuels, lubricants, soaps and detergents,  
 appropriate to make a powdered formulation of the compound. Such a formulation is easily prepared by making an intimately mixed, finely powdered mixture of the compound with dispersing agents, such as cutting oils, and paints to prevent the growth of mold 5 naphthalenesulfonates or ligninsulfonates, to which a and the degradation of the products which results from surface-active agent such as those described above may attack by microorganisms. be added. Inert fillers such as finely divided clays may Another useful variant of the method is the use of the also be used in some instances. Such formulations discompounds as coatings or impregnants for products perse easily through liquid environments, or disperse ,such as textiles, paper and other cellulose products, evenly over a solid environment, and thereby effecwood, and wall panelings and plaster to protect such tively bring the active compound into contact with misubstances from mold and decay caused by microbial croorganisms which may be present in the environinfestation. The method is especially useful for the ment. Use of such easily dispersing powders also haspreservation of such products as cosmetic formulatens the dissolution of the compound in the environtions. ment, thereby allowing the compound to come into Further, a particularly useful variation of the method contact still more readily with microorganisms. of killing such microorganisms is the u e of the com- The method of this invention is also effective against pounds as disinfectants and sterilizing agents for sur- 3 number of aquatic Weeds- For p application faces such as floors, walls, hospital equipment, kitchen 70 0f 10 PP of 4&#39;dlehlorohitrothethylz,e&#39;hltidihe yequipment d h lik drochlorlde to a body of water killed or effectively con- A h examples b l i i the new Compounds trolled the noxious weeds hydrilla, coontail, and duckare effective at very low concentrations. In general, the Weed- Shhilar effects were also Ptoclueed for e compounds are used in the practice of the microbio- 2-{11chloronltromethyl-t-plcohne hydroehlonde, cidal method in concentrations of from about 0.] ppm. and z&#39;dlb0mohltromethyl6plcohhe&#39; to about 100 ppm The example below reports the results of testing a In many cases, no special formulation of the new representat&#34;? g of the compounds a broad compounds is necessary to use them in the microbio- Spectrum annmlcroblal screen&#39; cidal method. The compounds may be simply added to the environment in which bacteria, fungi, or algae are 3 EXAMPLE 4 to be killed, usually with adequate mixing. For example, the compounds may be added to and intimately In VitfO g dilution test blended with the other ingredients of such composi- Bacteria and fungi in an agar medium were Stamped tions as adhesives, polymers paints, cosmetics, and the on a plate to which one drop of a 100 MgymL or a 10 like when the Compounds are to be dispersed in a pg/ml. solution of the compound was surface applied. y of Water, as for use in Cooling Water or are to be The agar plates were then incubated at 35C. for 12 used as a Coating Oh P p or the like, it is desirable to hours, at which time the antibacterial activities were P p a liquid formulation of the eompouhd- Such 3 evaluated. The fungi were incubated at 25C. for an adformulation. well known in th is readily P p ditional 60 hours before evaluation. The procedure was y dissolving the Compound in Water or an Organic essentially that recommended in Acta Par/ml. Mivent, to which surface-active agents such as alkyl-benbi L S d, B,, Suppl. 217, 11 (197]). A rating of zenesulfonates, alkyl sulfates, sorbitan esters, and eth- 100 indicates that the compound prevented growth of ylene oxide adducts of alkylphenol may be advantathe microbe at 100 pg/ml. and 10 indicates prevention geously added. at the 10 ,ug/ml. level. NT indicates the compound was At times, it is difficult to find a solvent for the desired not tested for activity against an organism, while a compound which is acceptable in the environment to blank space indicates the compound was not active at, be protected from microorganisms. In such cases, it is the highest level tested (100 ug./ml.).  
 a a. a 3  
  f: t t: l: .2 s t ,5 2;? s m 5% s .5 E a 3 3m 3 llii :13 i 33 IQ-fit; 35 L 26 w 2 P15 H 0H m we m 00 -H Etc: 0:! 510 m0 EH its an. we 0 as: $4; a :2 a: a s2 :2 as s as. ,0 as as as 22 2.: 2a a: 2 H a as as s as assesses :2 5 as :2 2a 2:: 2a .2: a; 2&#39;; m 8 $5 23 81 $3 &#39;Qfififi E23 3 25 3 5 53%, 83 5232183 eon a I 10 10 10 1o 10 10 10 10 1o 10 10 1o 10 10 1o 1o 10 10 II 10 1o 10 100 10 lo 100 100 100 10, lo 100 100 10 IV 10 100 100 10 100 100 10 10 100 100 10 10 1o 10 1o 10 1o 10 x111 lo 100 10 100 100 100 100 100 10 1o 10 1o XXI 100 100 100 100 100 100 100 100 100 100 100 100 10 100 100 100 100 100 100 mwpmenoopwnoo m mdimaw mdaawmnwmm mwax Esnwwoocdaow mmeoaoudowm E eaaweoaaem ma wu pmwm soconn daampm hom mmmx mwozamshow mesoioudmmm Pa a 8 mama H nmmhoB mdwponm wnuoooopmonvm III VII 100 100 100 100 100 100 100 100 100 1.00 100 100 100 100 100 100 100 100 100 VIII 100 XII XVl 100 XVII 100 XVLII 10 XIX  under sterile conditions. One hundred ag/ml. of the compound to be tested was added to a tube of broth.  
 and the treated broth was serially diluted with un- PL treated broth in which the culture had been inoculated. EXAM E 5 The tubes were observed, and the results for each com In vitro tube dilution test The organisms against which the compounds were to be tested were grown in nutrient broth in test tubes pound were recorded as the lowest concentration, in micrograms per milliliter. in which the compound prevented the growth of the microorganism.  
 adneasempw 2 E aaweamouzz aa a u m mm mwcoaoedmwm Edo vmwm em mEweamoozz cm mm mnooooopmonpm msouoooahnmmpm cesomsoo III VII  
 VIII 50 XIII 1.56  
 XVI  
 XVII  
 XIX  
 XXI  
  A generally similar test was performed to evaluate some representative compounds against additional species of fungi.  
 EXAMPLE 6 In vitro antifungal test In a different antifungal test, the compounds to be tested were absorbed on paper discs, and the discs were laid on plates of fungus-infected agar medium. The results were reported as the least amount of compound per disc which produced a measurable inhibition of the fungus.  
 C ompou nd Trir&#39;hoplrvlon Candida No. menlugrop/zylcs albir&#39;ans I 0.625 ,ugJdisc 0.156 p-gJdisc Representative compounds of this invention were tested in vitro to determine their ability to control protozoa and algae. The examples below report the results of such tests.  
 EXAMPLE 7 In vitro protozoa inhibition tests The tests were run against four representative protozoa, Terrahymena pyrlftn&#39;mix (T). Oclzromonas mallzamensis (O). Euglena gracilis (E), and Trichomonas vaginalis (TV). The protozoa were grown in the laboratory in nutritive media. When a test was to be run. nu-  
 tritive medium containing the test protozoa was mixed with sterile agar medium, and the mixed medium poured into plates.  
  Absorbent discs were treated with 0.02 ml. of a solution containing a known concentration of the compound to be tested. The concentrations used are indicated in the table below. The discs were placed on the surfaces of the protozoacontaining agar plates.  
  The plates were incubated for a time. and were then inspected to determine if the test compound absorbed on the discs had inhibited the growth of the protozoa. Inhibition, if present. was measured as the diameter in millimeters of the zone of inhibition around the disc.  
 The table below reports the zones of inhibition pro-- duced by representative compounds of this invention. The notation TR indicates that the compound gave a trace of inhibition. NT indicates that the compound was not tested against a given organism, and a blank space indicates that the compound was inactive.  
 Compound No. Cone. T O E TV I 2000 2O 18 500 l2 l3 10 I 10 ll 2000 l8 l5 l3 13 500 I2 TR TR 125 TR IV 2000 34 NT 25 26 500 18 NT l2 l3 l25 TR NT TR Xlll 2000 25 17 18 IS 500 15 14 10 125 12 TR XIV 2000 23 l3 15 500 ll TR TR XV 2000 26 30 32 TR 500 ll l5 I0 125 TR XVI 2000 24 22 23 10 500 l3 l4 l0 XXI 2000 30 NT l7 17 500 10 NT TR 125 NT.  
 EXAMPLE 8 In vitro algae inhibition test The activity of representative compounds against a typical alga was determined by conducting a test substantially identical to the test described above against Chlorella vulgaris. The results are reported below.  
  The following tests showed the ability of typical compounds of the invention to eradicate established microorganisms.  
 EXAMPLE 9 In vitro eradication test Aqueous dispersions of several compounds of this biocidal method were inoculated with known concentrations of organisms which are named below. Counts of the viable organisms per milliliter were made initially. The cultures were then incubated at 25C. and counted on subsequent days as shown in the tables below. The notation NC indicates that the organism was not counted on that day.  
  The compound 6-dibromonitromethyl-Z-picoline was tested with the following results.  
 0.1% of Compound 0.1% of Compound R Bram/0111011113 Axpzrgillm- Duvs 51: Days acrlminusu Iligz&#39;l&#39; 2 1; 7 5 0 5.5 x 3.5 X 10&#39; 5 2,13 26 100 100 7 100 100 7 100 100 s 100 100 Z8 100 100 A similar test against a broader range of microorganisms was performed with 6-dibromonitromethyl-2- picoline as the test compound. In this test. the fungi were inoculated in Sabouraud dextrose agar, and the The hydrochlonde 0f the above Compound was 11150 bacteria in soybean-casein medium. The test was othertestedwise similar to the test described immediately above.  
  One particularly useful embodiment of this invention is the preservation from deterioration by microorgan- 0 W f C d isms of compositions such as cosmetic and pharmaceuo Ompoun tical formulations. The tests reported in the example 70 below demonstrate the value of the compounds of the D. f W present method in such preservation.  
  dyS llL&#39;l llglIlObU &#34;1110! 0 1000 5.8 X 10 EXAMPLE 10 l [0 5 O X 7 8 Cosmetlc base cream preservatlon test 1 :88 The creams used as substrates for the tests reported here were typical eosmetlc creams composed of approximately 25 percent of heavy mineral oil and about percent of water, with small percentages of cetyl alcohol, wax, and lanolin. Creams were tested which were emulsified with both anionic and nonanionic sur- Another compound effective in the new method, 4- faetants. dichloronitromethyl-2,6-lutidine hydrochloride, was The first tests to be reported were of the compound also tested. 6-dibromonitromethyl-2-picoline.  
 0.1% of Compound Sluplrvlncm&#39;r&#39;11x lzirclu&#39;ric/riu Farm/0111011113&#34; Cum/ilk! -t\purgillu\ SIruplm&#39;occux Days UHIUHS (u/i m&#39;ruginusu ulhicum&#39; Niger jiu&#39;rulix 0 4.3 x 10&#34; 11.3 x 10 43 x10&#34; 3.1 X 10&#34; 3.0 X 10- 3.4 x 10 1 100 100 100 3.4 x 10&#39; 100 7 100 100 100 100 7 x 10- 100 l4 NC NC NC NC (IOU NC El NC NC NC NC 100 NC 221 100 100 100 100 100 100 0.0571 of Compound Slap/11101&#39;m&#39;t&#39;us lzlrt&#39;lwriz&#39;hiu Farm/mummy (&#39;umlizlu :lAPUFgi/IHJ .S&#39;il&#39;e Ilm&#39;ullllA Days durum wli m&#39;ru uinuxu ulhimm nigur jur&#39;m/h 0 5.0 10&#39; 7.3 x 10&#34; 5.0 x 10 1.: x 10&#34; 3.11 x 10-- 2.5 X 10&#34; 1 100 100 100 100 4.3 x 10* 100 7 100 100 100 100 3.0 X 10&#39;-&#39; 100 14 NC NC NC NC 10!) NC 21 NC NC NC NC H10 NC 28 100 100 1.0 X 10&#34; 100 100 100 0.171 in Anionic Cream Slap/111m&#39;nu&#39;ux Escherichia Rl&#39;l&#39;lIl/OIHUHUA&#39; Candida Axpcqui/IHS :Lrpcrgillus .S&#39;Ir&#39;cplm&#39;m&#39;t&#39;111 Days um&#39;cux (&#39;0/1&#39; (10171511110111 (II/JICUIIA Niger flurix fiu-mlis 0 1.5 x10 8.3 x10 8.5 10- 1.0 x 10&#39; 60x10&#34; 17 x10 1.1 10 1 100 100 1 X 10 100 4.7 x 10 4 x 10 100 7 100 100 100 100 100 100 100 14 NC NC 100 NC 100 100 NC 28 100 100 100 100 100 100 100 0.0571 in Nonionic Cream .S&#39;lup/rvloma&#39;ux l-jsvlu&#39;riclriu PM&#39;IIt/UIHUIHM Cum/121a :Lvpurgillua A.1&#39;/)01&#39; qil[u.\ Slrr&#39;plmou&#39;m Days uurvm L&#39;U/i ar&#39;l&#39;uginm&#39;u ullwit&#39;unx nigt&#39;l flm&#39;ix flux-(11111 0 9.0 x 10&#39; 3.9 x 10 4.4 x 10&#39; 3.0 X 10&#34; (1.0 x 10 4.0 x 10 5.0 x 10 1 100 100 100 100 3.5 10&#39; 4.5 x 10 a x 10 7 100 100 100 100 1.9 X 10- 100 100 14 NC NC NC NC 1.1 X 10- 100 100 1 NC NC NC NC 2.0 X I0 NC NC 100 100 100 100 4.0 x 10&#39;- 100 100 Similar tests were also performed with 4- 0.1% in Anionic Cream 1. A method of killing microbes selected from the group consisting of bacteria, fungi, and protozoa in vitro which comprises contacting the bacteria, fungi, and protozoa with an antimicrobially-effective amount of a compound of the formula wherein X represents bromo or chloro; each of the R groups independently represents C C;,  
  alkyl; :1 represents 0-2; provided that when the CX NO group is in the 3- position, n represents 0; or the l-oxides or hydrohalide salts thereof. 2. The method of claim 1 wherein the amount of the compound is from about 0.1 ppm. to about ppm. 3. The method of claim 2 wherein the compound is 6-dibromonitromethyl-Z-picoline.  
  4. The method of claim 2 wherein the compound is 6-dichloronitromethyl-2-picoline.  
  5. The method of claim 2 wherein the compound is 4-dichloronitromethylpyridine.  
  6. The method of claim 2 wherein the compound is 4-dichloronitromethylpyridine- 1 -oxide.  
 7. The method of claim 2 wherein the compound is 4-dichloronitromethyl-3-picoline.