United States Patent 0
`
`3,370,957
`Patented Feb. 27, 1968
`
`1
`
`2
`Generally, the invention comprises antifun-gal composi
`tions where an active ingredient has the general formula
`
`N
`H
`(I)
`wherein R represents a heterocyclic radical containing
`nitrogen and sulfur as the hetero atoms. In addition the
`Z-heterocyclic benzimidazole antifungal. agents described
`herein may, if desired, be further substituted on the benz
`imidazole nucleus, and particularly at the 1, 5 and/or 6
`position of such nucleus. The preferred substituents are
`conveniently described by reference to the formula
`
`Rs
`3.0-
`
`‘ -—N
`\N/”——R
`
`10
`
`15
`
`20
`
`25
`
`R1
`(11)
`wherein R is thiazolyl, isothiazolyl, or thiadiazolyl; R1
`is hydrogen, loweralkyl, acyl, alkenyl, aralkyl or ar
`alkenyl; and R5 and R6 are hydrogen, phenoxy, lower
`alkoxy, loweralkyl, halo, amino, loweralkylamino, dilow
`eralkylarnino, imidazolyl, thiazolyl, isothiazolyl, thiadi
`azolyl, thienyl, furyl, pyrryl, naphthyl, phenyl, halo
`phenyl, loweralkylphenyl, loweralkoxypheny-l, loweralkyl
`thiophenyl, loweralkylaminophenyl or diloweralkylamino
`phenyl.
`_
`According to a still further aspect ‘of the invention, the
`heterocyclic group at the 2-position may be further sub~
`stituted by, for instance, a loweralkyl group.
`“
`As illustrative of the compounds within the scope of
`this invention which are particularly effective as antif
`fungal agents, there may be mentioned:
`
`‘
`2-(4’-thiazolyl) benzimidazole,
`2-[3’-( 1',2’,5’-thiadiazolyl] benzimidazole
`2-(4'-thiazolyl)-5-methoxy benzimidazole,
`2-(4’-thiazolyl)-5-phenoxy benzimidazole
`hydrochloride,
`2~(2'-methyl-4’-thiazolyl) benzimidazole,
`2-[4'- ( l ',2’,3'-thiadiazolyl)] benzimidazole,
`l-acetyl-Z-(4'-thiazolyl)~5-phenyl benzimidazole,
`2-(4’.-isothiazolyl) benzimidazole,
`2-(4’~thiazolyl)-6-?uoro ‘benzimidazole,
`2-(4’-thiazolyl)-5-amino benzimidazole,
`2- ( 2'-thiazolyl ) -5-( 1'-imidazolyl) benzimidazole,
`2- (4’-isothiazolyl ) -5-chlorobenzimidazole,
`2-(4’-thiazolyl)-5-phenyl benzimidazole,
`2-[4"-(1’,2f,3'-thiadazo1yl)]-5-(4'-tolyl) benzimidazole,
`1-acetyl-2- ( 2’-thiazolyl ) -5-phenyl benzimidazole,
`l-methyl-2-(2'-isothiazolyl)-5-(2' - methoxyphenyl) benz
`2-(4'-isothiazolyl)sS-furyl benzimidazole,
`2-(4f-thiazolyl)-5-(4'-?uorophenyl) benzimidazole hydro
`chloride,
`-
`2-(4’-thiazolyl)-5-bromo benzimidazole,
`2-(4’-thiazolyl)-5-chloro benzimidazole,
`2-(2’-thiaZolyl)-5-methoxy benzimidazole,
`'
`‘
`2- (4’-thiazolyl)-5-(2’-?uorophenyl) benzimidazole hydro
`
`imidazole,
`
`I
`
`'
`
`3
`
`‘
`
`_
`
`60
`
`chloride,
`
`\
`
`<
`
`2- [3 '-( l ’,2’,5’-thiadiazolyl) ] -5-methylthio benzimidazole,
`2-(4’-thiazolyl)-5,6'di?uoro benzimidazole,
`>
`1~benzoyl-2-(4’-thiazolyl) benzimidazole,
`'
`2-(2’-thiazolyl)-5-(2’-pyrryl) benzimidazole,
`l-methyl-Z-(4’-isothiazolyl) benzimidazole hydrochloride,
`2- (4'-thiazolyl).-5-phenoxy benzimidazole,
`'
`-
`'
`
`70
`
`3,370,957
`ANTIFUNGAL COMPOSITIONS AND METHODS
`FOR THEIR USE
`Joseph R. Wagner, Moraga, Calih, Thomas W. Hum
`phreys, London, Ontario, Canada, and Herbert H.
`Royse, Oakdale, Calif., assignors to Merck & -Co., Inc.,
`Rahway, N.J., a corporation of New Jersey
`No Drawing. Continuation-impart of application Ser. No.
`282,551, May 23, 1963. This application May 12, 1964,
`Ser. No. 366,883
`19 Claims. (til. 99-90)
`This application is a continuation-in-part of our pend
`ing application, Wagner, Humphreys and Royse, Serial
`No. 282,551, ?led May 23, 1963, now abandoned.
`This invention relates to compounds active as fungicides
`and to methods for their use. More speci?cally, this in
`vention relates to 2~substituted benzimidazoles effective as
`fungicides. Still more particularly, this invention is di
`rected to novel fungicides comprising compounds of the
`formula 2-R~benzimidazole, where R is a thiazolyl, iso
`thiazolyl or thiadiazolyl radical, to compositions contain
`ing such compounds and to methods of killing fungi or
`controlling their growth by the use of such compositions
`and compounds.
`Fields of technology adversely affected ‘by the lack of
`effective fungicides are many and include the paint, wood,
`textile, cosmetic, leather, tobacco, fur, rope, paper, pulp,
`plastics, fuel, rubber and food industries. Fungicidal
`agents or materials may ?nd use in medical therapy such
`as the treatment of mycotic infections of man andv ani
`mals where the skin, hair, nails and other areas of the
`body are infected. Fungicides are also utilized for agri—
`cultural application, for instance in preventing or mini
`mizingfungus growth on plants, fruits, seeds or soil. In
`addition, fungicides are useful in preventing Mycotoxi
`cosis, an animal disease which may cause internal lesions,
`tumors and death and which results from ingestion of
`food contaminated by toxins of fungal origin. We have
`discovered that certain 2-substituted benzimidazoles are
`effective in controlling this undesired fungus growth.
`Although many antifungal agents have been described
`and used heretofore in an effort to control fungi, none are
`entirely satisfactory and continued losses resulting from
`fungal attack make the problem of control a serious and
`lasting one. The number of fungicides practically useful
`in combatting fungal growth have been small and only
`in a few cases have synthetic organic chemicals been
`found applicable.
`It is an object of this invention to provide novel anti
`fungal agents. It is a further object of this invention to
`provide new and improved methods of controlling the
`growth of fungi. Another object of this invention is to
`provide compositions useful in the control of fungi in or
`on‘food, plants and animals. It is still a further object of
`this invention to provide a method for controlling and
`killing fungi with synthetic organic chemicals. Further ob
`jects and advantages will become apparent from the
`following description of the invention.
`7
`As used in the description of our invention, the ex
`pressions “fungicide” and “fungicidal” are intended to en
`compass control ‘of fungi broadly so as to include the
`killing of fungi as'well as the inhibiting of the growth of
`fungi.
`According to the present invention it has now been
`found that certain Z-heterocyclic benzimidazoles are
`highly effective antifungal agents. It will be appreciated
`by those skilled in the art that not all of the compounds
`de?ned hereinbelow have exactly the same degree of anti
`fungal activity and it should also be understood that a
`particular compound of the invention will vary somewhat
`in activity depending upon the species of fungus subjected
`to its action.
`
`30
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`35
`
`40
`
`CFAD Exhibit 1023
`
`1
`
`

`
`3
`
`idazole,
`2-(4’-thiazolyl')-4-?uoro benzimidazole hydrochloride,
`2-(2'-thiazol'yl) benzimidazole, and
`l-acetyl-2-(4'-thiazolyl) benzimidazole.
`
`The Z-substituted benzimidazoles of the invention are
`effective in controlling the growth of Aspergillus species,
`for example A. niger, A. ?avus, A. fumigatus, A. oryzae,
`A. luchensis, A. versicolor, A. sydowi, A. nidulans, A.
`?aucus and A. terreus, Penicillium species, for example
`P. notatum, P. roqueforti, P. chryso-genum», P. oxalicum,
`P. spz'nulosum, P. martensii, P. citrinium, P. digitatum,
`P. expansum, P. italicu'm, P. .cyclopium, and P. funicu
`losum, Neurospora species such as N. sitophila, Phorna
`species such- as P. terrestrius, Rhizopus species, Alternaria
`species such as A. solam', Chaetomium species such as C.
`globosum, Chaetomicum species, for example C. clivace
`um, and Monilia species such as M. sitophi'la and M.
`nigra. The above fungi may be found on fresh, preserved
`or frozen foods, such as cheese, cereals, grains, meats,
`?sh, poultry, fats and oils, fruits, vegetables, baked
`goods, syrups, confections and the like, or they may be
`found on cosmetics, leather, electrical insulation, tex
`tiles, and numerous other materials capable of supporting
`their growth.
`I
`The compounds of this invention may be employed in
`treatment of plants, soils, fruits, seeds, fur, wood and
`the like. The fungicidal effectiveness of these compounds
`has been demonstrated against soil fungi, such as Rhizoc
`t‘onia solani, Fmsarium‘ soilani, and Pythium ultim'um,
`plant fungi, for instance Erysiphe polygoni and Alternaria
`sol'ani as well as against saprophytes known to attack
`wood, pulp and lumber such as Lenzites trabea and Cera
`t'ocystis pilifera and the fungus Pullularia pullulans which
`attacks paint.
`The 2-R-benzimidazoles of this invention have also
`demonstrated their effectiveness against pathogenic fungi
`such as Trichophyton species, for example T. men
`tagrophytes', T. rubnumi, and T. gypseum, Microsporum
`species‘ such as M. .awdouim' and M. gypseum, Cryptococ
`cus species such as C. neoformans, Hormodendrum spe
`cies such as H. pedrosoi, and Geotrichum species.
`It should be understood that the compounds may be
`utilized in diverse formulations, solid, including ?nely
`divided powders and granular materials as well as liquid,
`such as solutions, emulsions, suspensions, concentrates,
`emulsi?able concentrate, slurries and the like, depending
`upon the application intended and the formulation media
`desired.
`Thus it will be appreciated that compounds of this in
`vention may be employed to form fungicidally active
`compositions containing such compounds as essentially
`active ingredients thereof, which compositions may also
`include ?nely divided dry or liquid diluents, extenders,
`fillers, conditioners and excipients, including various
`clays, diatomaceous earth, talc, and the like, or water and
`various organic liquids such as lower alkanols, for exam
`ple'ethan'ol and isop-ropanol, or kerosene, benzene, tolu
`ene and other petroleum distillate fractions or mixtures
`thereof.
`In general, the compounds of this invention have been
`found effective in cornbatting fungi which attack and
`are an annoyance to humans. Fungus infections of man
`can be divided into two large groups (1) the super?cial
`mycoses in which the parasites invade only the keratin
`layer of skin or its appendages, and (2) the deep mycoses
`in which various totally unrelated fungi infect the deeper
`organs of the body. The compounds of Formula II above
`have been found particularly effective against the super
`?cial mycoses.
`The clinical classi?cation of the super?cial mycoses is
`based on the site or structure of the body involved and
`
`3,370,957
`not on the organism causing the infection. Among the
`common terms used are T irzea capitis, also commonly
`known as ringworm of the scalp; Tinea barbae, or ring
`worm of the beard; T inea corporis (Tinea circinata),
`which refers to tinea of the nonhairy, nonintertriginous
`areas; T inea crurz's, referring to fungus infection of the
`upper, inner thighs; Onychomycosis, or fungus infections
`of the nails; and Dermatophytosis (dermatomycosis), a
`term usually restricted to super?cial infections of the feet,
`hands and nails.
`Athlete’s foot, which is one of the major types of the
`super?cial mycotic infections, is classi?ed among der
`matophytosis. It is variously estimated that from 50%
`to 90% of the population of the United States have
`dermatophytosis of some type during their lives. Among
`the common organisms which give rise to this disease are
`T richophyton mentagrophytes, T richophyton rubrum and
`Epidermophyton ?occosum. Tric'hophyton mentagrophyz‘es
`is believed to be the cause of the acute form of athlete’s
`foot and is the cause of most of the in?ammatory reaction
`between the toes. Athlete’s foot due to T richophyton
`rubrum is a many-faceted disease entity characterized by
`a low grade inflammatory reaction and includes what is
`commonly known as chronic dermatophytosis and ony
`chomycosis. Epidermophyton ?occosum is occasionally
`involved in the cause of athlete’s foot and gives rise to
`a subacute infection, however, this organism is more com
`monly involved in Tinea cruris or the infection involving
`the inner surface of the upper parts of the thighs.
`When the active agents are employed in preventing
`topical fungal growth, one or more of the compounds
`may be uniformly distributed in a vehicle that is chemi
`cally compatable with the particular compound selected,
`noninhibiting with respect ‘to the action of the antifungal
`and essentially noninjurious to body tissue under the con
`ditions of use.
`It should be understood that the 2-substituted benzimid
`moles of the invention may be used in combination one
`with the other as well as with other fungicidally active
`materials. For instance, a mixture of 2-(4-'-thiazolyl)
`benzimidazole and sorbic acid or its salts, propionic acid
`or its salts, mycostatin, sodium diacetate, trichomycin,
`amphotercin, griseotluvin, undecylenic acid, chlorquina
`dol, 5,7-dichloro-8-hydroxyquinoline (Vioform), sodium 0
`phenylphenate, o-phenylphenol, biphenyl, chlorinated phe
`nols, sodium benzoate, dehydroacetic acid and its salts
`or esters of parahydroxybenzoic acid, such as the methyl
`and propyl ester (parabens) can be used to give fungicidal
`effect when used in appropriate concentrations. It is quite
`clear, too, that the compounds de?ned according to For
`mula II above may be used in conjunction with effective
`antibacterial materials in appropriate instances so as to
`combine the action of each in such a situation as to be
`particularly useful, for instance, in applications where the
`presence of bacteria creates undesirable results alongside
`the detrimental action of fungi. Accordingly, a combina
`tion of antifungal and antibacterial agents will be useful
`in the preparation of germicidal soaps, in the production
`of cosmetics, and in food, such as beer, cheese, or meat,
`and leather applications.
`'
`It has been found that growth of various fungi exist
`ing in soil is limited or terminated by the addition to the
`soil of minor quantities of the benzirnidazole compounds
`described. The term soil as used herein is intended to
`include all media capable of supporting the growth of
`plants and may include humus, sand, manure, compost,
`arti?cially created plant growth solution, and the like.
`We have also found that the fungicides of the invention
`are effective against fungal diseases of plants, and may
`be effectively used either by direct contact with the foliage
`or systemically, by introduction through the roots.
`The compounds of this invention also have activity
`against bacteria and yeasts and may, at appropriate levels
`of concentration, be effectively used to inhibit or prevent
`the growth of these organisms. It will be quite clear to
`
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`60
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`2
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`

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`10
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`30
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`3,370,957
`5
`those skilled in the art that the benzimidazoles of this
`invention will vary in activity against a particular organism
`‘at a particular concentration. Consequently, while all
`the compounds of this invention are effective fungicides
`and bactericides, certain of the compounds will be more
`active than others against for instance yeast, mold or
`bacteria.
`The fungicides described by Formula II above are
`useful in the ?eld of food preservation where fungi are
`known to attack doughs, cakes, breads, pastries, meats,
`cheeses, fruits, vegetables, cereal, jams and jellies, brines,
`?sh, poultry, fats and oils, juices, honey, syrup, condi
`ments, alcoholic beverages, confections and other food
`products.
`The present antifungals have been found e?ective in
`controlling fungus growth on cheese. They may be applied
`to the wrapper thereof or may be admixed directly with
`the edible itself. Application of the active agents to the
`wrapper may be made by any method known in the art
`such as by immersing, spraying or otherwise depositing
`the fungicide. All types of cheeses, notably cream cheese,
`American cheese, swiss cheese, blue cheese, muenster
`cheese, gruyere cheese, cottage cheese, cheddar cheese,
`farmer’s cheese, parmesan cheese, ricotta cheese, moz
`zarella cheese and the like, may be effectively protected
`in this manner.
`The fungicides of the present invention are useful in
`inhibiting mold growth in fruit such as citrus fruit. The
`active agent may be applied at any time before consump
`tion and preferably after harvesting. For instance, the
`antifungal may be applied during initial storage, before
`or after shipping or during ?nal storage before consump
`tion. The benzimidazoles may be utilized in a number of
`ways in this regard and may be applied either directly
`to the fruit in an emulsion, solution, suspension or the
`like or it may be applied to the fruit container or Wrapper.
`Suitable carriers for the active agents are waxes and other
`materials presently known in the art.
`It has also been found according to the present invention
`that benzimidazoles described by Formula II above may
`be added to bread dough prior to mixing and baking and
`thereby protect the ?nal product from mold spoilage. The
`active agent may be added to the brew or to any of the
`dry ingredients such as the ?our as a concentrated solu
`tion, suspension or the like. A uniformly mixed dough
`is desirable in all cases. The active agent may also be
`added to the dough by dissolving it in shortening. For
`instance, when 0.02 gram of 2-(2’-thiazolyl) benzimid~
`azole is dissolved in 30 grams of shortening, the resulting
`solution may 'be added to 1000 grams of flour to give a
`concentration of benzimidazole of 20 p.p.m. based on
`?our. In order that theconcentration of the compounds
`of this invention be suf?cient, so as to properly prevent
`mold growth on bread, quantities of from about 5-1000
`p.p.m., preferably from about 30-200 p.p._m._ based on
`?our may be utilized. The 2-substituted benz1midazoles of
`this invention such as 2-thiazolyl benzimidazoles are par
`ticularly e?icacious in preserving bread in a manner su
`perior to that now technically feasible w1th propionates
`without adversely affecting ?avor, loaf volume or odor.
`In addition, there is frequently an increase in loaf vol
`ume when the compounds of this invention are employed
`as antifungal agents.
`'
`The use life of bread depends largely on its ability
`to resist mold spoilage. Suitable agents for preventing
`mold spoilage should at the same time have no speci?c
`inhibitory effect on any of the yeasts used for leavemng.
`In principle, the most desirable agents for protecting
`bread from mold spoilage are those having selective action
`against mold outgrowth. Surprisingly, the benzimidazoles
`of Formula II above possess this highly desirable char~
`acteristic.
`The compounds described by Formula II above may be
`prepared by treating an appropriately substituted nitro
`aniline with a heterocyclic carboxylic acid or a derivative
`
`6
`thereof such as the ester or acid halide in a suitable inert
`solvent such as pyridine. The nitro group on the resulting
`anilide is then reduced and benzimidazole formation ef
`fected by treatment of said anilide with a reducing-cycliz
`ing system such as zinc-hydrochloric acid, zinc'acetic
`acid, iron~hydrochl0ric acid and the like. Alternatively,
`the nitroanilide may be reduced by catalytic reduction
`and the product cyclized by use of a strong mineral acid
`such as hydrochloric acid.
`Alternatively, the benzimidazoles of the invention may
`be prepared by reacting an appropriately substituted 0
`phenylenediamine and a heterocyclic carboxylic acid or
`derivative thereof, in polyphosphoric acid, preferably at
`temperatures of from about 175-275 ° C. for about 2-6
`hours. The compounds described by Formula II above
`may also be synthesized by treating an o-phenylenedi
`amine with a heterocyclic aldehyde in a. reaction medium
`comprising nitrobenzene or in a suitable solvent such
`as a lower-alkanol. When the reaction is performed in
`solvent other than nitrobenzene, the intermediate product
`is treated with a suitable oxidizing agent such as cupric
`acetate, lead tetra-acetate, mercuric acetate, ferric chlo
`ride and the like. When a heavy metal reagent is used to
`bring about benzimidazole formation from an o-phenyl
`enediamine in the above process, an insoluble heavy metal
`salt of the Z-heterocyclic benzimidazole is formed. This
`material is readily converted to the free benzirnidazole
`by removal of the heavy metal salt by reagents suitable ,
`for this purpose, such as hydrogen sul?de, ammonium
`polysulfide, ammonium hydroxide and the like.
`According to another process for making the above
`benzimidazoles, an appropriately substituted aniline may
`be treated with a heterocyclic nitrile in the presence of
`a suitable catalyst such as aluminum chloride to form an
`N’-p-henylamidine derivative of the heterocyclic com
`pound.
`The above—mentioned N'-phenylamidine may then be
`chlorinated or brominated to produce an N-chloro or
`N-bromo-N'-phenylamidine. This halogenation is brought
`about by reacting said N’-phenylamidine with a positive
`halogenating agent capable of halogenating the nitrogen
`atom of the amidine group. The preferred halogenating
`agents are hypochlorous and hypobromous acid. These are
`conveniently ‘formed in situ by addition of an alkali or
`alkaline earth metal hypohalite to a solution of the N'
`phenylamidine acid addition salt, whereby neutralization
`of the acid addition salt and the generation of the haloge
`nating agent occur concurrently. Typical hypohalites use
`ful for this purpose are‘ sodium or potassium hypochlo
`rite, sodium hypobromite and calcium hypobromite.
`The N-halo-N’-phenyl amidine resulting from the above
`halogenation is converted to the benzimidazole by treat
`ment with a base, such as an alkali or alkaline earth metal
`hydroxide such as sodium hydroxide, potassium hydrox
`ide or calcium hydroxide.
`One method of obtaining a l-substituted benzimidazole
`of Formula II above is by converting the non-l-substituted
`compound to an alkali metal salt, preferably the sodium
`salt, by intimately contacting said compound with sodium
`hydride in a suitable solvent. A slight molar excess of
`sodium hydride gives satisfactory results and equim'olar
`quantities of .benzimidazole and sodium hydride may also
`be used if desired. The reaction is conveniently brought
`about by warming the reactants at slightly elevated tem
`peratures, but room temperature gives satisfactory re
`sults.
`A l-substituted benzimidazole may then be obtained
`by contacting the benzimidazole alkali metal salt with
`an acyl, loweralkyl, alkenyl, aralkyl or aralkenyl halide
`in an inert solvent. The reaction is allowed to proceed at
`a temperature of from about room temperature up to
`about 100° C.
`The Z-substituted benzimidazoles described herein are
`normally isolated as the free base. The l-unsubstituted
`benzimidazoles are readily converted to acid addition
`salts by treatment with acid. Examples of salts which
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`50
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`55
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`80
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`65
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`70
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`75
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`3
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`

`
`7
`may be formed in this manner are mineral acid salts such
`as the hydrohalides, e.g. hydrochloride, hydr'obromide and
`hydroiodide, the sulfates, nitrates, phosphates and the
`like, aliphatic acid salts, and salts of polycarboxylic acids
`and the like. Certain of these salts, such as the hydro
`halides, are much more water soluble than the free bases.
`Since the solubility may also be decreased by formation
`of an appropriate salt, it will be seen that the solubility
`properties of a particular compound may be conveniently
`adjusted by judicious selection of a salt.
`The examples following are given for the purpose of
`illustration and not by way of limitation.
`
`3,370,957
`8
`three portions each of which is molded, put in pans,
`proofed 35 minutes at 100° F. and 80-85% relative
`humidity, braked at 435—445° F. and cooked 30-40
`minutes. The three loaves obtained from each treatment
`are further treated as follows:
`
`10
`
`(1) One loaf serves as a control.
`(2) One loaf is sprayed with a water suspension of spores
`of Aspergillus niger.
`(3) One loaf is sprayed with water suspension of spores
`of Penicillium species.
`The loaves are wrapped in an appropriate moisture-proof,
`dust-proof barrier, placed in a 30° C. atmosphere and
`periodically examined for mold growth. The amount of
`growth occurring is evaluated as follows:
`
`EXAMPLE 1
`Bread is prepared by a brew process as follows:
`TRIPLE BREW FORMULATION
`
`(grams
`Water -------------------------------- " 1800
`Flour ___________________________________ __
`300
`Sucrose _________________________________ _._
`23 20
`
`—=no growth exident;
`+—_—slight mold growth;
`++++++=extremely heavy mold growth,
`
`The results are set forth in the table below.
`
`Control
`
`TABLE I
`Soduirn Propionate
`
`2-(4’-thiazolyl)
`benzimidazole
`
`Time
`(Days)
`
`Plain
`
`Penicillium
`
`A. niger
`
`Plain Penicil-
`lium
`
`A. niger
`
`Plain Penicil- A. m'ger
`lium
`
`4 ____ __
`
`——
`
`—
`
`+
`
`-—
`
`-—
`
`5 ---- --
`
`-—
`
`++
`
`++
`
`—
`
`—
`
`-
`
`—
`
`6 .... ..
`7... -
`ll-_---
`
`—
`—
`11)
`
`—
`—
`++++
`++++
`-
`+
`+
`++++~l + ++++++ —
`D
`D
`D ++++ ++++++
`
`-_
`
`-
`
`—
`—
`D
`
`._
`
`_.
`
`—-
`—
`—
`
`._
`
`_
`
`-
`—
`Y
`
`1 Discarded.
`1 Only a few spots on air bubbles in the crust, no tendency toward spreadlng.
`Similar tests are run using Penicillium chysogen'um, P.
`notatum, P. citrinum, P. digitatum, P. roqueforti, P.
`expansum‘, P. italicum, P. martensii, Aspergillus versi
`color, and A. sydowi and in each instance the benzimid
`azole is at least sixty times as effective in inhibiting growth
`as sodium propionate on a weight to weight basis.
`
`40
`
`15
`Arkady’s yeast food ______________________ __
`45
`Dry milk solids, nonfat ____________________ __
`98
`Yeast ................................... -_
`60
`Salt ____________________________________ __
`1 200 ml. of water are mixed separately to suspend the yeast.
`100 vm1. of water are used to dissolve the salt separately.
`Water is mixed with all the dry ingredients with the
`EXAMPLE 2
`exception of the salt and the yeast. A suspension of yeast
`In another experiment using the procedure of Example
`is added and the brew is then fermented at 80—84° F. with
`1 0.0075 weight percent 2-(4'-thiazolyl) benzimidazole
`contsant agitation. Salt solution (38%) is added after
`(based on ?our) and 0.25 weight percent sodium propi
`15 to 20 minutes and the agitation reduced. Fermentation
`is allowed to proceed for 90-120 minutes. The brew is 45 onate (based on ?our) are used. The results are shown
`divided into three equal portions.
`in Table II.
`TABLE II
`2-(4’-thiazolyl)
`benzlmidazole
`Plain
`Peni~ A. niger Plain
`cillium
`
`Time
`(Days)
`
`Control
`
`Plain
`
`Peni-
`cillium
`
`A. niger
`
`Sodium Propionate
`
`Peui-
`cillium
`
`A. niger
`
`:
`
`3.
`
`+3...
`
`I
`
`—
`+++++
`+++
`——
`++++ ++++++ —
`+
`+ ++++++ ++++++ -
`++ ++++++ ++++++ -—
`
`:
`
`:
`
`—
`—
`-|-
`—
`+
`—-
`— 1++
`
`:
`
`~—~
`—
`—
`+
`
`:
`
`:
`
`+++
`——
`++++
`—-
`+++++
`—
`— ++++++
`
`1 No growth on crumb, little tendency to spread.
`When 2-(2'-thiazolyl) benzimidazole is used in place of
`2-(4’-thiazolyl) benzimidazole, substantially identical
`results as are shown in Table II are obtained.
`
`60
`
`The dough is then prepared as follows:
`900 grams of ?our and 30 grams of sugar are inter
`mixed. 3.1 grams of sodium propionate is dissolved in 20
`ml. of water and 2.5 ml. of ‘dough conditioner solution
`EXAMPLE 3
`[one tablet of IDX (Food Industries Company) per liter
`To a clean, sterile ?ask is added 5 milligrams of 2-(4'
`of water] and added to one portion of the brew. In a
`thiazolyl) benzimidazole and 0.3 milligram dimethyl
`similar manner, to a second portion is added 0.3 gram of
`formarnide as solvent. Sabouraud’s dextrose agar (50 mg.
`2-(4'-thiaz-olyl) benzimidazole. To the ?our-sugar mix is
`Difco, pH 5.6) at about 50° C. is then poured into the
`added one of the three brews and 20 ml. of water used to
`?ask and the contents thereof agitated to obtain uniform
`rinse the brew container. Each of the remaining brews are
`distribution of the benzimidazole therein. The mixture is
`similarly added to an identical ?our mix. The three doughs
`then poured into Petri ‘dishes and solidi?es on cooling. A
`are mixed at low speed for 15 seconds. 40 grams of
`water suspension of the spores of Trichophyton mentagro
`shortening, melted by heating to 110° F. is added to each
`phyres, the fungus which causes “athlete’s foot,” is
`dough and mixing continued for 45 seconds. The mixing
`streaked onto the medium in the Petri dish. After three
`is continued for 3.5 minutes at a somewhat higher speed.
`days of storage at room temperature, complete inhibition
`Each portion of the dough is then fermented for 30-40
`minutes at 80° F. and 75% relative humidity, cut into 75 of the fungus is noted.
`
`70
`
`4
`
`

`
`9
`EXAMPLE 4
`In a manner similar to that in Example 3 a solution of
`2-(4'thiazolyl) benzimidazole in dimethylformamide is
`prepared. A portion is then diluted with water so as to give
`varying concentration of ‘benzimidazole such that when
`added to 50 mg. of agar, concentration of 7.8 to 250 mcg./
`ml. are obtained. After the agar is poured into Petri dishes
`and solidi?es, it is inoculated with spores of a fungus of
`the type shown in Table III below. The concentration at
`which fungicidal action takes place is noted.
`TABLE III.—ANTIFUKNGAL ACTION OF 2-(4’-THI
`AZOLYL) BE‘NZIMIDAZOLE
`
`1 Fungicidal concentration
`Culture:
`Cryptococcus neoformans ___________ __ 7.8-15.6
`Hormodendrum pedrosoi ___________ __ 31.2-62.5
`Microsponum audouini _____________ __
`<7.8
`Microsporum gypseum ______________ __
`<7.8
`T richophyton mentagrophytes ________ __
`<7.8
`Trichophyton gypseum _____________ __ 125-250
`Trichophyton rubrum ______________ .._ 15.6-31.2
`1Concentration in meg/ml; incubation at 37° C. for 8
`8.375.
`
`3,37 0,957
`10
`Concentration of 2-(4'-thiazolyl)
`Growth of mold
`benzimidazole (p.p.m.):
`0 ________________________ __ Positive.
`6.3 _______________________ __ Positive (1 spot).
`12.5 ______________________ __ Negative.
`25 _______________________ _..
`Do.
`50 _______________________ __
`Do.
`100 ______________________ __
`Do.
`EXAMPLE 8
`Into one of eleven sterile ?asks is placed 25 mg. of
`one of the eleven compounds noted below. Into each of
`the other ten ?asks are similarly placed the ten remain
`ing compounds. A small amount of dimethylformamide is
`added as solvent. The solutions are then diluted with
`water, so as to give concentration of active ingredient such
`that when added to 50 ml. of potato dextrose agar (pH
`5.6, Difco), concentrations of 3.1 to 250 meg/ml. are
`obtained. The agar is then poured into Petri dishes and
`allowed to solidify. Inoculation with spores of Aspergz'llus
`niger and Penicillium species is then effected and the con~
`centration at which fungicidal action takes place is noted
`after three days incubation at room temperature.
`
`10
`
`15
`
`20
`
`EXAMPLE 5
`
`(A) A water insoluble ointment for topical treatment
`of mycotic infection is prepared according to the follow
`ing formulation:
`
`Grams
`Cholesterol ________________________________ __ 30
`Stearyl alcohol _____________________________ __ 30
`White wax ________________________________ __ 80
`White petrolatum ___________________________ __ 860
`2-(2’-methyl-4’-thiazolyl) benzimidazole ________ __ 90
`
`water soluble ointment for treatment of topical
`(B)
`ly disposed mycotic infection is composed of:
`
`Grams
`Polyethylene glycol (M.W. 4000) ____________ __ 4000
`Polyethylene glycol (M.W. 400) _____________ __ 800
`2-[3'-(l’,2',5'-thiadiazolyl)] benzimidazole ____ __ 100
`
`The above ointments and the cream of Example 6 are
`formulated using techniques known to those skilled in the
`art.
`
`EXAMPLE 6
`A cream preparation for topical applicationof active
`agent is ‘prepared according to the following formulation:
`
`9.2
`Cetyl alcohol, grams ______________________ __
`9.2
`Stearyl alcohol, grams _____________________ __
`1.5
`Sodium lauryl sulfate, grams ________________ -_
`White petrolatum, mll _____________________ __ 30.0
`Propylene glycol, ml. ______________________ __ 10.0
`,Distilled water to make total of, grams _______ __ 100.0
`2-(4’-thiazolyl) benzimidazole, grams ________ -_ 11.1
`
`25
`
`‘CONCENTRATIONS EFFECTIVE IN CONTROLLING
`FUNGUS GROWTH
`
`Antifungal Agent
`'
`
`30
`
`35
`
`2-(4'-th1azolyl) benzimidazole ________________ _ _
`2-[3’-(1’,2',5'~thiadiuzolyl)] benzimidazole.
`_
`2—(2’-thiazolyl) benzimidazole __________ M
`2-(4’-thlaz0lyl)-5-methoxy benzimidazole"
`1-aeetyl~2~(4'~thiazolyl) benzimidazole ________ __
`2-(4’-thiazolyl)-5, G-di?uoro benzimidazole ____ . _
`2-(4’—thiazolyl)-5-?uoro benzarnidazole ________ _.
`2~(4’-thiazolyl)-5-(4’-?uorophenyl) benzimida~
`zole hydrochloride ______________________ _.
`_
`2-(4’-thiazolyl)-5-bromo benzimidazole.
`2~(4'~thiazolyl)~5-pheny1 benzimidazole_____ . ___
`2-(4’-th1azolyl)~5-(2’-?uorophenyl) benzimida
`zole h