CA2247348A1 - Antimicrobial phenazine compounds - Google Patents

Abstract

Phenazine compounds having antimicrobial activity are described. The phenazine compounds can be phenazine N-oxides, including phenazine-5,10-dioxides, which may optionally be substituted at one or more positions, preferably the 7-and/or 8-positions of the phenazine nucleus. Also described are methods for treating microbial infections, and methods for inhibiting the growth of a microbial cell. The microbes treated or inhibited can be multidrug resistant.
These compounds also inhibit the growth of tumor cells.

Description

A~TIMICROBIA~L PHENA~ZINE CO M POU~DS

Background of the Invention The al,~e~lce of drug-resistant microorg~ni~mc has caused concern among he~lthr~re practitioners around the wor}d. Drug-resistant microbes, especially bacteria, but also yeasts and fungi, have in some cases become multidrug resistant (MDR), that is, resistant to more than one drug. Clinical infections with such multidrug resistant strains can be rliffie~lt or impossible to treat with conventional antibiotics.

10 Summary of the Invention The ~e~ ce of multidrug ~ pathogens has prompted a search for new classes of antimicrobial drugs which are structurally and/or functionally different from ~Yicting drugs. Drugs having new m~rh~nicmc of action may be effective against resistant o~ ."c, where conventional drugs are no longer effective.
The invention provides methods and con.~o~ilions for treating infections, inr,hl-iing infections caused by drug-resistant microorg~nicmc In one embodiment, the invention provides a method for treating an infection due to a resistant microo~ l in a subject. The method includes the step of ~-lmini.ctPring to a subject in need thereof an effective amount of a ph~n~7:ine compound, such that the infection is treated.
In one aspect, the invention provides a method for treating an infection due to a multidrug-resistant microorganism in a subject. The method inch~ the step of ~-lminict~ring to a subject in need thereof an effective amount of a phen~7ine compound, or a ph~ c..l;c~lly-acceptable salt thereof, such that the infection is treated. In preferred embodiments, the ph~n~7ine compound is a phPn:~7in~ N-oxide, more preferably a phrn~7.ine 5,1 O-dioxide. In a particularly preferred embodiment, the phenazine N-oxide is a 7,8-~ bstitllted phenazine-5,10--iio~ e In ce~tain embo~lim.ont~, the multidrug resistant microorgalfisll, is a bacterium or other microbe, including a bacterium or other microbe sei~ted from the group consisting of Hemophilus spp., ~: coli, Enterobactcr spp., Citrobacter spp., Pro~eus spp., Morganella spp., Shigella spp., Yersinia spp., Salmonella spp., and other bacteria in the family Enterobacteriaciae, Neisseria spp., Burkholderia spp., Campylobacter spp., Helicobacter spp., ~'ibrio spp., Staphvlococcus spp., Enterococcus spp., Streptococcus spp., Chlamydia spp., Mycoplasma spp., Coryr.~ebacterium spp., and Clostridium spp., including azole- and amphotericin B-resistant strains.
In another aspect, the invention provides a method for treating an infection dueto a microo~ n in a subject, the method comprising the step of ~lmini.~tering to a subject in need thereof an effective amount of a phen~in~ compound, or a ph~nn~reu~ic~lly-acceptable salt thereof, such that the infection is treated, wherein the SUBSTITUTE SHEET (RULE 26) W O 98/27969 ~ PCT/CA97/00978 microorganism is selected from the group consisting of Hemop~ilt~s influenzae, l~emop*ilus parainflue~zae, Hemophilus aphrophilus, Campylobacter spp., Helicobacter pylori, Vibrio spp., coagulase-negative Staphylococcus spp., Cryptococcus spp., Histoplasma spp., Candida spp., Torulopsis spp., Blastomyces spp., Coccidioides S spp., Nocardia spp., ~ctinomyces spp., Aspergillus spp., Streptococci of the viridans group (a~pha-hemolytic Slaphylococci3, and Corynebacteril~m jeikeium.
In still another aspect, the invention provides a method for treating an infection due to a microorganism in a subject. The method includes the step of ~lministering to a subject in need thereof an effective amount of a phenazine compound. or a 10 pharmaceutically-acceptable salt thereof. such that the infection is treated, wherein the phen~7.ine compound is represented by the formula (R3)m R ~N~(R.~)n in which Rl and R are selected independcntly for each occurrence from thc group con.cicting of hydrogen~ halogen (including fluoro, chloro. bromo, or iodo), lower alkyl, 15 aryl (more preferably substituted or unsubslituted phen~ l). hydroxy, lower alko.Yy (such as mcthoxy), mercapto. Io~er alkylthio. nitro, cyano. amino. Iower alkylamino.
diall;ylamino. and trifluoromethyl; R3 and R~, are selected independentl- for each occurrence from the group consisting of halo~en. Iower all;yl. hydroxy. Iower alkoxy, mercapto. Iower all;ylthio, amino. Iower alkylamino. dialkylarnino. and trifluoromcthyl:
20 m is 0, 1 or ~': and n is an integer from 0 to 4: and X is, independently for each occurrence, oxygen or a pair of electrons. In certain preferred cmbodiments. m and n are each 0. In certain embodiments, at least one occurrcnce of.~ is oxygen; in particularly preferred embodiments. both occurrences of ~ are o.~;~ gen (i.e.. the compound is a ph~n~ine-S, I 0-dioxide!. In preferred embodiments. n is at least I, and one occurrence ~~5 of R4 is a l-hydroxy group (i.e.. the compound is a I -hydroxyphenazine (optionally a I -hydroxyphen~7ine-N-oxide). Rl and R can be the same or different; in certain embodiments. Rl and R2 are the same. and are both methyl or both chlorine. ln some embodiments. the organism is a multidrug resistant organism, including a bacterium.
In another aspect, the invention provides a method for inhibiting the growth of a 30 drug-resistant microorg~ni.~m 'rhe method includes contacting the drug-resistant microorganism with an effective amount of a phenazine compound, such that growth of the drug-resistant microorganism is inhibited. In preferred embo~liment~ the microorganism is a bacterium, and the phenazine compound has bacteriostatic activity SUBSTITUTE SHEET (RULE Z6~

against the bacterium. In another embodiment, the microorganism is a bacterium. and the ph.~?n~7ine compound has bactericidal activity against the bacterium. In certain embodiments, the phenazine compound is a phen~7.ine N-oxide, which can be a ph~n~7.ine 5,10-dioxide. In certain preferred embo-1iment.~. the phenazine compound is a 5 7,8-disubstituted phenazine compound. including, without limitation, a 7,8-dimethylphen~7in~ or a 7,8-dihalophenazine (e.g., a 7,8-dichlorophenazine. such as 7,8-dichlorophen~ine-5,10-dioxide). In eertain plef~ d embodiments. the phenazine is a l-hydroxyphen~7ine, more preferably a l-hydroxyphenazine-5,10-dioxide.
In another embodiment~ the invention provides a method for targeting a drug-10 resistant microorganism in a subject for the purpose of treating an infection. Themethod includes ~r~minictering an effective amount of a phen~7ine compound to a subject in need thereof. such that the drug is targeted to a drug-resistant microorganism known or suspected to be present in the subject and the infection is treated. In a preferred embodiment. the phenazinc compound also targets a non-dmg resistant 15 microorg~ni~m In a preferred embodiment. the phenazine compound is a phenazine-N-oxide. including a phenazine 5-oxide. a phenazine-10-oxide. or a phenazine-5.10-dioxide. ln certain preferred embodiments. the phenazine compound is a 7.8-disubstituted phenazine compound. including. without limitation, a 7,8-dimethylphenazine or a 7.8-dihalophenazine (e.g., a 7,8-dichlorophen~7ine. such as 7.8-20 dichlorophenazine-5, 1 0-dioxide). In certain preferred embodiments. the phenazine is a l -hydroxyphenazine. more preferably a l -hydroxyphenazine-5. 1 0-dioxide.
In another embodiment. the in~-ention pro~ ides a method for si~nificantly decreasing the infecti~ e ability of a drug-resistant microorganism. Ille method includes contacting the drug-resistant microorganism ~ ith an effecti~-e amount of a phenazine ~5 compound. such that the infective ability of the drug-resistant microorganism is significantlv decreased. In a preferred embodiment. the phenazine compound is a phenazine-N-oxide. including a phenazine 5-oxide. a phen~ine-l 0-oxide. or a phf n.~7ine-S, 1 0-dioxide. In certain preferred embodiments. the phenazine compound is a 7,8-disubstituted phPn~7inP compound, including, without limitation, a 7,8-30 dimethylphen~7ine or a 7.8-dihalophena~ine (e.ga 7.8-dichlorophena~ine, such as 7.8-dichlorophenazine-5, 1 0-dioxide). In certain preferred embodiments. the phenazine is a I -hydroxyphena_ine. more preferably a I -hydroxyphenazine-5, 1 0-dioxide. In certain preferred embodiments, the phen~7ine compound is able to better stabilize a positive charge compared to unsubstituted phenz~ine.
In another aspect, the invention provides a packaged drug, comprising a composition including a phenazine compound in a container, packaged with instructions for ~lmini~tering the composition to a subject for treating an infection due to a microorganism in the subject, which microorganism may be drug-resistant. In a SUBSTITUTE SHEET (RULE 26) W 09~27969 4 PCT/CA97100978 r~l.ed embodiment, the phçnzl7ine compound is a phen~7ine-N-oxide, including a phf-n:~7ine 5-oxide, a phçn~7ine-1 0-oxide~ or a phenazine-5,1 0-dioxide. In certain preferred embo~liment~7 the phenazine compound is a 7,8-disubstituted phenazine compound. including, without limitation, a 7,8-dimethylphenazine or a 7,8-5 dihalophenazine (e.g., a 7?8-dichlorophen~7ine, such as 7,8-dichlorophenazine-5,10-dioxide). In certain preferred embo(limçnt~ the phenazine is a 1-hydroxyphen~7ine more preferably a I -hydroxyphenazine-5. 1 0-dioxide.
In still another embodiment, the invention provides a method for treating a subject having a tumor, the method comprising ~mini~tering to the subject an effective 10 anti-tumor amount of a ph~:n~ine compound. such that the tumor is treated.

Detailed Description of thc Invention Antimicrobial drugs are the mainstay of treatment and control of infectious diseases in hnm~n~ and ~nim~l~. Antimicrobial agen~s have been ei7ec~ e against all 15 classes of pathogens: bacteria~ fungi, viruses and parasites. Ncvertheless. nc~ classes of antimicrobial molecules with therapeutic potential are still required to provide treatment for pathogens that have or will becomc resistant to e.Yistin~ antimicrobial agents The problcm of resistant microorg~ni~ms has been kno~n since the firs~ uses of antibiotics in the 1 920s. but in the last decade the number of org~ni~m~ resist~nt to currently available 20 antimicrobials has increased at an accelerated rate. So alarming has been this increase in microbial resict~nce that many governmcnt and nongovernment scientific or~ni7:~tions have published guidelines and position statements to draw attention to this problem (see.
e.g.~ Lcderberg J. et al. Emerging Infections: Microbial threats to health in the United States. Washington. DC: National Academy Press. 199'').
~5 Preventing microbial infections can be accomplished bv vaccination (e.g.,pertussis. measles)~ physical isolation of infected individuals from the rest of the cornrnunity (e.g., tuberculosis. measles. chickenpox). personal cleanliness (e.g., salmonellosis). reducing personal lifestyle risks (e.~.. Hl~'. I lepatitis C). proper food h~n~ling (e.g., enterohemorrhagic E. coli 0157.~I7), disposal of waste (e.g., salmonella.
30 shigella. cholera), m~int~inin~ clean. fresh. potable water (e.g., cholera). or use of antimicrobial agents for prevention (e.g., malaria) or for treatment of active disease (e.g., meningitis. pneumonia).
Preventive measures, while critically important in controlling infectious diseases, are not 100% effective and therefore antimicrobial agents will always be very important 35 in treating or preventing infectious diseases. This is especially true with an ever exr~nfling population of citizens undergoing aggressive treatment for diseases which, as a consequence of the disease itself, or the treatment, are immune compromised (e.g.
cancer chemotherapy, systemic lupus. AIDS, transplantation).

SUBSTITUTE SHEET (RULE 26) W O 98/27969 PCT/C~97/00978 _ 5 _ Despiee a wide variety of antimicrobial agents effective against bacteria. fungi, viruses and parasites, resistance of these microorgAni~m~ to currently availableantibiotics is increasing. Several recent examples (by no means exhaustive) are I ) penicillin-resistant Streptococcus pneumoniae (PRSP); 2) methicillin-resistant 5 Stap~ylococctls aureus (MRSA); 3) vancomycin-resistant Enterococci (VRE); 4) cephalosporin-resistant Enterobacteriaceae; 5) malarial parasites resistant to chloroquine. mefloquine and other AntimAlArials; 6) fungi resistant to azoles, triazoles.
and amphotericin B: 7) herpes simplex virus resistant to acyclovir and related drugs: and 8) HIV resistant to zidovudine and other antiviral drugs. The skilled artisan will 10 appreciate that MRSA are actually multidrug resistant bacteria (not merely resistant to methicillin); however, for convenience, these orgAnicmc are referred to herein as MRSA.
Similarly~ multidrug resistant S. epidermidis is conventionallv referred to as methicillin-resistant S. epidermi~lis; the latter terminology is used herein for convenience.
The causes of drug resistance can varv from one organism to another and from I ~ one drug to another. For examplc. it is generallv accepted that MRS~ and VRE are resistant to beta-lactarns and glvcopeptides. respectively. due to alterations in cell-wall proteins.
The present invention provides a class of antimicrobial drugs that is not being widely used for the treatment of infcctions and which shows significant antimicrobial ~0 properties against a broad range of microorganisms. bacteria~ viruses~ fungi and parasites~ some of ~vhich are known to be highl~ resistant to currentl~ available antimicrobial a~,cnts. The invention also provides phenazinc compounds useful as anti-tumor agents. The phenazine compounds will pro- idc neu- therapeutic options for the treatment of infections~ both those susceptible and those resistant to currentl~ available ~5 antimicrobial agents.

For con-enience~ certain terrns used throughout the spécification and claims uhich follou are defined.
The terrn "microorganism" as used herein~ refer to microbes~ including disease-30 causing microbes~ such as bacteria~ yeast. fungi. viruses~ and parasites (such as thesporozoan malaria). (The terms "microbe" and "microorganism" are used interchangeably herein) As the skillcd artisan will appreciate, the efficacy of the ~ phenazine compounds of the invention against microorgAnism~ will vary; that is, not every phenazine compound will have activity against all microorgAni~m~. A phenazine 35 compound having activity against a particular microorganism can be selected through use of assays such as are known in the art, including the assays described herein.
The term "inhibiting the growth of a microorgAni~m~ll as used herein, refers to the process of preventing, arresting, slowing, or decreasing the growth of a selected SUBSTITUTE SHEET (RULE 26) W O 98t27969 6 PCT/CAg7/00978 microorg~ni~m Thus, "inhibiting the growth of a microorganism" includes both destruction (i.e., killing~ of a microorg~ni~m, e.g., a microbial cell, and inactivation of a microbe, e.g., a virus, arresting, reducing or elimin~ting the ability of the microbe to grow, reproduce, or cause infection of a subject. A tre~tment capable of "decreasing 5 infective ability," as used herein, refers to a treatment which kills, inactivates, or otherwise inhibits or prevents a microorganism from infecting an uninfected subject or contimling to infect an infected subject. Exemplary microbes include, but are not limited to: Hemophilus spp. (including H. influenzae. H. parainfluen~-ae,and H.
aphrophilus), E. coli, ~nterob~cter spp, Enterococcus spp. (including E faecalis, E.
10 faecium, E. gallinarum, and E. cassilflavus), Ci~robacter spp., Pro~eus spp.. Morganella spp., Shigella spp., Salrnonella spp. (including S. tvphosa and S tvphimerium). ~eisseria spp., Burk~zolderia spp., Campylobacter spp.. ~ersini~l spp. (including ~ pestis and ~' entercolitica), Helicobacter spp. (including H pvlori'), T'ihrio spp. (including T' cholera and 11 ~~ulnificus). Staphvlococcz~s spp. (including coagulase-positive and coagulase-15 negativc Staphvlococcus spp. (including ~S epidermidi.s) and MRSA). .S~reptococcus spp.(including Strcptococci of the viridans group and ~S. pneum0niae (including penicillin and multidrug-resistant)), Chlamvdia spp (including C pneumoniac and C.
trac~omatis), A~vcoplasma spp. (including AI. pneumoniae), Corvnebacteriz~m spp.(including C jeikeium and C. xerosis), and Clostridium spp. ~including C~ difficile), 20 Nocardia spp., Actinom~ces spp.. Cr~ptococcus spp.. Histoplasma spp., Candida spp..
Torulopsis spp.. Blastomvces spp., C'occidioides spp. (including C. immitis), and Aspergillu~ spp. Such e,~emplary microbes also include multidrug resistant strains of the bacteria and fungi, including azole-. triazole- and amphotericin B-resistant strains of the fungi.
~5 The term "subject," as used herein. rcfers to a an animal. preferably a warm-bloodcd animal. more preferably a m~rnm~l, including. e.g.. canle. sheep, dogs, cats. and hnm~n~.
The term "effecti- e amount" is art-reco~nized. and. as used herein. refers to an amount of a phenazine compound which is effective to achie- e a desired result, e.g., 30 killing of a microbe. An "effecti~ e amount" of a particular phenazinc compound will vary according to such factors as the phenazine compound. the microorganism to be affected, the weight, age and condition of the sub3ect ~if any), and thc like. The skilled artisan will be able to determine an effective amount of a particular compound to achieve a particular result in accordance with standard practices of the ph~ eutical 35 art and the te~-:hings herein. In certain preferred embodiments, the phen~7ine compounds of the invention have a minimum inhibitory concentration (MIC) of lessthan about 5 ,ug/mL, more preferably less than about 10 ~lg/mL. more preferably less than about 5 ~Lg/mL. and most preferably less than about I ,ug/mL~ against at least one SUBSTITUTE SHE T (RULE 26) selected microor~,qnism, preferably including a bacterium, which can be a drug-resistant bacterium.
The term "treating" is art-recognized, and. as used herein, refers to ameliorating or preventing a microbial infection and/or at least one symptom associated therewith.
S For exarnple, treating a subject includes eradicating or arresting a microbial infection in a subject, preventing or slowing infection of the subJect by a microore,qni~m decreasing the progression or extent of microbial infection in a sub~ect (e.g., the duration, severity, or extent of tissue infected), and the like.
The term "contacting," as used herein, refers to both in ~itro and in vivo 10 contacting. The term "targeting," as used herein. refers to causing a micro-organism to be treated. e.g., by contacting the microor~anism with a phenazine compound.
The term "phenazinc compound" is kno-vn in the art and. as used herein. refers to substituted and unsubstituted compounds having the phenazinc (dibenzo- I . I-diqzine) nucleus, as wcll as analogs. deri-ativcs. and con_eners thereof: and salts thereof 15 (pref'erably pharmaceutically acceptable salts). Thus. the term "phert t~ine compound"
refers to the phenazine compounds set forth herein. includin~ nitrogen oxidcs ofphen,t~ines (referred to hcrein as "phenazinc N-oxides." and including phenazine-5-oxides, phenzine-10-oxides. and phenazinc-5~10-dioxides). Phenazine compounds can be substitutcd. e.g.. on thc phenyl rings, with one or more substitutents selected from the '~0 ~roup consisting of halogcn. all;yl. aryl, all;oxy. hydro.Yy. amino (including substituted amino), cyano, trifluoromethyl. carboxy, alkoxycarbonyl. aminocarbonyl, and the like.
Particularly preferred phenazine compounds include phenazine-l~'-oxides. including a phenazine 5-oxide. a phenazine-10-oxidc. or a phen~ine-5.10-dioxide. In certain preferred embodiments. the phenazine compound is a 7,8-disubstituted phenazine ~5 compound. including, without limitation. a 7.8-dimethylphenazine or a 7.8-dihalophenazine (e.g., a 7.8-dichlorophcnazine. such as 7.8-dichlorophenazine-5.10-dioxide). In certain preferred embodiments. the phenqzine is a l-hydro.Yyphenazine~
more preferablv a l-hydro:cyphenazine-5.10-dioxide.
A "drug-resistant microorganism" refers to a microorganism which is resistant to30 at least one drug which is commoniy used for treatment of infections caused by that microorg~tnicm, or which is effective against the ~vild-type microorganism. A
"multidrug resistant microorganism" is a microorganism which is resistant to two or more drugs. For example, many bacterial species have acquired resistance to older antimicrobial compounds such as penicillin. Other bacterial strains have acquired 35 resistance to all but a few of the drugs commonly used to combat infection; for example.
strains of Enterococcus spp. have been reported which are resistant to vancomycin, a preferred treatment for certain E~tterococcus spp. infections. The term "drug-resistant organism", as used herin. refers to any drug-resistant or multidrug resistant ore,qnism, SUBSTITUTE SHEET (RULE 26) e.g., as described herein. A drug-resistant organism may be resistant to one or more drugs ~depending on the organism) selected from the group consisting of vancomycin (and related glycopeptides such as teichoplanin and ramnoplanin), ampicillin (and related beta-lactarns), aminoglycosides (such as gentarnicin, streptomycin, tobramycin, S arnikacin, and kanamycin), macrolides (such as erythromycin and chloramphenicol), tetracycline, metronid~ole, cephalosporins. carbapenems, monobactams, fluoroquinolones, ~oles, tri~oles. and arnphotericin Band the like.

Phenazine Compounds As early as 1860, the production of a blue pigment by the gram-negative bacillusPseudontonas aer2~ginosa has been described (Fordos. Compt. Rend. 1860;51 :215).This was deterrnined to be a phenzl7ine molccule called pyocyanin (1-hydroxy-5-methyl-phen~7ine). During the ensuing 137 years. the structure of many naturally occurring phen:-~inec. their physiochemical properties~ and their cffects on prokaryotic and 15 eul;aryotic cells have been described. More than 50 phen~inc compounds have been described in the scientific literature and cven more variously substituted phenazines havc been dcscribed in patents (J. Berger. "Microbial phen:l~ines" in Antibiotics, Chemotherapeutics Scientific. and Antibacterial Agents for Disease Control. M.
Grayson. Editor. John Wiley and Sons, New Yor~;. 1982. pp. ~55-275) Many ofthe 20 phen~7ines described are produced by microorg7~nicmc and these orE~nicmC are the sole source of naturally occurring phen~ines. A wide ran~e of organisms produce phe~7i~es including P.seudomonas spp.. Brel~i*acl~?riZ~m spp.. Soran~ n~ spp., .5~rep~om~ces spp.. .SrreplosorangiZ~n~ spp.. ~Iierohispora spp.. and IVorearc~iaeeae.
Some phenazines have been found to possess antimicrobial. antiviral. antifungal.~5 an~iparasitic. and/or antitumor activity. The degree of antimicrobial activity against selectcd pathogens is highly variable. depending upon the phen~ine structure (Weigele ~1. et al.. Antimicrobial A~ents and Chemotherapy. 1970~ 6-49; and Milchenko ~;.. et al.. Mikrobiol. Zh (Kie~). 1976;38(1)~ lowe-er. prior to the prescnt in~ention.
phenazines have not been tested against recent clinical isolates of pathogens that are 30 rcsistant to currently available antibiotics Chemical Charaeteristies of Phena in~s All phen~inf c include the dibenzo- 1,4-di~ine skeleton. Many substitutions have been described in the scientific literature or in patents. An important subgroup to 35 point out are the 5, 10-N, N-dioxides of phen~ine and rclated compounds which have been found to possess some of the greatest antimicrobial activity. However, other phen~?7ines, including 1-hydroxyphen~7ine and pyocyanin, which are simple phenazine derivatives~ also possess significant antimicrobial activity.

SUBSTITUTE SHEET (RULE 26) Some phenazines are freely soluble in water (e.g., pyoeyanin), while others are known for their insolubility in non-polar and polar organic solvents (e.g., iodinin, myxin). The phen~7ines possess important redox properties whieh eonfer upon them the ability to funetion as aeid-base indieators or to interaet with the eleetron transport chain 5 of mieroorg~ni~mc (Bissehop A, et al., European Journal of Bioehemistry. 1 g79; 93:369-374).

Biosynthesis of Phena7ines Many substituted phenazine eompounds ean be easily extraeted from baeterial 10 eulture broth, either from the supernatant or as an insoluble preeipitate. These biosynthesized phenazines ean be purified and. if not possessing antimierobial activity, modified chemically into a moleeule with antibiotie properties (Weigele M~ et al. ibid~
Gerber N. Bioehemistry 1965;4(1):176-180). The synthesis and purifieation of a wide variety of phen~7ine compounds is well within the ability of one having ordinary sl;ill in I ~ the art using no more than routine experimentation.
The genetics of phenazine biosynthesis have been examined. and the phz R
(phe~ 7ine regulator) operon ha.s been cloned ~nd sequenced and shown to eontrolphenazine pigment production in response to environrnental stimuli (Pierson LS III~
lieppenne VD and Wood DW, Journal Bacteriology 1994; 176(13):396-374: and 20 Pierson LSII and Thomashow LS~ Mol. Plant. Mierobe. Interact 1992;5:330-339).
Antihiotic ~ffec~s of Phencl_ines It has been known for over 100 years that cenain phen~7ine compounds possess antimicrobial activity. Antibiotic activity has been deseribed against Gram positi- e and 25 Gram negative baeteria. as well as fungi~ yeasts~ viruses and p~rasites (Berger~ J.~ 1982 ibid: Bouchard C.C.R. Acad. Sci. Paris; 1888: 108:713; and Emmerlich R.. and Loeu O.~ Zbl. B~;t. 1899; 26:237). Hayes et al. isolated a crude e.~;tract of ~'seuc~omonas acru~inosa cultures that included pyocvanin (Hayes et al.~ Journal of BiologiealChemistry 1945). They showed that pyocyanin had antimicrobial acti- ity ~vhen 30 ~tlministered or~lly, subeutaneously and intravenously. Other studes from 1924 to 1989 have shown that pyoeyanin ean be purified from ~seudomonas aeruginosa eulture supernatants and that pyoeyanin has antibiotic activity against Gram positive and Gram negative bacteria including Staphylococcus aureus, ~Streptococcus pyogenes, Bacillus subtilis, Corynebacterium diphtheriae, and Neisseria gonorrhoeae, Yersinia pestis, and 35 Salmonella t~phimurium (Schoentel R.. Brit. J. Exp. Pathol., 1941; 22:137-147; Chang PC and Blackwood AC, Can J. Microbiol., 1969; 15:439; Baron, S., Rowe, J.~
Antimicrobial Agents Chemother, 1981; 20:814-820). Miee have been shown to tolerate 100 mg/kg intraperitoneally of pyocyanin. Pyocyanin is bactericidal. A closely related SUBSTITUTE SHEET (RULE 26) degradation product of pyocyanin, 1 -hydroxyphenazine (1 -HP), also known as hemipyocyanin, has been shown to possess activity against Baeillus subtilis, Staphyloeoccus aureus, and Candida albieans, at concentrations ranging from 10 to 100 ,~Lg/ml. T imite~l toxicity data suggests 1-hydroxyphenazine is less toxic in animals than 5 pyocyanin, where mice tolerated 10 mg intraperitoneal injections and chicken heart fibroblasts did not show toxic effects at 0.1 mg/ml of 1-HP ~Gerber. N., J. Org. Chem.
1967; 32:4055; ~choentel, R., Br. J. Exp. Pathol. 1941).
Iodinin was first isolated as a purple pigment in 193~ from a culture of Brevibaeterium iodinum This phenazine compound possesses the structure of 10 phenazine-5, 10-N. N-dioxide. Many substituted forrns of this basic skeleton have been described. some with signficant antimicrobial activity (Weigele. M., Antimicrobial Agents in Chemotherapy, 1971). Iodinin is notoriously insoluble in aqueous solution unless sodium hydroxide or sulfuric acid is added. Iodinin is easily produced from cultures of Br~ibacterium iodinum, .S~rep-on~vcc s t~liolutes, ~I 'aksn~ania aerata~
15 Nocardia hydrocarbonoxydans Pseudor/lonas aureofaciens and Pseudom~nas phena-inium as an insoluble precipitate. Methylation ~ ith dimeth~ l sulfate in all;ali or with diazomethane yields the best known of the phenazine antibiotics. myxin.
Iodinin is itself a weak antibiotic with minimum inhibitory concentrations (MIC)against Staphvlococcus aureus, E coli ~seudomonas aen-ginosa and Candida albicans 20 greater than 100 ~lg/ml, although some reviews cite a MIC of 0.08-0.5 ~lg/ml against Corynebacterium fimi (Weigelc, M., 1971 ~ Ber~er. J .. 1982) . Iodinin has not been used as an antibiotic in animal studies. It has been tested as an antihypertensive at doses of 0.1 -300 m~ll;g body weight orally in humans. althou~h it has nevcr been brought to marl~et as a treatment for hypcrtension. The LD50 is reported to be greater than I g/kg '~5 bodv weight (Jones, P. and Somani, P, U.S. Patent 3,764.679, October 9, 1973 to Abbott Laboratories ).
Myxin has been marketed as an antibiotic. although it is currently no lon~er produced. Mvxin. the l-methoxy deri-ative of iodinin. uas described in Canada as a product of Sorangium sp (strain 3C~ in 1966 (Peterson EA, Gillespie, DC, Cook FD, 30 Can. J. Microbiol. 1966; 1 :~21-230). It was shown to have a very broad spectrum of action against Gram positive and Gram negative bacteria as well as many fungi and plant pathogens (Peterson EA, et al., 1966, Weigele, M., 1971). ~inimllm inhibitory concentrations against Streptoeoeez~s agalactia~, Staphylococcus aureus and E. coli are less than 4 llg/ml, and against Candida albicans, I X ~lg/ml. Myxin is bactericidal.
35 Myxin was produced as a copper complex and form~ ted for topical use in veterinary skin infections. Unfortunately, myxin shows significant toxicity in mice with an LD50 of 40 mg/kg inll~peli~oneally~ 3.75 mg/kg intraperitoneally in mice was not effective SUBSl-ITUTE SHEET (RULE 26) W O 98/27969 ~ PCT/CA97/00978 against bacterial infections. However~ a topical cupric complex of mycin was found to be useful in bacterial and fungal pyoderma in f~nim~l.s.
~ ome 30 derivatives of iodinin and myxin have been described, several of which have significant antimicrobial activity, while others do not. Of particular interest are the 5 1 -methyl and I -methoxy derivatives of phenazine-5, 1 0-N,N-dioxide. which. according to Weigele, M. et al., possess antimicrobial activity equal to or exceeding that of myxin.
Lomofungin (also known as lomondomycin), the 6-formyl trihydro-l-carboxyphena_ine derivative~ with many highly substituted derivatives~ has been described (Bigge, C.F.~ et al.. U.S. Patent 4~657,909. April 14. 1987, Warner-Lambert 10 Company). A wide variety of compounds synthesized using the lomofungin nucleus is described in the above mentioned patent; however. the antimicrobial activity is not especially high. being up to 1000 times less potent than that of the phena_ines mentioned previously.
Griseolutein ~ and B. products o f Streprom~ cc.s griscollltet~.s. are also known to 15 have antibiotic activit~ although griseolutin B is more potent (Tausig~ F.~ Wolf. FJ.
Miller. AK Antimicrobial Agents and Chemotherap~ 196~:59: C~sato~ T.~ Maeda~
Ume7~wa. H.. J. Antibiot. 195~: 7:15: and Nakamura. S.. Maeda 1;. and Ume~wa. H..
J. Antibiot. 1964; 17:33) Griscolutein E3 has documented inhibitory conccntrations against ~. coli of 11 ~lg/ml. S. aureu.s of 0.~ ~lg/ml and Salmonellc~ ~allinarium of 0.7 20 g/ml. Interestingly, griseolutein B has documented efficacv in treating S. ~ ureus infections in mice~ with effective doses of 0.95 mg/kg intraperitoneally, ~3.5 mg subcutaneously and 850 m~ orall~ . The diacetyl derivative of yiseolutein B is 5 times more active against Stap~locc)ccz~s atJret(.s infections by thc oral routc than griseolutein B itself.
-Phena_ine Deri-atives For 7rearntent of AIicrohial I~?fection It has now been found that phe~7ine compounds are active antimicrobial agents against a variet~ of dru~-resistant organisms. and that certain phen~7ines are also active against wild-type microorganisms. We have tested selected strains of bacteria that 30 commonly cause human infection against several representativc phenazine compounds.
As described in more detail below (see. e.L~,., Examples 8 and 9, infra) phena_ine compounds of the invention are active antimicrobial compounds against a variety of microbes, including drug-resistant bacteria. fungi, molds, and the like.
The compounds of the invention can also be employed for purposes such as 35 selective gut decont"rnin~tion. Gut decont~min~tion is useful for preventing microbial infections in immunosuppressed patients; for example. a patient about to receivechemotherapy can be treated with a phenazine compound of the invention to decont~min~te the gut, to thereby prevent infection by gut pathogens after the patient has SUBSTITUTE Sl IEET (RULE 26) received chemotherapy. In this embodiment. it is preferred to use a phenazine compound which are not significantly absorbed through the gastrointestin~l wall, this increases the effective concentration, and therefore the apparent potency, of the ph~n~7ine compound in the gastrointestinal tract.
It would be apparent to one of ordinary skill in the art in light of the te~hings herein, that phenazine compounds which are not significantly absorbed through the gastroint.ostin~l wall, but rather are retained at or near the mucosa~ would be useful for the treatment of infections due to microorg~ni~mc which cause gastrointestinal diseases.
including, without limitation, C. dif~icile (diarrhea and colitis) and H pylori (gastric and duodenal ulcers).
In a related embodiment~ topical application of a phenazinc compound according to the invention, can be used to decont:~min~te the mouth, throat~ and upper rcspiratory tract to prevent microbial infection. For example~ infection bv microorg~nicm~ resident in the mouth can result in pneumonia in a susceptible indi- idual. especially if the patient is recei~ing respiratory support from a ventilator.
It has been found by the present invcntors that phenazine N-oxides te.g.~
phenazine-5-oxides. phenazine- I 0-oxides. and phen zine-5. 1 0-dioxidcs) are of ~en more active against microorg~ni~ms than the corresponding phenazines. Accordingl~. in a preferred embodiment. a phenazine compound of the invention is a phen~7ine-N-oxide, most preferably a phenazine-5~ 1 0-dioxide.
Without wishing to be bound by any theor~ it is believed th~t the phenazine compounds of the invention may exert an antimicrobial acti- ity at least in part due to the ability of phenazine compounds to undergo electron transfer rcactions, ~vhich rcsult in the forrnation of toxic activc oxygen species at thc site of action in ~ o. with rcsultant darnage to infcctious microorg~ni~mc (see~ e.g.~ I' W. Crawford et al. Chem. Biol.
Interact. ( 1986) 60:67-84~. The ability of a phenazinc compound to undergo clectroreduction in Vil~O may therefore bc correlated ~~ith the antimicrobial activit~ of thc phPn~ine compound (compounds with more positi~e rcduction polenti~ and which show rcversible reduction waves~ are often found to have greater antimicrobial activit~ ).
Accordingly~ in certain preferred embodiments~ a phenazine compound of the invention can be substituted, e.g., on the phenazine nucleus~ with a substituent(s) which increases the ability of the phenazine compound to be reduced in vivo. For exa~nple, in certain preferred embodiments, a phen~7ine compound of the invention includes at least one substituent which is capable of stabilizing a radical cation (e.g., of a phenazine) or radical anion ~e.g., of a phenazine-5, 1 0-dioxide, see, e.g., M.D. Ryan et al. J Pharm.
Sci. ( 1985) 74:492) resulting from a one-electron reduction of the phenazine nucleus.
Without wishing to be bound by theory, it is believed that such a radical anion (e.g., of a phenazine-S~ 1 0-dioxide) may be able to transfer electrons in a catalytic manner.

SUBSTITUTE SHEET ~RULE 26) Illustratively, the phen~7ine nucleus can be substituted, at the 7 and/or 8-positions, with electron-withdrawing substituents (such as, e.g., halogens, including fluoro, chloro~ bromo, and iodo), or methyl groups. Other groups which change the electron distribution of the ring structure are also acceptable. Especially preferrecl S embodiments of the invention are phenazine N-oxides which are substituted at the 7 and/or 8 positions. including, without limitation, a 7,8-dimethylphenazine or a 7,8-dihalophenazine (e.g., a 7,8-dichlorophenazine. such as 7,8-dichlorophenazine-5,10-dioxide). It is believed that substituents at the 7 and 8 positions of the phenazine ring system may reduce toxicity of the compounds in vivo (possibly analogous to toxic10 structure-function relationships of riboflavins) while ret~ining antimicrobial activity.
In certain preferred embodiments. the phenazine is a l-hydroxyphenazine. morc preferablv a l -hydroxyphen~zine-5~ 1 0-dioxidc. Without ~vishing to be bound bv any theory. it is belie~cd that the I -hydroxy group of a I -hydroxyphenazine- 1 0-oxide (or 5.1 0-dioxide ) mav interact with the 1 0-o:;idc oxygen atom to thereby render the 15 compound more bioacti-~e. possihl~ by enhancing charge transfer through hvdrogen bonding. Similarly, a 6-hvdroxy group may interact with the ~-oxide oxygen atom to enhance charge transfer Thus. in preferred embodiments. a phen~7ine compound useful in thc methods of the invention can be represented hy the structure:
(R3)m R~ ~ N ~3 ~0 in ~hich Rl and R, are selected independently for each occurrence from the groupconsisting of hydrogen. halogen ( including fluoro. chloro~ bromo. or iodo). Ic-~cr alkyl.
ar l (more preferably substituted or unsubstituted phenyl). hydroxy. Iower allioxy ~such as methoxy). mercapto. Iower alkylthio. nitro. cvano. amino~ lower all;ylamino.
~5 dial~;ylamino. and trifluoromethyl; R3 and R4 are selected indcpendently for each occurrence from the group con~istin~ of halogen. Iower alkyl, hydroxy, lower alkoxy, mercapto, lower alkylthio, amino. Iower alkylamino. dialkylamino. and trifluoromethyl:
m is 0, 1 or 2; and n is an integer from 0 to 4; and X is. independently for each occurrence, oxygen or a pair of electrons. In certain preferred embodiments, m and n are 30 each 0. In certain embodiments, at least one occurrence of X is oxygen; in particularly preferred embodiments. both occurrences of X are oxygen (i.e.. the compound is aphenazine-5.10-dioxide). In preferred embodiments, n is at least 1, and one occurrence of R4 is a l-hydroxy group (i.e., the compound is a 1 -hydroxyphenazine (optionally a I -SUBSTITUTE SHEET(RULE26) W O 98/27969 ~4 rCT/CAg7/0~978 hydroxyphenazine-N-oxide)). Rl and ~, can be the same or different; in certain embodiments, Rl and R2 are the sarne, and are both methyl or both chlorine. In some embodiments, the organism is a multidrug resistant org~ni~m, including a bacterium.
In ~r~ d embodiments, m and n are each 0 (i.e., the phenazine compound can S be substituted at the 7 and/or 8-positions, but is other~vise unsubstituted). Exemplary prefc~ d phen~ine compounds include 7,8-dichlorophenazine-5,10-dioxide (P5) and 7,8-dimethylphPn~7in~-5, 1 0-dioxide (P6).
It will be understood that a particular phen~7ine compound can be selected according to several criteria~ which will be routine to onc of ordinary skill in the art in 10 view of the teachings provided herein. For e:Yample. as described above. certain substituents on the phenazine nucleus are preferred to increase the antimicrobial potency of the phenazinc compound. In addition. substituents of the phenazine nucleus can be selected to improve thc solubility of the compound (e.g., by substitution with hydrophilic substituents such as amino. carbo.~;y, hydro.Yy. and the like). to improve the 15 biodistribution of the compound. and the like. One of o}dinar! sliill in the art will be able to select an appropriate phenazine compound using no more than routine experimentation in light of the teachings hcrein.
Moreover. the ef~lcacy of a particular phen~ine compound can be deterrnined.
e.g., by routine screening, e.g.. such as described infra. An effective antimicrobial 20 compound can therefore be readil~ selected for use against a selected microorganism of interest. ~ctivity of certain l;nown antibiotics is related to the ability of the antibiotic to forrn mctal complexes with metal ions; accordingl~. in certain embodiments. a phen~inP compound of the in~ ention is capable of forming a metal comple.~ (e.g., ~vith a metal center such as calcium. magnesium. zinc. iron. m~ng:~ncse, or copper) and/or is 25 ~ minictered to a subject in the form of a metal complex.
It has been found that the phenazine compounds of thc invention are active against bacteria (both Gram-positi~e and Gram-negative), including drug-resistant strains of bacteria. It is believed that the compounds of the in~ention are also acti~e against fungi, yeasts, parasites, and the like. Compounds of the invention exhibit both 30 bacteriostatic activit~,~ (i.e.. inhibit the ~rov.,th of a bacterium without l;illing the bacterium) and bactericidal activity (i.c.. killing the bacterial cell). In a preferred embodiment, a phPn~7in~ compound of the invention is ~lminictPred to a subject to have a bactericidal effect on the microorganism.
It has been found in preliminary experiments that phPn~7ine compounds 35 described herein are capable of inhibiting the growth of a hurnan tumor cell line in cell culture. Accordingly, the phen~7in~ compounds described herein are useful for inhibiting the growth of tumors and tumor cells. eithcr in vitro (ex vivo) or in vivo. In one embodiment, the invention provides a method for treating a subject having a tumor.

SUBSTITUTE SHEET (RULE 26) The method includes ~tlmini~tering to the subjeet an effective (preferably non-toxie) anti-tumor arnount of a phen~7ine compound, such that the tumor is treated.
Compounds of the invention can be obtained by methods well known to the skilled artisan. Certain ph~n~7ine eompounds are commereially available. or can be 5 obtained from cultures as described above. Additional compounds can be prepared by well known teehniques of organie synthesis (see, e.g., Examples 1-3, infra).

Pharmacezltical Cor~.~positions In another aspeet~ the present invention provides pharmaceutically acceptable 10 compositions which comprise a therapeutically-effective amount of one or more of the pherl,q7ine eompounds described above. optionally forrnulated together with one or more pharmaceutieallv acceptable carriers (additives) and/or diluents. As described in detail belou . the pharm,qcel-tical eompositions of the present invention may be specially formulated for administration in solid or liquid forrn. includin~ those adapted for the 15 follo-ving: ( I ) oral administration. for exqmple. drenches ~ aqueous or non-aqueous solutions or suspensions). tablets. boluses~ powders. ~ranules. pasles for application to the tongue; (')) parenteral administration. for example. h- subcutaneous. in~ramuscular or intravenous injection as, for example. a sterile solution or suspension; (3) topical applieation. for example, as a eream. ointment or spray applied to the skin; or (4) ~O intravaginal or intrarectal ~lminictration for example. as a pessary, cream or foarn.
The phrase "therapeutieally-effeetivc amount" as used herein means that amount of a compound. material. or composition comprisin~ a compound of the present invention which is effective for producinL~ some desired therapeutic effeet by treatin~
(i.e.. preventing or ameliorating) a bacterial. fungal. viral or parasitic infection in a ~5 subject. e.g., at a reasonable benefit/risk ratio applicable to any medical treatment.
The phrase "pharmaeeutically aeeeptable" is emploved herein to refer to those eompounds. materials. compositions. and/or dosa~e forms v~hieh are. ~ ithin the seope of sound medieal jud~ment. suitable for use in contact with the tissues of human beings and animals without exeessive toxieit~, irritation. aller~ie response. or other problem or 30 complication. commensurate with a reasonable benefit/risk ratio.
The phrase "pharrnaeeutically-acceptable carrier" as used herein means a pharmaceutically-aceeptable material, composition or vehicle~ such as a li4uid or solid filler, diluent, exeipient. solvent or encapsulating material. involved in suspending, or earrying or transporting a subjeet phenazine compound. e.g., from one organ, or portion 35 of the body, to another organ, or portion of the body. Eaeh earrier must be "aeceptable"
in the sense of being compatible with the other ingredients of the forrnulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: ( I ) sugars~ such as lactose, glucose and SUBSTITUTE S~IEET ~RUI E 26) W O 98/27969 -16- PCTt~ 97/00978 sucrose; (2) starehes, such as eorn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl eellulose and cellulose acetate; (4) powdered tr~ga-~nth, (~) malt; (6) gelatin, (7) talc; (8) excipients~ such as coeoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, S safflower oil, sesame oil, olive oil, eorn oil and soybean oil; (l û) glyeols, sueh as propylene glycol; (l l) polyols, such as glycerin. sorbitol, m~nnit-)l and polyethylene glycol; (12) esters, sueh as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents~ sueh as magnesium hydroxide and alllminl-m hydroxide; (15) alginie aeid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) 10 phosphate buffer solutions; and (~1 ) other non-toxic compatible substances employed in pharrnaeeutical formulations.
As set out above. certain embodiments of the present compounds can eontain a basic funetional group. sueh as amino or alkylamino. and are. thus. eapable of formin~
pharrnaeeutically-acceptable salts w ith pharmaceutieally-acceptable acids. The term I ~ "pharrnaeeutically-acceptable salts" in this respect. refers to the relati~el~ non-toxic, inor~anic and or~anic acid addition salts of compounds of the present invention. These salts can be p~epared in sit~ during the final isolation and purification of the compounds of the invention. or by separately reacting a purified compound of the in~ention in its free base form with a suitable organic or inorganic acid~ and isolating the salt thus 20 formed. Representative salts inelude the hydrobromide. hydrochloride. sulfate. bisulfate.
phosF~hale. nitrate. acetate. valerate. oleate. palmitate. stearate. Iaurate. benzoate. lactate.
phosphate. tosylate, citrate. maleatc. fumarate. succinate. tartrate. napthylate. mes~late.
~lucoheptonate. Iactobionate. and laur. lsulphonate s~l~s and the like. (See. C.L~.. Berge et al . ( 1977) "Pharmaceutical Salts". J pharnt. ~ci. 66:1-19) ~5 In other cases. the compounds of the present invention may contain one or more acidic funetional groups and. thus. are eapable of forming pharmaeeutieally-acceptable salts t ith pharrnaceutieally-acceptable b~ces. The term "pharmaceutically-acceptable salts" in these in.ct~nCes refers to the relati~el- non-to~ic. inor~anic and organic base addition salts of eompounds of the present invention. These salts can likewise be iO prepared in situ during the final isolation and purifieation of the compounds. or by separately reacting the purified eompound in its free acid form with a suitable base. such as the hydroxide, earbonate or biearbonate of a pharrnaceutically-acceptable metal cation~ with arnmonia, or with a pharmaceutically-acceptable organic primary, seeondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, 35 sodiurn, potassium, ealcium. m~necium, and aluminum salts and the like.
Representative organic amines useful for the forrnation of base addition salts include ethylamine, diethylamine, ethylen~ mine. ethanolamine, diethanolamine, piperazine and the like. (See, for example, ~erge et al., supra) SIJ~ ITE SHEET (RULE 26) Wetting agents, emulsi~lers and lubricants, such as sodium lauryl sulfate and m~necium stearate, as well as coloring agents, release agents, coating agents, sweet~ning, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Exarnples of pharmaceutically-acceptable antioxidants include: (1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate~ sodium metabisulfite, sodium sulfite and thc like; (2) oil-soluble antioxidants, such as ascorbyl palmitate. butylated hydroxyanisole (13HA), butylated hydroxytoluene (BHT), lecithin~
propyl gallate, alpha-tocopherol, and the li~e; and (3) metal chelating agents, such as citric acid. ethylene~ mine tetraacetic acid (EDT~), sorbitol. tartaric acid. phosphoric acid, and the like.
Formulations of the present invention include those suitable for orah nasal.
topical. transdermal buccal~ sublinguah rectah vaginal and/or parenteral administration.
The formulations may conveniently be presented in unit dosagc form and may be prepared by anv mcthods well l;nown in thc art of pharmacy. Thc amount of activcingredient which can be combined with a carrier material to produce a single dosage forrn will vary depcnding upon the host bcing treated~ and the particular mode of ~n~inistration. The amount of active ingredient which can be combined with a carricr material to producc a single dosage forrn will generally be that amount of the compound ~0 which produces a therapeutic effect. Generally. out of one hundred per cent. this amount will range from about I per cent to about ninetv-nine percent of active ingredient~
prefcrably from about ~ per cent to about 70 pcr cent~ most preferably from about 10 per cent to about 30 pcr cent.
Methods of preparing these formulations or compositions include the step of ~5 bringing into association a compound of the prescnt invention with the carrier and~
optionally. one or more accessor~ ingredients. In general, the formulations are prepared t~y uniformly and intimately bringin~ into association a compound of thc presentinvention ~ ith liquid carriers. or fincl- di~ ided solid carricrs~ or both~ and then~ if necessary. shaping the product.
Formulations of thc invention suitable for oral administration may be in the forrn of capsules, cachets, pills~ tablets~ lozenges (using a flavored basis. usually sucrose and acacia or tr~g~c~nth), powders, granules. or as a solution or a suspension in an aqueous or non-a~ueous liquid, or as an oil-in-water or water-in-oil liquid emulsion~ or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each cont~ining a predetermined amount of a compound of the present invention as an active ingredient. A
compound of the present invention may also be ~lminictered as a bolus, electuary or paste.

SUBSTITIJTE SHEET (RULE 26) In solid dosage forms of the invention for oral ~f~minictration (capsules. tablets.
pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more ph~ reutically acceptable carriers. such as sodium citrate or dicalcium phosphate, and/or any of the following: ( 13 fillers or extenders, such as starchest lactose, sucrose, glucose, mannitol, and/or silicic acid, (2) binders, such as, for example, carboxymethylcellulose, ~lgin~te~ gelatin. polyvinyl pyrrolidone. sucrose and/or acacia;
(3) humectants, such as glycerol; (4) disintegrating agents. such as agar-agar, calcium carbonate. potato or tapioca starch. alginic acid, certain silicates, and sodium carbonate;
(5) solution retarding agents. such as paraffin; (6) absorption accelerators. such as ~uaternary ammonium compounds; (7) wetting agents. such as~ for e~ample. cetyl alcohol and glycerol monostearatc; (8) absorbents. such as l;aolin and bentonite clay; (9) lubricants, such a talc. calcium stearate. magnesium stearatc~ solid polyethylene glycols.
sodium lauryl sulfate. and mixtures thereof: and ( 10) coloring a~ents In the case of capsulcs tablets and pills. the pharmaceutical compositions may also comprise buffcring 1~ agents Solid compositions of a similar type may also bc emplo- ed as fillers in soft and hard-filled gelatin capsules usin( such excipients as lactose or other mill; sugars. as well as high molecular weight polyethvlene glycols and the lil;e A tablet may be made by compression or molding. optionally with one or more accessory ingredients. Compressed tablets may bc prepared using binder (for example '~O gelatin or hydroxypropylmethyl cellulose), lubricant. incrt diluent. preser-ative, disintegrant (for example. sodium starch glycolate or cross-linked sodium carbo:;ymethyl cellulose), surface-active or dispersing agent Molded tablets may be made b~ molding in a suitable machine a mixture of thc po- dered compound moistened ~ith an inert liquid diluent The tablets~ and other solid dosage forms of the pharrnaceutical compositions ofthe prcsent invention. such as dragees. capsules, pills and granules. may optionally be scored or prepared with coatings and shells. such as en~cric coatings and other coatings ~ ~ell known in the pharmaceutical-forrnulatin~ art The~ ma- also be forrnulated so as to provide slow or controlled release of the active ingredient therein using. for example.
hydroxypropylmethyl cellulosc in varying proportions to provide the desired release profile other polymer matrices. Iiposomes and/or microspheres The- may be sterilized by, for exarnple, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water. or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s~ oniy, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner Examples of embedding compositions which can be used include polymeric substances and waxes SUBSTITUTE SHEET ~R~JLE 26) The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral :~firninistration of the compounds of the inventioninclude pharmaceutically acceptable emulsions. microemulsions, solutions, suspensions, 5 syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art. such as. for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol~
ethyl carbonate~ ethyl acetate, benzyl alcohoh benzyl benzoate, propylene glycol. 1,3-butylene glycol~ oils (in particular, cottonseed~ groundnut~ corn, germ~ olive, castor and 10 sesame oils), glycerol, tetrahydrofuryl alcohol, polyethvlene glycols and fatty acid esters of sorbitan, and mixtures thcreof.
Besides inert diluents. the oral compositions can also include adjuvants such aswetting agents~ emulsifying and suspending agents~ s~veetcning, flavoring, coloring"
perfuming and preservative agents Suspensions~ in addition to the acti- e compounds. may contain suspendin~
agents as. for example~ cthoxylated isostearyl alcohols. polyo~;vethylene sorbitol and sorbitan esters. microcrystalline cellulose. aluminum metah-dro:;idc. hentonitc. aL~ar-agar and traL~acanth~ and mixtures thereof.
Formulations of the pharm~re~ltical compositions of the invention for rectal or vaginal ~Aminictration may be presented as a suppository. which ma- be prepared by mixing one or more compounds of the invention with one or morc suitable nonirritating excipients or c~rriers comprising. for example. cocoa butter. polyethylcne ~Iycol~ a suppository ~ ax or a salicylate~ and ~ hich i!. solid at room temperaturc~ but liquid at body temperature and~ therefore~ will melt in the rcctum or vaginal cavity and release thc '5 active compound.
Formulations of the present invention which are suitable for vaginal :l-iministratjon also include pessaries~ tam}-ons. creams~ ~els~ pastes~ foams. troches~
vaginal tablets or spray formulations containin~ such c~Tiers as are l;no- n in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a compound of thisinvention include powders~ sprays. ointments. pastes~ creams. Iotions, g~els~ solutions, patches and inh~l~ntc. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier~ and with any preservatives, buffers~ or propellants which may be required.
The ointments, pastes, crearns and gels may contain, in addition to an active compound of this invention, excipients~ such as animal and vegetable fats, oils, waxes~
paraffins, starch, tr~g~r~nth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid~ talc and zinc oxide, or mixtures thereof SU~ ITE SHE~T(RULE 26) Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, alurninum hydroxide, calcium silicates and polyamide powder. or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted 5 hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the phenazine compound in the proper mediurn. Absorption enhancers can also be used to increase the flu?c of the subject phen~?ine across the skin.
10 The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the phenazine in a polymer matrix or gel.
Ophth~lmic formulations. eye ointments, powders. solutions and the like~ are also contemplated as being within the scope of this invention Pharmaceutical compositions of this invention suitable for parenteral 15 a lminictration comprise one or more compounds of thc invention in combination ~~fith one or more pharrnaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions. dispcrsions. suspensions or emulsions. or sterilc po~ders ~vhich may be reconstituted into sterile injectable solutions or dispersions just prior to use. which may contain antioxidants. buffers. bactcriostats. solutes which render the formulation isotonic '~0 with the blood of the inten(le~l recipient or suspending or thickcning agents.
Examples of suitable aqueous and nonaqueous c~rriers ~hich may be employed in thc pharmaceutical compositions of the invention include ~ atcr. ethanol. polyols ~such as glycerol. propylene glycol. polyethylene glycol. and the like)~ and suitable mi~;tures thereof. vegetable oils~ such as olive oil. and injectable or~anic esters. such as cth~ l oleate. Proper fluidity can be maintained. for examplc. b- the use of coating matcrials. such as lecithin. by the m~inten~nce of the required particle size in thc case ol dispersions. and by the use of surfactants.
These compositions may also cont~in adju-ants such as preser-~atives. ~ etting agents, emulsifying agents and dispersing agents. Pre~ention of the action of 30 microorg~nicms may be ensured by the inclusion of various antibacterial and antifungal agents. for example, paraben. chlorobutanol, phenol, sorbic acid. and the like. lt may also be desirable to include isotonic agents. such as sugars. sodium chloride, and the like into the compositions. ~n addition, prolonged absorption of the injectable pharm~centical form may be brought about by the inclusion of agents which delay absorption such as 35 ahlminllm monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline or amorphous material SUBSTITUTE SHEET (RULE 26) W O 98/27969 ~1 PCT/CAg7/00978 having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally-a~minictered drug forrn is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forrns are made by forrning microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide.
Depending on the ratio of drug to polymer. and the nature of the particular polymer employcd. the rate of drug release can be controlled. Exarnples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectablc formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
When the compounds of the present invention arc ~r~ministered as pharrnaceuticals, to humans and ~nim,.lc, they can be given per se or as a pharmaceutical composition containing. for example. 0.1 to 99.5% (more preferably, 0.5 lo 90%) of I 5 acti~e in~zredient in combination with a pharmaceuticallv acceptable carrier.
The preparations of the present invcntion may be gi~cn to humans or other ,.nim ~Ic for therapy by any suitable routc of administration. including orall y ~
parcnterally, topically, vaginally or rectally. They are of course given by forms suitable for cach ,7~rnitlictratioh route. For exampic. ~vhen they are ~nminictered oraiiy they are ~0 formulated. e.g., in tablets or capsule forrn: for parenteral aflminictration~ the formula~ion is suitable for injection or infusion; for topical administratiom including buccally and sublin~ually~ in the form of a po~der~ drop. Iotion or ointment: nasal administration in spray form; and for va~inal and rectal administration the composition including a pherl.~.7ine compound can be ,.~lminictered in the form of suppositories. Oral ~5 or topical a~lminictration is preferred.
The phrases "parenteral ,.~iminictration" and '~nminictercd parenterally" as used herein mean modes of :~nminictration other than enteral and topical administration~
usually by injcction~ and includc. without limitation. intra~cnous. intramuscular, intraarterial, intrathecal. intracapsular. intraorbital. intraocular. periocular. intracardiac.
intradermal. intraperitoncal. transtracheal. subcutaneous. subcuticular, intraarticulare.
subcapsular, subarachnoid. intraspinal and intrasternal injection and infusion.
The phrases "systemic ~minictration," "~nminictered systemically," "peripheral ~(lminictratjon" and "~r1minict~red peripherally" as used herein mean the a~1minictration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for exarnple. subcutaneous ~rlminictration.
Regardless of the route of zl~iminictration selected, the compounds of the present invention, which may be used in a suitablc hydrated forrn, and/or the pharmaceutical SlJ~:j 111 UTE SHEET (RULE 26) WO 98127969 ~2 PCT/CA97/00978 compositions of the present invention, are form~ t.-A into ph~ reutically-acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the ph~ ceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which 5 is effective to achieve the desired therapeutic response for a particular patient, composition? and mode of ~qAministration, ~,vithout being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed. or the derivative (e.g., ester, salt or amide) thereof, the route of ~-lministration, the time of aArninistration~
10 the rate of excretion of the particular compound being cmployed. the duration of the treatment. other drugs, compounds and/or materials used in combination with the particular compound employed. the age, sex~ wei~,ht~ condition. general health and prior medical history of the patient being trcated. and lil;e factors well l;nown in the medical arts.
A phvsician or veterinarian having ordinarv sl;ill in thc art can readil~ dcterminc and prescribe the effective amount of the pharmaceutical composition required. For examplc. the physician or veterinarian could start doses of the compounds of theinvention employed in the pharrnaceutical composition at levels lo- cr than that required in order to achieve the desired therapeutic effect and graduallv increase the dosage until 20 the desired effect is achieved.
In general~ a suitable daily dosc of a compound of the invention will be that amount of the compound which is the lowest dose effec(i- e to produce a therapeutic ef~ect Such an effecti- e dose will generally dcpend upon the f~ctors described above.
Generally~ doses of the compounds of this invention for a patient~ ~~hen used for the '~5 indicatcd effects. will range from about 0.0001 to about 100 mg per l;ilogram of body weight per day. more preferably from about 0.01 to about 50 m~ per kg per day. and still more preferablv from about 0.1 to about 40 m~ er lig per day.
If desired~ the effective daily dose ofthc acti-e compound m~y be ~ rinistered as two~ three~ four~ five~ six or more sub-doses ;lAministcred separately at appropriate 30 inter~als throughout the day, optionally, in unit dosage forms.
While it is possible for a compound of the present invention to be ~Anninisteredalone, it is preferable to ~lrninister the compound as a pharmaceutical composition.

Exemplification Ill2~strati7~e Preparation of Compounds The following compounds were obtained from commercial suppliers or prepared as described herein: I -hydroxy--5-methylphenazine (Pyocyanin, P 1 );

SlJt~ ITE SHEET ~RULE 26) W O 98127969 -23- PCTtCA97/00978 1--hydroxyph~n~ine ~P2); phenazine methosulfate (P3); l-hydroxyphen~7ine-5,10-dioxide (P4); 7,8-dichloro--1-hydroxyphenazine-5.10-dioxide (P5); and 7,8-Dimethyl-1--hydroxyphenazine-5, 1 0-dioxide (P6).
P1, P2 and P3 are commercially available:
P1, 1-hydroxy-5-methylphenazine. was obtained from Colour Your Enzyme (Bath, Ontario, Canada); P2, l-hydroxyphenazine, was obtained from TCI America (Portland, Oregon, U.S.A.); and P3, phenazine methosulfale, was obtained from Aldrich Chemical Co, Milwaukee. WI.
As described in detail below, P4 was synthesized by the method of Issidorides etal [1]. In the synthesis of P5, 5,~dichlorobenzofurazan oxide and its con~lcn~tion ~~ ith 1 ,2-cvclohexanedione was accomplished by a modification of the method of Issidorides et al [ 1~. In the synthesis of P6, the condensation of S~dimethylbenzofurazan oxide with l .2~yclohexanedione was accomplished by a modification of the method of Issidorides et al ~ l ].
Example I
Pr~paration of l ~ vdro.r.~/tenazi~ 5. I ()-dioxicl~ (P~) l 0., g of benzofurazan oxide, dissolved in 90 mL diethylamine. were added dropwise to 4.'' g of 1.2--cyclohexanedione dissolved in a 10 mL diethylamine in a 250 ~0 mL tw~necl; round bottom flask. The reaction mixture was stirred by a magnetic stirrer and the flask was kept in an ice-bath. After the addition of the benzofurazan oxide was completed. the mixture was stirred for 30 min in the ice bath. follo- ~ed hy additional stirring for l hr at room temperature. The mixture ~vas transferred into a 500 mL round bottom flask. diluted with about 100 ~ crushed ice and acidified by drop-vise addition of ~5 ~lacial acetic acid. The brown-red solid obtained was filtered. washed with water and dried in a vacuum oven at 50~C for 2-3 hr.
5.0 g of the crude product ~vas dissolved in a minimum amount of benzene (50-80 mL) and oxidized by addition of a warm benzene solution of '--chloroperbenzoic acid (5.0 ~ in 50 mL benzene) to it. The reaction mixture was stirrcd at room temperature for 12 hr and transferred to a 500 mL separation funnel. The bcnzene solution was extracted three times with a total of 100 mL of 10% aqueous Na-~SO3 in order to reduce 3--chloroperbenzoic acid to 3-chlorobenzoic acid. Six additional extractions were r carried out with a total of 200 mL of 10% of aqueous NaHCO3 in order to make 3-chlorobenzoic acid water soluble and thus remove it from the benzene layer. The benzene layer was dried with 10 g of anhydrous Na2SO4 or MgSO4 for 10 min.
Evaporation of the dried benzene solution gave a dark red solid. The solid was dissolved in 200-250 mL of 10% aqueous NaOH with stirring and heating. The resulting blue solution was acidified with glacial acetic acid ~iving a bright red solid SUBSTITUTE SHEET (RULE 26) product. The solid was filtered and dried in a vacuum oven at 40~C for ~5 hr.
Recryst~11i7~tion from methanol-chloroforrn yielded about 0.9 g (12% yield) ofthe pure product. Dark red needles melted at 182--184~C, TLC confirrned the homogeneity of the product and FT--IR, 1 H NMR and UV-VIS spectra were consistent with reported spectra ~1]:
IRI~max3400,3100, 1590, 1550? 1470, 1400, 1380. 1345, 1320, 1300, 1260~
1245, 1095, 1060, 1040, 880, 835. 800~ 770, 750, 655 cm-1. 1H NMR 7 0 9 (m, 2H) 7.7 (m, 4H), 8.3 (m~ 2H) Example 2 Preparatio~z of 7 ~-dichloro-l-fzydrox~7~hena_ine-5 10-dioxidc (P5) a Sl~nlhesi~s ~7f5 ~ ichlor~hen-ofi~ra a~z oxide 5~dichlorobenzofurazan o~ide was svnthesized by the method used by Green and Rowe [2] to svnthcsi7P some benzofurazan oxides b- a hypochlorite o:~idation of o-nitroaniline derivativcs. Two other methods E3-~J- w hich were adapted from this 1912 procedure of Green and Rowe~ are reported for synthesis of several other derivatives of benzofurazan oxides.
A mixture of 10.5 g of KOH and 125 mL ethanol was stirred and heated on a water bath until the solid dissolved. I ~ of 4~5--dichloro~nitroaniline was dissol- ed in ~5 mL alcoholic TiO~I in a round bonom flasl;. The resulting red soiution ~vas stirred orously and cooled to 0~C in an ice bath; ~5-30 mL of 5% sodium hypochlorite solution was then added dropwise until the red colour had disappeared. The flocculent pale vellow precipitate was collected hy filtration using a Buchncr funnel. The crude product was washed with 15 mL water and dried in a vacuum oven at room temperature.
0.8 g of crude product was recrvstallized from dilutcd ethanol~ ~iving pale vellow crystals with a sharp melting point at 110~C.

b. Condcn~ation of ~, S~ichlor~ en of zJra-a~70xi~e ~if~z I 2-cl~cln~zcxa~7~dione This con~içn~tion was carried out by a modification of the method of Issidorideset al [1]. 0.6 g of 1.2-cyclohexanedion was added in a 100 mL two neck round bottom flask and dissolved in 3 mL triethylamine. The mixture was cooled in ice, stirred vigorously, and a solution of 1.95 g 5 ~dichlorobenzofurazan oxide dissolved in 30 mL
triethylamine was added dropwise. After the addition was complete, the reaction mixture was stirred for 10 hr in ice bath followed for additional 8 hr at room temperature. The mixture was diluted with 10 g crushed ice and acidified by dropwise addition of 25 mL glacial acetic acid. The brown- red solid was filtered, washed with Sl.lt~ 111 LITE SHEET ~RULE 26) water and dried in vacuum oven at 50~C for 5 hr. TLC indicated purple and olive green products.
0.35 g of crude product was dissolved in benzene and oxidized by addition of a warm solution of 0.71) g 3-chloroperbenzoic acid in 8 mL of benzene to it. The reaction 5 mixture was refluxed for 40 min. The solution was extracted with 10% aqueous Na2SO4 followed by a extraction with 10% aqueous NAHCO3. Dried benzene solution was evaporated giving dark red-purple solid. Recryst~11i7~tion yielded thc pure product with melting point of 207- 209~C. FT-IR. NMR and UV-VIS spectra are in agreementwith the reported ones [1]:
IR vmaX 3400~ 3085, 1620, 1580. 1540, 1490~ 1450 1420, 1395, 1355, 1340, 1240~ 1 _5. 1~05. 1180, 1140. 1110. 1095. 1060, 1040, 910. 900. 830. 785. 735. 720, 665~ 635 cm~ H NMR 7.5 (m) ExamPle 3 ~'reparation of ,.~-dir~clh~ drc).~ph~>na~ c-5. 1()-dioxidc fl'6) a. .~ nthe.~i.s of ~ . 6~imel h~ l~en-l~filrcl ~In ~7xidc~

2.0 g of 4,5-dimethyl~nitroaniline was dissolved in 70 mL alchocolic KOH.
Red solution was cooled to 0~C in ice bath and intensi~cly stirred; 30 mL of 5% sodiurn hypochlorite solution was added slowl-~ until the red colour had disappeared. The yellow precipitate was filtered. washed uith water and dried at room temperaturc. 1.5 g of crude product was recrvs~llized ~i~ in~ ~ cllo~ cr~ stals with mcltin~, point of 1 ~6~C.

h. Conden.~crtion of ~ 5-~imelhl lher?_ofilra~an ~7xidc .~ith 1. ~-c~clo~?exanedion~
This condcnsation was carried out by a modification of the method of Issidoridese~al. [1].
5.6 g of 4.5-dimeth~ lbenzofuraz~n oxide was dissol~ ed in 110 mL dieth~ l~nine and added dropwise to 2.0 g of l .2-c- clohcxancdione durin~ ~ igorous stirrin~. After thc addition of the solution was completed. the mixturc was stirred for an sdditional 3 hr at room temperature. The mixturc was diluted with crushed ice and acidified withglacial acetic acid. The brown-red solid was filtered, washed with water and dried.
TLC indicated presence of red and olive green products.
1.7 g of the crude product was dissolved in a minim:~l amount of benzene and oxidized by dropwise addition of a warm benzene solution of 2.7 g 3-chloroperbenzoic acid (in a minim~l amount of benzene). The reaction mixture was refluxed for 24 hr.
The solution was first extracted with 10% aqueous Na2SO3 followed by extraction with 10% aqueous Na~CO3. Evaporation of the dried benzene solution produced a dark red solid which was treated with 10% aqueous NaOH. The solution was acidified with SUBSTITUTE SHEET ~RULE 26) W O 98/27969 ~6 PCTICAg7/00978 glacial acetic acid, producing 0.8 g red solid. Recry~t~l1i7~3tion of the crude product yielded the pure product having a melting point of 179--180~C. FT-IR, 1 ~ NMR, and UV-VIS spectra were in agreement with reported spectra fl]:
IR vmax 3400, 3100. 2900, 1620, 1550, 1500, 1475, 1410, 1385, 1340, 1280, 1265, 1180, 1135, 1100, 1060, 1040, 1010, 915, 805, 750, 680 cm-1. lH NMR 2.4 (s, 6H), 7.5 (m, 6H) Re ferences 10 1. C.H. Issidorides. M.A. Atfah.3.J. Sabounji. A.R. Sidani and M.J. ~ lin~
Tetrahedron, 34 (1978) 217--221 ? A.G GreenandI;.M.Rowe, J.Chen.. Soc. 101(191'')245?--?459 3. R.J. Gaughran. J.P. Picard and J V.R. ~ fm~n J. Am. Chem. 5'oc 76 (1954) ?733_??36 ~. F.B. Mallory. C)rg. ~5'vnth. Coll. Vol. IV (1963) 7-~ 7 E.Yample 4 .Su*ceptibili~l~ Tc.~tin~ of ~erol7ic Rac~ericl Suscep~ibility testing for aerobic bacteria was carried out usin~ either macrobroth dilution or microbroth dilution methods. It will bc appreciated that auger dilution or disk diffusion methods can also be lltili7f d The drug to be tcsted was prepared in absolute ethanol and dilu~ed such that thefinal working concentration of ethanol was lcss than 5~~O of the final ~vorking volume~ so ~5 as to minimi7f any effects of thc ethanol on thc microbial assay. The antibio~ic was diluted using cation-supplemented Mueller-Hinton broth (BBL~ Becton Dickinson Diagnostics~ Sparks~ MD). Two-fold serial dilutions of the antibioiic were preparcd in the concentrations indicated in Tables I and ~ belo~A. The antibio~ic dilutions were prepared immediately prior to the assay and not stored at their dilutions.
Preparatio~, of Inoculum A final inoculum of 5 x 105 colony-fo;ming units (CFU) per mL was used.
Isolates were inoculated into Mueller-Hinton broth and incubated until turbid and the turbidity ad3usted to match a 0.5 McFarland standard. Alternatively, 4-5 isolated colonies from overnight growth on Tryptic soy agar or Tryptic soy blood agar plates were directlv suspended into broth to match a turbidity of 0.5 McFarland standard This latter method was used for testing Staphylococcus aureus and Enterococcus species. A
portion of suspension was diluted 1: 100 with broth and I mL of this final dilution was SUBSTlTUTE SHEET (RULE 26j added to each tube cont~ining 1 mL of the prediluted drug so that a final inoculum of 5 x 105 CFU/mL was achieved. Tubes without antibiotics were likewise inoculated as growth controls and uninoculated broth was used as a sterility control.
A sample of a final inoculum was routinely serially diluted and plated in duplicate to determine colony counts to verify accuracy of the inoculum.
All inoculated tubes were incubated in room air at 35~C for 6-20 hours and this was extended to 24 hours for vancomycin-resistant Enterococci and methicillin-resistant Staphylococci.
For all MIC tcsting, appropriate ATCC control org~nicm~ were run in parallel as l O indicated below. The end points were sharp and there were no trailing end points detected. No skipped tubcs were ever detected. The lowest concentration that completely inhibited visible growth with the organism hy the unaided eye was recorded as the minimum inhibitory concentration.

I~Iicrobroth Dilution ll1ethodfc)rAerohic Organisn~.s Stocl; antimicrobial solutions are prepared as indicated abo~ e. Between 0.05 or0. l mL are dispensed to the wells of the microbroth dilu~ion tra~ . These travs are stored in plastic at -70~C.

lnoculation A final desired inoculum of S ?; l O5 CFU/mL is us~d. The inoculum is p.~ cd similarl~ to above b~ either direct colonies or from colonies ~ro~vn in broth prior to inoculation. A 0.5 Mc~'arland suspension of the test organism is diluted l: l O in broth and bet-veen O.OOl and 0.005 mL is inoculated into wells cont:-ining 0.1 mL of broth.
When a 0.05 mL of broth per well system is used~ 0.05 mL of inoculum is used. which is prepared by l: l 00 dilution of a 0.5 McFarland suspension. The inoculum density is routinely checked hy plate counting and an aliquot of the inoculum is routinel~ plated out to checl; for purit~. Wells not containinL~ antimicrobial a~ents are used as ~ro-~th controls.
Plates are covered with plastic tape or se~led in a plastic bag and incubatcd at35~C for 6-24 hours.
Quality control strains as mentioned above are used. The end point MIC is the same as for macrobroth dilution. Gro~vth is determined by comparison with that in the growth control well and it is indicated by turbidity throughout the well or by a button.
single or multiple, in the well bottom.
Quality control strains used are Escherichia coli ATCC 25922, Pseudo~770nas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213. and E. coli ATCC 25922.

SUBSTITUTE SHEET (RULE 26) Determination of Minimum Bac~ericidal Concentration After the minimllm inhibitory concentration was deterrnined. the miniml~m bactericidal concentration (MBC) was determined by subculturing tubes which had no visible growth, or, in the case of microtiter plates, wells that have no visible growth. In the case of macrobroth MB~s, after 20 hours of incubation the tubes were re-vortexed and incubated for an additional 4 hours and then 100 ,u~ of the broth was removed and spread across agar plates with a bent glass rod~ in duplicate.
In the case of microbroth MBC determination, 100 IlL is aspiratcd and dispensed to the surface of agar plates and spread evenly using a bent glass rod.
The MBC is defined as the lowest concentration of antibiotic that reduces the inoculum hy 99.9% within ''4 hours.

References 15 1. National Cornmittee for Clinical Laboratory ~tandards. 199~. Methodology forthe serum bactericidal test. Tentative Standard M~l-T. National Committee for Clinical Laboratorv Standards. Villanova. PA.
2. National Comrnittee for Clinical Laboratory Standards. 199~. Methods for deterrnining bactericidal activity of antimicrobial agents. Tentative Standard M26-T. National Committee for Clinical Laboratory Standards. Villanova. PA.

E~aml~le ~

~5 ~usceplihilih~ ~eshng of ,4naerobic Bac~cria The antibiotics are prepared as discussed above.

Inoculum Preparation The inoculum of anaerobic org~ni~mc is prepared by suspending colonies taken 30 from a 24-7' hour anaerobic blood agar plate directly to Thioglycolate broth or Brucella broth to a density of a 0.5 McFarland standard. Alternatively. initial suspension is prepared by inoculating S colonies into enriched Thioglycolate medium and incubating for 4-6 hours or occasionally overnight for slow-growing org~ni~ms. This suspension is then diluted to a density of a 0.5 McFarland standard.
Media The media for agar dilution anaerobic susceptibility testing is Wilkins-Chalgrenagar or Brucella agar base. Where appropriate, Schaedler's, Wilkins or Brain-Heart SUBSTITUTE SHEET (RULE 2~;) infusion broth are used. The broths are supplemented with vitamin K1, 1 ,ug/mL and Hemin 5 ,~Lg/mI,. Defibrinated sheep blood or ~ysed sheep blood is a~so used where ~ U~JI iate to supplement Wilkins-Chalgren agar and Brucella agar base. Both of these supplements are used at a final concentration of 5%. Where required, 1% Tween 80 for S Gram positive cocci, and rabbit serum or 2-3% Iysed horse blood for pigmented org~ni~m~, are used. The plates are not stored for more than 7 days prior to use.

Incul~ation Conditions Anaerobic jars with disposable hydrogen-carbon dioxide generators and palladium coated catalvst pellets are routinely used. The incubation atmosphere contains 4-7% carbon dioxide. Incubation is at 35~C for 48 hours. Control plates~
microdilution trays or broth macrodilution tubes are divided in duplicate, where one is incubated anacrobicall- as a growth control and the other incubated aerobically as a 15 cont~min~nt controi .

AS~ar Dilutions for Susceptibilit~ Testing oJAnaerobcs In agar dilution testing thc final inoculum is 105 CFU/spot.

'~0 Bro~hMicro<lilutionAnaerobicSuscep~ihili-~ Tes~ing Microdilution trays are prepared and frozen. and frozen trays are held at 70~C for up to 6 months prior to use. The final ~olurne pcr uell is not less than 100 ~L.The antibio~ics arc prepared as described above. The inocula are prepared similarly as for agar dilution with the final inoculation in each well of l o6 C~U/mL.
~5 The microdilution trays are pre-rcduced b~ holding them in an anaerobic environment for 2~ hours prior to inoculation.
Control uells include a ~ ell u ith broth but no drug as a ~routh control and anuninoculated well as a sterilit- control.

30 Interpretation of Results For anaerobic agar dilution testing, the end point is read as the concentration at which there is the most marked change from the growth control. This change is manifested as no growth, or as tiny colonies or a s1ight haze, or as significantly ~iimini~he~l ~rowth compared to that of the control. In broth microdilution, MIC35 anaerobic determination trays are examined with indirect transmitted light. That concentration at which the most significant reduction in growth is observed is chosen as the end point. This is either complete inhibition of growth or tiny, gradually ~limini~hing button of growth.

SUBSTITUTE SHEET ~RULE 26) The following quality eontrol strains are used for anaerobic suseeptibility testing:
Bacteroidesfiagilis ATCC ~5285, Bacteroides thetaiotaomicron ATCC 29741, and Eubacterium lentum ATCC 43055.

S References for Aerobic ~Example ~ and Anaerobic fExample 5) Susceptibility Testing 1. National Committee for Laboratory Clinieal Standards. 1993. Performanee Standards for Antimierobial Disk Susceptibility Tests. Approved Standards for Villanova, PA.
National Comrnittee for Clinieal Laboratory Standards. 1993. Methods for dilution antimierobial susceptibility tests for baeteria that grow aerobieally. Approved Standard M7-A3. National Comrnittee for Clinieal Laboratorv Standards, Villanova, PA.
3. National Committee for Clinieal Laboratory Standards. 1993. Evaluating production lots of dehydrated Mueller-Hinton agar. Tentative standard M6-T.
National Committee for Clinieal Laboratory Standards. ~'illanova~ P~.

4. National Committee for Clinieal Laborato.~ Stand~rds. 1994. Development of invitro susceptibility testing criteria and quality control pqrameters. NCCLS
doeument M~3-A. National Committee for Clinical Laborato~y Standards.
Villanova~ PA.

5. National Committee for Clinical Laboratory Standards. 1993. Methods for antimicrobial suseeptibility testing of anaerobie bacteria. 3rd Edition. Approved standard M I 1 -A3 . National Committee for Clinical Laborato.~ Standards~
Villanova. PA.

~5 Example 6 ~Iethoclology ff)r Testing SuscerJtibilit~ of Alycobacteria spp.
Souree of inoeulum: The source inoeulum for mycobaclerial susceptibility testing is derived from a mycobacterial subculture using an indirect method. Inoculum 30 is grown on Middlebrool; 7~I10 or 7HI I agar or another egg-based medium, but the sample is taken less than 4 weeks after growth on the solid media. Turbid growth in a liquid medium such as Middlebrook 7H9 broth or in BACTEC 1 B media is also used,as appropriate.

35 Media used Middlebrook 7H10 agar supplem.onted with oleic acid-albumin-dextrose-e:~t~l~ce (OADC) is used as a standard medium for susceptibility testing of slow growing myeobacteria by the proportion method. Where required to support growth of more SUBSTITUTE SHEET (RULE 26) W O 9~27969 -31- PCT/CA97/00978 fastidious strains~ Middlebrook 7H11 medium is substituted. The BACTEC method ~BBL, Becton Dickinson Diagnostics, Sparks, MD) is used preferentially for all susceptibility testing against mycobacteria. This uses an enriched Middlebrook 7H9 broth cont~ining 4 ~LCi of C-14 labelled palmitic acid per vial (also known as BACTEC
5 7B media).
The antibiotics are prepared as mentioned above. Generally, growth from a BACTEC 12B model of the test organism is used where it reached a growth index of500 or higher. After mixing to break up clumps, turbidity is adjusted to match aMcFarland standard of 0.5 and a 1:100 dilution of the final inoculum is used as a 10 control. Dilutions are prepared in distilled water or BACTEC diluting fluid but not broth. Purity is determined by platin~ the inoculum to blood agar and 7H 10 plates.

Incuhation BACTEC l~B vials arc pre-run in a BACTEC ~60 instrument in order to 15 establish a gas phase of 5% CO~ in air in the headspace of the ~ ial. An~ ~ ial ~ ith initial grouth inde.~c greater than or equal to ~0 is rejected. Eacll pre-run vial is inoculated ~ith 0.1 mL of the adjusted suspension of the test or quality control isolate and the ~ ials arc incubated at 37~C in the dark.
Thc~ACT~ vial~ are re~d on the 13ACTE~ ins~rument at 24 hour intervals for 20 a minimum of 4 d~ys and until thc control vial reached a growth index of ~reater than or equal to 30. When the gro-~,qh inde~; of the control is greater th~n or equal to 30 after a minimum of 4 da~ s~ the differencc in the ~ro-~qh indices from one datc to the ne:;t are examined. If the difference in grou-th inde~ of the control is ~reatcr than the differencc in the gro~th index of the drug. the organism is considered susceptible to the test dru~
~5 If the differencc in the grouqh index ofthe control is less than ~he difference in the gro- th index of the drug~ it is classif~ed to be resistant. If the grourth index is greater than or equal to 500~ and then is 500 on the ne.~t reading~ the isolate is considercd resistant to that drug regardless of the chanL!c in grouth inde.~;. If the change in differences in grouqh index between the control and the test drug are close and uithin 30 10% of each other~ thc trials are repeated.

Quality Control Referenced strains of known susceptibility pattern are tested with each new batch of drug and BACTEC 1 2B media. Quality control tests are perforrned with each test 35 isolate. M. tuberc2llosis ATCC''7294 (H37Rb) strain is used as a control.

SlJ~ JTE SHEET (RULE 26) W O 98~796g -32- PCTICA97/00978 Example 7 Susce~tibilitv Testin~ of Yeasts and Fun~i Test Medium A completely defined synthetic medium such as RPMI 1640 with L-glut~m~te cont~ining a pE~ indicator without sodium bicarbonate is the medium of choice. It is buffered to a pH of 7.0 at 25~C. The buffer used is morpholinepropanesulfonic acid (MOPS) at a final molarity of 0.165 for pI~ 7Ø
Stock Drug Solu~ions Solutions of antibiotic to be tested are prepared at 10 times the highest concentration to be tested. ~11 test solutions are freshly prepared.

rreparation of Inocula Spectrophotographic methods are used to prepare thc inocula. The test organisms arc grown on Sabouraud a~ar at 35~C and subcultured twice~ thus ensuring viability and purity. The inoculum suspension is created b~ selecting 5 isolated colonies of at least I mm in diamcter and suspending them in 5 mL of sterile 0.85% saline.
'~O Turbidity is measured at 530 nanometers and the culture is adjusted with saline to provide a 0.5 McFarland barium sulphate standard cquivalent. This resulted in a suspension of 1-5 x I o6 organisms per mL~ ~vhich is furthcr diluted ~000 times with I media~ giving a final test inoculum of 0.5-~.5 x 103 organisms per mL.
A 0.5 mL sarnple of the preparcd antibiotic solution is addcd to 4.5 mL of ~5 medium: for each of compounds E'1-P6 a I :10 dilution is similarly prcpared. A two-fold drug dilution scheme is used. The 10 times strength antibiotic solutions are dispensed in 100 ~lL volumes in round-bottom~ pol~styrene. sterile tubes and inoculated by addin~
900 ~L of the corresponding diluted fungus or ~ east inoculum. The growth control tube received 900 ~L of the inoculated suspension and 100 ~LL of the drug-free medium. A
quality control organism is tested in the sarnc manner and is included each time a test isolate is tested. 1 mL of uninoculated drug-free media is included as a sterility control.
The tubes are incubated at 35~C for 50 hours in ambient air and observed for thepresence or absence of visible turbidity or growth. Where appropriate~ incubation time is extended to 75 hours.
The minimnm inhibitory concentration endpoint was determined visually by F~x~minin~ for turbidity and the minimnm inhibitory concentration determined to be the lowest concentration of the test drug that subst~nti~lly inhibited the growth of the org~ni~m In general, this was the lowest concentration preventing any visible growth.

SIJ~ 111 ~)TE SHEET (RULE Z6) W O 98/27969 33 PCT/CAg7/00978 Alternatively, by visual ex~nin~tion~ thc MIC was defined as the lowest drug concentration reducing growth by 80% relative to the growth control.
Control or~ni~mc tested are Candida albicans ATCC90028 and Candida albicans ATCC90029, Candida parapsilosis ATCC900 l 8, Cryptococcus neoformans 5 ATCC90112 and ATCC90113, and Torulopsis glabrata ATCC90030. Candida parapsilosis ATCC22019 and Candida krusei ATCC6258 are also used as appropriate as reference strains.
A microbroth dilution method similar to the above but scaled down to microbroth dilutions is also used where convenient.
For testing filamentous fungi, the above method is altered as follows. The inoculum is prepared from growth on potato dextrose agar after 7 days at 35~C. Aconidial suspension in ~ mL of sterile 85% saline is prepared and the turbidity of the suspcnsion measured using a spectrophotometer at 530 nanometers and adjustcd to provide a conidial suspcnsion of 0.5-5.0 x l O~ C~U/mL.
Daily ex:~min:ltion of the tubcs for growth is carried OUI~ and w hen ~ isible growth is seen. each tube is vortexed f'or l O seconds to allow for the detection of small amounts of growth. Growth in tubes is compared to the s~eril:: control and scored as either showing no growth. 75% reduction in growth~ 50% reduction in growth. ~5%
reduction in ~rowth~ or no reduction in growth. The minimum inhibitory conccntration 20 for fun~i (rnolds~ is deterrnined as the lowest drug conccntration ~ hich inhibited 75% or more of the L~rowth compared to the growth control.

Refercncc I . National Committec for Clinical Laboratory Standard~. 199~. Reference Method'5 for Broth Dilution Susceptibility 1'csting of ~ east: Proposcd Standard. NCCLS
document M27-P. National Committee for Clinical Laborator~ Standards.
Vill.~nova. PA.

Examp~le Testing of Phena ine Compolmds Several compounds were tested to determine the minimum inhibitory concentration against a variety of bacteria. The results are shown in Table 1.

SU~STITUTE SHEET(RULE 26) Table 1:
~riniml~m Inhibitory Concentration (MIC) & Minimum Bactericidal Concentration (MBC~ of Selected Bacterial Pathogens to 4 Phenazine Compounds Bacterial Strain Pyocyanin 1-hydroxy- Phenazine Phen~7ine N.
(Pl) phenazine (P2) methosulfate N-dioxide ,ug/ml ,ug/ml (P3~ llg/ml ,ug/ml S. aureus29213 18.8/37.5 6-12.5P60 37.5/>300 >166 5. aureus 25923 30/150 15/>60 37.5/>300 94 5. aureus 30/nd 15/nd 60/nd nd (approximately 25 clinical strains) ~. coli. ~59~2 37.5/75 >60/>60 18.8/75 1~8 E. f~lecali.s ~921-' ~5/>300 75/>60 75/>300 ~1~5 r aerl(ginosc~ >300 >60/>60 300/>300 188 Methicillin- 12/~5 '1~.5/<25 12.5-~5/nd 2166 resistant S aure~s (MRSA) Methicillin- 12/25 <1'.5/<12.5 51''.5/nd 166 resistant S
epi~rmidis ( MRSE) Vancomycin- ~6/50 1 ~ .5/25 50- 100/>~00 nd resistant F,ntcr~( <~ccus (VRE) 5 Results are presented as MIC/MBC
nd= No data It can be seen from Table 1 that l-hydroxyphen~ine appears to be effective against several microorg~ni~m~1 with an MIC against S aureus of 6-12 ~g/ml and an 10 MIC against F:nterococci of 1~'.5-25 ~Lg/ml. 1 -hydroxyphena_ine is clearly bactericidal against S aureus, as well as MRSA and multidrug-resistant S. epidermidis. Most significantly, 1-hydroxyphen~7in~ is ver,v active against vancomycin-resistant Enterococci(V RE) and MRSA, with an MIC of 12.5 ~Lg/ml and a miniml-m bactericidal concentration of 25,ug/ml. These are 2 org~ni~m~ which are very difficult to treat SUBSTITUTE SHEET ~RULE 26) W O 98/27969 35 PCT/CA97tOO978 because of their resistance to beta-l~ct~m~. and in the case of VRE, resistance to vancomycin and related glycopeptides. MRSA, MRSE and VRE are increasing in ~requency and therefore any drug with activity against these pathogens could seewidespread applicability in human and veterinary medicine.
l-methoxyphen~7ine was tested in this assay and was found to be a relatively poor antimicrobial compound.

Exam~le 9 Compounds P 1 -P6 were tested against a sample panel of microorganisms to determine the effect of additional substituents on the phenazine nucleus on the antibacterial spectrum.
The rcsults are shown in Table Table 2:
Minimum Inhibitor Concentration (I\IIC) of Compounds P I -P6 A~ainst Selectecl Bacteria Bacterial P I P' P3 P4 P5 P6 Strain ~g/ml ~g/ml ~/ml ~Lg/ml ~g/ml ~/ml ~S. aureus 18-37 6-15 75 1.6 0.8 0.8 (methicillin-susceptible ) ,S. aurcus 1-' 512 '5 1.6-3.1 0.8 0.8 ( methicillin-resistant) ~S. epiclerrJzidis 12 ~6-1~ 12.5 1.3 <0.8 ND
E. faccalis ''5 25 7S-100 1.6 3.1 3.1 ( ~ ancomycin-susce~tible) E. faecalisand ~6-25 12-50 50-100 1.6-3.1 3.1 ND
E;: faecium (vancomycin-resistant) P. aeruginosa >300 >60 300 >100 12.5-25 12.5 E. coli 37.5 >60 18.7 <3.1 12.5 12.5 ND=No data SlJ~a l l l ~JTE SHEET (RULE 26) It can be seen from the data in Figure 2 that compounds PS
(7,8--dichloro-1-hydroxyphen~ine-5,10-dioxide) and P6 (7,8-dimethyl--1-hydroxyphenazine-S, I 0-dioxide) are especially effective against the tested bacterial strains, and that in several cases P5 and P6 are as effective against drug-resistant strains S as against the non-resistant wild-type or~ni~m~. Accordingly, PS and P6 (or equivalents thereof. including 7,8-disubstituted phen~ines) are particularly preferred compounds.

Example 10 Acute Toxicitv Studies Six mice were given compound P4~ mixed with a polvethvlene glycol carrier, in doses of 100 mg/kg, 500 mg/kg and 1000 mg/kg orally (two mice per group). Thc mice were observed for 48 hours. No acute toxicity was observed.
Another group of six micc werc ~mini~tercd compound P4 mixed with polyethylene glycol in doses of 100 mg/l;g, 500 mg/l;g~ and 1 00V mg/l;g by intraperitoneal injection (two mice per group). The micc were observed for 48 hours.
No significant toxicity was observed at 100 mgikg~ but some to~cicity was seen at 500 and 1000 mg/k~.

~0 Those skilled in the art will recognizc. or bc able to ascertain using no more than routine e~perimentation. numcrous cquivalents to the specific proccdures described hcrein. Such ec~ui~ alents are considered to be within the scope of this in~ention and are covered by the following claims.
The contents of all cited references (including literaturc references. issucd patents~ published patent applications~ and co-pending patent applications) cited throughout this application ( including the Bacl;ground Section I are herebv e!cpressly incorporatcd by reference.
Other embodiments are within the following claims.
What is claimed is:

SUBST3TUTE SHEET (RULE 26)

Claims (25)

1. A method for treating an infection due to a multidrug-resistant microorganism in a subject, the method comprising the step of administering to a subject in need thereof an effective amount of a phenazine compound, or a pharmaceutically-acceptable salt thereof, such that the infection is treated.

2. The method of claim 1, wherein the phenazine compound is a phenazine N-oxide.

3. The method of claim 2, wherein the phenazine N-oxide is a phenazine 5,10-dioxide.

4. The method of claim 3, wherein the phenazine N-oxide is a 7,8-disubstituted phenazine 5,10-dioxide.

5. The mcthod of claim 1, wherein the multidrug resistant microorganism is a bacterium.

6. The method of claim 4, wherein the bacterium is selected from the group consisting of Hemophilus spp.. E. coli. Enterobacter spp., Citrobacter spp., Proteus spp., Morganella spp.. Shigella spp.. Salmonellc spp.. Campylobacter spp.. Yersinia spp.. Helicobacter spp.. Yihrio spp.. Staphylococcus spp.. Enterococcus spp..
Streptococcus spp.. Corynebacterium spp.. Neisseria spp.. Burkholderia spp., Chlamydia spp.. Mycoplasma spp.. and Clostridium spp.

7. A method for treating an infection due to a microorganism in a subject, the method comprising the step of administering to a subject in need thereof an effective amount of a phenazine compound, or a pharmaceutically-acceptable salt thereof, such that the infection is treated, wherein the microorganism is selected from the group consisting of Henzophilus influenzae. Hemophilus parainfluenzae, Hemophilus aphrophilus. Campylobacter spp., Helicobacter pylori. Vibrio spp.. .Streptococci of the viridans group, Cryptococcus spp.. Histoplasma spp., Candida spp.. Torulopsis spp., Blastomyces spp., Coccidioides spp.. Nocardia spp.. Actinomyces spp., and Aspergillus spp., coagulase-negative Staphylococcus spp., and Corynebacterium jeikeium.

8. A method for treating an infection due to a microorganism in a subject, the method comprising the step of administering to a subject in need thereof an effective amount of a phenazine compound, or a pharmaceutically-acceptable salt thereof, such that the infection is treated. wherein the phenazine compound is represented by the formula:

in which R1 and R2 are selected independently for each occurrence from the group consisting of hydrogen. halogen, lower alkyl, phenyl, hydroxy, lower alkoxy, mercapto, lower alkylthio, nitro, cyano, amino, lower alkylamino, dialkylamino, and trifluoromethyl;
R3 and R4 are selected independently for each occurrence from the group consisting of halogen, lower alkyl, hydroxy, lower alkoxy, mercapto, lower alkylthio, amino, lower alkylamino dialkylamino, and trifluoromethyl;
m is 0, 1 or 2;
n is an integer from 0 to 4: and X is, independently for each occurrence. oxygen or a pair of electrons.

9. The method of claim 8, wherein X is, for each occurrence, oxygen: and m and nare each 0.

10. The method of claim 9, wherein at least one of R1 and R2 is selected from the group consisting of methyl and halogen.

11. The method of claim 8, wherein the organism is a multidrug resistant organism.

12. The method of claim 11, wherein the organism is a bacterium.

13. The method of claim 11, wherein the organism is a fungus or a yeast.

14. A method for inhibiting the growth of a drug-resistant microorganism, the method comprising contacting the drug-resistant microorganism with an effective amount of a phenazine compound, such that growth of the drug-resistant microorganism is inhibited.

15. The method of claim 14, wherein the microorganism is a bacterium. and the phenazine compound has bacteriostatic activity against the bacterium.

16. The method of claim 14, wherein the microorganism is a bacterium, and the phenazine compound has bactericidal activity against the bacterium.

17. The method of claim 14, wherein the organism is a fungus or a yeast.

18. The method of claim 14, wherein the phenazine compound is a phenazine N-oxide.

19. The method of claim 18, wherein the phenazine compound is a phenazine 5.10-dioxide.

20. A method for targeting a drug-resistant microorganism in a subject for the purpose of treating an infection, the method comprising administering an effective amount of a phenazine compound to a subject in need thereof, such that the drug is targeted to a drug-resistant microorganism known or suspected to be present in the subject and the infection is treated.

21. The method of claim 20. wherein the phenazine compound also targets a non-drug resistant microorganism.

22. The method of claim 20. wherein the organism is a fungus or a yeast.

23. A method for significantly decreasing the infective ability of a drug-resistant microorganism, the method comprising contacting the drug-resistant microorganismwith an effective amount of a phenazine compound. such that the infective ability of the drug-resistant microorganism is significantly decreased.

24. A packaged drug, comprising:
a pharmaceutical composition including a phenazine compound in a container.
packaged with instructions for administering the pharmaceutical composition to a subject for treating an infection due to a drug resistant microorganism in the subject.

25. A method for treating a subject having a tumor, the method comprising administering to the subject an effective anti-tumor amount of a phenazine compound, such that the tumor is treated.