Note: Descriptions are shown in the official language in which they were submitted.
CA 02247348 1998-08-24
W O 98/27969 PCT/CA97/00978
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)
CA 02247348 1998-08-24
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~
CA 02247348 1998-08-24
W O 98/27969 3 PCTICA97/00978
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
W O 98/27969 7 PCT/CA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 8 PCT/CA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 PCT/CA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 10 PCT/CA97/00978
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
W 0 98/27969 -12- PCT/CA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 -13- PCTICA97/00978
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
W O 98/27969 -15- PCT/CA97/00978
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
W O 98/27969 -17- PCT/CA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 -18- PCT/CA97/00978
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)
CA 02247348 1998-08-24
W 098/27969 19 PCT/CA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 PCT/CA97/00978 -20-
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
W O 98127969 - 24 - PCT/CA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 -25- PCT/CA97/00978
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)
CA 02247348 1998-08-24
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
CA 02247348 1998-08-24
W O 98/27969 -27- PCTICA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 -28- PCT/CA97/00978
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~;)
CA 02247348 1998-08-24
W O 98127969 29 PCT/CA97/00978
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)
CA 02247348 1998-08-24
W O 98/27969 30 PCT/CA97/00978
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
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)
CA 02247348 1998-08-24
W O 98/Z7969 34 PCT/CA97/00978
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
77853
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)
CA 02247348 1998-08-24
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
27853
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)
CA 02247348 1998-08-24
W O 98/27969 -36- PCT/CA97/00978
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)
