U.S. patent application number 14/531048 was filed with the patent office on 2015-09-24 for antibiotic tolerance inhibitors.
The applicant listed for this patent is The General Hospital Corporation, Inc., Institut National de la Recherche Scientifique. Invention is credited to Francois Lepine, Biliana Lesic-Arsic, Laurence Rahme, Melissa Starkey.
Application Number | 20150266827 14/531048 |
Document ID | / |
Family ID | 46721423 |
Filed Date | 2015-09-24 |
United States Patent
Application |
20150266827 |
Kind Code |
A1 |
Rahme; Laurence ; et
al. |
September 24, 2015 |
Antibiotic Tolerance Inhibitors
Abstract
The present disclosure relates to benzimidazole-benzamide
derivatives, and the use thereof, e.g., to treat infections.
Inventors: |
Rahme; Laurence; (Brookline,
MA) ; Lepine; Francois; (Lavaltrie, CA) ;
Starkey; Melissa; (Philadelphia, PA) ; Lesic-Arsic;
Biliana; (Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The General Hospital Corporation, Inc.
Institut National de la Recherche Scientifique |
Boston
Quebec |
MA |
US
CA |
|
|
Family ID: |
46721423 |
Appl. No.: |
14/531048 |
Filed: |
November 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14000600 |
Aug 20, 2013 |
8877940 |
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PCT/US2012/026028 |
Feb 22, 2012 |
|
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14531048 |
|
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61445448 |
Feb 22, 2011 |
|
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Current U.S.
Class: |
548/307.1 |
Current CPC
Class: |
A61K 31/496 20130101;
C07D 235/28 20130101; A61P 31/00 20180101; C07D 405/12 20130101;
A61K 31/4184 20130101; C07D 263/58 20130101 |
International
Class: |
C07D 235/28 20060101
C07D235/28 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under Grant
No. AI063433 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1. A compound of Formula I: ##STR00012## or a pharmaceutically
acceptable salt thereof, wherein: Y is (CH.sub.2).sub.p, O, or a
bond; Ring A is aryl, heteroaryl, heterocycloalkyl, substituted by
1 to 5 R.sup.1; R.sup.1 is H, --NR.sup.aR.sup.b, NO.sub.2, or
--NHC(O)R.sup.c; or 2 R.sup.1 together with the carbon atoms to
which they are attached form a heteroaryl or heterocycloalkyl;
R.sup.2 is H, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, --NR.sup.aR.sup.b,
--NHC(O)R.sup.a, NO.sub.2, --CN, --SR.sup.a, --S(O).sub.2R.sup.a;
R.sup.a and R.sup.b are each independently H or C.sub.1-6 alkyl;
R.sup.c is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl; m is 1, 2, 3,
or 4; and p is 1, 2, 3, 4, 5, or 6.
2.-28. (canceled)
Description
CLAIM OF PRIORITY
[0001] This application, claims the benefit of U.S. Provisional
Patent Application Ser. No. 61/445,448, filed on Feb. 22, 2011, the
entire contents of which are hereby incorporated by reference.
TECHNICAL FIELD
[0003] The present disclosure relates to benzimidazole-benzamide
derivatives, and uses thereof, e.g., in the treatment of acute and
chronic infections.
BACKGROUND
[0004] Hard-to-eradicate, often untreatable, infections including
chronic wounds and infections of medical devices pose increasing
threats to human health worldwide. Such infections are often
refractory to antibiotics due to antibiotic resistant bacterial
cells, and/or to antibiotic tolerance of a subpopulation of
bacterial cells that are not antibiotic resistant mutants bin
rather "dormant" cells that survive antibiotic killing. Antibiotic
tolerance is defined as the ability of a fraction of a susceptible
bacterial population to survive exposure to normally lethal
concentrations of bactericidal antibiotics. According to the
existing paradigm, many chronic infections are therefore
unbeatable.
SUMMARY
[0005] The present disclosure provides, inter alia, MvfR inhibitors
that are compounds of Formula I:
##STR00001##
or pharmaceutically acceptable salts thereof, wherein the
constituent variables are defined herein.
[0006] The present disclosure further provides pharmaceutical
compositions comprising a compound of the disclosure and a
pharmaceutically acceptable carrier.
[0007] The present disclosure further provides methods of treating
an antibiotic-tolerant infection, e.g., a gram-negative infection,
e.g., with a compound described herein, e.g., as described below or
shown in FIGS. 2A-L, 3A-C, or 4, or a pharmaceutically acceptable
salt thereof. The methods can also include administering one or
more additional antibiotics as known in the art.
[0008] The present disclosure further provides methods of treating
an infection, e.g., a Gram negative infection, e.g., an acute or
chronic infection, with a compound described herein, e.g., as
described below or shown in FIGS. 2A-L, 3A-C, or 4, or a
pharmaceutically acceptable salt thereof. The methods can also
include administering one or more additional antibiotics as known
in the art.
[0009] The present disclosure further provides methods of
identifying compounds that inhibit antibiotic-tolerant
infections.
[0010] In one aspect, the present disclosure features compounds of
Formula I:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein:
[0011] Y is (CH.sub.2).sub.p, O, or a bond;
[0012] Ring A is aryl, heteroaryl, heterocyeloalkyl, substituted by
1 to 5 R.sup.1;
[0013] R.sup.1 is H, --NR.sup.aR.sup.b, NO.sub.2, or
--NHC(O)R.sup.c; or 2 R.sup.1 together with the carbon atoms to
which they are attached form a heteroaryl or heterocycloalkyl;
[0014] R.sup.2 is H, C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
--NR.sup.aR.sup.b, --NHC(O)R.sup.a, NO.sub.2, --CN, --SR.sup.a,
--S(O).sub.2R.sup.a;
[0015] R.sup.a and R.sup.b are each independently H or C.sub.1-6
alkyl;
[0016] R.sup.c is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl;
[0017] m is 1, 2, 3, or 4; and
[0018] p is 1, 2, 3, 4, 5, or 6.
[0019] In some embodiments Y is O.
[0020] In some embodiments, Y is O and Ring A is phenyl substituted
by 1 to 5 R.sup.1.
[0021] In another aspect, the present disclosure features a
compound, or a pharmaceutically acceptable salt thereof, having
Formula Ia:
##STR00003##
[0022] In some embodiments, the compound is:
##STR00004##
[0023]
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(4-phenoxyphenyl)aceta-
mide, or a pharmaceutically acceptable salt thereof.
[0024] In some embodiments, the compound is:
##STR00005##
[0025]
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(4-phenoxyphenyl)aceta-
mide.
[0026] In some embodiments, the present disclosure features a
composition comprising a compound of Formula I or I-a, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier. Also provided herein are methods for using
these compositions for the treatment of infections, e.g.,
antibiotic-tolerant infections, in a subject.
[0027] In another aspect, the present disclosure features methods
of treating an infection, e.g., an antibiotic-tolerant infection in
a subject. The methods include administering to the subject a
therapeutically effective amount of a compound of Formula I-b:
##STR00006##
or a pharmaceutically acceptable salt thereof wherein:
[0028] X is O or N;
[0029] R.sup.3 is H, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, --NO.sub.2,
--NHC(O)R.sup.c;
[0030] R.sup.4 is H, halo, C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
aryloxy, C.sub.1-6 haloalkyl, --CN, --NO.sub.2, --SR.sup.a; or 2
R.sup.4 together with the carbon atoms to which they are attached
from a heteroaryl or heterocycloalkyl;
[0031] R.sup.ais H or C.sub.1-6 alkyl;
[0032] R.sup.c is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl; and
[0033] n and q are each independently 1, 2, 3, 4, or 5.
[0034] In some embodiments, the compound is:
[0035]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-cycnophenyl)acetamide;
[0036]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(3-methylthio)phenyl)acetamide-
;
[0037]
N-(4-bromophenyl)-2-((6-methyl-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide;
[0038]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-chlorophenyl)acetamide;
[0039]
N-(4-ethylphenyl)-2-((6-methyl-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide;
[0040]
N-(2,4-dimethoxyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)a-
cetamide;
[0041] 2-(benzo[d]oxazol-2-ylthio)-N-phenylacetamide;
[0042] 2-((1H-benzo[d]imidazol-2-yl)thio)-N-phenylacetamide;
[0043]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(2-methnoxyphenyl)acetamide;
[0044]
2-((1H-benzo[d]imidazol-2yl)thio)-N-(3-methylthio)phenyl)acetamide;
[0045]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-nitrophenyl)acetamide;
[0046]
2-((1H-benzo[d]imidazol-2yl)thio)-N-(4-isopropylphenyl)acetamide;
[0047]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-bromophenyl)acetamide;
[0048]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-chlorophenyl)acetamide;
[0049]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(3,4-dichlorophenyl)acetamide;
[0050]
N-(4-ethylphenyl)-2-((6-methyl-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide;
[0051]
N-(4-bromophenyl)-2-((6methyl-1H-benzo[d]imidazol-2-yl)thio)acetami-
de;
[0052]
N-(3,4-dichlorophenyl)-2-((6-methoxy-1H-benzo[d]imidazol-2-yl)thio)-
acetamide;
[0053]
2-((6acetamido-1H-benzo[d]imidazol-2-yl)thio)-N-(4-iodophenyl)aceta-
mide;
[0054]
2,2,2-trifluoro-N-(2-((2-((4-iodophenyl)amino)-2-oxoethyl)thio)-1H--
benzo[d]imidazol-6-yl)acetamide;
[0055]
2-((6-nitro-1H-benzo[d]imidazol-2yl)thio)-N-phenylacetamide;
[0056]
N-(2-methoxyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)aceta-
mide;
[0057]
N-(4-methoxyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)aceta-
mide;
[0058]
N-(2,4-dimethoxyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)a-
cetamide;
[0059]
N-(benzo[d][1,3]dioxol-5-yl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)t-
hio)acetamide;
[0060]
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(p-tolyl)acetamide;
[0061]
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(4-trifluoromethoxy)ph-
enyl)acetamide;
[0062]
N-(4-cyanophenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetami-
de;
[0063]
N-(4-bromophenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetami-
de;
[0064]
N-(4-chlorophenyl)2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetami-
de;
[0065]
N-(4-iodophenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetamid-
e; and
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(4-phenoxyphenyl)aceta-
mide, or a pharmaceutically acceptable salt thereof.
[0066] In another aspect, the present disclosure features a method
of treating a bacterial infection in a subject comprising,
administering to the subject a therapeutically effective amount of
a compound of Formula I-c:
##STR00007##
or a pharmaceutically acceptable salt thereof, wherein:
[0067] X is O or N;
[0068] R.sup.3 is H, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, --NO.sub.2,
--NHC(O)R.sup.c;
[0069] R.sup.4' is H, bromo, chloro, C.sub.1-6 alkyl, C.sub.1-6
alkoxy, aryloxy, C.sub.1-6 haloalkyl, --CN, --NO.sub.2, --SR.sup.a;
or 2 R.sup.4 together with the carbon atoms to which they are
attached form a heteroaryl or heterocycloalkyl;
[0070] R.sup.a is H or C.sub.1-6 alkyl;
[0071] R.sup.c is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl; and
[0072] n and q are each independently 1, 2, 3, 4, or 5.
[0073] In some embodiments, R.sup.4' is H, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, aryloxy, C.sub.1-6 haloalkyl, --CN, --NO.sub.2,
and --SR.sup.a.
[0074] In some embodiments, the compound is:
[0075]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-cyanophenyl)acetamide;
[0076]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(3-(methylthio)phenyl)acetamid-
e;
[0077]
N-(4-bromophenyl)-2-((6-methyl-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide;
[0078]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-chlorophenyl)acetamide;
[0079]
N-(4-ethylphenyl)-2-((6-methyl-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide;
[0080]
N-(2,4-dimethoxyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)a-
ccetamide;
[0081] 2-(benzo[d]oxazol-2-ylthio)-N-phenylacetamide;
[0082] 2-((1H-benzo[d]imidazol-2-yl)thio)-N-phenylacetamide;
[0083]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(2-methoxyphenyl)acetamide;
[0084]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(3-(methylthio)phenyl)acetamid-
e;
[0085]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-nitrophenyl)acetamide;
[0086]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-isopropylphenyl)acetamide;
[0087]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-bromophenyl)acetamide;
[0088]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(4-chlorphenyl)acetamide;
[0089]
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(3,4-dichlorophenyl)acetamide;
[0090]
N-(4-ehtylphenyl)-2-((6-methyl-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide;
[0091]
N-(4-bromophenyl)-2-((6-methyl-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide;
[0092]
N-(3,4-dichlorophenyl)-2-((6-methoxy-1H-benzo[d]imidazol-2-yl)thio)-
acetamide;
[0093]
2-((6-acetamido-1H-benzo[d]imidazol-2-yl)thio)-N-(4-iodophenyl)acet-
amide;
[0094]
2,2,2-trifluoro-N-(2-((2-((4-iodophenyl)amino)-2-oxoethyl)thio)-1H--
benzo[d]imidazol-6-yl)acetamide;
[0095]
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-phenylacetamide;
[0096]
N-(2-methoyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide;
[0097]
N-(4-methoxyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)aceta-
mide;
[0098]
N-(2,4-dimethoxyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)a-
cetamide;
[0099]
N-(benzo[d][1,3]dioxol-5-yl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)t-
hio)acetamide;
[0100]
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(p-tolyl)acetamide;
[0101]
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(4-(trifluoromethyxy)p-
henyl)acetamide;
[0102]
N-(4-cyanophenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetami-
de;
[0103]
N-(4-bromophenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetami-
de;
[0104]
N-(4-chlorophenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetam-
ide; and
2-((6nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(4-phenoxyphenyl)acet-
amide; or a pharmaceutically acceptable salt thereof.
[0105] Also provided herein are pharmaceutical compositions
comprising a compound of Formula I-c as described herein, and a
pharmaceutically acceptable carrier.
[0106] In some embodiments, the bacterial infection or
antibiotic-tolerant infection is caused by a Gram-negative
bacterium.
[0107] In an embodiment of any of the methods of the invention, the
microbial infection is the result, of a pathogenic bacterial
infection, fungal infection, or viral infection. Examples of
pathogenic bacteria include, without limitation, bacteria within
the genuses Aerobacter, Aeromonas, Acinetobacter; Agrobacterium,
Bacillus, Bacteroides; Bartonella, Bortella, Brucella, Burkholderia
Calymmatobacterium, Campylobacter, Citrobacter, Clos]ridium,
Corynebacterium, Enterobacter, Escherichia, Francisella,
Haemophilus, Hafnia, Helicobacter, Klebsiella, Legionella,
Listeria, Morganella, Moraxella, Proteus, Providencia, Pseudomonas,
Salmonella, Serratia, Shigella, Staphylococcus. Streptococcus,
Treponema, Xanthamonas, Vibrio, and Yersinia. Specific examples of
such bacteria include Vibrio harveyi, Vibrio cholerae, Vibrio
parahemolyticus, Vibrio alginolyticus, Pseudomonas phosphoreum,
Pseudomonas aeruginosa, Yersinia enterocalitica, Escherichia coli,
Salmonella typhimurim, Haemophilus influenzae, Helicobacter pylori,
Bacillus subtilis, Borrelia burgfdorferi, Neisseria meningitidis,
Neisseria gonorrhoeae, Yersinia pestis, Campylobacter jejuni,
Deinococcus radiodurans, Mycobacterium tuberculosis, Enterococcus
faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, K.
pneumonia. A. baumannii and Staphylococcus aureus.
[0108] In some embodiments, the gram-negative bacterium is a
Pseudonumas, e.g., P. aeruginosa, Kelbsiella, e.g., K. pneumonia,
or Acinetobacter, e.g., A. baumanni. In some embodiments, the
gram-negative bacterium is Burkholderia species.
[0109] In some embodiments, the infection is a polymicrobial
infection, e.g., an infection comprising more than one organism. In
some embodiments, the infection comprises at least one of the
organisms listed above, e.g., one or more of Pseudomonas, e.g., P.
aeruginosa, Kelbsiella, e.g., K. pneumonia, and/or Acinetobacter,
e.g., A. baumanni.
[0110] In some embodiments, the methods further include
administering an antibiotic selected front the group consisting of
penicillins, cephalosporins, carbacephems, cepltamycins,
carbapenems, monobactams, quinolones, tetracyclines,
aminoglycosides, macrolides, glycopeptides, chloramphenicols,
glycylcyclines, licosamides, lipopeptides, oxazolidinones and
fluoroquinolones.
[0111] In some embodiments, the bacterial infection is pneumonia,
septic shock, urinary tract infection, a gastrointestinal
infection, or an infection of the skin and soft tissue.
[0112] In some embodiments, the subject is a mammal, e.g., a human
or non-human mammal, or plant. In some embodiments, the methods
include treating one or more cells, e.g., cells in a culture
dish.
[0113] In one aspect, the present disclosure features a method of
treating a Gram negative infection in a subject, the method
comprising administering to said subject in need of such treatment
a therapeutically effective amount of a compound described
herein.
[0114] In some embodiments, the Gram negative infection is caused
by Pseudomoms aeruginosa.
[0115] In some embodiments, the subject is a trauma patient or a
burn patient suffering from a burn or skin wound.
[0116] In a further aspect, the present disclosure features a
method of reducing bacterial tolerance in a subject, the method
comprising administering to said subject a therapeutically
effective amount of a compound described herein.
[0117] In some embodiments, the method further includes identifying
said subject suffering from a bacteria tolerant infection.
[0118] In some embodiments the method further includes
co-administering to said subject an antibiotic.
[0119] In some embodiments, the antibiotic is a quinoione
antibiotic.
[0120] By virtue of their design, the compositions and methods
described herein possess certain advantages and benefits. First,
the compounds described herein can target virulence factor
production and therefore decrease the incidence of resistance.
Second, the compounds described herein target factors specific to
pathogens (e.g., P. aeruginosa) and therefore do not kill
beneficial commensal bacteria. Additionally, the compounds
described herein can treat both acute infections as well as chronic
infections that plague immune-compromised individuals.
[0121] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In ease of conflict, the present
specification, including definitions, will control.
[0122] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0123] FIG. 1 is a diagram showing how the MvfR inhibitors were
chosen.
[0124] FIG. 2A-2M are tables showing the effect of compounds on
4-hydroxy-2-alkylquinoline (HAQ) levels, including the MvfR ligands
4-hydroxy-2-heptylquinoline (HHQ) and
3,4-dihydroxy-2-heptylquiuoline (PQS), as well as
2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) and
2,4-dihydroxyquinoline (DHQ). Levels of the phenazine, pyocyanin,
were also determined, as were levels of anthranilic acid (AA) and
2-amino acetophenon (2-AA).
[0125] FIGS. 2N-2P are bar graphs showing the effects of selected
compounds on levels of HHQ, PQS, HQNO, and pyocyanin.
[0126] FIGS. 3A-3C and 4 are tables showing the effect of compounds
tested on HAQ, PQS, HQNO, and pyocyanin levels.
[0127] FIG. 5 is a bar graph depicting HAQ's levels in the presence
of compounds from the high throughput screen.
[0128] FIG. 6A is a bar graph depicting the cytotoxicity of the
compounds M64, M62, M59, M51, M50, and M27.
[0129] FIG. 6B is a bar graph depicting the cytotoxicity of
infected cells in the presence of the compounds M64, M62, M59, M51,
M50, and M27.
[0130] FIG. 7 are images of yeast plated in a yeast killing assay
in the presence of the compounds M34, M50, M51, M57, M58, M59, M61,
and M64.
[0131] FIG. 8 is a graph depicting percent survival of a thermal
injury mouse model.
[0132] FIG. 9A is a line graph showing percent survival in a murine
burn and infection model after administration of M64 plus
Ciprofloxacin.
[0133] FIG. 9B is a pair of line graphs showing bacterial loads
were lower in M64+ ciprofloxacin mice in muscle samples taken
adjacent to burn wound (adjacent muscle, right graph) than in mice
treated with M64 or ciprofloxacin alone.
[0134] FIG. 10 is a bar graph showing that M64 interferes with MvfR
binding to pqsA promoter by competing with MvfR ligands.
[0135] FIG. 11 is a bar graph showing that Exogenously addition of
2-AA to exponentially growing cultures of A. baumannii (A. baum) or
Klebsiella pneumoniae (K. pn.) (green) increases the antibiotic
tolerant cell fraction.
DETAILED DESCRIPTION
[0136] The present disclosure provides, inter alia, compositions
and methods for treating and/or preventing an infection caused by a
pathogen such as a bacierium (e.g. Pseudomonas aeruginosa) by
blocking bacterial virulence mechanisms and pathogenesis in vivo.
Population density-dependent signaling, generally referred to as
quorum sensing (QS), is one such mechanism. QS is a cell-cell
signaling density-dependent communication system that is achieved
through the production and regulation of low molecular weight
molecules as a means to activate virulence factors critical for
full virulence in mammals and regulation of multiple aspects of
virulence. It is important for the development of acute infections
as well as for the formation of antibiotic-tolerant cell
populations, a process underlying pathogen persistence in chrome
infections. Bacteria cells that are able to tolerate antibiotic
killing and host defense mechanisms can persist in the body and
provide a reservoir for re-initiation of infection. Etiological
agents of some serious chronic infections are often refractory to
antibiotics due to antibiotic tolerance, and are therefore very
difficult to treat.
[0137] QS signaling circuits are evolutionarily conserved and play
central roles in modulating virulence mechanisms in many different
human pathogens. For example, without wishing to be bound by
theory, the compounds described herein may target the QS
cell-to-cell signaling systems used by many pathogens to
communicate and activate multiple virulence factors. The QS systems
of the Gram-negative bacterium Pseudamonas aeruginosa, have been
recognised for their importance in virulence and thus can serve as
an excellent target for the development of novel therapeutics that
are urgently needed.
[0138] P. aeruginosa is a recalcitrant Gram-negative bacterium that
defies eradication by antibiotics and exemplifies current
problematic pathogens in hospitals and intensive care units. This
pathogen causes difficult to treat infections such as urinary tract
in infections, respiratory system infections, dermatitis, soft
tissue infections, bacteremia and a variety of systemic infections,
particularly in victims of severe burns and individuals with cystic
fibrosis, cancer, and AIDS who are immunosuppressed.
[0139] P. aeruginosa QS regulated virulence functions are
controlled by the low molecular weight signaling molecules,
acyl-homoserine lactones (HSL) 3-oxo-C12-HSL and C4-HSL produced
through the Las and Rhl systems; and by the
4-hydroxy-2-alkylquinolines (HAQs) produced through the MvfR
system. The compounds described herein can inhibit the production
of the low molecular weight compounds, HAQs, and thus QS signaling
that controls virulence and antibiotic tolerance.
[0140] Conventional antibiotics target a narrow spectrum of
activities that are essential for bacterial growth in culture.
Pharmacological targeting of non-essential functions such as
virulence factor production through inhibition of QS signaling may
decrease the incidence of resistance since bacterial growth per se
is not directly challenged and subjected to selection pressure.
[0141] Thus, QS systems are suitable targets for new
antimicrobials, since they are not imperative for bacterial growth
or survival, and they play important roles in pathogenesis. As
such, there exists a need for new antimicrobial compounds which
target virulence factor production, and antibiotic tolerance. The
compounds described herein help fulfill these and other needs.
Compounds
[0142] At various places in the present specification, substituents
of compounds of the invention are disclosed in groups or in ranges.
It is specifically intended that the invention include each and
every individual sub-combination of the members of such groups and
ranges. For example, the term "C.sub.1-6 alkyl" is specifically
intended to individually disclose methyl, ethyl, C.sub.3 alkyl,
C.sub.4 alkyl, C.sub.5 alkyl, and C.sub.6 alkyl.
[0143] For compounds of the invention in which a variable appears
more than once, each variable can be a different moiety selected
from the Markush group defining the variable. For example, where a
structure is described having two R groups that are simultaneously
present on the same compound; the two R groups can represent
different moieties selected from the Markush group defined for R.
In another example, when an optionally multiple substituent is
designated in the form:
##STR00008##
then it is understood that substituent R.sup.2 can occur m number
of times on the ring, and R.sup.2 can be a different moiety at each
occurrence.
[0144] As used herein, the term "substituted" or "substitution"
refers to the replacement of a hydrogen atom with a moiety other
than H. For example, an "N-substituted piperidin-4-yl" refers to
the replacement of the piperidinyl NH with a non-hydrogen
subsiituent, such as alkyl. In another example, a "4-substituted
phenyl" refers to replacement of the H atom, on the 4-position of
the phenyl with a non-hydrogen substituent, such as chloro.
[0145] It is further intended that the compounds of the invention
are stable. As used herein "stable" refers to a compound that is
sufficiently robust to survive isolation to a useful degree of
purity from a reaction mixture, and preferably capable of
formulation into an efficacious therapeutic agent.
[0146] It is further appreciated that certain features of the
invention, which are, for clarity, described in the context of
separate embodiments, can also be provided in combination in a
single embodiment. Conversely, various features of the invention
which are, for brevity, described in the context of a single
embodiment, can also be provided separately or in any suitable
sub-combination.
[0147] As used herein, the term "alkyl" is meant to refer to a
saturated hydrocarbon group which is straight-chained or branched.
Example alkyl groups include methyl (Me), ehtyl (Et), propyl (e.g.,
n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl),
pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An
alkyl group can contain from 1 to about 20, from 2 to about 20,
from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to
about 4, or from 1 to about 3 carbon atoms. The term "alkylenyl"
refers to a divalent alkyl linking group.
[0148] As used herein, "haloalkyl" refers to an alkyl group having
one or more halogen substituents. Example haloalkyl groups include
CF.sub.3, C.sub.2F.sub.5, CHF.sub.2, CCl.sub.3, CHCl.sub.2,
C.sub.2Cl.sub.5, and the like.
[0149] As used herein, "aryl" refers to monocyclic or polycyclic
(e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as,
for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl,
indenyl, and the like. In some embodiments, aryl groups have from 6
to about 20 carbon atoms.
[0150] As used herein, "aryloxy" refers to an --O-aryl group. An
example aryloxy group is phenoxy.
[0151] As used herein, "cycloalkyl" refers to non-aromatic cyclic
hydrocarbons including cyclized alkyl, alkenyl, and alkynyl groups.
Cycloalkyl groups can include mono- or polycyclic (e.g., having 2,
3 or 4 fused rings) ring systems as well as 2-ring, 3-ring, 4-ring
spiro system (e.g., having 8 to 20 ring-forming atoms). Example
cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl,
cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl,
adamantyl, and the like. Also included in the definition of
cycloalkyl are moieties that have one or more aromatic rings fused
(i.e., having a bond in common with) to the cycloalkyl ring, for
example, benzo, pyrido or thieno derivatives of pentane, pentene,
hexane, and the like. Carbon atoms of the cycloalkyl group can be
optionally oxidized, e.g. bear an oxo or sulfildo group to form CO
or CS.
[0152] As used herein, "heteroaryl" groups refer to an aromatic
heterocycle having at least one heteroatom ring member such as
sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic
and polycyclic (e.g., having 2, 3 or 4 fused rings) systems.
Examples of heteroaryl groups include without limitation, pyridyl,
N-oxopyridyl, pyrimidinyl, prazinyl, pyridazinyl, triazinyl, furyl,
quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,
pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl,
isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl,
1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl,
carbazolyl, benzimidazolyl, indolinyl, and the like. In some
embodiments, the heteroaryl group has from 1 to about 20 carbon
atoms, and in further embodiments from about 3 to about 20 carbon
atoms. In some embodiments, the heteroaryl group contains 3 to
about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some
embodiments, the heteroaryl group has 1 to about 4, 1 to about 3,
or 1 to 2 heteroatoms.
[0153] As used herein, "heterocycloalkyl" refers to non-aromatic
heterocycles including cyclized alkyl, alkenyl, and alkynyl groups
where one or more of the ring-forming carbon atoms are replaced by
a heteroatom such as an O, N, or S atom. Also included in the
definition of heterocycloalkyl are moieties that have one or more
aromatic ring fused (i.e., having a bond in common with) to the
nonaromatic heterocyclic ring, for example phthalimidyl,
naphthalimidyl, and benzo derivatives of heterocycles such as
indolene and isoindolene groups. Heterocycloalkyl groups can be
mono- or polycyclic (e.g., having 2, 3, 4 or more fused rings or
having a 2-ring, 3-ring, 4-ring spiro system (e.g., having 8 to 20
ring-forming atoms)). Heteroatoms or carbon atoms of the
heterocycloalkyl group can be optionally oxidized, e. g., bearing
one or two oxo or sulfido groups to form SO, SO.sub.2, CO, NO, etc.
In some embodiments, the heterocycloalkyl group has from 1 to about
20 carbon atoms, and in further embodiments from about 3 to about
20 carbon atoms. In some embodiments, the heterocycloalkyl group
contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms.
In some embodiments, the heterocycloalkyl group has 1 to about 4, 1
to about 3, or 1 to 2 heteroatoms. In some embodiments, the
heterocycloalkyl group contains 0 to 3 double bonds. In some
embodiments, the heterocycloalkyl group contains 0 to 2 triple
bonds. Example "heterocycloalkyl" groups include morpholino,
thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl,
2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane,
piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl,
pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, as well
as radicals of 3H-isobenzofuran-1-one, 1,3-dihydro-isobenzofuran,
2,3-dihydro-benzo[d]isothiazole 1,1-dioxide, and the like.
[0154] As used herein, "halo" or "halogen" includes fluoro, chloro,
bromo, and iodo.
[0155] As used herein, "alkoxy" refers to an --O-alkyl group.
Example alkoxy groups include methoxy, ethoxy, propoxy (e.g.,
n-propoxy and isopropoxy), t-butoxy, and the like.
[0156] As used here, "haloalkoxy" refers to an --O-haloalkyl group.
An example haloalkoxy group is OCF.sub.3.
[0157] As used herein, "arylalkyl" refers to alkyl substituted by
aryl and "cycloalkylalkyl" refers to alkyl substituted by
cycloalkyl. An example arylalkyl group is benzyl.
[0158] As used herein, "amino" refers to NH.sub.2.
[0159] As used herein, "alkylamino" refers to an amino group
substituted by an alkyl group.
[0160] As used herein, "dialkylamino" refers to amino group
substituted by two alkyl groups.
[0161] As used herein, "dialkyiamiuo" refers to an amino group
substituted, by two alkyl groups.
[0162] The compounds described herein can be asymmetric (e.g.,
having one or more sterocenters). All stereoisomers, such as
enautiomers and diastercomers, are intended unless otherwise
indicated. Compounds of the present invention that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically active starting materials are
known in the art, such as by resolution of racemic mixtures or by
stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present invention. Cis and trans geometric
isomers of the compounds of the present invention are described and
may be isolated as a mixture of isomers or as separated isomeric
forms.
[0163] Resolution of racemic mixtures of compounds can be carried
out by any of numerous methods known in the art. An example method
includes fractional recrystallization using a "chiral resolving
acid" which is an optically active, salt-forming organic acid.
Suitable resolving agents for fractional recrystallization methods
are, for example, optically active acids, such as the D and L forms
of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,
mandelic acid, malic acid, lactic acid or the various optically
active camphorsulfonic acids such as .beta.-camphorsulfonic acid.
Other resolving agents suitable for fractional crystallization
methods include stereoisomerically pure forms of
.alpha.-methylbenzylamine (e.g., S and R forms, or
diastereomerically pure forms), 2-phenylglycinol norephedrine,
ephedrine, N-methylephedrine, cyclohexylethylamine,
1,2-diaminocyclohexane, and the like.
[0164] Resolution of racemic mixtures can also be carried out by
elution on a column packed with an optically active resolving agent
(e.g., dinitrobenzoylphenylglycine). Suitable elution solvent
composition can be determined by one skilled in the art.
[0165] Compounds of the invention also include tautomeric forms,
such as keto-enol tautomers.
[0166] Compounds of the invention can also include all isotopes of
atoms occurring in the intermediates or final compounds. Isotopes
include those atoms having the same atomic number but different
mass numbers. For example, isotopes of hydrogen include tritium and
deuterium.
[0167] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgement,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0168] The present invention also includes pharmaceutically
acceptable salts of the compounds described herein. As used herein,
"pharmaceutically acceptable salts" refers to derivatives of the
disclosed compounds wherein the parent compound is modified by
converting an existing acid or base moiety to its salt form.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines; alkali or organic salts of acidic residues such as
carboxylic acids; and the like. The pharmaceutically acceptable
salts of the present invention include the conventional non-toxic
salts or the quaternary ammonium salts of the parent compound
formed, for example, from non-toxic inorganic or organic acids. The
pharmaceutically acceptable salts of the present invention can be
synthesized from the parent compound which contains a basic or
acidic moiety by conventional chemical methods. Generally, such
salts can be prepared by reacting the free acid or base forms of
these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two; generally, nonaqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and
Journal of Pharmaceutical Science, 66, 2 (1977), each of which is
incorporated herein by reference in its entirety.
[0169] The present disclosure also includes prodrugs of the
compounds described herein. As used herein, "prodrugs" refer to any
covalently bonded earners which release the active parent drug when
administered to a mammalian subject. Prodrugs can be prepared by
modifying functional groups present in the compounds in such a way
that the modifications are cleaved, either in routine manipulation
or in vivo, to the parent compounds. Prodrugs include compounds
wherein hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded
to any group that, when administered to a mammalian subject,
cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl
group respectively. Examples of prodrugs include, but are not
limited to, acetate, formate and benzoate derivatives of alcohol
and amine functional groups in the compounds of the invention.
Preparation and use of prodrugs is discussed in T. Higuchi and V.
Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the
A.C.S. Symposium Series, and in Bioreversibie Carriers in Drug
Design, ed, Edward B. Roche, American Pharmaceutical Association
and Pergamon Press, 1987, both of which are hereby incorporated by
reference in their entirety.
Synthesis
[0170] Compounds of the present disclosure, including salts,
hydrates, and solvates thereof, can be prepared using known organic
synthesis techniques and can be synthesized according to any of
numerous possible synthetic routes, e.g., as described herein.
[0171] Reactions for preparing compounds of the present disclosure
can be carried out in suitable solvents which can be readily
selected by one of skill in the art of organic synthesis. Suitable
solvents can be substantially nonreactive with the starting
materials (reactants), the intermediates, or products at the
temperatures at which the reactions are carried out, e.g.,
temperatures which can range from the solvent's freezing
temperature to the solvent's boiling temperature. A given reaction
can be carried out in one solvent or a mixture of more than one
solvent. Depending on the particular reaction step, suitable
solvents for a particular reaction step can be selected.
[0172] Preparation of compounds of the disclosure can involve the
protection and deprotection of various chemical groups. The need
for protection and deprotection, and the selection of appropriate
protecting groups can be readily determined by one skilled in the
art. The chemistry of protecting groups can be found, for example,
in T. W. Green and P. G. M. Wuts, Protective Groups in Organic
Synthesis, 3rd. Ed., Wiley & Sons, Inc., New York (1999), which
is incorporated herein by reference in its entirety.
[0173] Reactions can be monitored according to any suitable method
known in the art. For example, product formation can be monitored
by spectroscopic means, such as nuclear magnetic resonance
spectroscopy (e.g., .sup.1H or .sup.13C) infrared spectroscopy,
spectrophotometry (e.g., UV-visible), or mass spectrometry, or by
chromatography such as high performance liquid chromatography
(HPLC) or thin layer chromatography.
[0174] Example Synthetic routes to compounds of the invention are
provided in Scheme 1 below, where constituent members of the
depicted formulae are defined herein.
##STR00009##
[0175] Compounds of the invention can be assembled as shown in
Scheme 1. Functionalized anilines of Formula I-1 can be reacted
with chloroacetyl chloride in the presence of a base to produce the
chloro-acetamide compounds of Formula I-2. Subsequent reaction with
thiobenzimidazoles of Formula I-3 in the presence of a base yields
compounds of Formula I.
Methods of Treatment
[0176] The present disclosure provides methods for treating and
preventing acute and chronic infections by administering to a
subject a therapeutically effective amount of a compound described
herein. For example, the compounds described herein can be used to
treat an acute infection caused by a pathogen and, as a result of
treatment, shut down the infection. In addition, the compounds
described herein can be used to treat chronic, persistent
infections caused by pathogens such as bacteria (e.g., gram
negative bacteria, e.g., P. aureginosa) that have become tolerant
to antibiotic treatment, e.g., as a result of activation of a QS
system. The compounds described herein can treat individuals
suffering from such chronic infections, e.g., by targeting the
virulence factor pathways of these tolerant bacteria. In general
the methods can be used to treat any organism that is susceptible
to bacterial infections, e.g., animals, including mammals, e.g.,
humans and non-human mammals, as well as plants.
[0177] Patients suitable for such treatment may be identified by
methods known in the art, e.g., by the detection of symptoms
commonly associated with infection, such as fever, pain, pus,
culture of organisms, and the like. Infections that can be treated
with the compounds described herein include those caused by or due
to pathogens. In some embodiments, the pathogen is a bacterium
(e.g., a gram-negative bacterium, e.g., Pseudomonas, e.g., P.
aureginosa).
[0178] Clinical indications can include, but are not limited to: 1)
burn and/or wound infections; 2) nosocomial pneumonia; 3) cystic
fibrosis; 4) osteomyelitis; and 5) sepsis in an immunosuppressed
host. In some embodiments, the subject has an acute infection. In
some embodiments, the subject has a chronic infection. A chronic
infection can last three weeks or more, or if the infection is
recurrent despite completion of antibiotic treatment. In some
embodiments, the following pathogenic infections are treated using
the compounds described herein.
[0179] Invasive burn wound infections remains the most common cause
of morbidity and mortality in extensively burned subjects.
Infection is the predominant determinant of wound healing,
incidence of complications, and outcome of burn subjects. The main
organisms responsible are Pseudomonas aeruginosa, S. aureus,
Streptococcus pyogenes, and various Gram-negative organisms.
[0180] Nosocomial pneumonias account for nearly 20% of all
nosocomial infections. Subjects most at risk for developing
nosocomial pneumonia are those in intensive care units, subjects
with altered levels of consciousness, elderly subjects, subjects
with chronic lung disease, ventilated subjects, smokers and
post-operative subjects. In a severely compromised subject,
multiantibiotic-resistant nosocomial pathogens are likely to be the
cause of the pneumonia. The main organisms responsible are P.
aeruginosa, S. aureus, Klebsiella pneumoniae and Enterobacter
spp.
[0181] Cystic fibrosis (CF) is the most common genetic disorder of
the Caucasian population. Pulmonary disease is the most common
cause of premature death in cystic fibrosis subjects. Optimum
antimicrobial therapy for CF is not known, and it is generally
believed that the introduction of better anti-pseudomonal
antibiotics has been the major factor contributing to the increase
in life expectancy for CF subjects. The most common organisms
associated with lung disease in CF are S. aureus; P. aeruginosa and
H. influenzae, P. aeruginosa is the loading pathogen.
[0182] Osteomyelitis causes the vascular supply to the bone to be
compromised by infection extending into surrounding tissue. Within
this necrotic and ischemic tissue, the bacteria may be difficult to
eradicate even after an intense host response, surgery, and/or
antibiotic therapy. The main organisms responsible are S. aureus,
E. coli, and P. aeruginosa.
[0183] Treatment of infections in subjects who are
immune-compromised by virtue of chemotherapy-induced
granulocytopenia and immunosuppression related to organ or bone
marrow transplantation can be a challenge. Neutropenic subjects are
especially susceptible to bacterial infection. Organisms likely to
cause infections in granulocytopenic subjects are: S. epidermidis,
S. aureus, S. viridans, Enterococcus spp. E. coli, Klebsiella spp.
P. aeruginosa and Candida spp.
[0184] Small bowel bacterial overgrowth syndrome (SBBOS), or small
intestinal bacterial overgrowth (SIBO), also termed bacterial
overgrowth; is a disorder of excessive bacterial growth in the
small intestine. Certain species of bacteria are more commonly
found in aspirates of the jejunum taken from patients with
bacterial overgrowth. The most common isolates are Escherichia
coli, P. aeruginosa, Streptococcus, Lactobacillus, Bacteroides, and
Enterococcus species. See e.g. Kopacova et al. "Small Intestinal
Bacterial Overgrowth Syndrome" World J. Gastroenterol, 16(24):
2978-2990, 2010, which is incorporated herein by reference in its
entirety. In some embodiments, the compounds-described herein can
be used to treat small intestinal bacterial overgrowth syndrome
(SIBO).
[0185] In some embodiments, the compounds described herein can be
used to preventatively treat patients undergoing endoscopy. These
patients are often found to be infected by Pseudomonas aeruginosa
after undergoing endoscopic procedures.
[0186] In some embodiments, the compounds described herein can be
used in combination with an antibiotic agent. The combination may
be used to affect a synergistic result, to overcome an acute or
chronic infection, or overcome bacterial tolerance.
[0187] Examples of classes of antibiotics that can be used in
combination with the compounds described herein include
penicillins, cephalosporins, carbacephems, cepbamycins,
carbapenems, monobactams, qninolones, tetracyclines,
aminoglycosides, macrolides, glycopeptides, chloramphenicols,
glycylcyelines, licosamides, lipopeptides, oxazolidinones and
fluoroquinolones.
Essential Structure of Quinolone Antibioties
##STR00010##
[0189] In some embodiments, the antibiotic that can be used in
combination with the compounds described herein is a quinolone
antibiotic. Example quinolones include without limitation
cinoxacin, flumequine, malidixic acid, oxolinic acid, piromidic
acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin,
fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin,
perfloxacin, rufloxacin, balofloxacin, grepafloxacin, levofloxacin,
pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin,
clinafloxacin, gatifloxacin, gemifloxacin, moxifloxacin,
sitafloxacin, trovafloxacin, prulifloxacin, garenoxacin,
delafloxacin, danofloxacin, difloxacin, enrofloxacin, ibafloxacin,
marbofloxacin, oribfloxacin, and sarafloxacin.
[0190] In some embodiments, the antibiotic that can be used in
combination with the compounds described herein is riflaximin to
treat SIBO.
Plants
[0191] Resistance of plant pathogens to antibiotics such as
oxytetracycline is rare, but the emergence of
streptomycin-resistant strains of Erwinia amylovora, Pseudomonas
spp., and Xanthomonas campestris has impeded the control of several
important plant diseases.
[0192] In some embodiments, the compounds described herein can be
used to treat plant bacterial diseases. As used herein, "plants"
refer to photosynthetic organisms, both eukaryotic and prokaryotic.
Plants include trees and shrubs (e.g., conifers), herbs, bushes
(greater than 100 different families), grasses (e.g., Gramineae,
Cyperaceae, and Juncaceae), vines (any number of families using any
climbing method), ferns (e.g., a species from the Psilotopsida,
Equisetopside, Marattiospida or Polypodiopsida class), mosses
(i.e., bryophytes), fungi (e.g., edible and/or commercially useful
varieties), and green algae (e.g., unicellular, flagellates, and
filamentous).
[0193] Representative species of plants that may benefit from
application of the antibiotic described herein, many of which are
grown around the world for agronomic purposes, include, without
limitation, corn (Zea mays), wheat (Triticum spp.), rice (Oryza
spp.), tobacco (Nicotiana ssp.), potatoes (Solanum tuberosum),
cotton (Gossypium hirsutum), rapeseed and canola (Brassica spp.),
and sunflower (Helianthus annus), as well as any number of fruits
(e.g., Malus spp., Citrus spp., Vitus spp., and Musa spp.) or
legumes (e.g., soybean (Glycine max), peas (Pisum salivum), and
beans (from the Leguminasae family)). There are a number of
flowering species (e.g., species of asgiosperms) not included in
any of the above-indicated plants that also may benefit from
application of the antibiotic described herein.
[0194] In some embodiments, the compounds described herein can be
used in combination with antibiotics that are used to treat plant
bacterial diseases. Examples of antibiotics that can be used in
combination include, but are not limited to, streptomycin,
oxytetracycline, gentamicin, and oxolinic acid. See e.g. McManus et
al. "Antibiotic Use in Plant Agriculture" Annu. Rev. Phytopathol.
40:443-65, 2002, which is incorporated herein by reference in its
entirety. See also the worldwide websites
"apsnet.org/publications/aspsnetfeatures/Pages/AntibioticsForPla-
nts.aspx,"
"apsnet.org/edcenter/intropp/topics/Pages/PlantDiseaseManagment-
.aspx," and "plantmanagementnetwork.org/pub/php/review/antibiotic/"
for examples of plant disease control and antibiotic usages, each
of which are incorporated by reference in its entirety.
[0195] In some embodiments, the compounds are applied to the leaves
of a plant (e.g., as part of a foliar spray or dust); in some
embodiments, the compounds are applied to the soil surrounding a
plant, or into which a plant, seed, or seedling will be placed.
Compositions for use in plants can contain other agriculturally or
horticulturally-acceptable or useful ingredients.
Pharmaceutical Formulations and Dosage Forms
[0196] When employed as pharmaceuticals, the compounds of the
present disclosure can be administered in the form of
pharmaceutical compositions. These compositions can be prepared in
a manner well known in the pharmaceutical art, and can be
administered by a variety of routes depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration can be topical (including ophthalmic and to mucous
membranes including intranasal, vaginal and rectal delivery),
pulmonary (e.g., by inhalation or inusslation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal intramuscular or injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Parenteral administration can be in the form of a
single bolus dose, or can be, for example, by a continuous
perfusion pump. Pharmaceutical compositions and formulations for
topical administration can include transdermal patches, ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids and
powders. Conventional pharmaceutical carriers, aqueous, powder or
oily bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful.
[0197] The present disclosure also includes pharmaceutical
compositions which contain, as the active ingredient, one or more
of the compounds described herein in combination with one or more
pharmaceutically acceptable carriers. In making the compositions of
the invention, the active ingredient is typically mixed with an
excipient, diluted by an excipient or enclosed within such a
carrier in the form of, for example, a capsule, sachet, paper, or
other container. When the excipient serves as a diluent, it can be
a solid, semi-solid, or liquid material, which acts as a vehicle,
carrier or medium for the active ingredient. Thus, the compositions
can be in the form of tablets, pills, powders, lozenges, sachets,
cachets, clixirs, suspensions, emulsions, solutions, syrups,
aerosols (as a solid or in a liquid medium), ointments containing,
for example, up to 10% by weight of the active compound, soft and
hard gelatin capsules, suppositories, sterile injectable solutions,
and sterile packaged powders.
[0198] In preparing a formulation, the active compound can be
milled to provide the appropriate particle size prior to combining
with the other ingredients. If the active compound is substantially
insoluble, it can be milled to a particle size of less than 200
mesh. If the active compound is substantially water soluble, the
particle size can be adjusted by milling to provide a substantially
uniform distribution in the formulation, e.g. about 40 mesh.
[0199] Some examples of suitable, excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, and methyl cellulose. The formulations can
additionally include: lubricating agents such as talc, magnesium
stearate, and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and
propylhydroxy-benzoates; sweetening agents; and flavoring agents.
The compositions of the present disclosure can be formulated so as
to provide quick, sustained or delayed release of the active
ingredient after administration to the subject by employing
procedures known in the art.
[0200] The compositions can be formulated in a unit dosage form,
each dosage containing from about 5 to about 1000 mg (1 g), more
usually about 100 to about 500 mg, of the active ingredient. The
term "unit dosage forms" refers to physically discrete units
suitable as unitary dosages for human subjects and other mammals,
each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient.
[0201] In some embodiments, the compounds or compositions of the
present disclosure contain from about 5 to about 50 mg of the
active ingredient. One having ordinary skill in the art will
appreciate that this embodies compounds or compositions containing
from about 5 to about 10, from about 10 to about 15, from about 15
to about 20, from about 20 to about 25, from about 25 to about 30,
from about 30 to about 35, from about 35 to about 40, from about 40
to about 45, or from about 45 to about 50 mg of the active
ingredient.
[0202] In some embodiments, the compounds or compositions of the
present disclosure contain from about 50 to about 500 mg of the
active ingredient. One having ordinary skill in the art will
appreciate that this embodies compounds or compositions containing
from about 50 to about 75, from about 75 to about 100, from about
100 to about 125, from about 125 to about 150, from about 150 to
about 175, from about 175 to about 200, from about 200 to about
225, from about 225 to about 250, from about 250 to about 275, from
about 275 to about 300, from about 300 to about 325, from about 325
to about 350, from about 350 to about 375, from about 375 to about
400, from about 400 to about 425, fern about 425 to about 450, from
about 450 to about 475, or from about 475 to about 500 mg of the
active ingredient.
[0203] In some embodiments, the compounds or compositions of the
present disclosure contain from about 500 to about 1000 mg of the
active ingredient. One having ordinary skill in the art will
appreciate that this embodies compounds or compositions containing
from about 500 to about 550, from about 550 to about 600, from
about 600 to about 650, from about 650 to about 700, from about 700
to about 750, from about 750 to about 800, from about 800 to about
850, from about 850 to about 900, from about 900 to about 950, or
from about 950 to about 1000 mg of the active ingredient.
[0204] The active compound can be effective over a wide dosage
range and is generally administered in a pharmaceutically effective
amount. It will be understood, however, that the amount of the
compound actually administered will usually be determined by a
physician, according to the relevant circumstances, including the
condition to be treated, the chosen route of administration, the
actual compound administered, the age, weight, and response of the
individual subject, the severity of the subject's symptoms, and the
like.
[0205] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical
excipient to form a solid pre-formulation composition containing a
homogeneous mixture of a compound of the present disclosure. When
referring to these pre-formulation compositions as homogeneous, the
active ingredient is typically dispersed evenly throughout the
composition so that the composition can be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid pre-formulation is then subdivided into unit
dosage forms of the type described above containing from, for
example, 0.1 to about 1000 mg of the active ingredient of the
present disclosure.
[0206] The tablets or pills of the present disclosure can be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0207] The liquid forms in which the compounds and compositions of
the present disclosure can be incorporated for administration
orally or by injection include aqueous solutions, suitably flavored
syrups, aqueous or oil suspensions, and flavored emulsions with
edible oils such as cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0208] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described supra. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in can be
nebulized by use of inert gases. Nebulized solutions may be
breathed directly from the nebulizing device or the nebulizing
device can be attached to a face mask tent, or intermittent
positive pressure breathing machine. Solution, suspension, or
powder compositions can be administered orally or nasally from
devices which deliver the formulation in an appropriate manner.
[0209] The amount of compound or composition administered to a
subject will vary depending upon what is being administered, the
purpose of the administration, such as prophylaxis or therapy, the
state of the subject, the manner of administration, and the like.
In therapeutic applications, compositions can foe administered to a
subject already suffering from a disease in an amount sufficient to
cure or at least partially arrest the symptoms of the disease and
its complications. Effective doses will depend on the disease
condition being treated as well as by the judgment of the attending
clinician depending upon factors such as the severity of the
disease, the age, weight and general condition of the subject, and
the like.
[0210] The compositions administered to a subject can be in the
form of pharmaceutical compositions described above. These
compositions can be sterilized by conventional sterilization
techniques, or may be sterile filtered. Aqueous solutions can be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile aqueous carrier prior to
administration. The pH of the compound preparations typically will
he between 3 and 11, more preferably from 5 to 9 and most
preferably from 7 to 8. It will be understood that use of certain
of the foregoing excipients, carriers, or stabilizers will result
in the formation of pharmaceutical salts.
[0211] The therapeutic dosage of the compounds of the present
disclosure can vary according to, for example, the particular use
for which the treatment is: made, the manner of administration of
the compound, the health and condition of the subject, and the
judgment of the prescribing physician. The proportion or
concentration of a compound of the present disclosure in a
pharmaceutical composition can vary depending upon a number of
factors including dosage, chemical characteristics (e.g.,
hydrophohicity), and the route of administration. For example, the
compounds of the invention can be provided in an aqueous
physiological buffer solution containing about 0.1 to about 10% w/v
of the compound for parenteral administration. Some typical dose
ranges are from about 1 .mu.g/kg to about 1 g/kg of body weight per
day. In some embodiments, the dose range is from about 0.01 mg/kg
to about 100 mg/kg of body weight per day. The dosage is likely to
depend on such variables as the type and extent of progression of
the disease or disorder, the overall health status of tire
particular subject, the relative biological efficacy of the
compound selected, formulation of the excipient, and its route of
administration. Effective doses can be extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0212] The compounds of the present invention can also be
formulated in combination with one or more additional active
ingredients which can include any pharmaceutical agent such as
antibodies, immune suppressants, anti-inflammatory agents,
chemotherapeutics, or drugs used for the treatment of a bacterial
infection and the like.
Kits
[0213] The present disclosure also includes pharmaceutical kits
useful, for example, in the treatment or prevention of acute and
chronic infections which include one or more containers containing
a pharmaceutical composition comprising a therapeutically effective
amount of a compound of the present disclosure. Such kits can
further include, if desired, one or more of various conventional
pharmaceutical kit components, such as, for example, containers
with one or more pharmaceutically acceptable carriers, additional
containers, etc., as will be readily apparent to those skilled in
the art. Instructions, either as inserts or as labels, indicating
quantities of the components to be administered, guidelines for
administration, and/or guidelines for mixing the components, can
also be included in the kit.
EXAMPLES
[0214] The present disclosure will be described in greater detail
by way of specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of noncritical parameters which can be changed or modified
to yield essentially the same results.
Example 1
2-((6nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(4-phenoxyphenyl)acetamide
##STR00011##
[0216] To 3.55 g of 4-phenoxyaniline in 40 ml of methylene chloride
containing 2.9 ml of triethylamine is added 1.6 ml of chloroacetyl
choride in 20 ml of methylene chloride. After two hours at room
temperature, the mixture is extracted with water to yield 2.74 g of
N-(4-phenoxyphenyl)-2-chloro-acetamide.
[0217] To 2.74 g of N-(4-phenoxyphenyl)-2-chloro-acetamide in 180
ml of a 1M NaOH solution containing 130 ml of MeOH and 60 mL of
water is added 2.06 g of 6-nitro-2-thiobenzimidazole and the
mixture is heated at 70.degree. C. for 3 h. The mixture is
extracted with ethyl acetate and washed with 0.5M NaOH. The oily
residue is recrystallized in ethyl acetate and hexane to produce
0.138 g of
N-(4-methoxyphenyl)-2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)acetamide.
[M+H.sup.+]=421, MS/MS 236 and 208
Example 2
High Throughout Screening (HTS) of 284,256 Compounds
A Bacterial Cell Growth Assay Aids in the Discovery of
Anti-Virulence Compounds
[0218] Experiments were performed to identify compounds that
inhibit the MvfR regulon without altering growth, ultimately
attenuating P. aeruginosa infection. MvfR is a LysR-type
transcriptional regulator that directs HAQs synthesis, including
that of its ligands, 4-hydroxy-2-heptylquinoline (HHQ) and
3,4-dihydroxy-2-heptylquinoline (PQS). MvfR regulates the
production of many virulence factors including pyocyanin, elastase,
and lectins as well as a myriad of low molecular weight molecules;
and both MvfR and PQS have been demonstrated as essential for
pathogenesis in several host models.
[0219] MvfR promotes the production of HAQs by bninding to and
activating the pqs operon, which encodes enzymes for HAQ synthesis.
Anthranilic acid (AA), derived from the phnAB, kynABU, and trpEG
pathways, is the precursor for HAQs. pqsA encodes an
anthranilate-coenzyme A ligase, which activates anthranilic acid
and catalyzes the first committed step to HAQ production. The exact
roles of PqsB and PqsC are unknown, though both show homology to
acyl-carrier-proteins and both are required for HHQ and PQS
production. PqsD is a condensing enzyme that along with PqsA has
been shown to be necessary and sufficient for the production of
2,4-dihydorxyquinoline (DHQ), a molecule whose biological role has
yet to be determined. The final gene of the operon, pqsE encodes
for a putative hydrolase, and while the protein is not required for
the synthesis of HAQs, it is necessary for pyocyanin
production.
[0220] To generate a biological reporter assay suitable for HTS,
the fact that ligand-bound MvfR binds to and activates the pqsA
promoter was exploited. PqsA is an anthranilate-coA ligase which
catalyzes the first step in the HAQ biosynthetic pathway. The pqsA
promoter was fused to the Bacillus subtilis sacB gene and
incorporated this construct stably into the PA14 chromosome. The
sacB gene product levansucrase is toxic when cells are grown in the
presence of sucrose, and this gene has been previously incorporated
into allelic exchange vectors as a means of counter-selection. In
this system, the bacteria cells die when the pqsA promoter is
activated by MvfR, allowing the identification of compounds that
suppress pqsA activity by measuring growth. Using a plate reader
with OD.sub.600nm as a readout for the assay, the construct was
tested and proved successful in both laboratory and pilot
high-throughput settings; 4-CABA, an AA analog which has been shown
to effectively inhibit MvfR was used as a positive control.
[0221] P. aeruginosa PA14 cultures were grown overnight and
subcultured in the morning until reaching mid-logarithmic phase.
Cells were centrifuged, washed and resuspended in LB+10% sucrose to
a final OD.sub.600mm of 0.05. 30 .mu.l of cells were aliquoted into
384-well plates using Matrix WellMate. 1.5 mM 4-CABA was added to
one column as a positive control, and another column was reserved
free of compound for a negative control. 300 nl of library
compounds in DMSO were added to each plate via an Epson compound
transfer robot, representing a final concentration of 50 .mu.g/ml.
Each library plate was screened in duplicate. After 8 hours
incubating at 37.degree. C., OD.sub.600mm was determined for each
well using an EnVision.RTM. plate reader (Perkin-Elmer). The
strength of each compound-containing well was determined by
calculating its z-score.
Bacterial Strains and Growth Conditions
[0222] PA14 is the wild-type P. aeruginosa strain. Burkholderia
thailandensis is closely related to Burkholderia pseudomallei. All
strains were routinely cultured in LB at 37.degree. C., with
antibiotics where necessary: 75 .mu.g/ml tetracycline, 100 .mu.g/ml
rifampicin, and 300 .mu.g/ml carbenicillin.
Quantification of Pyocyanin
[0223] Pyocyanin levels were determined by measuring OD.sub.520mm
of chloroform-extracted cultures.
LC/MS Analyses for HAQ Determination
[0224] The quantification of HAQs in bacterial culture supernatants
was performed. The HAQs were separated on a C18 reverse-phase
column connected to a triple quadrupole mass spectrometer, using a
water/acetonitrile gradient. Positive electrospray in MRM mode with
2.times.10.sup.-3 mTorr argon and 30 V as the collision gas and
energy was employed to quantify HAQs, using the ion transitions HHQ
244>159, HHQ-D4 248>163, HQNO 260>159, PQS 260>175, and
PQS-D4 264>179. B. thailandensis HAQs were assessed as above.
The pseudomolecular ions of each compound were monitored in full
scan mode, using the unsaturated PA14 HAQ response factors.
Identification of Novel Lead HAQ Inhibitors
[0225] A total of 284,256 compounds were screened in duplicate from
the available libraries of the Institute of Chemistry and Cell
Biology (ICCB)-Longwood screening facility (Harvard Medical School,
250 Longwood Avenue, Seeley Mudd Room 604, Boston Mass. 02115)
(FIG. 1). The strength of each hit garnered in the screen was
determined by calculating its z-score. This statistical analysis
normalizes hits on a plate to plate basis and represents the
standard deviation from the mean plate value. 532 compounds with a
strong z-score were identified. The strongest hits with structures
having limited potential liability based on structural analysis,
392 in total, were available as aliquots for further analyses. The
most promising compounds were tested in a secondary screening assay
using a reporter construct with the Pseudomonas aeruginosa (pqsA)
promoter fused to a short half-life green fluorescent protein
(GFP), screening for compounds that quench fluorescence. This
construct was used to confirm the repression of pqsA, to determine
the compound's influence on bacterial growth, and to rule out
potential false positives, such as the possibility that the
compound negatively affects SacB. Two concentrations of compounds
were tested in the secondary screen, representing final
concentrations of .about.50 .mu.g/ml and .about.25 .mu.g/ml, and
both growth (OD.sub.600nm) and fluorescence were measured. A total
of 33 commercially available compounds that did not impact growth
and that completely eliminated fluorescence at the lower
concentration were purchased for further studies.
[0226] Compounds were tested at various concentrations for their
impact on 4-hydroxy-2-alkylquinoline (HAQ) levels, including the
MvfR ligands 4-hydroxy-2-heptylquinoline (HHQ) and
3,4-dihydroxy-2-heptylquinoline (PQS), as well as
2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) and
2,4-dihydroxyquinoline (DHQ) (FIGS. 2A-2C). Levels of the
phenazine, pyocyanin, an MvfR-regulated virulence factor, were also
determined. At 50 .mu.g/ml, the concentration used in the original
screen, seventeen compounds decreased HAQ levels to less than 15%
of the control (FIG. 3).
[0227] Six of these compounds were effective at concentrations as
low as 10 .mu.M (M34, M27, M29, M26, M31, and M4), a concentration
150 times lower than required for AA analogs previously identified.
All compounds resulted in increased anthranilic acid (AA), likely
since this molecule is not being utilized tor HAQ production.
Interestingly, some compounds inhibited HAQ production while not
greatly impacting pyocyanin. Twelve of the 17 compounds (B1, M7,
M8, M17, M18, M19, M23, M26, M27, M33, M34, M41) also significantly
restricted pyocyanin levels to less than half of the control (FIG.
4). The impact of various compounds on the acyl-homoserine lactone
3-oxo-C12-HSL and C4-HSL concentrations was also determined, though
the levels of these molecules were similar to wild-type PA14.
Example 3
Structure-Activity Relationship
[0228] Benzamide-Benzimidazole Molecules art the most Potent Mvfr
Regulon Inhibitiors
[0229] Several compounds (M4, M24, M23, M26, M27, M34, and B1),
including the best inhibitors identified in the HTS, share a basic
backbone of a benzamide-benzimidazole structure (FIGS. 3 and 4).
Thus efforts were focused on commercially available variations of
this backbone in an effort to understand what substitutions are
critical for activity and to ultimately design more potent
inhibitors. FIG. 4 compares various benzamide-benzimidazole
structures assessed and their impacts on HAQ and pyocyanin levels
when tested at 10 .mu.M. Compound M56, which possesses the
unsubstituted benzamide-benzimidazole structure, has little impact
on pyocyanin or HAQ production. The presence of a methoxy group at
the ortho position (M46) of the benzamide ring did not improve the
activity, however a thiomethyl group at the meta position (M4)
improved HAQ inhibition. A strong electron withdrawing group such
as a nitro group in the para position of the benzamide ring (M45)
did not improve the activity, but the isosteric isopropyl group at
the same position (M49) did yield a more active compound. The
presence of a cyano (B1), a bromo (M55) or a chloro (M26)
substituent at the same para position produced active compounds. A
second chlorine atom at the meta position on the benzamide ring, as
in M22, decreased the activity as compared to M26. Substitution of
the benzimidazole ring with a methyl group and of the benzamide
ring with an ethyl group (M23) provided a moderately active
compound as compared to M27 which shares the same methyl
benzimidazole group but which contains a para-bromobenzamide
function. Interestingly, M27 is more active than M55, which lacks
the methyl group on the benzimidazole ring. A methoxy group instead
of a methyl decreased the activity, when comparing M24 and M22.
Attempts to substitute the benzimidazole ring with a
trifluoroacetamide, as in M63, or with an acetamide group, as in
M65, yielded inactive compounds compared to their nitro substituted
counterpart M59.
[0230] The most active commercially available inhibitors generally
all had a nitro substituted benzimidazole ring and a benzamide ring
substituted at the para position with a methoxy (M61),
trifluoromethoxy (M58),a cyano (M62), a hromo (M50), a chloro (M51)
or a iodo (M59). The nitrobenzimidazole derived compound M53 with
its benzamide group substituted with an ortho methoxy group was not
very active while the ortho and para dimethoxy analogue M34 was
quite active. In addition to the aforementioned commercially
available molecules,
2-((6-nitro-1H-benzo[d]imidazol-2-yl)thio)-N-(4-phenoxyphenyl)acetamide
(described above in Example 1) was synthesized, which has a nitro
substituted benzimidazole ring and a para-phenoxy substituted
benzamide ring (the compound is also referred to herein as M64).
This compound proved to be one of the most effective at reducing
both HAQ and pyocyanin at very low concentrations. Overall the most
active inhibitors were found to be M64, M59 and M50, which
considerably inhibited HAQs and pyocyanin in the nanomolar
range.
[0231] Disrupting virulence factor production without impacting
cell viability can provide an alternative to traditional
antibiotics which promote the emergence of resistant microbes,
MvfR-controlled QS system of P. aeruginosa was targeted as
mutations in MvfR or the pqs operon do not impact bacterial cell
growth. HTS yielded several novel MvfR pathway inhibitors that were
not toxic to mammalian cells and were effective at micromolar
concentrations. Analysis of the structure of these compounds showed
that specifically substituted benzamide-benzimidazole molecules
were potent MvfR regulon inhibitors, and by testing various
modifications of this structure, potency was improved. These
molecules effectively reduced HAQs and pyocyanin production at
nanomolar concentrations, far exceeding the potency of previously
reported inhibitors.
[0232] A variety of active structures was identified in the HTS,
with only the substituted benzamide-benzimidazole core structure
appearing in multiple hits. Several compounds, particularly M21 and
M32, were effective HAQ inhibitors while not impacting pyocyanin
levels at all. The regulation of pyocyanin production was complex
and under the control of various environmental and genetic factors,
so the explanation for this phenomenon remains unclear.
Example 4
Elucidating the Mechanism of Inhibition
[0233] The structures of the identified potent inhibitors did not
resemble precursors or intermediates in the HAQ synthesis pathway,
thus their mechanism of inhibition is nuclear. However, since these
compounds diminish or abolish P. aeruginosa HAQ production, it is
possible that they target either the MvfR protein itself or enzymes
in the pqs operon that are required for HAQ synthesis. To further
understand the target(s) of these compounds, a .DELTA.mvfR mutant
constitutively expressing the pqs ABCD genes was used. This strain
is capable of synthesizing HHQ, HQNO, PQS and DHQ independently of
MvfR. Using LC/MS, the production of HAQs was measured in the
presence of 100 .mu.M of selected inhibitors; many of them do not
significantly alter HAQ levels compared to the solvent control
(M18, M19, M34, M43, M50, M53, M57, M60, M61, and M62), suggesting
that these compounds target MvfR or other components upstream of
MvfR (FIG. 5). As shown in FIG. 5, another group of inhibitors
reduced HHQ and HQNQ levels but increased DHQ when present in this
strain (M26, M27, M23, M4, M55, and M52). Since PqsA and PqsD are
necessary and sufficient for DHQ production, it is possible that
these inhibitors target either PqsB or PqsC enzymes, which are
required for HHQ and PQS production. Finally, several inhibitors
exhibited an intermediate phenotype in between those that inhibit
MvfR and those inhibiting PqsB or PqsC (B1, M51, M59, and M58),
implying that they may have dual or alternative targets.
[0234] Some compounds target MvfR directly, while others seem to
target enzymes of the pqs operon, likely PqsB or PqsC, therefore
these inhibitors worked via a novel mechanism. Those compounds that
likely inhibited PqsB or PqsC also limited
4-hydroxy-3-methyl-2-alkylquinolines (HMAQs) in Burkholderia
thailandensis, which had homologs of these genes; while compounds
targeting MvfR had no impact in this species, likely since there
was no B. thailandensis MvfR counterpart.
[0235] The nature of MvfR inhibition was unclear, but the compounds
did not appear to render the protein less stable. It is possible
that these molecules compete for PQS or HHQ ligand binding, though
experiments to address this issue have been inconclusive.
Structural information about the MvfR protein would be useful to
identify a potential binding pocket for the compounds.
Interestingly, all MvfR-specific inhibitors sharing the
benzamide-benzimidazole basic structure contain a nitro group, and
incidentally these molecules are the most effective in eliminating
virulence in the yeast model. However, the nitro substitution on
the benzamide-benzimidazole ring alone is not enough to confer
MvfR-target specificity, as several molecules with tins basic
structure are clearly not targeting MvfR directly.
Example 5
Inhibitors are not Cytotoxic and can Effectively Attenuate Death in
PA14-Exposed Macrophage Cell Lines
Cell Culture Assays
[0236] Raw264.7 macrophage cells were cultured in Dulbecco's
modified eagles medium (DMEM) containing 10% FCS, 2 mM glutamine
and antibiotic-antimycotic solutions. Before infections cells were
washed with PBS (without Mg2+ and Ca2+) and antibiotic-free medium
was used for infections. The cells were infected with PA14 and PA14
isolates grown in the presence of compounds at an MOI of 100 for 3
h at 37.degree. C. in a 5% CO2 atmosphere. During infections cells
were incubated with 100 .mu.M of compounds as well. After 3 hours
of PA14 infection, the cells were washed and incubated with DMEM
medium containing polymixin B and gentamicin in order to kill the
extracellular bacteria. The viability of Raw264.7 was assessed
using the MTT assay, which detects the early sign of eukaryotic
cell death. The formazan crystals formed in viable, metabolically
active cells were dissolved by the addition of 100 .mu.l DMSO and
optical density was measured at 570 nm in a microplate reader.
[0237] In order in determine whether or not the potent inhibitors
are cytotoxic in eukaryotes, Raw 264.7 macrophage cells were
exposed to selected compounds. No significant cytotoxicity was
observed following compound exposure for 3 hours compared to the
DMSO solvent control (FIG. 6A). The cytotoxicity of the macrophage
cells was also determined when infected with PA14 bacterial cells
alone or in the presence of the compounds. Incubation with PA14
cells resulted in only 33% viable macrophages, whereas addition of
the anti-infective compounds significantly reduced cytoxicity (FIG.
6B). M64 resulted in the greatest improvement of viability with 80%
of cells surviving post-infection. Both compounds that target
PqsB/C (M27) or MvfR (M64, M50, M62) effectively reduced
cytotoxicity in this system. These results suggest that the
identified MvfR inhibitors can effectively reduce P. aeruginosa
pathogenesis in vivo.
[0238] The most potent compounds were not cytotoxic to eukaryotic
cells in the conditions tested, making them plausible
anti-virulence factor pro-drugs. Furthermore, these compounds can
significantly decrease cytotoxicity in macrophage cells infected
with PA14 bacterial cells, and they eliminate killing in yeast
incubated with PA14. The molecules identified in this study can
provide the framework for the design of effective anti-virulence
therapeutics at a time when new antimicrobials are in great
demand.
Example 6
MvfR-Inhibitors Eliminate Killing in a Yeast-Psendomonas
Pathogenesis Assay
Yeast Virulence Assays
[0239] The Cryptocaccus neoformans KN99 .alpha., yeast strain was
used to initially assess the impact of the compounds on virulence.
Yeasts were mixed with YPD top agar and poured onto YPD plates. 1
.mu.l of compound or DMSO solvent control was spotted onto the
plate and allowed to dry. P. aeruginosa PA14 or control mvfR mutant
cells were inoculated as 1 .mu.l spotted directly on top of the
dried solvent or compound. The impact of compound on virulence was
assessed by comparing the clearing zone around the bacteria to
PA14+solvent (large zone, control for killing) or the mvfR
mutant+solvent (no killing zone).
[0240] A yeast killing assay was used to initially determine the in
vivo impact of the most potent compounds on pathogenesis. In this
system, a .DELTA.mvfR mutant was completely avirulent compared to
PA14, thus allowing for a clear comparison with MvfR inhibitors.
Cryptococcus neogormans was used as the model yeast and plated as a
top agar suspension. 1 .mu.l of a 10 mM DMSO solution of the
inhibitors or DMSO control was spotted onto the plate followed by a
1 .mu.l inoculation of either PA14 or .DELTA.mvfR cells in the same
location. Compounds were also spotted alone to determine toxicity
to the yeast. After 2-3 days of growth, the zone of inhibition
surrounding the bacteria and compound inoculum was compared to PA14
with DMSO (zone of clearing) and .DELTA.mvfR with DMSO (no clearing
zone). Several compounds, including M34, M50, M51, M59, and M61
eliminated bacterially-induced yeast killing, while M58 and M57 had
reduced clearing zones (FIG. 7), Several potent HAQ inhibitors,
including M26, M27, M52, M53, and M55 were not effective at
reducing virulence in the yeast model. Interestingly, all compounds
effectively eliminating virulence contained a nitro group,
suggesting that this substitution is critical for in vivo activity,
however not all nitro-containing compounds were effective,
indicating that substitution on the benzamide ring was also
important for attenuating virulence.
Example 7
MvfR Inhibitors Attenuate Virulence in a Murine Burn and Infection
Model
Mouse Burn and Infection Model.
[0241] A thermal injury mouse model was used as described
previously to assess bacterial pathogenicity in 6 week old BALBC
mice (Charles River Laboratories). Following mouse anesthetization,
a full-thickness thermal burn injury involving 5%-8% of the body
surface area was produced on the dermis of the shaved abdomen. An
inoculum of 4.times.10.sup.4 PA14 cells, corresponding to a lethal
dose of 50%, was injected intradermally into the burn eschar along
with the compound or solvent, prepared as follows: 5 .mu.l of a 50
mM stock in DMSO of compound was added to a solution of 5%
CremophorEL/Ethanol (50/50% vol) in 50 .mu.l saline. Assuming each
moose has approximately 2.5 ml of blood, this injection corresponds
to .about.100 .mu.M inhibitor/mouse/injection. Each mouse also
received IV injections of 50 .mu.l of compound or solvent, as
prepared above at 6 hours and 24 hours post-infection.
A murine thermal injury and infection model was used to assess the
impact of the compounds on P. aeruginosa pathogenesis in animals.
Due to the hydrophobic nature of the compounds, 5%
CremophorEL/Ethanol (50/50% vol) was used as an emulsifier to
dissolve the compound in saline prior to injecting into animals.
The bacterial cultures were grown in the presence of 10 .mu.M of
the inhibitor. The bacteria and either 100 .mu.M. compound/mouse or
DMSO control, wore co-injected at the time of infection. Compounds
were also injected 6 hours and 24 hours post-infection. M64
resulted in 84% overall surviving mice compared to the solvent
control, for which only 38% survived (FIG. 8). These results
indicated that this compound has an important anti-virulence effect
in vivo, and supports the notion that MvfR inhibitors could be
developed into novel anti-infective therapeutics.
Example 8
MvfR Inhibitor M64 Plus Ciprofloxacin Attenuates Virulence in a
Maurine Burn and Infection Model
[0242] A murine thermal injury model (Stevens et al., J Burn Care
Rehabil. 1994;15:232-235) was used as previously described to
further assess bacterial pathogenicity in 6-wk-old DC-1 mice (Rahme
et al., Science, 268(5219): p. 1899-902 (1995)) in the presence of
combination of Ciprofloxacin (Bayer AG) and M64. Following mouse
anesthetization, a full-thickness thermal burn injury involving
5%-8% of the body surface area was produced on the dermis of the
shaved abdomen, and an inoculum of about 6-7.times.10.sup.3 PA14
cells was injected intradermally into the burn eschar along with
DMSO or 100 .mu.M M64 compound. 100 .mu.M (4 mg/Kg) of M64, 0.4
mg/Kg of ciprofloxacin (lower than the recommended dose of 10
mg/Kg), or a combination of M64+ciproliocaxin was also injected IV
via tail vein at 6,20,28, 40, 60, 68 h and 80 h post-infection.
Mice survival was subsequently assessed over the course of 7 days.
Ten animals per treatment were used. In this set of experiments the
concentration of ciprofloxacin used was lower than the therapeutic
levels recommended to permit assessment of the possible synergistic
effect between M64 and ciprofloxacin. The recommended dose of 10
mg/Kg ciprofloxacin is expected to clear infection.
[0243] FIG. 9A shows that M64 and Ciprofloxacin limited P.
aeruginosa virulence in vivo, wherein mice were administered
multiple intravenous injections of M64 or ciprofloxacin alone or in
combination at 6, 20,28, 40, 60, 68 h and 80 h after injury and
infection. While the un-injected, controls exhibited 30% survival
to P. aeruginosa infection, mice injected with M64, ciprofloxacin
or the combination of thereof, exhibited significant increased
survival rates, 75%, 80% or 100% respectively. The combination
M64+ciprofloxacin promoted 100% survival. Furthermore,
M64+ciprofloxacin synergized in restricting bacterial dissemination
in mice better than M64 or ciprofloxacin alone. FIG. 9B shows that
bacterial leads are lower in M64+ciprofloxacin mice in muscle
samples taken adjacent to burn wound (adjacent muscle) than in mice
treated with M64 or ciprofloxacin alone.
[0244] These results indicate that the anti-virulence effect of M64
can increase survival rate of infected animals even when low
concentrations of antibiotic are used in combination.
Example 9
Chromatin Immunoprecipitation (ChIP) Studies
[0245] To probe the mode of action of M64 and determine the
inhibitors' involvement in MvfR binding to the pqs and phnAB operon
promoter. MvfR was fused to a vesicular stomatitis virus
glycoprotein (VSV-G) epitope tag (Castang et al., Proc Natl Acad
Sci U S A. 2008;105(48): 18947-52) and introduced into PA14 cells.
As a negative control, the ability of the tagged MvfR protein to
bind non-MvfR regulated gene promoters (i.e. rpoD) was tested. ChIP
studies were performed in the presence and absence of M64
inhibitors and in the absence and presence of exogenously added
ligand PQS using the VSV-G-tagged MvfR in PA14 cells, LB broth
aliquots (5 ml) were inoculated (at OD.sub.600.apprxeq.0.03) and
grown at 37.degree. C. to an OD.sub.600 of 1.5-2.5, in the presence
or absence of each inhibitor (10 and 100 mM). Cross-linking and
ChiP were performed as described previously (Castang et al. Proc
Natl Acad Sci USA. 2008:105(48):18947-52). Quantitative PGR was
performed using oligonucleotides corresponding to known MvfR
binding sites in the pqs operon region. (Cao et al., Proc Natl Acad
Sci USA. 2001;98(25):14613-8, Xiao et al. Microbiology. 2006;152(Pt
6):1679-86). Using iTaq SYBR green with RQX (Bio-RAD) and an
Applied Biosystems StepOne-Plus detection system, ChiP fold
enrichment values were calculated (Gastang et al., Proc Natl Acad
Sci (USA, 2008;105(48):18947-52). These values represent the
relative abundance of a sequence of interest versus a negative
control region and in absence of inhibitors. All ChiP fold
enrichment values represent the average of at least three
biological replicates. The results, shown in FIG. 10, suggest that
M64 affects MvfR binding to pqsA promoter by competing with the
MvfR ligand PQS. It is likely that the same occurs with the other
MvfR ligand HHQ.
Example 10
Polymicrobial Infections and Tolerance in Klebsiella and
Acinelobacter
[0246] Human chronic wound infections involving Pseudomonas
aeruginosa are typically polymicrobial, which impair healing and
clearance compared to monomicrobial infections (Dalton et al., PLoS
One, 2011. 6(11): p, e27317). Moreover, bacteria isolated from
polymicrobial wound infections display increased antimicrobial
tolerance in comparison to those in single species infections
(Dalton et al., PLoS One, 2011. 6(11): p. e27317). This suggests
possible synergistic interactions among bacteria in polymicrobial
communities.
[0247] To investigate this, 2-AA was added to exponentially growing
cultures of A. baumamii and Klebsiella pneumonia. 2-AA is a
volatile, low molecular weight molecule, 2-amino acetophenone
(2-AA), produced by the opportunistic human pathogen Pseudomonas
aeruginosa that reduces bacterial virulence in vivo in flies and in
an acute mouse infection model. (Kesarwani et al., PLoS Pathog.
2011 August; 7(8):e1002192. Epub 2011 Aug. 4). The results, shown
in FIG. 11, show that exogenous 2-AA increases the antibiotic
tolerant cell fraction.
[0248] These results suggest that 2-aminoacetophenon, an excreted
small molecule whose synthesis is regulated by MvfR and the enzymes
encoded by the pqs operon genes may promote antibiotic tolerance in
polymicrobial settings (e.g., wounds, lungs) in both K. pneumoniae
and A. baumannii (FIG. 11). These two bacterial pathogens are
frequently isolated from polymicrobial infections, together with P.
aeruginosa (Rezaic et al. Burns, 2011. 37(5); p. 805-7). Since
exogenous addition of 2-AA enriches the accumulation of antibiotic
tolerant cells in these pathogens, and since the inhibitors
described herein target MvfR and inhibit the synthesis of the
molecules synthesized by the pqs operon, it is expected that the
inhibitors described herein, including M64, decrease antibiotic
tolerance also in these organisms.
Other Embodiments
[0249] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *