U.S. patent application number 16/948227 was filed with the patent office on 2021-04-01 for methods for treatment of clostridium difficile infections.
This patent application is currently assigned to The Scripps Research Institute. The applicant listed for this patent is The Scripps Research Institute. Invention is credited to Kim D. Janda.
Application Number | 20210093605 16/948227 |
Document ID | / |
Family ID | 1000005266198 |
Filed Date | 2021-04-01 |
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United States Patent
Application |
20210093605 |
Kind Code |
A1 |
Janda; Kim D. |
April 1, 2021 |
METHODS FOR TREATMENT OF CLOSTRIDIUM DIFFICILE INFECTIONS
Abstract
In this study, we capitalized on the antimicrobial property and
low oral bioavailability of known salicylanilide anthelmintics
(closantel, rafoxanide, niclosamide, oxyclozanide) to target the
gut pathogen. The anthelmintics displayed excellent potency against
C. difficile strains 630 and 4118 (with MIC values as low as
0.06-0.13 .mu.g/mL for rafoxanide) via a membrane depolarization
mechanism. Interestingly, closantel, rafoxanide and compound 8 were
bactericidal against logarithmic- and stationary-phase cultures of
the BI/NAP1/027 strain 4118. Further evaluation of the
salicylanilides showed their preferential activity against
Gram-positive over Gram-negative bacteria. Moreover, the
salicylanilides were non-hemolytic and were non-toxic to mammalian
cell lines HepG2 and HEK 293T/17 within the range of their in vitro
MICs and MBCs. The salicylanilide anthelmintics exhibit desirable
bactericidal and pharmacokinetic properties and are amenable to
repositioning as anti-C. difficile agents.
Inventors: |
Janda; Kim D.; (La Jolla,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Scripps Research Institute |
La Jolla |
CA |
US |
|
|
Assignee: |
The Scripps Research
Institute
|
Family ID: |
1000005266198 |
Appl. No.: |
16/948227 |
Filed: |
September 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16317914 |
Jan 15, 2019 |
10792272 |
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PCT/US17/42056 |
Jul 14, 2017 |
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16948227 |
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62362675 |
Jul 15, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 31/166 20130101; A61K 31/277 20130101; A61K 31/167
20130101 |
International
Class: |
A61K 31/277 20060101
A61K031/277; A61P 31/04 20060101 A61P031/04; A61K 31/166 20060101
A61K031/166; A61K 31/167 20060101 A61K031/167 |
Claims
1. A method of treatment of a Clostridium difficile infection in a
mammal, comprising administering to the mammal an effective dose of
a compound of formula (I) ##STR00003## wherein X is halo or H,
provided at least one X is halo, wherein the ring bearing X is
optionally further substituted with halo; wherein Ar is benzyl,
naphthyl, or indanyl, unsubstituted or independently substituted
with one or more halo, (C1-C4)alkyl, cyano, or nitro groups.
2. The method of claim 1, wherein X is chloro or iodo.
3. The method of claim 1, wherein Ar is substituted with halo or
(C1-C4)alkyl, or both.
4. (canceled)
5. A compound having the formula: ##STR00004##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application and claims
the benefit of priority to U.S. Ser. No. 16/317,914, filed Jan. 15,
2019, which is a national stage application filed under 35 U.S.C
.sctn. 371 from International Application Serial No.
PCT/US2017/042056, filed on Jul. 14, 2017, and published as WO
2018/013890 on Jan. 18, 2018, which claims the benefit of priority
to U.S. provisional application Ser. No. 62/362.675, filed on Jul.
15, 2016, the disclosures of which are incorporated herein by
reference in their entirety.
BACKGROUND
[0002] Clostridium difficile infections (CDI) has plagued nearly
half a million Americans that resulted in 29,300 deaths in
2011,.sup.1 and the propensity of nosocomial CDI recurrence has
been observed in up to 50% of patients..sup.2 The growing epidemic
of CDI has been largely attributed to the emergence of the
hypervirulent strain BI/NAP1/027,.sup.3-5 coupled with the paucity
of therapeutics that specifically target the gram-positive,
spore-forming bacillus as well as, prevent the recrudescence of the
disease. Although current treatment options (metronidazole and
vancomycin) are still able to manage moderate cases of CDI, the
escalating rates of fulminant and recurrent infections pose a
significant threat that warrant immediate attention. Fidaxomicin is
a non-absorbed oral macrocyclic antibiotic that was recently
approved by the FDA for the treatment of CDI. It demonstrated
similar rates of clinical cure as vancomycin.sup.6,7 and
significantly lowered the rate of recurrence of non-NAP1-associated
infections.sup.6--a finding that is attributable to its high
selectivity against C. difficile.sup.8,9 and its ability to inhibit
toxin and spore production in the offending pathogen..sup.10,11
However, there was no difference in outcomes observed for patients
that were infected with the hypervirulent BI/NAP1/027 strain..sup.8
Although resistance is not widespread as of yet, C. difficile
strains with reduced susceptibility to metronidazole, vancomycin or
fidaxomicin have already been noted..sup.12-14
[0003] The persistence of CDI is alarming in its breadth and points
to the pressing need to identify effective treatment options. As a
result, the scientific community has risen to the challenge of
developing alternative small molecule and biotherapeutic strategies
to combat the infectious malady..sup.15 It is evident that
anti-difficile agents with low oral bioavailability (to localize
the drug at the site of infection) and a narrow antimicrobial
spectrum (to minimize collateral damage to the resident
gastrointestinal microbiome) are preferable. Hypervirulent C.
difficile isolates have been shown to produce robust amounts of
lethal toxins (TcdA and TcdB) and spores primarily during the
stationary phase of growth..sup.4 This sets an impediment because
quiescent stationary-phase cells are especially resilient to
antimicrobial chemotherapy..sup.16 An emerging strategy to combat
refractory dormant C. difficile is to target the vulnerability of
its membrane. The clinical relevance of such concept lies in the
essentiality of the microbial membrane in both metabolizing and
non-growing cells, and the associated multifactorial mechanism of
action that could limit the likelihood of bacteria to develop
resistance..sup.17 Indeed, membrane-active agents have demonstrated
potential in eliminating quiescent C. difficile cells, which
subsequently led to a substantial decrease in toxin production and
sporulation..sup.16,18,19
[0004] The salicylanilides have been reported to exhibit
antimicrobial properties.sup.20,21 albeit they are chiefly
exploited as antiparasitic agents. Closantel (1), rafoxanide (2),
niclosamide (3) and oxyclozanide (4) represent four of the widely
used salicylanilide anthelmintics (FIG. 1). Iclosamide is an
FDA-approved drug for the treatment of tapeworm infections, while
the other three are marketed as veterinary drugs for liver
fluke/roundworm infections in ruminants..sup.22 The exact
antibacterial mode of action of salicylanilides is not well defined
but is thought to involve dissipation of the (trans)membrane
potential or the proton motive force (pmf). The pmf modulates the
spatial organization of morphogenetic proteins.sup.23 as well as
ATP homeostasis that is vital for bacterial survival..sup.24 These
functions of the pmf offer an explanation for the effects observed
with certain membrane-active compounds, albeit depletion of which
does not always result to cell death in many bacterial
pathogens..sup.25 The potential use of salicylanilides as
antimicrobials has drawn considerable interest as exemplified by
recent studies demonstrating the anti-staphylococcal properties of
closantel, niclosamide and oxyclozanide..sup.26,27
[0005] A limiting aspect is the low oral bioavailability of
salicylanilides, which may render them ineffective in treating
systemic infections. For instance, niclosamide was found to be only
partially absorbed from the GI tract (with a maximal serum
concentration ranging from 0.25 to 6 .mu.g/mL after oral
administration to human volunteers) and was also poorly distributed
to tissues..sup.28 Closantel, rafoxanide and oxyclozanide exhibited
similar pharmacokinetic (PK) attributes and were minimally
metabolized and mostly excreted unchanged (up to .about.90% for
closantel) in the feces in ruminants..sup.22
SUMMARY
[0006] The invention provides, in various embodiments, a method of
treatment of a Clostridium difficile infection in a mammal,
comprising administering to the mammal an effective dose of a
compound of formula (I)
##STR00001##
[0007] wherein X is halo or H, provided at least one X is halo,
wherein the ring bearing X is optionally further substituted with
halo;
[0008] wherein Ar is phenyl, benzyl, phenethyl, biphenyl,
benzyhydryl, phenoxyphenyl, naphthyl, or indanyl, any of which can
be unsubstituted or independently substituted with one or more
halo, (C1-C4)alkyl, cyano, or nitro groups.
[0009] More specifically, X can be chloro or iodo. More
specifically, Ar can be phenyl, phenethyl, or phenoxyphenyl, any of
which can be substituted with halo or (C1-C4)alkyl or both.
[0010] For instance, the compound of formula (I) can be any one of
compounds closantel (1), rafoxanide (2), niclosanide (3),
oxyclozanide (4), or of any one of a compound of formula (5a),
(5e), (5f), (5g), (6a), (7a), (7b), (7c), (7d), (7e), (7f), (7g),
(7h), (7i), or (8).
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1. Structures of salicylanilide anthelmintics useful
for practice of a method of the invention.
[0012] FIG. 2. Structures of salicylanilide analogues useful for
practice of a method of the invention.
[0013] FIG. 3. Time-kill kinetics against stationary-phase cultures
of BI/NAP1/027 strain 4118. Various concentrations of A) closantel,
B) rafoxanide, C) compound 8, and D) metronidazole or vancomycin
are shown. Data plotted as mean log.sub.10 cfu/mL.+-.s. d. versus
time in h (n=2).
DETAILED DESCRIPTION
[0014] Prolonged use of broad-spectrum antibiotics disrupts the
indigenous gut microbiota, which consequently enables toxigenic
Clostridium difficile species to proliferate and cause infection.
The burden of C. difficile infections was exacerbated with the
outbreak of hypervirulent BI/NAP1/027 strains that produce copious
amounts of enterotoxins and spores. In recent past, membrane-active
agents have generated a surge of interest due to their bactericidal
property with a low propensity for resistance.
[0015] We show that the salicylanilide derivatives efficiently
inhibited the growth of C. difficile via membrane depolarization,
and more importantly, killed both logarithmic- and stationary-phase
cells in a concentration-dependent manner. The bactericidal
property against quiescent C. difficile could in principle lower
the production of toxins and spores, which would in turn mitigate
disease severity and recurrence.
[0016] We initially tested the known anthelmintics closantel,
rafoxanide, niclosamide and oxyclozanide for their activities
against C. difficile strains 630 (CD630, ATCC BAA-1382) and 4118
(CD4118, ATCC BAA-1870). CD630 is a virulent, multidrug-resistant
strain whose genome has been completely sequenced,.sup.29 while
CD4118 is a BI/NAP1/027 hypervirulent pathogen. All four
salicylanilides displayed excellent potency with MIC values as low
as 0.06-0.13 .mu.g/mL for rafoxanide (Table 1). In comparison,
metronidazole had an MIC value of 0.25 .mu.g/mL, whereas that of
vancomycin was significantly higher at 1-2 .mu.g/mL (Table 1). In
order to ascertain that the observed activity of the
salicylanilides occurs through dissipation of the bacterial
membrane potential, we prepared analogues 5 and 6 (FIG. 2) as
previously described,.sup.30 and evaluated their growth inhibitory
activity against CD630 and CD4118. We have earlier delineated the
structural features that are necessary for protonophoric activity
of salicylanilides, requiring both a dissociable phenolic OH group
and an amide proton that forms an intramolecular hydrogen bond to
maintain hydrophobicity and stabilize the anionic form of the
molecule..sup.30 The MIC values that were determined for 5 and 6
are consistent with a membrane depolarization mechanism as the
compounds devoid of protonophoric activity [i.e. analogues that
lack either the weakly acidic OH (5b, 5c, 5h, 5i, 6b and 6c) or the
amide proton (5d)] were inactive, whereas protonophores 5a, 5e, 5f,
5g, and 6a exhibited high in vitro potency against CD630 and CD4118
(Table 1). Encouraged by these results, we explored several other
derivatives, which harbor the diidosalicylate moiety coupled to
varying substituents including biphenyl (7a), halogenated mono-aryl
rings (7b-d), a fused-ring fluorenyl core (7e) and the more
flexible ethylbenzenes (7f-i). Compounds 7a-i demonstrated low MIC
values (.ltoreq.2 .mu.g/mL), except for the ortho-chloro analogue
7c, which showed reduced activity against CD630 and CD4118 (MIC=8
.mu.g/mL). Replacement of the diiodosalicylate with its dichloro
congener (compound 8) resulted in a 4-fold enhancement of potency
relative to 5g and metronidazole, and .about.32-fold improvement of
activity over vancomycin (Table 1).
TABLE-US-00001 TABLE 1 MIC against Clostridium difficile strains
630 and 4118. All minimum inhibitory concentration (MIC) values are
expressed in pg/mL. MIC Cmpd C. difficile 630 C. difficile 4118
Closantel 0.13 0.25 Rafoxanide 0.06 0.13 Niclosamide 1 4
Oxyclozanide 0.5 1 5a 0.5 1 5b >32 >32 5c >32 >32 5d
>32 >32 5e 0.13 0.13 5f 0.13 0.25 5g 0.13 0.25 5h >32
>32 5i >32 >32 6a 0.5 0.5 6b >32 >32 6c >32
>32 7a 0.5 1 7b 0.25 0.5 7c 8 8 7d 0.13 0.25 7e 0.25 0.5 7f 2 2
7g 1 1 7h 0.25 0.5 7i 0.25 0.5 8 .ltoreq.0.03 0.06 Metronidazoe
0.25 0.25 Vancomycin 1 2
The foregoing observations led us to probe other ionophores such as
tropolones and .beta.-carbolines as well as other structurally
related compounds lacking the salicylanilide moiety; however, none
of these were found to be active against CD630 and CD4118
(MIC>32 .mu.g/mL).
Salicylanilides are Bactericidal Against Logarithmic- and
Stationary-Phase Cultures
[0017] The superb growth inhibitory potency exhibited by the
salicylanilides spurred us to further investigate their
bactericidal activities against C. difficile. Although ionophores
are known to dissipate the pmf that is crucial for bacterial energy
metabolism, they do not always display bactericidal
activity..sup.25,27 We were particularly interested in determining
the cidal effect on stationary-phase C. difficile cells, because
these quiescent cells are the primary producer of toxins and spores
that contribute to the severity and recurrence of CDI..sup.4 We
selected the more potent compounds (closantel, rafoxanide and 8)
and assayed them for minimum bactericidal concentration (MBC,
defined as the lowest concentration of the antibacterial agent
required to kill.gtoreq.99.9% of the initial inoculum) against
growing and non-growing cells of the BI/NAP1/027 pathogen CD4118.
As shown in Table 2, all three compounds displayed bactericidal
activities against both logarithmic- and stationary-phase cells of
CD4118 at concentrations close to their MIC values. The MBC.sub.log
values of the protonophores were determined to be 0.25-2 .mu.g/mL
(.about.4 to 8-fold greater than their respective MIC values).
Significantly, the salicylanilides retained bactericidal activities
against dormant stationary-phase C. difficile cells, in stark
contrast to metronidazole and vancomycin, which did not result in
.gtoreq.3-log reduction of CD4118 cells at 32 .mu.g/mL (Table
2).
[0018] Next, we determined the time-kill kinetics of closantel,
rafoxanide and 8 (at 1.times., 4.times., and 16.times. their
respective MICs) against stationary-phase cultures of CD4118. As
depicted in FIG. 3, all three salicylanilides showed a
concentration-dependent mode of killing of the quiescent cells. At
16.times. the MIC of each protonophore, rafoxanide (at 2 .mu.g/mL)
eradicated >99.9% of viable cells in 6 h (FIG. 3b), while
closantel (at 4 .mu.g/mL) and compound 8 (at 1 .mu.g/mL) achieved a
similar potency in 24 h (FIGS. 3a and 3c). At four-fold lower
concentrations (i.e. 4.times.MIC), rafoxanide caused a 2.7-log
decrease in the number of CFUs in 24 h, comparable to those of
closantel and 8, which reduced bacterial cell viability by 2.2- and
2.4-log, respectively. In comparison, neither metronidazole (at 4
.mu.g/mL) nor vancomycin (at 32 .mu.g/mL) reached .gtoreq.3-log
killing of CD4118 stationary-phase cells, even at 16.times. their
respective MIC values (FIG. 3d). The rapid bactericidal property
demonstrated by closantel, rafoxanide and 8 is a significant
finding because quiescent C. difficile cells are notoriously
recalcitrant to antibiotic-mediated killing..sup.16 We surmise that
the cidal effect of such protonophores on stationary-phase C.
difficile cells would ameliorate the effect of toxin production and
spore formation, similar to what was observed with other
membrane-active compounds..sup.16
TABLE-US-00002 TABLE 2 In vitro activity against Clostridium
difficile strain 4118. Cmpd MIC MBC.sub.log MBC.sub.stat Closantel
0.25 2 4 Rafoxanide 0.13 0.5 1 8 0.06 0.25 1 Metronidazole 0.25
>32 >32 Vancomycin 2 8 >32 Abbreviations: MIC, minimum
inhibitory concentration; MBC.sub.log, minimum bactericidal
concentration for logarithmic-phase cells; MBC.sub.stat, minimum
bactericidal concentration for stationary-phase cells, All MIC and
MBC values are expressed in .mu.g/mL.
Salicylanilides Mainly Target Gram-Positive Bacteria
[0019] In an effort to assess the ant bacterial spectrum of
protonophores we evaluated representative compounds (closantel,
rafoxanide, 6a, 7b, 8) against a panel of aerobic and anaerobic
organisms. All five agents were generally more selective against
Gram-positive bacteria, displaying high potency against B. subtilis
ATCC 6051, S. aureus RN4220 and S. epidermidis 1457
(MIC.ltoreq.0.25 .mu.g/mL;) and modest activity against other
anaerobic clostridial species C. sporogenes ATCC 15579 and C.
clostridioforme ATCC 25537 (MIC=1-16 .mu.g/mL). By comparison, the
compounds were ineffective against aerobic Gram-negative bacteria
(MIC.gtoreq.32 .mu.g/mL against A. baumannii M2 and P. aeruginosa
PAO1) and had modest MIC values of .gtoreq.4 .mu.g/mL against gut
commensals B. thetaiotaomicron ATCC 29148, P. distasonis ATCC 8503
and P. nigrescens ATCC 33563. These results are consistent with
those of niclosamide and oxyclozanide, which were shown to
primarily target Gram-positive bacteria..sup.27 Compound 5i, which
does not possess protonophonc activity,.sup.60 lacked antibacterial
activity whereas metronidazole and vancomycin mainly targeted
anaerobic bacteria and Gram-positive organisms, respectively. The
complex multilayered cell envelopes of Gram-negative organisms
impose a permeability barrier to microbial agents and most likely
account for the diminished potency observed for the salicylanilide
molecules. Of note, rafoxanide and 8 had MIC values of .ltoreq.0.13
.mu.g/mL for C. difficile, which rendered .gtoreq.32-fold
selectivity over the Gram-negative gut commensals that were
tested.
In Vitro Cytotoxicity and Hemolytic Activity of Salicylanilides
[0020] Although the salicylanilides have been used extensively in
veterinary medicine, there is little information available
concerning their biological effects on humans, except for
niclosamide, which is FDA-approved for treatment of intestinal
cestode infections. In order to gauge potential cytotoxicity of the
salicylanilides, hemolysis using sheep erythrocytes and MTS.sup.33
assay using two human cell lines (liver carcinoma HepG2 and
embryonic kidney HEK 293T/17) were performed. A significant finding
was that the salicylanilides (closantel, rafoxanide, niclosamide,
oxyclozanide and compound 8) did not cause rupture of red blood
cells when tested at 32 .mu.g/mL. However, treatment of human cell
lines with niclosamide led to a significant decrease in viability
even at a low concentration of 0.125 .mu.g/mL. Despite its high in
vitro cytotoxicity, niclosamide is considered a "safe drug" because
of its minimal absorption from the GI tract and high plasma protein
binding,.sup.28 thus sparing the host cells from its uncoupling
property. An intriguing observation was the comparably lower in
vitro toxicities of compound 8 and the veterinary drugs (closantel,
rafoxanide, oxyclozanide) toward HepG2 and HEK 293T/17. Both
closantel and rafoxanide had no apparent effect on mammalian cell
viability even at a concentration of 8 .mu.g/mL, which is
.gtoreq.32-fold higher than their corresponding MIC values against
C. difficile (Table 1). These results do not guarantee drug safety
(relative to niclosamide) but nevertheless indicate the potential
for repositioning of the veterinary anthelmintics as human
drugs.
[0021] A common cause of antibiotic failure is the inadequate
penetration of the target infection site. In the case of CDI, it is
imperative that the active drug achieves therapeutic levels in the
colon to repress or eliminate the outgrowth of toxigenic C.
difficile. This places the salicylanilide anthelmintics at a
definite advantage; their low oral bioavailability and high fecal
excretion (as observed in ruminants and humans).sup.22,28 would in
theory result in adequate gut concentrations necessary to disarm
the target pathogen. A substantial feature of the salicylanilides
(as we have shown for closantel, rafoxanide and 8) is their
bactericidal activity against stationary-phase cultures of
hypervirulent C. difficile--a property that is not exhibited by
many antibiotics including metronidazole and vancomycin..sup.16
Killing of dormant and hypervirulent C. difficile could likely
suppress toxin production and inhibit sporulation, which in
principle would lead to an improved sustained response and reduced
recurrence rate. The clinical potential of membrane-active agents
is demonstrated by daptomycin and telavancin, which function
through permeabilization/depolarization of bacterial membranes and
are FDA-approved to treat complicated skin and skin structure
infections..sup.34,35 Our results exemplify notable attributes of
the salicylanilide anthelmintics and demonstrate their potential
for repurposing as anti-Clostridium difficile agents. Work is
ongoing in our laboratory to exploit the salicylanilides as
alternative therapies to combat CDI.
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[0057] All patents and publications referred to herein are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference in its entirety.
EXAMPLES
[0058] Bacterial strains. Clostridium difficile 630 (ATCC.RTM.
BAA-1382-FZ.TM.), Clostridium difficile 4118 (ATCC.RTM.
BAA-1870.TM.), Clostridium sporogenes (ATCC.RTM. 15579.TM.),
Clostridium clostridioforme (ATCC.RTM. 25537.TM.), Bactercides
thetaiotaomicron (ATCC.RTM. 29148.TM.), Parabacteroides distasonis
(ATCC.RTM. 8503.TM.) Prevotella nigrescens (ATCC.RTM. 33563.TM.),
and Bacillus subtilis (ATCC.RTM. 6051.TM.) were purchased from ATCC
(Manassas, Va. USA). Pseudomonas aeruginosa PAO1 was provided by
Dr. Kendra Rumbaugh. [0059] Bacterial culture. Clostridium species
were routinely cultured either on blood agar base II plates with 5%
sheep blood (Teknova), or in brain-heart infusion broth/agar plates
supplemented with 0.5% yeast extract (BHIS) containing 0.03%
L-cysteine. Bacteroides thetaiotaomicron, Parabacteroides
distasonis, and Prevotella nigrescens were grown on Brucella
broth/agar plates supplemented with hemin (5 .mu.g/mL), vitamin
K.sub.1 (1 .mu.g/mL) and 5% lysed horse blood. Anaerobic bacterial
culture was performed in an anaerobic cabinet (Coy Lab Products) at
37.degree. C. in a reducing anaerobic atmosphere (8% H.sub.2, 8%
CO.sub.2, 84% N.sub.2). All broths and 96-well plates were
pre-reduced (incubated anaerobically overnight) prior to use for
anaerobic culture. Aerobic bacteria were routinely cultured on
Mueller-Hinton broth/agar plates. [0060] Determination of minimum
inhibitory concentration (MIC). All MICs were determined in 96-well
plates using the broth microdilution method. Two-fold serial
dilutions of test compounds were inoculated with
.about.5.times.10.sup.5 cfu/mL bacteria. MIC was recorded as the
lowest concentration of the test compound that inhibited visible
bacterial growth after 20-24 h of incubation at 37.degree. C. MIC
assays were performed in duplicate. [0061] Determination of minimum
bactericidal concentration (MBC). Clostridium difficile strain 4118
was grown to OD.sub.600.about.0.4-0.5 (logarithmic phase) or for 24
h (stationary phase), and thereafter added to two-fold serial
dilutions of test compounds. Cultures were incubated for 20-24 h at
37.degree. C., and then viable counts were enumerated on BHIS agar
plates. The MBC was determined as the lowest concentration of the
test compound that resulted in .gtoreq.3-log reduction of the
initial cell inoculum. MBC measurements were performed in
duplicate. [0062] Time-kill kinetics assay. Stationary phase
cultures of Clostridium difficile strain 4118 were treated with
closantel, rafoxanide, compound 8 at 1.times., 4.times.,
16.times.MIC or with metronidazole and vancomycin at 16.times.MIC.
At various time points, sample aliquots were taken and determined
for bacterial viability on BHIS agar plates. Kinetic experiments
were performed in duplicate. [0063] In vitro cytotoxicity assay.
Cell lines Hep G2 [HEPG2] (ATCC.RTM. HB-8065.TM.) and 293T/17 [HEK
293T/17] (ATCC.RTM. CRL-11268.TM.) were purchased from ATCC and
cultured according to manufacturer's instructions. HEPG2 or HEK
293T/17 cells were plated in 96-well plates, and incubated at
37.degree. C. in a 5% CO.sub.2 humidifying chamber for 24 h. Cells
were then treated with test compounds at varying concentrations,
and an MTS assay was performed at 16-h post-incubation at
37.degree. C. in a 5% CO.sub.2 humidifying chamber, using the
CellTiter 96 aqueous non-radioactive cell proliferation assay kit
(Promega, Madison, Wis., USA) per manufacturer's instructions. MTS
assays were performed in duplicate. [0064] Hemolysis assay. Sheep
red blood cells (Innovative Research, Novi, Mich., USA) were washed
three times with PBS pH 7.4. A 3% cell suspension in PBS (100
.mu.L) was added to test compounds in PBS (100 .mu.L), and then
incubated at 37.degree. C. for 1 h. The plate was centrifuged at
500.times.g for 10 min, and supernatants (100 .mu.L) were
transferred to a clean 96-well plate. Hemolysis was determined by
measuring absorbance at 540 nm, with 1% Triton X-100 as the
positive control and 0.5% DMSO in PBS as the negative control.
Hemolysis assays were performed in triplicate.
[0065] Tables 3 and 4 provide an indication of the bioactivity of
compounds (5i), (6a), (7b), and (8) versus a selection of aerobic
and anaerobic bacteria, respectively.
TABLE-US-00003 TABLE 3 In vitro activity against select aerobic
bacteria MIC.sup.a (.mu.g/mL) Clo- Ra- metro- van- strain santel
foxanide 5i 6a 7b 8 nidazole comycin B. subtilis .ltoreq.0.03
.ltoreq.0.03 >32 0.06 0.06 .ltoreq.0.03 >32 0.13 ATCC 6051 S.
aureus RN4220 0.25 0.25 >32 0.25 0.06 0.13 >32 1 S.
epidermidis .ltoreq.0.03 .ltoreq.0.03 >32 0.06 .ltoreq.0.03
.ltoreq.0.03 >32 2 1457 A. baumannii M2 >32 >32 >32
>32 32 32 >32 >32 P. aeruginosa >32 >32 >32
>32 >32 >32 >32 >32 PAO1 .sup.aPerformed in
duplicate. For clarity, MIC values against Gram-positive and
Gram-negative bacteria are shown in blue and red, respectively.
TABLE-US-00004 TABLE 4 In vitro activity against select anaerobic
bacteria MIC.sup.a (.mu.g/mL) strain Closantel Rafoxanide 5i 6a 7b
8 metronidazole vancomycin C. sporogenes 1 1 >32 8 16 4 0.25 2
ATCC 15579 C. clostridioforme 4 1 >32 4 16 4 0.06 0.5 ATCC 25537
B. thetaiotaomicron >32 >32 >32 >32 >32 >32 1
>32 ATCC 29148 P. distasonis 16 8 >32 16 8 4 2 >32 ATCC
8503 P. nigrescens 8 8 >32 8 4 4 2 >32 ATCC 33563
.sup.aPerformed in duplicate. For clarity, MIC values against
Gram-positive and Gram-negative bacteria are shown in blue and red,
respectively.
Synthesis and Characterization of Compounds
[0066] Closantel (Sigma), rafoxanide (TCI America), niclosamide
(Combi-Blocks), oxyclozanide (Sigma), metronidazole (Combi-Blocks),
and vancomycin hydrochloride hydrate (Sigma) were used as
received.
[0067] Compounds 5a-i, 6a-c, 7a-d, 9a-b, 10a-b, 11a-f and 12 were
prepared as previously described..sup.2-4 Compounds 7e-i and 8 were
synthesized according to published procedure..sup.2 Briefly.
3,5-diiodosalicylic acid (or 3,5-dichlorosalicylic acid, 1 eq) was
heated to reflux with SOCl.sub.2 (5 eq) for 7 h, and thereafter
concentrated under reduced pressure. The corresponding acyl
chloride product was precipitated with cold hexanes, filtered and
air-dried. Coupling with the respective amine (1 eq) was performed
in DMF in the presence of DIPEA (3 eq) at rt for 1 h. All
salicylanilide products were purified by preparative HPLC. Reagents
and solvents were obtained from commercial sources, and reactions
were carried out using technique known to those having ordinary
skill in the art.
##STR00002##
[0068] .sup.1H and .sup.13C NMR spectra were recorded on Bruker
DRX-600 equipped with a 5 mm DCH cryoprobe. Purity of all tested
products were generally >95% as assessed by HPLC.
[0069] N-(9H-Fluoren-2-yl)-2-hydroxy-3,5-diiodobenzamide (7e).
Yield: 40%. .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 3.94 (s, 2H),
7.30-7.34 (m, 1H), 7.39 (t, J=7.3 Hz, 1H), 7.44-7.48 (m, 1H), 7.56
(d, J=7.4 Hz, 1H), 7.75-7.81 (m, 2H), 7.81 (d. J=1.8 Hz, 1H), 7.89
(s, 1H), 7.98 (s, 1H), 8.20 (d, J=1.8 Hz, 1H). .sup.13C NMR (151
MHz, CDCl.sub.3) .delta. 37.2, 80.4, 89.1, 116.9, 118.5, 120.0,
120.3, 120.5, 125.2, 127.0, 127.1, 134.3, 134.8, 139.9, 141.0,
143.4, 144.6, 151.1, 160.5, 166.4. HRMS-ESI (m/z): [M+H].sup.+
calcd for C.sub.20H.sub.14I.sub.2NO.sub.2, 553.9114; found, 553.
9110.
[0070] N-(2Chlorophenethyl)-2-hydroxy-3,5-diiodobenzamide (7f).
Yield: 49%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 2.99 (t,
J=7.2 Hz, 2H), 3.54 (q, J=7.0 Hz, 2H), 7.24-7.31 (m, 2H), 7.34 (dd,
J=2.1, 7.2 Hz, 1H), 7.44 (dd, J=1.9, 7.3 Hz, 1H), 8.16 (d, J=1.9
Hz, 1H), 8.18 (d. J=1.9 Hz, 1H), 9.26 (t, J=5.6 Hz, 1H). .sup.13C
NMR (151 MHz, DMSO) .delta. 32.3, 81.4, 88.8, 116.2, 127.4, 128.4,
129.3, 131.2, 133.2, 135.1, 136.4, 149.4, 159.8, 168.1. HRMS-ESI
(m/z): [M+H].sup.+ calcd for C.sub.15H.sub.13ClI.sub.2NO.sub.2,
527.8719; found, 527. 8706.
[0071] N-(3-Chlorophenethyl)-2-hydroxy-3,5-diiodobenzamide (7g).
Yield: 53%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 2.87 (t,
J=7.2 Hz, 2H), 3.53 (q, J=7.0 Hz, 2H), 7.19-7.22 (m, 1H), 7.26-7.29
(m, 1H), 7.30-7.35 (m, 2H), 8.16 (d, J=1.9 Hz, 1H), 8.17 (d, J=1.9
Hz, 1H), 9.22 (t, J=5.5 Hz, 1H). .sup.13C NMR (151 MHz, DMSO)
.delta. 34.0, 40.5, 81.4, 88.9, 116.2, 126.3, 127.5, 128.6, 130.2,
133.0, 135.1, 141.6, 149.4, 159.8, 168.1. HRMS-ESI (m/z):
[M+H].sup.+ calcd for C.sub.15H.sub.13ClI.sub.2NO.sub.2, 527.8719;
found, 527. 8717.
[0072] N-(4-Chlorophenethyl)-2-hydroxy-3,5-diiodobenzamide (7h).
Yield: 51%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 2.85 (t,
J=7.2 Hz, 2H). 3.51 (q, J=7.2 Hz, 2H), 7.27 (d, J=8.5 Hz, 2H), 7.35
(d, J=8.5 Hz, 2H), 8.16 (d, J=1.9 Hz, 1H), 8.18 (d, J=2.0 Hz, 1H),
9.21 (t, J=5.4 Hz, 1H). .sup.13C NMR (151 MHz, DMSO) .delta. 33.7,
40.7, 81.4, 88.9, 116.2, 128.3, 130.6, 130.9, 135.1, 138.1, 149.4,
159.8, 168.0. HRMS-ESI (m/z): [M+H].sup.+ calcd for
C.sub.15H.sub.13ClI.sub.2NO.sub.2, 527.8719; found, 527. 8713.
[0073] N-(2,4-Dichlorophenethyl)-2-hydroxy-3,5-diiodobenzamide
(7i). Yield: 59%. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 2.97
(t, J=7.0 Hz, 2H), 3.53 (q, J=6.9 Hz, 2H), 7.35-7.39 (m, 2H), 7.60
(d, J=1.1 Hz, 1H), 8.14-8.19 (m, 2H), 9.23 (t, J=5.5 Hz, 1H).
.sup.13C NMR (151 MHz, DMSO) .delta. 31.8, 81.4, 88.8, 116.2,
127.5, 128.7, 131.9, 132.5, 134.1, 135.1, 135.7, 149.4, 159.8,
168.2. HRMS-ESI (m/z): [M+H].sup.+ calcd for
C.sub.15H.sub.12Cl.sub.2I.sub.2NO.sub.2, 561.8329 found, 561.
8319.
[0074]
3,5-Dichloro-N-(4-(4-chlorophenoxy)phenyl)-2-hydroxybenzamide (8).
Yield: 55%. .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 6.96 (d,
J=8.8 Hz, 2H), 7.04 (d, J=8.9 Hz, 2H), 7.31 (d, J=8.8 Hz, 2H), 7.50
(d, J=2.3 Hz, 1H), 7.53 (d, J=8.9 Hz, 2H), 7.56 (d, J=2.3 Hz, 1H),
8.02 (s, 1H), 12.16 (s, 1H). .sup.13C NMR (151 MHz, CDCl.sub.3)
.delta. 116.6, 119.6, 120.3, 123.5, 123.7, 124.2, 124.6, 128.8,
130.0, 131.6, 134.3, 154.9, 155.8, 156.0, 166.6. HRMS-ESI (m/z):
[M+H].sup.+ calcd for C.sub.19H.sub.13Cl.sub.3NO.sub.3, 407.9955;
found, 407. 9955.
SYNTHETIC METHODS DOCUMENTS CITED
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