Salicylanilide Analog Minimizes Relapse of Clostridioides difficile Infection in Mice

Article abstract of DOI:10.1021/acs.jmedchem.0c00123

Clostridioides difficile infection (CDI) causes serious and sometimes fatal symptoms like diarrhea and pseudomembranous colitis. Although antibiotics for CDI exist, they are either expensive or cause recurrence of the infection due to their altering the colonic microbiota, which is necessary to suppress the infection. Here, we leverage a class of known membrane-targeting compounds that we previously showed to have broad inhibitory activity across multiple Clostridioides difficile strains while preserving the microbiome to develop an efficacious agent. A new series of salicylanilides was synthesized, and the most potent analog was selected through an in vitro inhibitory assay to evaluate its pharmacokinetic parameters and potency in a CDI mouse model. The results revealed reduced recurrence of CDI and diminished disturbance of the microbiota in mice compared to standard-of-care vancomycin, thus paving the way for novel therapy that can potentially target the cell membrane of C. difficile to minimize relapse in the recovering patient.

Full text of DOI:10.1021/acs.jmedchem.0c00123

Subscriber access provided by Uppsala universitetsbibliotek
Article
Salicylanilide analog minimizes relapse
of Clostridioides difficile infection in mice
Steven Blake, Rajani Thanissery, Alissa J Rivera, Mark S
Hixon, Mingliang Lin, Casey Michelle Theriot, and Kim D Janda
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00123 • Publication Date (Web): 01 Jun 2020
Downloaded from pubs.acs.org on June 2, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 17
Journal of Medicinal Chemistry
1
2
3
4
5
6
Salicylanilide analog minimizes relapse of
7
8
9
Clostridioides difficile infection in mice
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Steven Blake,† Rajani Thanissery,‡ Alissa J. Rivera,‡ Mark S. Hixon,∫ Mingliang Lin,† Casey M. Theriot‡
and Kim D. Janda*,†
†Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm
Institute of Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, California 92037, United States
∫Mark S. Hixon Consulting, LLC., 11273 Spitfire Road, San Diego, CA 92126, United States.
‡Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina
State University, Raleigh, NC 27607, United States.
ABSTRACT
Clostridioides difficile infection (CDI) causes serious and sometimes fatal symptoms like diarrhea and
pseudomembraneous colitis. Although antibiotics for CDI exist, they are either expensive or cause
recurrence of the infection due to their altering the colonic microbiota which is necessary to suppress the
infection. Here, we leverage a class of known membrane-targeting compounds that we previously
showed to have broad inhibitory activity across multiple C. difficile strains while preserving the
microbiome to develop an efficacious agent. A new series of salicylanilides was synthesized and the
most potent analog was selected through an in vitro inhibitory assay to evaluate its pharmacokinetic
parameters and potency in a CDI mouse model. The results revealed reduced recurrence of CDI and
disturbance of the microbiota in mice compared to standard-of-care vancomycin, thus paving the way
for novel therapy that can potentially target the cell membrane of C. difficile to minimize relapse in the
recovering patient.
INTRODUCTION
cytoskeletal structures.2 The host then
experiences excruciating and sometimes fatal
symptoms such as diarrhea (3 or more times a
Clostridioides difficile (formerly Clostridium
difficile), an anaerobic, sporulating, Gram-
positive bacterium, is the major cause of hospital-
acquired infections in the US with estimated
costs of $4.8 billion in acute care facilities alone.1
The pathogenesis of C. difficile infections (CDI)
typically stems from an opportunistic exposure of
dormant, yet robust C. difficile spores to an
individual whose gut microbiome has been
compromised by antibiotic treatment for a recent,
unrelated infection. Upon germination of these
spores, the nascent bacteria colonizes the colon
within 36 hours. Primarily during their stationary
phase, the pathogenic strains secrete multiple
toxin proteins (TcdA / TcdB) that cause cell
death by disrupting cellular tight junctions and
day), abdominal cramping and toxic megacolon.3,
4
Due to the scarcity of effective treatments,
CDI has become the leading cause of
gastroenteritis fatalities, claiming 14,000 lives in
2007 and resulting in approximately 29,000
deaths within 30 days of diagnosis in 2011.5
Moreover after treatment, up to 30% of patients
experience recurrence of CDI.6 This drastic
distribution of CDI across the population arises
largely from the emergence of NAP1/BI/027
isolates that exhibit robust toxin production,
sporulation and fluoroquinolone resistance.7-9
Although, for over 30 years, metronidazole and
vancomycin have remained the best therapy for
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 17
CDI,10 they are now linked to high recurrence
Because multiple sites are being targeted
1
2
3
4
5
6
7
8
rates11, 12 and a gradual increase in resistance
across some C. difficile strains.13-15 A key to
minimizing recurrence is immediate recovery of
the pre-CDI bacterial distribution of the gut
microbiome; however metronidazole and
vancomycin delay the recovery,16-18 making it
easier for C. difficile to re-colonize after their
resistant spores re-germinate upon cessation of
treatment. Fidaxomicin, which was FDA-
approved in 2011, significantly reduces the risk
of recurrence for non-NAP1-mediated infections,
yet only minor differences in the outcomes of
patients infected with the hypervirulent
compared to vancomycin which only targets cell
wall synthesis, a lower chance for resistance
against these agents can be expected.27 Finally,
we have previously shown that a salicylanilide, 1,
displays broad activity against 16 C. difficile
isolates with 3-fold lower minimum inhibitory
concentration (MIC50 and MIC90) than
vancomycin. Importantly, 1 displayed a low
propensity for resistance and a 1000-fold
reduction in CD4118, a hypervirulent strain
(ATCC BAA-1870), at its MIC that both
metronidazole and vancomycin failed to do.
These positive observations were noted with
minimal impact to gut commensals.28
These attractive antimicrobial properties of
salicylanilides incentivized us to embark on a
campaign to synthesize in a facile manner more
potent analogs. Herein, we detail this new series
of analogs, their MICs, PK and therapeutic
evaluation in a mouse model of recurrent CDI.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
BI/NAP1/027 strain are observed relative to
those treated with vancomycin.17 Based on the
drawbacks of these current drugs, antimicrobials
that display the following parameters are needed
to combat CDI: 1) bactericidal across multiple
strains especially virulent ones; 2) low propensity
for resistance; 3) high gut restriction; 4) narrow
antimicrobial spectrum to preserve the gut
microbiome; and 5) activity against stationary-
phase where toxin release and sporulation
primarily occur.
RESULTS AND DISCUSSION
Screen for potent salicylanilide.
Salicylanilides are known proton ionophores29
that require both a dissociable phenolic OH and
an amide proton (which forms an intramolecular
hydrogen bond to maintain hydrophobicity and
stabilize the anionic form of the molecule) for
activity.30 Initial studies to characterize the mode
of action of salicylanilides against C. difficile
(CD630 / CD4118 strains) infers a membrane
depolarization mechanism as analogs devoid of
proton ionophoric activity (i.e. lacking either the
weakly acidic OH or the amide proton) were
inactive, whereas the proton ionophore with
intact phenolic OH and amide proton displayed in
vitro activity against CD630 and CD4118.28 A
major advantage to protonophoric antimicrobials
is their selective bactericidal activity against C.
difficile in the presence of gut commensals.27 For
example, diarylacylhydrazones that carry a
phenol functional group can target C. difficile and
decouple oxidative phosphorylation.31 Given that
salicylanilides possess a similar structural motif
to these hydrazones (an amide flanked by an aryl
group on one side and a phenol on the other), it
suggests that a similar mechanism of action may
underlie selective anti-C. difficile activity in the
presence of gut commensals.28
One class of compounds that potentially
adheres to the aforementioned parameters is the
salicylanilides. For example, niclosamide, is
FDA-approved for intestinal cestode infections
and has recently been demonstrated to diminish
the toxicity of C. difficile’s TcdA and TcdB
proteins to the host.19 Moreover, other
salicylanilides such as closantel, rafoxanide and
oxyclozanide are approved for veterinary use
against certain worm infections in ruminants.20
Furthermore, exploiting salicylanilides to directly
target infectious bacteria such as Gram-positive
Staphylococcal and Enterococcus strains has
been explored.21-23 From these studies, the
supposed mode of action is attributed to the
detrimental impact on energy metabolism,
damage to the cell membrane and leakage
brought upon by the disruption of transport and
respiratory processes.24, 25 One proposed
mechanism behind the impact on energy
metabolism involves the salicylanilides’ role in
shuttling protons from the extracellular space to
the less acidic intracellular space, disturbing not
only the intracellular pH but all dependent
biochemical processes such as ATP synthesis.26
ACS Paragon Plus Environment

Page 3 of 17
Journal of Medicinal Chemistry
R
O
Cs2CO3, DMSO
F
OH
1
2
3
4
5
6
7
8
9
Cl
NO2
Cl
O2N
R
Fe, NH4Cl, THF
EtOH, H2O
P(OPh)3,
toluene
R
O
R
O
OH
Cl
O
Cl
Cl
N
H
Cl
Cl
NH2
1 - 13
Cl
COOH
HO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. General route for the synthesis of
salicylanilide analogs (1-13).
Figure 1. Graphs depicting plasma
exposure of 11 from A) i.v. and B)
p.o. administration.
To further probe the molecular mode of action
and improve the bactericidal activity against C.
difficile, we assessed the inductive effect of
substituents on the anilide ring that is also known
to have a strong bearing on its uncoupling
activity32 of salicylanilides in addition to
hydrophobicity. Thus, a series of salicylanilide
analogs was synthesized (Scheme 1) with
substituents ranging from strongly electron-
donating (methoxy, 7 and 8) to electron-
withdrawing (dinitro, 13) (Table 1). The VPI
10463 (CD10463) strain was chosen for these in
vitro experiments because of its high toxin
production compared to other strains.33, 34 We
observed a trend toward better activity than 1
when electron-withdrawing groups such as chloro
(4), cyano (9), fluoro (6), bromo (2 and 3), iodo
(5 and 10), diiodo (12) and bis(trifluoromethyl)
(11) were placed on the anilide ring. Following
this trend, we synthesized analog 13 which
carries two nitro groups but observed diminished
antimicrobial activity. From these findings, we
reasoned if the salicylanilides’ anti-C. difficile
properties originate from the same protonophoric
activities against other organisms,29 then there
should be an ideal pKa for the amide proton
Table 1. MIC90s of vancomycin and various
salicylanilides with 11 showing the best activity
against CD10463
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 17
of 76% in mice. Although higher than
1
2
3
4
5
6
7
8
anticipated, high bioavailability alone is not
problematic if the therapeutic window is wide.
The maximum cecal concentrations of 11 at 50
µM was obtained 4 hours after gavage.
Minimum cecal concentrations over 24 hours
were in excess of 1 µM thus exceeding the in
vitro MIC90 by at least 100-fold (Figure 2).
In vitro cell viability was determined using an
MTS (cell proliferation) assay with HEK 293T /
17 human kidney cell lines and hemolysis assay
with sheep erythrocytes. Although the MTS assay
indicated cytotoxicity at concentrations of 11
exceeding 2.5 µM (Figure S1), cytotoxic levels
were lower than niclosamide, an FDA-approved
salicylanilide. A more gut-restricted analog of 11
can mitigate toxicity that might arise from
systemic exposure. To probe cytotoxicity further,
we carried out a hemolysis assay, which
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
where transmembrane proton shuttling is
maximized in the intracellular and / or
displayed negligible hemolysis (< 2%) in the
range of 0.02 – 40 µM of 11.
extracellular spaces flanking the membrane.
Therefore based on this reasoning, we believe the
bis(trifluoromethyl) functional groups instilled
11’s amide with the necessary acid / base
properties to yield the highest potency (lowest
MIC) against CD10463 among the 12 other
candidates. As such, this compound was
In vivo efficacy of 11 in a mouse model of
recurrent CDI. After elucidating the PK
parameters of 11, it was examined in a mouse
model of recurrent CDI. According to the
experimental design (Figure 3), one of four
groups cefoperazone-treated mice (C57Bl/6J,
n=6) were exposed to either: 1) no C. difficile
advanced for further studies.
Pharmacokinetic and cytotoxic properties
of 11. As a next step in this medicinal chemistry
campaign, PK experiments were undertaken to
evaluate the absorption, distribution, and
excretion of 11 in C57Bl/6J mice. These
experiments enabled the quantification of local
gut concentrations after oral administration,
which then determined the dosage and dosing
frequency that would be most appropriate for the
in vivo efficacy studies. To mirror the CDI mouse
model, mice were pre-treated with the antibiotic
cefoperazone to alter their gut microbiota. Then
11 was examined at an oral dose (p.o.) of 1
mg/kg. In addition, it was dosed intravenously
(i.v.) at 0.3 mg/kg in non-antibiotic treated mice
to determine oral bioavailability and clearance
(Table 2). The plasma exposure data (Figure 1)
illustrates an expected longer retention of
compound in plasma after p.o. administration
than after i.v. administration over 24 hours.
While a gut restricted asset is favored over one
with systemic distribution, 11 has a bioavailablity
Figure 2. Graph illustrating the
change in drug concentration of 11
(administered at 1 mg / kg) in the
cecum through a 24-hr period
relative to their in vitro MIC90
Figure 3. Mouse model of recurrent C. difficile
infection. Mice were treated with cefoperazone
in drinking for 5 days then a two day wash out,
before being challenged with C. difficile spores
on day 0. Treatment with vancomycin, vehicle,
or 11 was done from day 4-9, and then mice
were monitored for recurrent CDI, which
occurred on days 13-14 in the vancomycin
ACS Paragon Plus Environment
treated group.

Page 5 of 17
Journal of Medicinal Chemistry
spores and vancomycin (no C. difficile); 2) C.
1
difficile spores and vancomycin (vanco); 3) C.
difficile spores and vehicle (vehicle); and 4) C.
difficile spores, vehicle and 11 at 5 mg / kg (11).
As expected, antibiotic-treated mice challenged
with C. difficile spores developed clinical signs
of disease including weight loss (Figure 4A) and
lethargy by day 4 post challenge. Challenged
mice had significantly lower body weight when
compared to the no C. difficile group by day 3 for
the vanco (p = 0.033) and 11 (p= 0.019), and by
day 4 for the vehicle (p = 0.032, 2- way repeated
measure ANOVA with Holm-Sidak’s multiple
comparisons post hoc test). High levels of C.
difficile total vegetative cells plus spores (Figure
4B) and spores only (Figure 4C) were present in
the feces of mice on day 1 and 4 post challenge.
As anticipated in the vanco-treated group, during
the 5-day treatment (from day 4-9), C. difficile
load decreased below the limit of detection by
day 7 (Figure 4B and C). However, 3 days after
the cessation of vancomycin treatment, mice
began to show clinical signs of disease again
including weight loss, and the return of C.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
difficile in feces, similar to pre-treatment levels,
signifying recurrent CDI (Figure 4A-C). On the
other hand, the 11-treated group showed a
Figure 4. Percent baseline weight and C. difficile
fecal load throughout in a mouse model of
recurrent CDI. (A) Baseline weight loss of
cefoperazone treated C57BL/6J mice challenged
with C. difficile strain 630 (n=6 per treatment
group), then treated with vancomycin, vehicle, or
11 from days 4-9. (B) Total vegetative cells plus
spores and (C) spores only of C. difficile in
colony forming units (CFUs) per gram of fecal
content throughout infection. Significance was
determined by Mixed-effect model test followed
by Tukey’s multiple comparisons post test (*, p
<0.05; **, p < 0.01; ****, p < 0.0001). Error
bars represent the standard deviation from the
mean. The black line from day 4-9 represents
duration of treatment.
significant reduction in C. difficile load (total
vegetative cells plus spores) when compared to
the vehicle group during treatment (Figure 4B,
Mixed-effect model with Tukey’s multiple
comparisons post hoc test , p = 0.0096) which
remained low even after cessation of treatment.
By day 13, 50% of the mice from this group had
a C. difficile load below the limit of detection.
The other 50% that still had detectable levels of
C. difficile correlated with a higher body weight
indicating a need to enhance the dosing to
observe complete clearance. Interestingly, the 11-
treated group showed clearance of spores (below
detection limit) on day 9 (Figure 4C) suggesting
11 could directly affect sporulation or
germination of spores, which needs further
investigation. However, spores became
detectable upon cessation of treatment. The mice
from the vehicle group remained persistently
colonized with C. difficile (both total vegetative
cells plus spores and spores only; Figure 4B and
C) throughout infection suggesting that the
vehicle had no effect on C. difficile. On the day
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 17
of necropsy (day 14), vanco-treated mice
exhibited 10-26% weight loss which was
1
2
3
4
5
6
7
8
significantly lower compared to no C. difficile
control (p = 0.031), vehicle (p = 0.035) and 11 (p
= 0.035; Figure 4A). The 11-treated group had
significantly less total vegetative cells plus spores
in cecal content when compared to both the
vanco-treated group (p = 0.004) and vehicle
group at necropsy (p = 0.002, Student’s
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
parametric t-test with Mann-Whitney post hoc
test) (Figure 5A). The number of spores in mouse
cecal content from the 11-treated group was
significantly lower than the vanco-treated group
(p = 0.002), but not the vehicle group (Figure
5B). Treatment with 11 also resulted in a
significant decrease in toxin activity in cecal
content to below the limit of detection in all but
one mouse when compared to the vanco-treated
group (p = <0.0001) and the vehicle group (p =
<0.0039, 1- way ANOVA with Mann-Whitney
post hoc test) (Figure 5C). Spearman’s
correlation (r) of the C. difficile load CFU/ml and
toxin activity in the cecal content of mice was
significantly correlated (r = 0.8385, p < 0.0001),
suggesting low toxin activity is due to decreased
colonization or growth, and this is most evident
in the 11-treated group (Figure 5D). Vanco-
treated mice were clinically ill on day 14 and
histopathological evaluation revealed submucosal
edema, inflammation, and epithelial damage in
the ceca (Figure 5E and F). The histopathological
summary scores were significantly lower for the
11-treated (p ≤ 0.01) group and the no C. difficile
controls (p ≤ 0.05) when compared to the vanco-
treated group. In contrast, by day 14 the vehicle
group revealed high C. difficile burden, but lower
toxin activity correlating with lower
Figure 5. Colonization levels, toxin activity,
and histopathological evaluation of the cecum
oFnigDuraey 164.. (AIm) pTaocttalovfeg1e1taotinvegcuetllsdiavnedrssiptyoreasnd
abnadcte(Bri)alScpoomrems ounliytyinmCemFUbesrpsheripgroanmdoayf c1e4c.al(A)
cAolnptheant.d(iCve)rTsiotyxinmaecatsivuirteydinbtyheincvecearslecoSnitmenptson
(ind=e6xpperretsreenattemdefnotrgdrioffuepr)e.n(tDtr)eCatomrreenltagtiroonups. (B)
bCehtawnegeenstotxoinbaaccttievriitaylancdomC.mduifnfiitcyilemembership
caomlonigzadtiifofenr.e(nEt)trReeaptmreesnetngtarotiuvpesi.mBaagrepslotfs depict
htheemmateoaxnylpineracnednteaobsuinn-dsatanicneesdocfecthael ttiosspubeafcrtoemrial
dFaym1il4iefsorsowrtheidchbtyhePhreyslulmts.anre=sh4owfonr iunnpinafneeclted
F(N. BoaCr.sd, i5f0f )0gµrmou.p(,Fa)nTdonta=l 6hifsotor laollgoicthalersctroeraetsmoefnt
cgerocumps.harKvreusstekdala-Wt daalylis14 oSnieg-nwifaiycanacneawlyassis of
dveatreiarmnciene(Ad NbyOMVAix)edfo-ellfofwecetdmboydDeluwnnit’hsTmuukletiyp’lse-
mcoumltpipalreiscoonms ppaorsitsohnoscptoest hwoacsteusstewd ains uAse(d*,inp ≤
A0.0a5n;d *B*.,Stpud≤ent0’.s0p1a).ramErertorricbt-atresstrwepitrhesMenatnnt-he
Wstahnidtnaerdy dpeovstiahtioocntefrsotmwathseusmeedainn. Kruskal-
Wallis one-way analysis of variance (ANOVA)
followed by Dunn’s multiple-comparisons post
hoc test was used in F (*, p ≤ 0.05, **, p ≤ 0.01;
***, p ≤ 0.01; ****, p ≤ 0.0001). Error bars
histopathological changes to the cecum.
To define changes in the gut microbiota,
mouse cecal content from day 14 was
represent the standard deviation from the mean.
characterized by 16S rRNA illumina sequencing
(Figure 6). 11-treated mice had the highest alpha-
diversity compared to all other groups followed
by the vehicle group. Corresponding changes in
the microbial community structures were also
seen in mice treated with 11 and the vehicle when
compared to the no C. difficile and vanco-treated
groups. Five days after cessation of 11 and
vehicle treatment there was an increased
abundance of the Phylum Firmicutes, mainly
ACS Paragon Plus Environment

Page 7 of 17
Journal of Medicinal Chemistry
members of the Family Lachnospiraceae and
mmol) were dissolved in 2.4 mL DMSO. Cesium
1
Ruminococcaceae, when compared to the other
groups. Members of this Family are associated
with C. difficile resistance in the murine gut35.
Family Bacteroidaceae was overrepresented in
the vanco-treated mouse gut (no C. difficile and
vanco groups), while a loss or reduced proportion
were noticed in vehicle and 11 treated groups
respectively. It has also been shown that the
relative abundance of C. difficile is positively
associated with Proteobacteria,36 37 the presence
of which was evident in all treatment groups
except those treated with 11.
carbonate (847.13 mg, 2.6 mmol) was added to
the mixture. The resulting suspension was stirred
at either room temperature or at 80°C for 4 hours
when TLC monitoring indicated completion of
the reaction. The solution was diluted with 50 mL
of DCM, was washed with H2O (4 x 75 mL) to
remove DMSO and salts and was washed with 75
mL of brine. The organic layer was dried with
MgSO4 was then filtered and concentrated in
vacuo to dryness. The concentrate was carried to
the next step without further characterization.
General Procedure B: Synthesis of a 4-chloro-
(4-aminophenoxy)benzene. A 4-chloro-(4-
nitrophenoxy)benzene (1.2 mmol) was dissolved
in 3 mL THF, 3 mL ethanol (EtOH) and 1 mL
H2O. To this solution was added elemental iron
(670 mg, 12 mmol) and NH4Cl (193 mg, 3.6
mmol). The mixture was refluxed for 2 hours
when TLC indicated completion of the reaction.
The mixture was cooled to room temperature,
diluted with 20 mL ethyl acetate (EtOAc) and
was passed through a pad of Celite which was
washed with EtOAc. The organic solution was
washed with 40 mL of saturated NaHCO3, 40 mL
of H2O, and 40 mL of brine. The organic layer
was dried with MgSO4 which was then filtered
and the filtrate was concentrated in vacuo to
dryness. The concentrate was carried to the next
step without further characterization.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
The findings from this study highlight the
importance of developing therapy that can
presumably target the Clostridial membrane to
not only reduce the deleterious impact on the gut
microbiota but the likelihood of recurrence in the
host as well. Pursuing a potent analog of the
salicylanilide class has yielded a molecule that
can reduce bacterial cell and spore counts in a
mouse model as well as reduce relapse upon
completion of treatment. Further exploration into
improving the salicylanilide formulation is
warranted to reduce the bioavailability of the
therapeutic agent in order to increase its effective
concentration at the site of infection in the colon
and thereby improve its potency and reduce
systemic toxicity.
General Procedure C: Synthesis of a
salicylanilide. A 4-chloro-(4-
aminophenoxy)benzene (1.2 mmol) was
EXPERIMENTAL SECTION
dissolved in 5 mL of toluene. To this solution,
3,5-dicholorosalicylic acid (323 mg, 1.56 mmol)
and then triphenylphosphite (403 μL, 1.56 mmol)
were added. The resulting suspension was
refluxed for 12 hours. The solution was
concentrated in vacuo to dryness. The crude
mixture was subjected to silica chromatography
using a gradient of 0 to 10% DCM in methanol.
Because some analogs co-elute with an impurity,
further purification must be performed by first
dissolving the concentrated, eluted crude mixture
in a minimal amount of DCM and then
precipitating the desired product with excess
hexanes. After filtration, a white powder is
obtained.
3,5-dichloro-N-(4-(4-chlorophenoxy)phenyl)-
2-hydroxybenzamide (1). Procedures A, then B
Chemistry. General Information. 1H and 13C
NMR spectra were recorded on a Bruker DRX-
500 or a Bruker AV-400. The purity of the
salicylanilides was determined to be 95% or
greater by measuring the absorbance at 365 nm
with HPLC (Supporting Information). HPLC
spectra were recorded on a Agilent Systems 1260
using a Poroshell 120 EC-C8 column. Mobile
phase gradient went from 50% H2O in
acetonitrile to 100% acetonitrile over 7 min and
then isocratic at 100% acetonitrile for 3 min at a
0.5 mL / min flow rate. Synthetic scheme for
compounds 14 – 17 can be seen in Figure S2.
General Procedure A: Synthesis of a 4-chloro-
(4-nitrophenoxy)benzene. 4-Chlorophenol (334
mg, 2.6 mmol) and a 4-halonitrobenzene (2.1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 17
and finally C. 54% yield over three steps. 1H-
124.3, 124.6, 126.6, 128.7, 130.7, 134.7, 135.7,
149.6, 157.1, 157.4, 168.3. HRMS-ESI (m/z):
[M+H]+ calculated for C19H11Cl4NO3, 441.9566;
found 441.9407. Purity by HPLC was found to be
100%.
1
2
3
4
5
6
7
8
NMR (500 MHz, Acetone-d6) δ 7.05 (d, J = 9.0
Hz, 2H), 7.10 (d, J = 9.0 Hz, 2H), 7.41 (d, J =
9.0 Hz, 2H), 7.67 (d, J = 2.4 Hz, 1H), 7.79, (d, J
= 9.0 Hz, 2H), 8.04 (d, J = 2.3 Hz, 1H), 10.01 (s,
1H), 13.06 (s, 1H). 13C-NMR (125 MHz,
Acetone-d6) δ 117.8, 120.2, 120.9, 123.5, 124.2,
124.5, 126.4, 128.7, 130.7, 134.1, 134.5, 154.9,
157.3, 157.5, 168.2. HRMS-ESI (m/z): [M+H]+
calculated for C19H12Cl3NO3, 407.9955; found
407.9800. Purity by HPLC was found to be
100%.
N-(2-bromo-4-(4-chlorophenoxy)phenyl)-3,5-
dichloro-2-hydroxybenzamide (2). Procedures A,
then B and finally C. 75% yield over three steps.
1H-NMR (500 MHz, Acetone-d6) δ 7.14 (d, J =
8.7 Hz, 3H), 7.37 (d, J = 2.5 Hz, 1H), 7.46 (d, J =
8.8 Hz, 2H), 7.71 (s, 1H), 7.82 (d, J = 8.8 Hz,
1H), 8.08 (s, 1H), 9.96 (s, 1H), 12.61 (s, 1H).
13C-NMR (125 MHz, Acetone-d6) δ 118.1, 119.0,
121.2, 121.9, 123.3, 124.1, 124.3, 127.1, 129.7,
130.0, 131.0, 131.6, 134.6, 156.2, 156.7, 157.1,
168.0. HRMS-ESI (m/z): [M+H]+ calculated for
C19H11BrCl3NO3, 485.9061; found 485.8909.
Purity by HPLC was found to be 100%.
N-(3-bromo-4-(4-chlorophenoxy)phenyl)-3,5-
dichloro-2-hydroxybenzamide (3). Procedures A,
then B and finally C. 62% yield over three steps.
1H-NMR (500 MHz, Acetone-d6) δ 7.00 (dt, J =
2.3, 9.0 Hz, 2H), 7.19 (d, J = 8.8 Hz, 1H), 7.41
(dt, J = 2.3, 9.0 Hz, 2H), 7.70 (d, J = 2.4 Hz,
1H), 7.80 (dd, J = 2.6, 8.9 Hz, 1H), 8.04 (d, J =
2.4 Hz, 1H), 8.22 (d, J = 2.6 Hz, 1H), 10.07 (s,
1H), 12.79 (s, 1H). 13C-NMR (125 MHz,
3,5-dichloro-N-(4-(4-chlorophenoxy)-3-
iodophenyl)-2-hydroxybenzamide (5). Procedures
A, then B and finally C. 52% yield over three
steps. 1H-NMR (500 MHz, Acetone-d6) δ 6.99 (d,
J = 8.95 Hz, 2H), 7.09 (d, J = 8.8 Hz, 1H), 7.41
(d, J = 9.0 Hz, 2H), 7.84 (dd, J = 2.5, 8.8 Hz,
1H), 8.04 (d, J = 2.4 Hz, 1H), 8.38 (d, J = 2.5 Hz,
1H), 10.01 (s, 1H), 12,83 (s, 1H). 13C-NMR (125
MHz, Acetone-d6) δ 89.3, 117.8, 120.0, 121.1,
123.6, 124.2, 124.3, 126.6, 128.7, 130.8, 133.6,
134.7, 135.8, 153.9, 157.1, 157.4, 168.3. HRMS-
ESI (m/z): [M+H]+ calculated for C19H11Cl3INO3,
533.8922; found 533.8758. Purity by HPLC was
found to be 100%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3,5-dichloro-N-(4-(4-chlorophenoxy)-3-
fluorophenyl)-2-hydroxybenzamide (6).
Procedures A, then B and finally C. 69% yield
over three steps. 1H-NMR (500 MHz, Acetone-
d6) δ 7.03 (d, J = 8.9 Hz, 2H), 7.25 (t, J = 8.9 Hz,
1H), 7.40 (d, J = 9.0 Hz, 2H), 7.58 (d, J = 8.8 Hz,
1H), 7.69 (d, J = 2.4 Hz, 1H), 7.88 (dd, J = 2.3,
12.8 Hz, 1H), 8.02 (d, 2.25 Hz, 1H), 10.11 (s,
1H), 12.77 (s, 1H). 13C-NMR (125 MHz,
Acetone-d6) δ 111.5, 111.7, 119.0, 119.1, 119.2,
123.4, 123.7, 124.3, 126.5, 128.5, 130.7, 134.7,
135.8, 135.9, 153.7, 155.7, 157.3, 157.5, 168.3.
HRMS-ESI (m/z): [M+H]+ calculated for
C19H11Cl3FNO3, 425.9861; found 425.9701.
Purity by HPLC was found to be 100%.
3,5-dichloro-N-(4-(4-chlorophenoxy)-3-
methoxyphenyl)-2-hydroxybenzamide (7).
Procedures A, then B and finally C. 57% yield
over three steps. 1H-NMR (500 MHz, Acetone-
d6) δ 3.80 (s, 3H), 6.89 (dt, J = 3.5, 9.1 Hz, 2H),
7.12 (d, 8.65 Hz, 1H), 7.32 (dt, J = 3.5, 9.1 Hz,
2H), 7.40 (dt, J = 2.8, 8.7 Hz, 1H), 7.66 (t, J =
2.6 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 8.04 (d, J =
2.3 Hz, 1H), 10.08 (s, 1H), 12.98 (s, 1H). 13C-
NMR (125 MHz, Acetone-d6) δ 56.3, 108.2,
115.1, 117.9, 118.4, 123.0, 123.6, 124.2, 126.5,
127.2, 130.3, 134.5, 136.1, 141.8, 152.8, 157.4,
158.4, 168.2. HRMS-ESI (m/z): [M+H]+
calculated for C20H14Cl3NO4, 438.0061; found
437.9902. Purity by HPLC was found to be
100%.
Acetone-d6) δ 115.4, 117.8, 119.9, 122.4, 123.4,
123.7, 124.3, 126.6, 127.6, 128.7, 130.7, 134.7,
135.8, 150.8, 157.0, 157.4, 168.3. HRMS-ESI
(m/z): [M+H]+ calculated for C19H11BrCl3NO3,
485.9061; found 485.8906. Purity by HPLC was
found to be 100%.
3,5-dichloro-N-(3-chloro-4-(4-
chlorophenoxy)phenyl)-2-hydroxybenzamide (4).
Procedures A, then B and finally C. 39% yield
over three steps. 1H-NMR (500 MHz, Acetone-
d6) δ 7.00 (dt, J = 2.2, 9 Hz, 2H), 7.21 (d, J = 8.9
Hz, 1H), 7.41 (dt, J = 2.2, 9 Hz, 2H), 7.70 (d, J =
2.4 Hz, 1H), 7.75 (dd, J = 2.6, 8.9 Hz, 1H), 8.04
(d, J = 2.5 Hz, 1H), 8.07 (d, J = 2.6 Hz, 1H),
10.09 (s, 1H), 12.78 (s, 1H). 13C-NMR (125
MHz, Acetone-d6) δ 117.8, 119.7, 122.7, 123.7,
ACS Paragon Plus Environment

Page 9 of 17
1
Journal of Medicinal Chemistry
3,5-dichloro-N-(4-(4-chlorophenoxy)-2-
(m, 2H), 7.73 (d, J = 2.5 Hz, 1H), 8.10 (d, J = 2.5
Hz, 1H), 8.59 (s, 2H), 10.65 (s, 1H). 13C-NMR
(125 MHz, Acetone-d6) δ 117.8, 122.3, 123.9,
124.4, 124.5, 125.2, 126.8, 126.9, 127.2, 128.3,
130.3, 135.11, 137.0, 157.3, 159.2, 168.7.
HRMS-ESI (m/z): [M+H]+ calculated for
methoxyphenyl)-2-hydroxybenzamide (8).
Procedures A, then B and finally C. 59% yield
over three steps. 1H-NMR (500 MHz, Acetone-
d6) δ 3.90 (s, 3H), 6.65 (dd, J = 2.4, 8.7 Hz, 1H),
6.87 (d, J = 2.3 Hz, 1H), 7.07 (d, J = 8.9 Hz,
2H), 7.41 (d, J = 9.0 Hz, 2H), 7.69 (d, J = 2.4 Hz,
1H), 7.99 (t, J = 8.4 Hz, 1H), 8.05 (s, 1H), 9.69
(s, 1H), 12.65 (s, 1H). 13C-NMR (125 MHz,
Acetone-d6) δ 56.6, 104.1, 111.1, 118.8, 120.9,
122.8, 124.0, 124.2, 125.7, 127.1, 128.7, 130.7,
134.3, 153.7, 155.9, 156.4, 157.2, 167.1. HRMS-
ESI (m/z): [M+H]+ calculated for C20H14Cl3NO4,
438.0061; found 437.9902. Purity by HPLC was
found to be 95%.
2
3
4
5
6
7
8
9
C21H10Cl3F6NO3, 543.9703; found 543.9543.
Purity by HPLC was found to be 100%.
3,5-dichloro-N-(4-(4-chlorophenoxy)-3,5-
diiodophenyl)-2-hydroxybenzamide (12).
Procedures A using 15 as the 4-halonitrobenzene,
then B and finally C. 51% yield over three steps.
1H-NMR (500 MHz, DMSO-d6) δ 6.80 (d, J =
8.5 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.82, (s,
1H), 8.02 (s, 1H), 8.33 (s, 2H), 10.82 (s, 1H).
13C-NMR (125 MHz, Acetone-d6) δ 90.9, 117.7,
117.9, 123.8, 124.3, 126.7, 127.8, 130.6, 133.7,
134.9, 137.9, 151.6, 156.2, 156.9, 157.3, 168.4.
HRMS-ESI (m/z): [M+H]+ calculated for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3,5-dichloro-N-(4-(4-chlorophenoxy)-3-
cyanophenyl)-2-hydroxybenzamide (9).
Procedures A, then B and finally C. 52% yield
over three steps. 1H-NMR (500 MHz, Acetone-
d6) δ 7.14 (d, J = 9.1 Hz, 1H), 7.22 (d, J = 8.9 Hz,
2H), 7.50 (d, J = 9.0 Hz, 2H), 7.70, (d, J = 2.4
Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 2.3
Hz, 1H), 8.25 (s, 1H), 10.20 (s, 1H), 12.71 (s,
1H). 13C-NMR (125 MHz, Acetone-d6) δ 105.1,
115.9, 117.7, 119.6, 121.9, 123.8, 124.3, 126.6,
127.4, 129.1, 130.4, 131.1, 134.2, 134.8, 155.5,
156.6, 157.3, 168.5. HRMS-ESI (m/z): [M+H]+
calculated for C20H11Cl3N2O3, 432.9908; found
432.9754. Purity by HPLC was found to be
100%.
C19H10Cl3I2NO3, 659.7889; found 659.7720.
Purity by HPLC was found to be 100%.
3,5-dichloro-N-(4-(4-chlorophenoxy)-3,5-
dinitrophenyl)-2-hydroxybenzamide (13). 3,5-
dichlorosalicylic acid (200 mg, 0.97 mmol) was
dissolved in 6.8 mL of dry methylene chloride
under argon. After adding thionyl chloride (140
μL, 1.94 mmol), the resulting solution was stirred
at 40°C overnight. The mixture was concentrated
in vacuo to dryness to yield the acid chloride.
The product (309 mg, 1 mmol) of Procedure A
using 17 as the 4-halonitrobenzene was dissolved
in 2 mL of ethanol along with
3,5-dichloro-N-(4-(4-chlorophenoxy)-2-
iodophenyl)-2-hydroxybenzamide (10).
Procedures A, then B and finally C. 72% yield
over three steps. 1H-NMR (500 MHz, Acetone-
d6) δ 7.11 – 7.14 (m, 2H), 7.15 (dd, J = 2.8, 13.2
Hz, 1H), 7.43 – 7.47 (m, 2H), 7.59 (d, J = 2.8 Hz,
1H), 7.66 (d, J = 8.8 Hz, 1), 7.70 (d, J = 2.5 Hz,
1H), 8.10 (d, J = 2.5 Hz, 1H), 9.90 (s, 1H), 12.82
(s, 1H). 13C-NMR (125 MHz, Acetone-d6) δ 98.0,
117.7, 119.8, 121.9, 123.9, 124.3, 126.8, 129.5,
129.7, 130.0, 130.1, 134.7, 156.2, 157.2, 168.5.
HRMS-ESI (m/z): [M+H]+ calculated for
C19H11Cl3INO3, 533.8922; found 533.8761.
Purity by HPLC was found to be 96%.
diisopropylethylamine (540 μL, 3 mmol) and
cooled to 0°C. This cooled solution was added to
the acid chloride. The resulting solution was
refluxed overnight. Upon cooling the solution to
room temperature, it was concentrated in vacuo
and subjected to silica chromatography using
hexanes and ethyl acetate (1:1) as the mobile
phase to yield an off-white powder (7 mg, 1.4%
yield). 1H-NMR (400 MHz, Acetone-d6) δ 7.03 –
7.08 (m, 2H), 7.36 – 7.41 (m, 2H), 7.70 (d, J =
2.5 Hz, 1H), 8.08 (d, J = 2.5 Hz, 1H), 8.88 (s,
2H). 13C-NMR (125 MHz, Acetone-d6) δ 117.9,
118.0, 122.8, 123.4, 124.7, 127.1, 129.0, 130.6,
135.1, 136.9, 137.7, 145.4, 157.4, 157.7, 168.7.
HRMS-ESI (m/z): [M+H]+ calculated for
3,5-dichloro-N-(4-(4-chlorophenoxy)-3,5-
bis(trifluoromethyl)phenyl)-2-hydroxybenzamide
(11). Procedures A using 14 as the 4-
halonitrobenzene, then B and finally C. 57%
yield over three steps.1H-NMR (500 MHz,
Acetone-d6) δ 6.88 – 6.93 (m, 2H), 7.35 – 7.39
C19H10Cl3N3O7, 497.9657; found 497.9496.
Purity by HPLC was found to be 100%.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 17
2-bromo-5-nitro-1,3-
4-fluoro-3,5-dinitroaniline (17). After cooling
1
bis(trifluoromethyl)benzene (14). 2-Bromo-1,3-
bis(trifluoromethyl)benzene (2 g, 6.82 mmol)
was added to a mixture of 4.2 mL of concentrated
H2SO4 and 3 mL of fuming HNO3. The solution
was heated at 70°C for 2 hours, cooled to room
temperature and poured onto 200 mL of ice
water. The precipitate was filtered and dissolved
in 100 mL of ethyl acetate and washed with 100
mL of H2O to remove residual acid. The organic
layer was dried with MgSO4 which was then
removed with filtration and the filtrate was
concentrated in vacuo to dryness to afford an off-
white powder (2.19 g, 95% yield). 1H-NMR (400
MHz, CDCl3) δ 8.73 (s, 2H).
2-fluoro-1,3-diiodo-5-nitrobenzene (15). 4-
Nitro-1-fluorobenzene (113 μL, 1.06 mmol) was
mixed in triflic acid (1 mL, 10.6 mmol) and
cooled to 0°C. N-iodosuccinimide (525 mg, 2.33
mmol) was added in portions to the cooled
solution. The solution was warmed to room
temperature and stirred for 4 hours, at which
point an additional portion of N-iodosuccinimide
(137 mg, 0.61 mmol) was added and stirred
overnight. Since TLC monitoring didn’t show
completion of reaction, an additional portion of
N-iodosuccinimide (200 mg, 0.89 mmol) and
triflic acid (1 mL, 10.6 mmol) were added and
stirred over a second night. The reaction mixture
was quenched with cold H2O, extracted with
methylene chloride, washed with 10% sodium
bisulfite, H2O and then brine. The organic layer
was dried with MgSO4 which was then removed
with filtration and the filtrate was concentrated in
vacuo to dryness to afford a yellow powder (393
mg, 94% yield) which was carried to the next
step without further characterization.
H2SO4 (15 mL) containing 20% SO3 to 0°C, 16
(5.5 g, 24.3 mmol) was added, followed by
ethylene chloride (20 mL). Sodium azide (1.81 g,
27.9 mmol) was added in portions to the cooled
solution to not exceed 20°C. The solution was
then heated at reflux for 1 hour, cooled to room
temperature, poured over ice water and the
resulting precipitate was collected by filtration to
yield a orange powder (4.0 g, 82% yield). 1H-
NMR (500 MHz, MeOD) δ 7.51 (d, J = 5.4 Hz,
2H). ESI-TOF (m/z): [M+H]+ calculated for
C6H4FN3O4, 202.02; found 202.1.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pharmacokinetic (PK) Studies. PK studies
of 11 were conducted in 18 C57Bl/6J mice (9
mice for the i.v. arm and 9 mice for the p.o. arm
with n = 3 at each time point).
For the p.o. arm, mice were pre-treated with
antibiotics (cefoperazone, 0.5 mg / mL in
drinking water) for 10 days. Following a 2-day
wash out period, 11 was administered at 1 mg/kg
each day for 5 days. On first and final day of the
period of administration of 11, the mice were
bled 5 min, 10 min, 30 min, 1 hour, 2 hours, 4
hours, 10 hours and 24 hours post administration.
Moreover, on the final day the mice were
euthanized 30 min, 4 hours and 24 hours post
administration and their cecal contents were
collected.
For the i.v. arm of the PK study, 11 was
administered at 0.3 mg/kg and the mice were bled
5 min, 10 min, 30 min, 1 hour, 2 hours, 4 hours, 8
hours and 24 hours post administration. The mice
were euthanized 4 hours, 8 hours and 24 hours
post administration and their cecal contents were
collected.
Plasma samples were processed by mixing
with two volume equivalents of acetonitrile
containing an internal standard, centrifuged, and
the supernatant was analyzed by LC/MS-SRM
(liquid chromatography / mass spectrometry –
selected reaction monitoring). Fecal samples
were weighed and homogenized (in four volume
equivalents of acetonitrile containing an internal
standard) using a bullet blender (Next Advance),
centrifuged and the supernatant was subsequently
analyzed by LC/MS-SRM.
4-fluoro-3,5-dinitrobenzoic acid (16). A
mixture of H2SO4 (22.5 mL) containing 30%
SO3 and fuming HNO3 was cooled to 0°C . To
the cooled solution, 4-fluorobenzoic acid (5 g,
35.8 mmol) was added slowly to not exceed
20°C. The solution was heated at 85°C for 10
min, cooled to 40°C and then heated at 95°C for
an additional 3 hours. After cooling the mixture
to room temperature, it was poured over ice water
and the resulting precipitate was collected by
filtration to yield a yellow powder (5.6 g, 68%
yield). 1H-NMR (400 MHz, Acetone-d6) δ 8.96
(d, J = 6.3 Hz, 2H).
Determination of minimum inhibitory
concentration (MIC). A single overnight colony
of C. difficile strain VPI 10463 (streaked onto a
ACS Paragon Plus Environment

Page 11 of 17
Journal of Medicinal Chemistry
plate of blood agar base II with 5% sheep blood)
10ꢀmin and supernatants (100ꢀμL) were
1
2
3
4
5
6
7
8
was suspended in brain-heart infusion broth
growth medium supplemented with 0.5% yeast
extract (BHIS) containing 0.03% L-cysteine as
previously described38, 39 and grown to OD600 of
0.4–0.5 (logarithmic phase). MIC assays for C.
difficile were conducted in BHIS using an
anaerobic cabinet (Coy Lab Products Inc., Grass
Lake, MI, USA) at 37°C in a reducing anaerobic
atmosphere (8% H2, 8% CO2, 84% N2). All
broths and 96-well microtiter plates were pre-
reduced (incubated anaerobically overnight) prior
to use with the C. difficile culture. All MIC
determinations were performed in 96-well
microtiter plates using the broth microdilution
method. Briefly, two-fold serial dilutions of test
compounds were inoculated with ~5ꢀ×ꢀ105
cfu/mL bacteria. After 20–24ꢀhr of incubation of
the test compound with the bacteria at 37°C,
MICs were determined using the Lambert-
Pearson method.40 MIC assays were performed in
duplicate.
In vitro cytotoxicity assay. The cell line
293T/17 [HEK 293T/17] (ATCC® CRL-
11268TM) was cultured according to the
manufacturer’s protocol. HEK 293T/17 cells
were adhered to wells in 96-well plates by
incubating 2 x 104 cells per well in 100 μL of
Dulbecco's Modified Eagle Medium (DMEM) at
37ꢀ°C in a 5% CO2 humidifying chamber for
24ꢀhours. Media was then discarded and the cells
were then either treated with 11 or niclosamide in
the range of 0.020 – 40ꢀμM in DMEM. After 16
hours of incubation, an MTS assay was
transferred to a clean 96-well plate. Hemolysis
was determined by measuring absorbance at
540ꢀnm, with 1% Triton X-100 as the positive
control and 1.6% DMSO in PBS as the negative
control. Hemolysis assays were performed in
duplicate.
Ethics statement. This study was approved
by the University committee on the Animal Care
and Use (IACUC) at North Carolina State
University College of Veterinary Medicine
(NCSU). The NCSU Animal Care and Use policy
follows the standards and guidelines set forth in
the Animal Welfare Act and Health Research
Extension Act of 1985. Laboratory animal
facilities at NCSU adheres to guidelines set forth
in the Guide for the Care and Use of Laboratory
Animals. The animals were assessed daily for
physical conditions and behavior, and any
moribund animals were humanely euthanized by
CO2 asphyxiation followed by cervical
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
dislocation. Trained animal technicians or a
veterinarian performed animal husbandry in an
AAALAC-accredited facility.
Animals and housing. C57BL/6J mice (5
weeks old; females and males) purchased from
Jackson Laboratories (Bar Harbor, ME) were
used for the experimental infections. Mice were
housed with autoclaved food, bedding, and water.
Cage changes were performed weekly in a
laminar flow hood. Mice had a 12 hr. cycle of
light and darkness.
Mouse model of recurrent C. difficile
infection and sample collection. C. difficile
strain 630 spores were prepared and tested for
purity as described previously.34, 36 Groups of 5
week old C57BL/6J mice (n=24, 12 males and 12
females) were given cefoperazone (0.5 mg/ml),
in drinking water ad libitum for 5 days to render
them susceptible to C. difficile. This was
followed by a 2-day wash out with regular
drinking water (Gibco Laboratories, 15230).
Viable spores were enumerated by plating for
colony-forming units (CFU) per mL on
taurocholate, cefoxitin, cycloserine, fructose agar
(TCCFA) to determine the challenge dose. At
day 0 all mice except the antibiotic only control
group (n= 6) were challenged via oral gavage
with 105 spores of C. difficile 630. All mouse
stool tested culture negative for C. difficile before
performed at 37ꢀ°C in a 5% CO2 humidifying
chamber for 1 hour using the CellTiter 96
aqueous non-radioactive cell proliferation assay
kit (Promega, Madison, WI, USA) per
manufacturer’s instructions. The data was
normalized to a positive control (DMEM with
cells only and no drug) and negative control (0.2
mM digitonin in DMEM with cells). MTS assays
were performed in duplicate.
Hemolysis assay. Sheep red blood cells
(Innovative Research, Novi, MI, USA) were
washed three times with PBS pH 7.4. A 3% cell
suspension in PBS (100ꢀμL) was added to 11 in
PBS (100ꢀμL) to create a concentration range of
0.625 – 80 μM and then incubated at 37°C for
1ꢀh. The plate was centrifuged at 500ꢀ×ꢀg for
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 17
challenge. From day 4 to 9, mice were treated as
at 65°C for 20 min to ensure killing of vegetative
1
2
3
4
5
6
7
8
follows (n=6 per group): 1) uninfected control; 2)
vanco (0.4 mg/mL) in drinking water ad libitum;
3) vehicle control (2-(hydroxypropyl)-
cells and then plated on TCCFA to enumerate
viable spores. All plates were incubated
anaerobically at 37°C for 24 hr. Bacterial load
was expressed as colony forming units (CFUs)
CFU/g of feces or cecal content for total
vegetative cells and spores from the unheated
samples and spores only from the heat-treated
samples.
Vero cell cytotoxicity assay. Toxin activity
was measured using a Vero cell cytotoxicity
assay.34 Vero cells were grown and maintained in
DMEM media (Gibco Laboratories, 11965-092)
with 10% fetal bovine serum (Gibco
Laboratories, 16140-071) and 1% Penicillin
streptomycin solution (Gibco Laboratories,
15070-063). Cells were incubated with 0.25%
trypsin (Gibco Laboratories, 25200-056) washed
with 1X DMEM media and harvested by
centrifugation 1,000 RPM for 5 min. Cells were
plated at 1 × 104 cells per well in a 96-well flat
bottom microtiter plate (Corning, 3596) and
incubated overnight at 37°C / 5% CO2. Cecal
content was diluted (1:10) with PBS and filtered
before testing. The samples were further diluted
10-fold to a maximum of 10−6. Sample dilutions
were incubated 1:1 with PBS (for all dilutions) or
antitoxin (performed for 10−1 and 10−4 dilutions
only, TechLabs, T5000) for 40 min at room
temperature. Following incubation, these
admixtures were added to the Vero cells and
plates were incubated overnight at 37°C / 5%
CO2. Vero cells were viewed under 200X
magnification for rounding after overnight
incubation. The cytotoxic titer was defined as the
reciprocal of the highest dilution that produced
rounding in 80% of Vero cells for each sample.
Vero cells treated with purified C. difficile toxins
(A and B) and antitoxin (List Biological Labs,
152C and 155C; TechLabs, T5000) were used as
controls.
cyclodextrin) by oral gavage; and 4) 11 (5
mg/mL) by oral gavage. Body weight was
measured daily and fecal pellets were collected
throughout the experiment as seen in Figure 3 for
C. difficile enumeration. Clinical signs of C.
difficile infection including lethargy, diarrhea,
and hunched posture were monitored for 14 days
post challenge. Animals were humanly
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
euthanized by CO2 asphyxiation followed by
cervical dislocation prior to necropsy. Cecal
content was collected at necropsy for bacterial
enumeration. Cecal tissue snips were flash frozen
in liquid nitrogen, and stored at -80°C until
further analysis. The cecum was prepared for
histology by placing the intact tissue into
histology cassettes, which were stored in 10%
buffered formalin for 48 hr and then transferred
to 70% ethyl alcohol. Tissue cassettes were
processed and paraffin embedded, and sectioned.
Hematoxylin and eosin stained slides were
prepared for histopathological examination
(LCCC Animal Histopathology Core Facility at
the University of North Carolina at Chapel Hill).
A board-certified veterinary pathologist (S.
Montgomery) conducted the histopathological
evaluation in a blinded manner. For tissue
scoring, a 0-4 numerical scoring system was
employed to separately assess edema,
inflammatory cell infiltration, and epithelial cell
damage in the cecum and colon based upon a
previously published scoring scheme.41
Photomicrographs were captured on an Olympus
BX43 light microscope with a DP27 camera
using cellSens Dimension software.
Bacterial enumeration of C. difficile
vegetative cells and spores. Bacterial
enumeration was done on fecal pellets and cecal
content using previously published protocols.34
Briefly, a 1:10 dilution of 1X PBS was used to
re-suspend the samples. The samples were then
incubated anaerobically at room temperature for
30 min, to allow the contents to settle. Ten-fold
serial dilutions for each sample in 1X PBS
followed by plating on TCCFA was done to
determine the total number of vegetative cells
and spores. The dilution plates were heat treated
DNA extraction, 16S rRNA-based bacterial
community sequencing using Illumina MiSeq
platform, and bioinformatic analysis
Cecal tissue snips collected at necropsy were
subjected to community 16S rRNA gene
sequencing. Microbial DNA was extracted from
the cecal tissue snips using the Mag Attract
Power Microbiome kit (Mo Bio Laboratories,
ACS Paragon Plus Environment

Page 13 of 17
Journal of Medicinal Chemistry
Inc.). The DNA libraries were prepared as
Impact of 11 and niclosamide on HEK
293T / 17 cell viability (PDF)
1
2
described previously.42
Analysis of the V4 region of the 16S rRNA
gene was done in the statistical programming
environment R.43 using the package DADA2.44
Version 1.8 of the DADA2 tutorial workflow
(https://benjjneb.github.io/dada2/tutorial.html)
was followed to process the MiSeq data. Raw
sequences have been deposited in the Sequence
Read Archive (SRA) with SRA accession number:
3
4
5
6
7
8
9
Synthetic scheme for 14 – 17 (PDF)
Analytical HPLC traces denoting final
purity for 1 – 13 (PDF)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AUTHOR INFORMATION
Corresponding Author
PRJNA625021
and
SAMN14591013
to
SAMN14591034.
*Kim D. Janda - The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla,
California 92037, United States. Phone: (858)
785-2515. Fax: (858) 784-2595. E-mail:
kdjanda@scripps.edu.
Statistical analysis. Statistical tests were
performed using Prism version 7.0b for Mac OS X
(GraphPad Software, La Jolla California USA.
Statistical significance was set at a p value of <
0.05 for all analyses (*, p <0.05; **, p < 0.01; ***,
p < 0.001; ****, p < 0.0001). Two-way repeated
measure analysis of variance (ANOVA) test with
Holm-Sidak’s multiple comparisons post hoc test
was used to calculate significance between
treatments for baseline weight loss. Mixed-effect
model with Tukey’s multiple comparisons post
hoc test was used to compare differences in C.
difficile load (CFU/g) in feces. Kruskal-Wallis
one-way analysis of variance (ANOVA) followed
by Dunn’s multiple-comparisons post hoc test was
used to calculate significance between treatment
groups for histopathological scores of C. difficile
infection and alpha diversity comparisons using
inverse Simpson indices. A Student’s t-test
corrected for multiple comparisons followed by
Mann-Whitney post hoc method was used to
calculate significance between treatment groups in
C. difficile load (CFU/g) and toxin activity in cecal
content.
Notes
CMT is a scientific advisor to Locus Biosciences,
a company engaged in the development of
antimicrobial technologies. CMT is a consultant
for Vedanta Biosciences and Summit
Therapeutics.
ACKNOWLEDGEMENTS
No funding to declare
ABBREVIATIONS USED
Clostridioides difficile, C. difficile;
Clostridioides difficile infections, CDI; cell
proliferation assay, MTS; vancomycin, vanco;
analysis of variance, ANOVA; colony forming
unit, CFU; ethanol, EtOH; ethyl acetate, EtOAc;
selected reaction monitoring, SRM; brain-heart
infusion broth growth medium supplemented with
yeast extract, BHIS; Dulbecco's Modified Eagle
Medium, DMEM; taurocholate / cefoxitin /
cycloserine / fructose agar, TCCFA; fourth
hypervariable, V4
ASSOCIATED CONTENT
Supporting Information
The following files are available free of charge at
Molecular formula strings (SMILES) are
available for 1 – 17 (CSV)
REFERENCES
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 17
1.
Dubberke, E. R.; Olsen, M. A. Burden of
10.
Gerding, D. N.; Muto, C. A.; Owens Jr,
1
2
3
4
Clostridium difficile on the healthcare system.
Clin. Infect. Dis. 2012, 55, S88-S92.
2.
R. C. Treatment of Clostridium difficile
infection. Clin. Infect. Dis. 2008, 46, S32-S42.
11.
Di Bella, S.; Ascenzi, P.; Siarakas, S.;
Musher, D. M.; Aslam, S.; Logan, N.;
Petrosillo, N.; Di Masi, A. Clostridium difficile
toxins A and B: insights into pathogenic
properties and extraintestinal effects. Toxins
(Basel) 2016, 8, 134.
Nallacheru, S.; Bhaila, I.; Borchert, F.; Hamill,
R. J. Relatively poor outcome after treatment of
Clostridium difficile colitis with metronidazole.
Clin. Infect. Dis. 2005, 40, 1586-1590.
5
6
7
8
9
3.
Crobach, M. J.; Vernon, J. J.; Loo, V. G.;
12.
Pépin, J.; Valiquette, L.; Gagnon, S.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Kong, L. Y.; Pechine, S.; Wilcox, M. H.;
Kuijper, E. J. Understanding Clostridium difficile
colonization. Clin. Microbiol. Rev. 2018, 31,
e00021-17.
Routhier, S.; Brazeau, I. Outcomes of
Clostridium difficile-associated disease treated
with metronidazole or vancomycin before and
after the emergence of NAP1/027. Am. J.
Gastroenterol 2007, 102, 2781.
4.
Furuya-Kanamori, L.; Marquess, J.;
Yakob, L.; Riley, T. V.; Paterson, D. L.; Foster,
N. F.; Huber, C. A.; Clements, A. C.
Asymptomatic Clostridium difficile colonization:
epidemiology and clinical implications. BMC
Infect. Dis. 2015, 15, 516.
13.
Adler, A.; Miller-Roll, T.; Bradenstein,
R.; Block, C.; Mendelson, B.; Parizade, M.;
Paitan, Y.; Schwartz, D.; Peled, N.; Carmeli, Y.
A national survey of the molecular epidemiology
of Clostridium difficile in Israel: the
dissemination of the ribotype 027 strain with
reduced susceptibility to vancomycin and
metronidazole. Diagn. Microbiol. Infect. Dis.
2015, 83, 21-24.
5.
Lessa, F. C.; Mu, Y.; Bamberg, W. M.;
Beldavs, Z. G.; Dumyati, G. K.; Dunn, J. R.;
Farley, M. M.; Holzbauer, S. M.; Meek, J. I.;
Phipps, E. C. Burden of Clostridium difficile
infection in the United States. New Engl. J. Med.
2015, 372, 825-834.
14.
Freeman, J.; Vernon, J.; Morris, K.;
Nicholson, S.; Todhunter, S.; Longshaw, C.;
Wilcox, M. H. Pan-European longitudinal
surveillance of antibiotic resistance among
prevalent Clostridium difficile ribotypes. Clin.
Microbiol. Infect. 2015, 21, 248. e9-248. e16.
6.
Aslam, S.; Hamill, R. J.; Musher, D. M.
Treatment of Clostridium difficile-associated
disease: old therapies and new strategies. Lancet
Infect. Dis. 2005, 5, 549-557.
7.
Loo, V. G.; Poirier, L.; Miller, M. A.;
15.
Spigaglia, P. Recent advances in the
Oughton, M.; Libman, M. D.; Michaud, S.;
Bourgault, A.-M.; Nguyen, T.; Frenette, C.;
Kelly, M. A predominantly clonal multi-
institutional outbreak of Clostridium difficile–
associated diarrhea with high morbidity and
mortality. New Engl. J. Med. 2005, 353, 2442-
2449.
understanding of antibiotic resistance in
Clostridium difficile infection. Ther. Adv. Infect.
Dis. 2016, 3, 23-42.
16.
Louie, T. J.; Cannon, K.; Byrne, B.;
Emery, J.; Ward, L.; Eyben, M.; Krulicki, W.
Fidaxomicin preserves the intestinal microbiome
during and after treatment of Clostridium difficile
infection (CDI) and reduces both toxin
reexpression and recurrence of CDI. Clin. Infect.
Dis. 2012, 55, S132-S142.
8.
Merrigan, M.; Venugopal, A.; Mallozzi,
M.; Roxas, B.; Viswanathan, V.; Johnson, S.;
Gerding, D. N.; Vedantam, G. Human
hypervirulent Clostridium difficile strains exhibit
increased sporulation as well as robust toxin
production. J. Bacteriol. 2010, 192, 4904-4911.
17.
Louie, T. J.; Miller, M. A.; Mullane, K.
M.; Weiss, K.; Lentnek, A.; Golan, Y.; Gorbach,
S.; Sears, P.; Shue, Y.-K. Fidaxomicin versus
vancomycin for Clostridium difficile infection.
New Engl. J. Med. 2011, 364, 422-431.
9.
Warny, M.; Pepin, J.; Fang, A.; Killgore,
G.; Thompson, A.; Brazier, J.; Frost, E.;
McDonald, L. C. Toxin production by an
emerging strain of Clostridium difficile
associated with outbreaks of severe disease in
North America and Europe. The Lancet 2005,
366, 1079-1084.
18.
Tannock, G. W.; Munro, K.; Taylor, C.;
Lawley, B.; Young, W.; Byrne, B.; Emery, J.;
Louie, T. A new macrocyclic antibiotic,
fidaxomicin (OPT-80), causes less alteration to
the bowel microbiota of Clostridium difficile-
14
ACS Paragon Plus Environment

Page 15 of 17
Journal of Medicinal Chemistry
infected patients than does vancomycin.
Microbiology 2010, 156, 3354-3359.
19. Tam, J.; Hamza, T.; Ma, B.; Chen, K.;
Beilhartz, G. L.; Ravel, J.; Feng, H.; Melnyk, R.
A. Host-targeted niclosamide inhibits C. difficile
virulence and prevents disease in mice without
disrupting the gut microbiota. Nat. Commun.
2018, 9, 5233.
phase Clostridium difficile. Sci. Rep. 2016, 6,
1
2
3
4
5
6
7
8
33642.
29.
Martin, R. Modes of action of
anthelmintic drugs. Vet. J. 1997, 154, 11-34.
30. Gooyit, M.; Tricoche, N.; Lustigman, S.;
Janda, K. D. Dual protonophore–chitinase
inhibitors dramatically affect O. volvulus
molting. J. Med. Chem. 2014, 57, 5792-5799.
9
20.
Swan, G. The pharmacology of
31.
Chen, C.; Dolla, N. K.; Casadei, G.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
halogenated salicylanilides and their anthelmintic
use in animals. J. S. Afr. Vet. Assoc. 1999, 70, 61-
70.
Bremner, J. B.; Lewis, K.; Kelso, M. J.
Diarylacylhydrazones: Clostridium-selective
antibacterials with activity against stationary-
phase cells. Bioorg. Med. Chem. Lett. 2014, 24,
595-600.
21.
Kratky, M.; Vinsova, J. Salicylanilide
ester prodrugs as potential antimicrobial agents-a
review. Curr. Pharm. Des. 2011, 17, 3494-3505.
32.
Terada, H.; Goto, S.; Yamamoto, K.;
22.
Rajamuthiah, R.; Fuchs, B. B.; Conery,
Takeuchi, I.; Hamada, Y.; Miyake, K. Structural
requirements of salicylanilides for uncoupling
activity in mitochondria: Quantitative analysis of
structure-uncoupling relationships. Biochim.
Biophys. Acta, Bioenergetics 1988, 936, 504-512.
A. L.; Kim, W.; Jayamani, E.; Kwon, B.;
Ausubel, F. M.; Mylonakis, E. Repurposing
salicylanilide anthelmintic drugs to combat drug
resistant Staphylococcus aureus. PLoS One 2015,
10, e0124595.
33.
Theriot, C. M.; Koumpouras, C. C.;
23.
Rajamuthiah, R.; Fuchs, B. B.; Jayamani,
Carlson, P. E.; Bergin, I. I.; Aronoff, D. M.;
Young, V. B. Cefoperazone-treated mice as an
experimental platform to assess differential
virulence of Clostridium difficile strains. Gut
Microbes 2011, 2, 326-334.
E.; Kim, Y.; Larkins-Ford, J.; Conery, A.;
Ausubel, F. M.; Mylonakis, E. Whole animal
automated platform for drug discovery against
multi-drug resistant Staphylococcus aureus. PLoS
One 2014, 9, e89189.
34.
Winston, J. A.; Thanissery, R.;
24.
Denyer, S. P.; Stewart, G. Mechanisms of
Montgomery, S. A.; Theriot, C. M.
action of disinfectants. Int. Biodeterior.
Biodegrad. 1998, 41, 261-268.
Cefoperazone-treated mouse model of clinically-
relevant Clostridium difficile strain R20291.
JoVE-J. Vis. Exp. 2016, e54850.
25.
Macielag, M. J.; Demers, J. P.; Fraga-
Spano, S. A.; Hlasta, D. J.; Johnson, S. G.;
Kanojia, R. M.; Russell, R. K.; Sui, Z.; Weidner-
Wells, M. A.; Werblood, H. Substituted
salicylanilides as inhibitors of two-component
regulatory systems in bacteria. J. Med. Chem.
1998, 41, 2939-2945.
35.
Reeves, A. E.; Koenigsknecht, M. J.;
Bergin, I. L.; Young, V. B. Suppression of
Clostridium difficile in the gastrointestinal tract
of germ-free mice inoculated with a murine
Lachnospiraceae isolate. Infect. Immun. 2012,
IAI. 00647-12.
26.
Lee, I.-Y.; Gruber, T. D.; Samuels, A.;
36.
Theriot, C. M.; Koenigsknecht, M. J.;
Yun, M.; Nam, B.; Kang, M.; Crowley, K.;
Winterroth, B.; Boshoff, H. I.; Barry III, C. E.
Structure–activity relationships of antitubercular
salicylanilides consistent with disruption of the
proton gradient via proton shuttling. Biorg. Med.
Chem. 2013, 21, 114-126.
Carlson Jr, P. E.; Hatton, G. E.; Nelson, A. M.;
Li, B.; Huffnagle, G. B.; Li, J. Z.; Young, V. B.
Antibiotic-induced shifts in the mouse gut
microbiome and metabolome increase
susceptibility to Clostridium difficile infection.
Nat. Commun. 2014, 5, 3114.
27.
Wu, X.; Cherian, P. T.; Lee, R. E.;
37.
Reeves, A. E.; Theriot, C. M.; Bergin, I.
Hurdle, J. G. The membrane as a target for
controlling hypervirulent Clostridium difficile
infections. J. Antimicrob. Chemother. 2012, 68,
806-815.
L.; Huffnagle, G. B.; Schloss, P. D.; Young, V.
B. The interplay between microbiome dynamics
and pathogen dynamics in a murine model of
Clostridium difficile Infection. Gut microbes
2011, 2, 145-158.
28.
Gooyit, M.; Janda, K. D. Reprofiled
anthelmintics abate hypervirulent stationary-
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 17
38.
Bouillaut, L.; McBride, S.; Sorg, J. A.;
1
2
3
4
5
6
7
8
Schmidt, D. J.; Suarez, J. M.; Tzipori, S.;
Mascio, C.; Chesnel, L.; Sonenshein, A. L.
Effects of Surotomycin on Clostridium difficile
Viability and Toxin Production In Vitro.
Antimicrob. Agents Chemother. 2015, 59, 4199-
4205.
39.
Cherian, P. T.; Wu, X.; Yang, L.;
9
Scarborough, J. S.; Singh, A. P.; Alam, Z. A.;
Lee, R. E.; Hurdle, J. G. Gastrointestinal
localization of metronidazole by a lactobacilli-
inspired tetramic acid motif improves treatment
outcomes in the hamster model of Clostridium
difficile infection. J. Antimicrob. Chemother.
2015, 70, 3061-3069.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
40.
Lambert, R. J. W.; Pearson, J.
Susceptibility testing: accurate and reproducible
minimum inhibitory concentration (MIC) and
non-inhibitory concentration (NIC) values. J.
Appl. Microbiol. 2000, 88, 784-790.
41.
Theriot, C. M.; Koumpouras, C. C.;
Carlson, P. E.; Bergin, II; Aronoff, D. M.;
Young, V. B. Cefoperazone-treated mice as an
experimental platform to assess differential
virulence of Clostridium difficile strains. Gut
Microbes 2011, 2, 326-34.
42.
Theriot, C. M.; Bowman, A. A.; Young,
V. B. Antibiotic-induced alterations of the gut
microbiota alter secondary bile acid production
and allow for Clostridium difficile spore
germination and outgrowth in the large intestine.
mSphere 2016, 1, e00045-15.
43.
Team, R. C. R: A Language and
Environment for Statistical Computing. In
Vienna, Austria, 2019.
44.
M. J.; Han, A. W.; Johnson, A. J.; Holmes, S. P.
DADA2: High-resolution sample inference from
Illumina amplicon data. Nat. Methods 2016, 13,
581-3.
Callahan, B. J.; McMurdie, P. J.; Rosen,
16
ACS Paragon Plus Environment

Page 17 of 17
Journal of Medicinal Chemistry
Table of Contents graphic
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17
ACS Paragon Plus Environment