Ultrapotent Inhibitor of Clostridioides difficile Growth, Which Suppresses Recurrence in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.0c01198

Clostridioides difficile is the leading cause of healthcare-associated infection in the U.S. and considered an urgent threat by the Centers for Disease Control and Prevention (CDC). Only two antibiotics, vancomycin and fidaxomicin, are FDA-approved for the treatment of C. difficile infection (CDI), but these therapies still suffer from high treatment failure and recurrence. Therefore, new chemical entities to treat CDI are needed. Trifluoromethylthio-containing N-(1,3,4-oxadiazol-2-yl)benzamides displayed very potent activities [sub-μg/mL minimum inhibitory concentration (MIC) values] against Gram-positive bacteria. Here, we report remarkable antibacterial activity enhancement via halogen substitutions, which afforded new anti-C. difficile agents with ultrapotent activities [MICs as low as 0.003 μg/mL (0.007 μM)] that surpassed the activity of vancomycin against C. difficile clinical isolates. The most promising compound in the series, HSGN-218, is nontoxic to mammalian colon cells and is gut-restrictive. In addition, HSGN-218 protected mice from CDI recurrence. Not only does this work provide a potential clinical lead for the development of C. difficile therapeutics but also highlights dramatic drug potency enhancement via halogen substitution.

Full text of DOI:10.1021/acs.jmedchem.0c01198

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Article
Ultrapotent inhibitor of Clostridioides difficile
growth, which suppresses recurrence in vivo
George Naclerio, Nader S. Abutaleb, Daoyi Li, Mohamed N. Seleem, and Herman O. Sintim
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01198 • Publication Date (Web): 22 Sep 2020
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Ultrapotent Inhibitor of Clostridioides difficile Growth, which Suppresses
Recurrence in vivo
a
b
b
George A. Naclerio †, Nader S. Abutaleb , Daoyi Li , Mohamed N. Seleemb,c,d*, and Herman O.
†
Sintima,d*
a
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Chemistry Department, Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907,
United States
b
Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West
Lafayette, Indiana 47907, United States
c
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary
Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24060, United States
d Purdue Institute of Inflammation, Immunology, and Infectious Disease, West Lafayette, Indiana 47907,
United States
†Equal contributions
Abstract:
Clostridioides difficile (C. difficile) is the leading cause of healthcare-associated infection
in the U.S. and considered an urgent threat by the Centers for Disease Control and Prevention
(CDC). Only two antibiotics, vancomycin and fidaxomicin, are FDA-approved for the treatment
of C. difficile infection (CDI) but these therapies still suffer from high treatment failure and
recurrence. Therefore, new chemical entities to treat CDI are needed. Trifluoromethylthio
containing N-(1,3,4-oxadiazol-2-yl)benzamides displayed very potent activities (sub-µg/mL
minimum inhibitory concentration (MIC) values) against Gram-positive bacteria. Here, we report
remarkable antibacterial activity enhancement via halogen substitutions, which afforded new
anti-C. difficile agents with ultrapotent activities (MICs as low as 0.003 µg/mL (0.007 µM)) that
surpassed the activity of vancomycin against C. difficile clinical isolates. The most promising
compound in the series, HSGN-218, was non-toxic to mammalian colon cells and is gut
restrictive. In addition, HSGN-218 protected mice from CDI recurrence. Not only does this work
provide a potential clinical lead for the development of C. difficile therapeutics but also it
highlights dramatic drug potency enhancement via halogen substitution.
Introduction:
Clostridioides difficile (C. difficile) is a spore-forming Gram-positive anaerobic
bacterium and the leading cause of nosocomial infections as well as antibiotic-associated
diarrhea in the United States1. In 2017, the Centers for Disease Control and Prevention (CDC)
determined that in the U.S., 223,900 patients were hospitalized with C. difficile infection (CDI),
resulting in 12,800 deaths and more than $1 billion in healthcare costs2. CDI causes severe
diarrhea along with life-threatening complications such as toxic megacolon, pseudomembranous
colitis, and systemic inflammatory response syndrome3. Manifestations of the disease are
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credited to the toxin-mediated damage produced by two major toxins: toxin A
(TcdA/enterotoxin) and toxin B (TcdB/cytotoxin), which catalyze the inactivation of Rho
GTPases, ultimately causing intense inflammation of the gut, accompanied by necrosis and
apoptosis of colonic mucosal cells4-5. Furthermore, C. difficile’s ability to produce spores hinders
the clinical management of CDI because these spores are very resistant to environmental
conditions, antibiotics, and disinfection processes. C. difficile spores can spread throughout the
environment and once ingested by vulnerable hosts, they develop into vegetative cells that
colonize the intestines, thereby producing toxins and establishing infection6-7. Therefore, C.
difficile spores serve as the major cause of CDI circulation and recurrence.
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CDI is typically caused from the use of antibiotics, which disrupts the reproduction of
normal and protective gut microbiota, ultimately allowing C. difficile to grow in the colon and
produce infectious toxins8. Although the overuse of antibiotics is one of the main reasons
contributing to CDI, the management of CDI requires antibiotic treatment. Currently, there are
only three drugs used to treat CDI: metronidazole, vancomycin, and fidaxomicin. Yet only
vancomycin and fidaxomicin are approved by the FDA for treatment of CDI. Although,
metronidazole was previously recommended as a first-line therapy for CDI, its use is now only
limited to non-severe CDI cases when patients are unable to be treated with vancomycin or
fidaxomicin9. Moreover, other limitations with metronidazole treatment are its potent activity
against a wide spectrum of protective normal microbiota, as well as its high absorption (100%
bioavailable) from the intestinal tract, restricting its concentrations in the colon10-11. Although
oral vancomycin is minimally absorbed into the systemic circulation12, it has broad spectrum
activity against Gram-positive bacteria, leading to a reduction in microbiome diversity13.
Furthermore, both vancomycin and metronidazole treatments are inadequate due to high
treatment failure (14% with vancomycin and 22% with metronidazole) and high recurrence rates
(25% to 30%). This is because both antibiotics are ineffective against spores and also they cause
disruption of the beneficial gut microbiota14-15. Fidaxomicin is the only new drug approved for
CDI in the last 30 years. Fidaxomicin has lower recurrence rates compared to vancomycin and
metronidazole because of its selectivity towards C. difficile; however, its high cost limits its
use16-18. Even though vancomycin and fidaxomicin are FDA-approved therapies for CDI,
emerging resistance or reduced susceptibility are evident to these antibiotics17, 19. In addition, one
emerging alternative non-antibiotic therapy for CDI is fecal microbiota transplant (FMT), which
restores the disrupted normal microbiome, leading to renovation of the colonization resistance to
C. difficile20. While FMT appeared to be successful in the treatment of some CDI cases, it has
many restrictions and poses a serious risk of transmitting infectious pathogens to the patients;
especially immunocompromised and elderly patients21-22. Therefore, due to the increase in
treatment failure and recurrence rates with the commonly used anti-CDI drugs, along with
growths of CDI, efforts to develop novel anti-CDI therapeutics have intensified23.
Our program focuses on the discovery of new N-(1,3,4-oxadiazol-2-yl)benzamides to
combat the urgent threats of antibiotic-resistant bacteria24-25. We previously reported the
trifluoromethylthio-containing (1,3,4-oxadiazol-2-yl)benzamide, compound 12, as a potent anti-
MRSA agent26. Compound 12 was found to have bactericidal activity as well as being non-toxic
to mammalian cells26. Compound 12 was however not evaluated in vivo as it was not deemed an
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ideal lead due to the presence of a potential thiophene toxicophore (Figure 1). In this report, we
describe the generation of a new series of trifluoromethylthio containing (1,3,4-oxadiazol-2-
yl)benzamides, which leads to the identification of N-(5-(3,5-dichlorophenyl)-1,3,4-oxadiazol-2-
yl)-4-((trifluoromethyl)thio)benzamide (HSGN-218), which does not contain a thiophene
(Figure 1). HSGN-218 was tested for its activity against a panel of clinical pathogenic C.
difficile strains. Cytotoxicity against mammalian cells, bi-directional Caco-2 permeability and
activity against normal gut microbiota were also investigated. Moreover, the activity of HSGN-
218 treatment was evaluated in an in vivo CDI mouse model and its ability to prevent C. difficile
recurrence in vivo was also investigated.
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Figure 1. Compound 12 contains a potential thiophene toxicophore but was found to be potent against C.
difficile. Utilization of halogen substitution led to the discovery of an ultrapotent anti-C. difficile agent
(HSGN-218) with a 70-times imporvement in potency (from 0.5 µg/mL (1.4 µM) to 0.007 µg/mL (0.02
µM).
Results and Discussion:
Halogenation, a High-Level Medicinal Chemistry Design Strategy
Halogens (X = F, Cl, Br, and I) are commonly used substituents in medicinal chemistry
and drug discovery27-30. For instance, around 40% of the drugs currently FDA-approved or in
clinical trials are halogenated and about 25% of the published medicinal chemistry papers and
patents contain the late stage addition of halogen atoms28. Likewise, 35% of the top-15 selling
drugs from 2010 to 2016 are halogenated31 (Figure 2A). Of the halogenated drugs, 57% contain
fluorine, 38% contain chlorine, 4% contain bromine, and only 1% contain iodine28. The addition
of halogen substitutents has been shown to have a major effect on a drug’s potency and
pharmacological properties. Regarding pharmacological properties, addition of halogen
substituents to lead compounds has been shown to increase lipophilicity, permeability,
membrane binding and metabolic stability32-33. Likewise, insertion of halogen atoms into lead-
like compounds also showed enhanced drug metabolism because the carbon-halogen bond is not
easily metabolized by cytochrome P45028. Concerning potency, halogen atom substitution’s
effect has been documented. For example, L86-8276, a cyclin-dependent kinase 2 (CDK2)
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inhibitor was shown to have an IC50 value of 2.4 µM (Figure 2B)34. Yet, the addition of a
chlorophenyl group to give Flavopiridol showed a six-fold improvement in potency to give an
IC50 of 0.4 µM against CDK2 (Figure 2B)34.
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Figure 2. Importance of the addition of halogen substituents to lead compounds. A. Examples of the top-
15 selling drugs that are halogenated. B. Addition of chlorophenyl to CDK2 inhibitors led to a six-fold
enhancement in potency.
Synthesis and Anti-C. difficile Activity of Trifluoromethylthio Containing (1,3,4-oxadiazol-
2-yl)Benzamides
We previously reported that compound 12 was potent against a panel of clinically
important Gram-positive bacteria26. Based on its broad-spectrum Gram-positive activity, we
wondered if it would be active against C. difficile. Compound 12 inhibited C. difficile ATCC
BAA 1801 with an MIC of 0.5 µg/mL (1.4 µM) (see Figure 1 and Table 1), which is
comparable to vancomycin. However, compound 12 contains an unsubstituted thiophene
moiety, which can lead to toxicity concerns (Figure 1). For instance, thiophene metabolism,
caused by cytochrome P450 mediated oxidation, can lead to the formation of reactive
metabolites, thiophene-S oxides35-36, thiophene epoxides36, and sulphenic acids37, which have a
high propensity to react with nucleophiles such as water and glutathione38. We were however
encouraged that compound 12 showed good activity against C. difficile, so we proceeded to
make new analogs, which did not contain thiophene but instead substituted phenyl groups.
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Scheme 1: General Route for the Synthesis of trifluoromethylthio-containing N-(1,3,4-
oxadiazol-2-yl)benzamidesa
6
CF3
S
7
8
9
NH2
NH
H2N
CF3
S
N
O
O
O
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O
N
c.
a.
HN
b.
N
+
N
R
O
N
R
R
O
OH
I.1 I.17
-
R
1-17
aReagents and Conditions: (a) Semicarbazide hydrochloride, NaOAc, MeOH:H2O (1:1), rt, 30 min,
95% (b) Bromine, NaOAc, AcOH, 60 °C, 1 h, 40% - 70% (c) BOP Reagent, DIPEA, DMF, rt, 12 h, 16%
- 33%.
In our previous report26, we determined that the 4-(trifluoromethylthio)phenyl group is
vital for optimal activity so we kept this constant. The synthesis of the compounds began with a
substituted benzaldehyde followed by the addition of semicarbazide and sodium acetate to give
the corresponding semicarbazone. Then, using bromine and sodium acetate, the semicarbazone
was cyclized into the subsequent 1,3,4-oxadizol-2-amine (Scheme 1). Amide coupling between
the 1,3,4-oxadiazol-2-amine and 4-trifluoromethylthio benzoic acid using benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent gave the desired
trifluoromethylthio containing (1,3,4-oxadiazol-2-yl)benzamides (Scheme 1).
With the compounds in hand (see Table 1), we proceeded to evaluate them against C.
difficile. Halogen substitutions (especially the Cl, F or CF3 groups) resulted in the most active
compounds. Substitution with OMe, Me, and i-propy groups showed only moderate to no actvity
(see Table 1 for MICs of compounds 6, 11, 12, and 13 against C. difficile ATCC BAA 1801).
For halogen substituents, the position on the phenyl ring was also important. For example, the
MIC for meta-Cl (5) was 0.03 µg/mL (0.08 µM), whereas that for the ortho- (3) and para- (9)
analogs were 4 µg/mL (10.0 µM) and 2 µg/mL (5.0 µM) respectively against C. difficile ATCC
BAA 1801 (Table 1). Additionally, for di-substituted halogen containing compounds, the
position of the halogens affected actvity. For instance, the 3,5-dichlorophenyl analog (15,
HSGN-218) was more than four times more potent than the 2,4-dichlorophenyl (14) analog
(MIC = 0.06 µg/mL and 0.007 µg/mL for 14 and 15 respectively). We also proceeded to
investigate subsitution of the phenyl group with heteroaromatics, such as pyridinyl (17) which
had only moderate activity (Table 1) allowing us to conclude that the phenyl ring is needed for
optimal activity.
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Table 1. MICs in µg/mL (µM) of HSGN-218, analogs, and vancomycin, against C. difficle
ATCC BAA 1801.
6
7
8
9
O
N
N
S
CF3
NH
R4
R3
X
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11
12
13
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O
R1
R2
Compound/Control
-R1
-R2
-R3
-R4
X
MICs in
µg/mL
(µM)
Antibiotic
Compound 12
-
H
F
Cl
H
-
-
-
-
0.5 (1.4)
2 (5.5)
4 (10.4)
4 (10.0)
0.03
1
2
3
4
H
H
H
F
H
H
H
H
H
H
H
H
CH
CH
CH
CH
(0.08)
0.03
5
H
Cl
H
H
CH
(0.08)
4 (10.1)
0.015
6
7
H
H
OMe
CF3
H
H
H
H
CH
CH
(0.04)
4 (10.4)
2 (5.0)
0.125
8
9
10
H
H
H
H
H
H
F
Cl
CF3
H
H
H
CH
CH
CH
(0.29)
4 (10.1)
4 (10.5)
128
11
12
13
H
H
H
H
H
H
OMe
CH3
i-Propyl
H
H
H
CH
CH
CH
(314.2)
0.03
(0.07)
0.007
14
Cl
H
H
Cl
H
H
CH
CH
15; HSGN-218
Cl
Cl
(0.02)
2 (4.7)
8 (21.8)
1 (0.7)
0.25
16
17
OMe
H
H
-
H
H
-
Cl
H
-
CH
N
-
H
-
-
Vancomycin
Metronidazole
-
-
-
-
(1.46)
0.06
Fidaxomicin
-
-
-
-
-
(0.06)
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Comprehensive antibacterial profile of HSGN-218 against various C. difficile clinical
isolates
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After the initial screening of HSGN-218, we assessed its antibacterial profile against a
panel of C. difficile clinical isolates. As depicted in Table 2, HSGN-218 exhibited exceptional
actvity against C. difficile clinical isolates with MICs ranging from 0.003 µg/mL (0.007 µM) to
0.03 µg/mL (0.07 µM). Vancomycin displayed MICs ranging from 0.25 µg/mL (0.2 µM) to 1
µg/mL (0.7 µM) against all the tested strains (Table 2). With regard to micromolar
concentrations, HSGN-218 is between 2.5 to 100 times more potent than vancomycin in
inhibiting clinically relevant C. difficile growth in vitro. Metronidazole inhibited the growth of
the tested C. difficile strains at concentrations ranging from 0.125 µg/mL (0.7 µM) to 0.25
µg/mL (1.46 µM). Fidaxomicin displayed MIC values ranging from 0.015 µg/mL (0.01 µM) to
0.06 µg/mL (0.06 µM).
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Table 2. MICs in µg/mL (µM) of HSGN-218 and vancomycin against various C. difficile
clinical isolates.
Compound/Control
C.
C.
C.
C.
C.
C.
C.
Antibiotic
difficile
NR-
13432
difficile
NR-
13435
difficile
NR-
32883
difficile
NR-
32891
(P13)
difficile
NR-
32895
(P19)
difficile
NR-
32904
(P30)
difficile
ATCC
43255
(isolate 6) (isolate 9) (P2)
HSGN-218
0.03
(0.07)
0.25 (0.2) 1 (0.7)
0.25
(1.46)
0.06
(0.06)
0.003
(0.007)
0.007
(0.02)
0.5 (0.4)
0.125
(0.7)
0.007
(0.02)
0.5 (0.4)
0.125
(0.7)
0.007
(0.02)
1 (0.7)
0.25
(1.46)
0.03
0.007
(0.02)
1 (0.7)
0.25
(1.46)
0.015
(0.01)
0.015
(0.04)
1 (0.7)
0.25
(1.46)
0.015
(0.01)
Vancomycin
Metronidazole
0.125
(0.7)
0.06
Fidaxomicin
0.03
(0.03)
0.015
(0.01)
(0.06)
(0.03)
Antibacterial profile of HSGN-218 against vancomycin-resistant enterococci and Gram-
negative bacteria
Next, the antibacterial activity of HSGN-218 was assessed against vancomycin-resistant
enterococci (VRE) and Escherichia coli that are highly common bacteria in the gut. The
overgrowth of VRE and colonization of the gut are one of the major issues associated with the
vancomycin and metronidazole treatment of CDI39-40. Thus, anticlostridial agents capable of
inhibiting the growth of VRE are highly desirable. On the other hand, E. coli is the predominant
aerobic bacteria colonizing in the gut which remains resident throughout the life of the host41. As
depicted in Table 3, HSGN-218 exhibited potent actvity against VRE clinical isolates with
MICs ranging from 0.06 µg/mL (0.14 µM) to 0.125 µg/mL (0.29 µM) outperforming
vancomycin and metronidazole. When tested against E. Coli, HSGN-218 was found to be
inactive against E. coli BW25113 (wild-type strain). Conversely, the compound showed
moderate activity (MIC = 4 µg/mL (9.2 µM)) against E. coli JW55031 which is deficient in
AcrAB-TolC efflux pump. Thus, the lack of activity against the wild-type E. coli could be
attributed to that HSGN-218 may be a substrate for AcrAB-TolC efflux pump.
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Table 3. MICs in µg/mL (µM) of HSGN-218 and control antibiotics against vancomycin-
resistant enterococci (VRE) and Escherichia coli isolates.
6
7
8
9
Compound/Control
Antibiotic
E. faecium
ATCC
700221
E. faecalis
ATCC 51299 JW55031
E. coli
E. coli
BW25113
(wild-type
strain)
(TolC
Mutant
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HSGN-218
Vancomycin
Metronidazole
Linezolid
Gentamicin
NT1, not tested
0.125 (0.29)
32 (22.1)
>64 (>373.9) >64 (>373.9)
1 (3.0)
NT
0.06 (0.14)
>64 (>44.2)
4 (9.2)
>64 (>44.2)
NT1
16 (47.4)
0.25 (0.52)
>16 (>36.8)
>64 (>44.2)
NT
>64 (>189,7)
0.25 (0.52)
1 (3.0)
NT
HSGN-218 is highly tolerable to human cell lines
Prokaryotic cell selectivity is a vital attribute for any antibiotic candidate. Thus, HSGN-
218 was assessed for toxicity to mammalian cells. HSGN-218 showed an excellent safety profile
against human colorectal cells (Caco-2) (Figure 3). It was highly tolerable to Caco-2 cells at
concentrations higher than 64 µg/mL. This concentration is more than 9,000-times higher than
the compound’s corresponding MIC value against C. difficile ATCC BAA 1801 used in the
initial screening.
100
4 µg/mL
8 µg/mL
16 µg/mL
32 µg/mL
64 µg/mL
DMSO
50
0
HSGN-218
Figure 3. In vitro cytotoxicity assessment of HSGN-218 (tested in triplicate) against human colorectal
cells (Caco-2) using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium) assay. Results are presented as percent viable cells relative to DMSO
(negative control). Error bars represent standard deviation values. A one-way ANOVA, with post hoc
Dunnet’s multiple comparisons test, determined statistical difference between the values obtained for the
compound and DMSO (denoted by the asterisk) (P < 0.05).
HSGN-218 demonstrates low Caco-2 permeability:
In order to treat CDI, it’s vital that a compound does not cross the gastrointestinal tract
but instead stays localized in the gut. Thus, we assessed whether HSGN-218 would permeate
across the gastrointestinal tract via a Caco-2 bidirectional permeability assay42. The assay
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(performed as a service at Eurofins Panlabs (MO, USA) demonstrated that HSGN-218 showed
limited ability to permeate across Caco-2 bilayers (Papp = 0.2 × 10−6ꢀcmꢀs−1 from the apical to
basolateral and Papp = 0.1 × 10−6ꢀcmꢀs−1 from the basolateral to apical, see Table 4). This
permeability is comparable to rinitidine (Papp = 0.5 × 10−6ꢀcmꢀs−1 from the apical to basolateral
and Papp = 1.3 × 10−6ꢀcmꢀs−1 from the basolateral to apical, see Table 4), a drug that is known to
have low permeability across Caco-2 bilayers. Propranolol was used as a high permeability
control as its Papp = 37.2 × 10−6ꢀcmꢀs−1 from the apical to basolateral and Papp = 22.7 ×
10−6ꢀcmꢀs−1 from the basolateral to apical (Table 4). Therefore, the Caco-2 permeability results
indicate that HSGN-218 will not cross the gastrointestinal tract and instead concentrate in the
gut, the site for C. difficile infections.
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Table 4. Caco-2 Permeability Analysis for HSGN-218 and Control Drugs.
Compound/Control Drug
Mean A → B Papp
(cm s−1)
Mean B → A Papp
(cm s−1)
Notes
HSGN-218
Ranitidine
0.2 x 10-6
0.1 x 10-6
Low Permeability
Low Permeability
Control
High Permeability
Control
0.5 x 10-6
1.3 x 10-6
Propranolol
37.2 x 10-6
22.7 x 10-6
In vitro antibacterial evaluation of HSGN-218 against normal microflora.
Antibiotics administration (especially broad-spectrum ones) causes alteration of the
normal intestinal microbial composition, resulting in gut colonization by opportunistic pathogens
like C. difficile43. Consequently, we investigated whether HSGN-218 has a deleterious effect on
important representative members of the normal gut microbiota such as Lactobacillus spp and
Bacteroides spp. Bacteroides spp comprise a large proportion of the intestinal microbiota, which
were reported to contribute to bile acid-mediated inhibition of C. difficile and prevent CDI in
mouse model44-45. Additionally, lactobacilli were reported to interfere with C. difficile both in
vitro and in vivo46-47. As depicted in Table 5, HSGN-218 exhibited weak antibacterial activity
against Lactobacillus strains (MIC = 16 µg/mL (36.8 µM)) and inhibited growth of species of
Bacteroides (MIC=1-2 µg/mL (2.3-4.6 µM)). Similarly, vancomycin inhibited Lactobacillus
strains (MICs = ≤1-2 µg/mL (0.7-1.4 µM)) and exhibited weak activity against Bacteroides spp
(MICs = 32-64 µg/mL (22.1-44.2 µM)). Although HSGN-218 was similar to vancomycin, the
anti-CDI drug of choice, in inhibiting the growth of certain species of the normal microbiota, it
must be noted that HSGN-218 inhibits C. difficile at concentrations that are 100-times less than
what is needed to inhibit Bacteroides (compare Table 2 with Table 5). On the other hand,
vancomycin inhibited both C. difficile and Lactobacillus strains with comparable MIC values of
1-2 µg/mL. Metronidazole and fidaxomicin (to a lesser extent) also inhibit certain members of
the normal intestinal microbiota48-50
.
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Table 5. MICs in µg/mL (µM) of HSGN-218 and vancomycin against human normal gut
microbiota.
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7
8
9
Bacterial strains
Lactobacillus
gasseri HM-400
Lactobacillus
crispatus HM-103
Lactobacillus
crispatus HM-371
Bacteroides fragilis
HM-711
Bacteroides fragilis
HM-709
Bacteroides dorei
HM-719
HSGN-218
16 (36.8)
Vancomycin
≤1 (≤0.7)
Metronidazole Fidaxomicin
>64 (>373.9)
>64 (>373.9)
>64 (>373.9)
≤1 (5.84)
>64 (>60.5)
>64 (>60.5)
>64 (>60.5)
>64 (>60.5)
>64 (>60.5)
>64 (>60.5)
16 (36.8)
16 (36.8)
2 (4.6)
2 (1.4)
2 (1.4)
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64 (44.2)
32 (22.1)
64 (44.2)
1 (2.3)
2 (11.68)
2 (4.6)
≤1 (5.84)
Frequency of mutation.
The promising results of HSGN-218 led us to investigate the likelihood of C. difficile to
develop resistance to HSGN-218. No resistant mutants were isolated at a concentration of 15 ×
MIC and 20 × MIC in the presence of a high inoculum of C. difficile (Table 6), indicating that C.
difficle is unlikely to form rapid resistance to HSGN-218. Likewise, vancomycin exhibited low
frequency of mutation (<1.1× 10-9) and no resistant mutants were isolated, in agreement with a
previous report51.
Table 6. Frequency of mutation of HSGN-218 against C. difficile ATCC 43255
Test agent
Frequency of mutation
15  MIC
<1.1× 10-9
<1.1× 10-9
20  MIC
<1.1× 10-9
<1.1× 10-9
HSGN-218
Vancomycin
In vivo efficacy of HSGN-218 in a CDI mouse model52
The potent antibacterial activities of HSGN-218 against C. difficile prompted us to
investigate its efficacy in a CDI mouse model and its potential to protect mice from CDI
recurrence, as described before. As shown in Figure 4, vancomycin (10ꢀmg/kg) protected 100%
of mice up to 5 days, as previously reported53-54. HSGN-218 (50ꢀmg/kg), was able to
significantly protect 66.7% of the mice against C. difficile during the 5-days treatment period.
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*
*
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HSGN-218
Vancomycin
Vehicle
0
1
2
3
4
5
Days
Figure 4. In vivo efficacy of HSGN-218 in a CDI mouse model. Kaplan–Meier survival curves were
analyzed using a log-rank (Mantel–Cox) test. Asterisks (*) denote statistically significant difference
between mice treated with either HSGN-218, or vancomycin in comparison with the vehicle-treated mice.
After testing the efficacy of HSGN-218 in the CDI mouse model, we sought to
investigate this promising activity of HSGN-218 in preventing C. difficile recurrence. C. difficile
recurrence is challenging to treat. In addition to the subsequent prolongation of C. difficile
shedding and transmission, 1 out of every 5 patients experienced C. difficile recurrence episode
died within 30 days of diagnosis55. Therefore, we sought to investigate this promising activity of
HSGN-218 in preventing C. difficile recurrence. Mice were infected and treated for 5 days and
then they were monitored for survival and possible C. difficile recurrence until the 21st day.
Vancomycin-treated mice survived the first 5 days (similar to prior reports)53, but in accordance
with previous studies52, 54, mice treated with vancomycin were susceptible to C. difficile
recurrence and 83.3% of vancomycin-treated mice died after stopping vancomycin treatment. In
contrast, HSGN-218 (50 mg/kg), significantly protected mice from CDI recurrence with 100%
survival after 5- days treatment period (Figure 5).
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*,#
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HSGN-218
Vancomycin
Vehicle
0
2
4
6
8
10 12 14 16 18 20
Days
Figure 5. In vivo efficacy of HSGN-218 against CDI recurrence. Mice were treated with HSGN-218 (50
mg/kg), vancomycin (10 mg/kg) or the vehicle for 5 days and treatments were stopped thereafter.
Kaplan–Meier survival curves were analyzed using a log-rank (Mantel–Cox) test. Asterisks (*) denote
statistically significant difference between mice treated with either HSGN-218, or vancomycin in
comparison with the vehicle-treated mice. Pound (#) denotes statistically significant difference between
mice treated with compound HSGN-218 in comparison with vancomycin-treated mice
Conclusion:
In conclusion, we have identified HSGN-218 as a highly potent small molecule inhibitor
of C. difficile growth. HSGN-218 is up to 100-times more active (MICs ranging from 0.003
µg/mL (0.007 µM) to 0.03 µg/mL (0.07 µM)) against C. difficile clinical isolates than
vancomycin, the drug of choice for CDI. The compound is also non-toxic to mammalian cells as
well as demonstrates low Caco-2 bidirectional permeability, indicating that HSGN-218 would
have minimal systemic absorption. Even though HSGN-218 inhibited the growth of certain
representative members of normal microbiota, excitingly, HSGN-218 protected mice from CDI
as well as it showed significant efficacy against C. difficile recurrence. Therefore, compound
HSGN-218 is considered as a lead compound to develop as anti- C. difficile therapeutic and
deserves serious consideration.
Experimental Section:
Chemistry:
General Information: unless noted otherwise, all reagents and solvents were purchased
from commercial sources and used as received. The 1H, 13C, and 19F NMR spectra were obtained
in DMSO-d6 as solvent using a 500 MHz spectrometer with Me4Si as an internal standard.
Chemical shifts are reported in parts per million (δ) and are calibrated using residual
1
undeuterated solvent as an internal reference. Data for H NMR spectra are reported as follows:
chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). Multiplicities are
reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, or
combinations thereof. High resolution mass spectra (HRMS) were obtained using electron spray
1
ionization (ESI) technique and as TOF mass analyzer. Compounds were characterized by H
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NMR, C NMR, F NMR, and HRMS data. The purity of compounds was determined to be
greater than 95% by measuring the absorbance at 260 nm with high performance liquid
chromatography (HPLC) (See supprting information). HPLC spectra were recorded on an
Agilent 1260 Infinity system using a ZORBAX RR Eclipse Plus C18 column. The mobile phase
gradient went from 50% H2O : 50% MeOH over 5 minutes and then 40% H2O : 60% MeOH for
5 minutes, followed by 10% H2O : 90% MeOH for 2 minues and lastly 50% H2O : 50% MeOH
for 3 minutes at a 1 mL/min flow rate.
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Synthesis of 1,3,4-oxadiazol-2-amines [I.1 – I.17]:
The synthesis of I.1-I.17 was performed using a literature reported procedure56. Obtained 1H,
13C, and 19F spectra were in agreement with literature reported data.
Amide Coupling Procedure for the Synthesis of Compounds 1-17:
A 20 mL screw caped vial, charged with the corresponding acid (1 eq.), amine (1 eq.),
BOP reagent (2.7 eq.) and diisopropylethylamine (1.5 mL) in DMF solvent (5 mL) was stirred at
room temperature for 16 h. After completion, the reaction mixture was concentrated under
reduced pressure, followed by flash column chromatography (hexanes:ethyl acetate 90:10 to
70:30) to give the desired product.
N-(5-Phenyl-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (1):
Off-white solid (46 mg, 28%). 1H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 8.0 – 7.9 (m, 2H),
13
7.9 (m, 2H), 7.6 (m, 3H). C NMR (126 MHz, DMSO-d6) δ 165.6, 161.1, 158.6, 136.0, 135.8,
132.2, 131.1 (q, J = 308.7 Hz), 130.1, 129.8, 128.6, 126.6, 123.8. 19F NMR (471 MHz, DMSO-
d6) δ -42.4 (s, 3F). HRMS (ESI) m/z calcd for C16H11F3N3O2S [M + H]+ 366.0524, found
366.0522. Purity by HPLC was found to be 96%.
N-(5-(2-Fluorophenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (2):
1
Off-white solid (38 mg, 22%). H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 8.0 (td, J = 7.6,
13
1.8 Hz, 1H), 7.9 (m, 2H), 7.7 (tdd, J = 7.4, 5.1, 1.8 Hz, 1H), 7.5 – 7.4 (m, 2H). C NMR (126
MHz, DMSO-d6) δ 165.6, 160.6 (d, J = 257.0 Hz), 158.7, 157.7, 136.1, 135.7, 134.5 (d, J = 8.82
Hz), 131.1 (q, J = 308.7 Hz), 130.1, 129.7, 128.6, 125.8, 117.6 (d, J = 20.2 Hz), 112.2 (d, J =
11.3 Hz). 19F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F), -112.0 (d, J = 5.7 Hz, 1F). HRMS
(ESI) m/z calcd for C16H10F4N3O2S [M + H]+ 384.0430, found 384.0429. Purity by HPLC was
found to be 96%.
N-(5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (3):
Off-white solid (41 mg, 23%). 1H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 8.0 – 7.9 (m, 1H),
13
7.9 (m, 2H), 7.7 (m, 1H), 7.6 (td, J = 7.8, 1.6 Hz, 1H), 7.6 (t, J = 7.6 Hz, 1H). C NMR (126
MHz, DMSO-d6) δ 165.2, 159.2, 158.8, 136.1, 135.6, 133.5, 132.3, 131.6, 131.5, 131.1 (q, J =
308.7 Hz), 130.1, 128.7, 128.3, 123.0. 19F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F). HRMS
(ESI) m/z calcd for C16H10ClF3N3O2S [M + H]+ 400.0134, found 400.0135. Purity by HPLC was
found to be 96%.
N-(5-(3-Fluorophenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (4):
1
Off-white solid (55 mg, 32%). H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 7.9 (m, 2H), 7.8
(m, 1H), 7.7 – 7.6 (m, 2H), 7.5 (td, J = 8.5, 2.7 Hz, 1H).13C NMR (126 MHz, DMSO-d6) δ
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165.3, 163.7 (d, J = 245.7 Hz), 160.2, 158.7, 136.0, 135.5, 132.4 (d, J = 8.82 Hz), 131.1 (q, J =
308.7 Hz), 130.2, 128.7, 125.9 (d, J = 8.82 Hz), 122.8, 119.3 (d, J = 21.4 Hz), 113.3 (d, J = 25.2
Hz). 19F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F), -112.5 (q, J = 8.5 Hz, 1F). HRMS (ESI)
m/z calcd for C16H10F4N3O2S [M + H]+ 384.0430, found 384.0429. Purity by HPLC was found
to be 98%.
N-(5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (5):
Off-white solid (35 mg, 19%). 1H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 7.9 – 7.8 (m, 4H),
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7.7 (dd, J = 23.9, 7.9 Hz, 2H). C NMR (126 MHz, DMSO-d6) δ 165.4, 159.8, 159.0, 136.1,
134.6, 132.0, 132.0, 131.1 (q, J = 308.7 Hz), 130.2, 128.9, 128.5, 126.0, 125.8, 125.2. 19F NMR
(471 MHz, DMSO-d6) δ -42.4 (s, 3F). HRMS (ESI) m/z calcd for C16H10ClF3N3O2S [M + H]+
400.0134, found 400.0135. Purity by HPLC was found to be 96%.
N-(5-(3-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (6):
Off-white solid (42 mg, 24%). 1H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 7.9 (m, 2H), 7.6 –
7.5 (m, 2H), 7.4 (s, 1H), 7.2 (d, J = 7.9 Hz, 1H), 3.8 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
165.5, 160.9, 160.2, 158.6, 136.0, 135.7, 131.2 (q, J = 308.7 Hz), 130.1, 128.7, 128.6, 125.0,
118.9, 118.3, 111.5, 55.9. 19F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F). HRMS (ESI) m/z
calcd for C17H13F3N3O2S [M + H]+ 396.0630, found 396.0632. Purity by HPLC was found to be
98%.
N-(5-(3-Trifluoromethylphenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide
(7):
1
Off-white solid (53 mg, 27%). H NMR (500 MHz, DMSO-d6) δ 8.2 (d, J = 7.9 Hz, 1H), 8.2 –
13
8.1 (m, 3H), 8.0 (m, 1H), 7.9 (m, 3H). C NMR (126 MHz, DMSO-d6) δ 165.4, 159.8, 159.1,
136.1, 135.8, 131.4, 131.1 (q, J = 308.7 Hz), 130.8 (q, J = 31.5 Hz), 130.5, 130.4, 130.1, 128.6,
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125.2 (q, J = 272.2 Hz), 125.0, 122.8, F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F), -62.8 (s,
3F). HRMS (ESI) m/z calcd for C17H10F6N3O2S [M + H]+ 434.0398, found 434.0399. Purity by
HPLC was found to be 98%.
N-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (8):
1
Off-white solid (43 mg, 24%). H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 8.0 (dd, J = 8.7,
5.5 Hz, 2H), 7.9 (m, 2H), 7.4 (t, J = 8.7 Hz, 2H).13C NMR (126 MHz, DMSO-d6) δ 165.5 (d, J =
250.7 Hz), 160.4, 158.6, 136.1, 135.7, 131.1 (q, J = 308.7 Hz), 130.1, 129.3 (d, J = 8.82 Hz),
128.6, 120.5, 117.2 (d, J = 22.7 Hz). 19F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F), -108.8. (s,
1F). HRMS (ESI) m/z calcd for C16H10F4N3O2S [M + H]+ 384.0430, found 384.0431. Purity by
HPLC was found to be 96%.
N-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (9):
Off-white solid (36 mg, 20%). 1H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 8.0 – 7.9 (m, 2H),
13
7.9 (m, 2H), 7.7 – 7.6 (m, 2H). C NMR (126 MHz, DMSO-d6) δ 164.5, 160.4, 158.6, 137.0,
19
136.1, 135.6, 131.1 (q, J = 308.7 Hz), 130.1, 128.6, 128.4, 127.2, 122.7. F NMR (471 MHz,
DMSO-d6) δ -42.4 (s, 3F). HRMS (ESI) m/z calcd for C16H10ClF3N3O2S [M + H]+ 400.0134,
found 400.0132. Purity by HPLC was found to be 97%.
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N-(5-(4-Trifluoromethylphenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide
(10):
6
7
8
Off-white solid (42 mg, 22%). 1H NMR (500 MHz, DMSO-d6) δ 8.1 (dd, J = 14.0, 8.0 Hz, 4H),
8.0 (m, 2H), 7.9 (m, 2H). C NMR (126 MHz, DMSO-d6) δ 165.2, 160.0, 159.0, 136.1, 135.6,
13
132.0 (q, J = 31.5 Hz), 131.1 (q, J = 308.7 Hz), 130.1, 128.7, 127.6, 127.4, 126.9, 125.3 (q, J =
272.2 Hz).19F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F), -62.8 (s, 3F). HRMS (ESI) m/z calcd
for C17H10F6N3O2S [M + H]+ 434.0398, found 434.0397. Purity by HPLC was found to be 99%.
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N-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (11):
1
Off-white solid (48 mg, 27%). H NMR (500 MHz, DMSO-d6) δ 8.1 (d, J = 8.0 Hz, 2H), 7.9 –
13
7.8 (m, 4H), 7.1 (m, 2H), 3.8 (s, 3H). C NMR (126 MHz, DMSO-d6) δ 164.0, 162.5, 161.4,
19
158.1, 136.1, 131.1 (q, J = 308.7 Hz), 130.1, 128.4, 127.3, 116.1, 115.4, 115.1, 56.0. F NMR
(471 MHz, DMSO-d6) δ -42.4 (s, 3F). HRMS (ESI) m/z calcd for C17H13F3N3O2S [M + H]+
396.0630, found 396.0631. Purity by HPLC was found to be 99%.
N-(5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (12):
1
Off-white solid (35 mg, 21%). H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 7.9 (m, 4H), 7.4
(m, 2H), 2.4 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.7, 161.3, 158.4, 142.5, 136.1, 135.8,
131.1 (q, J = 308.7 Hz), 130.4, 130.1, 128.5, 126.6, 121.1, 21.6. 19F NMR (471 MHz, DMSO-d6)
δ -42.4 (s, 3F). HRMS (ESI) m/z calcd for C17H13F3N3O2S [M + H]+ 380.0681, found 380.0682.
Purity by HPLC was found to be 98%.
N-(5-(4-Isopropylphenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (13):
Off-white solid (39 mg, 21%). 1H NMR (500 MHz, DMSO-d6) δ 8.2 – 8.1 (m, 2H), 7.9 (dd, J =
13
8.3, 2.5 Hz, 4H), 7.5 (m, 2H), 3.0 (h, J = 6.9 Hz, 1H), 1.2 (d, J = 6.9 Hz, 6H). C NMR (126
MHz, DMSO-d6) δ 165.5, 161.2, 158.4, 153.1, 136.1, 135.8, 131.2 (q, J = 308.7 Hz), 130.1,
19
128.5, 127.9, 126.7, 121.4, 33.9, 23.9. F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F). HRMS
(ESI) m/z calcd for C19H17F3N3O2S [M + H]+ 408.0994, found 408.0993. Purity by HPLC was
found to be 97%.
N-(5-(2,4-Dichlorophenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (14):
Off-white solid (35 mg, 18%). 1H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 8.0 (m, 1H), 7.9 –
7.8 (m, 3H), 7.7 (dd, J = 8.5, 2.1 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 165.1, 158.8,
158.5, 137.5, 136.1, 135.5, 133.3, 132.6, 131.2, 131.1 (q, J = 308.7 Hz), 130.1, 128.7, 122.0. 19F
NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F). HRMS (ESI) m/z calcd for C16H9Cl2F3N3O2S [M +
H]+ 433.9745, found 433.9747. Purity by HPLC was found to be 99%.
N-(5-(3,5-Dichlorophenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide
(15,
HSGN-218):
1
13
Off-white solid (37 mg, 19%). H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 7.9 (m, 5H). C
NMR (126 MHz, DMSO-d6) δ 165.4, 159.7, 158.7, 136.1, 135.7, 131.4, 131.2 (q, J = 308.7 Hz),
130.1, 128.4, 127.2, 126.3, 124.9. 19F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F). HRMS (ESI)
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m/z calcd for C16H9Cl2F3N3O2S [M + H]+ 433.9745, found 433.9744. Purity by HPLC was found
to be 99%.
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9
N-(5-(5-Chloro-2-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide
(16):
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1
Off-white solid (46 mg, 24%). H NMR (500 MHz, DMSO-d6) δ 8.1 (m, 2H), 7.9 (m, 2H), 7.8
(d, J = 2.7 Hz, 1H), 7.6 (dd, J = 9.0, 2.8 Hz, 1H), 7.3 (m, 1H), 3.9 (s, 3H).13C NMR (126 MHz,
DMSO-d6) δ 165.8, 158.7, 156.8, 136.1, 135.8, 133.3, 131.2 (q, J = 308.7 Hz), 130.1, 129.4,
128.6, 126.3, 124.9, 115.4, 114.4, 57.1. 19F NMR (471 MHz, DMSO-d6) δ -42.4 (s, 3F). HRMS
(ESI) m/z calcd for C17H12ClF3N3O3S [M + H]+ 430.0240, found 430.0241. Purity by HPLC was
found to be 97%.
N-(5-(Pyridin-2-yl)-1,3,4-oxadiazol-2-yl)-4-((trifluoromethyl)thio)benzamide (17):
Off-white solid (26 mg, 16%). 1H NMR (500 MHz, DMSO-d6) δ 8.8 (d, J = 4.7 Hz, 1H), 8.1 (dd,
J = 8.2, 2.7 Hz, 3H), 8.0 (td, J = 7.8, 1.8 Hz, 1H), 7.9 (m, 2H), 7.6 (ddd, J = 7.6, 4.8, 1.2 Hz,
1H). C NMR (126 MHz, DMSO-d6) δ 164.9, 160.8, 159.0, 150.7, 143.2, 138.3, 136.1, 135.5,
131.1 (q, J = 308.7 Hz), 130.1, 128.7, 126.6, 122.9. F NMR (471 MHz, DMSO-d6) δ -42.4 (s,
3F). HRMS (ESI) m/z calcd for C15H10F3N4O2S [M + H]+ 367.0477, found 367.0475. Purity by
HPLC was found to be 98%.
13
19
Bacterial strains media, cell lines and reagents
Bacterial strains used in this study (Table 1S) were obtained from the Biodefense and Emerging
Infections Research Resources Repository (BEI Resources) and the American Type Culture
Collection (ATCC). E. coli BW25113 and JW25113 were obtained from the Coli Genetic Stock
Center (CGSC), Yale University, USA. Brain heart infusion broth was purchased from Becton,
Dickinson and Company (Cockeysville, MD, USA) and was purchased from Fisher Scientific.
Yeast extract, L-cysteine, vitamin K, hemin and phosphate buffered saline (PBS) were all
obtained from commercial vendors. Human colorectal adenocarcinoma epithelial cells (Caco-2)
(ATCC HTB-37) was obtained from the American Type Culture Collection (ATCC) (Manassas,
VA, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and
phosphate-buffered saline (PBS) was purchased from Corning (Manassas, VA, USA).
Vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO, USA), linezolid and
gentamicin sulfate (Chem-Impex International, Wood Dale, IL, USA), metronidazole (Alfa
Aesar, Ward Hill, MA, USA), and fidaxomicin (Cayman Chemical, Ann Arbor, MI, USA) were
purchased commercially. Compounds were synthesized from commercial sources in our
laboratory.
Determination of the MICs against C. difficile clinical isolates:
The minimum inhibitory concentrations (MICs) of tested compounds and control drug;
vancomycin, were determined using the broth microdilution method, as previously described57-60
against C. difficile clinical isolates. Briefly, 0.5 McFarland bacterial solution was prepared and
diluted in brain heart infusion supplemented (BHIS) broth (to an inoculum size ~5 x 105
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CFU/mL). Test agents were added and serially diluted before plates were incubated
anaerobically at 37°C for 48 hours. MICs reported are the lowest drug concentration that
completely suppressed the growth of bacteria, as observed visually.
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Determination of the MICs against vancomycin-resistant enterococci (VRE) and
Escherichia coli strains
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The MICs of HSGN-218 and control drugs were determined using the broth microdilution
method, according to guidelines outlined by the Clinical and Laboratory Standards Institute
(CLSI61) against Enterococcus faecium, Enterococcus faecalis and Escherichia coli strains.
Bacterial strains were grown aerobically overnight on tryptone soy agar (TSA) plates at 37° C.
Afterwards, a bacterial solution equivalent to 0.5 McFarland standard was prepared and diluted
in cation-adjusted Mueller-Hinton broth (CAMHB) (for E. coli) or tryptone soy broth (TSB) (for
enterococcal strains), to achieve a bacterial concentration of about 5 × 105 CFU/mL. Test agents
were added in the first row of the 96-well plates and serially diluted along the plates. Plates were
then, incubated as previously described. MICs reported in Table 3 are the minimum
concentrations of the test agents that completely inhibited the visual growth of bacteria.
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In vitro cytotoxicity analysis of HSGN-218 against human colorectal cells.
Compounds were assayed for potential cytotoxicity against a human colorectal adenocarcinoma
(Caco-2) cell line, as described previously62-63. Briefly, tested compounds were incubated with
Caco-2 cells for 2 hours. Then, cells were incubated with MTS (3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent for 4 hours before
measuring absorbance values (OD490).
Caco-2 permeability assay
Assay and data analysis were performed by Eurofins Panlabs (MO, USA) according to a
previously reported protocol64-65. The apparent permeability coefficient (Papp) of the tested
agents was calculated using the equation below:
where VR is the volume of the receiver chamber. CR,end is the concentration of the test compound
in the receiver chamber at the end time point, Δt is the incubation time and A is the surface area
of the cell monolayer. CD,mid is the calculated mid-point concentration of the test compound in
the donor side, which is the mean value of the donor concentration at time 0 minute and the
donor concentration at the end time point. CR,mid is the mid-point concentration of the test
compound in the receiver side, which is one half of the receiver concentration at the end time
point. Concentrations of the test compound were expressed as peak areas of the test compound.
In vitro antibacterial evaluation of HSGN-218 against normal microflora.
The broth microdilution assay was utilized to determine the MICs of HSGN-218 against
commensal organisms that compose the human gut microflora, as described elsewhere48, 61, 66. A
bacterial solution equivalent to 0.5 McFarland standard was prepared and diluted in BHIS broth
(for Bacteroides) or in MRS broth (for Lactobacillus) to achieve a bacterial concentration of
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about 5 × 105 CFU/mL. Test agents were added and serially diluted along the plates. Plates were
incubated for 48 hours at 37°C before recording the MIC by visual inspection of growth.
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Frequency of spontaneous mutation.
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HSGN-218 was tested against C. difficile to determine the likelihood of development of
spontaneous mutation as previously described51, 67. Briefly, HSGN-218 and vancomycin were
added to BHIS agar to achieve a final concentration of 15 × MIC and 20 × MIC and poured in
plates and left to dry out. An inoculum of ~ 109 CFU/mL of C. difficile ATCC 43255 was spread
over the plates and incubated anaerobically at 37°C for 48ꢀhours before plates were checked for
the possible bacterial growth.
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Preparation of C. difficile spores for mice infection
C. difficile spores were prepared as described earlier 68,52. Briefly, C. difficile ATCC 43255 was
inoculated onto BHIS agar and incubated anaerobically for 5 days. Spores were collected
anaerobically using PBS containing 10% bovine serum albumin, heated at 70°C for 20 minutes
to get rid of vegetative cells and counted by dilution and plating onto BHIS supplemented with
0.1% taurocholic acid. Spores were then, stored at 4℃ overnight before infecting mice.
C. difficile infection (CDI) mouse model
The study was reviewed, approved and performed following the guidelines of the Purdue
University Animal Care and Use Committee (PACUC) and according to the recommendations in
the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice
were housed in individually ventilated autoclaved cages and received sterile food and water ad
libitum throughout the duration of the experiment. CDI mouse model was performed as
described previously52. Eight-week-old female pathogen-free C57BL/6 mice (Jackson, ME,
USA) were pre-treated with an antibiotic cocktail in sterile drinking water to disrupt the mice
normal intestinal microflora, reducing the colonization resistance and facilitating infection with
the toxigenic strain of C. difficile. Afterwards, mice were switched to regular autoclaved water
for 2 days and they received a single dose of clindamycin (10 mg/kg) intraperitoneally 1 day
prior to C. difficile challenge. For infection, mice were restrained and infected via oral gavage
with 1.3 x 106 spores of C. difficile ATCC 43255. Following infection, mice were randomly
allocated into groups (n=6) for treatment. Two hours post-infection, one groups were treated
orally with HSGN-218 (50 mg/kg), one group was treated with vancomycin (10 mg/kg) via oral
gavage, and one group was treated orally with the vehicle (10% DMSO, 10% tween 80, 80%
PBS). Treatments were continued once daily for five days and mice were closely monitored for
disease signs (including weight loss, behavioral changes, hunched posture, decreased activity,
wet tail and diarrhea).
In vivo efficacy of HSGN-218 in C. difficile recurrence
In order to investigate the activity of HSGN-218 in preventing C. difficile recurrence, mice were
infected, as described above and one group was treated orally with HSGN-218 (50ꢀmg/kg), one
group was treated with vancomycin (10ꢀmg/kg) via oral gavage, and one group was treated orally
with the vehicle for 5 days. Treatments were stopped after 5 days, and mice were monitored for
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disease signs and recurrence of infection till the 21st day. Then, mice were humanely euthanized
using CO2 asphyxiation.
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Statistical analyses
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The survival data were analyzed by Log-rank (Mantel-Cox) test utilizing GraphPad Prism
version 6.00 for Windows (GraphPad Software, La Jolla, CA, USA).
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In Silico PAINS Analysis
All synthesized analogs were subjected to PAINS filters by using the SwissADME
program69. Molecular formula strings of analogs were manually entered into the program, which
indicated no PAINS were found.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI: 10.1021/acs.jmedchem.xxxxxxx.

Various bacterial strains used in this study; and 1H NMR, 13C NMR, and 19F NMR
spectra of analogs
 Molecular SMILES strings and MIC values
Author Information:
 Corresponding Authors:
o Email: hsintim@purdue.edu
o Email: seleem@vt.edu
 ORCID
o Herman O. Sintim: 0000-0002-2280-9359
 Notes:
o The authors declare no competing financial interest.
Acknowledgments
Research reported in this publication was supported by Purdue University, the National Institute
of Allergy And Infectious Diseases of the National Institutes of Health under Award Numbers
T32AI148103 and R01AI130186.
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Abbreviations:
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ATCC
BHIS
BOP
American type culture collection
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brain heart infusion supplemented
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate
Clostridium difficile infection
clinical and laboratory standards institute
diisopropylethylamine
CDI
CLSI
DIPEA
DMEM
DMF
DMSO
FBS
dulbecco’s modified eagle medium
dimethylformamide
dimethyl sulfoxide
fetal bovine serum
H2O
water
MeOH
MIC
methanol
minimum inhibitory concentration
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3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium)
MTS
7
8
9
NaOAc
PAINS
PBS
sodium acetate
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pan assay interference compounds
phosphate buffered saline
room temperature
RT
TSA
tryptic soy agar
TSB
tryptic soy broth
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