Structure-Activity Relationship for the Picolinamide Antibacterials that Selectively Target Clostridioides difficile

Article abstract of DOI:10.1021/acsmedchemlett.1c00135

Clostridioides difficile is a leading health threat. This pathogen initiates intestinal infections during gut microbiota dysbiosis caused by oral administration of antibiotics. C. difficile is difficult to eradicate due to its ability to form spores, which are not susceptible to antibiotics. To address the urgent need for treating recurrent C. difficile infection, antibiotics that selectively target C. difficile over common gut microbiota are needed. We herein describe the class of picolinamide antibacterials which show potent and selective activity against C. difficile. The structure-activity relationship of 108 analogues of isonicotinamide 4, a compound that is equally active against methicillin-resistant Staphylococcus aureus and C. difficile, was investigated. Introduction of the picolinamide core as exemplified by analogue 87 resulted in exquisite potency and selectivity against C. difficile. The ability of the picolinamide class to selectively target C. difficile and to prevent gut dysbiosis holds promise for the treatment of recurrent C. difficile infection.

Full text of DOI:10.1021/acsmedchemlett.1c00135

pubs.acs.org/acsmedchemlett
Letter
Structure−Activity Relationship for the Picolinamide Antibacterials
that Selectively Target Clostridioides difficile
Enrico Speri, Yuanyuan Qian, Jeshina Janardhanan, Cesar Masitas, Elena Lastochkin,
Stefania De Benedetti, Man Wang, Valerie A. Schroeder, William R. Wolter, Allen G. Oliver,
Jed F. Fisher, Shahriar Mobashery,* and Mayland Chang*
Cite This: ACS Med. Chem. Lett. 2021, 12, 991−995
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ABSTRACT: Clostridioides difficile is a leading health threat. This
pathogen initiates intestinal infections during gut microbiota
dysbiosis caused by oral administration of antibiotics. C. difficile is
difficult to eradicate due to its ability to form spores, which are not
susceptible to antibiotics. To address the urgent need for treating
recurrent C. difficile infection, antibiotics that selectively target C.
difficile over common gut microbiota are needed. We herein
describe the class of picolinamide antibacterials which show potent
and selective activity against C. difficile. The structure−activity
relationship of 108 analogues of isonicotinamide 4, a compound that is equally active against methicillin-resistant Staphylococcus
aureus and C. difficile, was investigated. Introduction of the picolinamide core as exemplified by analogue 87 resulted in exquisite
potency and selectivity against C. difficile. The ability of the picolinamide class to selectively target C. difficile and to prevent gut
dysbiosis holds promise for the treatment of recurrent C. difficile infection.
KEYWORDS: Clostridioides difficile, recurrent C. difficile infection, picolinamide, antibacterial, microbiota
agents that selectively target C. difficile are needed to tackle
persistent and recurrent CDI.
Clostridioides (formerly known as Clostridium) difficile is a Gram-
positive anaerobic bacterium which causes intestinal infections
that can lead to diarrhea, sepsis, and life-threatening colitis.1−3
Since the outbreak of the hypervirulent strain NAP1/027 in
2000,4,5 the incidence of C. difficile infections (CDIs) continues
to trend upward.6 In 2017 alone, CDI resulted in 223 900
hospitalizations and 12 800 deaths in the United States, with
nearly $6.3 billion of estimated healthcare costs.7,8 A challenge
in the treatment of CDI is the formation of spores that are not
susceptible to antibiotics, resulting in 24−27% recurrence of
CDI.9,10
CDI is currently treated with metronidazole (MTZ),
vancomycin (VAN), and fidaxomicin (FDX), with VAN and
FDX recommended for initial CDI and MTZ used only in mild
cases where VAN or FDX is unavailable. Furthermore, MTZ is
no longer recommended for repeated or prolonged CDI
treatment due to neurotoxicity.11 The failure rates of these
antibiotics are 22.4%, 14.2%, and 12%, respectively.9,12 In
addition, treatment of CDI with MTZ and VAN promotes
emergence of vancomycin-resistant enterococci.13 The pertur-
bation of gut microbiota (gut dysbiosis) caused by antibiotics
allows for C. difficile colonization.14 While VAN and FDX have
similar clinical cure efficacy, patients treated with FDX had lower
recurrence (15.4% for FDX compared to 25.3% for VAN),
which was attributed to the higher selectivity of fidaxomicin
toward C. difficile than to gut microbiota.15 New antibacterial
We discovered the isonicotinamide antibacterial 4 during the
structure−activity relationship (SAR) exploration on the
cinnamonitrile class of antibiotic adjuvants (compounds 1 and
2, Figure 1) against methicillin-resistant Staphylococcus aureus
(MRSA).16 Cinnamonitrile 1, a protein kinase(s) inhibitor,
showed promise as an adjuvant for β-lactam antibiotics and also
exhibited modest antibacterial activity.17 Since azoles are
common among protein kinase inhibitors,17,18 the oxazole was
introduced as a favored moiety (compound 3, Figure 1). Upon
insertion of the oxazole, the resulting series of molecules showed
respectable Gram-positive antibacterial activity (minimum
inhibitory concentration (MIC) ≤ 4 μg·mL−1 against MRSA
strain NRS70). Further modifications of 3 (amide linker,
pyridine core, and removal of the bridging oxygen) led to the
potent isonicotinamide antibacterial 4.
Compound 4 was evaluated against the ESKAPE panel
(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumo-
Received: March 5, 2021
Accepted: May 3, 2021
Published: May 7, 2021
https://doi.org/10.1021/acsmedchemlett.1c00135
© 2021 American Chemical Society
ACS Med. Chem. Lett. 2021, 12, 991−995
991

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Letter
Table 1. Activity of Picolinamides Analogues against MRSA
and C. difficile (SAR3 and SAR4)
Figure 1. Evolution of the structural template of the cinnamonitrile
class of potentiators into the isonicotinamide antibacterial 4.
Figure 2. Left, the four sites of structure modification for the SAR study
as highlighted by the boxes. Right, the structure of picolinamide 5.
Scheme 1. Synthetic Routes for Accessing (A) Biaryl and (B)
Biarylether Derivatives
niae, Acinetobacter baumannii, Pseudomonas aeruginosa, and
Enterobacter species). These bacteria account for the majority of
human problematic infections.19,20 The compound was active
only against the Gram-positive members of the panel (E. faecium
and S. aureus). We further evaluated 4 against C. difficile ATCC
43255, also a Gram-positive bacterium, and obtained an MIC of
0.25 μg·mL−1. These results provided the impetus for an SAR
investigation around this structural template. We report herein
the preparation and evaluation of 108 analogues of compound 4
(Figure 2), of which the prototype picolinamide 5 was reported
recently.21 As will be outlined, several of these analogues
exhibited exceptional activities against C. difficile, both in
potency of the antibiotics and in selectivity toward the organism.
The aforementioned selectivity in targeting C. difficile is
extremely important, as it would avoid gut dysbiosis and
mitigates recurrent infections, which are at the root of the bad
outcome in the clinical setting.
The initial compound of interest, 4, was prepared in three
steps following the general Scheme 1A. The first step was a
Suzuki-Miyaura coupling reaction22 between the chloro-
cyanopyridine and arylboronic acid 6, using Pd(PPh3)4 as
catalyst and potassium carbonate as a base, yielding the cyano-
derivative 7. Alkaline hydrolysis of the nitrile gave the carboxylic
acid 8. A coupling reaction between 8 and 1,3-oxazol-2-amine
a
b
NRS70 strain, resistant to erythromycin and spectinomycin. ATCC
c
43255, ribotype 087. Selectivity is defined as MICMRSA/MICC.difficile
.
d
Reported previously21 and included for comparison.
(or other amines) using propanephosphonic anhydride (T3P)23
as the coupling reagent led to derivatives 9. The preparation of
compounds possessing the diarylether moiety followed a similar
synthetic route, but using an initial nucleophilic aromatic
substitution of a chloro-cyanopyridine with a phenol derivative
(Scheme 1B). We used the general synthetic route to derivative
1321 for syntheses of compounds 112−119.
The syntheses produced 108 analogues corresponding to
diversification at four structural regions (Figure 2, left structure
with four different colored boxes). A representative C. difficile
strain (ATCC43255) along with MRSA strain NRS70 were
chosen for MIC evaluation. Compounds with MICs of ≤8 μg·
mL−1 against C. difficile were considered active.
SAR1 involved modifications of the oxazole ring in lead
compound 4, a main substructure that defines the antibacterial
activity of the isonicotinamides. Compounds 14−31 were
prepared. The majority of these compounds retained activity
against both bacteria, albeit with poor selectivity (Table S1).
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Table 2. XTT IC50 of Selected Picolinamides Analogues with
HeLa Cells
Table 4. Drug Concentrations in Fecal Samples of Selected
Compounds
IC50 (μg·mL−1
)
MIC,
dose,
μg g−1
conc. feces/
MIC
a
b
μg·mL−1
selectivity
mg·kg−1
feces
4
5
87
89
90
92
93
94
79.9 1.0
179.3 3.6
a
4
5
87
107
108
116
0.25
0.125
0.125
1
1
0.50
1
128
1024
≥256
64
20
20
10
20
20
20
2.8
13
0.79
2.4
49
11
108
6.6
2.4
49
c
95.2 1.0
170.2 2.5
b
NT at 64
b
NT at 256
NT at 64
b
128
3.7
7.4
a
b
C. difficile strain, ATCC 43255, ribotype 087. Selectivity is defined
as MICMRSA/MICC. difficile. Reported previously; included for ease of
comparison.
40.1 1.6
135 8.6
114.8 2.2
37.0 1.0
105.9 2.1
88.0 2.3
93.3 1.7
77.4 2.3
91.6 1.3
53.3 3.6
c
97
98
99
106
107
108
109
110
114
116
118
119
Moreover, compounds 87−98 lacked activity against MSSA and
E. faecium (Table S3).
Introduction of an ether linkage between the pyridine core
and the phenyl ring resulted in MIC values for C. difficile of ≤1
μg·mL−1. The picolinamides (106−111, Table 1) had
significantly higher selectivity for C. difficile than the
isonicotinamides (99−105, Table S2). Unfortunately, the
introduction of the ether linkage did not increase the
antibacterial activity of the picolinamides. These SAR studies
led us to introduce a carboxylate, either as a salt or as an ester
(compounds 112-119), which improved water solubility of the
family while still maintaining potent activity against C. difficile
(0.12 μg·mL−1 for 5 and 0.50 μg·mL−1 for 116).
The XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-
carboxanilide) assay24 with HeLa cells was performed on
representative compounds (Table 2). Most of the compounds
tested had IC50 values >64 μg·mL−1 (except for picolinamides
94 and 114, and isonicotinamide 99). Compound 87 had an
IC50 of 95.2 1.0 μg·mL−1, 760-fold higher than its MIC (0.125
μg·mL−1).
b
NT at 128
79.9 6.3
123.6 3.3
Reported previously21 and included here for comparison. NT, no
detectable toxicity at the specified concentration.
a
b
The SAR study then focused on optimizing the phenyl ring
(SAR3) with respect to the central pyridine ring (SAR2), while
leaving the oxazole group intact. Most of the compounds
containing isonicotinamide (32−74, Table S2) were active
against C. difficile, however the selectivity between MRSA and C.
difficile was <16, except for the nitro-containing analogues (49−
50). The 2,6-substituted picolinamides (75−80) were inactive.
The most compelling modification was 2,4-substitution of the
picolinamide (87−98). This substitution imparted the desired
selectivity toward C. difficile compared to MRSA. Picolinamide
87, a constitutional isomer of compound 4, was inactive against
MRSA (MIC = 128 μg·mL−1) yet active against C. difficile (MIC
= 0.125 μg·mL−1). This exquisite selectivity of 1024-fold was
achieved by the mere repositioning of one nitrogen atom.
We evaluated the activity of the picolinamides against the
common gut bacteria: Bacteroides fragilis, Bifidobacterium
longum, Corynebacterium spp., Fusobacterium nucleatum, Lacto-
bacillus reuteri, Lactobacillus gasseri, Veillonella sp., and
Eubacterium sp.25−30 An optimum antibacterial will target
selectively the pathogenic bacterium and not the natural gut
Table 3. MIC Values (μg·mL−1) for MTZ, VAN, FDX, and Compounds 4, 5, 87, and 108 against Major Gut Bacteria
a
MTZ
VAN
FDX
4
5
87
108
C. difficile ATCC43255
0.25
1
1
>32
2
>32
>32
2
0.5
16
0.25
0.5
0.25
>32
1
>32
2
0.0625
>32
<0.01
<0.0625
<0.0625
>32
2
8
0.25
1
>16
0.5
8
8
>16
4
>16
0.125
4
16
8
4
4
>16
16
16
0.125
4
>16
16
8
4
>16
16
8
1
8
16
>16
8
a
Bacteroides fragilis
Bifidobacterium longum
Corynebacterium spp.
b
c
d
Fusobacterium nucleatumi
e
Lactobacillus reuteri
4
f
Lactobacillus gasseri
>16
16
8
g
Veillonella sp.
h
Eubacterium sp.
1
16
a
Strain HM-709, a Gram-negative, anaerobic bacterium that is commensal and critical to host immunity and a minor component of the human gut
b
microflora (<1%). Strain HM-846, an anaerobic, nonsporulating Gram-positive bacterium commonly found in the normal human intestinal
microflora isolated from human feces. Strain HM-784, an aerobic or facultatively anaerobic Gram-positive bacterium that occurs in the mucosa
and normal skin flora of humans and animals. Strain HM-992, an anaerobic, nonsporulating Gram-negative bacterium commonly found in the
gastrointestinal tract. Strain HM-102, an anaerobic Gram-positive bacterium commonly found in the normal human gastrointestinal tract, and
c
d
e
f
used frequently as a probiotic to maintain the balance of gut microbial flora. Strain HM-644, a facultative, anaerobic Gram-positive bacterium
commonly found in the normal human gastrointestinal tract, used frequently in yogurt production as a probiotic to suppress Helicobacter pylori
g
h
infections. Strain HM-49, a nonsporulating Gram-negative bacterium commonly found in the intestinal tract of humans and animals. Strain HM-
178, an anaerobic, nonsporulating Gram-positive bacterium commonly found in the gastrointestinal flora of humans and animals.
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Letter
flora. The selectivity (MICgutbacteria/MICC.difficileATCC43255) ranged
from 4 to 128 for MTZ, 0.5 to 64 for VAN, 1 to 512 for FDX, 2 to
64 for 4, 32 to 128 for 5, 32 to 128 for 87, and 8 to 16 for 108
(Table 3). Picolinamides 5 and 87 were more selective than 4
and more selective than the clinically used antibiotics VAN,
MTZ, and FDX. The selectivity for C. difficile ATCC43255 with
respect to Bifidobacterium longum, a major gut bacterium that is
reported to help repress CDI,27,31 was 128 for 87 compared to 4
for MTZ, 0.5 for VAN, and 0.16 for FDX.
Recurrence of CDI for the less selective VAN is 25%, while
that for the somewhat more selective FDX is 15%.15
Picolinamide 87 is >1000-fold selective. As gut dysbiosis
contributes to CDI recurrence, the importance of this high
selectivity is self-evident. Selectivity in targeting C. difficile
emerges as an important attribute as mere potency (low MIC)
might become less significant given that a compound such as 87
has already a remarkably very low/potent MIC of 0.125 μg·
mL−1 (nanomolar range).
For the treatment of CDI, compounds that are poorly
absorbed or not absorbed and achieve high concentrations in the
gut are desirable. We selected compounds 4, 87, 107, 108, and
116 for in vivo PK studies in mice based on their potency, safety
profile, solubility, and selectivity against C. difficile. Both plasma
and feces were collected and analyzed for levels of the
compounds and compared to those of compound 5. While all
the compounds showed nonquantifiable concentrations in
plasma, levels in feces were higher (Table 4). The selected
compounds showed 2- to 50-fold higher concentrations in the
feces over the MIC values.
The lack of selective antibiotics for the treatment of CDI
contributes to gut dysbiosis and recurrence of CDI. The
picolinamide family of antibacterials shows exquisite potency
and selectivity in targeting C. difficile. Starting with isonicotina-
mide 4, a compound that is equally active against MRSA and C.
difficile, structure optimization gave the new picolinamide 87
with >1000-fold selectivity for C. difficile compared to MRSA
(strain NRS70). Compound 87 shows exceptional selectivity
against C. difficile while lacking activity against other, normal
Gram-positive and Gram-negative gut microbiota. As gut
dysbiosis contributes to CDI recurrence, the picolinamides
have the potential for treatment of recurrent CDI.
Phone: 574-631-2933; Email: mobashery@nd.edu;
Fax: 574-631-6652
Mayland Chang − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46556,
United States; orcid.org/0000-0002-4333-3775;
Phone: 574-631-2965; Email: mchang@nd.edu; Fax: 574-
631-6652
Authors
Enrico Speri − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46556,
United States
Yuanyuan Qian − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46556,
United States
Jeshina Janardhanan − Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame, Indiana
46556, United States
Cesar Masitas − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46556,
United States
Elena Lastochkin − Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame, Indiana
46556, United States
Stefania De Benedetti − Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame, Indiana
46556, United States
Man Wang − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46556,
United States
Valerie A. Schroeder − Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame, Indiana
46556, United States
William R. Wolter − Freimann Life Sciences Center, University
of Notre Dame, Notre Dame, Indiana 46556, United States
Allen G. Oliver − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46556,
United States
Jed F. Fisher − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46556,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00135
ASSOCIATED CONTENT
■
sı
* Supporting Information
Author Contributions
The manuscript was written through contributions of all
authors. All authors approve the manuscript.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00135.
General synthetic procedures; synthetic experimental
procedures and characterization data; X-ray data
collection method; crystal data and structure refinement
for 87; general biological methods; activity of picolina-
mide analogues against C. difficile and MRSA, MSSA
(ATCC 29213), and E. faecium (NCTC 7171); PK
studies; NMR spectra for representative compounds; and
molecular formula strings for the synthesized compounds
(PDF)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institute of Allergy and
Infectious Diseases Grants AI116548 (to M.C.) and AI104987
(to S.M.). E.S. was the recipient of an ECK Institute for Global
Health Graduate Student Fellowship and of a Berry Family
Foundation Graduate Fellowship in Advanced Diagnostics &
Therapeutics. Y.Q. is also a recipient of an ECK Institute for
Global Health Graduate Student Fellowship and was a fellow of
the Chemistry-Biochemistry-Biology Interface Program (Train-
ing Grant T32GM075762 from the National Institute of
General Medical Sciences) at the University of Notre Dame.
The normal gut bacterial strains and the C. difficile strains were
AUTHOR INFORMATION
■
Corresponding Authors
Shahriar Mobashery − Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame, Indiana
46556, United States; orcid.org/0000-0002-7695-7883;
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Letter
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provided by BEI Resources. The antibiotic-resistant C. difficile
strains were generously provided by Dr. Curtis J. Donskey at the
Cleveland Veterans Affairs Medical Center, Case Western
Reserve University and Drs. Ellie J. C. Goldstein and Diane M.
Citron at the R. M. Alden Research Laboratory.
ABBREVIATIONS
■
C. difficile, Clostridioides difficile; CDI, C. difficile infection;;
ClogP, calculated logP value; FDX, fidaxomicin; MDZ,
metronidazole; MRSA, methicillin-resistant Staphylococcus
aureus; MSSA, methicillin-sensitive S. aureus; Pd(PPh3)4,
tetrakis(triphenylphosphine)palladium(0); PK, pharmacoki-
netics; SAR, structure−activity relationship; T3P, propylphos-
phonic anhydride; TLC, thin layer chromatography; VAN,
vancomycin; XTT, (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-
2H-carboxanilide).
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