Chemical Space Exploration around Thieno[3,2- d]pyrimidin-4(3 H)-one Scaffold Led to a Novel Class of Highly Active Clostridium difficile Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01198

Clostridium difficile infection (CDI) is the leading cause of healthcare-associated infection in the United States. Therefore, development of novel treatments for CDI is a high priority. Toward this goal, we began in vitro screening of a structurally diverse in-house library of 67 compounds against two pathogenic C. difficile strains (ATCC BAA 1870 and ATCC 43255), which yielded a hit compound, 2-methyl-8-nitroquinazolin-4(3H)-one (2) with moderate potency (MIC = 312/156 μM). Optimization of 2 gave lead compound 6a (2-methyl-7-nitrothieno[3,2-d]pyrimidin-4(3H)-one) with improved potency (MIC = 19/38 μM), selectivity over normal gut microflora, CC₅₀s > 606 μM against mammalian cell lines, and acceptable stability in simulated gastric and intestinal fluid. Further optimization of 6a at C2-, N3-, C4-, and C7-positions resulted in a library of >50 compounds with MICs ranging from 3 to 800 μM against clinical isolates of C. difficile. Compound 8f (MIC = 3/6 μM) was identified as a promising lead for further optimization.

Full text of DOI:10.1021/acs.jmedchem.9b01198

Subscriber access provided by Caltech Library
Article
Chemical Space Exploration Around Thieno[3,2-d]pyrimidin-4(3H)-one
Scaffold led to a Novel Class of Highly Active Clostridium difficile Inhibitors
Xuwei Shao, Ahmed AbdelKhalek, Nader S. Abutaleb, Uday Kiran
Velagapudi, Sabesan Yoganathan, Mohamed N. Seleem, and Tanaji T Talele
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01198 • Publication Date (Web): 04 Oct 2019
Downloaded from pubs.acs.org on October 6, 2019
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 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Chemical Space Exploration Around Thieno[3,2-
d]pyrimidin-4(3H)-one Scaffold led to a Novel Class
of Highly Active Clostridium difficile Inhibitors
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†
‡
‡
†
Xuwei Shao , Ahmed AbdelKhalek , Nader S. Abutaleb , Uday Kiran Velagapudi , Sabesan
Yoganathan , Mohamed N. Seleem *,‡,, Tanaji T. Talele*,†
†
†
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's
University, Queens, New York 11439-0001, United States
‡
Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine,
West Lafayette, Indiana 47907-2027, United States

Purdue Institute of Inflammation, Immunology, and Infectious Disease, West Lafayette,
Indiana 47907-2027, United States
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT: Clostridium difficile infection (CDI) is the leading cause of healthcare-associated
infection in the United States. Therefore, development of novel treatments for CDI is a high
priority. Toward this goal, we began in vitro screening of a structurally diverse in-house library of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7 compounds against two pathogenic C. difficile strains (ATCC BAA 1870 and ATCC 43255),
which yielded a hit compound, 2-methyl-8-nitroquinazolin-4(3H)-one (2) with moderate potency
MIC = 312/156 µM). Optimization of 2 gave lead compound 6a (2-methyl-7-nitrothieno[3,2-
(
d]pyrimidin-4(3H)-one) with improved potency (MIC = 19/38 µM), selectivity over normal gut
microflora, CC s >606 µM against mammalian cell lines, and acceptable stability in simulated
5
0
gastric and intestinal fluid. Further optimization of 6a at C2-, N3-, C4- and C7-positions resulted
in a library of >50 compounds with MICs ranging from 3 – 800 µM against clinical isolates of C.
difficile. Compound 8f (MIC = 3/6 µM) was identified as a promising lead for further optimization.
INTRODUCTION
Clostridium difficile is a Gram-positive spore-forming anaerobic bacterium that causes diarrhea
and serious intestinal conditions. Toxigenic strains of C. difficile produce two glycosylating toxins:
toxin A (TcdA/enterotoxin) and toxin B (TcdB/cytotoxin), both of which initiate damage of the
colon, life-threatening inflammation of the gut (C. difficile colitis), and a spectrum of intestinal
pathologies ranging from mild diarrhea to pseudomembranous colitis in the infected host.1-3 The
CDI, confined to the gastrointestinal tract, is usually triggered by the use of antibiotics, which
disturbs the reproduction of normal and protective gut microflora allowing C. difficile to proliferate
4
in the colon and to produce toxins. According to Centers for Disease Control and Prevention
(CDC), about half a million cases of CDI occur each year in the US hospitals and long-term health
care facilities with an estimation of 29,000 deaths. The prevalence and severity of CDI appear to
be rising, partly due to a larger elder population with high risk factors, an increasing use of
ACS Paragon Plus Environment

Page 3 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
antibiotics, a higher proportion of hypervirulent bacterial isolates with increased production of
5
6
lethal toxins A and B, and the emergence of hypervirulent epidemic strains (BI/Nap1/027).
Currently available therapeutics (metronidazole, vancomycin and fidaxomicin) for CDI are
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
inadequate in efficacy and/or tolerability.3
Both metronidazole and vancomycin treatments encounter substantial disease relapse.7
Metronidazole, an antibiotic with activity against a wide spectrum of anaerobic bacteria and
parasites, is only recommended for the treatment of mild-to-moderate episodes and is inferior to
8
vancomycin. Moreover, it is essentially 100% bioavailable leading to limiting concentrations in
9
the colon, the prime location of CDI. Unlike metronidazole, vancomycin is minimally absorbed
into the systemic circulation upon oral administration, thereby resulting in a high concentration in
10
the colon. However, its broad spectrum of action against Gram-positive bacteria leads to a
reduced microbiome diversity and the potential selection of vancomycin-resistant enterococci.11
In addition, recurrent infection caused by newer and stronger C. difficile strains is a formidable
12
preclinical challenge. Both metronidazole and vancomycin treatments can worsen the condition
of patients due to the loss of beneficial gut microbiota, and subsequent recurrence at an alarming
rate. Selectively targeting C. difficile over normal gut flora has been considered as a strategy to
achieve prevention of recurrence.13 Compared with vancomycin, fidaxomicin (a macrolide
antibiotic) demonstrates a narrower spectrum of activity and selectivity towards C. difficile;
however, it does not greatly improve sustained clinical responses especially against hypervirulent
strains BI/NAP1/027.14 In view of the transient efficacy of these antibiotics, particularly of
metronidazole and vancomycin, patients are predisposed to ~25% relapse rate as compared to 15%
for fidaxomicin and a subsequent prolongation of C. difficile shedding and transmission.15, 16
Although fidaxomicin treatment showed significantly lower rates of CDI recurrence compared to
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
metronidazole and vancomycin, it does so only in non-NAP1 CDI patients. In addition, clinical
1
5
resistance to fidaxomicin has already been documented. Although 93% of fidaxomicin remains
unabsorbed after oral administration, it is detectable in the range of 25-50 ng/mL in the plasma of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
17
patients, which leads to a serious concern of potent cytotoxic effect. Moreover, the cost of
fidaxomicin treatment is prohibitively expensive partly due to complexity in synthesizing a large
molecule with molecular weight beyond 1000 Da. Although ridinilazole (NCT02784002) is
currently undergoing clinical trials for the treatment of CDI, it remains to be seen whether it would
1
8
offer any benefit over current treatment. Despite unmet medical need, progress toward anti-C.
difficile drug development has been very limited.19-23 Therefore, the discovery of new “best-in-
class” drugs to fight against C. difficile is needed to adequately address CDI.
To identify highly selective novel C. difficile inhibitors, we conducted whole-cell screening of
a set of 67 in-house compounds (see Supporting Information, Table S1), comprising diverse
structural classes (valine-, proline-, phenylalanine-, and tyrosine-derived thiazole peptidomimetics
and quinazolinones, benzoxazines, indazoles, benzodioxines, imidazopyridines, and
benzodioxepines) with molecular weights (MW) ranging from 164 to 652 Da. This screening
method allowed us to ensure penetration of the C. difficile cell membrane as well as to obtain
minimum inhibitory concentrations (MICs) as biological readouts to identify promising hit
compounds. Each scaffold in the library had a sufficient number of structurally close analogues to
produce robust results while capturing key SAR trends. This screening test identified two
2
4
previously reported quinazolinone analogues as hit compounds, 6-nitroquinazolin-4(3H)-one (1,
MIC = 335/335 µM, hereafter, against two C. difficile clinical strains; ATCC BAA 1870/ATCC
4
3255) and 2-methyl-8-nitroquinazolin-4(3H)-one (2, MIC = 312/156 µM) (3% success rate) that
showed moderate to weak MICs (Figure 1). In vitro MIC values were compared with three FDA
ACS Paragon Plus Environment

Page 5 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
approved drugs, vancomycin, metronidazole and fidaxomicin. Compound 2 was established as a
promising fragment hit for further medicinal chemistry optimization because of its selectivity
profile toward multiple clinical strains of C. difficile over human normal microflora (MIC >1248
µM) such as Lactobacillus, Bifidobacterium, Escherichia coli, and Enterobacter cloacae) (Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
). Medicinal chemistry optimization of hit 2 via analogue synthesis produced compounds 3-5
with a loss of potency suggesting the contribution of the C8-nitro substituent. Next, we decided to
implement scaffold hopping strategy, which led to identification of small MW scaffolds 6a (MIC
=
19/38 µM), 6b (MIC = 41/41 µM) and 6c (MIC = 38/38 µM). Compound 6a was prioritized for
further SAR study based on its structural novelty, ease of synthetic derivatization, favorable
potency, selectivity, and in vitro mammalian cell toxicity as shown in Figure 1. Herein, we report
the synthesis, antibacterial screening, bactericidal investigation, cytotoxicity, and physicochemical
property evaluations of analogues based on a newly identified lead scaffold 6a as C. difficile
inhibitors.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Antimicrobial profile of HTS hits 1-2, hit analogues 3-5, and scaffold hopping
compounds 6a-6c along with positive standards and the plan for chemical space exploration of 6a-
6
c. VAN = vancomycin; FDX = fidaxomicin and MTZ = metronidazole.
RESULTS AND DISCUSSION
Chemistry. Fused pyrimidinone derivatives were synthesized starting from amino and methyl
ester substituted aromatic/heteroaromatic intermediates (Scheme 1). Acetylation of commercially
available chemicals 14-16 and 20-22 was performed using acetic anhydride, and subsequent
treatment of intermediates with ammonium hydroxide to obtain compounds 2-4 and 7a.24
2
5
Hydrogenation of the nitro group of 2 yielded 5. The reaction conditions used for the preparation
of 7b and 7c required an alternate strategy due to varying reactivity of starting materials. As
illustrated, intermediates 24b and 24c were prepared by the same procedure that was used for the
synthesis of compound 2 and heated in a mixture of NaOH, and aqueous methanol to obtain the
ACS Paragon Plus Environment

Page 7 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
26
cyclized compounds 7b and 7c. The nitro group was introduced on 7a-7c by treating with fuming
nitric acid and concentrated sulfuric acid mixture to obtain nitro derivatives 6a-6c.27
Scheme 1: Synthesis of Fused Pyrimidinone Derivativesa
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
OCH3
a
O
b
NH
X
NH2
X
N
X
N
14-16
17-19
2: X=C-NO2
3: X=N
4: X=C-F
1
4, 17: X=C-NO2
15, 18: X=N
16, 19: X=C-F
c
5: X=C-NH2
O
O
N
O
S
S
b
NH
NH
O
O
O
N
2
3a
7a
O
a
e
NH
A
A
O
O
NH2
O
O
NH2
20-22
A
A
6a-6c
b
d
NH
N
A
NH
NH
O
O
2
3b-23c
24b-24c
7b-7c
S
S
20: A=
6a: A=
O N
2
O
O
21, 23b, 24b, 7b: A=
6b: A=
O N
2
S
6c: A=
2
2, 23c, 24c, 7c: A= S
O N
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aReagents and conditions: (a) acetic anhydride, 14 (for 17), 15 (for 18), 16 (for 19), 20 (for 23a),
2
2
7
1 (for 23b), 22 (for 23c), rt, 12-24 h, 65-85%; (b) 30% NH OH, 17 (for 2), 18 (for 3), 19 (for 4),
4
3a (for 7a), 23b (for 24b), 23c (for 24c), rt, 6-8 h, 62-70%; (c) H /Pd/C, MeOH, 50 psi, rt, 8 h,
2
0%; (d) Aq. NaOH, MeOH, 24b (for 7b), 24c (for 7c), reflux, 4-6 h, 35-40%; (e) HNO , conc.
3
H SO , 0 °C - rt, 4-12 h, 40-82%.
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To investigate the role of C2-methyl group, 7d-7g with C2-H, -ethyl, -propyl, and -phenyl
substituents were prepared from 20 and 23e-23g, respectively.28, 29 The nitrated derivatives 6d-6f
2
7
were prepared from 7d-7f by following the same method used for the preparation of 6a. Target
compound 6g was obtained from nitration of 7g wherein two nitro groups (C7 of the bicyclic
scaffold and meta-position of the C2-phenyl substituent) were introduced in the same reaction. To
analyze the role of C7-nitro group, compounds with C7-methyl group (7h and 7i) were synthesized
from commercially available 25 over two-steps for 7h and one-step for 7i (Scheme 2).
Scheme 2: Synthesis of Thienopyrimidinone Derivativesa
ACS Paragon Plus Environment

Page 9 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
N
O
S
S
NH
NH
a
c
b
N
O
7
d
O2N
6
d
S
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
O
NH2
23e, 7e, 6e: R = C H
2 5
O
S
S
S
NH
20
NH
d
b
2
3f, 7f, 6f: R = C H
NH
3 7
R
N
R
O N
2
O
R
,
23g 7g
: R = phenyl
2
3e-23g
7e-7g
6e-6g
6g: R = 3-nitrophenyl
O
O
O
S
d
e
S
NH
NH
O
N
O
S
O
23h
7h
NH2
O
N
25
S
NH
a
7i
a
Reagents and conditions: (a) formamide, rt, 6-8 h, 60-65%; (b) HNO , conc. H SO , 7e (for 6e),
3
2
4
7
f (for 6f), 7g (for 6g), 0 °C - rt, 6-8 h, 50-70%. (c) triethylamine, DCM, propionyl bromide (for
23e), butanoyl bromide (for 23f), benzoyl chloride (for 23g), rt, 4-8 h, 80-85%; (d) 30% NH OH,
4
2
3e (for 7e), 23f (for 7f), 23g (for 7g), 23h (for 7h), rt, 6-8 h, 28-60%; (e) acetic anhydride, rt, 14
h, 75%.
To pursue more potent compounds, lead 6a was condensed with (un)substituted
aromatic/heteroaromatic aldehydes using microwave conditions to obtain C2-styryl derivatives
a-8n (Scheme 3). 30, 31
8
Scheme 3: Synthesis of C2-Styryl Derivatives of Thienopyrimidinone Corea
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
O
S
S
NH
a
NH
N
R
O N
O N
2
2
6a
8a-8n
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
j: R=
8
a: R=
CN
Cl
8
f: R=
F
O
S
8
b: R=
8k: R=
8l: R=
F
8
g: R=
h: R=
i: R=
8
c: R=
d: R=
F
8
8
8
m: R=
8
OH
O
COOH
COOH
O
NO2
8n: R=
8
e: R=
aReagents and conditions: (a) benzaldehyde (for 8a), furfural (for 8b), 2-
thiophenecarboxaldehyde (for 8c), 4-formylbenzoic acid (for 8d), 5-formyl-2-furoic acid (for 8e),
-fluorobenzaldehyde (for 8f), 2-fluorobenzaldehyde (for 8g), 3-fluorobenzaldehyde (for 8h), 4-
nitrobenzaldehyde (for 8i), 4-cyanobenzaldehyde (for 8j), 4-chlorobenzaldehyde (for 8k), 4-
ethynylbenzaldehyde (for 8l), 4-hydroxylbenzaldehyde (for 8m), 4-methoxybenzaldehyde (for
n), AcOH, MW, 180 °C, 5-6 h, 20-37%.
4
8
A wide range of substituents were installed at N3-position of 6a as shown in Scheme 4 to
generate a library of N3-alkylated analogues. For example, 9a-9h were prepared by reacting 6a
with various (un)substituted phenylalkyl halides in the presence of potassium carbonate in DMF.32
To avoid complexity associated with multiple nitrated products during the synthesis of target
compounds 10a-10d, we began the synthetic sequence with a selective nitration of the thiophene
substrate 23a. Thus, intermediate 23a was nitrated using concentrated sulfuric acid and fuming
nitric acid to produce 26. Intermediate 26 was hydrolyzed in the presence of lithium hydroxide to
ACS Paragon Plus Environment

Page 11 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
yield 27, which was subsequently cyclized using acetic anhydride under reflux condition to obtain
isatoic anhydride-like intermediate 28. Intermediate 28 was then condensed with substituted
3
0
anilines to produce target compounds 10a-10d. To prepare analogues with N3-ester (11a and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1b), -carboxylic acid (11c), and -amide (11d-11f) substituents, 6a was subjected to N-alkylation
using respective halides in the presence of potassium carbonate and DMF to yield 11a and 11b.32
The ester 11a was subjected to lithium hydroxide-mediated hydrolysis to obtain 11c, and the
resulting carboxylic acid was coupled with aromatic/aliphatic amines to yield 11d-11f.33
Scheme 4: Synthesis of N3-Substituted Derivatives of Thienopyrimidinone Corea
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
O
9a: n=1, R =H
1
S
S
9b: n=2, R =H
NH
a
N
n
1
R1
9
c: n=3, R =H
1
N
9d: n=1, R =4-NO
O N
O2N
1
2
2
9
9
9
9
e: n=1, R =2-NO
1
2
6a
9a-9h
f: n=1, R =3-NO
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
g: n=1, R =4-CN
1
h: n=1, R =4-OCH
1
3
O
O
O
O
O
R2
O
S
b
O
c
OH
d
e
S
S
S
S
N
O
NH
NH
NH
N
N
O N
2
O N
2
O N
O N
2
O
2
O
O
2
3a
26
27
28
10a-10d
1
1
1
1
0a: R =4-OMe
0b: R =3-COOH
0c: R =4-Isopropyl
0d: R =4-Phenyl
2
2
2
2
O
O
O
N
OH
R3
O
S
S
S
c
N
f
N
N
C H
2
5
O
O
O
N
N
O N
2
O N
2
O N
2
a
6a
11a
11c
11d-11f
H
O
N
N
1
1d: R3=
O
S
N
O
O N
2
O
N
1
1b
11e: R3=
11f: R3=
N
N
aReagents and conditions: (a) benzyl bromide (for 9a), (2-bromoethyl)benzene (for 9b), 1-
bromo-3-phenylpropane (for 9c), 4-nitrobenzyl bromide (for 9d), 2-nitrobenzyl bromide (for 9e),
-nitrobenzyl bromide (for 9f), 4-cyanobenzyl bromide, (for 9g), 4-methoxybenzyl bromide (for
h), ethyl bromoacetate (for 11a), tert-butyl 2-chloroacetate (for 11b), K CO , DMF, rt, 8-12 h,
42-60%; (b) HNO , conc. H SO , -40 °C - rt, 4 h, 41%; (c) LiOH, THF, H O, 26 (for 27), 11a (for
11c), rt, 8-10 h, 65-87%; (d) acetic anhydride, reflux, 3 h, 84%; (e) 4-methoxylaniline (for 10a),
-aminobenzoic acid (for 10b), 4-isopropylaniline (for 10c), 4-aminobiphenyl (for 10d), AcOH,
3
9
2
3
3
2
4
2
3
reflux, 6-8 h, 24-35%; (f) aniline (for 11d), morpholine (for 11e), N-methyl piperazine (for 11f),
EDC, HBTU, triethylamine, DCM, rt, 6h, 33-40%.
ACS Paragon Plus Environment

Page 13 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
As depicted in Scheme 5, compound 12a was prepared from the reaction of 6a with phosphorus
oxychloride. The C4-chloro group was then nucleophilically displaced by aniline or morpholine
under microwave condition to obtain 12b or 12c.34
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5: Synthesis of C4-Substituted Analogues of Thienopyrimidinone Corea
O
N
Cl
N
R
N
NH
N
S
S
S
12b: R=
NH a
N
b
N
O N
O N
O N
2
2
2
12c: R=
O
6
a
12a
12b-12c
aReagents and conditions: (a) phosphorus oxychloride, reflux, 18 h, 40%, (b) aniline (for 12b),
morpholine (for 12c), DMF, MW, 150 °C, 50-70 min, 35-45%.
Target compound 13a with C2- and N3-disubstitution was prepared from the reaction of 9a and
4
-fluorobenzaldehyde. The 2,4-disubstituted compound 13b was obtained by treating 8f with
phosphorus oxychloride, which upon nucleophilic substitution by morpholine led to 13c as shown
in Scheme 6.
Scheme 6: Synthesis of Disubstituted Analogues of Thienopyrimidinone Corea
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
O
S
N
S
N
a
O N
N
2
O N
2
9
a
13a
F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
O
N
Cl
S
S
S
NH
N
N
b
c
N
N
O N
O N
O N
2
2
2
F
F
F
8
f
13b
13c
aReagents and conditions: (a) 4-fluorobenzaldehyde, AcOH, MW, 180 °C, 4 h, 22%, (b)
phosphorus oxychloride, reflux, 18 h, 72%; (c) morpholine, DMF, MW, 150 °C, 1 h, 32%.
Elucidation of Regioisomers from Nitration Reaction. Variety of techniques, including X-ray
1
13
crystallography, 1-D ( H and C) and 2-D (HSQC and HMBC) NMR analyses, were employed to
determine the regioselectivity of products obtained from the nitration reaction. We hypothesized
that the position of the nitro group in representative compound 6a would be at C7 instead of C6.
This hypothesis was tested initially based on determination of the single X-ray crystal structure of
1
6
a-derived analogue 11b, and supported by H and 13C NMR chemical shifts analyses.
Unambiguous assignment of regioisomer 11b was accomplished by solving its single X-ray crystal
structure (Figure 2) (for the details of crystallography see Supporting Information, Table S2-S9).
The C6-thiophene proton chemical shift of 6a is 9.30 ppm and 11b is 9.36 ppm, and the thiophene
C6 chemical shift of 6a and 11b are 138.99 and 139.98, respectively. These similar chemical shifts
provide evidence for identical regioisomerism within compounds 6a and 11b.
ACS Paragon Plus Environment

Page 15 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Crystal structure of compound 11b.
There is no distinct sets of HMBC correlation present to differentiate the nitro group’s position
on the furan ring of compound 6b (see Supporting Information, Figure S1). It was noted that the
reactivity of the starting material 7b is similar to the sulfur-isostere 7a, from which compound 6a
was derived. Therefore, we believe that the nitro group is at 7-position in 6b similar to that
observed for 6a. The HMBC spectra of 7b illustrates that both C6 and C7 protons have same
number of HBMC correlations (see Supporting Information, Figure S2).
Both HSQC and HMBC experiments were performed to investigate correlations between the
carbons and protons within compound 7c, which was the starting material for the nitration reaction
to generate 6c. The proton chemical shifts of thiophene protons in 7c are assigned using the HSQC
experiment (see Supporting Information, Figure S3). The correlation between individual carbon
atom and the proton attached to it was evidenced by the HSQC spectrum of 7c (see Supporting
Information, Figure S3). Additional HMBC analysis of 7c shows four HMBC correlations for the
hydrogen at 5-position, and three HMBC correlations for the hydrogen at 7-position as expected
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
see Supporting Information, Figure S4). The HMBC spectrum of compound 6c shows four
HMBC correlations for a single thiophene proton (see Supporting Information, Figure S5). These
observations lead to the conclusion that the nitration reaction afforded the 7-nitro analogue as the
exclusive regioisomer.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For analogues 6d – 6g, which are structurally analogous to 6a, we observed that the thiophene
proton (9.31 – 9.41 ppm) and the carbon atom (138.89 – 139.21 ppm) with proton attached, have
1
13
very similar chemical shift values compared to compound 6a ( H 9.30 ppm, C 138.99 ppm)
indicating the presence of a nitro group at 7-position.
Structure-Activity Relationship. An in vitro antibacterial screening protocol was designed to
identify compounds with potent inhibitory activity toward C. difficile, and minimal effect on
normal human microflora and mammalian cells. To achieve this goal, we tested target compounds
against two pathogenic strains of C. difficile (ATCC BAA 1870 and ATCC 43255) as shown in
Tables 1-5. MIC values (µM and µg/mL) are used to describe antibacterial activities of the
compounds and used for the interpretation of SAR data. An initial SAR study was focused on
elucidation of the role of nitro substituent on hit-to-lead scaffolds 6a-6c toward C. difficile
inhibition as shown in Table 1. Des-nitro analogues of 6a-6c gave 7a (MIC >770/385 µM), 7b
(
MIC >852/852 µM), and 7c (MIC = 192/768 µM) with a substantial loss of anti-C. difficile
potency. Other des-nitro derivatives such as 7d (MIC = 420/840 µM), 7g (MIC >560/>560 µM),
h (MIC >710/>710 µM), and 7i (MIC >770/>770 µM) were also manifested with a loss of
7
potency. These results allowed us to conclude the essential role of a nitro substituent at the C7-
position. Next, we decided to elucidate the contribution of a C2-methyl group on anti-C. difficile
potency (Table 1). Toward this goal, we prepared and tested C2-desmethyl (6d, MIC = 20/10 µM),
C2-ethyl (6e, MIC = 35/35 µM), C2-propyl (6f, MIC = 67/67 µM) and C2-meta-nitrophenyl (6g,
ACS Paragon Plus Environment

Page 17 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
MIC = 50/50 µM) analogues exhibiting potency comparable to that of 6a. Therefore, we
hypothesized that further extensions at the C2-position may be tolerated while expanding chemical
space to obtain potent compounds.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. The Minimum Inhibitory Concentration (MIC) of Compounds with Core
Modifications against Pathogenic C. difficile Strains
MIC
Compound
Structure
µM (µg/mL)
ATCC BAA 1870
ATCC 43255
O
O N
2
1
NH
335 (64)
312 (64)
335 (64)
N
O
NH
2
3
4
156 (32)
N
O
NO2
NH
NH
>794 (>128)
>718 (>128)
>794 (>128)
>718 (>128)
N
N
O
N
O
F
NH
5
>730 (>128)
>730 (>128)
N
NH2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
S
NH
N
6
a
19 (4)
41 (8)
38 (8)
20 (4)
35 (8)
67 (16)
38 (8)
41 (8)
38 (8)
10 (2)
35 (8)
67 (16)
O N
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
NH
N
6
b
O N
2
O
NH
N
6c
S
O N
2
O
S
NH
N
6
d
O N
2
O
S
NH
N
6e
O N
2
O
S
NH
N
6
f
O N
2
O
S
NH
N
6
7
g
a
50 (16)
50 (16)
O N
2
NO2
O
S
NH
>770 (>128)
385 (64)
N
ACS Paragon Plus Environment

Page 19 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
O
7
b
NH
NH
NH
>852 (>128)
>852 (>128)
768 (128)
840 (128)
N
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
c
192 (32)
420 (64)
S
N
O
S
7
d
N
O
N
S
NH
7
g
>560 (>128)
>560 (>128)
O
S
S
NH
7h
7i
>710 (>128)
>770 (>128)
>710 (>128)
>770 (>128)
N
O
NH
N
Vancomycin
Metronidazole
Fidaxomicin
-
0.7 (1)
0.3 (0.5)
1.5 (0.25)
-
-
0.7 (0.125)
0.1 (0.0625)
0.1 (0.0625)
Table 2 shows the MICs of C2-styryl analogues. The C2-styryl derivative 8a yielded a promising
MIC of 13/52 µM. Considering the favorable result, we replaced the phenyl ring with isosteres
such as furan-2-yl (8b, MIC = 27/27 µM) and thiophen-2-yl (8c, MIC = 13/13 µM) which
suggested the tolerance for these isosteric replacements. Next, we prepared carboxy substituted
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
derivatives of 8a and 8b leading to 8d (MIC = 46/184 µM) and 8e (MIC >384/>384 µM) with a
considerable loss of potency. This loss of potency may be attributed to unfavorable interactions
with the target and/or poor permeability. On the contrary, 4-fluorophenyl analogue (8f, MIC = 3/6
µM) showed excellent potency. A fluoro-scan was conducted to identify the most favorable
position for a fluoro group, which led to 2-fluoro (8g, MIC = 6/12 µM) and 3-fluoro (8h, MIC =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3/13 µM) analogues. Since 4-fluoro analogue yielded the potent MIC value, we explored
substitutions of different electron-withdrawing/-donating groups at the 4-position. While 4-nitro
analogue (8i, MIC = 24/12 µM) gave comparable activity, the 4-cyano analogue (8j, MIC
>
197/>197 µM) proved detrimental. The 4-chloro analogue (8k, MIC = 6/6 µM) being a classical
isostere of 4-fluoro group retained potency similar to that observed for the 4-fluoro analogue.
Retention of potency by 4-acetylene derivative (8l, MIC = 6/12 µM) indicated a tolerance for the
conformationally rigid acetylene group. Similar to the electron-withdrawing groups, electron-
donating groups also showed favorable potency as exemplified by 4-hydroxy (8m, MIC = 26/13
µM) and 4-methoxy (8n, MIC = 12/12 µM) analogues.
Table 2. The Minimum Inhibitory Concentration (MIC) of Compounds with C2
Substitutions against Pathogenic C. difficile Strains
MIC
Compound
Structure
µM (µg/mL)
ATCC BAA 1870
ATCC 43255
O
S
NH
N
8
a
13 (4)
52 (16)
O N
2
ACS Paragon Plus Environment

Page 21 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
S
NH
NH
8
b
27 (8)
13 (4)
27 (8)
13 (4)
O
S
N
O
O N
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
S
8
c
N
O N
2
O
N
S
NH
8
d
46 (16)
>384 (>128)
3 (1)
184 (64)
>384 (>128)
6 (2)
O N
2
COOH
O
N
S
NH
O
8
e
O
COOH
O N
2
S
NH
8
f
N
O N
2
F
O
N
S
NH
F
8
g
6 (2)
12 (4)
13 (4)
O N
2
O
N
S
NH
8
h
13 (4)
F
O N
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
S
NH
8i
24 (8)
>197 (>64)
6 (2)
12 (4)
>197 (>64)
6 (2)
O N
2
NO2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
S
NH
NH
NH
8
j
N
O N
2
CN
O
S
8
k
N
O N
2
Cl
O
S
8l
6 (2)
12 (4)
N
O N
2
O
N
S
NH
NH
8
m
26 (8)
12 (4)
13 (4)
12 (4)
O N
2
OH
O
N
S
8
n
O N
2
O
Vancomycin
Metronidazole
Fidaxomicin
-
0.7 (1)
0.3 (0.5)
1.5 (0.25)
-
-
0.7 (0.125)
0.1 (0.0625)
0.1 (0.0625)
ACS Paragon Plus Environment

Page 23 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The next SAR included investigation of various N3-substituents in lead 6a as shown in Table 3.
At the onset, we inserted N3-substituents such as benzyl (9a, MIC = 7/56 µM), phenylethyl (9b,
MIC = 13/13 µM) and phenylpropyl (9c, MIC = 12/12 µM) to investigate the influence of varying
linker length and obtained potency that was comparable to that observed for 6a. Based on these
analogues, it may be suggested that the binding pocket of N3-substituents is located further from
the N3-position. Based on the favorable MIC values and straightforward derivatization, we chose
benzyl analogue 9a for further SAR study. Scanning of the nitro group on the benzyl moiety at
different positions produced 4-nitro (9d, MIC = 6/12 µM), 2-nitro (9e, MIC = 23/23 µM) and 3-
nitro (9f, MIC = 23/23 µM) analogues. Considering favorable contribution of 4-substituents, we
prepared 4-cyano analogue (9g, MIC = 25/50 µM), which showed 4-fold reduced potency. The
electron-donating 4-methoxy analogue (9h, MIC = 24/24 µM) also suffered a loss of activity
compared to 9d. To determine the role of flexible methylene linkers in N3-phenylalkyl analogues,
we directly linked N3 with substituted phenyl rings leading to the synthesis of four derivatives
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(10a, MIC = 202/101 µM), (10b, MIC >386/193 µM), (10c, MIC >389/>389 µM) and (10d, MIC
=
352/176 µM) with rigid aniline substitutions. However, these analogues proved less potent than
the benzyl counterparts, and thus suggesting the importance of the methylene bridge. Loss of
activity of N3-aryl derivatives 10a-10d suggests the presence of a geometrically restricted target
binding pocket at the N3-vector and allows only for arylalkyl substitutent to bind effectively as
seen in 9a-9c. These findings prompted us to investigate untapped chemical space around 6a,
which led to N3-ethoxycarbonyl methylene analogue (11a, MIC = 54/54 µM), N3-tert-
butyloxycarbonyl methylene analogue (11b, MIC = 49/98 µM), and methylene carboxylic acid
(11c, MIC >475/>475 µM). This data suggested the tolerance for an ester group at N3-position.
The detrimental effect of a carboxyl group in 11c may be due to poor permeability through C.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
difficile cell membrane and/or unfavorable interaction with the target, which is consistent with the
activity loss observed for the other carboxylic acid analogues 8d, 8e, and 10b. Thus, we decided
to convert unfavorable carboxyl group to an amide by coupling it with an aniline, morpholine, and
N-methylpiperazine, respectively, to produce 11d (MIC = 46/23 µM), 11e (MIC = 47/94 µM), and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1f (MIC = 91/91 µM) with a >5-fold recovery of activity compared to the carboxyl analogue 11c.
Table 3. The Minimum Inhibitory Concentration (MIC) of Compounds with N3
Substitutions against Pathogenic C. difficile Strains
MIC
Compound
Structure
µM (µg/mL)
ATCC BAA 1870
ATCC 43255
O
N
N
S
9
a
7 (2)
13 (4)
12 (4)
6 (2)
56 (16)
O N
2
O
N
N
S
9
b
13 (4)
12 (4)
12 (4)
O N
2
O
S
N
N
9
c
N
O
O N
2
S
9
d
N
NO2
O N
2
ACS Paragon Plus Environment

Page 25 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
NO2
S
N
9
e
23 (8)
23 (8)
25 (8)
24 (8)
23 (8)
23 (8)
50 (16)
24 (8)
N
N
O N
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
NO2
S
9
f
O N
2
O
S
N
N
9g
N
O
CN
O N
2
S
9
h
N
O
O N
2
O
O
S
N
1
0a
202 (64)
101 (32)
193 (64)
N
O N
2
O
S
N
COOH
1
0b
>386 (>128)
N
O N
2
O
S
1
0c
>389 (>128)
>389 (>128)
N
N
O N
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
1
0d
352 (128)
176 (64)
S
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O N
2
O
O
O
S
N
N
1
1a
54 (16)
49 (16)
54 (16)
98 (32)
O
O
N
O
O N
2
S
11b
N
O N
2
O
OH
S
N
1
1c
>475 (>128)
46 (16)
>475 (>128)
O
N
O N
2
O
N
H
N
S
N
1
1d
23 (8)
O
O N
2
O
O
N
S
N
1
1e
47 (16)
94 (32)
O
N
O N
2
O
N
N
S
N
1
1f
91 (32)
91 (32)
O
N
O N
2
Vancomycin
-
-
0.7 (1)
0.3 (0.5)
Metronidazole
0.7 (0.125)
1. 5 (0.25)
ACS Paragon Plus Environment

Page 27 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Fidaxomicin
-
0.1 (0.0625)
0.1 (0.0625)
Our next SAR involved expansion of a chemical space around C4-position of 6a, which led to
synthesis of C4-chloro derivative (12a, MIC = 17/34 µM) as shown in Table 4. We noticed that
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
1
2a featured a significant change in the scaffold structure (transforming sp -hybridized N3 to sp -
hybridized N3) with increased planarity, and still exhibited comparable activity to that of 6a. We
took advantage of the ease of nucleophilically displacing the C4-chloro with aniline and
morpholine to obtain 12b (MIC = 28/14 µM) and (12c, MIC = 14/14 µM) with essentially retention
of activity as that of 12a.
Table 4. The Minimum Inhibitory Concentration (MIC) of Compounds with C4
Substitutions against Pathogenic C. difficile Strains
MIC
Compound
Structure
µM (µg/mL)
ATCC BAA 1870
ATCC 43255
Cl
S
N
1
2a
17 (4)
34 (8)
N
O N
2
HN
1
2b
S
28 (8)
14 (4)
N
N
O N
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
N
N
1
2c
14 (4)
14 (4)
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O N
2
Vancomycin
Metronidazole
Fidaxomicin
-
-
-
0.7 (1)
0.3 (0.5)
1.5 (0.25)
0.7 (0.125)
0.1 (0.0625)
0.1 (0.0625)
The SAR data gathered so far indicated the favorable contribution of C2-arylidene, N3-benzyl
and C4-chloro derivatives for C. difficile growth inhibition. Next, we decided to combine these
structural features into a single molecule to evaluate whether additive effect on potency could be
achieved (Table 5). This has led to the synthesis and testing of C2-, N3-disusbtituted analogue
(13a, MIC = 157/157 µM) with substantial loss of activity. The C2-, C4-disubstituted analogues
1
3b (MIC = 95/95 µM) and 13c (MIC = 166/166 µM) were also proved detrimental. This data
suggests that the disubstitutions oppose each other’s productive binding within respective pockets
of the target. Overall summary of the SAR findings is depicted in Figure 3.
Table 5. The Minimum Inhibitory Concentration (MIC) of Compounds with C2-and N3- or
C2- and C4-Disubstitutions against Pathogenic C. difficile Strains
MIC
Compound
Structure
µM (µg/mL)
ATCC BAA 1870 ATCC 43255
ACS Paragon Plus Environment

Page 29 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
N
S
N
13a
13b
157 (64)
95 (32)
157 (64)
95 (32)
O N
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
Cl
S
N
N
O N
2
F
O
N
1
3c
S
166 (64)
166 (64)
N
N
O N
2
F
Vancomycin
Metronidazole
Fidaxomicin
-
-
-
0.7 (1)
0.3 (0.5)
1. 5 (0.25)
0.1 (0.0625)
0.7 (0.125)
0.1 (0.0625)
ACS Paragon Plus Environment