N-(1,3,4-oxadiazol-2-yl)benzamides as antibacterial agents against neisseria gonorrhoeae

Article abstract of DOI:10.3390/ijms22052427

The Centers for Disease Control and Prevention (CDC) recognizes Neisseria gonorrhoeae as an urgent-threat Gram-negative bacterial pathogen. Additionally, resistance to frontline treatment (dual therapy with azithromycin and ceftriaxone) has led to the emergence of multidrug-resistant N. gonorrhoeae, which has caused a global health crisis. The drug pipeline for N. gonorrhoeae has been severely lacking as new antibacterial agents have not been approved by the FDA in the last twenty years. Thus, there is a need for new chemical entities active against drug-resistant N. gonorrhoeae. Trifluoromethylsulfonyl (SO₂CF₃), trifluoromethylthio (SCF₃), and pentafluorosulfanyl (SF₅) containing N-(1,3,4-oxadiazol-2-yl)benzamides are novel compounds with potent activities against Gram-positive bacterial pathogens. Here, we report the discovery of new N-(1,3,4-oxadiazol-2yl)benzamides (HSGN-237 and-238) with highly potent activity against N. gonorrhoeae. Additionally, these new compounds were shown to have activity against clinically important Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and Listeria monocytogenes (minimum inhibitory concentrations (MICs) as low as 0.25 μg/mL). Both compounds were highly tolerable to human cell lines. Moreover, HSGN-238 showed an outstanding ability to permeate across the gastrointestinal tract, indicating it would have a high systemic absorption if used as an anti-gonococcal therapeutic.

Full text of DOI:10.3390/ijms22052427

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International Journal of
Molecular Sciences
Article
N-(1,3,4-Oxadiazol-2-yl)Benzamides as Antibacterial Agents
against Neisseria gonorrhoeae
George A. Naclerio 1, Nader S. Abutaleb 2 , Marwa Alhashimi 2, Mohamed N. Seleem 2,3,4,* and
Herman O. Sintim 1,4,
*
1
2
3
Chemistry Department, Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA;
gnacleri@purdue.edu
Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette,
IN 47907, USA; nabutale@purdue.edu (N.S.A.); alhashim@purdue.edu (M.A.)
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine,
Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA
Purdue Institute of Inflammation, Immunology, and Infectious Disease, West Lafayette, IN 47907, USA
Correspondence: mseleem@purdue.edu (M.N.S.); hsintim@purdue.edu (H.O.S.)
4
*
Abstract: The Centers for Disease Control and Prevention (CDC) recognizes Neisseria gonorrhoeae as
an urgent-threat Gram-negative bacterial pathogen. Additionally, resistance to frontline treatment
(dual therapy with azithromycin and ceftriaxone) has led to the emergence of multidrug-resistant
N. gonorrhoeae, which has caused a global health crisis. The drug pipeline for N. gonorrhoeae has
been severely lacking as new antibacterial agents have not been approved by the FDA in the last
twenty years. Thus, there is a need for new chemical entities active against drug-resistant N. gonor-
rhoeae. Trifluoromethylsulfonyl (SO2CF3), trifluoromethylthio (SCF3), and pentafluorosulfanyl (SF5)
containing N-(1,3,4-oxadiazol-2-yl)benzamides are novel compounds with potent activities against
Gram-positive bacterial pathogens. Here, we report the discovery of new N-(1,3,4-oxadiazol-2-
yl)benzamides (HSGN-237 and -238) with highly potent activity against N. gonorrhoeae. Additionally,
these new compounds were shown to have activity against clinically important Gram-positive bac-
teria, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Naclerio, G.A.; Abutaleb,
N.S.; Alhashimi, M.; Seleem, M.N.;
Sintim, H.O. N-(1,3,4-Oxadiazol-2-
yl)Benzamides as Antibacterial
Agents against Neisseria gonorrhoeae.
Int. J. Mol. Sci. 2021, 22, 2427.
https://doi.org/10.3390/ijms
22052427
(VRE), and Listeria monocytogenes (minimum inhibitory concentrations (MICs) as low as 0.25 µg/mL).
Both compounds were highly tolerable to human cell lines. Moreover, HSGN-238 showed an out-
standing ability to permeate across the gastrointestinal tract, indicating it would have a high systemic
absorption if used as an anti-gonococcal therapeutic.
Academic Editor: Tanneke Den
Blaauwen
Keywords: Neisseria gonorrhoeae; 1,3,4-oxadiazole; antibiotic; antimicrobial resistance
Received: 8 January 2021
Accepted: 22 February 2021
Published: 28 February 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Drug-resistant bacterial infections have become a serious global threat. Neisseria
gonorrhoeae is a Gram-negative bacterial pathogen which causes gonorrhea, a sexually
transmitted infection (STI) [
the urethra, endocervix, pharynx, and rectum) [
complications, such as pelvic inflammatory disease, ectopic pregnancy, and increased
susceptibility to HIV infections [ ]. The Centers for Disease Control and Prevention (CDC)
considers N. gonorrhoeae an urgent threat, as it accounts for 550,000 infections per year and
$133.4 million dollars in medical costs in the United States alone [ ]. Globally, N. gonorrhoeae
is also devastating. The World Health Organization (WHO) listed N. gonorrhoeae as a
priority 2 (high) pathogen, as it is has caused 87 million new cases as well as an estimated
total treatment cost of $5 billion dollars [5–8].
1
]. N. gonorrhoeae infects a variety of mucosal surfaces (i.e.,
2] and, if left untreated, can cause drastic
3
Copyright:
© 2021 by the authors.
4
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Efforts to develop novel antibiotics against urgent threat pathogens, especially N.
gonorrhoeae, have intensified [
line therapies such as penicillin, fluoroquinolones, and cefixime, which are now deemed
9]. For instance, N. gonorrhoeae is now resistant to former front-
Int. J. Mol. Sci. 2021, 22, 2427. https://doi.org/10.3390/ijms22052427
https://www.mdpi.com/journal/ijms

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Int. J. Mol. Sci. 2021, 22, 2427
2 of 12
ineffective as treatment options [10]. This increased resistance rate prompted a global health
scare, leading the CDC to recommend treating N. gonorrhoeae with dual therapy involving
ceftriaxone and azithromycin [
leading to the rise of multidrug-resistant N. gonorrhoeae (commonly referred to as super
gonorrhea) [ ]. To make matters worse, no new classes of antibiotics to treat N. gonorrhoeae
1,11]. Yet, resistance to this dual therapy has been reported,
2
have been FDA-approved over the last two decades, warranting the public health concern
that once easily treated gonorrhea infections will soon become deadly [12–14]. Therefore,
the rise in multidrug-resistant N. gonorrhoeae infections necessitates intense research efforts
to identify and develop new antibiotics.
Our program focuses on the development of N-(1,3,4-oxadiazol-2-yl)benzamides to
treat drug-resistant bacterial pathogens [15,16]. We recently reported the discovery of
trifluoromethylsulfonyl (SO2CF3), trifluoromethylthio (SCF3), and pentafluorosulfanyl
(SF5) containing N-(1,3,4-oxadiazol-2-yl)benzamides that exhibited potent antibacterial
activities against clinically important Gram-positive bacterial pathogens [17]. These agents
were found to be active against clinical isolates of drug-resistant Gram-positive bacteria,
were non-toxic to mammalian cells, and effectively reduced the burden of intracellular
methicillin-resistant Staphylococcus aureus (MRSA) [17]. Here, we describe a new generation
of N-(1,3,4-oxadiazol-2-yl)benzamides with potent activity against N. gonorrhoeae. The
antibacterial activity against N. gonorrhoeae, cytotoxicity against mammalian cells, and
bi-directional Caco-2 permeability were investigated.
2. Results and Discussion
2.1. Synthesis and Antigonococcal Activity of N-(1,3,4-oxadiazol-2-yl)benzamides
We previously reported that trifluoromethylsulfonyl (SO2CF3), trifluoromethylthio
(SCF3), and pentafluorosulfanyl (SF5) containing N-(1,3,4-oxadiazol-2-yl)benzamides
(
compounds 6
Gram-positive bacteria [17]. We wondered if these compounds would be active against
N. gonorrhoeae and discovered that compounds 6 12, and 13 have quite potent activity
against N. gonorrhoeae strain 181, with minimum inhibitory concentrations (MICs) of 0.5,
0.06, and 0.06 g/mL, respectively (see Table 1). While all three compounds have favorable
, 12, and 13, respectively) were potent against a panel of drug-resistant
,
µ
CLogP values, they also contain an unsubstituted thiophene moiety (Figure 1) which can
cause toxicity. For example, the cytochrome P450-mediated oxidation of thiophene moeities
can lead to reactive metabolites such as thiophene epoxides [18], thiophene-S oxides [18
and sulphenic acids [20], which can react with nucleophiles such as glutathione and/or
water [21]. However, since Compounds 6 12, and 13 have shown excellent activities
,19],
,
against N. gonorrhoeae as well as adequate CLogP values, we desired to further optimize
these compounds via the synthesis of new analogs. We proceeded to use computational
methods to guide our synthetic strategy. We began to substitute the benzamide ring with
the trifluoromethoxy (OCF3) group due to its importance in medicinal chemisty [22,23].
For instance, it was reported that the electronegativity of the OCF3 group allows for en-
hanced in vivo uptake and transport in biological systems [22]. Thus, utilizing this strategy
has led to the synthesis of HSGN-235, which contained a fluoro atom ortho to the OCF3
group as well as a trifluoromethyl phenyl. Yet, HSGN-235 was found to contain a much
larger CLogP value compared to previously synthesized analogs (Figure 1). Since LogP
shows a positive correlation between low aqueous solubility and compromising bioavail-
ability (an extremely important attribute when creating antibacterial agents against N.
gonorrhoeae) [24], we replaced the thiophene moiety with a substituted thiophene or
phenyl group as unsubstituted thiophene could be a toxicophore, as mentioned above.
Considering that the addition of halogens to compounds has been shown to improve drug
properties and metabolic stability [25–29], our new analogs were made up of compounds
with halogen substitutions to a phenyl ring (Figure 1).

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Figure 1. Previously reported analogs as well as newly synthesized N-(1,3,4-oxiadizol-2-
yl)benzamides for this study. Note: CLogP was calculated using SwissADME.
The synthesis of these compounds started with a substituted aryl aldehyde, fol-
lowed by the addition of semicarbazide and sodium acetate to give the corresponding
semicarbazone. Then, using bromine and sodium acetate, the semicarbazone was con-
verted into the subsequent aryl 1,3,4-oxadizol-2-amine. Amide coupling between the
aryl 1,3,4-oxadiazol-2-amine and 4-trifluoromethoxy benzoic acid using benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent gave the de-
sired N-(1,3,4-oxadiazol-2-yl)benzamides (Scheme 1).
Scheme 1. Synthesis of N-(1,3,4-oxadiazol-2-yl)benzamides. Reagent◦s and Conditions: (
a) Semicarbazide hydrochloride,
NaOAc, MeOH:H2O (1:1), rt, 30 min. (b) Bromine, NaOAc, AcOH, 60 C, 1 h. (c) BOP Reagent, DIPEA, DMF, rt, 12 h.
Trifluoromethoxy containing (1,3,4-oxadiazol-2-yl)benzamides with the substitution
of the thiophene moiety with a fluorophenyl (HSGN-237) or chlorothiophenyl (HSGN-238
)
groups had potent activity against N. gonorrhoeae strain 181 with MICs of 0.125 g/mL
µ

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(Table 1). Interestingly, the substitution of the 4-trifluoromethoxy phenyl group with a
fluorine, as well as the substitution of the thiophene moiety with trifluoromethylphenyl
(
HSGN-235) only had moderate activity when tested against N. gonorrhoeae strain 181
(Table 1). Since both HSGN-237 and HSGN-238 contained aromatic rings bearing a halo-
gen atom, we speculate that the loss of activity for HSGN-235 is due to the addition
of the fluorine atom ortho to the trifluoromethoxy group (see Figure 1 and Table 1 for
comparisons).
Table 1. MICs (µg/mL) of the previously reported analogs (compounds 6, 12, and 13) and the
new compounds (HSGN-235, -237, and -238) against N. gonorrhoeae strain 181. The experiment was
repeated 3 independent times and the same MIC values were obtained. Compounds tested are in
bold while control drugs are in regular script.
Compound/Control Drug
N. gonorrhoeae Strain 181
Compound 6
Compound 12
Compound 13
HSGN-235
HSGN-237
HSGN-238
0.5
0.06
0.06
16
0.125
0.125
256
2
Azithromycin
Tetracycline
After the initial screening against N. gonorrhoeae 181, the anti-gonococcal activity
of HSGN-235 -237, and -238 was explored against a panel drug-resistant pathogenic N.
,
gonorrhoeae strains, including one WHO reference strain (N. gonorrhoeae WHO L) which
has a well-characterized antibiogram and phenotypic and genetic markers [30]. As depicted
in Table 2, HSGN-237 and -238 exhibited potent activity against the tested strains, with
inhibitions of their growth at concentrations ranging from 0.03 to 0.125 µg/mL. Both
were superior to azithromycin and tetracycline against the tested isolates. On the other
hand, HSGN 235 inhibited the growth of the tested strains at concentrations ranging from
1 to 2
µg/mL. Interestingly, the minimum bactericidal concentration (MBC) values of
HSGN235
,
-237, and -238 were the same as or one-fold higher than their corresponding
MIC values, indicating that the compounds exhibit bactericidal activity against the tested
N. gonorrhoeae strains.
Table 2. MICs and MBCs (
µg/mL) of HSGN-235, -237, and -238 against N. gonorrhoeae clinical isolates. The experiment was
repeated three independent times and the same MIC values were obtained.
HSGN-235
MIC MBC
HSGN-237
HSGN-238
Azithromycin
Tetracycline
MIC MBC
Bacterial Strains
MIC
MBC
MIC
MBC
MIC
MBC
N. gonorrhoeae 165
N. gonorrhoeae 166
N. gonorrhoeae 194
N. gonorrhoeae 197
N. gonorrhoeae 200
N. gonorrhoeae WHO L
2
2
0.06
0.06
0.03
0.03
0.06
0.06
0.125
0.06
0.06
0.06
0.06
0.06
0.125
0.125
0.125
0.125
0.125
0.06
0.25
0.25
0.125
0.125
0.125
0.125
1
4
1
0.5
2
0.5
1
4
2
1
2
2
8
2
1
1
2
1
2
1
2
2
2
0.5
0.25
0.5
0.5
0.5
8
4
4
8
2
0.5
2.2. Antibacterial Activity of N-(1,3,4-oxadiazol-2-yl)benzamides against Other Bacterial Species
While the focus of these new N-(1,3,4-oxadiazol-2-yl)benzamides is towards N. gonor-
rhoeae, we proceeded to test their activity against other Gram-positive and Gram-negative
pathogens. Intriguingly, HSGN-235
against the tested Gram-positive bacterial pathogens. For instance, all three compounds had
potent activity against the staphylococcal strains, with MICs ranging from 0.25 to 1 g/mL
(Table 3). Furthermore, HSGN-235 HSGN-237, and HSGN-238 maintained potent activity
, HSGN-237, and HSGN-238 had potent activity
µ
,